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,
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A,k no\1i lco.l~emenIS
•
Preface
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InuothK110D
7
r an 1
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Pari 2
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The red cells and their precursors ,\'0 , ,"<11fo rms and ahnomwl \'arjaTlfJ ., Granu lUl;)tes. monocyte» and ,ne!lilk ,t TytX-Y' CS N urnllll and nhnormal [n rm'
...
"
Parl3 ...yrnphocy tcs , plasma cells a nd their de rivatives and prccu r""rs in t>1
f'JI
Part 4 :\h lol:c ll.:incous cell~ trom hone marro.. or Mood S OU:"IS. relicul()oenJot he lial cc 11s. oaeoclasts and O!ItlNh!as,s. foret,," cells and parasites
PanS
32<
Im prin ts a nd sections 0 1lymph nodd>
aed spleen APf'C odi x: staining techniq ues
A hbrc\ iat ion s
Index
.i T·
Part 1 The red cells and their precursors Norm al fo rms and abnormal variants
The nome nclat ure of red-cell prec ursors is co nfusing. The earliest recognizable member of the red-cell series. the procryrhro btast , has the cytoplasmic basophilia. the nuclco latcd and moderately lcptochrornatic nucleus and the large cell size gene rally characteristic of primitive ce lls. It gives rise to a seq ue nce of nucleat ed cells. the erythroblasts , which progressively develop increasing ly pac hychromatic nucl ei . lose their nucleoli and their cyto plasmic basophili a and acq uire a rising haemoglobi n con te nt, Th is seq uence is subject 10 an arbi trary divisio n into stages. the co mmonest division being into three : l. Th e baso philic o r early ery throblast . or no rmoblast A 2. Th e polychro matic o r inte rmed iate e rythro blast . o r normob last B 3. Th e o rthoc hromatic or late ery thro blast. o r norm oblast C.
Reti culocytes spe nd up to two days in the bo ne marrow before being released into the peripheral blood. T here they make up nor mally less than I % of the redcell populatio n and within a no ther o ne to two days lose the remn ant s of cytop lasmic basophilia which give them their cha racte ristic staining properties. and become orthochromati c mature red ce lls. Mature red cells survive some 120 da ys before destruction. They ar e normally circula r and fairly uniform in diam eter. but arc readil y distorted by exte rna l pressure . as fro m neig hbo uring cells in a smear. Their stru ctu re as bico ncave discs leads to wea ker eosinophil staini ng at the centre than at the pe riphery. a fea ture which is least prominent at the tail of a blood smea r whe re the ce lls are most spread o ut and flatten ed . In the body of the smea r it becom es more conspi cuou s and this normal phenomenon must be appreciated and distinguished from hypochromia . Abnor ma l var iants: nucleated prec ursors
T he re are objectio ns to the use of many of these ter ms. but the y are all so firmly entrenched in co mmon usage that they must he accepted . When autho rs use diffe rent or more elaborate staging and nomenclature they usually de fine their terminology. but those who use any of the syno nyms abov e expect them to he under stood without furt he r explanatio n. T he proeryth robla st is not itself the function al ste m cell se rving as a self-maintai ning progenito r of the nor mo blast series. but is de rived from an earlier func tiona l myeloid stem cell of unide ntified ' mo rphology. having pluri pote nt ial capacity for giving rise to cells of eryt hro id. gra nulocytic . monocytic and megakaryocyte -plate let lines. Kine tic studies with radi o-isotopically labelled ce lls suggest that four cell cycles culminating in mitoses occur duri ng developme nt from proerythroblast to late no rmoblast. three at the procr yth robl ast and ea rly basophilic no rmoblast stages. and the last at the po lychro matic intermediate nor moblast . stage. Nests of erythroblasts of diffe re nt stages of maturity co mmo nly occur in appositio n around a ce ntrally situated reticuloendothelial cell . T ra nsfer of iro n may he effect ed in o nc or ot her d irectio n. and the ce ntra l macrophage is ofte n rich in stai nable free iron . Late normoblasts do not unde rgo a furt he r cell cycle but lose their nucl ei by ext rusion and give rise to marrow re ticulocytes .
Th e chief cytological variants involving altera tion in morph ology rathe r than numbers or relative pro portions o f c ryth rob lasts are as follows:
Macronormoblasts. Cells havin g the general nuclear and cyto plasmic morpho logy of normoblasts but an increased average cell diamet er. Th ey occur especially in sta tes of active ery throhlast ic hyperpl asia without haem atinic defects. such as haemolytic anae mias , but may be seen also in the early stages of d isorders that subseque ntly become megalob lastic, Mcgaloblasss. The esse ntial morphological cha nge in mcgnlobla sts as co mpared with normobl asts lies in the more open chromatin pattern of the nucleus at all stages of developm ent. T he re is often a de gree of haemoglobini zation of the cytoplasm which appea rs excessive for the sta te of nucle ar maturation . and later megaloblasts may appea r fully or thoc hromic. Mcgaloblasts are lar ger than no rmo blasts of comparable maturi ty_ Th ey may sho w mitoti c irregularities . with multipolar mitoses.' sometimes asymmetrical. producing un equal daughter ce lls.'Cyto kinesis may not occu r and giant cells with two or mor e nucle i. so metimes uneq ual in size . result . Chromosomes or fragments of chromatin may become separated from the spindle and constitut
accessory small nuclear masses : Th ese ' Howcll-Jolly bodies' may rem ain in the red cells afte r the mai n bulk of nuclear materia l has bee n extrude d. Rosette formation. wit h several chrom atin masses linked by bridges. may follow a halt of the mito tic process in metaph ase . Megalo bla ts are present in the marrow in- state s of B 1:, and folic acid deficie ncy. the com mo nest of which are Add isonian pernicio us anae mia and cond itions of gastro-i ntcsti nal malabso rption d ue eithe r to disease or to ga_trie o r intestinal resectio ns. Th e folic acid deficiency of pregnancy may lead to megalob lastic anae mia. Megalo blasts arc see n also in so me cases of refracto ry siderob lastic anaemia and in £!)'1bmclJliL m~s . The y may be seen in acute leukaemi as. especially followin g treatme nt with antimet abolitcs .
Micronor moblasts. Sma ll normob lasts with a tcnd ency ragged cyto plasmic ou tline a nd irre gular sta ining a re found in slates of iro n deficien cy. Th e cha nges a re most evident in interme diate , and late normob lasts. which also show defective haemog lohini zation .
10
Side roblasts. Normobl asts containing free . non -haemoglobin, iron are best detected by the use of Prussian blue staining , but occasionally coarse accumulat ions of free iron may be visible in Romanowsky preparations.
Bizarre cytological variants. G ross mitoti c and nuclear abno rmalities. of the same kind as describ ed und er megaloblas ts but much more ex tre me in degree . occ ur in e rythraem ic myelosis. Reticule cytes
Blood normally contai ns less than I % of ret iculccyt cs and an increase in numbers indicates a heighten ed o utput of young red cells from the bon e marr ow. Th is may occur durin g the co rrectio n by increase d marrow activity of any anaemic sta te . whet her spo ntaneo usly (as afte r an acute haem orrhage ) or following trea tment (as in pern iciou s anae mia aft er BJ2 or iro n deficiency anae mia afte r iron) . Reticul ocyto sis is especia lly promine nt in haem olytic ana e mias , whe re great e rythropoie tic activity att empts to co mpe nsate for the shorte ned life spa n of periphe ral erythroc ytes . In Ro manowsky prepara tion s reticulocyrcs may sometim es be detected as po lychro matic cells. with a blue or pu rple tinge. -or much less often may show a scatte ring of fine blue ' baso philic stipples'. Th ey arc best recognized . ho wever . by the use of supravit al staini ng with brilliant cresyl blue . which reveals a netwo rk of fine filaments and dots. Re ticulocytes a rc ofte n a little lar ger in diam ete r than mature red cells. Matu re red cells Red ce lls may diffe r from norm al in their appea rance in stained films by varia tio ns in size (a nisocytos is. microcytosi s. macrocytosis). in shape (po ikilocyt osis. elliptocytosis. sickle cell formatio n. cre scent formati o n. crenatio n) and in dept h of sta ining (hypoc hro mia . ani sochrnmi a . spheroc ytos is. target cell formatio n) . Th ey may also und ergo fragme ntatio n (schisto cytosis)
or co nta in abnormal inclusio ns. such as the r chro mat in materia l of the Howell -Jolly bodies. All these ab nor malities may be seen in vaf)i n ~ deg ree and frequ ency and severa l of the m may occur togeth er in the same smea r. Disorders tnvotving chiefly the red- cell series. and thri r associated main cytological features
Megalo blastic anaemias. These disord ers are characterized by striking erythro blastic abnormalities arising from deficiencies of e ither vitamin B l 2 or folic acid . The und erl ying bioch emical defect involves a slowing of DNA synthes is in (he S phase of the cell cycle . due principally to defective formation of thymidylate . which is normally depend ent on bot h BJ2 a nd folate as eofacto rs in its synthesis. Nuclear division is thus retarded . and the patt ern of nucle ar chro matin disposition and nuclear- cytopl asmic synchro niza tio n in develop ment is affected . with the result s illustrat ed in 39-69. In fact, the changes of megalohl asto sis. with increas e in ce ll size and opening of the nuclear chroma tin network . arc detect ahie in most prol iferating cells. such as those of the gut mucosa. and includi ng granulocyte and platelet precur sors in the bo ne marrow. The findings in bo ne marrow aspirates and biopsies include high ce llularity . the hyperplasia involving mainly the red -cell series. with mycloid-erythro id (M :E) ratio comm onl y I :1 or even 1:2 - co mpared with the nor mal range of 5-20: 1. The re is a shift to the 1eft in red -eell pre cu rso rs. with a n increased proport ion of large proe rythro blasts and ea rly basophil ic e rythrohl asts, num e rous mito tic figures - often sho wing Howell-J olly bodies - and the typ ical prem ature haemoglobinizat ion best seen in interme di ate c rythrob lasts. G iant myclocytes and mct am yelocyte s ca n be fo und. as can multinuclea ted po lymorphs ( 7~) . and par allel changes involving asynchrony of nuclear- cytoplasmic maturation . with gra nular cyto plasm and relatively primitive nuclear structure. so metime s in mitosis. occur in promegak aryocytes , while nuclear hypcrse gment at ion may be co nspicuo us in mature mega karyocy tes. Macrop hagcs usu ally show an increase d load of haem osiderin . with the fast turnover patt ern of small particle s. resulting from the con sider able co mpo nent of ineffective erythro poiesis and co nseq uent intra med ullary breakdown of non- viable red-ee ll precurs ors. The periphe ral blood find ings include a more or less severe ana emi a with normoc hromic but ma rkedly macrocytic erythrocytes. the mean cell volume (MCV ) usually exceedi ng 115 fe mtolitres (ft) and reaching as high as 150ft. Th e mean ce ll haemoglob in (MCII ) is raised abo ve 32 pico grams (pg) . but the mean cell haemoglobin concent ration (MC HC) is normal . not over 36% . A nisocyto sis and poi kilocytos is appear ea rly in the co urse of the anae mia and become increasingly seve re . large ovalocytcs bei ng particularly characteri stic. but other shapes . including tear-drop poiki locyte s, may also be see n (711-73) . Th e red cells may occasion ally show basophilic stippling ( 126) and I lnwcllJolly nucle ar fragmen ts ( 146). but reticulocytes arc not usually increase d and may be depresse d. As the anaemi a
l
pro gresses. occasional late eryt nrob lasts with residual meg alobl ast ic features appea r in the circ ulation . Leuc openia aro und 2-t x 10"'/1 is usual in mega loblast ic an aemias. pe rha ps especia lly in perniciou s anaemia . with neutrop e nia and a relative Iymp hocytosis . The neutrophils show a shift to the right with increase d nuclear lobulation. most cells having fOUT lobes a nd some hypcrse gmentc d cells having five or six. Neutrophils may also manifest cellular gigantism - these exce ptio nally large cells are known as macrop olycytes . Platelets may be present in normal number s. but thrombocytope nia is more commo n and is sometimes severe . while giant platelet s and occas iona l megak ar yocyte fragme nts provi de furthe r evide nce of megaloblastic dyst hro mbopoi esis. Haemol ytic anaem ias. Excessive destruction of red cells arises from two ver y broad gro ups of causes. intri nsic and ext rinsic. In the first group are defec ts of the cell constitution. ranging from heredit ary spherocy tosis ( HS). enzyme defects such as pyruvate kinase (PK) and glucose -6-phos pha tc de hydroge nase (G6 PD) deficien cies, and paroxys mal noctu rnal haemoglobinuria (PNH) . to haemoglobin opathi es such as the th alassae mias and sickle cell disease. The secon d grou p includes acqui red auto- immune hae molytic anaemias (A IHA) due to warm lytic antibod ies active at the redcell surface: cold hae magglut inin disease (C HAD) due to high titre agglutinating antibod ies. usually with ant i-I specificity: and hae molysis seconda ry to toxins. foreign an tibodies as in mismatched transfusions, and circulatory impedime nts such as occur in microangic pathy. thromh otic thrombocytopen ic purpura (TIP) . ren al glomerular disease . dissemina ted intravascu lar coagulation ( DIC) and artificial vascular prost heses . All these conditions share features in common. arising from the increased breakdown of red cells. with resulting raised levels of seru m bilirubin and disappe ara nce of haptoglobins, and attempt s at compensatory marrow erythro poietic hyperpl asia. with norm oblastic proliferation. a redu ced M:E rat io. and an outpo uri ng of rcticulo cytes, sometimes accompa nied by late normcblasts. into the pe ripheral blood . The intense normoblastic hyperplasia. with oblite ration of fat spaces and 100% cellularity in trephin e sections . from a seve re hae molytic anaemia due to PK deficiency . is shown in 19 and 20. and a less marked but substantial erythro id component in sections from a case of AIHA seco nda ry to non-Hodgkin's lymphom a (N HL) is shown in 21 and
22. Tbe anae mia of hae molytic disease is usually normocbromic and neither macrocytic nor microcytic. although ~1~ ~1osb
and poikilocytosis are common. Neutrop hil may be seen. especially in acute haemolytic reaction . but the count is ofte n normal. Apa rt from (he acqu ired hae molytic processes associated with microan giopa thy, TIP and DIC. where there is seve re thrombo cytope nia . platelet levels show no consiste nt trend . The chief morpho logical findings of differential diagnostic value among this wide range of diseases are therefore to be found in the circu lating ery throcytes themse lves. alth ough they are rare ly pathognomonic and need confirm ation or supplementation by appro-
priate immunologica l studies. e nzymology. or haemoglobin electrop horesis. as the case may be . Nevertheless. red-cell morph ology can offe r some useful pointers . Spherocy tes ( 135-137). apparen t in stained blood smears as small. deeply stained red cells. are always present in HS and commo nly de tectab le in A IHA . Elliptocytcs (138) formin g more than 5% of the redcell populat ion are virtually confined to her editary e lliptocytosis. but a few marked ly oval or cigar-sh aped red cells may be found wheneve r gross anisocytosis is presen t, as in seve re megalobla stic or iro n deficien cy anae mia and in thalassa emia. and a localized elllprocytotic appe arance may be prod uced in some areas of a smear as a spread ing artefact (139). Echinocytes or 'burr cells' , erythrocytes with numerou s surface undul at ions or spicules ( 141), may be produced by exposu re of nor mal 'discocytes' to an alka line pH or to vario us cchinocytoge nic factors such as bile acids, lysolecithins and fatty acids. Although the change is rever sible, it is associa ted with a tend ency to sphe ring a nd subseq ue nt haemolysis. In path ological states. echinocytes occu r especia lly in uraem ia. chronic liver disease , and acutely in severe bums and toxic infections. Acanth ocytes or 's pur cells". erythrocy tes with sha rp spiny irreg ular projections ( 1.13). arc also associated with a haemolytic tende ncy. and occur in heredita ry acanthocytosis or congenital abeta ltpopro teinacm ia. and may develop in liver disease or after splenec tomy. Stomatocytes , cup-shaped erythrocytes which in stained smears sho w an elo ngated slit of central pallo r instead of the norm al circular area ( 128 and 158). are found conspicuously in heredita ry stomatocyrosis. and not infrequently in haemolytic anaemias with altered membra ne pe rmeabil ity. A few may usually be found in normal blood smea rs. and thei r numbers may be increase d in hep atic cirr hosis and in many generalized neoplastic sta tes . D repanocytes or sickle cells ( 155) are characteristic of haemoglobin S disease , being produ ced by the crystallization of reduced HbS within the cell. Codocytes or target cells . bell-sha ped eryt hrocytes with a striking target appeara nce in stai ned smea rs (150. 151 and 153). are especia lly promin ent in thalassaemia, but are found also in othe r haemoglob inopat hies , such as HbC and HbSC disease . Leptocytes, or thin flat ery throcytes (150) . occur in the same conditions as target cells. and also in liver or gall bladd er disease with biliary obstruc tion. and after splenecto my. Schistocytes , red cells with irreg ularly fragme nted sha pes due mostly to mechan ical da mage (134. 152. 163 and 164). a rc found especia lly in micro angiopath ic haem olytic anae mia and the related states o f DIC . TIP and microva scular nep hropath y. Heinz bodies (160) may be dem onstrable in the red cells in supravitally stained preparation s of blood from patient s with haemol ytic anae mias due to G6PD deficie ncy. unstable hacmoglobins, chemical dam age or thalassa emia. They arc someti mes remov ed by the spleen a nd therefor e are parti cularl y conspicuous after splenec to my.
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Haemoglobin H inclusio ns. which stain with supravital cresyl blue a fte r prolo nged exposure ( 162), are found in the red cells of alpha-thalassaemia. A part fro m these vario us pointers. the differential diagnosis bet ween haemolytic disorders is not greatly assisted by morphological featu res present in the pe ripheral blood. and the only contributory differential findi ng in the bone marro w. where normo blastic or macrono rmoblastic hyperplasia is the co mmon ru le. is the existence of strong periodic acid-Schiff ( PAS) posiuvity in the erythroblasts of beta-thalassaemia major and to a lesser exte nt in th ose of be ta- tha lassaemia minor. Ot her haemo lytic sta tes o nly ra rely sho w any eryt hro blastic PAS pc sitivity. altho ugh this was a fea ture of foetal haemo lytic d isease . now fortunately preventable and therefore uncommon where there are adeq uate medical services.
Aplastic and hypoplastic anaemias. Despite the titular e mphasis o n anae mia. these diseases arc usuall y pancyto penias, with acco mpa nying neut ropen ia and thrombocytopenia. all beco ming progressively mo re severe as the disease adva nces . and associated with failure of myelopoiesis. The bone marrow is poorly ce llular. with a predominance of fatty spaces and a reactive o r inflammatory inliltrat e ranging from a few residual islands of Iymphocytes and plasma cells to widespread d iffuse Iympho plasmacytosis. Marrow aspirate s are generally acellular. and firm diagnosis req uires trephine biopsy. Typical appearances at diffe rent stages of disease arc shown in 24--27. The anae mia itse lf is usually no rmocytic and normo chromic with litt le anisocytosis o r poikilocytosis . and re ticulocyres arc scanty. Pure red-cell aplasia . witho ut concomitant decrease of granulocyt es and plate lets. and wi th a cellular marrow de void of red -cell precurso rs. occ urs o nly rarely. Primary and seconda ry po tycytnaemios. Polycythaemia rubra vera ( PRV) o r primary proliferative polycytbae mia is characte rized by high haemoglobin . haematocrit and red-cell count . a raised red -cell mass. oonnal arterial oxygen saturation. rommonly significant plenomegaly. and usually elevat ed to tal leucocyte and platelet counts. It is thus a pancytosis. and the high peripheral blood figures reflect a chron ic neopl astic myeloprolife ra tive state involving all cell series in the bone marrow (28 and 29). whe re fat spaces arc much reduced in trephine sectio ns and trilineage hyperplasia ma) approach 100% ceJl ularity. Cytogenetic st udies in the stable ph ase of PR V re vea l non -random chro mosomal abnormalities in many patients. trisomy H. trisomy 9. 5q- and. especially. 2Oq- being the most com mon . thus co nfinning the clo nal neoplastic nature of the disea se . Morpho logica lly. there arc few changes from normal. with e rythro poiesis normoblastic and eryt hrocytes no rmoc hro mic and normocytic. and with bot h gra nulopoiesis and th rom bopoiesis regular and cytolo gically typical. apart from an occa sional increase in basophils and a few gian t plat elets. at least during the main course of the disease . The one abnorm al cytochemical fea ture almost invariably fo und is a raised
leucocyte alkaline phosph atase (LA P) sco re. A late transition to a myelo fi brotic. state occ urs in around 15-20% of PRY cases . and when this change ta kes place the marrow sections show . of course. increasing reticulin and fibrosis (as in 30). and the peripheral red cells manifest anisocy tosis and teardrop poikilocytosis. Terminal tr ansformation to a preleu kaemi c myelodysplastic state or to fra nk acute myelo id leukae mia (A ML) may also occur. Seco ndary polycythaemia or erythrae mia resembles PRY in having raised haemoglobin. haematocrit . erythrocyte count and red -cell mass. bu t differs in the absence of leucocytosis and th rom bocytosis. and . when d ue to chro nic cardiovascul ar or pulmonary disease , in showing decrea sed arterial oxygen saturation. Morphologically. erythroid hypcrpla-..ia predominates in the bone marrow . withou t paralle l increase in o ther lineages. and the overall cellularity of sections is ge nerally less marked than in PR Y. as exemplified in 23.
Congenital dyserythrop oietic anaemias (CDA s). This group of relatively uncommon disorders is of parti cular interest to the cytologist because the dyseryt hro poie nc abnormalities manifest in the erythroolasts are so striking and bizarre . There are thr ee main varieties of CDA . Type I affects the later basophilic and intermediate erythroblasts predominan tly. with megaloblastoid features and spo ngy chromatin patterns sometimes giving an appearance of irre gularity in the nuclear membranes. and with occasion al internuclear chromatin hridges. Type 11 . the com mone st form . affects later ervthroblasts especia lly. with conspicuous muitinuclea n ty and with double nud ear membranes seen b)' electro n microscopy. In this type of CDA the erythrocytes are susceptible to haemolysis in an acid medium. hence the alte rnative na me . HEMPA S - hereditary erythroblastic multi nuclearity with positive acidified serum test. Type III is characterized by giant eryth roblasts. up to 70 micro ns in d iamet er. with either num ero u separa ted nuclei or a single rnultiplo id lobul ated nucleu . Exa mples of all these vari an ts of CDA and their mai n cytological and cytochemical features are illustrated in 78-88 . Erythruemic myelosis and erythroleukaemia. Alt hough an acute neoplastic state with vast preponderance of erythroblasts has lon g been recognized as erythrae mic myelo sis or Di Gu glielmo's disease. it is probable that this occ urs rarely if ever as a pu rely monolineage disorder. but that it is almost always. if not always. a bilineage o r trilineage disease from the sta rt, with involve ment to a greater or less exte nt of the gra nu locytidmonocytic line and the megakar yocytic line . The name 'eryth roleukaemia' is the refore strictly more co rrect in all cases. although erythrae rnic myelosis is still co mmonly us-ed for cases where the erythroid co mpon ent is clearly dominant. Cytoge netic abno rma lities are found in the maj ority of cases . including (he co mmones t AM L alte ra tion. trisomy 8 (8 + ), and defects of 5 and 7. but multipl e nume rical and structural abe rra tions are usual. Th e cyto logy of the neoplastic erythroblasts in the bo ne
marrow and in the blood . where they may be numerous. vane... fro m relauvelv normal to zrosslv bizarre (89-95) . C~ tochemica llv. the~ usually show conspicuous PAS IX Iti\ ity (98- 102). and ofte n strong localized acid pho-phatasc reactions ( 103 and I~ ) .
Refractory sideroblastic illJaemias (RSA 'i ). These diso rde rs are characterized by a n overload of iro n in the bo ne marrow ( 112) . and by hypercellular micronormobla stic or occasiona lly rne galo basti c e rythropo iesis. Coa rse sidero tic gra nules . demon strabl e by the Prussian blue stain. a re found a rra nge d in a rin g around th e nucleus of man y c rythro btas rs. T he a ppea ra nce is mo st st riking a nd involves the highest proportion o f c ryth rob lasts in cases of primary acquired RSA . a neepla...tic mye lodys plastic sta te which may tran sform to A ~ lL. Exa m ples o f the cyto logy a nd of ringed sidcroblu ... ts are shown in 113-116. In hereditary RSA and in acq uired cases seco nd a ry to age nts such as iso niazid, cyclose rine . chloram phe nicol. a lco ho l a nd lead. th ere a rc ue ne ra l!v fewe r sidc roblasts. a nd ontv interme dia te a nd ~I ate ervth roblas ts a rc affected. . PAS posit ivity is found in a p ropo rtio n of the crythroblas ts in so me cas es o f RSA . alt ho ug h the reaction is usua lly m uch we ak er than in c rythracrnic crvthroblasts
•
•
cvt osi s and poikilocytosis ( 0 1 and 133
indices, ~t C \' .
•
~ICH
and MCHC. aD O«_I
platelet count is often raised . U ntil ,rOIl U·. ....... started the rericulocvte count is 10.... and pe ripheral polyc hromasia . Ervthropoese, iD ma rrow is hype rp last ic. but with a bnonnal ~106~ e ryt hroblasts being small. poorly hac TTlOgIobiDizrd. ofte n with irregular and ragged outlines ( I aDd I The y may contai n PAS-po siti\·e mat e rial (l J marrow is not abl y de void of free iron whether in ~ phages o r e xtra-ce llula rly in part icles a nd flee s ( I l l and there ar c virtually no sideroblasts or siderocy These cyto logical features a re really diagnostic. co nfirmat ion is give n by the findin g of lo w serum lTOO a nd rai sed total iro n-binding ca paci ty (TIBe) , .... ltb decreased sa tura tio n and a low se ru m ferritin level. Other microcytic hypochromic a na e mia- generally have clear distinguishing features : the anaemia of chronic disorders. for e xa mp le. shows a n increase o f largeparticle free iron in th e mar ro w. decreases in both serum iro n a nd TIB e . and norm al or ra ised ferritin . while thalassaemia a nd HbC a nd H bS C diseases show conspicuo us target ce lls a nd increased marrow iron .
1118).
Leucoervshrobtastic anaemia (myelophthisic anaemia. myeloid me/aplasia). T his co mp lex form of a naemia -
A1W('mia ofblood loss und iron deficteucv. Acute blood lov, insuffici e nt to produce ca rd iovascular collapse re... ults in haemodilution and th e de velop me nt. o ver a pe riod of 2-1. hours or so. o f an anaemi a which is normoch ro mic and normocyti c. hut usu all y associa te d with come increase in rcti culocytc s and a pa ralle l rise in le ucoc yte and platelet co unts as marrow act ivity is -tim ulare d . Ch ro nic blood loss leads to the developme nt of iro n dcficic ncv a naem ia. a nd is th e commonest cause by fa r of this d;s0rde r. cha rac te rize d by fallin g hae moglobin .... 1Ih initia lly microcy tic a nd lat er hypoch ro mic red cells .... ith increasing ce ntral pall or a nd progressive aniso-
with th e a ppea ra nce of bot h e ryth roblas ts a nd granulocytc prec urso rs in the periphe ral blood . and with moderat e ly seve re a nisocytosis a nd po ikilocytos is . the latt er ofte n including dacr yocytcs - a rises when the marrow is infiltra ted with met ast ati c tumour cells or othe r forei gn tissue . Megakaryocyt e fra gmen ts may he see n in the blood , and platele t morphology is frequ e ntly a bnormal with distorted o r giant forms . Bone marrow aspira te s o r trephine biopsies re vea l the nature of the infiltrate . Exampl es of ca usative invasion a rc illu strat ed and de scr ibed in Part -1.. but the resulting red-cell morphology ge nera lly resem bles that show n in 1.' 1.
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(
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2
I
I
t
1-12 Romanowsky stains of bone marrow aspirates.
3 •
1. A sequence from procrythroblast through early. intermediate and late normoblasts to non-n ucleated red ce lls. The gradual progression in nuclear and cytoplasm ic maturation shown here indicates th e art ificial nature of the arbitrary division into stages . 2. A proerythroblast and six intermediate-t o -late no rm oh lasts . The procrythroblast has deeply basophilic cytoplasm with a paler area to one side of the nucleus. commonly seen at this cell stage. as in the proerythroblasts in I and 3 . and probably representing the Golgi region . The nucleus has seve ra l poorly defined nucleoli . recognizable as bluer areas in the centre . The normoblas ts have pachychrom atic nuclei and cytoplasmi c hacmoglobinization. giving a polychromatic-to orthoc hromatic co lo ur in co mpariso n with the fully hacmoglob inizcd and or thoc hroma tic co lour of the ncig hhouring erythrocytes. T he field also conta ins two lyrnphocyrcs and an eos inophil po lymorp h. 3 . A procrytbrobl ast and two ea rly normoblasts.
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A proeryt nro btasr in early prophase of mitosis. The MKkar chroma tin i ~ Ilegmning to conde nse into distinct cbromo<;omes an d the integrit y o f the nuclear membrane a divupllng. sho wing a reas of cytoplasm through t he eeeee of the chromatin rna..s. Below a re a late no rmobIMt a nd a Iympbocyte. a nd a bove is a cell of unce rtain ..mre, ~ ..ibly 11 poorly gra nular myeloc yte . S . A proe ryt hro bta st . a [;IIC normoblas t and an early .... nlObla..1 o r proeryth rnblast in mitosis (metaphase ).
A group of normoblas ts with acco mpa nying granulo(or compa riso n. The ten normoblasts ra nge from ~ basophilic tolntc orthochrom atic stages . illustrat•
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reduction in size. progressive nuclear coodt=-Uon a nd inc reasing cytoplasmic hacm oglohiniPM__ tha t occur with mat urati on . The gra nulocytes - heck a n eosmopt nt. two neut rophil stab cells, an early ."Dphil myelocyte a nd two mc ta mye locytes. The -aIUR' granu joc ytcs have' lighte r nuclei than the d}rt ~l~. and pale , wea kly gran ular cytopla..m .
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7. A not her example of proph ase o f mito sis in a proe rvt hro blast. Th er e is evide nce of abnorrnulitv in th e irregular loss of cytoplasmic basophilia and" in the separa tio n of fragments of cytoplasm by a n exagger ated process of buddi ng or exocytosis. The mature red cells sho w rouleaux for ma tion. The cell below th e proeryt hro blast is a no rmal neut rophil pol ymor ph. but the one ab ove is a weakly granular mature neu troph il with a single nuclear lobe - an examp le of pseud o Perger -Huct phenomeno n - sugges ting tha t granulopo iesis is also dysplastic. This ma rro w aspir ate is in fact fro rn a patient with a refractory anae mia and dys myelopoiesis - ;'1 myelodysplas tic sta te (MDS) . a nd 9. Two furt he r fields from th e sa me marrow aspirate. both showing the dimo rp hic red -ce ll picture comrnonlv found in MDS. with a mixtur e of eithe r macrocytic or normocytic norm ochrom ic cells and microcytic hypochrom ic ones. Ea ch field also co ntains a proc rvt broblast o r ea rl)' basophilic normoblast with con spicuous cvtoptasrnic budding - an exaggeration o f a feature often sho wn less markedly by normal early red cell pr ecu rsors. In 8 the re is a neutrophil with fragmenting cytop lasm. and in 9 a pseudo Pelger- Huct polymorph and an early myeloc yte with a hand-mirror disto rt io n of tr ailin g cyto plasm.
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9
11
10
I A group of normoblasts with two gra nulocytc precu rso rs and a plasma ('C II. for comparison . The ~ a ce ll is at the upper left of the field and differs in appeara m:c from the early and intermediate norrnoby the more marked eccentricity of its nucleus and 1bc' greater amo unt of cytop lasm. Nevertheless. these cxII types may o ften be difficult to distingui sh . since the -=leaf chro matin patterns tend to he similarly coa rse , .cl no rmo blast nuclei arc often eccentric. as shown in 11 md I ! . 11 aDd 12. Nests o f normoblasts 'a round a central oohagc. These er ythroblast island s ar e a common in norma l ho ne marrow. serving the purpose o f e cha nge betwee n the macrophage and the ..."oan"ing e rythrobJasts. There arc probably two "Ol""l3g~ in the island sho wn in 11, their cyto plas m v and with ind istinct outlines. but their nuclei ...-,_zable in the central a rea and at th e right -hand of the ce ll clu mp by their light reddish-purple ..._..,. nd lcp toc hro matic density . as compar ed with -black co lo ur and pachychromatic density o f _rmobla t nuclei. Th e macrophages in both th ese co ntain o ne o r two large iron particles. "'-III! e- an o ra nge co lour in th ese Romanowsky prcp60. . ._ .. bu t I~, grossly particulate free iron is not lbout special stai ning . as in 14 and 15.
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13. Nor mo blasts aro und a macrophage - alkaline phosphatase reaction . Th e stro ng po sitivity of the macrophage spills ove r to give thr eads of posit ive reaction over the surface of contiguous normoblasts. 14 and 15. Nests o f no rmobl asts around a centra l macro phage - Prussian blue stain for free iron. The iron co ntaining cyto plas m of the macro phage e xte nds as a long broad tail across the field in 15. Th e central macrophagcs ofte n co ntain a few remn ant s of ingested cellular fragments. and in the upper island of 14 these are more striking tha n usual . with three o r four ghost areas proba bly representi ng the sites o f inges ted erythrocytes, and the encircled nucleu s of a lat e normoblast. The lower island in the same field shows the
macrophage cyto plasm exte nd ing around and between the neighbouri ng late no rmobl asts, hut not convincingly ingesting them. If multiple red cells and norm o blasts were in fact being engulfed. (he existence o f the path ological state of malig na nt histiocytosis wou ld have to be co nsidered .
15
16. Nor mal mature r ' ells. from the tail of a blood mea r... trior var iatio ns in size and depth of sta ining are seen. toget he r with occasio nal pre ssur e distortion of hape , I . ~ 9rmal mature red ce lls from the hod y of a blood
mear. The re is a grea te r te nde ncy her e for ce ll!'> to o verlap o ne ano ther. and the weaker staining of thc ce ntral a rca is more evide nt. Mixture of normal adult a nd cord blood red ce lls - Kle ihauer reaction. The HbF-containing co rd ce lls remain untysed. while the Hb A-cont ainin g adult ce lls . hae mo lysis. Acid and alkali resistance less mark ed than chat of co rd cells but greater than that of DOI111al red cells may be sho wn by the red cells in beredital)' persistence of HbF and in some thalassaernias.
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IX
20
19
19 and 20. Trephine biopsy of bone marrow , sta ined by H& E . ffi)m a- patient with an auto -immune haemolytic an ae mia (A l l tA ) . showing i nten~~ryth rQb lm;t ic hyperplasia, Th e low-power view ( 19) shows the suhstantially complete re placeme nt o f fatt y spaces by predominan tly ery th ro poietic marrow, with o nly a few clumps of gra nulocyte precursors having light er nuclear sta ining and mor e cyto plasm than the eryt h roblasts . and three o r fou r mcg aka ryocy tcs . In the high er -po we r field (20). there is an ove rw.. helming preponder an ce of eryt hro blasts. with all stages of maturity rep resent ed . from proeryt hroblnsts to late normohlasts. There is no uggestion of megalob lastic cha nge . Nuclei freq ue nt ly appear ecce ntric. so me times sufficic ntly so as to mimic plasm a ce lls. hut it is doubtful whether there are act ually any plasma cells present. A sma ll number of neu trophil polymorp hs, with twisted and seg me nte d nucle i. ca n be made o ut . 21. Trephine biopsy of bone marrow . sta ined by H&E. from ano ther patient with AIHA. in this case secondary to a lymphoma wit h sple no mega ly. The overall cellularity here is less than in the previou s sections. with about 40% of the marrow occ upied by fat. but erythropoiesis again predomina tes . includ ing er yth ro blasts of all stages and . in this field . conside ra ble num be rs of mature red cells . There is no evidence of infiltratin g lymph om a cells in th is speci me n.
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• 22. A higher-power view of part of the field sho wn in 21 herevirtually all of the ce lls can be c1carl y identified as o f the erythroid series. Th e largc proeryth ro blasts with leptochro matic nuclei and conspicuo us nucleo li arc pa rticularly well shown . 'A
23. H& E-staincd section of marrow trep hine biopsy from a patient with polycyt haemia (or more properly eryt hrae mia) seco ndary to chro nic lung disease. The IT O""" sho ws increased cellu larity . with reduction in fat and a prcdominance of eryt hropoiesis. and with OlbC'r cell series present in red uced amount . Contrary to u ual finding in polycythaemia rubra vera (PRV) . daere is no increase in mega ka ryocytes . Th e re is co nof sinuses and conside rable haem o rrhage.
1-1 In co mplete contras t to the previou s hyperplastic this treph ine biop sy of iliac cres t ma rrow fro m a _ ng: wom an with a dru g-induced severe aplastic aaacmia shows loss of virtuall y all haemopoi eti c tissue . ooJ ~· a few rcsid ual lymphocytcs distri buted among e ensive fat SP;;ll'CS~ and little to suggest any reactive mato ry increase in perivascular Iymph ocytes plasma cells - thus grade 0 histology. T his non reoct"·'" ma rrow picture may indicate a reduced likeliof response 10 anti-lymphocyte globu lin or o the r ____ ppr ive therapy.
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25. An iliac crest tr ephine biopsy from a you ng woman with pe riphe ral blood pancytopenia whose marrow shows moderately se vere hypoplastic changes. Fatty spaces co nstitute over 90% o f th e mar row and. apa rt from mature erythrocytes. residual haemi c ce lls ar e scanty. wit h merely a scattering of Iymphocytcs and plasma ce lls. and no gran ulocytes in this field. Ncve rthele ·S. there are severa l norrn o hlasts present and this offe rs so me prospect of recover y. wheth er spo nta neo us or in response to trea tme nt. Bone marrow aspirate at this tage yielded on ly blood .
27
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26. A furt he r biopsy from the same pa tient during the recovery pha se. following treatmen t with nnti-lymphoeyre globuli n (A LG ). Active haem opoi esis now occupies 7~% o f th e tot al marrow . wit h nume ro us island of normob lastic hyperplasia and of early gra nulocvte precursors. 27. T rephine biopsy from a you ng adult with severe a plastic anaemia. sho wing almost co mplete loss of haemopo ietic acti vity. Th e few residu al nucleated ce lls are chiefly lymphocyt cs . Th is ma rro w picture . gra de 0 histo logy. when associa ted with very sev ere pe riphe ra l pancytopenia . ma y· indicate less cha nce of response to ALG than o ne with a more co nsiderab le increase in inflamm ato ry infiltr ation by Iymphocytes an d p lasma cells - whe the r modest (gr ade I ). mar ked hut irregularly distributed (grade 2). o r marked and diffusely distributed {grade 3) .
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2S and 29. Respecti vel y thi ck (6-micro n) and thin (2micro n) sec tions o f iliac crest tr ephi ne biopsy from a patient with PRY in a stable erythrocyto tic phase. Both show overall increased cellularity with trilincagc involveme nt - erythroi d . gra nulocytic and megakar yocytic. In 2S the black nuclei of the thicklv sectioned nor moblasts allo w their relat ive proport ions to be assessed easily. and the megakaryocytosis can be readily appreciated. while in 29 the finer details of cvtoloav in the thinner section permit more accurate identification of individual cells. •"'L Section of marr ow trephi ne biopsy fro m a patient with long-sta nding PR Y . now moving into a phase of mvelo fibrosis. as occurs in abo ut 15- 20% of case s. The sectio n shows persiste nce of trilin eage hyperpl asia , hut (here is now a strong com po nen t o f fibro blustic proliferatio n , and th e picture is beco ming ind istinguisha h le fro m that of idio pa thic myelofibrosis ( I~1F) or agnoge nic myelo id met apl asia ( A M M). At this stage the raised haemoglob in and red-ce ll co unt ten d to fall towa rd s normal. while splenomegaly increases. and tea rd rop po ikilocytes appear in the blood . A ttem pts to asp irate marrow arc likely to yield a d ry tap .
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32. Ear ly and intermediate rnacronormobl asts . showing cytoplasmic maturation with accumulation of haemoglobi n in some cells 10 a degree more advanced th an would no rmall y accompany their stat e of nuclear matu ration. 33. Macron orrnobl asts. from early to late stages . Th er e is a lymphocyte o n the right. 3-$. A gro up of four basophilic norrnoblasts derived
from a single cell as a result of two recent successive mitot ic divisions. with the cytoplasm of each pair still linked by fine th rea ds of ce ll membrane. and with a further cross-loo ping of the threa ds. To the left arc two inte rmediate-l a-late normobJ asts with dense nucle ar chro matin and polychro matic. partially haemoglobin izcd cytoplasm . and at the bottom right is a late norm ob last shedding its nucleu s.
26
35
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35. Late telo phase of mito tic division in a macronormoblast. with daughter cells eac h containi ng two uneq ual nuclear masses. A mature neutrophil and a myeloc yte are also present.
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Periodi c acid-Schiff (P.AS) reac tion on a no rma l bo ne marrow as~rale. shcmiilg an erythrob lastic island ;Iiound a macroPhage. Th e macrophage nucleu s. with an o ~ n chromatin network . is in the midline a third of the way from the bottom . and its cytoplasm . with a scatte ring of PAS-positive gran ules especially towards the ma rgins. spreads o ut between the ce lls cluste ring around. Among these are 14 cryt hroblasts. all PASnegati ve. as are four others in the top-ri ght corn er. Across the lower part of the field arc several gran ujocyt e-s. with a gro up of immat ure cells on the lef t. and a stab ce ll and a matur e segmented neut rophil o n the right. These show the typ ical increasi ng PAS positlvity l ib increa sing cell maturity. A single plasma cell just \ e the pro mye locyte shows moderately intense PAS pesitivny. _ . Macro no rmo blastic hype rplasia in foeta l haemo~tic disease. sta ined by the PAS reaction . Normal ~I) throblasts are PAS-ne gativc. but positive material is f~nd in foetal haemolytic disease. tha lassaemia. iro n deficiency anaemia. refr actory sidc rob lastic anae mia and erythrae mic myelosis or eryt hro leukaemia. Here . late ervr hrob lasts sho w moderately heavy positive stippling.
37
39
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.lX. Haemorrhage and hyperplastic areas of ery thropoie tic ac tivity. tog ether with an increase in rncg akuryocytcs. in it trephine biopsy of bon e marrow from an elderly patient with idiopathic (auto- immune ) thrombocytope nic purpura (fT P) . Two of the megakarvocyrcs arc immat ure . wit h single ""Iohed nuc~ i"i romhwn in thisdisorder. I here IS no (hcl all ma rrOw hype rpl;:i'Si'aiif t hi~ rx:um c n . and a normal proportion o f fat spaces rema ins.
39 and .w. Respectively low- and high-power fields from a marrow biopsy sec tio n taken from a patient with Brc deficiency seco ndary to int rinsic facto r deficie ncy pernicio us anae mia. T he first field (39) gives a good impre....ion of the moderate degree of hyperpl asia . with no great diminution in rat spaces in this case . but with a predominance of e ryth ro po iesis. In the second (.&0) . the shift to the Icft in c rythro hlasts is appa re nt . with nume rou ... nucleoi atcd proery throh lasts. and a suggestio n of rncgalobla..tic cha nge can he appreciated . Neverth eles.... the histological de tai l is much inferior to the cytological detail evident in smear preparations fro m aspirated marrow . as sho wn in the illustr at io ns that follow .
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ra , vie w o f a marrow sme a r from a patien t E rythro blasts grea tly predo minate . and c rylh m po i" sis is ..megalob laWc. Early tag es In th e seq ue nce from procrythrohlast onwards arc
~.... it h pe rn icious anaemia. pa n icularly co m m o n .
-&2. Proerythro blasts (nuclco la te d ) and ea rly a nd inte rmed iate mcgalobl asts. While the nuclear pattern of proeryt ro lasts In perniciou s anaemia and ot he r megaloblastic a nae rnias is no t d istinct ive lv differe nt !ro;n that o f no rma l proerythrobl asts . th ere is a ten delle); for cyto plasm to be more ab unda nt and nucleoli larg er and more co nspicuo us . Th e field con tain s a late mega loblast o f large size and havin g an irr egular pycnotic nucle us. Th er e is also a se pa ra ted fragment of polychro matic megalo blast cyto plasm.
42
-13
-16
-15
• ·0 . An ea rly megalo blast with nuclcol ur traces and deep cytoplasmic basophilia . and an inte rme d iate megaloblast o f gigan tic size. -1.4. A mat ure neutrophil with a proer yth rohlast , an ea rly-to-intermedi ate megalo blast and a late megaloblast. This last cell has not quite reached the stage of complete nuclear pycnosis. hut contains a Howell -JoJly body .
4S and -U.. T wo fields fro m a per iphe ral blo od h uffy coat smear from a pat ient with chronic mye loid leukaemia (C t L) treated with the antimetabolite hyd roxyurea . In -15 two basophil's and two neut roph il stab ce lls accompany two lar ge intermedia te mc galo blasts . a nd in..u, arc sho wn a sta b ce ll. two neutrophil polymorphs. a met am yeloc yte and two late mcg!Uohl asts. widely sep arated but with inco mple te nuclea r' reconstitu tio n . / after a p rece ding mitosis.
~ Early . int ermedi ate and late rncgalobl usts. th e last
containing two Howell -Jollv bodies . At the nuclear margin ~re fo ur addi lion~ 1 chroma tin fragmen ts. appa ren tly still attached to th e nuclear membrane but of the sa me character as Howell -Jolly bodies.
30
-17
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57. This series of fields showing mcgulo blasts with cvtotogicat obno nna ttues ( '0"U'5 from marrow or pnip heral MoOtI sam ples from patients with pernicious -.ocmia. and illustrates thut gross abnormalities may be in rem ediable B I ! 1/(jid ellcy ami are not confi ned "J throleukuemias .
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A giga ntic early megaloblast with four nuclei. bly res ulting from two co nsecutive incomplete • ""''''''. with nuclea r divis ion unacco mp an ied by cyto- division.
•
An intermed iate and a late megalobl ast . the former ..,,...__ nuclear distortion of minor de gree .
intermed iate megaloblast with two nucl ei . 52
megaloblasts. Onc is gigantic. with two con irregular nucle ar masses and a free Howc ll-Jo lly ~
in
ular nuclea r mass a pproachi ng extrusion in 3&oblast.
3
55
53
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53 and 54. Late megalo blast!'. showin g typical nuclear rosette for mation . probably arising from an incomplete mitos is with hold- up at metaphase .
56
55. Metaphase of mitosis in an early megaloblast. The chro moso mes are notably long and thin and unusu ally wide ly scatte red . 56. Telophase in late mcgalo blasts. T he mitotic process has bee n defect ive and several chromat in fragments (pe rhaps whole chromatids) have been lost fro m the spindle and appear separate from the ma in bodies of the reconsti tu ted nuclei . This illustrates the genesis of Howe fl-Joll y bod ies. 57. An abno rmal tr ipolar mitosis. approaching telophase . in a baso philic megaloblast. T wo more early megaloblasts. o ne already con taining a Howell-Jolly body . usually see n mostly in lat er mcgaloblasts. and a late erythroblast with pycnoti c nucle us ma ke up the field .
57
61
n intermediate megaloblast and a Iymphocytc (fo r
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in the blood ,
intermediate megalo blast with nuclear distorThe eccentric pos ition of the nucleus and the ">CnollCDlr basophilia of the cyto plasm give a resem to a plasma cell. 6 t , Late megaloblasts in the peri phera l blood . 'S almost co mple te extru sion of the pycnoti c ........._ With loss of the nucleu s a polychromatic "'XlI""'~_'le o r young erythrocyte will remain.
proe ryrhro btast and a late megaloblast. sur. erythrocytes showing macrocytosis and ytosis and poikilocytos is. A few sma ll _""'"JoII ~ bodi es are visible in several of the mature
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6.'. A range o f cryr hrob lasts with minimal mc galo blastic cha nge . T he confus ing terms ' intermed iate' and ' tra nsitio nal" rncgalobl asts arc so metimes applied to such c rythroblasts, whet her ea rly or late in the rnuturutiu n sequ en ce . A late megaloblast with densel y pycnou c pai red nuclei shows hasop hilic stippling of th e orthochro matic cyto plasm . At the top o f the field is a ne utro phil metamyelocyte . an d h5. Further gro ups of crythroblasts with minor rnegalobla...tic changes. The nuclear pattern is mo re open tha n that of co mparable normoblast s. hUI less so than in tlo rid mcgal oblasts. The cell at the bottom of (H is a lymphocyte . and the loss of nucleus from a lute normoblast
(H
65
67
,\ se ries of fields illustr atin g recogn izable but galo blastic cha nges in cryth roblusts from the 8i11TOW of a pa tient in an ear ly stage of perni ciou s _~_ It i-, interesting to not e tha t ther e is alre ady red nuclea r-cyto plasmic asynchron y of de_. ~_..... ~ wit h conspicuous premature haemoglobin iz.-x:om panying mod erate nuclear chro matin co niIe. . . . . . . alt ho ugh the nuclear pattern do es not sho w . open and fully megalohlastic chro matin netic abno rmalities arc also alr ead y occ urring. .......tri·ng Howell-Jolly bodies in two lat e megalo-
6H
70
69
71
69. Further minor megaloblastic changes in e rythroblasts. The large cell in the ce ntre is a ' giant mctam c loc~h a ract e ris t ica ll y found in perni cious anae mia. 70. Polych rom asia in a Lcishman-stained prepa ration from the per iphe ral bloo d of a pat ient with a megaloblastic anae mia responding to specific treatment. Such polychromatic eryt hrocy tes tend . as her e . to be larger than more mature o rt hoc hro mat ic cells . and with crcsyl blue supravital staining are shown to be rcticulocytes. 71. A simila r specimen to 70. stai ned with cresyl blue supravitally. ote that the haemoglo bin co ntent of the rcticulocvtes appears high - an element of hypochrom ia accompanied the megaloblastic changes in this case. 'Dimorphic' pa tterns with macroc ytosis and hypo chromia and de ficiencies of both vitamin Bc! (or folic acid) and iron occur especially in association with intest inal malabsorption. j
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71. Peri pheral bloo d in pernicious ana emia . There arc late mcgalob tasts with nuclear rose tte form ation Md basophil stip pling (a not he r man ifestation of rei yte material). The red cells show macroc ytosis. .-rosis and poikilocytosis. - 3.
imila r changes in red cells from ano the r ar ea in th e specimen as appea red in 72. Thi s field includes late meg alo blast and a mult i-lo bed neu trophil
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Another field showing macrocytosis. and minor ~ tosis and poi kilocytos is. with a striking multioe utro phil po lymor ph.
74
76
75
75. A hon e marrow aspirat e sta ined by Sudan black (SB). showing vacuolation in iroc rythrobla sts following toxic react ion to chlora mp lel11CO . le proe rythrohlast s are SR negative . while the sudano philic grunulocytcs sho w no vacuo lation. A simila r vacuo lated
77
a nd 79. Co ngc nita l d yserythr op oict ic a naem ia (C OA l. type I. Late r oa ~o p h i l ic and po lychromatic e rythro blasu chief! affect ed : mt;galoolastoid cha nges; bi n~d ce lls with intern uclear chromatm bndges ; ~ngy ch romatin with irregular nuclear o utline , ed : . CO A, typ£ 11. Late cfvthroolasts chi' . . ma O'" bi nucleate" cells. double nuclear membra ne is isible only o n electro n microscopy. In type 11 COA (he red itary eryth roblastic multin uclcarity with positive ...cidified se rum test - II E!\tP A S) the red ce lls ar c ptib le to ad d haem olysis.
82 81
81. CD A, type II. A furthe r example showing two erythrob lasts with doubl e nuclei and on e with four uneq ual nuclei.
82 and 83. C J:) A. typc Ill. Gian t crytbroblasts...-wil b multiple nuclei or a sillgle large lobulated nucleus predominate.
83
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Cytochemical reactions oferythroblusts in ClJA . Tile patte rns iliustruted app ear com mon 10 all types .
84 . PAS reacti on shows weak diffu se . and some finely gra nular. positivity . much less tha n usual in er ythrae mic eryt hroblusts. • . Prussia n blu e stain. Excess free iron in eryt hrobla sts. hut no ringed sideroblasts see n. 86. Klciha ue r reaction . C DA ervt hrohl asts and som e erythrocytes show acid-resista nt ft bF. . Acid phosp ha tase : nor mal posinvit v in ;'1 1l1;'ICrophage an d an eosi no phil myeloc yte . C DA ery throblasts are esse ntially negativ e. in co ntras t to crythrucmic I) throh lasts . Double este rase: normal chlo roace tare est erase (C E ) positivity in a neut rophil sta b cell : negative reaction in CDA cryt hrob lasts, unlike th e po sitive tyra te este rase ( BE) in crythrac mic cr yth ro btasts .
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89 . Bizarre nucl ear ab norma litie s in c rvt hr o b tas ts fro m t he hone marrow of a pat ient wit h c ryt hracmi c mye losis. Most cells p resen t are erythro h las ts (apart fro m thr ee pla sma ce lls) a nd se ve ra l sho w m ultiple nucle ar ma sses of irr e gul ar size a nd sha pe . as well as sma lle r Hc we llJoll y bodi es . At th e to p-rig ht co rne r is a gro up of thre e ce lls with light -stai ning nucle i. posxihlc nucl eo li. a nd weak ly ba sophil ic cyto plas m. ,,\..hich ma y he mvcloblasts. rep rese nting a rel a tivel y inconspicu o us gra nuloeyre pre cursor co mpo ne nt o f Ihis pr edo mina nt ly c ryt hrac m ic e ryth ro leukae m ia. 90 . Mul tinuc le a ted giant cryt hroblas ts or pr oerythroblasts in c ryt hrac rnic my elosis . Th e lntcr e ryth ro bla sts to th e right. wi th o rt hoc hro mat ic cy to plas m . sho w seve ra l e xce ptio na lly lar ge nucle ar frag men ts or Ho wcll-Jull y bodi es se para te d fro m t he ma in 1ll1(.' !cU S.
..
• 91- 94. Hi ghly ab no rmal e rythro hlas ts fro m c ryt hrac micrnvclosis. Cc llula"r gigantism. mu ltiple a nd irr egu la r nucl ear ma..-.c:s and ab norma l mitotic figures occ ur in c rythro bla cts at d ifferent stuucs of nuclear ma tur at ion a nd ~ to plasrnic haem oglobin izat io n . Ab norma lities as st riking and bizarre as these are virt ua llv restricted 10 c rvth racrnic mvc losis and e rvt hro Icul acmias. hut they ' are probab ly not cssc~( ia lly diffe re nt in kind fro m the simi lar but much less frequent nd promine nt nuclea r ab norma lities illust rat ed earlier a, occ u rring in the co mmon megalo blastic anae rnias , or the freq ue nt hut more reg ular rnulu nuc lca rity of C DA .
'"
- .'
96
95
95. A gro up of procrythroblasts with so me later crythroblasts of var ious stages of maturity surr ound ing the m . fro m a pati ent with crythracmic myelosis . Mitotic ab no rmalities were not gross or frequent in this case. hut e rvthro blas ts vastly predominat ed . 96. Another example of bone marrow cytology in
cryth rac rnic myelosis. Althou gh the ce lls arc mostly proeryt hroblasts. they show a suggestion of megaloblastic cha nge . Ery thro hlasts a rc always negative to S8 and for pero xidase . Thi s smear sho ws a perox idase sta in. with strong pociuvity in two ncut rophils but a co mplete absence of reactio n in the red -cell precurs ors. 97. B stai n on a fur the r marrow aspirate from eryth raemic myelo sis. The e rythro blasts are negati ve . \\ hilc three gra nulocytc precu rsors show characte ristic posttivitv .
97
99
-.
and 99. Two examp les of PAS positivity in eryt hroIS • from erythraernic myelosis. Coa rse granul ar positiviry is conspicuous in earl y erythroblasts. with diffuse positivity. sometimes very intense . in later eryt hroblasts. Both these fields contain gran ulocytcs , four mctarnyclocytes at the top and a possible myeloblast ne xt to the diffusely PAS-positive late eryt hroh last . . and three myelobla sts or early prom yelccyres and a tab cell in 99. The se cells show the expect ed pattern of PAS positivity for their respective stages of maturity. and thei r presence again demonstrates the existence of multilineage involve ment in eryth raemic myelosis.
.----
..
•
_ PAS positivity in two grossly abnormal giant eryt roblasts from erythracmic myelosis.
-5
ilK)
102
101
10-'
101 and 102. Further examp les o f PAS posmvny in cryth rae mic e rythrohlasts, o ne in mitosis. Ea rly gran ulocytc precurs ors in th ese two fie lds sho w negative reactions. Of . as in the promyelocyte in 102. on ly a weak diffu se tinge o f positivh y.
HU. Acid phosp hatase In crytbrne rnic myelosis . Although normal er yt hro blasts also show para nuclear acid phos phatase positlvity . the reacti on tend s to he mo re ma rked in certain prol iferat ive sta te s. no tabl y erythroleu kae mias and megalohlast ic a nac mias . Th e enzyme in erythroblasts. unlike that in 01(')'1,1 other haemic cells hut like the acid p hos phatase en zyme in hairy ce lls. is tart rate resistant . It.... . A nother case of ervthracmic mvclo sis showing eve n coarse r acid phosphatase positivity in e rythro -
blu...ts.
105. Do uble esterase in ery thrac rnic myelosis. The early crvthrohlas ts show a mixture of bot h RE and CE po...itivity , chiefly the forme r. Normal red-ce ll precursors arc usually negativ e for hot h tiE and CE . although thc v may exhibit a weak reac tion for acetate este rase . Th ~ re · is·a neut rophil polymorp h at the lower left. showing norm al ...tr on g positivity for CE .
-16
1115
106 ~1 07. Erythroblasts fro m th e bo ne marro w in ~de~i~ ncy anae mia. Erythro poiesis is normoblastic.
IIIK
the no rmo hlasts tend to he sma ll. with ragge d arid defective hacmoglon mlzation . The Ill. joio-crvt broid ratio appea rs no tab ly low in the se fiellh. with few gra nuloc ytcs prese nt . bu t although this is ot en th e case in iro n deficiency a nae mia. ery thro id b~pe rp l as i a is not always cons p icuo us . 1
lliI1t.~
I PA S positivity in the small and ragged latc eryt bro blasts of iron deficie ncy anae mia . This is a Ir ue nt hut not inva riab le findin g in iron deficien cy. Ea rlie r red -cell precursors in thi s field show littl e or no pcsiu vitv. while th e two metamyclocytcs and onc stab cdl prese nt sho w the expected stro ng reactions.
•
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109
110
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109. Lc ishman stai n of a marro w fleck co nta ining scattered brow nish-black gran ules , prob ably mostly within rc ticulo-cn dothc lial ( RE) ce lls.
110. Co nsecutive free iron stain on the same field show ing that the granules reac t for iron '\ ,. 111. A cellula r fleck fro m a bo ne ma rrow smea r in iron deficie ncy anaemia . stai ned for free iron by the Prussi an blue m thod . Free iron is abse nt.
112. A similar preparation fro m a patient with a
sideroblastic anaemia and increased iron stores. (. 'o rmal subj ects have stainable free iron in amounts mid way bet we e n these two extremes.)
112
11
IU
•
I
•
,
. ...
•
•
• 115
.....""'....an sta in. bo ne marrow from a pati ent obIas-tic anaemia. Th ere is a suggestion of nucle.ilLc~a1]~ S and irregular cyto plasmic the ery throhlasts. for free iron o n the same marrow speci men. Iy positive ringed sideroblasts - cr ythrotree iro n gra nules arra nged as a co ntinuo us. .nuo us. ring aro und the nucleu s. Thi s iron ntrated in mitoc hond ria . Prussian blue stain for free iron. showing es of ringed sideroblasts. /
Y1 116
/ -
•
•
11 . Free iron in erythroblasls (siderohlasts) with a
117
on-ringed distribution. Th is exa mple is of the 'normal' pattern of free iron dist ribution in ery throblasts , hut the positive cells (sideroblasts) arc mo re nu merous than usual.
I ·--~nsecutivc reaction s for PA S po sitivity a nd free
~ ~Olthc
marrow fro m ..I pa tient with refractory sideroblastic anae mia. T her e arc ringed slder o blasrs present a nd a lso gra nular PA S positivity in ea rly cryt hro hlasts. Simil ar pict u res may be see n in erythraemic myelosis. e xcept that the iron tends to be less abu ndant a nd ra rely in ringed for m. and the PAS posuivity is often stronger. especially in later cryt brob lusts.
118
50
11
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. 51
123
122
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122. Hey) stain of a peripher al blood buffy coa t prepa ra tion fro m a pati ent with a n ~IDS . havin g a stro ng compo nent of sidero blastosis hut also an excess of blast ce lls. and mark ed evide nce of dysmcgak ar yocyto poiesis. Th e field sho ws granulocytcs at all stages of maturi ty. including seve ral basophils and an eos inophil as well as nc utrophils, a nd a numbe r o fJate nor mobl asts: gro ups of abno rmal giant platelet s are particularly co nspicuo us.
I
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•
123. H& E stain of a trephine biopsy bone marrow section fro m the same pa tient as fea tured in 122. The specimen is inte nsely ce llular. with primitive cells. including binuclea ted and multinucleated megakaryoblasts> widely distributed throu ghout the field . and with numerous macrophages heavily lade n with hae mosiderin . Eryth roblasts and cells of the granutocyte series can also be easily recognized . Th is marrow picture suggests imminent trilinea ge leuk ac rnic tran sform ati on . 12-'. Prussian blue stain o n the same biopsy speci men as 123. Th ere is free iron as haemosiderin in the nume rous macro phagcs, as seen in the previous H& E sta in. but in addition , the ervt broblasts ca n now be see n to contain the hcavv rings 'o f mitochondrial free iron cha racte ristic of ringed sideroblasts.
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124
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I _'-I!S . \ 'ariOllS app m f(lfl CeS of reuc ulocvses.
Polvc hro masta. In this Ro manowsky-stained mea r fro m a patien t with a mega loblastic _oauia respo nding with a brisk ret iculocytosis to B 1:!. nz red ce lls freshlv released from the bone -~- ap pear a little larger than mature red cells and ~ a purple tinge to their no n-nucleated cytoplasm . colour resu lts fro m a deficit in haemoglobin. still vnth ecized. and a residuum of the earlier cY',. .~..b·l3s.t cyto plasmic basophilia due to RNA. Basophilic stippling. T he same residu al riboem may form discrete aggregated deposi ts to baso philic stippling. This appea rance is ~_......,t in lea d poison ing and in thalassaem ias. but is as a mino r component of the ret iculocyte ..."" in ot her anaernias. as in this case of li S. with .-<"", Iai Somali de nse sphcrocytcs contrasting wit h the macrocyte. cliculln material afte r sup ravital brilliant cresyl -;:;;",...~.=i-,,"ii':-th ;; Leishma n counte rsta in . Th is met hod ~ = preci pitates the residual R NA mater ial 10 partially clum ped network of blue- staini ng Diff rent stages of reticulocyte maturation recognized accor ding to the q uantity and - - - - - of the preci pita ted mate rial. the earlier bein g genera lly restricted to the marrow.
127
I2S
128Aticulocytcs sta ine d supra vita lly. hut uncounte rsta ined . So me ery throcy tes manif est a precipitat ed re ticulin ne two rk . while seve ra l othe rs in this field show a po lychromasia with the crcsy l blue dye . accompan ying minimal de pos it at th e cell pe ripher y. Am ong the re d cells illustrated here a rc seve ra l with a ce ntral slit of pallor inste ad of the usual circular area stomatocyfcs \"'/Wfl ich ma he see n occas IOna lly In man y sta tes of cryt rop OIetlc yperp asia.
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I
129
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54
129. Gms ' loe tosis - appco
•
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Ani socytosis. Th e red ce lls show var iat io ns in size.
Ifis rnear Illustrates minim al changes . Grosser aniso. is usually acco mpanied by o the r defects, such as alocyt osis.
ut.
Poi kilocytosis. The red cells show variations in _ This smea r also illustr ates mini'mal changes , )' of ' tf.j;lrd n,p' ch 'It"J!'t (~r dacryocyto sis: G rosse r "-lex') tosis usua lly occ urs togci her with o ther . Mild teardrop poikilocytos is of this sorf'rnoy _
~n~::'hromia and micro cyto sis - a gene ral low·
vi ew. A few cells stai n normall y and are of normal ....... (arc normoch romic and no rmocytic) but most have s ce nt ral pallor (hypoc hro mia) and small ';';'_de r (microcytosis). This is the picture of severe deficie n" .. anae mia . The red-cell indice s, Mev: aod MCH C. would all be much below nor ma l.
133
133. H YQQ~hromi a anisocyl o sis ~and _poik iloc yt osis ; from a pa tient with iron deficienc y an aemi a . All th e red cells show mark ed hypochromic central pallor . A few also display an c lliptoc ytic or t ear~ f.Qp-di storti on of a fre q ue nc y and ki ntf oftcn se e n III simple iron deficien cy anaemia. and no t indicat ing th e existe nce o f anothe r pathological disord er . 134. Mark ed hypochromia . anisocytosis and poikilocytosis. Wh er e . as here . i~re glllar fra gments of red cell s or ver y grossly d istorted cells ar e see n. the term 'sctli:itocvto sis' may he a plied . TIle relatively mod erate degre e of hypochromia an t le extent of hizarr~)ik\!QcytoS! S mak e thi s unlikely to he an e xa mple of iron deficiency and rath er sugges t a hacmoglohin opathy (despite th e ah sen ce of targ et cells). or possibl y onc of the micro an giopathi c d iso rders.
56
136
•
•
•
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IJS a nd 136. Respective ly low - and high-power views ol~ sDherocytcs. Th ese are red cells wiffi more sphe rica l IX tha ll no rma l. reco gnizabl e in stai ne d smears by lbcir apparent small diam eter a nd dense stainin g. More half the ce lls in these fields are acceptable as soemat: o nly the small . vcry dee ply stain ed cells can he regarded with fai r ce rta inty as spherocytes. IJ . Anisocvtosis due to a mixture of 01 'chro matic cclicuTocytcs and sma ller s£ lCroCytcs from a G ISC o f li S. SUpra\'lta l staining With n lh;'lnt c rcsyl~ conhrms I the po lychro matic red cells co nta in residual RNA JWCCipitab le as a network . with a n overall reticulocy te coao t around 60"'/0.
1.17
•
•
139
138
•
• •
•
140
U8 . Elliptocy tos is. An inhe rited ano ma ly of th e red cells which Involves. in th is case . some 50% of th e red cell po pulation. T he affected ce lls appc~val O f cigarshaped.
~
139. PscudO-CllipHlChlosis . A co mmon artef act o f siile'3n ng . rcc ogmi..a b c as suc h by two ch ief po ints : (a) th e psc udo-elliproc ytcs ar e fo und in ce rta in areas of the smea r o nly. usually nea r th e tail. and ~ h) their Ion axes are eoerall . acallet or nearl y so, ""hcr~~urnC-cllip locyt cs
dis v
•
show ran om scattcn n of
"ilio n (a s in 138).
l -W. (rescent cells - d isrupted erythrocytes drawn o ut into a crcsccnnc form . Thi s is a fairly co mmo n a rtefact of smearing. especially in anae mic blood. ---
•
•
•
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1 ~2
I ~ I. ' Cre nation', Cre nated erythrocytes show an eregutar undulation of th ~~.1LJncmbf.ill1c in fhed smears. Th e;;ppc.lfancc may be indu ced by exposing redcells to hype rto nic s
14.1
1 ~2 .
Ery throcytes showing a tend ency to cre nat ion and eVidence o t ph ySIcal da mage o r distortIOn--ing the ce ll mem br ane. o ne red cell in the up per pan of the field showing a co nstrictive defect so that it rubles a co ttag e loaf in sectio n and a lower cell ari ng to have lost a bite from the pe riphe ry. Prooablv bo th these defects result from mechanical ma (ro m fibrin thr eads. ~
So-ca lled 'spll[ cdls' or 'If''lO thoq·tcs. showing rese mblance to cren ated cells. hut with sharper ions. ey occur. 0 tcnmassociation as here fragm ented schisrocytes . particularly in uraemia mkroangio pathy. bu t may also be see n afte r -:::om~' and in abe tali po prote inaemia and • rpti ve states.
59
1+1
145
14.4 . Mode rate rou leaux formation; some hypochro mia:-RQ!llcaux arc ag~a tlOns or piles of erythro C)'les a rra nged like rolls or sac ks 01 ca ms . I he re is a tende ncy for red ce lls to adopt this arra ngeme nt within the circu lation an d to manifest it in the thicker part s o f blood sme ars. but the presen ce o f ro ulea ux in areas where red ce lls are well dist ribut ed suggests some ad ditional pre disposi ng factor. A mon g the most impo rta nt of these a TC co nditions associated with a high .,./ erythrocyte sed imentatio n rate such as chronic inftamma t;lry or malig na nt d isease. as in the p resen t slide from a pa tient with Hod gkin's disease . and sta tes of hvpe rgammag lobulinacmia and hyperfibrinogcn ac mia. The com mo nes t cause of marked ro uleaux form ation is multiple myelo ma. mac roglobulinae mia or other monoclonaJ ga mmopathy. 145. .MQ!~m ;uke
~re(,Cn ce of a
l.ui . Macrocytic er ythroc ytes from pe rn icious anaem ia. with Ho well-Jolly bod ies in two o f th~CfCiS'3slig ht suggestio n of o ~.~ valocno"-J(. and r-~ anIsoc~osis . but most ~ e........... disto rt io ns of shape appea r to be tne cs ult of co mpression by neigh bo uring. cells rather than true poi kilocytosis . The red ce lls are well haemoglobinized .
00
146
•
•
,~
1..$1. Romanowsky stain showing a HowclI-JolI body and a blue-staining side rotic granule o r Pappcnhcim er y in the same red cell. fro m th e peripher al blood of a patie nt with megaloblastic anaemia. The red cells arc macrocytic. wit h an occasi o nal stomatoc yte .
1 ~9
_ A rema rkable giant binucleated lat e erythroblast
"m poly chro masia and coa rse baso phil stippling o f th e
irregularly stained cyto plasm. in the blood o f a patient ith A ~ f L and mega loblastic e rythropoiesis ind uced by a co mbinatio n of purine and pyrimidine antime ta bo lite cbe motherapy. Co nspicuo us basophjlic stippling du e -"" 322rc e:ates of RNA is a i.lfticular feature of er thro~ e pyrimidinc-5-nucleoti e- mta l o r acq ulTcd. as In
ase e ctcncy. wether con-
lead or o the r heavy met al ing: its occur rence following the use of a pyrimidiDe ana logue may indicate interference with the action t enzy me and conseq uent failure to complete the do wn of cyto plasmic RNA .
•
,
PAS reac tio n o n a slide of bo ne marr ow aspirate a patien t with glycogen sto rage disease . which as a conseq uence of congenita l deficiency of ......glucosidase . Var ious granulocytes show substa n~ eo rmal patte rns of glycogen distrib ution . while a DOIlIlOblast in the ce ntre of the fi eld cont ains a single of glycogen. resem bling the localized PAS....._ ..... vacuoles see n in lymphocytes in this disorder.
J
151
150
152
ISO. Ta rget ce lls or codocytcs. with accompanying an isocytos is and poikilocytosis. and hypo chromia - from ~l case o f he. it-thalassaemia minor . Th e field also sho ws thin flat cel ls or leptocytcs. Sever al elliptocytcs a rc present. 151. Anot he r field from th e sa me spec ime n. wher e ta rget cells greatly predominat e . The striking appea rance o f these red ce lls. with wel l-haernoglobinized cent re and periphery. and a pale ring betwee n. re flects \,../ thei r be ll-like o r'Mexican hat d istortion . TliCfrcq uc ncy see~ figure is almost restricted to haem oglo binopathics. especially beta-tha lassaemia . hut the appearance affects a smaller proportion of ce lls in live r d isease. in obs tructive jau ndice. in her ed itar y lecithincholesterol acyl- tr ansferase ( LeAT) deficien cy, and after splenectomy . 152. Po i .itocytcsis, with conspicuo us sharp-angled ' helmet' cc us. lro m a case of thrombotic m rom bocvt o~ mc purpura . Th is type o f deformity , probably du e to mcchamcal damage fro m fo rced passage through a fibrin meshwork , is co mmonly found in any of the micronngiopat hic haemolytic anae mias and in other conditions associated with the intravascul ar deposition of fibrin . such as d isseminated intravascul ar co agulation ( D ie).
~>
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ce lls, macrocytcs, spherocytcs, schistocyt cs
luiJing helmet ce lls - a nd sma ll cell fra gment s. from o f seve re bet a-th ala ssaemia .
155
,
L..... Leishman-staincd fres h fixed blood smea r. from pa tie nt with sickle ce ll disease. Elongated or sickled are rare .. but anisocytos is an d poik ilocytos is. and .~t ~I; are mo re co m mo n .
-
1
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cells. drepanocy tcs, fo rmed by exposing rocytcs fro m a pa hcnt with sickle ce ll di se ase (0 the
ing actio n of sod ium met abisulphite under a sealed rslip. As the reduced HbS crysta llizes with in the . they all co me to assume the disto rted elo ngated
sha pe .
C" J,. !' M
•
JuJ/1/ t/10 JjT n" t~~
lJJ
158
156
•
157
•
156 and 157. Cahot riggs. respectively r~d and in form , In stippled red cells. The y are pro"b3D y an RNA--of protein precipitation artefac t of little diagnostic significance but sho uld not be co nfuse d with malaria parasites. fi~urc- o f-e ig h t
158. Lcishman-staincd erythrocytes from a patient with haemolytic anae mia and incre ased iron sto res . Th er e is faint polychromasia present and also Howell-JoJly bodies. Gra nules of free iron are occasio nally de tectable. Several sto matocytes are prese nt with elo ngated areas of central pallor.
159. Prus ian blue stain for free iron on the same blood speci me n as in 158. Siderocytes (eryt hrocy tes with free iron) are seen to be numerou s.
•
159
161
,
•
••
•
0n.z bo dies in ery throcy tes from haem olytic of alucosc-e... .. ~ ' rogenasc . ese Ot ICS, show n her e in methyl viole t sta in. a re pr ecipi tat es of _ ed_hacm<: gIQ ~_m r~sulti n g fro m the lack 0 reducme . Heinz bo dies a ppear to e rmli f ixed to me mbra ne . Th e y occur in small nu mb er s .. but arc remo ved by the splee n. and thus some de ected in most pat ients ~ft e r splenectomy.
162
........:ei::l assocIa ted wJlh deficle nc
la
nte rstai ned pr e pa rati on of re d ce lls from
......-t5.iG3 soae mla expIT"ed to cresylbluc s t arrr-for ten
•
- tettcu locyrcs have ta ke n up th e stai n . 'Ja r p repa ra tio n fro m the sa me blood sa mp le r's exposure la sta in. Th e mult iple do tted - "go lf-ball' pattern in ma ny ce lls is that of in H inclus ions. A high proport io n of the red H dis ease co ntai n te tr a rner s o f th e be ta chain - ~ o xidize a nd p rec ipitate as th ese inclusion quite small numb e rs of ce lls with HbH can be . supra vita l staining a nd allo w th e dia gnosis of ...............sae mia var iants with minima l H bH formade.
•
,
,
6,
IM
163
163 and 164. Targe t ce lls. sp ur ce lls o r acant hocyte s with mult iple sharp pro ject ion s. schistocytcs of irregular frag mented shape and 'sput nik' cells. aca nthocytes with two or three elongated spurs. Such changes are see n in micro angiopathy especially and may he conspicuo us in sple necto mized pati ent s. Th ese preparat ions are in fact fro m a patient with congenita l PK deficiency and severe haemolytic a nae mia. who had nee" sple nectomized 20 years previously, and had recen tly developed obstructive jaund ice as a result o f the accum ulat io n of pigm ent sto nes in the gall bladd er . 165. Freque nt co nspicuous te ar drop_poikilQc'y'!cs in mycl fibro sis. Suc h ce lls m ay he fo und in most conditions of m~o w infiltration causing the
dacO'CKytC5
de\'elopment of a le ucoc rytFiiUblas IC anacmla_. anOTh smalle r numbers in megalob lastic anacmias .
66
165
Part 2 Granulocytes, monocytes and megakaryocytes 'ormal and abnorm al forms
blasts or eve n from pro cr ythrobt a,c, in so me leukacmi c and cry thrac mic states. Cyto che mical and immunocyto che mical reaction s will usuullv resolv e such di fficulties. C ha rac te ristic cytoche mical ' p
A cwe leukuemius of myeloid origin. Leuka emias are clonal neoplastic proliferations arising from stem ce lls.
67
Fo r the A"ILs. the origi na ting cell m3Y be the ge ne ral mye lo id precursor. the CF - G E ~ I ~ 1. capable of giving rise 10 colonies ill vitro containing grunulocytcs . ".10noC)tc_ macrophugcs . erythrob last- and mcgakaryocy tes : o r perhaps the mo re narrowly co m mi tted granuloeyte monocyte macrophage pre cur sor , the C FU-G ~ I : or the corre pend ing e ryt hroblas t or mega ka ryocy te preCUN.l~, HF U-E a nd C FU- Meg , res pective ly. Pur e ervt hrob lastic or mcgak aryo blast!c -lcukacmias' mus t be \eT) rare , if they occur at 90'X) myc fob tasts}: r\'12, mye lo blastic wit h gr an ul.ocy.!iE.-matura tio n (> 10°(0 pnj myclocytcs or Inter granulocytcs] : ~t3 . p-.IQ!TIyeloc}tic (either coarsely gra nular or rnicrogranular promyclocyrcs prcdominating): ~.I-t , myelornonocy tic (>20% gran ulocytcs , or pr ecursors a nd > 20'Yo mo nocytcs o r precurso rs) ; :\15, monocyt ic (M5a predomina nt ly monoblastic. M5b wit h diffe re ntia tio n): ~1 6 , erythro leukaemi a (>50% erythroblasts}: a nd ~1 7 , mega kuryo blastic (>SO% mega kuryoblasts}. A M L with mini ma l differentiation hut po sitive antige nic markers ma y be gro uped as MO. Th is classification is based on a rb itrary borde rlines bet ween the gro ups, a nd alt hou gh broadly -in co nformity with thc ge ne ra l usage of pred omin a ting cel l classifica (io ns in the e a rly IlJ70s it has two import ant defect s . One conce rn !'! its fa ilure to discr iminate bet wee n mon o lineage (co unting th e pro geny of the CF U-G~ I as of one lineage ) and m ulti lincage cases (with granukx... yte / mo noc yte (G ~ 1l involve men t hut a lso erythroid an d/o r mc ga ka rvoc vn c ). Thi s ma y he of so me im po rtance in pr ogno sis . y, ith m ulrilineagc ca se- po ssibly surviving less well overall , although with adva ncing cfficacv of treat ment this diffe renl:e may well dimLini sh . Of greater sig nifican ce , how ever . ma y be the different pat hoge neti c mec han ism , ac tive in pr oducing. resp ectively. monoline ae c or multilincaee disease , with e it he r a d ifferent 1~\·e1 o f stem cell 'affected or a d ifferen ce in ste rn ce ll e xpression dict at e d by biologica l co ntr o lling mech ani sms. A s bioloeical ac tiva to rs such , IS colo nvstimulati ng factor!'! (G~ I -CSF etc.) co me mo re widel\'.
-
into clinical use, different factors rnav we ll be found to have more appropriate use in mo nolincage and multi lineage cases , res pectively. The second broad de fect of the FAH class ification is ~_u!1.9ue. rel~nc~ o.n Rqm anowsk~' ~)10!ogy as a n ind~x 7" of ce ll d lftCrentlatJOn kSudanophlha , often shown by mat uring rnyclo blasts before azurophil granula tion becomes e vide nt in Rom an owsk y sta ins . is pr o babl y the ) hest sing le prognost ic factor in AML. a part fro m age . Surface antigcnic markers hav e not proved of gre at discri mi natory value in A I\IL. a lt ho ugh po sitivitv to the major G M marke rs (CR..t.~ and/or C D33) hel ps to d isti nguish poorly dif fer enti at ed AML from Al. T'(ucut e lym phoblastic leukaemi a ). a nd reactions with antibod ies to glycophorin A or to platelet glycoproteins m,ly furt her aid the recognition of c rvt hrob lastic or rnc aaku ryo blast ic co mpo n"'e nts. · "Cy toge netic cha nges ma y we ll ha ve pa t hog e net ic significance a nd arc of some pr ognostic va lue , t( :21). t( 15:17) , im'( 16 ), und trisomy X as a n isol ated defect. all havi ng rel a tivel y good prognos is , ttv : 11), monosorny 7, del e tion s of 7 o r 5, a nd most m ultiple ab norma lities, ha vin g .1poor one, a nd ot her str uct ura l ah nor ma lities having inte rmed iate prognostic significance. How eve r . diffe rences o f this kind tend to d iminis h as treatme nts becom e mo re effec tive . In a nv case . since so me SO% of AML cas es do nut e xhibit an o bvio us chro mos oma l abno rmality. cytogenet ic da ta do nu t a t pr esent provide a satisfactory bas is for a gene ra l cla ssifica tion . The a uthors hav e deve loped a simple cla ssificat io n of AML whi ch tak es th ese vari ou s co nsiderat ions int o account . Mon olin cagccascsu rc gro uped .tS T ).:r.£J and mult ilineuge a~ Ty~_U . Each type is furthe r sP.!!t into A and B uccordinu to whet her > or < 50% oft he lcukucrnic G M cc lls a re ;uda no philic. T ype lA thus incl udes .all t(X;21) . inv (16) a nd 1(15 ;17) cascs . a nd may be o f an y FAB gro up fro m ~1l _ 1o--,\ I5 . Type IIJ. with little diffe rent iation , includes most t(\' ;1 I) , and again crosses the FA ll groups widel y from Ml to MS, excluding ~ 13 . T ype 11 cas es include most wit h poo r progn osis cytoge net ics, as we ll as FAB groups !\.16 a nd M7 a nd
Chro nic mvctoid leukucmiu (CM I.) , This d isease is a not he r clo na l neoplasttc state , probably a rising in t he
mined plu ripote ntial haemopoietic ste m cell. but lh differentiatio n exp ression initi ally co nfined to th e doid ce ll lines . Som e X5 ft!c. of cases mani fest th e Ph cmosome. a sho rt ch ro moso me 22 resu lting fro m a reci procal tra nslocation betw een parts o f its I~Jn g arm - ' the long arm of chromosom e 9.1(9 ;22) (q 34;ql I) . lh the transfe r o f th e c-ahl on cogenc from 9q to a site ~ q .nown as th e breakpoint cluster region (b.£r ). Tbe combi ned chime ric gene at thi s site produces an .iboormal protei n kinase . characte ristic for C ~ 1 L Most the rem aining 15% of -cases with the cyto log ical ure of C ~ I L - with a high leu cocyte co unt and nee o f granutocvtc p rec ursors in the blood , ~ ulocy t i c hyperplasia of th e bo ne marrow . freq ue nt philia and basophilia. and a low leu cocyte ine phospha tase (LA P) sco re in the neu tro phils evide nce that a morph ologicall y inapparen t transtio n has occ ur red. with production of the bcr-ab l cbimeric gene . 10 the ext ent that th e same abnormal pro tein kinas e ge ne product as in Ph +vc Cfvl l, can be de tec ted and c-ab l probes locat e this on cogcn c at the her le o n chro moso me 21. C ML is well co ntrolled by single agent che mo the ra py. ally busulphan o r hyd roxyurea . for var iable pe riods from a few mon ths to many years. unt il a change in th e pa tern o f disease happen s. with mal ignan t progression hypop las ia . myelofi brosi s. or an ac ute lcuk acm ic pecture , usually th at o f Al\1L but occasionally ALL in . -pe. In tra nsfo rma tio ns to an AML·like sta te there is lten an eryt h rae mic and/o r a megak .eyocyric co mpone nt. with a multiline age T ype 11 cyto logy - althou gh , som etimes. a more purel y monolm eage GM form [Type It develo ps. A t thi s stag e furth er chro moso me ub nor IDalilies arc freque ntly found . not abl y one or mor e ex tra .22 trans locatio ns, struct ural ano ma lies o f 17q. and trisomies o f S or 19. Th e hae matolo aical findi ngs in bo th chro nic and transfo rmed phases of C ~ ll . a~l" illustrated in 320-37.$. be rc cha racteristic features of histology, cytology and . tochc mistrv arc fur ther descr ibed in the ca ptions. c-
JIII enile C \I L. Thi s is a form o f subac ute ncopl astic ~oIi fe ra t i o n with Ic.uurcs inte rmedi ate be twee n th e It type of Ph +ve C~lL (which itself occurs in dliIdIiKx)(J. hut o nly rarely) a nd a myelom on oc ytic acut e ae mia . This d isease is not associated with tl 9:22). about 50% of cases sho w chro mosomal ub no rmalimuch the co mmo nes t being mOllosom y 7. Th e is illustrated in 375-383. where ad dit ional PtI\C material is included in the captions. vsplassic states (.\ 1DS ) ami other preleukaemic rs. A mong the conditions gro uped toge the r me title o f ~1 D S arc refr acto ry a nae mia ( RA) and -dn.:to~ a nae mia with side ro hlas ts (RA S), bo th o f ......1 . D,,~ e bee n discussed and illustrated alr ead y in Part prese nt sectio n includes the remaining MDS . re fracto rv anaemia with e xcess o f blast.. .-..E8 . the- ~m~ with a hi-g:hc"'r- prorXlTli on of bElsts
and/or the presence of A uer n
S -
hence presumed to
be close r to transformation to A~1L ( RAE Bt) - and.
fina lly. chronic rnyelomonocytic leuk aem ia (C ~ I ~ 1L ) . Th is nom en clature is tha t propo sed hy th e FA R group for con d itions descr ibed over man y yea rs under var ious nam es. which pro bably mak e up a bro ad spec tru m uf indolent or smouldering c1on al1cukaernic states, alread y associated with non -random cytoge netic abnormali ties. Th e way in which th ese d iso rder s evo lve is errat ic: transformation to a floridl y lcukae mic p ictur e o f multilineage (Type 11 ) AM L m;;I Y occ ur in a ny of them , perhaps more commonly in RA EB and RA EB t. but many patients with MDS die from chronic anaemi a. infectio n or haemorrhage without any suc h overwhelm ingly blast ic meta morphosis . T he histo logy. cytology and cytoche mistry o f RAEB and CMML
Reactive chunges ill granu tocytes and monocytes. Leu cocytosis with incre ase in the WBC above the normal up pe r limit o f I I x 10"'/1 occurs chiettv in infe ctive and mftammato ry states with jI reacii\'e inc rease In neu trophils accompanied by a ' shift to the left ":""'wtth a IJ ised pro po rt ion Of stab cells and po ly· morphs with fcw nuclear lobes . Occasiona lly. mor e immature pre cursor s. mctamyclocytes - o r even a n odd myelocyte - may be found in the peripheral bloo d. Th e neutrophils may sho w coa rse darkly _stain ing 'to xic' granules. and ther e may he bluish Dohlcbodics visible ift ~Romanowsky p!.cpa ra ti(H1S -in- th cir cyto plas m, r~pre~entin g ureasof. hj-peractive aggregated ro ugh or smoo th end oplasmic.reticulum . Le ucopemas with gra nulocyte co unts < 1.5 x IU"/l arc seen in states o f pri mary or secondary marrow hypoplasia or aplasia . with generally defective myclopoicsis and assoc iated ana e mia and thro mbocvtopcrna : ho wever. selective neut ro pe nia may occur in o ver whelmingly seve re infec tions. whether bac terial. viral. rickett sial o r pro tozoal, an d may proceed to ag ra nulocytos is. with circula ting neut rophil s < O.S x 10"/1 . A similar selective neutropenia may result from se nsitivity to certa in dru gs. includi ng sulpho namides , ant i-thy roid agents and some anti -con vulsan ts. Cytoplas mic inclusio ns o f degeneratin g nuclear
material resulting from the action of an anti-nuclear facto r (ANF) form a special feature of systemic lupus c rvt hc rnatosi-, (SL E) . and are of cyto logical inte rest as we ll as pro vidi ng a rapid diagnostic test. although their pract ical impo rtance has been largel y replaced hy immunological testing for ANF. Examples arc illustrated in .'S-a and 385. The reverse of the infective ' left shift" in neutrophils is seen in megaloblastic anaemia». where deficiency of B l.:! o r folat e leads to nuclear changes involving a ' right shif t' with an increase in the ave rage number of nuclear lobes in neutrophil polymorph, and the presence of some cells with six or more nuclear lobes. macropolycytes or hvpcrscgmcntcd ne utrophi ls. Eosinophilia . with blood levels over O.5 x 10"/1. may he seen a, a familial ano maly. but occurs as a reactive phenome non - especia lly in skin disorders. allergic states, and pu rasitic infestat ion - and has add itio na lly Peen fou nd wit h associated mya lgia in pa tie nts la king dietary supple me nts of Ltrypro ph nn. Buso ph ilia. with counts ovcr u.zx 10<)/1 . may also be seen in so me allergic or inflammatory states, altho ugh it is relat ively uncom mo n. Mo nocytosis. above the nor mal upper level of I x 10"/ 1. may occur in more chro nic infective states or in those d ue to non -pyogenic bacteria . viral or rickettsia l infections and so me p rotozo al or oth er pa rasitic infes tation s. A reacti ve mon ocytosis may a lso he seen in so me auto- immune disorde rs and co llage n diseases, and in so me cases o f disseminated malignancy.
Congr nitul anom alies and acquired parallels. Alth ou gh ma ny an o ma lies o f leu coc ytc s have bee n described . most a rc ve ry uncommo n; on ly the four most important arc described and illustr ated her e . The l)l'IJ,:l·r·llut't nuclear ano ma ly occu rs as a ra re benign familial inh erited diso rder P S7) . hut it is see n very co mmon ly as an acqui red o r ' pse udo' fo rm in man y cases of myelo dys plasia an d bo th ac ute (e .g . 215) and ch ro nic (3AA) myeloid leuk aem ia . Th e Ma~' - l h1:.g l i n ano maly. like the Pe tge r-Hu ct anomaly. is a ge nera lly be nign co ndition inherited as an autoso mal domi nant . It is associated with the for ma tio n of large oval or crcsccnuc basophilic inclusions (394 and 395) in th e cytoplasm of gra nulccyrcs and occasionally also o f monocytcs. These represe nt a special type of inclusio n material rather than an aggregation of no rmal cytoplasmi c structures. and arc thus essentially different horn Doh le bodies. Giant platelets may also be found in this ano ma ly. and there is sometimes thrombocytopenia and leucopenia . The Aldrr-Rrilh' a no ma lv. which mav occur in associanon with various mucopolysaccharidcscs. involves the development of coarse azurophific granules in the cytoplasm of gr anulocyrcs (389) . The material of the gra nules is derived from the mucopolysaccharide and resem bles basophil granules somewhat. in reacting met ach rom atically with toluidine blue.
711
T he Ched lak-H jgashi-Steinh rlnck syndro me is a ra re inherited d isease . usually fatal in early life . with severe cytopc nias and a lyrnphorna-Iike clinical picture. associatcd with rem ark abl y lar ge cyto plas mic gra nules in va rious tissue ce lls - incl ud ing man y lcu coc ytcs o f blood a nd bo ne marrow . especially gra nulocy tcs (398). T hese coa rse gran ules arc probably lysoso mal in nature . Pseu do-C hed iak inclusions may develop as an acq uired phen o menon in so me acute lcukaemias. notably cases of A ML with the 8;2 1 tra nslocation (211 and 213 ).
Platelet disorders , \Vhile qualitative defects of platelets arc best det ected by platelet funct ion tes ts and arc not reliably recogn ized by changes in pla telet morphology. quanti tative cha nges thrombocytopcnias and thrombocvtosis or th rombocvt haernia - arc associated with char~cteristic cyto logica l findings . not on ly in ter ms of numbers of circulating platelets hut a lso with regard to the cytology of mcgakarvocyrcs and the process of thrombopoiesis as observed in marrow smears and sections. T hro mbocytopenia. with reduction in circ ulat ing p latelets be low 150x 10"/1. arises in most instances fro m o ne or two broad causes: defective formation of plate lets (as in prim ary or secondary aplastic or hypopl astic sta tes . inclu din g th e effects of myelotoxic che mo the rapy and so me less co mmo n d rug sensitivities) . and increased de struction o f plat elets in the periphera l circulat ion and splee n (a rising ge ne ra lly from auto-i mmune sc nsitizution of platel ets. render ing them liable to phag ocy tos is by macrop hagcs ). Th e fo rm er type of thru mbocy to pcnia is associated with a reduction or abse nce of mcgakaryocytcs in the hon e marr ow - an amcgakaryocy tic thrombocytop eni a - wher eas, in the latt er . mcgak ar yo cytcs arc gene ra lly plentifu l and ofte n incre ase d in nu mb er s, alth ou gh they may a ppear cyto logica lly unu sual in hav ing immature nuclea r feat ures and defective gra nularity. with often promi nent glycoge n inclusion bod ies and few peripheral p latele ts (467- *73) . Thrombocytos is. with counts over 500 x 1tI
C'f'JII blood smears . where megakaryocyte nuclei and -=lear fragments may also sometimes be present. Icga .arvoblastic proliferation in the bone rnarrov . be promine nt in some trilineage acute leukaemiamay occasionally predominate. This occurs in some . apparently arising de nm 'o (48R--l95) . hut also rg as a form of blastic transfo rmation or malignant eeceression in the ter minal phases of polycythemia
rubru vera (PRV) or CML (339-348). More mature
megakaryocyte proliferation . arising in a s.ir:nilar fashion as either a primary or a secondary condition. ap~a rs often to stimulate the development of myelofibroti c or osteomyelosclerotic changes. prohahly. thr,!ugh t~e production of growth factors and other biological a~tI\"· atcrs of fihroblast proliferation and collagen formation .
•
11>6
•
•
•
.
#, I
167
, ~
••
•
166. Tre phine biop sy scsJion of norm al bo ne marrow from a c h iJ~). showing somcc:9ll% ovcrntt cellu > la rity a nd good rep resen tatio n of m o ~.or '.!!~I1l
.
•
•
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•
16H
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•
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169. A
r1!1~ h ) ~ l as t w i t h _1 W O-C( mspi c:.uo us....!!.~ c1co l i .
a
\acuo lcy nd a few uzuro philic gran ules. a larger-promyelocyte . a myelocyte and a sta b cell o f th e neutro ph il series. toge the r with a lat e no rmoblast. Bot h th e cells at the pro mye locyte and early myelocyte stages show fad ing nucleo li and azurophil gra nules. th e smaller and mo re ma ture myelocyt e also having less sharply de fined and less stro ngly coloured neutrophil spec ific gra nules. li .e tho se in the acco mpa nying stab cell. 1 O. A seque nce of granu locyrcs. with a myelob last. pro myelocyte . myelocyte (with neut rophil gra nules). and a late neutrophil meta myelocyte or early stab cell. The myeloblast. o n the left o f the gro up . has a clearly defi ned nucleolu s within a dar k ring of nucleolu sa.....sociated chromatin: th e prom yelo cyte . in th e centre . ha three less distinct nucleo li visible in th e upper part of the nucleus and numerou s coa rse azu rophil gra nules: hilc the two maturing ncutro ph ils show a de nse r eucl ear ch romatin patt ern and have man y speci fic ~n u l c s. Th e red cells arc abno rmal. with anisoc ytosis and irregular hypoc hro mia. 1/ 1. A pro myelocyte. two myclocyrcs. a rnetamyelo. e and a st ab ce ll. o f the neu trophi l se ries . Th e red cells in sho w anisocytosis and marked hypo chro mia .
\ j
171
173
172
•
17~
172. A myelocyte and four metamyelocytcs of in Teasing maturit y (with increa sin gly ind ented nucl ei) . Th e myelocyte is the sma llest cell in thi s gro up and has the most basophilic cytop lasm . th ou gh without any det ectable azu ro phil granu les. and its nucl eu s has sev era l sma ll but fad ing nucleoli . Spe cific granules are inconspicuous in th is ce ll but numerous in all the metamyelocytes. The re is aga in hypoch romia of the red cells and a stomatocyte with a nei ghbouring dacryocyrc towards the upper left.
173. A myelobla st and an earl y neutrophil myelocyte . The cyto plas mic basophilia , due to residual RNA . and the three o r fou r pale b lue nucleoli of th e myeloblast are well sho wn her e an d in 174 and 175. 174. A myeloblast with a monocyte for co mpariso n. The monocyte sho ws a coarse r nucl ear chromatin pattern. a n absence o f nucleo li, and a gre yish rather tha n a basophilic cytoplasm . !
I
~
177
1 S. A myeloblast with a neutrophil stab cell . To the right o f the myeloblast-and impinging on its cyto plasm is .Ji rat her large platelet. Three small er platelets ar c prese nt at the top of th e field. li6. Below is a promyelocyte with num erou s azuro phil ~an u l e s
in its mod er ately basophilic cyto p las m and mooth leptochrornatic nucleus witho ut clea rly inguishable nucl eoli. while above is a neut roph il ~ e n t e d po lymo rp h. There a re three platelet s present .ad the re d cells sho w o nly slightl y exagger ated ce ntra l Ior. sca rcely amo unting to hypochrom ia .
a
. Two rnyelob lasts. the sma ller showing diminut ion cyt o plasmic baso philia and the first appea ra nce o f azurophilic gra nulatio n. a promyelocyte with coa rse ulari ty and co nside ra bly larg er size th an the loblasts (a frequent finding) . and a monocyte . for
parison.
5
179
17
IHO
178. A seq uence of neutrophil grunulocytcs with a myelobl ast . pro myelocyte (with vac uo le ) . myelocyte . met am yelocyte and two stab cells. Th e myelobl as t and the promyelocyte eac h contain three or four nucleo li. so me with co nspicuo us nucle olus associated chromatin. while the late r ce lls h ;'IVC lost th cir nucl eoli a nd sho w increasingly pachychrom a tic nu clei.
179. Perox id ase reaction on norm al ho ne marrow cells; a seq ue nce o f gra nulocytc precursors shows stro ng positiv ny: c rythroblusts
)
J
76
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, IK3
' - I. SB stain o n norm al buffy coat smear. T he coarse uvirv in neu trophil po lymorphs con trasts with the er reac tio ns in mo nocvtcs - o ne negativ e . o ne with fine era nulcs and thr ee with ~a tt cred granules. disc rete than in the neut rophils. Th er e is an no phil with hollow positi ve granu les. a negative hil and a negative Iymphocyte . I3I."Side the basophil . the top left of the fie ld . is a clump of negati ve platelets. ~
B sta in on norma l peripheral blood , illustr ati ng ntras t betwee n the den se and coarse posit ivity in re era nutocvtcs and the d iscrete scatte red granul e m ~)f monocytcs. Lyrnphocyt es arc nega tive.
,
B stain o n norm al buffy coat showing the presence myelo blast. as can usually he found in no rma l cirg blood if carefully so ught . Two negative base. 0-. 0 normally reac ting neu trophil po lymo rphv. a _le and a Iymphocytc co mplete the field.
•
'7
185
I~
•
IH4. Peroxidase stain on no rma l pe riph er al blood . T wo ne utrophil polymo rphs arc stro ngly posit ive: a basophil is negati ve and on c of two rnonocytcs sho ws loca lized cytoplasmic positivity co ntrasting with the discre te scatte red granules o f the SB rea ction in mo nocytcs.
ISS. Peri odi c acid-Schiff (PAS ) reaction on a normal Pone marrow smear. to illustrat e the gra dual increase in positivi ty found in the gra nulocytc se ries with increa sing cell mat urity. Erythrohlasts arc nega tive. Th e d isrupted cell with cen trally situa ted nucleus and co arse gra nules of material co nta ining free iron . sp reading out between neighbouring cells. is a ret iculo-en do theli al ( RE ) ce ll. 186. PA S reaction o n no rma l huffy coa t . sho wing two positive neutrophlts. a coa rsely reactin g basophil . two negative Iymphocytcs, two monocyte, with fain t diffuse reaction . and an eosi no phil with ne gativ e gra nules against a PA S·pos itive bac kgro und.
7S
186
.
;
..' -, "
,
.. .., ... ".'~
.
~,
,.
. A cid phosphatase positivitv in the norm al cell is the rclore generally most conspic uo us an ) cloc vtes.
Dual este rase on normal peripheral blood , CE- positivc polymo rph and BE· positive
_ _0) .1<.
. IH9
I . Acid pho sphatase in grunulocytcs in normal aarr m" ; myelobl ast . promyelocyte , st ab and two ~ e n t c d neutrophils. All these gra nulocytcs sho w a - ueri ng of fine granules . probably represent ing the prima ry lysoso mal azuro phil granules which first appea r • the late mye loblast to ea rly promyelocyte stage and in in the cyto plasm of later gra nulccyres . altho ugh led in co nce ntratio n by division at the myelocyte
Dual esterase. o n normal marrow ce lls - seve n op hil granulocytcs. includin g two myclocyrcs. o nc itosis, all showing chlo roac etat e este rase (eE) tivitl. two negative eosino phil rnyelocyt cs. a _ _ x-..le o f mixed butyrate es te rase ( BE) a nd CE -eactIon. and a negative late normoblast and Iymph o-
,
."
.~
191
)'iO
192
t9(~19-l , A ser ies of stains on bone mar ro w smears fro m a case of acu te mve lob lastic leu kaem ia. show ing re lative ly litt le c~;i dc ncc- 0 1 differentia tion an d non e o~f erythroblas tic or rnegak ar yobl astic involve me nt . a nd thus classifiab le as T ype III ~ Th ere a re neit her gra nules nor Au cr ro ds visib le In th e Hc vl stai n ( 190)
193 j , .. I
\
and < 10% of blast cells are 58 positive (i91). There is wea k d iffuse PAS positivi tv in most blast cells in 192. and o nly a late r neu trophil and onc of the grou p of rnyelo blasts sho w CE positi vitv in 193. In 19-1 . ho we ver. there is clear positivi ry to the myeloid MA b C DD in this al ka line ph ospha tase-a ntialkaline phos pha tas e (A PA A P) immunocytoche mica l preparation. About 20% of cases of acute rnveloid leukae mia (A \ tL) fall into th is category . and [bcv have a relatively poorer pro gnosis tha~ mo re clearly differenti ated ea scv. with 58 pcsi nvit v in > 5U% of the blast cells or with Aucr rod.. or other inclusions readily found . Wh e n few sign of myelo id differentiation a re present. apart from positive antige n marke rs . the FA R clasvilica tio n \111 may be appl ied.
19 ~
193
196
195-198 . An oth er examp le o f a hon e mar ro w smear from a case of Type III AM L. aga in witho ut multi -
lineage involvement and with only poor ly differentiated blast cells present . where the cytology in the Roma no wsky stai n ( 195) sho ws so me inde ntati on of nuclea r me m branes .•md t he identifica tion of the blas ts as mve loblasts rat her tha n monohlast s (l\11 rat her tha n
~tS) 'is uncert ain. Th e prese nce of localized rath er than scat tered SB posirivity. eve n in onl y < 10% of ce lls in 196 . suggests th e form er. Th e weak d iffuse PA S rea ctio n with an occasiona l posi tive gra nule (197) is equivocal on this issue . as is the positive surface membrane APAA P reaction to C Ol3 s ~own in 198. Reac tions to bo th d ua l cst c rnscs and the mon ocvt c ~ I Ah C D 1-1. were negative . T he cas e is ther efor e almost ce rtainly myelo blastic (7\11) - but in term s o f the Type IB classification th e possible d ifference is un imp ort an t. the prese nce of < 50% S lr-positi ve bla st ce lls ca rry ing a poor pro gnosis. whateve r the precise ba lance be twee n the gra nulocytc an d mon ocyte precur sor co mpo ne nts of an es se ntially mo nolin cugc AM L
19H
199
200
201
202
199. A gro up of myelob lasts from an AM L. sho wing Auc r rod s and az uropt-ilic inclus ions . T here is also a myelocyte and two lyrnph ocyrcs in this field . ZOO. Perox idase reaction in A ML. show ing pos itivity virtua lly co nfined to Au cr rod s whic h are prese nt in all th e blast cells in thi s field . A sing le lymph ocyte is negative .
•
201. Mvet ob lasts and pol ymorph s fro m a similar case o f AM L. Several mye lob lasts show pero xida se posinvity. and inclu sio ns ap pear pos itively stained. Th e poly morp hs in th is case display stro ngly positive reaction as in norma l polymorphs - although sometimes in AML the mature po lymo rphs show wea k o r
even negat ive reactio ns. 202. 5B reaction in AML . showing severa l stro ngly po sitive A ucr rod s and loca lized reac tion. 203. SB reaction in myelob lasts of A ML. Stro ng positivity is mostly localized to cytoplasm with a positive Auer rod in one myelobl ast.
S2
203
•
2117
PAS reaction in a simila r acut e leukaemi a . Th e po lymo rph is normall y po sitive . whi le the liv e cells give a reaction ra nging from negat ive to
a· diffuse positive tinge ove r most of the cyto. So me times fine gra nules a re also prese nt . but o n gro und o f diffuse tingeing unlik e th e clear bac kseen in Iympho blasts .
PAS reac tio n in marrow cells from ano the r case A ~ 1 L.
T he field sho ws a ce ntra l group of four _ .doollasts a nd a met am yelocyte . all with diffuse to gra nula r po sirivity . and a nega tive la te norrnoa t the uppe r right. One of the mye loblasts ;:::~ a wea kly positive Au cr rod o verlying the
Acid phos phatase reaction in a similar case . Id)' coa rse gra nula r po sitivity is present.
Dua l ester ase reaction in the same case. The IS sho w a weak . lincly gra nular . sca tte red CE . ~'. A single ne utrophil po lymorph sho ws stro ng
I lQCtion .
'.
2119
2f1K
20K-215. Humun n wsky stains of how! mllrrow and blood SUI i' ll rS f rom cases of A M L wilh tilt' X;21 l rans locution - 1(8;21) (q22;q22). In thi... A MI. variant the b last cells ar e gen era lly la rge hut ... how vunab ilit v in size and in the amount of cvtoplu ...m. which is basop hilic , oft e n con tai n... lo ng a nd ...lender A ucr rod s, and may also co ntain vacuole ... and ulcbular indu... ion s. T he nucleu s may he eccent ric. with commonly an inde nta tion o n the side next to the mai n mass 01' cvtopla... m . v. here t he re may he a pale -... raining area representi ng th e Gnlgi body and where inclu... ions o ften tend to he localized. Th ere arc so met ime ... uro.....er disto ru o n-, of the nucleus and sepa ra ted nuclear trua mc m , mac he foun d . N ucle o li are ac ne ra llv laru e and. pale. and- tw 0 or three are co mmo nly de tectable , u... u .J1I~ ",idd~ spaced. Th e- pro mye locy te ... o fte n po.........·...... many coa r-e azurophil (p rima ry ) gra nules and may ...how large pveudo-Chcdiak inclusions as well as slende r Auer rod .... u...uallv ...inulc . Later staces o f maturati on . fn~m myelocyte... ~mv.;rds . show defect ive pro d uction (lCs~cifiC' gr -notc ~ o plas-m--- t h a l -a p pc a r ... pJ!lkish:)dlo", aitht=myeTocyfe sfagC ...omcti mc... with a residual pcnpherartlmofl1eejJoa...opfii~ he re arc par allel defects in nuclear maturation. with occasio na l ring nuclei and. at the end of the maturation ...cquc ncc . some neu tro phil... with pscudo- Pclgc r-Huct nucle i in the ir agra nula r cytoplasm . Occasional abnor ma l cosinophib may he see n. with atypical greeoic h-grcv or blue-staining gra nules (St'e also 220).
210
/
21-l
•
liS
2 17
216
216-225. Cytochemical reactions in cases of1(8;21). Peroxidase and Sudan black reactions (2 16-2 19) always sho w stro ng localized cytoplasmic positivity in the great majo rity of blast s. Generally . this is concentrated at the nuclear indentatio n hut often it involves most of the cytoplasm and. in the case of Sudan black espec ially. tends to obsc ure the frequent positively reactin g Auer rods . which a re therefore best see n in diam inobe nzid ine (DA B)-pc roxidase prepar ations. but may occasio nally be found in maturing eosi nophils. too (see 217). The inclusions sccn in Romanowsk y stai ns a re also usually peroxidase positive and sudanophilic. Occasio nal nega tively rea ctin g neutroph il po lymo rphs may be seen . PAS st ains show the usual weak diffus e o r finely granula r positivity of lcuk aemi c myeloblasts, but may also sho w coarse gran ules o r block s of positive reaction (223). an o therwise q uite unu sual fea ture for AML with granul ocyt e lineage predo minance . Auer rod s may sometimes stain positively (222) . Th e atypical granules seen in eos inophils in Rom anow sky stain s (220) may show PAS posit ivity (221), unlike normal eosinophil granules. which are PAS · negative against a background of cytoplasmic positiviry . Dual esterase staining with eit he r do uble (224) or single (225) capture agents shows CE positivity in most leuk aemic cells with posit ive Auer rods co mmo nly visible . someti mes with a negative co re, and similar reacti ons in other inclusions. CE positivity of Auer rods is not often see n in cases of A ML ot her tha n the 8;21 and 15:17 translocat ion s
2211
221
~I
22-1
225
•
227
226
228
226-256 . Cytology and cytochem istry of AM I. cases with the / 5: / 7 translocation, t! /5: 17l (lJ22;q21) - (K w e promvelocvtic leuka emia (A PL ). 226. A n A PL with ea rly gra nula rity in th e urcho pluvrruc lone next to the nucleu s in most cells. Th e cells arc Icukac mic pr omycloc ytcs. hut do not show mult ip le Auc r rods. althou gh th er e is it sugges tio n o f the coarsely lo bular nuclea r changes of th e typ e ofte n seen in APL. Striking vac uo lation was a feature o f thi s case .
227. 58 reaction in the same ea-c . TIle den se cytoplasmic poviti vity resembles that seen in no rm al late r gran uk:~te"S from myelocyte to po lymo rph. In this prepar at ion the I ndcncy towa rds nuclear ind e ntat ion to produc e twinned o r unevenly pai re d lobes . a strikin g fea tur e of APL. can be discerned in seve ral cells. 228 . The PAS reaction o n these ce lls sho ws diffu se po..itivitv with some increased gra nula rity in the a rchcplasmic zones . Thiv i-, the PAS pictu re of AML wit h pr o m vcloc ytic prepondera nce . 229 . D ual c.....tcra-e reaction in the same cells, with strong CE posi tivi tv . including a Cli-po-itivc Aucr rod .
•
229
231
A PL with coa rse azurop hil (prima ry) gra nu les _e1 ~' filling the cyto plasm of all the le ukaem ic pro· jocytcs. Nucleo li arc co nspicuo us and va riab le in bcr hut the ove rlapping o r dumb-bell type of ar shape often see n in A Pt is no t shown he re , a nd Iller rods arc not visible in this l.cis hma n-staincd ration.
1..
.=J I. Ano ther preparation from the same case . stained \t GG . Co nspicuo us multiple Au er rod s are now in seve ra l cells. The differen ce in staining ion of A uc r rod s in APL to Leishman and ~ IGG . thoug h no t always shown . is frequently striking .
Anothe r case o f APL with granular promyclo.. . seve ral ha ving multiple Auer rod s and so me __ typical nuclea r lobulati on .
232
233
233. Myc lobla sts a nd prornyelocytcs from the peripheral blood of anoth er case of APL. all showing the virt ua lly diagno stic nuclea r patt ern altho ugh only the ce ntra l ce ll is he avily gra nular.
2..\4 . Another case of APL. showing few o r no cyto plasm ic gra nules a nd no Auer rods with e ither Lcishm an or MGG sta ins. hut with the char acter istic and diagnostic nuclea r shape with dumb-bell appea rance o r ove rla pping of twinned nuclear lo bes. Thi s var iant of A PL is sometimes d istinguished as a sepa rate 'agra nular' o r ' microgranula r' for m. although usually as her e and in 235 - a n occas ional norma lly granu lar promyelocyte may be seen . 235. Anothe r example of microgranul ar APL. with minimal granula rity. hut aga in with seve ral cells showing the nuclear struct ure typical of APL. Th at these ce lls arc not ntially different from those of the more co mmon coarsely granular var iant is indicated by the ir havin g the sa me chromosomal defe ct . the 15:17 tr anslocat ion . and also by thei r capacity to de velo p coa rse azuroph il granul ation afte r short pe riods of culture ill vitro (.'it·f· 236).
90
235
I )
136. T he sa me ma rrow as see n in 234. here af ter 20 hours in culture: coarse azuroph i l gran ules have now appeare d in the cytoplasm of all the promye locytes . 237. A ma rro w fro m AP L:SS sta in showing heavy ove rall positivity. with positive ly sta ined Aue r rods . 238. Anothe r case o f A PL :SS sta in of ma rrow cells
sho ws similar ove ra ll positivit y. but here the Aue r rods . seen in the ce ll at th e extreme right of th e field. appear
boIlo w with a sudanop hilic outer coat but a negative core .
!J9. A similar S H prepa rat ion from another case of ~Pl . again showing stro ng positivity and with bo th -solid and hollow Auer rods visib le in cells to the right ~ left of the field . respect ively. T here is also a pt ing promye locyte show n. with sca ttered suda no- gra nules among which several positively reacting r rods can he made o ut. Multiple Auer rods . limes a rranged in leashes o r fasce s. arc diagnosticrJ cha racte ristic of APL. and can often be best ed in SB sta ins. especial ly in thinner parts of the ~ . ~ he re they are less likely to be obscure d by heavy granula r posi tivity. He re they may show up as e rods (if they have hollow SS-nega tive cores) , or d amo ng the scatte red gra nules of disrupting
I
241
240
242
240. PA S reaction in APL. showing mod er ately stro ng. diffuse and finely gra nular po sitivity. Occasional nucl ei sho w the A PL type of twinning or distortio n. 24 1. An other field fro m the sa me pr ep ar at io n sho wing unus ua lly stro ngly PAS-positive Au er rods. including se veral in the cyto plas mic rim and othe rs ove rlying the nucle us in the cell at th e right of the field . 242. Anot he r exa mple o f stro ng PA S pos itivity in an Aue r rod overlying th e nucleus in a lcukae mic promyelocyte in A PL. 2-lJ. Acid phosp ha tase reactio n in APL: th e leuk acm ic
pro mvc jocytcs sho w only a very weak gran ular reac tio n. con tra ling with th e stro nge r reacti on in a neig hbouring plasma cell. As normal promye locyt e azurophil granules arc u ua lly q uite rich in acid phosp hatase . this weak reaction may indicate a lysosomal functional defic ie ncy in APL.
92
243
• 0
,
, .•..0;
'.
Dua l este rase reaction in APL: th er e is a negative . throhlast pr esent hut all live promyclocytc s e xhihit g gra nula r Cfi po sitivity a nd two of them contain In ple Aue r rod s which ar e her e int en sel y CEtivc. T he strong reactio n for CE, with or without _ativc or Bfi-posuivc ce ntra l cores, is not usuall y see n Auer rods except in the 15: 17 a nd 8:2 1 tru nstocati on s.
_...a..I
_
~
Dual esterase reaction in AP L: a negati ve ea rly hro b last a nd two Cli-positivc promye locyte s con g mu ltiple A ucr rods sho wing Cfi-posuive outl ines ne gative cor es. D ual este rase react ion in APL: the leukaemic sho w C E positivity with nume rous strongly CEu ve A ucr rods: the de nse circula r or ring -like Cfive structur es, some with a hollow Cfi- nega rivc ue, see n es pec ia lly in o nc of th e prom yelocyrcs. bly represen t early stages in th e form ati on of Au e r A seq ue nce betw een these hollo w rings and the co mmo n holl ow rod s (as shown in 2~5 ) ca n he o ur.
.-
A dual es te rase react io n in a nother case o f APL: lo w rings and short Auer rod s in seve ra l o f th e . elocvtes here d isplay a CE-pos itive envelope with -pos n ivc co re .
'
4
2 ~7
249
2511
248. Section of bone marrow trephin e biop sy ( H& E sta in) from a pati ent with APL. showing high overall ce llularity with almost complete replace ment of norm al marrow cells by nuclcolat cd prim itive ce lls in which it is just poss ible to discern the granularity of their cytoplusm. and among which some show signs of the double lohu latio n nuclear pat ter n. T here are a few residual e rythro blasts.
U9 . Anot her sect ion from the same biops y sta ined with Gie msa. hut not providin g better gra nule definiti on o r clea re r cell differentia tion . although the lcptochrom utic nucle i with pro minent nucleoli arc well visua lized and the prese nce of occas io nal nuclear twinni ng can be detected . 2: . A thin plastic-embedded H&E-stained section of another trephine biopsy from a patient with APL. in which this high-po wer view reveals the cytop lasmic azurophil gra nules of the pro myelocytes more clearl y. 251. A still-higher-power view of the same section. where several leashes of Auer rods ca n be see n in the poorly defined cytoplasm of the leu kaemic cells. While this sequence of histological sections does include diagnostic material and illustr ates the power of modern histologica l techniq ues. especia lly with the use of plastic embeddin g, there ca n be no compa rison with the much supe rior cytology of smea r prepa rations .
94
...
• 25J
~-
255
151-256 . A [urthcr APL variant. 1S1. APL variant- the cells show numerous mod eratel y rse granules hut no Auer rod s were visible in eithe r Leish rnan (here ) o r MGG stains.
':3 . Heavy granular SB positivity typical of prom yclobut again no Au er rods. tro ng diffuse tinge of PAS positivity - the eristic A PL patt ern . -,
'egative reac tion o r fine scattered gra nules o nly of phos phatase positivity. Dual este rase - a rem arkable mixtur e of strong
256
same granule (ef. 2-17 ). A mixtur e of CE and BE -C} is quite commonly found in APL. eit her in - , ; : : ce lls making up a du al populatio n with 01: • e1}' CE and BE posi tivity. o r with both kinds of .",...... prese nt in the same cells. Ab out a third of cases feature . hut it is accompa nied. as in this ease . 'ise typ ical pro myelocytic cytoc hemistry and IIIaJ .er immunology and does not ind icat e the ...~.,. of a mo nocytic compo nent.
95
• 258
257
•
257. Exce ptio nally lar ge pro mycloc ytcs. myclo cytcs and rnctu myelocytes may he enco untere d in the marrow of pernicio us anae mia ( PA) and someti mes of other megaloblastic (and rar ely nor mob lastic) anacmias. Her e arc e xamples of gia nt pro myelocyte and myelocyte from
rA.
258. A neutrophil sta b cell and a segmented cell from nq rmal periphera l blood . 259. Th ree segmented neutrophil po lymorphs, onc showing a drumstick appendage . Two small sessile appe nda ges. not co untable as drum sticks. arc visible in the uppe r ce ll. Well-separated small nuclear appendage . attached to the main nucleu s by a short narrow strand. arc found in fem ale neutrophils o nly and rep resent the same inactive late- replica ting X-chromosome material as is visible in the Barr bod ies of buccal muco sal or other epithelial cells . They are not to be confused with the smaller prot ru sio ns. which have a broad attachment rath er than a nar row stalk. and which occur quite often in the ne utrop hils of either sex.
96
259
16 1
160. Th ree segmented neu trophil polymorphs a nd a myelocyte . The myelo cyte still retains a degree of . to plasmic basophi lia as co mpared with the late r eeutrophils. and the nucleus - although showin g the irreg ularly dense chro matin patt ern associated with mat uration - is less pachychro mati c tha n in the case of the polymo rphs. 161. A dru mstick appe ndage attached to the nucleus of a tab cell. A lthough the connecting strand is a litt le . ker than in the typical appe ndag e . the size is abo ut ~ t and the body is accept able as a fema le dr umstick appenda ge. ~.
A segmented neutr op hil pol ymorp h. with dru mappe ndage and a monocyte . T he lighter grey ilaining of the mono cyte cyto plasm and the mor e op en chro matin patte rn of the nucleus contrast clea rly with me sta ining colour and inte nsity of the neutrophil. ite the granularity of this monocyte cytoplasm. Th e ulev are also different , bei ng generally finer and a mo re reddish and less purple colour in the ~..e as co mpared with the neut rop hil polymorp h.
262
263
265
263. Three segment ed neut roph il polymorphs in the centre o f th e field. with an eos ino phil pol ymorph and a mo nocy te . T wo more neu trophils appear at the edges of the field.
266
UM. Nuclea r twinning in neu tro phil po lymorphs: there appears to be so methi ng approaching a mirror image disposition of nuclear lobes . This specimen was from an AML following chemot herapy. 265. A multi-lobed polymorph from the peripheral blood in a megaloblastic anaemia. These macropolycytes may be found in megaloblastic states d ue to defic iencies of eit her cya nocoba lamin or folic acid. Th ere is anisocytosis and macrocytosis of the accompanying red ce lls visib le in thi s field .
266. Coa rse gra nularity in stab cells from an infective state wit h leucocy tosis and left shift. Th is appearance is so metimes ca lled toxic gra nularity. 267. An other neu troph il showing very coa rse toxic gran ulation. in this case a bilobed po lymorph. aga in fro m a severe infection with a ma rked left shift . i.e . a relat iv·e increase in th e numbers of stab ce lls an d neu trophil po lymorp hs with onl y two lobes as compared with thr ee- and fou r-lobed cells.
261
normally gra nular and a non-gra nular neutrophil ....in""'rph. toge the r with a Iymphocytc . Ab senc e of ~_lI3rit )" in po lymorphs is most co mmon in leukacmi c but may occasio nally he sce n in mo st a naemic or .nlaJpenic states.
270
-\ poorly gra nula r mult i-lobed po lymorph fro m th e in a case of pe rnicious anae mia . A second ~~:cd~ cell with four nuclear lobes, a hasophil .... and various earlier gra nulocytes ma ke up the ........oder of the field . 8laJTOW
smear fro m the buffy coat of a blood sample _.al.-\In>ma pat ient who developed se vere deficien cy o f .. and folic ac id while o n parenteral nutrition in
."''''·C the rapy uni t. The field shows an ea rly
';::::;me ~ galoblast and a mult i-lobed pol ymo rph or
•
le. Two red cells co ntain Howell-Jo lly
99
27J
271. LClU..ocytc a lkaline pho sphatase ( LA P) reaction . w eak and stro nger povitivity in neu troph ils. with a nega tive lymphocyte . Positive reaction s in hae mic cells arc virtually confined to neutrophil stabs and segme nted cells. although macropbagcs may also show positivity. 272. Grades of posinvity in polymorphs. rang ing from 1 (+ ) to -t ( + + + + ). Summation of ratings on 100 neutrop hils gives a sco re with possible range fro m 0 to 400. T he norm al range is betwee n 15 and 100. 273. Increased LAP sco re. with man y strongly po sitive ce lls from an inllammatory leucocytosis. Similarly high scores may be found in po lycytha emi a vera . myelofibrosis. hairy cell leukaemi a (Hf. L} and Hodgkin's disease (11 0) .
) )
<'
G
I I
•
276
_ -_
Cytology ami cysochemiu rv of bon e m llrrow om a patient witlt tm hereditary leucocyte defect ell: yme deficiencies and the Peiger-Huet
Romanowsky sta in illustra ting var iou s sta ges o f mat ura tion and a single late no rmoblast at om o f t he field . A ZUTOp hil gran ule s a rc visible in vclocvrc just above the normoblast. and specific !'""..._ ..... a re prese nt in th e three ma ture eos lno phils. but trophils show litt le or no gra nu larity. A ll the a n uloc yte s , nc utro phil s a nd cosinoph ils a like . gme nted coa rsely pac hychro matic nuclei with o r cu rved ba nd o r dumb be ll-like structure . of the ra re homozygous form of the Pelger -Huct Thi s is a be nign familial disorder . inherited as ...._ omal do minant. In th e comm on er hcter oncutrophils have mostly bilob cd nuclei . ed form of this ano maly . which occurs q uite roliferative slates and generally resembles . _o tic form has already been illustr at ed in th the S::!t tr anslocation vari an t of AML
•
~,_"".l e
277
•
B and DA B-pero xidase . respectively . IIOI1Jl3l1y positive eosinophils hut negative Tbe deficie ncy of myclopc roxida sc (MPO) . m the familial form which . like the Pclgcr _ _.... . is inhe rited as an autoso ma l do mina nt. commonly as an acqu ired fea ture in eve stat es. especially in AML. CM L. aDd mvelod vsplastic sta tes . -e-
P
278
react io n is nor mally positive in the taini nz reve als a red uctio n in exthe ncutrophils for CE. although n t.
101
279
280
281
2M2
2H
279 and 280. Respectively low- and high-power views o f a bo ne marrow trephi ne biops y from a patient with an infective leucoc ytos is. T he first sho ws the den se ove ra ll cell ula rity of the spec imen and the seco nd allows the co nstit uent ce lls to be recognized as predo minant ly later gra nulocytcs, mostly at th e metam yeloc yte to po lymorph stages . A few late norrn o blasts with dense black round nuclei and minimal cytoplasm can be distinguished. but the M:E rati o is o ver 20:1. well above the norm al ra nge of 2: 1 to 8: 1. 281 and 282. Similar low- and high-po wer views of ano ther tre phine biopsy fro m a pat ient with a reactive leucocy tosis in HD agai n showing de nse cel lularity , but with a mor e mixed cytological pictur e . including erythroblasts and cosinophils as well as the predominating neut rop hil gra nulocyte s. which here include a higher proportion of ea rlier stages. bo th myelocyte, and some nucle olated promyelocytes . Th e M:E ratio is again high . around 10: J. 283. Another tre phine biopsy section fro m a pat ient with HO and a reac tive eos ino philia . Thi s high-power view shows a generally pleo morphic cyto logy, with various e rythroblast and gra nulocyte stages and a mononuclear mega karyocy te . together with numerous eosi no phile. mostly at the myelocyte stage. alt houg h a few have more mature bilobed nuclei . Th e cosi ncphils tend 10 be gro upe d along the line of a cap illary blood vesse l in the lower left part of the field . whe re the vascula r end ot helial cells are also conspicuous.
•
I _
•
• 286
Eosi no phil myelocytcs and met am yelocyte in bon e ....... _The re is also a neutro phil myelocyte , various -.:I segmented ncutrophils, and a late normoblast.
i c ::;: :
no rmal eos inophil po lymorph. showing the 'spectacle' arrangement of the nuclear . which are usua lly two in nu mber. The surred ce lls show slight hypochromia and there is relet o verlying one of them .
me _ _Ird
pted eosino phil. In this and the next two rela tively lar ge size of eosino phil as -ith neutro phil specific granu les ca n be
Eosi no phil po lymor phs with agran ular i.be cyto plasm. This appearance is not . but beco mes most freque nt a nd co n• Ie1J .acmias. The lower cell has a typica lly but the upper has an annular or ring tu re on ly occasionally see n in norm al ca..."",ohi l o r neutrophils although more form. of A ML. not ably case. with 8;21
•
-, -l-
287
289
2'10
289 . Fami lia l eosino philia : ge ne ra l " icy.' of ho ne mar ro w; a ll st ages o f ma turation in the eos ino phil se ries
•
arc present. 290 . Th e sa me case: high -powe r view to show 'a mp ho phil" a ppe a rances (hot h ba so phil a nd eos ino philstai ning gra n ules) in eo sinop hil p ro myelocyt e and mycl ocytcs . T he ' basophil" gra nu les probabl y rep rese nt pri mary eos inophil gra nules. Th is a mphophi l ar rea ra nce of granules in cosinophils. see n he re in an her ed ita ry eos ino philia. is a striking a mi co nspic uo us fea ture o f ce rtai n va ria nts o f Ar..1L. es pecial ly cas es with chro meso ma l a b nor mal ities af fec ting the lon g a rm o f 16 . a nd als o to a lesser e xte nt those wit h S:2 1 tra nsloca tion .
•
291 . Pe riph eral blood in th e sa me case as in 289 a nd 290 . Nu me ro us mature e os inop hil pol ymorphs ar c presen t.
•
• I •
291
,
_ 1-304 . Cvtologv and cytochemistry of bone marrow from cases of A AI L with abnormalities - deletions , eruons or translocutions - involving the long arm of , mosome 10 - del(l6) ('1221, il/l'(l6)(1'13;q22) , or [0111'13;'122 ).
_ :-_ -1 . Ge ne ra l low -power views of hone marrow I fro m three cases orA ~ l L with chro moso me 16 _Such cases show a highly ce llula r mixed pop ulauh a predominan ce of blast ce lls. usually. hut not __. includ ing some with nu cle a r twistin g or i lld c n ( a ~ 2\!.C'sti n g mOl1ocvtic linea ge a nd o thers with kxvtic ~)f Iute r g~an ulocvt lc features. T he cases led he- re all shll\\':"" these mixed features . but abo ut cases have 111 0re purely gra nulocytic pre cur sors . sio na l cas t's arc esse ntia lly mo nocytic . A mo ng cells a re scatt e re d var iable numbe rs of . a t all stages of ma turatio n . wit h freque nt i ie-, o f gra nule sta ining a nd d ispos itio n . In 292 rous eos inophile mostly have a blue tinge . 1'93 a nd 29.J the colo u r of mos t granu les is more I so me ce lls sho w an adm ixtur e of da rke r. ilic, gra nules . illustr a te d in more deta il in res.
105
296
295
29H
.
,
.. . \ ... ......: '-.... .. . . s : t- ..
106
/~
299
295-298. Higher-power views of the marrow ce lls in A~1L with 16q abno rma lities. T here are abnormal eosi no phils in each field. Th ey arc least prominent in 295. which has five cosinophi ls. three with normal granules. o ne disrupting. and the fifth with so me amp hophilia. 111e lowest cell shows lack of seg me nta tion of its o the rwise mature nucleu s. a pseud o-Pelgcr-Hu et phenomeno n frequently see n in these cases . The two eos ino phils in 296 arc grossly ab no rmal. with a scattering of coarse deep red or basophilic granules and a blueblack colour replacin g the norm al eosin sta in of the pre do minant granules . Similar features arc sho wn in variable degrees in 297 and 298. the granule colour repl acing eos in rang ing from hlue to greenish-gre y and the baso philic granu les varyin g widel y in numbers. These fields also demonstr at e the variable size of blast cells and the common presence o f both granulocyte and mono cyte precursors . 299 and J ( H) , Low- and high-power views of SB sta ining in a case of inv( 16). In 299 the presenc e of strong localized or heavy overall posit ivity of the granulocytic type in six blast cells con trast s with the mo nocytic type of discrete scatte red granules of positivity in four. In this field. and also in 300. the eosinophil granules show a mixture of the norm al reaction - positive she ll with negative centre - and solid sudanophilia. the latter probably in the coarse r and more basophilic ah normal granules.
~
•
300
•
- I
_ I and .l02. PAS react ion s in marrow cells from AML inv( In). sho wing negat ive o r weak finely granular -.:I diff use pos itivity in blast cel ls. but stro ng and ariable positivity in the atypical cos inop hils. Some ules show the usual negat ive reaction against a uve cyto plasmic bac kgro und. while o. hcrs rea ct a solid positivity. A d isru pting ear ly eos inophil in t has pos itive granules of this type , so me bein g rered wide ly acro ss th e whole field . Dual es terase reaction in a similar case. showing - e e ryt hro blasts. one apparently co nta ining ~'_""'''' i l granules within a vacuole. severa l rnyeloo r pro mye locytcs with weak -to -strong CE posi_. o nc mo noblast with stro ng BE re actio n. and two ring degene rating and partially disrupted ils at the centre left of th e gro up. o ne with weak uivity in so me o f th e coarse r gra nules . a feat ure ..-.all ~ seen in cos lnoptuts. 1.
Daal
terase reaction co mbined with Chlorazo l (a dye reacting spec ifically with eosinophil -0_ ......) to show th e va riable pa ttern o f CE posit ivity ~ules . seve ral co sinop hils showing onl y th e Plo.\. ..... in but ot he rs. notably the ce ll at the to p left. for CE. Blast ce lls of granu locytic and sho w respective ly CE and BE positivity,
107
30(,
J05. Basophil precursors. together with neutrophil prec urso rs . in the blood o f a pa tient with chroni c mye loid leuk aemia (C i\tl . ). •'06 . A basophil polymorph . to gether with two neutrophil s , an eos inop hil and a lyr nphocytc .
.'07. Another basophil polymorph . These cells do not show the clear separation of nuclear lobes seen in mat ure polymorph ... of o the r kinds . but o verlapping lobes mav he distingui shed . Here. thc coarse granules do not e nt ire ly obsc ure the nud e-m struct ure . whic h ca n be seen to have th ree inco mpletely separated lob es. •\t n~ . A disrupted degeneratin g bu...ophil. with 10...... of nuclea r structure and ... caucrina of er anulcs. The individ ual basophil granules, scattere d in thi s way and u...uallv seen at the tai l o f the smear , va rv con siderahlv in size and shape. often being large r tha n nor mal ncuirophit or eosino phil granul es and sometimes having a rod ...hapc .
307
310
.111
•
313. Bone marrow sme ars fr om leukaemia with basophilia .
all
unusual case of
A Romanowsky prepa rat io n showing a collectio n eecleo tated pr imitive cells with a ge ne rally low ar-cyto plavmic ratio . the ample cyto plasm co n~ nume rous vacuo lcs and fine basop hil granules. re is a neutro phil sta b ce ll, a lyrnphocyte and a nak ed -'~""' Iast nucle us also pre sen t in th is field.
"
..
312
A ...imilar field to that in 309 but sta ined with "ne bl ue. which rea cts with the basop hil granules a bluis h-purple coloration . One blast cell he re is c. as
,.\ "tide fro m th e sa me case as in .'10 . stained with prim itive cells show the usual positi ve reactio n o f neu tro phil pr ecursor s. while seve n others give romatic reac tion with the dye . resu lting in a a in . a pict ure not infreq uentl y see n in~ both MId pat ho logical baso phils.
313
ber slide from this marrow stained with the . The re is o ne negat ive blast cc ll and a reacting ne utrophil metam yelocyte : th e ~-_. etaht vacuo lated blast ce lls show inte nse and ~ula r povitivit y. characte ristic o f basop hils. stain fro m this seq ue nce is a d ual es te rase g CE pos itivity is present in a single -eecursor with a poss ible Cti- pc sit ivc Auer the lowe r borde r o f the nucl eu s. but the ne ighbouring vacuo lat ed pr imitiv e cclis eithe r C E o r BE activity. as ar c nor mal
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314. PAS reaction in mature polymorphs of neutrophil. eos ino phil and basophil se ries . T he ce ntral neut rophi l has dense stro nggra nula r pos itivity. packin g the cytoplasm to leave no visible back grou nd . Th e eosino phil
shows granules with unstained. negative appearance agai nst bac kgro und positivity, The baso phil gra nules are discretely scattered . very heavily positive , against it negative o r weak ly stai ned bac kground . Salivary amylase removes most positivit y. but not that of the baso phil granules. which presum abl y do no t cont ain glycoge n. T he unstained gra nules in the eosi no phil are ce rta inly the specific gra nules scc n in Rom anows ky sta ins. but the I)AS-posit ive granules in the basophil ar e not ide ntical with th, pccific gra nules in that ce ll. 315. 5B reaction in neut rophil. eosinophil and basophil po lymo rphs. Neut rophil granules are positive . eos inophil gra nules show positivity with a ho llow cent re . and basophil gra nules are negat ive (as her e) or occasio nally show positi ve or met achromatic sta ining. 316. Peroxi dase react ion in a similar trio . with appea rances ge nerally similar to those in the SB reaction .
110
J ib
318
317. Al kaline phosphat ase reactio n in a similar cell ~p:'on ly the neut roph il shows positivity . Th e eo slnoimmediately below the neutrophil has a typical d nucleus. while th..1t of the lower basophi l has -=omplete ly separated lo bes. Neith er sho ws any ine phosphatase activity. .3
319
cid phosphatase reaction in neutro phil. eos ine and baso phil polymo rp hs and a monocyt e. neutrophil and mon ocyte show no rmally po sitive r rea ctio ns: the eosinophil and basophil sho w litt le pos itivity with o nly a ra re positively reacting
Dual esterase in a group of basophil. eosi nophil. eeatrophits a nd a mon ocyte. Th e baso phil and il cells arc esse ntially negativ e . while the .....,pllils show typical CE pos itivity and the monoc yte BE positivity.
111
32 1
310
320. A low-pow er view of pe riphera l blood fro m a pati ent with CML. Gran ulocytes o f all stages of maturation ca n be seen, mostly neut rophil s hut with occasio nal basophils. Alt ho ugh myclcbl asts a nd pr omyeloc yrcs arc distinguishable . they arc relati vely few and much o utnum be red hy later grunulocyrcs from myelocy te o nwards. A periphe ral blood smea r with as many lcucocytcs of differe nt stages of mat urity as shown he re is found o nly in CML. wher e the wue at present ation is co mmonly above 50 and may exceed 500 x 10"/1. A higher-power view of anot he r prep aration sho wing va riou s granulocyte precur sors . includ ing severa l baso phils. In additio n to the grunutocyres. two nor moblasts arc present. Th ere is a single myelobl ast in the field but lat er gm nulocyrcs grea tly predom inate .
/J "A
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322. A chro moso me spread fro m a male patient with at 12 C ~ I L. The Philade lphia (Ph) chromoso me. seen o'cloc k. results from a translocation hetvece n the: lon g arms of chromoso mes 22 and 9 (usually) visible in unba nded preparatio ns as a loss of materia l fro m the long arms of on e of the small acrocen tric chro moso mes (no . 22). T he translocati on is a reciproc al one. involving the exchange of mater ial between the two chro moso mes - t(9 :22) (q34:q l l) - and is found in virt ually all haemic marrow cells. including those of arunulocvtic. e rythro id and megakaryocytic se'-ries . thu s confirm ing that t ~ I L is a multilineage neoplas m.
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325
-po wer view of a hone marrow smear from a "rh C ~ I L. sho wing a ce llular pr eparation with a t :E ratio and preponde ra nce o f neut rophil grunui th all stag es o f developmen t rep resen ted . scat te red cryth roh lasts and a proc ryrhroblast
bottom of the Iiekl can be seen . and an il myelocyte a nd po lymorph . whi le the two ,...ith dee ply stained nuclei and coarse gran ules hab le in t he na rrow rim of cytoplasm . 10 either side of a sma ll cent rally place d _.-.... myeloc yte. arc baso phil poly morphs.
...,...."'""'i
rnea r from a pa tient with C M L. showing granules. A basophil is on the left phil is to the upper rig ht of the central : the re ma ining cells arc all neutrophils gra nula rity. from a n a lmost agranular tbe partly sectioned cell at the bottom . to . _ranularity in the polymorph at the top and h o r the eosi no phil. Variations of this kind _ _mo o in CM L. ~ mo rph
Iv there rnav be an almost total abse nce later ne utro phils in C~tL. as in this sample ......he re the central promycto_""';>0111 granules but the remaining late r cells. metam veloc vtcs and two matu re c de void of specific gra nules . The dense "",...._1JIl but minimal segmentation or th e suggests that a pscu do-Pelgcr -Huet ID
lbC
Dl.
113
1ioI'!7'~ · 327
J 16
326. A section of a trep hine biopsy from the hone marrow of a patient with CML. Th e spec imen shows intense cellularity with ob literation of fat spaces. and eve n at this low power it is appa rent that all cell seri es are well represented. with a scatte ring of mcgakar yocytcs . num erous gran ulocytc precursor stage s with lightly staining nuclei and ample cytoplasm. and later granulecytcs a nd erythrob lasts having pac hychro matic nuclei. the for me r more irregular in o utline . 327. A further example of bone marrow trephine histology fro m ano ther pati ent with CML. In this case the re arc num erous residual fat spaces and less de nse ove rall cc llula rity. and later granulocyecs predomin ate. but there is also ano ther feature sometime s see n in ma rrow biopsies from CML. namely the marked proliferation of megak aryocytes . In this fic ld at least a dozen ca n be seen . with various degrees of nuclea r segmenta tio n and complexity. but most often with single o r bilobcd nuclei. Such cells. and eve n smalle r ones some times called micro mcgakaryocyrcs. are characIcristica lly fou nd in trephine sections fro m ahout X(llYo of cases of CML. although not necessa rily associated with increased thrombopoiesis. 328. A sect ion of a needle biopsy of liver from a patient ....it h CML. showing an area of leukae mic infiltration around a po rta l tract . with a dense accumulation of granulocytcs compressing the su rro unding liver ce lls.
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I A se ries of views at low, intermed iate and rs o f sectio ns made from a biopsy o f skin and _ ' . . . .""'''' tissue tak en from a pati ent with C ML. a k ukaemic infiltration. The first two figur es a section stained with H& E and th e third is "~~~~::,-stJ i ned section. Th e first field shows the . with the infiltrate ext ensively involving .....rt.!iolg_ de rmis. producing vascular erosion and - . . orrhagc . In the second field the nature of :::~.~ceIlS can be d istinguished mor e clea rly, ntly nuclcolatcd myeloblasts and fewer ytes spreading bet ween th e subcutaneous _ _oeti,..e tissue . Th e third field shows th e edge e as it reaches the epide rmis. where the ma tu re granulocyte precursors interco llagen fibrils and macrophagcs lad en .... .. .L eithe r free iron or possibly ca rbon particles. __ .... «11 is visible towards th e lower left co rne r. no w skin deposits arc no t unco mmo n in _ times nume ro us and wide spread so a,s -,......, a lru aemic skin rash . es pecially. as in the ~ at the time o f gene ral metamorphosis o f fro m the chro nic to a more acute appearance may therefo re have a poor . nee. More bulk y a nd restri cted "",,,,,,,... may also occu r. less co mmon ly. at any . and are sometimes referred to as
-'-' I
:
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115
333
JJ2
332. LAP reaction in CML. Th e polymo rph s are virtually devoid of enzyme . This is a striking and almost uniform characte ristic of ne utr ophils in C ~1L. 333. Unde r effectiv e trea tme nt . whe n the blood pictur e returns qu antit atively to nor mal. the LAP score rem ains low (and precursors still have the Ph chro mosome) . bu t an improve me nt towards the lower no rmal range often occu rs. Th is preparati on shows the pre sence of faint positivity in so me pol ymorphs fro m .1 buffy coat in well-co ntrolled C ~ I L. although nearly all of the neutrophils remain quite negat ive. 334. When blastic crisis emerges. the LAP sco re usuallv rises sharply. This figure shows + to ++ reactions in polymorphs in a pe ripheral blood film from a pa tient at this stage of the disease. Surroundin g the gro up of po lymor phs arc pre dom inant ly blast ce lls.
116
33-1
3J6
·T'Tu.Jlase re action in C~t L. Except ionally strong the rule in polymorp hs in this disease . pe roxidase-negative cells can be found in and mav occasionallv be numerous. Th e ....""ou\< polymo rphs may' includ e so me basors of them are certa inly neutrophils.
3.17
. n in CM L. The pol ymorphs may react tro ng posi tivity. as here . but th e reaction ea "er than nor mal and neg ative poly fou nd . includi ng so me baso phils (cf. a proportio n of the neutro phils. which allel absence of peroxida se reac tivity. mav show a metachro matic redd ish field from CM L progressi ng to ward s hich also shows typical positivity in ce utro phits with S8 . C\ IL showing S8 positivity in ncut rohile four baso phils show varying ~.""""' . partly met achrom atic. O ne o f the B-negative.
11
3-10
339
•
p rogressio n to myelofib rosis/megakaryocytic myelopr oliferative state.
339-34H. CM L
339. Section of bon e marr ow trephi ne biopsy from a patie nt with CML who showed progression to a phase of hacmor rhagic hypopl asia afte r a peri od of prolong ed co ntro l of the chro nic disea se by pu lse chemo therapy with busulphan. Th e marrow is poorl y cellular. with a backgro und of necrotic and haemorrh agic suppo rting tissue co nta ining a scatterin g of iron -laden macrop hagcs and bot h lymphocytcs and plasma ce lls. but few rcco gnizab le myelo id cells. A picture such as this may arise in CM L as a co nseq uence of mycloto xicit y fro m undul y aggress ive chemot he rapy. but may also he a spon taneous phase in disea se progression . genera lly pre ced ing a myelofi bro tic transfor mation.
3-W . A re ticulin-stained thi n section from the same marr ow biopsy as in 339 . showing that ther e is already a clear increa se in reticulin fibrils. and that the disease is undergoing a meta mo rphosis to a mye lofibrotic phase . This. in turn . is co mmo nly supe rseded after a variable peri od of weeks o r months by a more tloridly ce llular acute leuk aernic type of bloo d a nd bone ma rrow picture . no t infreque ntly sho wing at lea st a tr ansient megakaryo blastic o r megakar yocytic co mpo nent . ..l41 . Sectio n of bone marrow tre phine biopsy from a pati en t who showed the type of tr ansfo rmation de scribed in 3-W. with the replacemen t of successive chr on ic leukaem ic and myelofibro tic ph ases by a highly cellul ar acute tr ansform ation . the primitive ce lls having megaka ryo blastic featu res. sho wn furthe r in the next three fields (3-12--3+1).
11 8
,
•
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Thi n plastic-e mbedde d sec tions fro m th e ma rro w trep hine bio psy as in 341 . stai ned ~--"CI~ with H&E. Giemsa and the PAS reaction. ~ an area has bee n selected for photomicromowa sectioned hlood vessel . with ncopl ast ic it hin and surro undi ng it. Wh ile th ese are .......... all me mbe rs o f the same ncoplastic clo nal ......111<111. the ir loca lization affects their cytological :~:~.; \\ith the intramedullary blasts. despite the . of their cytop lasm. having more unifonnly - --.... fea tures o f Iept och rom at ic nuclear staining, ible nucleoli. especia lly in the G iemsa ".,- - - • • hereas the intravascular cells have a r nuclea r pattern and also show the • topIasmic o utlines cha rac te ristic of mcga aod early mcgaka ryoc ytcs when see n in ......ontions. Alt ho ugh th e PAS reac tion whe n CMl plastic sectio ns usually gives much less .[\ in haemic cells than is found in PA S "(d oJ.Ul). some peri phe ra l cytop lasmic disce rned in th e primitive cells in 344 . ....,_.... in the intravascular than in th e intr atio ns. The differences in cyto logy ites may be partly a consequence of _ ......... from contiguo us cells in the marrow the vascula r lume n. but may also ease in maturity associated with vessel wall.
:
119
345-348 . Cyto logical and cytoche mical details of m egakarvoblastic and m egakaryocytic transformation in CM L as seen in smear preparations of bone marrow aspirates.
345. Rom anow sky stain shows two hlast cells. to left and right. several megak aryocyte nuclei and masses of variably sized platelets. 346. PAS reaction sho ws the coa rse pos itivity in plat elet and megakar yocyte precur sors. 347. Acid phosphatase is strongly posit ive in megakaryocyte precursors . 348. Dual estera se shows strong CE reaction in polymorphs hut o nly weak BE reaction in a megakaryocyte and so me platele ts. and in a blast cell. A stronger reaction might be expec te d in cells of the megakaryocyte series if alpha naphthyl aceta te rather than but yrate is used as the subst rate for the esterase reaction.
120
,.
..
1. C \ f L progression to myeloblast-typ e crisis.
of plastic-embedded bone marrow trephine sectio ns (3-19- 35 7) and cYIOI01-:Y and cy lO-
ry of smears fro m bone marrow aspinnrs 1 ).
-powe r view of a thin sectio n of ma rro w from a pa tient und ergoin g myeloblastic tr ansof C ~ t L. with islan ds o f pr imiti ve cells through the marrow . amo ng residu al are as o f -phase cytology. I n this fie ld th e lower right area the myelo blastic component while the upp er \loS resid ual chronic-phase mixed cytology. :s.IOO o f CM L co mmo nly develops with a pat ch y - _ " " o f blast-ce ll island s. and a more reliabl e the process may he obt ained from trephine lhom fro m aspira tes.
351
•
• •
•
r-po we r view o f th e upper left part of ~9 . chronic-p hase cyto logy with num erous polvmorphs. metam yeloc ytes and myelo blast cells. occasiona l cryrhrob lasts . and a ryocytes, includ ing typical micromega-
contr ast is this view. at th e sa me increased the lower left area of ~~9 . wher e th e e- cells are rnycto blasrs with fine lcptoar chroma tin contai ning co nspicuo us only o ne or . less oft en . two visible in ~ sectio ned cells). Th e few scatte red raining nuclei are either erythroh lasts
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351
o
• Ano ther exa mple of myelob lastic transfo rmatio n develo ping in CML. In this thin sectio n of plasticembedd ed trephine biopsy mater ial fro m the iliac crest bone marrow . the edge of a blastic nod ule is on the right . betw een bon y trabeculae above and be low. with resid ual chro nic phase cells havin g gene rally da rker nuclei o n the left.
~'5 2 .
353. A higher -powe r view of the same section as in 352. showing a ton gue of myelobl astic cyto logy penetra ting be tween chronic phase ce lls to the uppe r right and lower left. Th e finely Icpt och romatic nuclear structur e and nucleo li of the myelobl asts arc well shown. contras ting with the da rker a nd mo re polymo rphic nuclea r patt ern of the late r granulocytes of the chro nic phase. 3,5.4 . A similar thin sectio n fro m anot her patient with eme rging myelobl astic transformation. her e illustrating the dest ructive trabecular erosion sometimes produ ced b)' the myelob lastic proli fer at ion . which in this section ca n be see n to be co nce ntrated around the dam aged trabecu lae . Th ere arc resid ual lat er granulocytes and erythro blasts fro m the chro nic phase of the disease in the upper left and lower right co rne rs of the field . Increase d bo ny destr uctio n. associated with the onset of bo ne pain and the developm ent of radi ological changes. is o ne of the more commo n for ms of present ation of blastic met amorph osis in C ~l L.
122
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-S-J57. T hree furt he r thin sectio ns from the same ine bio psy as in 354. Th e first (355) shows at higher "ficatio n an area similar to that reproduced at low in 3.5-4 . with a clea rly visib le co ntrast bet wee n the _ _dve nucleo latcd and lep toch rom atic myclobJasts ... the irregularly eroded trabecular margin and the e of more mature cells space d furt her away . The reil wit h large darkly stai ned nucleus. direc tly ........ a sem i-circular bo ny erosion at the middle of I ma rgin of the field. is probably a poly ploid _:udlast . The second field. in 356. from a Ponceau S· pre paratio n. reveals an absence of collagenous aDd fibro b lasts fro m an area of myeloblastic ....n1>e<" lar proliferation very similar to that shown preceding two figures . T he third similar field (357) 3 prepa ratio n sta ined for argyro phil retic ulin ~ n . the re is a notable absence of such fibrils in _ urhood of the tr abecula e , wher e the myeloproliferatio n is co ncentra te d.
357
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, 123
359
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.,
358 , A smea r pre parat ion fro m a bon e ma rrow aspi rate take n fro m a pa tien t in the blastic phase o f CM L. A Ro ma nows ky sta in shows a grou p o f blas t lTUS with little evide nce of differ enti ati on . 359. Th e Sll stain sho ws th at all th e blast ce lls co ntain localized st ro ng cytop lasmic positivity of gra nu locy tic type . 360 . Th e blast ce lls arc largely PA S-negative or wea kly react ing with di ffuse posit ivity and fine gra nules . A mo nocy te/m acrophage shows some coarse PA S-positive gra n ules - a po lymorp h is norm ally pos itive,
-,
•
361. Th e acid pho sphatase react io n shows a few coa rse granules of positivit y in most blast cells a nd a tr ail of stro ngly po sitive granules from a parti ally d isrupting ma cro phag e .
362
362. The dua l es terase reaction displ ays mod eratel y stro ng CE po sitivity in most of the blast ce lls.
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\...370. C \! L progression to a lymphobla st-type crisis. C·A L L untigrnic findin gs. Histology of trephine (363- 365 ) ami cytology and cytochemistry of _ ....Don , m ears (J 66-J70) .
•
th in sec tio n showing a sca tte red infiltration of lis amo ng resid ual later ura nulo cvtcs of the phase o( CM L. Alth ou gh this view includes spaces. the overall ce llulari ty of the specime n is ...."'~~. de nse . with closel y packe d cells a nd litt le to .I prece d ing stage o f myelofibrosis. :=-
hi_he r-po we r view 'If the same preparation . the inte rmingling o f scatte red nuclco latcd cells with late r . .ura nuloc vrcs - rnvelocvt cs. . te-, and poly·fllorp hs. · Onl y a' few' late 5 ca n be recognized . Th e p ro port io n o f blast 50°0. exceeds th at to he found during the o f C~ H .. and ind icates the emergence of o rmatio n.
r e xamp le of blas tic tran sformat ion o f in a thin section of plastic-em bedded ~ specime n of ho ne marrow. In this case n depicts an almost comple te repl acement ....,..,.,.pha...-.e cyto logy hy a unifor m sheet of blast )e nucleo li and ver y op en nucl ear chro--..... The re is a mitotic figure at the lower probable late neut rophils. and a plasm a ot the blast cells ca nnot he determined o&og:ical preparat io ns. but para llel _De....·_" f) s-ho ws the m to he Iymp hoblasts . ill
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367
366. Roma nowsky stai n shows agran ular blast cells with high nuclear-cytopl asmic ratio . in this smear of bone marrow aspirate from blastic crisis of CML.
369
367. Th e blast cells are negative to 58. Th ere is a single suda no philic myelocyte in the ce ntre of the field next to a mito tic figure . 368. The PAS sta in shows occa sional block o r coa rse gra nula r positivity of lymphoblasti c type . 369. Acid phosphatase stai ning sho ws scattered posi tive reaction . not espec ially concentrated at o ne pole of the nucleus. 370. Dual esterase sta mmg shows a strongly CE positi ve granulocyte out o nly weak scatte red BE posiu vity in the blast cells.
126
370
J 1
373
Ji-l Basophil predo m inance in malignant progresO IL.
ano wsky stai n sho ws a mixture of gra nuloaDd their p rec urso rs i tcluding two blast ce lls,
.......""e>. stab cells and fnur basophils .
Slain in the same case , showing a myc tone utro phil myelocytes with no rma lly strong _ _ "'P","lia and four bascphils from promyelocyte to with mos tly reddish met achromatic staining hil gra nules. react io n in the same case , sho wing coa rse _ pos itivity in four basop hils at vario us stages " ~ fro m promyelocyte o nwa rd s. Ne utro phil _ _ ....rs sho w nor mal diffuse tinge. reaction in anothe r exa mple of basophil ........_ _ece duri ng malignant prog ression ofCML. In ~""- re are two early neut roph il precu rso rs at the ;I neutro phil metamye locyte at the bottom , remai ning cell s are of th e basophil se ries and _ degrees of coarse gra nular PAS positivity cell series. '
37~
,
127
-
•
376
• 37K
~\75-JHJ .
Preparations f rom cases of juvenile CM L.
375 . A Leishrna n sta in o f blood smea r showing promine nt mon ocytic co mpo ne nt. seve ral monocyte - being vacuola ted . 376. Leishman sta in of bon e marrow smear fro m th e same patient . showing resem blance to C ML. with various grunulocytc pre curso rs predo minant . 377 a nd 378. Ro manowsky (Heyl) stai ns of bo ne ma rrow aspira te smea rs fro m ano ther case of j uve nile C ~t L. showing respectively low- and higher -power views of the cvroloav. which falls betw een tha t of an acute ffiyc hlffil; nocvtfc leukaemia (A MML ) and a C I\IL. This COItditinn is S()~cti ml."S called 'A~I ~I L of childhood' and is proba bly best trea ted as an ac ute leu kaem ia . Unlike CML pro per . which doe s occur rarely in childhood . so-called j uvenile C ML does not show th e 9 :22 tra nslocatio n. alt ho ugh the LAP sco re is usua llv low. There is typically a high level of H bF and low Hb A 2 • and the infantile rati o (3:1) of glycine to ala nine at position 136 o n the gamma cha ins of Hb F is ret ained . J 79 . S8 stain o n 5100d smea r: coarse posi tivity in neutro phile an d d iscrete sca tte red gra nules in several mo noc yto id cells.
379
. I
• · ...·,
•
"
-PA S stain on blood smear: a blast cell with r AS-positivity pattern, two vacuolated a nd two neutrophil polymorphs with normal rea ct io ns.
382
_",,~·t ic _""~'t es .
P
I. Acid phos pha tase : three polymorpbs and three _ _xytes in peripheral blood show moderately strong
Iar positivity, Dual este rase: two polymorphs show CE positivit y
- t 'x mo nocytes and precursors show moderate BE . -ity.
Anothe r exa mple of the du al esterase react ion on a ~
of bone marrow cells from (he same case of
_nik C ML as shown in 377 and 378 . The mix ture of --~.1ic a nd gra nulocytic precursors with respect BE and CE positivity is well shown . Several later .......>phil also show CE positivity.
383
•
385
38-4. An ' LE' cell . The characteristic inclusion body of ingested nuclea r materi al prod uced in leucocyres especially polymorphs - as a result of the action of a factor prese nt in the sc ru m of patients with dissem inat ed lup us erythe matosus. Th e factor is an antibody to nucle oprotein. chiefly histone . which acts in the presence of complement as an opso nizing age nt. rende ring the sensitized nuclei susceptible to phagocytosis by neutrophil polymorphs and to a lesser exte nt by eos inop hils and mon ocytes. T he smoo th and swollen appearance of the ingested nuclear material. resulting from the imm une process . is characteristically different from the smalle r and often denser nuclear remnants engu lfed by polymorphs o r monocytcs following the death of senesce nt cells. as see n. for exa mple . in the ' tart' cell illust rated in 386. Alth ou gh the demon str at ion of LE cells remai ns a useful and rapid diagnostic procedure . it has now largel y been replaced by o ther immunological tests for anti-nuclea r facto rs. and mor e specific tests for the antibodies to dou ble-str and ed D NA that especially disting uish syste mic lupu s ery thematos us (SLE) from othe r collagenoses . Neverthe less. the LE ce ll remains a st riking cytological phenomenon . 385. An oth er exa mple of an LE cell with a lar ge but partially d ivided inclusion . 386. A ' tart' cell . as commonly found in preparations made in the sea rch for LE cells . T he inclusio n is usuallv. as he re. in a mon ocyte . and co nsists of a ce ll nucleus, mos t often of a Iymphocyte . The inclusion stains more deeply th an does the LE body . These tar t ce lls ar e of no kno wn pathological significance and must be d istinguished from LE cells.
1.-
3116
•
•
,
e-
r-Huet phe no meno n. In the hom ozygou s pol yrno rp hs sho w a single ro unded dense the zra nulocvtes arc affected in the her ed iof this disorder. but the change ma y a ffect in myeloid leuk aemi a . giving a 'p se udoranee .
389
-HUCl phe no me no n. Het ero zygous for m pol yrno rphs showing a ' ba nd' o r bilo bed structur e. Th is a ppea ra nce ma y occ ur as a rma lity. but may be mimicked in the ~~ eer' poly morphs so me times see n in acute ..,. .., mye loid leuk aerni as and in myelofibrosis. a_pie illustrated here is from a patient with of the ce lls shows twinning of bilobed _~I'd~,r nuclei.
.p,."'_
red d ish-vio let gra nules in leucocytcs in ___ ~', a fa milial diso rde r o f leucocyte gra nuarance in the neutro phi ls resem bles the ,...,_.... commo nlv see n in leucocvtosis of in 390-392) , but the acco ';' pa nying ) phOC~l CS. some times with in vac uo les . f . sho winga 'co mma' shape . arc stro ngly r's a no maly.
tJ--
Dl
391
3
•
393
392
390-392. Examples of the blue-staini ng areas in the cyto plasm of neutrophil polyniorphs sometimes see n in infections. especially pneumonia . -Th ey arc known as Dohle bodies. The polymorphs also show some coa rse toxic gran ularity . Th e D ohte bodi es of infec tion rep resent area s of aggregat ed ro ugh endo plasmic ret iculum, as see n in the electron microscop e. and are essen tially different from the inclusion bod ies, sometimes incorrectly given the same name , which occur in the May-Hegglin ano maly . .
393. Further examples of Dohlc bodies in neut rophil polymo rphs. In this Instance the specific gra nules are wea k rath er than coarse , and the Do bte inclusio ns more readily visible . Do hle bod ies may be found in poly mo rphs in any fo rm of leukaem ia , but appea r to arise as a co nseq uence of associated infection rat her th an be ing intrinsic to the neopl astic process. 3 9~ and
395. Examples of the May-H egglin ano maly. an inherited disorde r with basophilic inclusions. 2-5 microns in diameter. in gra n ulocyies . Th e inclusio ns arc larger than Dohle bodies and not relat ed to infectio n. Ult rastructurally. they con ta in particulate material includ ing rod- like structures unlike any nor mal cytoplas mic co mpo nent.
395
•
... m . Furthe r unusual inclusions . some 3-5 ;;;iII~diam ~.~ eter, weakly basophilic and apparently
:
-bound, in granulocyte precursors and in from a child with a transient pancytopenia . syndrome. The material seems likely to ..._ _,i) protein.
granulation. with both red and pale blue .. a neutrophil polymorph . Similar giant - _ ...... kucocytes of all kinds may be seen in the rare <.1:Irdiak· Higashi·Steinbrinck anom aly. These -::.;::::~a~re thought to be deriv ed by the fusion of .. a l azurophil granules at the promyeloc:arl)" myelocyte stages. There are similar _ ...._ be found in many oth er tissues in this ed polymorphs appear hyperactive in but ineffective in killing and destroying ria or other material.
-
398
~.
399
inclusion par ticles. probably carbon . ,::::~::: bkxxI monocyte . Such material may be - _.....
a pe riod of temporary sequestration rory tract.
133
~O l
0$02
400 . Bone marrow as pirate fro m a patie nt with seve re ag ran u lo~ tosis a nd pe ripheral neu tro phil co unt < O. Sx 10 11. T her e is not a single neutro phil polymorph to be see n. and few neutroph ils beyo nd the p rom yelocyte and ea rly mye locyte stag es. alt ho ugh crythroblasts. eosl nop hils. Iymp hocytes and an occas ional mo nocyte ca n be recognized . 0$0 I and 0$02. Lo w- and highe r-po wer views o f bo ne ma rrow tr ephine bio psy sectio n fro m a patient with leu kopc nia due to dr ugs. Th e re is adequate ove rall ce llulari ty with plenti ful rncgak aryocytes and numerous mono nuclea r cells. shown in 402 to inclu de norm o blasts. lymphocy tes and plasma cells. but a virtu ally co mp lete absence of po ly morphs and recognizable earl ier granulocytes . ..ao3 and 0$0-1 . Two views at incr easingly higher powers of thin sections from a bone marrow sa mple take n at post rnortem from a you ng woman who died fro m an o verwhelming streptococcal septicae mia. unresponsive to antibiotics. Her peri phe ral neutrophi l co unt had fallen precipitously to < O.2 x 109/1 . The bone marrow sho ws complete abse nce of gra nulocytes at any stage of maturity. the relatively high ce llula rity bei ng made up o f mononuclear cells including erythroblasts but also nu merous plasma cells.
1: .
~07
Various mOlOcytes f rom norm al periph eral
ocvte with some reddish granules in the grey . togethe r with a lymphocyte , a stab cell. and .......ophil segmented polymorphs. The extent of - "_ _11 or more gross convolution of the monocyte ite variable . A ve rv few rnonocvtes with ....,,,,,,,,,,>liccytoplasm and nud ear chromatin pattern i witho ut inde ntation. perh aps 20% have inde ntation. as in the uppe r cell in 406. but _ _lC).l es in the periph eral blood show more -..dear bending or twisting. as in the remaining in .w~ 07 . Cytoplasmic gran ularity is also little or none to relatively coarse . weakl y ~ul es as in the central cell of 406 and the ""JO(~.- t es .
one with vacuoles. and a lyrnpho..c1_" are probably all phagocytic. one ' till ~ _ in :- ted platele t and the other two having d( este d their contents.
,.
•
_ _lC).lCS and. a stab cell. The m onocyte-
cytoplasmic granularity and typical
135
•
408 and 409. Co nsecutive Rom anow sky and d uplicate Prussian btue sta ins on the same field , fro m a buffy coat smea r made from the peripheral blood of a pat ient with chronic myelomon ocytic leuk aem ia (C MML). la show more bizarr e forms of nucl ear convo lutio n than are
usua lly see n in normal monocytes. The furth er tr ansfor mation of mon ocytes to macrophages normally occu rs in tissues. but may occasionally. as her e , take place in the blood. Th e transforming mon ocyte co ntai ns phagocytosed nuclear fragments and also a heavy load of free iron . as demon str ated in 409.
410. Promo nocytes from a case of AML with predo minance of the mon ocytic series . Th e cells. tho ugh nucleolared. show nuclear indentations and a cytoplasmic colour tending towards the mon ocyt ic grey rat her than the baso philia of less differ en tiated ce lls. Pure ' mc noblastic' o r "monocytic' leukaem ias are very unco mmo n; there is nearly always some gra nuloeytic element present. so that the name ' mye lomonocytic' migh t strictly be used , but, as in this case. ce lls of the monocyte se ries may great ly predom inate . and it is con venient to retain the term ' acute mon ocyti c leukaemia' (A Mo nL) for such cases. When recognizable gra nulocyt es make up less than 20% of the marrow cells the FA B classificatio n wou ld be M5. .$11. S8 staining in the same case as in 410. The promonocytes show discre te scattered gra nules of pos itivity, here chiefly co nfined to the cyto plasm, but not densely clumped as in gra nulocyte precursors.
411
11
l~ . AMonL. with monocyte precursors of variable -.rpbology and state of maturity. This field also iIIusa scattering of plasma cells. An incre ase in plasma generally focal or patchy in distribution , is ca_.ooly observed in acute leukaemias of any kind and seen when o the r malignant processes invade the
...
.".
B reaction in the same case . A single stro ngly =~~ i1ic polymorph contrasts sha rply with the . 1 C precu rsors. with their reactions ranging from .,..iYe to moderately strong positivity of the discrete .,....red granule type.
:
Three pri mitive cells from an acute leukaemia with
...-..aI morphological signs of differentiation in ky preparation. The SB reaction, shown the typica l distribution of positive granules dI..,,,,,,rn-,,ic of the monocytic series . The primitive are the refore monoblasts.
•
~1 6
-lI S. AM onL: peroxidase reaction . Monocytes and
~1 7
the ir p rec ursors arc genera lly negat ive for peroxidase. hut may show a faint localized cytoplas mic reaction . Auer rods. as shown in th e upper left corner . arc strongly peroxidase -pos itive . T hei r prese nce co nfirms the myelomonocyti c nature of th e disease . since Au er rod s probab ly do not occur in th e monocyte line hut ar e confined 10 granutocytc pre cursor s. bein g derived chiefly from primary azu rop hil granules.
•
•
-116. An other exa mple of peroxidase stai ning in an
A Mo nL. Th e mon ocytes ap pear ra the r mo re di fferentiated th an in the previous exa mple . and have a negative reaction .
.. ..
-Il7. 5B reaction in the same case: the monocyte , mos tly show discret e sca tte red gra nules . A myelobl ast with two posi tive Au er rod s is also see n.
J
•
.-.
.
,
41
419
'
.
•
. -lIK. A gro up of mo nocyte pre cur sors from an
A~lo llL.
.l)9. The same licit! . co nsec utively stained with the PAS react ion. Mon ocyte precur sor s show co nsiderable variability in thei r positivity to PAS . fro m co mp letely negative reaction s to coarsely gran ular. heavy pos itive ones. Her e the ce lls show a mixt ure of d iffuse cytoplasmic stai ning and fine granules.
421
•
00 o f mon ocytcs and prec ursors from an
illustrate the range of variatio n e ncounte red rC'aCti'"i ty in this cell ~se rics . . (0
(1)
stro ng PAS pos ltivitv with coarse blocks in .. fro m a furthe r case of mvclornon ocvtic with predomina nce of well: di ffcr cntia'tcd
, I
• 139
423
.'
....... ,
.:
..
.
, ',
• '
..
• •
I
424
422. A cid phospha tase reaction in acute mon ocytic leukae mia : most cells sho w stro ng and coarsely gra nular positivity. 423. Du al esterase reac tio n in acute mo nocytic leu kaemia: mon ocyte precur so rs sho w stro ng but yrat e esterase (BE) po sitivity in this case , while a gra nulocy te shows strong chloroacctate esterase (CE) positivity . 424. Dual este rase reaction in a case of acute myc lomonocytic leuk aem ia. In this insta nce the monocyte precursors sho w o nly weak reac tions with scanty scatte red gra nular BE positivity. Two myclo blasts sho w chie fly CE positivity, but so me granules of BE reaction a re also detectable.
'"
•
, •• ' 4j.
•
425--427. Rom anowsky. PAS and dual esterase reactio ns on the cells of an AMML. with predominantly monocytic morphology and typ ically mono cytic mixed diffuse and granular PAS react ion . but with weak CE rather than BE positivity in the rnonoc ytes. Thi s is a ver y unco mmo n picture out illustrates the potential variability of mon ocytic este rase conte nt.
427
141
~2 9
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,
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·U H- B O. Var iou s histological fields fro m thin sections of bone marro w tre phine biopsi es fro m cases of acute mo nocytic le uk ne mlas. In 428 and 429 . respectively lowan d higher -power views of th e same sect ion ( H&E stai n ). co nside rab le resid ual fat spaces re main . and islands o f erythropoietic tissue can be d istingui shed : but the cel lular a reas arc predo mina ntly occu pied by lar ge . poo rly di ffer ent iated blast cells wit h nucle i sufficie ntly widely spaced 10 ind icate a mp le cytoplasm . and wit h several nucleo li o fte n visible under the high er po wer . A suggestion of nuclear twisting ca n be d iscerned in ·*29. This is a more rcadil v recog nized feat ure of ·B O. a thin ne r section sta ined 'with Gie msa fro m anot her case. whe re there is denser ce llula rity wit h a sca ttering of cryt hroblasts having da rk nuclei a nd eosi no philic cytoplas m - but . aga in. a preponder an ce of nuclco lat cd pr imitive ce lls wit h nuclear twist ing of the mo nocyto id type . T hes e sectio ns prov ide valuable supple mentary info rmatio n o n o vera ll ce llula rity and relat ive pro po rtions of differen t ce ll types. but for cytologi cal deta il and co mpre hensive cytoc he mistry it is nece ssar y to stud y smea r prepar at ion s in parallel.
•
~31
(
431-B3 . Sections of a trephine biopsy from a case of A ~ IL with mon ocytic fea tures . In aspiration smea rs th e cyto logy showed a well-di ffer entiated mon ocytic patte rn; this is reflected in the predo minant cell appeara nce in these sections. wher e the H&E stain in 431 a nd ·H 2. a t respectively low a nd higher magnificati o n .
,
shows moderately dense nuclear chro matin. occasio nal nuclea r inde ntat ion . nu cle oli. a nd a ge nerally low nuclea r-cytoplasmic ratio . Residu al nest s of e rythro blastic ac tivity a rc apparent in -131, a nd sca tte red no rmo blasts with small dark nuclei are present in ·H2 . but the striking a nd unexpect ed fea ture in these
sectio ns. not previously note d in the marrow smears. is the conspicuous increase in macro phages. many heavily laden with haemoside rin and some conraininz other phagocytosed ce llular mate rial. T he sectio n appearing in 0$33 is of an acetate esterase stain o n plastic-em bedded mate rial. with reddi sh positivity in a proporti on of the
•
leukac rnic monocyt es . The nor mal seq uence of monocyte developm ent proce eds from the common granulocyte-rnonocyte pr _en itor to a co mmitted mon obl ast. then pro mono~ e and mat ure mo nocyte . with a final potentia l tran s[ion to tissue macrophage (or o the r rela ted cell as osteoclast or Lan gerhans cell) occu rring afte r the _ _1<'_ te has migrated from the pe riphe ral blood into (sues. Ncop lastic disord ers chiefly involving hagcs arc dealt with in Part 4. but the present \\~ the emergence of some differentiat ion to that III an o the rwise typical mon ocytic AML.
1 ~3
• 435
434-413. Examples of cytology and cytochemistry of A M L cast's with abnormalities involving / Jq23. Leuka emias with tr ansl ocation s o r deleti ons involvin g chromosome llq23 occur in young patient s especiallyoften childre n - and are mos tly of monoblastic o r monocytic cytology. MyeJomonocytic cases arc less commo n, while o nly a very few have been reported with grunulocytic predominance .
437
-'34. A blood smear from a child of nine months with AML and t(9; 11). In this low-power view all the ' nucleated ce lls are of the mon ocyte seri es . ran ging fro m sma ll undifferentiated blasts with minimal cyto plasm (a gro up of three arc at the lower right) , to well-differentiated prom onocytes with multi-lobulated nuclei and ample cyto plasm.
·435. A higher-po wer view of the same smear . showing a good seq uence of maturati on stages from small monoblast to late prom onocyte. again with marked lobulation . ·U 6. Ro manowsk y-stained bon e marr ow ce lls from another case of AML with t(I1 ; 19) and predominantly monoblastic or prom onocytic cytology. 437 and 438. S8 stains on bone marrow smears from two patients with AML a nd . respectively. t(II ;17) and t(9 ;11). The leukacmic ce lls arc Sls-nega tivc . There is a single Sls-positive myelocy te in 438. Weak or negative stai ning for S8 and peroxidase is usuall y found in the se detects of I Iq2J.
1-44
438
J5
~39
i ~.
'"
~I
36
•
37
138
439. PAS reaction o n the same speci men as in 434. T he re rsa negati ve o r weak reaction in the least mature blast cells. increasing to moderately coa rse gra nular positivity against a diffusely positive hackgro und in the more mature pro mon ocytes.
and 441. PA S reac tions fro m two furt he r cases of AML with 1(9;11). showing a similar pattern of incj eas-
.ut)
ing positivity wit h increasing ma turity. but in ge neral co nfo rmity with the pa tterns of PA S react ion us ually encountered in leuk ae mic monocytes. In 4-10 there is a ty pica lly mon ocytic pattern of positivity in t he promono-
cytc s and a negative eryth roblast . and in 441 six almos t nega tive monoblasts and one very coarsely positive
pro monocyte. 442 and 443. Dual esterase reaction s in I( 11; J7) and 1(9; 11). respect ively . The low-po wer view in 442 sho ws the variable positivity for BE in the leukaem ic cells. with gene rally negative blasts and mor e marked posi tivity in the most mature-looki ng promonocytes. The higherpo wer view in 443 illustrates the same pattern . with four nega tive monoblasts and a BE -pos itive promo nocyte .
"
-
•
444. A n AML with mixed gran ulocyte and monocyte precurso rs; when ad mixture is marked . as in this case (suggested by the Romanowsky stain and confirmed by the Sudan black) . the term ' rnyelomonocytic' is conveniently applied. The conspicuous Auer rods in two blast cells and the localized accumulation of azurop hil granules to the left of the nucleus in another iden tify myeloblasts and a promyelocyte . However. the remaining cells might be of either granulocytic or monocytic lineage . and cytochemistry is required to asce rtain which, ~5 . SB reaction from the same case; the localized cytoplasmic positivity in the early granulocyte precursors contrasts with the discrete scattered granule pattern of the monocyte precursors. ~6 .
Dua l esterase reaction in a similar case. with typica l BE react ion in the mono cyte precursors and CE reaction in gran ulocytic precurso rs. An erythrob last with distorted nucleus is esterase-negative.
~7 . An A ML with mixed e rythro id. granulocytic and mo nocytic prec urso rs - an crythro -myelo-mcnocytic jeu .ae mia. Such mixed leukac mias are qu ite com mon. an o bservatio n which supports the concept of a myeloid e m ce ll with multiple potentialities. Such a stem cell is pre umab ly the donogenic leuk ae mic cell with multilineage expression in cases like this o ne . whe re the gran ulocytc and mo nocyte involvement is acco mpa nied b~ erythroid and/or megakar yocytic ncopl astic cells de mo nstrable . by cytoge net ic or biochemical rech Diques. as heing of the same clona l orig in. All multiage cases are mo re sensibly grouped toget her as Type H AML (HA with > SO% SB positive blasts and liB with less) rather than scatte red among a wide range of FA B groups, M I. M2, M4, MSa or b. M6 or M7, acco rding to precise differenti al counts.
e.
•
The SB reaction helps to differentiate the negative rvth ro blasts from the myelob lasts I with dense ..x:alized posit ivity and the monoblasts with scattered ules.
A zeneral view of a bone marrow smea r from the case as shown in the previo us figures. sta ined by PAS reaction. T he diffuse and finely gra nular - -ity of granulocyte precurso rs. increasing in in: with increasing maturity. is shown ; the stro ng . reaction in some ery throblasts is very characterof erythraernic involvem ent in a mixed leukac mic
.'
...
147
~51
"'50. AML. with mixed proli fe ration of erythroid and granulocytc precur sors - an 'e rythro rnyeloid' lcu kae rma. Most cases of eryt hrac mic myelosis have from the ea rliest stages so me component of gran ulocytic or mo nocytic ce ll-line involvement . which . tho ugh it may be minim al
"'52. Th e SB reaction on a smea r from the same case of erythro leukae mia as sho wn in the last two figures . Eryt hroid precur so rs are negative . while gra nulocyte prec urso rs show typical coa rse locali zed pos itivity.
~52
•
•• •
..tSJ. PAS reaction in the same case. Stron g positivity .. red-ce ll precursors at differ ent stages of maturity is ecespicuous. while myclohlasts and prom yeJocytes show their custo mary negative reactions or faint diffuse . _e of the cytop lasm. At upper mid-left is another buddi ng cell with PA S-positive fragme nts. again suggcsIiBg megakaryocyte line involvement.
455
s . Acid phosp hatase reaction in mixed eryt hro.teloid leukaemia. Coa rse positivity. especia lly para-=lear. in all cells presen t.
'5. Dua l esterase react ion in the same case. An ea rly _ ulocyte precursor shows CE positivity and the erythrobtasts have tine granular positivity of mixed BE _ CE ,ype.
149
2 ~S 7
., •
•
~59
., .$56 . A normall y granular mature mega ka ryocyte . with minimal plat elet formation at the periphery.
457. A megakaryocyte with disrupting cyto plasm which has been actively for ming plat elet s. 458 . A poo rly gra nula r meg akaryoc yte with minim al platelet formation .
459. A pair of megakaryocytes. very actively releasing plate lets . and almost devoid of cytoplasm.
460. Stages in the for matio n of megakar yocytes. Th e large ce ll with thr ee nuclei and fragmenting cyto plasm might he ca lled a 'promega ka ryocyte ". and the acco mpanying prim itive cell. like an unusually large myeloblast. may be a ' megakaryo blast' .
mcgakaryocytcs with stro ng gra nularity of ..·.....,..m.
~ 63
aryocyte fragment in the peripher al blood crisis in CML). Th is may represent a precur sor . of the megakaryo blast or -.c:"'...."!'~.·l e stage . since the re is com monly a (he mega ka ryocyte line involved in blastic and the nucle ar pattern of this cell look s the prese nce of a few platelets on the right ea er mat urity. e-
platelet of snake-like form. beside various _:aII""'rs in the bo ne marrow of a pati ent with anaemia.
151
46 S
•
464. Phagocytosis of platel ets by neutrophil polymorphs in an auto-immune disorder with circulating Immuno blasts. two of which are see n in this field. 465. PAS reacti on in a normal megakaryocyte. Diffuse cytoplasmic positiv ity, weak in intensity . is accom panied by occas ional stro ngly po sitive glycogen inclusion bodies . -166. \Vhen plat elet formation is acti ve or imminent. a peri phe ral rim of denser positivity may also be o bserved .
In this low-power view of normal marr ow, the megakaryoc yte is surro unded by o ther marrow cells which show the increas ing positivit y with grea ter matu rity in the granulocyte series. Erythroid precursors are negative.
•
•
IS ~
•
•
i
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-169
-167. Num erou s megakar yocytes without peripheral platelets from the mar row of a pati ent with chro nic idiopathic th rombocytopen ic purpura (ITP). -168 . An immature megak aryocyte from another pat ient
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480--482 . Thin sect ions of bon e marrow trephine biopsies from thr ee differ en t examples of megak aryocytic hyperplasia . In 4HO the background co ndition is hairy cell leukaemia (HCL) with a secondary infection prod ucing o nly minimal neutrophil leucocytosis (re flecting the severe neut ropenia co mmo n in HCL). but an at te mpt at megak ar yocyte prolifera tio n which resulted in so me platele t increase . Th er e are no mo re than a few recognizable HCs present . but there are two lar ge megakaryocyres - the lower with seve ral se para ted nuclei and the uppe r in po lyploid mitosis with three o r perhaps fou r sepa rate spindles. G ross hype rplasia involving bo th gra nulocytic and mega karyocytic lines is visible in 481. with disa ppea rance of fat spaces a nd an almost co mplete absence of erythro blasts. Th e small cells with deeply sta ined and often lobulate d nuclei are neu trophil polymorphs: the large pale-staining cells are all megakar yocytes at va ri o us stages of matur ity, most with poorly lobulated nucle i, the few with densely stained nuclear mat erial bei ng in variou s stages of mitosis . T his is another exa mple of CML with rncgakary ocytosis, many of these cetls being classifiable as micromegak aryocytcs. The third example. 482. is from a patient with a chronic myelopro liferat ive disea se (C MPD) associated "'lh a high platelet count (> lOO()x 109/1) and numerous mature . sometimes almost gigantic, megakar yocytes multiple nuclear lobes and ample cytoplasm present iD the bo ne marro w. This disor der is vario usly called essential thrornbocythemia' or ' megaka ryocytic ..,d lSi's· of mature ce ll type .
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A th in section of bon e marrow trephin e biop sy fro m a patient with C ML. now movin g into a blastic tra nsfo rma tion. The re is a very mixed and pleo morphic cytological picture. with ery throblas ts , gra nulocy tes at various stages of maturation . and a subs tant ial component of primitive-looki ng cells with leptoc hromalic a nd sometimes nuclcol at ed nuclei . sho wn by cytoche mistry and immunology of their counte rpa rts in marr ow smears (cf . 487) to be immature ce lls of the megaka ryocyte line. -184. j ume rous mega karyocyt e fragments and probable megak aryocyte precursors in the pe riph eral blood of a patient with an Al\.IL with megak ar yocytic preponderance - the te rms acut e megakaryocytic myelo sis o r megakaryoblast ic leukaemia may be applied . Large and fully developed megak ar yocyte s are not pre sent. but these arc multiple mega ka ryoc yte fragme nts or ab no rma l sma ll cells of tha t se ries. wher e the usual po lyplo idy has not occurred .
485 . A furth er field fro m acute megak ar yocytic myelosis. The nucleolated primitive ce lls ha ve a similarity to rnyelo blasts. but may be ' Ieukaemic' megakaryoblasts.
158
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486. S8 reaction in the same case . A megakaryocyte wi th thre e phagocytosed lymphocytes or erythroblasts is negative. as is the neighbour ing megakaryoblast. . PAS reaction in acute megakaryocytic myelosis. TIle tro ng and coarse irregular positivity in the 'Ieu·3 mic' megakaryocytic cells resemb les that in platelets. The diffuse tinge in certain precursors does not allow a dear distinction from myeloblasts to be made .
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48~95 .
Examples ofthe cytology and cytochemistry of multilineage (Ty pe /I) A M L with chromosome abnormalities involving inversion or insertion 3;3.
T hese chromoso me changes arc associate d with an creas e in megakaryoblasts and later megaka ryocyte stages in the marrow. together usuall y with both eryt hroblastic and granulocytidmon ocytic involvement. 488. High-power view of bo ne mar row smea r from a case of inv(3;3) . showin g predominance of megakaryoblasts and prcmegakaryocytes . with cytoplasmic disruption in so me ce lls and. at the upper part of the field . forma tion of seve ral giant platelets. Th e blast at the top left of the lower ce ll clump con tains an Au er rod . and is probably a myeloblast . as is the blast with minimal cyto plasm near the upp er left corn er of the field .
-189. Another exa mple of AML with inv(3:3). showing the very characteristic cyto logy of megakaryobl asts. with freq uently irre gular cytoplasmic o utlines and a tendency to form bud s which often cont ain vacuoles. 490. Alt ho ugh these ce lls arc negative 10 myelopero xidase sta ining. they do possess platelet peroxidase (PPO ). hest demon strated by electron mieroscopy. This enzyme activity is confine d to the endoplasmic retic ulum and perinuclear space . as illustrated here.
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491. 58 stain o n the same specimen as in 488. showi ng granulocyte- type positivity in several myeloblasts and negative reaction s in two megakaryobl asts. excep t for a stro ngly positive Au er rod in the upper megaka ryob last and seve ral possi ble but mo re weakl y sudano philic Auer rods in the lower one. probably resu lting from phagocytosis. 492. PAS reaction on the same bone marrow as in 488 and 491, sho wing mixture of granulocytic and megakaryocy tic pa tte rns of positivity. 493. PAS reaction on the same bone marrow as in 489. whe re all the blast cells in the field arc megakaryoblas ts with coa rse granular positivity , especially in the pe riphe ral cvaginations.
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494. A furt her exa mple of PAS stai ning in a case of ins(3;3), with a gro up of megakaryohl asts to the left , toget her with a pair of probable mono blasts to the right. Between the two grou ps is a late neut rophi l with mat ure ly conde nsed nuclea r chro ma tin but an unsegmented nucleu s, possibly a normal stab cell but perhaps exe mplifying the pseudo-Pelger phen omenon ofte n encou ntered in these cases . 495. A PA S reaction o n clumped ce lls from a nothe r case of ins(3:3), to show both multinuclearity and gross phagocytic activity of some megak aryocytes in this form of A ML. Th ere are also several gro ups of PAS-positive ery thro blasts pres ent in this field.
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-196-502 . Examples uf the cytology and cytochemis try of AM L with trisom y 8. T riso my 8 is the co m mo nes t chro moso ma l abnorma lity found in AML. and of te n co-exists with o ther cytoge netic abe rratio ns. pe rhaps chiefly as a seconda ry phe no me no n in cases whi ch a rc usu ally of muhiline age (Ty pe 11) e xp ress io n a nd which hav e a ge ne ra lly poor prog nos is. W he n tr iso my 8 occ urs a lone . as it does in ab out a th ird of all cases with this ab normality. t he cyto log ica l picture is predomin antl y gra nulocytic with ma rked evide nce o f dysplastic maturation . Rom an owsky stai ns of bo ne marrow sme a rs fro m th re e d iffe re nt cases o f trisomy 8 appea r in -I96--l9S . -196 . T ypicall y va riab le matu rat ion . with e noug h gra nular ity in the more primiti ve cells to indi cat e the ir gra nulocytic rath er tha n monocytoid linea ge. hut with ag ra nular lat er nc ut roph ils show ing acquired Pclg crHuct nucle i.
..197 . M veloblasts , one with an unstai ned A ucr rod ove rtvine the nucleus. defective neut ro phil myelocyte ara nularitv . and various eoslnophils. some wit h greenish granul es . ..I9g . A more primit ive blast ic picture. with de e p nu clear lob ulatio n in on e cell a nd lar ge pinkish inclu sions or vac uolcs overlvinu the nucle us and in th e cyto plasm of a no the r. A pt)(; rl )~g.ra n u l a r neutrophil poly mo rph is also p rese nt.
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B sta in on ho ne marrow smea r from th e case trated in ~ 98 . thre e primitive blasts showing localizIed cytoplasmic pos ni vu v o f myeloblastic pattern ina a dense Au cr rod in the lower blast and eit her Auer rods or o the r inclu sion s in th e upper ce ll of group. Th e fourth blast cell has an o vera ll granul ar iadanophilia together with stro ng localized positivity . . xc gra nulocytic tha n mo nocytic in pattern. A norm. react ing eo sinophil is in the lower right corner o f the
S02
B stain o n the bo ne mar row of ano the r pat ien t and triso my 8. illustratin g the fre que nt g in th is co ndition of unexpectedly poor sudano_ The central de nsely positive cell. probably a ophil pol ymo rp h. shows a strong norm al reaction . promye locyte at the left . with an Au er rod . and lbe eosino phil a nd neu trophil myelocytes, arc very _ posi tive with a gre enish-brown rath er th an black A ~ IL
PA reaction in th is form of A ML gene rally has I findings. the blast cells having negative or reac tio ns. as her e . dual este rase rea ctio n sho wn here illustra tes .ngs. with mos t blast cells negative but C E appearing in lat er gra nulocyres from proon wards. T he re is BE positivit y in two plasma m severa l e rythro blas ts,
163
503-5 20. Examples of the cyto logy and cytochemistry of AM L associated with an interstitial deletion of 'he 10nK arm of chromosome 9 between bands ql 3 to q22. 9q(q/3 ;q22)(Type /lA ).
• 505
Although most cases of9q- have predominantly mycloblastic cytology . they show evidence of erythroid and megakaryocytic dysplasia .
503 and 5().l. Typical low-power fields (Heyl slain). with blasts of variable size. dysplastic erythrobl asts and. in 503. an occasional plasm a cell and eosinophil. 505-510. High-power details of the bone marr ow cell cyto logy from seve ral different cases of9q-A ML. In 505 there are two mycloblasrs with vacuol ar inclusions and a poo rly granula r stab cell. In 506 a myelo blast sho wing nuclea r co nvolution resembling that commonly seen in A PL is acco mpanied by two very coa rsely granular co sino phils; 507 shows ten myeloblasts, two with Auer rods. and a pro myelocyte , 508 an eosinop hil pro myelocyte with mixed granules. and four myclob lasts, 509 six blasts, one with a bilobed nucleus. anot her with a twisted mon ocytoid nucleus, one with primary granules, and o ne with a vacuole, and 510, four variably sized blasts with vacuolcs or small azurophilic inclusions . 511-513 . DAB peroxidase reactions. with posit ive Auer rods , ot her coa rse inclusions and granular positivity in most blast cells. 51~ . SS positivitv of chunky inclusions. usually with nega tive cores. in vacuo lated myelobl asts.
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• 5 15-5 18. Furth er illustr ation of the striking pa ttern s of SB posltivity in 9q- A ML. with very coarse granules or inclusions usually sho wing a positive peripher y..and a hollow negative centr e . Con spicuou s vacuolcs in some blast cells mani fest weak suda nophilia - especially at the circumfe rence. as in 516 - while Au er rod s may ap pea r as nee dle- o r spindle-shaped structures . or as shor t thick rods with either solid or hollow ce ntres (517 and 5 18). In 518 there arc also two c rvthroblasts. o ne binu cleutcd with a Howcll-Jollv body. and with a suggestion of residual megalo blastic nuclear features . 519. PAS reactio n on blast cells fro m 9q- AM L. The positivitv is typically weak . as here. with no mor e than a faint diffuse tinge or fine gran ular reaction . Nucleo li are revealed ver y clea rly in this prep aratio n. and their dispo sition. when pai red. at oppos ite poles of the nucleu s is well see n. T wo late crythrob lasts are present in this field. ho th with evidence of d ysplasia . one having it separated nuclear fragmen t o r Howet l-Jolly body and the o ther a trifolia te nucleus. Neit her shows convinci ng PAS positivity. 520. Dual esterase reaction in ano the r case of 9qA~1L. showing the presence of strong CE pos invitv in ea ch of thr ee blast cells. with even it possible positive Auer rod in the upper most.
166
518
519
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521-523 . Examples of A ML associated with mOllosomy 7. 7q- and 5q-( Type 11,1). These chromoso me abnor ma lities are co mmon in mvclo dysplastic states ( ~ I DS) and occur in bo th prima ry ( de " Ol'O) a nd seco ndary AML. Monosom y 7 and 7qare o fte n fo und toget he r with other ch romoso mal defects . Cases usuall y show dysp lasia of both erythro id and megak aryocytic lines as well as eithe r myclo blasts o r monoblasts. or bot h. 5q- is rela tively uncom mon as a sole abno rmality in AM L but has usually been associated with a rath er similar picture so far as er yth ro id and mc aakarvocvuc invo lvem ent is concerned . but with mos tly myelobl astic and later gra nulocytic cells ra the r tha n those of the mo nocyte line.
523
521. A lo w-power view of a hone marrow smea r [Hcyl ta in) fro m ;'1 patient with Af\.lL and mon osom y 7. sho wing blast cells with so me granulocytic matu rati on . a gro up of ab nor mal mcgakar yocytcs includin g mononuclear. binuclea r and tr inuclear form s. a nd ten e rvthroblasts. seve ral o f which shoev nuclear deformities o r me galoblastic cha nge. There is a central plasma ce ll. 522. An exam ple of th e bone marro w cyto logy in A f\. IL rh 5q- . showing a predominan tly myelobl astic a nd peom ve locyuc pictu re with ;'1 nor ma lly granular segmen ted neut ro phil and two eryth roblas ts. o nc biDUdeated. 513. A smea r fro m a case o f AML with mult iple osomul defects. includ ing mo nosomy 7 and y 8. The re are myeloblasts with nuclear ring no n and a sugges tio n of both def ective gra nularity P lger-H uct nucle i in later neut rophils.
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525
524-532 . Examples ofthe cytology and cytochemistry of A M L with 6;9 /ranslocation. t(6;9) (p2J;qJ./) . This chro mosomal defect is usua lly. though not quite always . associa ted with a stro ng basophilic component. including mature basophil s and numero us baso phil precursors from prom yelocytcs o nwards. and freq uently with abnor mally coarse o r poorl y staining granules o r aty pical nuclear forms. Cases have bee n classified mostly in FA B gro up M2. le55 commonly in M l. M4. or as MDS. but as there are often minor components of bo th erythroid and megakaryocytic involvemen t with d ysplasia and microm egakaryocytes. most cases probably fit Type HA. 524-527. Various fields from the bo ne marrow of a patient with t(6;9) A ML. stained by Romanowsky dyes to illust rate these abnormalities. In 524 the re are two late basophils with recognizable nuclear structure but poo rly stained gra nules . while two of the blast ce lls show a suggestio n of baso philic granulation . Four myeloblasts . a neut rophil stab cell and a coa rsely granular basop hil myelocyte can be see n in 525. while 526 has three blast cells with little differentiation. a binuclea tcd er ythro blast and . at the top . a poor ly granular basophi l pol ymo rph. There are no basophils present in 527. but there a re four erythrobtasts, one with some megaloblastic change. and a binucleated micromegakaryocyte.
168
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518 and 529. Fields stained with toluidine blue, which fC'3C1S spe cifically with the mucopolysaccharide of ph il granules. Po sitively reacti ng cells can be picked t with case and gene rally prove to be mo re numerous man had been thought fro m the Rom anowsky stains . Here. the coarse granularity of more mature basophils aDd the finer gra nules in basophil myclocytcs are clea rly . jble. and 531. S8 stains. 5,\() showing ;'1 nega tively reacti ng segmented basophi l. a negat ive myelobl ast and a sormally positive eosinophil myelocyte. 531 a metadIroma rica lly stai ning basophil. normally reac ting K8Uo phil-prccurso rs - o ne with a pos itive A uer rod nega tive crythroblasts.
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•
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533, A sectio n of bone marrow trephine biop sy from a patie nt with a multilin cage AML and co mplex chro moso me abno rmalities. seco ndary to a preceding MDS. T he predo mina nt cells are blasts. pro bab ly myelo blasts , but there are islands of crythrob lastic pro lifera tion and several dys plastic mcgakaryocytes. Ove rall ce llularity is high . 5~
and 535. Low- and higher-power views. respect ively. of a trephine biopsy sect ion fro m anot he r patient with multilin eage AM L. showing again a mar ginal predominance of nuclco tatc d blast cells. many with twiste d mo nocytoi d nuclei . amo ng a substa ntial co mpo nent of eryt h roblasts of d iffere nt stages of maturity. A few mcgak ar yocytcs. some appare ntly mo nonuclear. can be distinguished . T here is an occasional late neutro phil with dense but seg mented o r twisted nucleus. Sections of this kind help to estab lish the overall cellularity and broad differential proport ions of ce ll lines in cases of AM L. but smea r prepa ra tions arc required for more accurate recognition of dysplas tic changes and det ailed differentiation of pri mitive cell types.
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5~'6-S47 . Earlv cytological signs of emerging myeloid haemopoietic ;lctivity during remission development in aClde leuk aemia. The fea tures illustrated are usually fo und in ma rrows which otherwise remain hypoplastic fo llo wing cytotoxic therapy,
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539. A more mature megakaryocyte . bu t still with patc hes of residua l baso phi lia.
540, R E cells may appear relatively numerous at this phase - the marrow fleck here is composed almost entire ly of them . Elsewhere the marrow sho wed gross hypo plasia . yet remis sion e nsued within a few days.
541. A higher -pow er view of the cells in this fleck - the nuclear patte rn is characteristic of R E ce lls.
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5-1-1 . A similar seq uence of gran ulocyte precursor s toge ther with stab and segment ed neu trop hils of nor mal appea rance.
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174
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S-4K-550 . Three fur ther illustr a tion s o f ph ases of eme rging re missio n in ho ne marrow as pira tes from pat ien ts with AML afte r successful indu ction th e rap y. In the first (5-lK). a n islan d of e rythrob lasts with cy to plas m rang ing from baso philic to po lychr om ati c surro unds a
macrop hage. as active erythropoiesis e merges . In the sec o nd (549 ), a single neut roph il m ye locyt e is presen t in a field co ntain ing pr edom ina ntly e rythroblast s, fro m pro-e rythroblast to late norm oblast. Th er e is evide nt po lychromasia amo ng the red ce lls. no do ubt re flected by a retic ulocytosis in the pe rip he ral blood. T he thi rd field (550) shows a ra nge o f ne ut rophil grunu locytcs. illus tra ting maturat io n stages fro m nucle ol a ted mveloblast at t h~ bottom . through promyelocyte and myelocyte . to met amvctocvte and stab cell. Fo ur ervthrob lasts. a pos....ible h;ISOphil. and a co mpressed lymphocyt e nucleus ar e a lso present .
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and 555, Low- and higher -power views. res pectiv·:ly. o'f a bo ne mar row trephine biopsy sectio n ta ken from a pat ie nt with seco ndary AML. afte r repeated courses of re mission induction therapy were followed by a pro lo nged period of marrow hypoplasia and pe riphe ral pancytope nia, A t this stage the marrow under low power (55-4) shows a reticular stroma with recoven ng <;,Ci.l tlered ce llularity and fresh cell uli.lr proli ferati on . e!'oopccially along cap illaries . Th e higher magnifici.ltion of 555 revea ls that the cytology is pleom orphic. with plasma cells along the cap illaries and cells of vi.lrio us lineages sci.lttered through the stro ma . a mong them. primitive immi.lture cells. possibly represent ing leu kac mic recurre nce . hut possibly nor mal e merge nt prec urso rs. 55~
556. A fur ther biopsy ta ken a week lat er from the same patient as in 554 and 555 resolves the qu estion of re lapse or early remi ssion , Thi s thin sectio n shows a few resid ua l plasma ce lls but also goo d mat ur ation in the gra nuloeyte ~ri c s. with all stages fro m pro myelocyte o nwards recognizable. toge ther with scatte red eryt hrob lastic pro liferation. Clearly. remission is now eme rging.
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• 557. A thin section o f ho ne marrow tr ep hine bio psy fro m a patient with A ML afte r inducti on o f a temporary remissio n . now showing evide nce o f re lapse . This high po wer field conta ins
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560-565. Examples of defecti ve granulogencsis - with a "use- eosinophilic staining of the cytoplasm of mye loami few specific granutcs. This appearance is most n observed in preleukaemic stales, including defective ~th ropoies is wnb excess of mvetobtasts. neutrophilic elogranulur dysplasia , smouldering leukaemia and all of mvetodvsptastic syndrome (,\/ DS) . A typical appea rance of myelocytic cytoplasm in conditio n as seen by Ro man owsky staining. L A myelo blast, two probable premit oti c pr crn yclo with scattered azur ophil granules and promin ent ..:Ieoli . five rnyelocyt es with diffuse eosi nophilic ing. and two later granulocy tes with a mo re nor ~ gran ular cytoplasm.
Further gra nulocytes from the sa me case . showing diffuse a nd finely granular eosinophilic staining in ce lls.
562
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563. S8 stain on the same marrow sample. showing strong sudanophilia , de spite the lack of clear specific granules in the Rom anowsky preparations. 564. A high-power view of the S8 sta in in this co ndit ion . The sudanophilic granules are of the same size as those seen in myelocyte s with normal specific granules.
565. Dual este rase stain showing norm al Cfi -positive gra nules in three of these unusu al myelocytes, despite (heir poo r granularity with the Rom anow sky stai ns.
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566- 568. A n exam ple of transient myeloproliferative disorder (T MD) of Down 's syndrome, trisomy 21.
568
Patie nts with Down 's syndro me are at much increased risk of acu te leuk aemi a , including a form of A ML with a megakaryo blastic compo nent. They are subject to a tra nsient non-clon al blastic pro liferatio n, with a strong megakaryoblastic compo nent, which is man ifest in the peripheral blood and persists for a few week s before spo ntaneously disappearing witho ut trea tment . Forruna tely. this transien t disorder (TMD) can usually be eas ily distinguished fro m acu te megakar yobl astic leukaemia (AMegL) by its earl ier age incidence , < 1 month as compared with > 3 months, the presenc e of a higher proportion of blasts in the blood than in the marrow and the relatively nor mal levels of haemoglobin and platelets. T hese three field s are fro m the bo ne marro w aspirate in a neon ate with TM D and blast cells in the periphe ral hlood. The first (566) has a typica lly 'ariable blast cell population, with generally Iow nuclearcyto plasmic ratio , vacuolation, nuclear twisting or lobu lation with multiple nucleoli, a giant prom yelocyte rith coa rse azuro phil gra nulation , a plasma cell and a aeutrophil segmented cell with nuclear twinning . T he secon d field (567) sho ws ano ther area to illustrate especi ally the varied gran ulocyte developmen t , with 'eral eosinophils as well as lat er neut rophils; while the - d (568) shows the cdge of a dense clump of cells of . xc unifo rm mo rphology consiste nt with megakaryo, with occasional intermingled macroph ages.
181
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:;:69-571. Thin sections of bon e marr ow treph ine bio psy fro m a pa tient with a n MDS involvi ng compo nents o f ref rac tory sidc roblustic anaemia a nd excessive blastic pr oliferat ion toge ther with a n increase in mcgak a ryocytcs . In 569 th e low-powe r view sho ws genera lly high ce llula rity with a very pleo morphic picture. including numerous scatte red macro phagcs laden with haem osiderin a nd seve ral multin ucleated rnegakaryocytes. T he higher -pow er view in 570 is of a neig hbo uring area with more residu al fat spaces . whe re the megakaryocytic cytology is well shown . a nd where there a rc several areas of haemo rrh age of the kind from which the haemosidcr in in the macroph agcs in 569 was do ub tless de rived . T he rem aining ce lls in this field include grn nulocyrcs chie fly fro m myeloc yte o nwa rds and ervt hroblasts with densclv sta ined nuclei. In the reti cultn-staincd prepa ration ' at still high er power illustrated in 571 . the abse nce o f a ny increase in re ticuli n a nd the pre se nce o f lade n macroph ages. a nd also o f nuc lco la tcd blast cells with lightl y sta ined nucl ei . a rc clea rly evide nt.
182
571
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572. Prussian blue stain for free iron on cells in a hone marro w smear made from the same patient whose trephine sections arc shown in 569-571. Th e cluste r of ervthrc biasts in this field shows a high conte nt of free iro n with occasional ringed distributi on . 573. In this Rom anow sky stain. a buff y coat prepa ration from pe ripheral blood - agai n. from the same patient - shows a myelobl ast , a prom yelocyte . later neutrophils, two lat e erythrohlasts on eith er side of a megakaryocyte nuclear fragment . a nd a mass of giant platelets. This who le sequence of slides (569-573). tak en at the '503.me time from o ne patient. illustr ates the complex . totogy of the multilineage myclod ysplasias a nd the com mon ove rlap bet wee n the recognized subd ivisions. 57'" An other bo ne mar row sample fro m a pat ient with iDS. this time classified as refr actorv anae mia with cess blast' (RAE B). Th ere is a shift in the left in the eranulocyte series . hut. as this field reveals. there is picuo us granular dysplasia. with defective ncutrogranulatio n fro m myelocyte to polymorph . and 8Bdear defects ranging from pseud o-P elger-Hu et lack segmentatio n (in the three upper neut rophils ) to persegmentatio n in the lower polymorph .
183
576
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• 57:-577. Sections of trephine biopsies from furthe r pa tients with MDS . In 575 and 576. an exampl e is shown of the mor e hypocellu lar variant of MDS . in this case associa ted with mark ed dysmega karyocytosis (microforms and multinu cleat ed ce lls be ing especially conspicuo us in 575) ; the presence of erythrob lastic island s and the abse nce of collagenous fibrosis are mani fest in the Van G ieson stai n of 576. Th e section appearing in 577 is fro m anot he r pat ient with the RAEB form of MDS , associa ted also with the chro moso me anomaly 5q-. This defec t occurs most common ly in elde rly females . as in this case . and is usually found in association with ref racto ry a naemia. dysgranulo poiesis with left shirt. and a cha racte ristic megakaryocyte picture with mononuclear or bilobed ce lls. Typical dysplastic megakaryoc yres showing this feature are seen in the upper part of the field. A mo ng the scattered islands of e rythroblastic hyperplasia and later granulocytes occupying much of the lower part of the field are several small groups of blast cells. well away from the endosteal surfaces of bony trabeculae whe re early myeloid pre cursors are norma lly concent rated . Th is 'abno rmal local ization of immature precursors' (All ?) may pe rhaps predicate immi nen t leukaemic transfo rmat ion.
184
577
578-580. Two low-powe r views and a higher-power field. respectiv ely . from a thin sec tio n o f bon e mar row tre phine biopsy from a pat ient with MD S. showing RAEB with trilin eage dysp lasia . In 578 there is a generally pleo mo rphic picture with mon onuclear mega.aryocytcs and ot hers with multip le perip heral nuclei. an evident increase in early granulocytes - though with poorly gra nular cytoplasm - a few later neutrophi ls and inophils. and a sca ttered c ryth roblastic component ' th deeply stai ned co mpact nuclei. A broadly similar field is shown in 579 . but w ith several abnormal rnegaryocytcs having lar ge sing le o r do uble nuclei. In both fields occas iona l cells with elo ngated nuclei ~ est the prese nce of fibroblasts . The high-po we r view of 5SO clearly illustrat es the nucleo lat cd myelo blasts ich predo mina te . toge the r with eryt hroblastic islands • top a nd bottom of th e field showing some rncga lotic feat ures. and var ious ce lls of th e megakar yocyte . includ ing a hi nucleat ed cell at mid -centre left and a -Kro megakaryocyte at the right edge . a thi rd up from bo tto m.
5110
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.-., • 5KI-583. Two furt her examples of typica l bo ne marrow trephine b iopsy appea ran ces in va ria nts o f MDS with incre ase in blasts (RA Ell) . Both show hyperplastic ccllularity with few rat spaces a nti marked pleomorphism . the first case - with la\\'- and highe r-power field s shown in 581 and 582 - havin g all myelo id cell series well represent ed. but with an evide nt increase in immatu re cells. especially of the gra nulocyte se ries. bo th neutrophil and eosinophil. as revealed in more det ail in 582. The thicker section from anothe r case shown in 583 is agai n highly cellu lar. with all cell series pre se nt . hut with a co nspicuou s shift to the left in the grun ulocy res. with se ve ra l clumps of nuclcol a te d myc loblasts. Both these cases illustrate A LII' , possibly suggestive of early progression to florid trilin cagc A~t L.
5
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584--587. A success io n of fields from bo ne mar ro w aspira tes take n fro m pa tie nt s with RA E B to dem o nstra te so me o f the co mmo n cyto logica l a nd cytoc he mica l fea tures o f dysp lasia in th e myelopo ie tic se ries seen in ~tDS . In 584 c ryt hrob lasts show irregula r. twisted o r fragme nting nuclei. the re is an immature bin uclca tcd megakar yocyte with ingested red ce lls. a nd seve ra l ne utro phil rnyelocytcs show th e yellow cyto plasm ic co lour a nd defective gra nulation ofte n seen in myelodysp lasia (cf . 561 ). Figure 585 sho ws a not he r slide from the sa me asp irate . sta ined wit h SB. whe re a du al po pula tio n o f gra nulocyres is ap parent . wit h six strongly po sitive rnyelocyres or prcmyclocyrcs and three totally negative cells . a myeloblast. a pro myelocyte or early myeloc yte . a nd a neu trophil stab cell . Figures 586 and SS7 a re from slides of a nothe r bone marrow aspira te take n from a second patient with RA EB and stained r pccnvc ly for peroxidase and with S8 . In 586 a promyeloc yte is strongly peroxidase-pos itive , while th ree poorly gra nu lar segmented neutrophils arc qui te ecga nvc. In 587 one ne utroph il polymorph is normally sudano philic , while two others show on ly weak sca ttered ~ nula r positivity.
586
587
187
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588-591. Buffy coat prep arations from the peripheral blood of a pat ient with CMML . ano the r variant ofte n class ified amo ng Ihe myelod ysplastic sta tes . T he blood picture in this disorder shows a substa ntial mo nocytosis . com monly 5-lO x 10'1/1. and the monocytes may have ma rked ly convo luted or hypcrscgmenled nuclei. as illust rated here. Th ere may also be occas ional immal ure gra nulocytes in the bloo d . and . as at the lo p of the field in 588. dysplastic megak ar yocytes o r naked nuclei. and irregular o r giant platelet s. In 589 and 590 the perox ida se co nte nt and SB reactions arc revealed as generally normal for bo th gra nulocytcs and mon ocytcs in this case. altho ugh erratic sta ining is so metimes found . and mon ocyte, may give negative o r weak reactions. As shown in the dua l esterase reactio n depicted in 591. the monocytcs arc usually stro ngly positive for BE. while neu trophil s react norm ally for CE. T he re arc two negatively reactin g basophil s in this field .
59 1
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592
•
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592 and 593. T he lo w- and higher-po wer views. respectively. shown here arc from a bone marrow trephi ne biopsy taken from a patient with CMML and a similar pe riphe ra l blood picture to that illustrated in 5~59 1 . Mo nocy tos is is usuall y evide nt in th e marrow . but is less co nspicuous than in the blood. and is acco mpa nied by ot her fea tures o f MDS . including vari abl e signs o f tri lineage dyspla sia . Th e sec tion illust rated here has bee n stai ned fo r the mon ocyte e nzyme mur amidase (lysoz yme). which allows th e mon ocyte co mponent ( 0 be distinguishe d more clearly tha n is po ssible in simple H& E o r Giemsa stai ns . In 592 seve ra l mult inucl eat ed megakaryoc ytes ca n be see n. and in 593 the re is a pleo mo rp hic picture with num erous gran ulocytes at diffe re nt stages of maturation. 594. T his section o f a trephine biopsy is from a patient with a long-stand ing C MML. now transforming to a more florid acute leukaem ia . with increasing numbers of blas t cells in the blood . The histology shows an almost uniform replacement of marrow tissue with immature CC'lIs havi ng promonocyti c features. including relatively . nuclea r-cyto plasmic ratio with poorly defin ed cytoplas mic o utlines and occasio nal nucle ar twist ing or I8de ntatio n. Th er e ar c two mitotic figure s visible .
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Part 3 Lymphocytes, plasma cells and their derivatives and precursors in blood and bone marrow ormal and abnormal fo rms Lym phocytes are form ed chiefly in the lymph glands , Peycrs patches and other nodal sites . among which must be included nodules of lymphocytic nature in the bone marrow. Precursors. 1)1npfiO blastrand peolyrn phocyt cs. arc difficult to recognize in norm al, marrow aspirates. however . and are illustrated from pathological conditions, principallY.;ll'Ute lymphoblastic an dyprolymphocytic leuk aemia . where the y beco me commo n in th e marrow and so met imes a lso in the peri pheral blood . Whi le the ea rliest stages of lymph oid ce ll dev elop ment from the probable co mmo n stem cell shared with the mye lo id series arc still uncertain . it seems likely tha t a funct ion al ste m ce ll for the lymph ocytic cell series exists which gives rise to two main so rts of lymp hocyte: T cells and B ce lls. Th e form er have a special role in ce ll-me diated immune responses and ma y have helper (TjL 'subset) or suppresso r (Ty subset) functio ns in relat ion to Hcell activity. B ce lls are larg e ly responsible for hum oral immune respon ses. These cells. and sma lle r gro ups of Iymphocytes which seem to fall into diffe rent . less clear . ca tegories are distinguishable by imm unological means. but also have certain morphological and cytochemical differ ences which are illustra ted in this sec tio n. and which are some times also manifest in neoplastic st ates . Imm unologically. T cells from th e earli est stages o f ma tu rity show a capacity to fo rm rosettes with she ep red cells, and reac t with the MAbs C D2, C D3 and C D? Th e helper subset reacts also with CD4 a nd th e uppressor subset with C D8 . From an ea rly stage. B· cell co mmitted precursors show immunoglobulin ge ne rea rra ngements, express io n of HLA-D r and ant igen s reacting with CD 19 and CD9. At a transient early phase o f development they react also with C D 10. and late r tra nsiently express cyto plasmic immunog lob ulin (cIg) before producing surface membran e immunezlo bulin (sm lg). T hese features are all o f importance in de termining the stage of ma turation at which neoplas tic cha nge e me rges. and ar e especially valu abl e in c1assitying lympho id leuk aemias. Unde r stimulatio n by phytoh aem agglutinin norm al T . mphocytes may undergo. in vitro. a process of dedi fferentiation o r transfonnation to prod uce primitive cells . with basophilic cytoplasm and leptochromatic leo lated nuclei . able to divide. A similar transrma tio n of B cells may occur following antigenic
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undergo furth er cyto logical transformation to give rise to plasma cells. Morphological variations in both Iymphocytes and plasma ce lts cover a much wider ran ge th an amo ng gra nulocytes . In infections . particularly th ose du e to viru ses . Iymphocytes show activated o r immunoblastic featu res intermedia te between mature and pr imitiv e cells. with increa se in cytoplasmic basophilia. and some time s the appeara nce o f visible nucleol i. Even in normal blood th er e may be co nsidera ble variation s in the size o f Iymphocytes and the amount o f cytoplasm . Cytoplasmic vacu ole s and inclusions are not infrequent. Fine or coar se gra nules and eve n blocks of glycoge n may occ ur in Iymphocytes and lymphoblasts, and o fte n appear espe ciall y co nspicuo us in leu kaemi as or other lymphoprolifer ativ e states. Plasma cells sho w even mor e abundant cytological variants. mostl y connected . there is litt le doubt . with thei r production of immunoglobulins and the accumula tion o f part o r the whol e of th ese protein molecules in vari ou s morphological guises in the cytoplasm . Here aga in. the cytological vari ant s are most conspicuous in mu ltiple myelom a . th e plasma-cell equivalent o f leukaemia . but an y o f the m may be see n occas ionally in plasma ce lls from othe r conditions and even in normal marrows. and non e appears to be spe cifical ly confined to myeloma .
A CUle l\'w phoblastic leuk aemias fA L Lr ). These. are monoclonal neoplastic d iseases arising from Iympho· blastic precursors of Iymphocytes . ALL makes up about 85% o f all acute leukacmic cases occurring below the age o f 16, a nd has been class ified morphologica lly by the FAB group into three subtypes according principally 10 th e size of the leukaemic cells : Ll cases have ho mogeneous small blasts with litt le cytoplasm . L2 cases hete rogeneous larger blasts with var iable amo unts of cyto plasm . and L3 case s homogeneous large blasts with vacuolated basophilic cytoplasm . Although this classificatio n doe s not correlate well with immu nolo gical or cytogenetic characteristics . except to an extent for th e uncom mo n L3 gro up, which includes most cases with the Bccell mark er of smIg production . and th e 8 ;14 tr an slocation. it has some prognostic significanc e . L2 cases .do ing less well th an Ll cases. This ma y pe rhaps
redectthe phaseofpro/ikrativeactivityratherthan an)'
19 1
ntial diffe re nce in cell type o r o rigin, since cases prese nting as II ofte n show a change to U in relapse . The disaimi nating morpho logical fea tures are neverillustrated in subsequ ent figures and furth er descri bed in the ir caption s. unologically, A LL ca n be subd ivided into man y I diffe rent types. but ther e arc five main gro ups: T-ALL derived from 'f-cell precursors (TdT+ , CD3+ , CD7+); ' null" ALL and C· ALL fro m ea rly B-<:ell precurs ors (TdT+ , CD 19+ and respecli vely CD I()- and CD I() + ); pre- B ALL from B precurs ors at a somewhat later sta ge (TdT + /-, cl gM + ); and B-ALL fro m a still late r stage of Bccell develop menl (TdT-, srnlg -i- ). CALL makes up abo ut 70% of childho od cases, T-ALL abo ut 15% , null ALL about 12% , and B-ALL < 2%, In ad ults. where only abo ut 15% of acute leukaem ias are lympho blastic , C-A LL is relatively infreque nt , co mprising perhaps 40% of all cases. o ther early B and pre B cases making up the differen ce . A small proport ion of cases «2%) with dubiou s or mixed reactions rem ain d ifficult to classify immun ologi cally . Ap art from the part ial association of B-AlL immuno logy with L3 mor phology. there are few distin guishing cyto logical feat ures in parallel with the rem ainin g sub type s, except for the pres ence of localized par anuclea r dot positi vity to acid phosphat ase and less markedly to o ther acid hyd rolases in T-ALL. Cytoge netically , seve ral non -random chro moso mal abno rma lities have been identified in ALL. The se includ e t(9:22 ). occurring in nearly 20% of adult ALL case s, h ut in only abo ut 5% of child hood cases; t(8;14) in most cases of B-ALL ; t(I ;19), t( v; 12) and 6q- in a small proport ion of earl y B-cell cases; t(4; 1I ) in uncomm on cases with a tenden cy to develop mixed lymphob lastic and mon ob lastic fea tures ; and t(11;14) in T-A lL. More import ant than these individual structural ano malies in terms of overall prognosis for the ALL gro up as a whole arc chromo some num bers. hyper diploidy with 50--60c hromos omes be ing associated with a relatively good response to treatme nt , dipl oidy and near haploid y with an inte rmed iate one , and minor hypodi ploid y o r pse udodi plo idy with most structura l ab no rma lities a worse o ne .
Chronic lympho cytic leukaemia (C LL) . Thi s disease is rare before middle years and has an increasing incidence with rising age . There is a periphe ral lymphocytosis usually over lSx 10' /1, with variable lymph adenopathy , sple no megaly, hepatom egaly a nd defe cts of marrow function manifest by anae mia and thromboc ytopenia. The extent of thes e clinical features forms the basis of various stagi ng systems, now in internat io nal use . Some 10% of pat ients with ClL also show a more o r less seve re auto-im mune haem olyt ic anaemi a with spherocytos is and a positive antiglo bulin test. Hypoga mmaglobulinaemia is co mmo n, especia lly in mor e advance d stages of disease, and a monocl onal par aprot ein may be produce d . proba bly always in case s with a Iymph oplasmac yto id co mpo nent to the cytology of both peripher al blood and lymph nod es. CLL is usually a Bccell disease with CDl9 positivity and at least wea k expression of smlg, but a small proport ion «5%) of cases with CLL morphology and sma ll ag ranular lymph ocyte s with non -convo luted nuclei show par anuclear focal acid phospha tase posi tivity. and react with the pa n-T MA bs
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CD3 and CD7 , and usually with CD4. Th ese cases are classifiabl e as T -Cl L and are to be disting uished from T-proly mphocytic leuk aemia and also fro m CD + granula r T -ce ll lymp hocytosis. wheth er in an ea rly no nmalignant sta ge . o r frankl y neoplas tic . Th ey co mmonl y show skin infiltrati on and a mo re aggressive clinical co urse th an B-CL L.
Prolymp hocytic leukaem ia (PL L) . This subacu te and clinicall y intracta ble varia nt may eme rge by a form of malignan t progres sion in Cl. L. or may arise de novo . usually in association with gross spleno megaly and ofte n also he pat ome galy. The periphe ral cell count is usually very high , with prolymp bocyr ic feat ures of nucleolat ed but moderately pachych rom atic nuclear chro matin and variable amount s of gran ular cyto plasm. Mosl cases of PLL sho w a Bcce ll phenot ype , with stro nger expressio n of sml g than in B-CLl, and with CD l9 positlvit y. but up to 20% of cases have a T-cell phenotype , most often CD4+ . Hairy cell leukaem ia (HC L) or leuk uem ic reticulo endosheliosis (LR E). Thi s cytolo gically fascinatin g leukaem ia of B-eell lineage occurs in middl e years or lat er . four times as commo nly in men as in women. and is associated with ge neral malaise , spleno megaly. occasio na l abdo minal lymph adenop ath y, mod er ate anae mia , th rom boc ytopeni a and neu tropeni a , and con spicuou s monocytop eni a . A few e ryth roblasts are usuall y to he found in the periphe ral blood . Th e bo ne marrow is d iffuse ly infiltrate d with hairy ce lls (HCs) a nd also shows d iffuse fibrosis. so that atte mpts at aspiration usually result in a dr y tap. T rephine biopsies sho w a very characte ristic picture . as illustrat ed in 767-772 . The HCs ca n be recog nized and studied in blood smea rs or in buffy coa t prepar at ions (7~766); the y have a striking and diagnostic cyto logica l appear ance in Rom anowsk y stains and have tartrat e-resistant acid phosph at ase (T RA P) positivit y and typica l PA S cytoc hemistry . Th e lA P score is not abl y high . lmmun ologically, He . , like B-CLL cells, have receptors for C3h (CD l1+) hUI , unl ike CLL ce lls, not for C3d (C D21- ). They express activatio n ma rke rs such as IU recepto rs (CD25 +) and have certa in relatively specific mark ers such as CD22 antigen , strongly pres ent o n HCs but o nly weak ly present o n a minori ty of normal B ce lls. Biochem ically, the ce lls produce mon oclon al Ig, and DNA analysis indicate s the pre sen ce of clon al rearr an gements of both light and hea vy chain genes . All these fea tures sugges t that HCs are activated B ce lls. at a stage before maturat ion to plasma cells. HeL is of furth er unique int erest in bein g the onl y haem atological malignancy to show almost inva ria bly a dr amatic therape utic response to the biological age nt alpha-in terferon . Sezary syndrom e (SS) . Thi s chronic T-eell neoplasm represe nts the leukaemi c ph ase of mycosis fungoid es . combin ing the skin infiltr ation . nodular and exfoli ative dermatitis and epide rmal Pautrier 's microabscesses, with the presence of typical Sezary cells in the circulating blood . Th e cha racteris tics of Sezary cells includ e , most importantl y. a deeply convoluted o r cerebrifo rm nucleus . not always clearly manifest . altho ugh it may be suspecte d . in smea rs viewed under the light micro-
sco pe . but striking in electro n microsco pic preparati on s (cf. 77&-788). Th ere is genera lly a high nuclear-cytoplas mic rati o . surface mem br ane reactivity with the pa n-T MAb CD3 and usua lly with the helper cell marker CD4. although rarely with the suppressor cell marke r COS instead. T he cells are usually PAS·positive but rarely show localized acid phosp hatase reaction . It seems likely that Sezary cells are the same as the convolu ted T cells of mycosis fungoides. alt ho ugh the latt er sho w some immunological differences. notably CD? and C 0 2S positivit y. suggesting that a possible activation effect may follow infiltration into the dermis.
Large granular lymp hocytosis (L C L) and granular lymphocytic leukaemia (CLL) . This chro nic diso rder with over lu x lU'J/l large gra nular Iymph ocytes in the peripheral blood, associated with splenomegaly but little other organ invo lvement. may have a ben ign co urse when found as a respo nse to variou s chro nic infective or auto-immune co nditions. but may also occur in an appa rently pri mary neoplastic form when it is best regarded as a form ofT·CLL. T he large granular Iymphocytes, with typically lymphocytic nuclei but with a mode rate ly low nuclear-cytop lasmic ratio and fine scattered azurophilic granules in the cytopla sm. as illustrated in 643. express the pan-T cell marke r CD? and are usuall y COR+ve. They may have cytotoxic functions, includin g natural killer (N K) cell activity. The y do not usua lly show focal paranuclear acid phosphatase positivit y. but scattered granul ar react ion (644 ). A dult T-cell leuka emia/lymphoma lA TL ). This neo plastic disease has cyto logical feat ures somewhat resemb ling. thou gh mo re exaggera ted than . those of SS. with circulating T ce lls having con spicuously co nvoluted nuclei . scattered acid phosp hat ase positivity. and an imm unophenotype includ ing CD3 and C0 4 positlvity. but unlike SS. the disease is du e to infection with a retroviru s. human Teccll Iymphotrophic virus 1 ( HT LV l) , and is largely restricted to ce rtai n areas in Japan and to Caribbeans of African descent. Clinically. the co ndition is associated with lymphadeno path y. hepatosple no megaly and co mmo nly hype rcalcaemia , and generally pu rsues an acute aggressive co urse . alt ho ugh chronic fo rms exist. Mvetoma and waldenstrom:s macroglob ulinaemia . T hese disorde rs involve a neoplastic gro wth of the plasma ce lls or Iymphoplasmacytoid cells at the end of the Bvccl l maturation chain . with the form ati on and secretio n of a mo noclon al immunoglobulin or its co mpo nent parts. In myelom a the plasma cells in an y individ ual case are usually of a single clone (rarely biclonal). pro liferating in and displacing the hon e marrow and producing a specific Ig. biochemically and immunologically uniqu e. This is usuall y IgG or IgA . rarely IgM and even mor e rarely IgD or IgE ; but in abo ut a third of cases . sometimes called Bence-Jo ncs myeloma. o nly the light chain. kap pa or lambda. is produced . There is associated bo ne destruction . d ue to increased osteocla stic act ivity. secondary to production by the neop lastic cells of an oste oclast activatin g facto r (O AF). Th e histological picture in treph ine biopsies is characte ristic and the cytology in aspirates usually
diagnostic. as illustrated in 798-830. although indivi cytological features such as mult inuclearity. fragme u . ing cells, flaming cells. the saurocytes. Mott cells. grape cells. cells with intranuclear or cyto plasmic inclusion and so forth are not in themselves indicative of neoplastic change . since any of them may be found in reactive plasmacytosis. An incr ease in plasma cells abov e 20% in the bo ne marrow . or a smaller increase with demon str ab le light chain restricti on . provides accept ab le evidence of myelo ma . confirmed when taken in conjunction with Xcray evidence of oste olytic lesions and the dem on strat ion of a circulatin g monoclonal paraprotein . Clinically. the bony destru ction ofte n leads to hypercalcae mia. and myelo matou s infiltratio n of the kidn ey freq uentl y produces renal failure and urae mia. Blood findings include a raised ES R and variable anaemia. but plasma cells are not often seen. except occasion ally in buffy coat preparations, and leucocytes and platelets are generally present in no rma l nu mbers. altho ugh ther e may be a mild leucoer yth roblastic picture with infrequent e rythroblasts and myelocytes de tect able . Levels of seru m beta 2 microglo bulin become increasingly elevated with advancing disease and probably pro vide thc best single prognostic index . although the degree of ana emia and of ren al failure are also of progno stic importance . w aldenstro m's macroglobulinaemia is a form of Iymphoplasmacytoid immunocytoma with bone marrow involvement and prod uction of a circulating monoclonal IgM paraprotein . As in myeloma, the re is usually a high ESR and mod erat e-to -severe ana emia. but leucocytes and platelets are genera lly normal. Th e bo ne marrow shows infiltration with Iympho plasmacytoid cells and com monly an increase in macrophage iron and in tissue mast cells. manifest in both aspirat es (834-842 and 844) and histological sections (843 and 845-851). where the large intranuclear inclusions known as Dut che r bod ies and characteristic of this dise ase may also be conspicuo us. Clinically. the course is ge ner ally more indolent than that of myeloma . and there is mor e resemb lance to a low-grade no n-Hodgkin's lymphoma. with lymphade nopathy and some degree of hepa tosplenomegaly mor e often fou nd .
Lym phoma. Th is malignant disease arising in lymph node s and involving ce lls of more or less matur e de gree in the lympho cytic line . commo nly extends at some stage to involve the bo ne marr ow and to liberate abno rmal lymph ocytes in the pe riphe ral blood . If the cell type is mature the disease is cytologically ind istinguishable from chronic lympho cytic leukaem ia (C LL), and, if very primitive. as it may be in children especially. it is equally indistinguishable from acute T- or B-cell lymphoblastic leukaemia. In some cases. however. abnormal 'lymphoma cells'. unlike either mature Iymphocytes o r the Iymphoblasts of acute le ukaemia bu t with characte ristics inte rmediate between the two . mav be see n. T hese cells arc usuall y of the B-cell line bu t 'J1l3.~ be of T-ce ll lineage and include neoplastic vari ants ot the centrocytcs . ccntrobl asts and immunoblasts preseee in lymph nodes and illustrat ed mor e fully in Pan S. The cytology and cytochemistry of all these and abnor ma l variants as seen in blood and man illustrated in the following pages.
l
S9S
59~39 . A cute lymphoblastic leukaemia (A L L). including examples of the range of m orph ology classifiable under the FAH system as LI (small blast cells with high nuclear-cytoplasmic ratio , regular nuclear outlines ap art f rom poss ible Rieder cell f orm ation , and poorly visible nucleoli} (596 and 598-#)9). as U (cells generally larger, with relatively more cytoplasm, more indentations in nuclear outline and more easily visible nu cleoli) (597 and 6 /()...(, 12J, o r as L3 (large cells with regular flue/ear outlines and homogeneous nuclear chromatin with readily visible nucleoli , and with ample basophilic cytoplasm sometim es containing vacuol es) (625-629). Immunoptienotypes co rrelate poorly with morphology. apart f rom an association betw een L3 and B-A LL and cytochem ical paranuclear localization of acid phosphatase positivity in blasts of T-A L L.
595. A lymphob last with a mature Iymphocyte aod a 'stab cell, from the peripheral blood of a patient with ALL.
596. Lyrnphoblasts from the com mon type of non-T 000 -8 acute lymphoblastic leuk aemia (e-A LL). snowing the high nuclear-cytoplasmic ratio and occasional nuclear cleavage - Rieder cell form ation . There are a few mature Iymphocyte s and some interm ed iate ' prolymphocytes' present. Despite the nuclear cleavage this cytological picture is pro bably best classified as Ll . 5fJ7. Another example of C-ALL. There are two granuloeytic cells present - a metamyelocyt e and an eosinophil myelocyte - and also some cells with denser nuclei maturing along the lym phocytic line. This field illustrates the difficulty of achieving the certain allocation of acute leukae mia to a specific type based ,pn the appea rances in Romano wsky stains alo ne. Cytoche mical staining in this case gave the typical findings shown in the following figures and estab lished the diagnosis unequiv ocally. Alth ough the cell size is much the same as in 596. the lower nuclear-cyto plasmic ratio and more conspicuous nucleoli confo rm with the criteria for L2.
194
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~
--
MMI
; 98. 5 B stain in C- A LL. There is a myelo cyte with no rmal positivity pre sent (for contrast) . hut the lymphoblasts and occ asionally mor e mature cells of the lyrnph ocyte line arc uniforml y negativ e . As well as the high nuclear--cyto plasmic rati o . occasion al vacuol ation ca n be
seen. 599 . Peroxidase rea ction in C-A LL. Lymphoblasts are uniformly ne gative . A polymorph pre sent is normall y positive.
!• I
. PAS sta in in acu te lymphobl astic leukae mia IC-A LL). T he Iymphobl asts mostl y sho w num erou s fine and coa rse gra nules of positivity. Note th at th e cryth rots present arc ne gat ive - unlik e th ose formi ng an erythraernic compo nent in mixed myeloid leu kaem ias. A . gle myelocyte shows norm al diffuse but wea k PA S itivity.
.;.
195
602
601
•
.
•
J
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/ 601. Another example of PAS staining in ALL. In this case so me ce lls co ntain blo cks in additio n to coa rse granules .
602. A group of Iymphob lasts from C-AL L. The cytoplasm shows frequent irregularities of staining and occasional vacuoles. particularly in cytoplasmic buds. 603 . The same field as abo ve . co nsccutivelv stained with the PAS reaction. Most of the lymphoblasts contain coarse granules of PAS· positive material. often eoinciding with vacuo les or areas of lighter staining within
J
'h e cytop lasm and. especia lly. in cytop lasmic protru bcrances - as shown in 602.
196
;( I
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6O-l. PAS reaction in a furt her case of C-A LL with
606
minima l positivity. Even such almo st entirely nega tive b mphoblasts commonly show so me granul ar positivity in a very occasional cell. Wea k reactions are per haps commoner in T-cell cases, but this is not a verv useful discriminatory feature since much variation exists. 5. Acid phosphatase reaction in the same case , wing little positivity. typically for C-A LL. There is a eormally positive polymorph presen t. Dual esterase reaction with weak scattered butyrate te rase (BE) positivity in lymph ob lasts of C-ALL. Tbls patte rn is usual in non-B non-T cases.
•
197
607
607. Immunocytochemical alkaline phosphatase-antialkaline phosphatase (A PAAP) reaction for the presence of terminal deoxynucleotide transferase (TdT) in the nuclei of lymphoblasts from the bone marrow in a case of C-ALL. Nuclear TdT is demonstrable in the primitive cells of more than 90% of cases of ALL, including C-A LL. null ALL. most pre -B ALL. and T-ALL. but is absent from B-ALL. TdT positivity in acute Icukacmic cells is not confined to ALL. however. being detectable also in the blast ce lls of around 20% of acut e myeloid leu kaemia (A ML) cases, most of them with no other fea tu re to suggest mixed phenotypic expression . 608 and 609. Further examples of immunocytoc hemical A PAA P rea ction s o n lympboblasts of C-A LL to demonstra te surface antigens of discrimin ative value in diffe re ntia l diagn osis. A posit ive reacti on is sho wn in 608 to the mon oclon al antibody (MA b) CD 10. and a negative reaction in 609 to the myeloid MA b C D I3 . Immunocytochemical reaction s for cell-surface antigens are not illustr ated extensiv ely in relat ion to all the varieties of leu kaemia becau se the appea ra nces do not reve al impo rta nt differences in d istribution patte rns of positivity . cells be ing simply positiv e o r nega tive . what ever MA b is used. Th e interpretat ion of positi vity patterns wit h different MAbs therefore depends esse ntially o n the cap tions. with C-A LL. for exa mple. showing typically cells tbat arc TdT+. CDIO +. CD 19+ and CD3-. CDI3-.
198
610
11
610. Roman owsky stain o n bon e marrow cells from an adult case of null ALL (TdT+, CD I9+, CD W-, CDJ--, CD IJ--), sho wing, mor e clearly than in the ea rlie r 597. th e patt ern of larger cell size. lower nuclearcytoplasmic ratio , mor e eas ily visible nucleo li and occa sio nal inde ntation of nuclear outline that is classifiable under the FAB syste m as the L2 variant. This variant is seen in adults more often than in child ren. at least at th e time of diagnos is. and carries a poo rer prog nosis than the more common Ll variant. There is no co rrelation, however. betw een immun ological phe notype and LI or L2 murphology, and cases IDi tially classed as Ll may sho w an L2 picture at relapse. 6 11. SB stain o n the sa me mar row sample. showing a
itive myelocyte but negative reactions in the remainblast cells .
.1 Ano ther example of an AL L bone marrow aspirate illustr ate the freq uent difficulty in applying the FA B ....ssification. The cells are generally large with -SCrale nuclea r--eytoplasmic ratio , regular nw:lear some conspicuous nucleoli. and moderately ilic cyto plasm with scatte red vacuoles. This 4'p<;arance conforms best to L3, but immun ophen o_ revealed a patt ern of null ALL as in the previous and reclassif ication as 12 was thought appropriate.
.""'le<;.
612
•
6 13
610
./
613-4j17. Cytology and cytochemistry of aspirated hone marrow from a case of pre-B ALL (TdT+ . CD IY+ . C D W+. c1gM +) . Th e Rom anowsky stain in 613 shows co nside rable variati on in ce ll size and irr egularit y of cyto plasmic o utlines. with a scatte ring o f per ipher al cytoplasmic fragment s. and variabl e nuclearcyto plas mic ra tio. Sever al cells show two or thr ee sma ll nucleoli . Th e cytopl asm is gene rally basophilic. with a suggestio n of vacuole form atio n. and in this area the appea ra nces woul d be co nsiste nt with L2 o r L3. T he Su -stained field (6 14) and the re ma ining cytoche mical stains (6 15-617) from the same prepar atio n. howeve r. show much mo re u nifor m cyto logy with smoo th nuclear chromatin. regular nuclear membranes. poorly visible nucleoli. and no more th an a minimal rim o f cytop lasm. ge nerally more co nsiste nt with an L1 classificatio n. Sudanoph ilic granulocyte precursors arc present in 61~ . and a neut roph il metamyelocyte and poly mo rp h with typical PA S po sjtivity in 615 . although the blast cells in caetfl'Uf these s have the characteristic rea ctions of lympho blasts.tnegan ve to S8 and with coa rsely gran ular positivi ty to PAS. The acid phosph at ase reaction in 616 shows scattered pos itivity in a neut rophil stab ce ll and a polymorp h, but the blast cells are essentially negative. The dual este rase stain in 617 Is also negative in the lymphoblas alt ho ugh a chloroacc tate esterase (CE)-positivc neu trophil myelocyte and a BE-positive mo nocyte are also prese nt in this field.
6 18
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618. Aci d pho sph atase reaction in a T-cc ll A LL. ho wing the stro ng locali zed paranuclear po sitivity in mos t Iympho blasts ch ara cteri stic o f thi s ALL vari an t. uch posi tivity is much less co mmo nly found in C- A LL an d null-cell ALL , and is absent in only a small mino rity of cases ofT-A LL.
,
620
•
' 19. Dua l esterase reactio n. sho wing localized BE itivity in Iymphob lasts of T-ce ll A LL. Scattered BE po sitivity in Iymph obl asts. with a It'Dlk ncy 10 loca lization o n o nc side of the nucl eu s (less . ing than in 619). T his pattern suggests aT-ce ll ge , but the cells are in fact fro m a C-A LL case .
;
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622
621
62 1~24 . T-ALL plus mark ed eosinophilia. (Curiously, eosinophilia if at least as common in ALL as in AML.)
621. Seven Iymphobl asts with seven eos ino phil or eos inophil pr ecur sor s and a lat e no rmobl ast ; from the ~~ of a patient with T -ALL. 622. A lymph o blast , a lympbocyte aod nine path ological eosi no phils; from the peripheral blood of the same case of T-A LL with gross eos inophilia. Th e eosinophil granules arc defective in num ber and smaller in size than no rma l. 623. Acid phosphatase reaction o n the same marr ow aspirate , showing scatte red gra nules of coarse positivity in three eosinop hils and strong localized paran uclear pos itivity in the T Iymphoblasls. 624. Du al este rase reaction o n the same aspir ate . showing a single Cli-positive neutrophil metamyelocyte but negatively rea cting eosino phil and blast ~e ll s .
202
6'
615
627
628
615-629. Bon e marrow and blood smear preparation s fro m an ad ult patient with B-ALL. Th e Heyl sta in in 615 shows a clump of marrow cells with mod er ate size, visible nucleoli , so mewhat irregular nucle ar outlines . high nuclea r--eytoplasmic rati o , and basop hilic but inimal cytoplasm. difficult 10 classify o n the FA H (em. There were num erous smea r ce lls presen t. T he B stain (626) showed the unu sual patte rn of coarsely ~an u l a r . glob ular suda no philia found in <2% of ALL #
The B lymphoblasts in the peripheral blood from this tie nt show a more typical L3 cytology than the ITO W blasts. with a greate r amoun t of basophilic qcoo(ated cytoplasm. as seen in 627. while the immuno. oche mical APAA P reactions sho w positivity to (D19 in 628 and nega tivity to C DW in 629. The ce lls TdT-and smIg+ .
629
203
631
63~2.
Cytochemical react ions. respective ly rnyeloperoxid ase (630) , SIl (63 1), a nd du al este rase (632). on a bone marrow asp irate from a pat ient variously diagnosed on morphological and cytochemical grounds by different haematologists as either AML or ALL. The Romanowsky appeara nces were as shown by the Heyl counterstain in 630. where peroxidase positivtty is confined to a single neutrophil stab cell and the negative blast cells show no features strongly suggestive of either myeloid or lymphoid lines. The SB stain reveals discrete sca ttered granula r positivity of monocytic character in some 30% of the blast cells. with two of seven blasts positive in 631. and the esterase reaction shows strong CE positivity in otherwi se recognizable but infrequ ent neutrophil granulocytes. like the myelocyte in 632. but quite strong BE positivity in most of the blast cells. resembling more the reaction of mono blasts than that usually seen in Iymphoblasts. Th e PAS reaction . not shown here . was negative . Immunocytochemistry revealed a mixed phen otype. with variable but overlapping surface expression of antige ns reacting with CO W. C0 19. COB and CD1S. indicating the probable existence of an acute leukaem ia of mixed lineage. Cytogenetic studies were not available. but this type of hybrid leukaemia has been found part icularly in associatio n with a 4;11 translocation or other abnormali ty involving a brea kpoint at I Jq23.
204
633
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633--635. A sequence of bone marrow tre phi ne biopsies fro m a pati ent with C-A LL. und er going remission ind uctio n. In 633. ta ken at the sta rt of therapy . the specime n shows an overwhelming prep ond er ance of primitive cells with large and leptochr o matic nuclei often co ntaini ng visible nucleol i. There are only a few cells of the ery thro id or later granulocyte series present. wit h more pach ych ro matic nucl ei. A fte r two weeks of 01010xic combination chemot herapy. marrow aspirates gave o nly a poorly ce llula r speci me n with too few cells for differential assess ment , but the tr ep hine biopsy sho wn in 634 revealed a severely hypopl astic picture still containing a substa ntial pr opor tion of blast ce lls. vith the co ntinuation of treatmen t . a furthe r trephin e biopsy two weeks later reveal ed the emergence of a remi ssio n picture with active pro lifera tion of normal mopoietic cells of all myelo id lines, alt hough with ervthroblasts predomina ting , as shown in 635. Th ese fields illustra te again the impo rtant additional -.formatio n to be o btained from mar ro w sect ions in e le ukaemias, especially with regard to ove rall rity and the differe ntial compos ition of hypo. marrow du ring phases of att empte d re mission
636
•
. n.
C-....LL plus normoblastic hyperplasia . The field some 20 blasts. 16 normobl asts and a reticulo........i!adlial ( RE) cell. Thi s type of mixed cytology is paases of e merging remi ssio n o r relapse in
205
6..'7
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• 637-639. Sections of bone marr ow tre phine biopsy fro m the iliac cres t of an ad ult pa tient with sm Ig+ BALL. moving from a period of chemo therapy-induced remission to relapse. T he H&E-stained paraffin sectio n show n in 637 illustrates the very mixed cell pop ulation at this stage . with a megakaryocyte at the left and numerous scatte red e rythroblasts and later gra nulecytes, but with blast cells having large leptochromatic and freque nt ly nucleolated nuclei making up some 50% of the whole. The PAS stai n in 638 reveals positively reacting neu trophi l polymorphs and earlier granulocyte precu rsors . but the blast cells appear negative. A furt her H& E stain , this time of a plasti cembedded thin section. again shows the mixed cytology. with cells of different lineage readily distinguishable . The B lymphoblasts. with their pale nuclear chromatin and conspicuous nucleoli . co nstitute about 50% of the pop ulation.
206
.
-
•
••
640
{HO. Three mature Iymphocytes , together with a basophil, an eo sinophil . and two segmented neutrophil pcl yr norphs.
642
{Ht. A hinucl eatcd Iymphocyte . Such ce lls ma y rarely be see n in norm al blood and are not. as at on e time tho ught. suggest ive of an irr adiation effect.
•
~2 . A Iymph ocytc with more cytoplasm th an th ose sho wn above , and with az uro phil gra nules . possibl y a suppresso r T cell (Ty) . A neutrophil pol ymorph is also prese nt.
, •
• 207
&B . Vari at ion s in lyrnphocyte cyto logy in a norm al buffy coat smear . On e of the five Iymphocytes has litt le cyto plasm and is prob ably either a B or a T I-l cell; o ne has some marginal hair-li ke processes . not infrequentl y seen in nor mal lymphocytes and not to be confused with the appeara nce of ha iry cells (see 748-750 ); and the remaini ng thr ee Iymphocytes have relatively large amo unts of cytopl asm with a few scatte red granules. and are probabl y T y cells. A persiste nt increase in ce lls of this cyto logical cha racte r. with positivity to the panT MAb CD? and the suppresso r subset MAh CD S. is found in large gra nula r lymphoc ytosis and granular lymp hocytic leuk aem ia. 644. Acid phosphat ase rea ction in buffy coat ce lls. Two of the thr ee lymph ocytes showloc al ized coa rse dot positivit y cha racte ristic of the TIJ. ce ll subset; the thir d is larger with more cytoplasm. and with a little scattered pos itivity o nly. and is probabl y a T y cell. (MS. Acid phosph atase rea ction in pe riphe ral blood lyrnpho cytes: the proport ion of cells with the localize d pos itlvity see n here in three Iymphocytc s closely pa rallels the Tit cell proport ion by surface mark er techniq ues . Thi s He ld also shows gra nular acid phosphatase positivit y in two neutrophils. a basophil and an eosinophil.
208
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•
646. Dual esterase reaction. Localized T-ccll positivity in o ne lymphocyte . probably a TJoL cell. and weak granular BE positivit y in a seco nd. Thi s may be a null cell or pe rhaps a cell of the 1'y subset. Th ere are four CE-positive po lymor phs and a BE-reacting monocyte . 6-17. T wo oormally rea ctin g (CE-positivc) polymorphs. a BE-positive mo nocyte and four Iymphocytcs - two showing the localized dot-like Tu-subset type positivity. o ne a sma ll. negative . probab le B cell. the o the r a large r lymphocyte with ample negative cyto plasm. probably of the T y subset.
MS. A mono cyt e and a polymorph, toget her with two lymphocytes of the T J.L subset .
209
649-660. Reactive 'immunoblusts' and 'virocytes' arising in various infective states. These activated lymphocy tes are generally PAS, acid and alkalin e ph osphatase und esterase negative. 649. A blood smea r from a pati ent with infectious mon onucl eosis. Ap art from a ce ntral mon ocyte (with phagocy tic vacuoles) and a seg mented neu trophi l. the nucleat ed cells are all lyrnphocytes. Th e mito tic figure (in te lophase) sho ws the increased cyto plasmic basophilia ofte n encounte red in the abnor mal Iymphocytes of this disease . Mitoses in mo no nuclear cells are very unco mmon in nor mal periphe ral blood. but are seen much mo re ofte n in activated Iymphocytes. 650 and 651. Fur ther examples . from two different patients. of the ab no rmal morphology of Iymphocytes in infectious mono nucleosis. Th e nuclear chro matin in the most abno rma l cells is still too coa rse and the cytoplas m too abunda nt for real confusion to exist be twee n these cells and Iymphohlasts.
210
652
654
652. A n immunoblast in the marrow of a pat ient with ALL ie remission. but with a viral infection. Such cells ust no t be confused with leuk aem ic blast cells.
653. A disrupted RE ce ll and a binucleated irnrnunot fro m the same case. Three stabs . two Iymphocytes and five immune-
. from an infective episode during remission c.ergence in A ML. One of the immunoblasts shows _ "flaming"of the cytoplasm (cf. 807-809)"
211
655. Two neutrophil s. two lymp hocytc s. a mo nocyte and four immun oblasts (activa ted Iymphocytes) - from the buffy coat of a leuk aemic pat ient in remissio n but with a mycop lasma infect ion. T hese cells must be distingu ished fro m ea rly mo nocytes o r leukaemic blast ce lls. 656. PAS reaction . showing two stab cells. o ne lyrnphocyte and eight immun o blasts. from the same buffy coa t as in 655. T he immun oblasts are almost ent irely PAS-nega tive . 657. Peroxidase reaction on a buffy coat from a pat ient with hairy cell leukae mia (Hel) (onc hair y cell at top right), showing 24% of immune reactive plasma cells resulting fro m acute bacterial infection. The ncutrophils show normal peroxidase positivity. while the remaining cells are negati ve.
212
,
6: A vacuolated imrnunoblast and two stab cells with io xic granules. mo re co nspicuous in one of them . fro m blood of a child with a pseud o mon as infection.
•
A binuclea ted immunoblast . showing a tendency nuclear distortion and peripher al cytoplasm ic baso- ia. fro m a pat ient with a viral infection . A mo no nuclear immunoblast with twisted nucleu s Md fine azurophil stippling of cytoplasm. but with philia at the periphe ry. from thc same case .
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213
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661-663. Sections ofbone marrow trephine biopsy from a patient with diffu se marro w involvement with angioim munoblustic lymphadenopathy (A IL) . A IL is generally regarded as a reactive process. although with a high risk of transformation to a floridly neoplastic non-Hodgkin :S lympho ma (NH L) . Mar row involvement may be either focal or diff use . The low- and higher-power views sho wn in 661 and 662 reveal the cha racte ristic morphological picture , with a swirling patte rn of branching endothel ium-lined blood vessels, an interspers ed proli feratio n of activated lymp hocytes plasmacytoid immunobl asts and plasma cells. and a n interstitial deposit of weakly staining amorphous eosinophilic materi al. Th ere is often a compo ne nt of haem olytic anaemia pr esen t in the se cases, and the marrow may show areas of nor moblastic hyperplasia . as in 662. wher e there is an island of normoblastic proliferation at the upper right corner. In the low-power view of a reticulin stain in 663. the characteristic increase in reticulin fibres and their distribution in a swirling patt ern around the pseudo-vessels is well sho wn. The periphe ral blood in AIL ofte n contains lyrnphoplasmacytoid immunoblasts and occasio nal mature plasma cells. and there is commo nly a polyclonal dysproteinaemia.
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6tH . PLL, Romanowsky stain: the cells show more
basophilic cyto plasm and more pro nounced nucleo li tha n is the case with lymphocytcs. but have moderately pachychromatic nuclei. unlike blast cells. "-5 , PLL. Romanowsky stain. a T-ccll case : the large size and moderately pachychromatic nuclear pattern . 'm occasional variab le nucleoli. contrast sharply with two normal lymphocytes present .
PLL. Leishman stain on another case; this was of 8-«11 origin. The nuclear cleavage and invaginations
times seen in PLL cells are well shown in this case.
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667. S8 on PLL: a nor ma lly reactin g eosinop hil with prol ymphocytes of var iab le cyto logy b ut un iformly negative rea ctions. 668. PLL. PAS reaction: the prolymphocytes are esse ntially PAS-negat ive in this 'J-ccll case . 669. PAS reaction o n a R-cell PLL case . Th e proIymphocytes are again essentially negative hut one , more immature . blast cell (top right ) shows a rim of granular PAS positivity.
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' -3 . Dual esterase reaction in the same case as 672; t half the pro lymphocy tcs showed moderately _ localized BE positivity. 675
Acid phosphatase reaction in a H-ccll PLL The "S a normally positive polymorph and seven ,:::::~;.tes. with occasional granules of coarse .. scattered mostly over the nucleus rather than _ _Ird in me paranuclear zone. ~ ..........
terase o n the same case as in 674. Two polymo rphs and a BE-positive norm al T~ with the virtually negative B prol ympho-
217
676
676. A general view of a peripheral blood smear from chronic lymphocytic leukaemia (Cl.L). The intact cells arc nearly all lymphocytes. showing some variation in morphology probabl y in parallel with maturit y. The smea red disrupt ed cells are typically numerou s in this disease . The spread nuclear remn ants arc sometimes know n as 'G umprecht's shado ws' ,
677
677. Another example of the blood picture in C t. L. The range ' of morphological variation is not great . but a few precursors .: pro lymphocytes or lymphohlasts - can be seen . and the smea red cells are again conspicuous.
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678
678. A high-power view of the perip heral blood io Cl.L. Again an occasional precursor is visible. 679. PAS reaction io e LL. The Iyrnphocytes show a pattern of positivity like that of normal lymphocytes. but a higher proportion of cells shows positivity than in a normal specimen.
Anotherexample of PAS reaction in Cl.L .
680
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6H1. Acid phosphatase in C l.L. Most cells show scatte red positivity without mar ked pa ranuclear concentration. 682. Dual esterase in e LL. Th e lymphocytes show mostly 'negative reactions; a few have scatte red weak BE pos itivity. Normal CE reaction in a polymorph and BE positivity in a mo nocyte a rc shown.
683. Dual esterase in another case of e l l . showing an occasional crcscc ntic positive reaction . similar to that usually sccn in HCL.
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from the peri pheral blood of a patient with B-CLL . stained for alkaline phosphatase . showing the rare finding of positivity for this enzyme in the neoplastic Iymphocytes. Four neut rophil pol ymo rphs show fro m 0--2+ positivit y. and most of the CLl cells show scattered positive granules. Altho ugh this findin g is quit e uncommon in e l l. the lympho id cells of NHL. especially the centrocytes and ce ntroblasrs of follicular lymphomas. show it rat her more often.
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.: ; . An immunocytochemical alkaline phos phatase u-alkaline phosphatase (A PA A P) prep ar ati on of BU l pe ripheral blood buffy coa t. showing a stro ng IDemb rane reactio n for IgM . A similar APAAP react ion o n peripher al blood fro m a T-CLL. sho wing membrane positiv ity to - T-
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687-692. Trephine biopsy sections from various cases of e L L to illustrate the common morphological patterns of infiltration. 687. A very low-power view of a marrow with about 50% overall cellularit y and with a single focus of eLL toward s the bottom left. The remain ing marrow cells represent esse ntially normal haemopoietic tissue . This type of focal involvement is commonly seen in the more indolent stages of eLL. 688. Another very low-power view of a thin section of marrow trephine biopsy from another pat ient with a so mewhat denser nodular infiltr ation , partly distributed randomly and partly of paratrabecular disposition. The residual normal marrow tissue, with its looser structure and recognizable scattered megakaryocytes , cont rasts with the dense focus of eLL cells in the upper part of the field and the similar paratrabecular focus at the lower right corner. 689. A third exampl e of eLL infiltration in the bone marrow , here with a more general diffuse distribution , as usually see n in more adva nced or rapidly prog ressive disease. Residual normal marrow in this specimen was virtually confined to the immediate paratrabecular areas . as illustrated in this field.
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692
691 and 692. Intermediate and high-power views, respectively. of a bone mar row trephine biopsy section fro m a patient with B-CLL and an accompan ying auto- immune haemolytic anaemia. Ther e is some _ ne ralized diffuse lymph oc ytic infiltrati on , but also a local collectio n of lymph ocytes at the lower left of the Id in 691, aga inst a cellular background of gross ervthroblastlc hyperplasia. The higher magnification of 2 reveals more cytological detail, and shows the :~~rp~h iC character of the interstitial cells, with ..res. various granulocyte stages, and a preof erythroblasts, including numerous nudeopeoe ryt hro blasts. There is a suggestion of megalochange among the early erythroblasts.
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693-697. Smears of a buffy coa t prepar at ion . made fro m the periphe ral hloo d of a pat ieo t with B-CLL in co urse of malignant progressio n to an immunoblastic leukae mia/lympho ma . T his type of tr ansform ation Richt er's syndrome - develops as a terminal manifestation in abo ut 5% of patients with Cl. L. and is usuall y characterized by recurrent lymphadenop athy . fever . weight loss . and resistance to previou sly effective chemot herapy. Immunoblasts arc usually pres ent in the peripheral blood and heavil y infiltrate the marr ow (see 69S-7(0) . "There are three residual lyrnphocytes and a neu trophil po lymo rph in 693. bu t the predom inant cell po pu lat ion is made up of immunohla sts with basophilic cyto plasm and so mewha t plasmacyto id nuclei . A part from the immun o hJasts in the blood . there may be other unu sual reactive eleme nts. like the rare tissue mast cell appea ring in 694. whe re the re are also seve ral neutroph ils. o ne of them polyseg memed. and a basophil. as well as a Iymphocyte and seve ral immunoblas ts of vari ed size. The S8 stain in 695 shows the pred om inat ing imm unoblasts to be negative . the positive cells in this field including two neutroph il stab ce lls. a baso phil to the right , and two probah le monocytes toward s the top left . Th e PAS reacti on in 696 reve als three no rmally posi tive neutrophil polymorp hs. a monocyte with peripheral gra nular positivit y, th ree negative lymp hocytes . and nine immuno blasts with either negative react ions o r no mor e than a faint tinge of pos itivity. The acid phosphatase stai n in 697 shows no rmal positivity in polymorp hs, a stro nge r reaction in a mon ocyte . a few positive gran ules in most of the immunoblasts . and paranuclear do ts of posi tivity in two T lymphocytes next to the monocyte .
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pbocytcs at the lower right half and large cells r nuclei and more ample cyto plasm occupying lbe upper left half. ... a low-po wer view of another transforming _ ....... sample reveals 3 less nodula r and more diffuse of infiltratio n with large immunoblastic cells . . also evide nce in this field of acco mpa nying .."....oIa!"'·ic react ion. a highe r-power view of the same section as in
s details of the cytology, including a
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of the large cells with vesicular nucl ei and co nspicuo us nucleoli amo ng a mixed po pu-
c idual small Cl.L lymphocytcs. occasional _lIP:" and erythroblasts, capillary endot helial scantv fibrob lasts. Several of the large ce lls ed nuclei and bear a gene ral resemblance "_C!'1O and it is not surprising that Richter' s at one time thought to represent a tic tra nsforma tio n of Cl.l.. but both aDd cytochemistry of these cells in smears :EqII
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Sections of bon e marrow tre ph ine biop sy "I'luO>e ns ta ke n fro m pati ents with e L L und er goin g "". .k>trmatio n to Richt er 's syndro me . field in 698 is of a jun ction al area with resid ual
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701. Four lymph om a cells fro m the hlood of a patien t with lo ngstanding N HL recently spreading to involve bo ne marrow and bloo d. The ce lls resem ble neither matur e lymphocytes nor leukaemic Iymphoblasts, but have intermediate characteristics . O ne ce ll shows a tendency to nuclear cleav ing. These ce lls are of 'centroeyrie' or 'follicular centre cell' (FeC) type . 702. Anot her exam ple of lymp homa cells in the blood : this represents a leukaemic phase of a secondary ce ntroblastic lympho ma. There are multiple small nucle oli visible in the irregular nuclei and cytop lasmic
basophilia is moderate. 703. A third example of lymphoma cells in the blood in this case the cell s have deep cytoplasmic basophilia
and multiple nucleoli. and are of immunoblastic character.
705
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706
7117
707 and 70H . Rom anowsky stains on two fur ther e xa mples of ce lls fro m the pe ripher al blood of a patient in a lcuk aemic ph ase o f irnmuno blastic lymphom a . Th e lar ge ce ll size . deep cyto plasmic basophilia , irregula r .
somewhat plasmacytoid. but generally primitive, nuclear chro matin patte rn . the presen ce of large centrally placed nucleoli in so me ce lls. and the variabi lity of nuclear shape from uniforml y round to deeply indent ed . all cha racte ristic featu res of ma lignant immu nob lasts . a rc well sho wn . 709. Scattered BE posi tivity in three neoplastic irnmuno.. blasts. a normally BE-positive mon ocyte . a T lymph ocyte with localized ' do t' BE positlvity. a nd a CEpositive neutrophil .
710
711
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, 710. A sec tion of bo ne marrow tre phine biopsy fro m a pa tient with a lar ge-cell di ffuse immunobl astic lymp ho ma. showi ng extensive dif fuse infiltratio n o f the marrow pa re nchyma with mos tly large immu noblastic cells. often having co nsp icuous cent rally placed single nucleo li. The remaining cells with darker nuclea r ch ro matin and less visible cytoplasm arc residu al hae mopo ietic e leme nts of erythroid and granulocytic lines.
712
.
111. A nother higher -power view of a tr eph ine biopsy o f ho ne marrow from a pat ient with a lar ge-cell lymphoma . thi s time aga in showi ng diffuse infilt ra tion but with cc nlro blasts rath er th an irnmun obl ust s. These ce lls ge ne rally show more twisted and indented nuclear ou tlines. and their nucleoli arc sma ller and more ofte n multiple and near the nuclear membra ne ; these cytologica l cha rac teristics arc those of ce ntroblasts or largecseaved follicular centre ce lls.
"'12. A high-po wer view of a section of marrow trephine y with centro blastic lymphomatous infiltration. lar ge. if so mewha t va riable. cell size and th e ~ Ia; nuclea r o ut lines. which give rise to the altere na me of ' large-cleaved cell". arc well shown . o
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7]3-715. Sections of bone marrow trephine biopsies from another patient with a diffuse follicular ce ntre cell or centrocytic lymphoma. showing response to treatment with an anti-lymphoid MAb. The respective lowand higher-power views of the pre-treatment bio psy in 713 and 714 illustrate the extensive nature of the marrow infiltration and the cytology of che cells as predominantly non-cleaved centrocytes . In 715. the fresh biopsy taken afte r a sho rt course of MA b therapy sho ws an entirely change d picture , with retu rn to the no rma l highly pleomorphic cell mixture of norm al haem opoietic tissue , including erythroblast s, granulocytes of all stages, and mcgakaryocytes, the only apparent abnormality being a rea ctive increase in eosinophils. Trephine biopsies are essential in the assessment of respo nse to therapy when marrow infiltration exists, since they allow the degree of residual tumou r-cell persistence to be measured with much greater accuracy than is possible in aspiration biopsies.
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7 16-7 18 . Blood smears at stages during the pr ogression
of a lymphoblastic lymphoma , convoluted (T-cell) typ e, 10 a picture virtually indistinguishable [rom ALL.
716. A lymphocyte and two lymphoma cells at a stage when involvement of the bloo d first occurred . with
small numbers- of cells. 717. The lymphomatous lymphohlasts now predominate and the total leucocyte co unt has risen above normal. In this field. as in the precedin g one , the twisted and co nvoluted nuclear o utlines of the T lymphoblasts are
apparent. 718. A fully leuka emic picture. The T lymphohlasts at this stage tend to lose their previousl y conspicuous nuclear convolution. and come to resemble the lympho-
blasts of primary, de no vo, T-A LL.
231
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719. A sec tion of bone marrow tr eph ine biopsy from a pati ent with co nvoluted T-celI lymphom a . prior to leukaemic progression. Th ere is a scatt ered diffuse infiltra te with lar ge ce lls. ofte n showing mark edl y twisted and co nvoluted nuclea r outlines , but with perhaps 30% of the whole cell populati on in this field composed of residu al no rmal haemopo iet ic tissue . 720. T he cells from the peripheral blood . du ring and after the emergence of the leu kaemic phase of convoluted T -cell lymph oma . sho w coarse PAS positivity of the lymp hoblastic patte rn. 721. Acid phosphatase reac tion sho ws strong localized pcsitivity in most cells.
722. Dual esterase reaction in the same cells shows negativity to BE but a few scatte red CE gra nules o nly. in the T lymphoblasts .
723
•
723-725 . Circulating lymphoma cells from a case of [ollicular lymphoma (mixed small and large Fee tumour , centro blastictcentrocy tic typ e],
725
723. Romanowsky sta in: a monocyte . two neu tro phils and seve n lymphoid cells of vari able cytology. includ ing a nucleolatcd ccntroblast. 724. PAS reacti on : variable positivit y in ce ntroblasts and ccm rocy tes. 725. Alk alin e phosph atase reacti on showing positivity in lympho ma cells - a most unu sual finding in the pe riphe ral blood . Th e seven neutrophils prese nt show positivity ranging from + to + + +.
233
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726
726-733 . Cytological ana cytochemical pr eparations from the peripheral blood buffy coat of a patient with circulating centroblostic lym phoma cells. 726 and 727. Romanowsky stains of ccntroblasts in the buffy coa t , accompa nied in 727 by two Iymphocytes and two neutrophil polymorphs. The distinctive cytological feat ures o f centroblasts as see n in smea r preparations are well shown ; large size , indented or cleaved nuclear outlines. moder ately dense nuclear chromatin, o nc to several, generally small, nucleoli, tending to be disposed at the nuclear membrane. grey rather than deeply basop hilic cytoplasm. and a nuclear-eytoplasmi c ratio ranging from high to medium. 728. S8 stain of these cells shows them to be negative . The two heavily sudanophilic cells in this field are neutrophil polymorphs, and the two with discrete scattered granules o f positi vity and co nspicuo us vacuo lation are monocytes. The negative cell to the right 01 the upper monocyte is prob ably a Iymphocyte , but all the remaining cells are centrobl asts.
729
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729. Acid phosphatase reaction on the same preparation revealsthat most cent ro blasts have no more tha n a few scatte red granules of positivity, without any paranuclear o r other parti cular localization . T he trio of cells at the top of this field are . fro m left to right , a moderately stro ngly positive monocyte. a T Iymph ocytc with a collection of paranucl ear gra nules. and a neu trophil polymorph with the usual scattering of positive granules. The rem aining cells are all centroblasts.
732
,
730 and 731. PAS sta ins for glycogeo on these cells show their reactions to be generally less stro ng than those see n in the ea rlier example of centroblastic PAS reaction (70S), here ranging from fine to moder ately coarse granular positivity. There is a nor mally reacting polymo rp h and a negative Iymph ocyte in 730. 733 732 and 733. T wo fields fro m a du al ester ase sta in on the same huffy coa t. Th e first (732) shows three CEpositive neutro phil pol ymorphs and a T Iymph ocyte with a do t of localized paranuclear react ion. but the remaining centroblasts are esse ntially negative with no more tha n a faint non -specific dustin g of mixed reactio n prod uct. In 733 the re are six negativel y reactin g centrablasts. six positive neutrophil s, a negati ve late normoblast. and , above it , a metamyelocyte with localized CE posi tivity to one side of the nucleus.
235
73-1
734-736. Sections of bone marrow trephine biopsies, from patients Wilh nodular [ollicular centre cell or cenuocytic-centrobtostic lymph oma and marrow inii itralion.
734 and 735. Low- and higher-power views, respectively. of a lymphom atous nodu le in the bo ne marrow pare nchyma . Th e nodu le occ upies most of the field in 7~ . The mixed cytology. with the larger centroblasts and smaller and more pachychro matic cem rocytes. is visible in 734. but can be sce n much mor e clearl y at the highe r magnification of 735, wher e the mo re leptochro matic. vesicular. and ofte n cleaved nuclei of the centroblasts, with their conspicuous perip herally disposed nucleoli. differ clearly from the denser and smalle r nuclei of the centrocytes. Occas ional no rmal marr ow elements are recognizabl e.
736 . A high-power view of a bone marrow treph ine biop sy section fro m another patien t with a more mark edly centrocytic follicular lymp homa. A minority of ce ntroblasts can be see n, bu t with a clear predominance of ccntrocytcs , with thc prese nce , in somc areas, of more mature cells with plasmacy toid features. including ecce ntric nuclei with cloc k-face chromatin marki ngs. This case illustrates the close relationship be tween ce nt rocytic-ce ntroblastic lympho mas and Iymphoplasmacyto id lymphom as. alt ho ugh the former arc mor e ofte n nod ular and the latter di ffuse .
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737-739. Sections of bone marrow trephine biopsies from patients with lymphocytic and Iymphocyticcentrocy ttc lymphohltlSana marrow mjJlrratlOn.
737 and 73S. Respectively low- and highe r-power views of a mixed nodul ar 3,Jld diffu.se lymp hocytic infiltrate . a bo rde r 01 less dcllsCiy mhltrated marrow rissue rem aini ng in the uppe r third of the field in 7 ~7 . which . is o the rwise lar gely occup ied hy a nodular foc us of lymp ho ma . In thts field. and mor e clea rly in 738. the namre of !b e infilt rating lymphom a cells is apparentas ma ture sma ll lymphoc ytcs. morphologically mdlstlO· ~is hab le from those found infiltrat ing the marrow In eLL.
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740
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740. A circul atin g lymph om a cell from lymphoblastic lymph om a , B-cell type , occasional lymphoma cells in the peripheral field also co ntains two Iymphocytes and a neutrophil.
a case of with only blood. The segmented
741. Marrow infiltra tion in the same case : the lympho ma cells show a more lymphoblastic appearance, with higher nuclear-cyto plasmic ratio . tha n those in the peri pheral blood . 742 and 743. Low- and higher-power views fro m a sect ion of bo ne marrow tre phine biopsy . taken fro m a patie nt with a B-Iymph oblastic lymphom a of Burk itt cell type . bo th showing almost complete replacem ent of the normal marrow elements by the lymphomatous infiltrate. The lymph oblasts are lar ge and pale-stainin g. with very lept ochrom atic nuclei mostly conta ining several co nsp icuo us small nucleoli. In bo th fields the re are to be seen macrophages containin g pha gocytosed ce llular materi al , similar to tho se giving the classical 'starry sky' appearance in histological sections of Burkitt 's lympho ma proper.
•
238
744
746
744-747. The first two figures are of Ro mano wsky stains and the latt er two of SB and PAS preparations. respec tively, from the bon e mar row of a child with Bur kitt's lymph om a. An appea rance comp arable with lympho matous or acute leu kaemic invasion , is. seen . This type of exte nsive mar row repl acemen t by tumour is unus ua l in Burkitt's lymphom a. T he mo rphology of the cells is unlik e that of typical leukaemic lymphoblasts; the nuclear chro mat in is coarser and more stranded , and most cells are PASnega tive. T he vacuola tion, charac te ristic of the tum our cells in this disease , is well shown, but similar vacuoles may so met imes be see n in acute leuk aem ias andthis feature is no t pathognomonic.
747
239
•
7 ~ 8-76 1. Preparations from typical H e L - leukuernic reticulo -endo theliosis (L R E). ln all these smears monocytes are conspicuous by their absence.
7..S . HCL. Leishm an stain of peripheral blood . Two neu trophils. a normal lyrnphocyte and four hairy cells (HC s). showing the typical eccentric nucle us with mode rate ly coarse chromatin . the pa le slate-blue cytoplasm. and the fine surface pro jections o r hai rs. A rodshaped nega tively staining inclusion in o ne of the HCs may represent a ribosom e-lamella (R-L) complex. 7~ 9 . HCL. Leishrnan stain of peripheral blood from ano the r case . A group of six HCs togethe r with one lymp hocyte a nd two segmen ted ncut roph ils. T he nuclear staining of the HCs sho ws the sponge-like or checke rboa rd pa ttern often see n .
750. HeL. Leishma n sta in of ano the r example. with an He showing the conspicuo us cyto plasmic vacuo lation which is often a fea ture of these ce lls. together with a nor mal lvmp hocytc. a neutrophil polymor ph and an irnmun ocyte o r plasm a ce ll. fro m a pat ient with HCL and a virus infection .
751
751
754
751. HCL. Lcishman stain of a bone marrow smea r. showing a cluster of HCs with the azurophilic inclusions so metimes present and seen. es pecially. after sho rtte rm culture .
752. Co nsec utive PAS stain o n the same field as in 751 .
The tinge and fine granu lar pos itivity are typical of HC, . 753 and 754. HC, in peripheral blood before and after a pe riod of 48 hou rs in culture . sho wing increase in cyto plasmic granularity and the occurrence of mitos is. 755. A further exa mple of HC, afte r 48 hours in culture. showing phagocytosis of red cells and bacteria.
755
n
756
756. HCL. SB reaction, showing normal positivity in two neutr ophils, a negative Iymphocyte and two negative HCs. 757. H CL. PA S reactio n, showing typ ical diffuse and granular positivity in HCs. A normally reacting neut rophil and a negative Iymph ocyte co mplete the field.
•
75H . HCL. Acid phosp hatase reactio n. Five polymorphs, four probable TI-' Iymphocytes and two HCs, all showing typical positivity. That in the HCs is tartrateresistant.
,
759
;
759. A further example of acid phosphatase staining in HCL , showing a neutrophil, two TI' cells and five HCs, several containing rod -like structures with positive staining, possibly representin g R-L complexes. Such inclusions are seen more commonly after acid phosphatase staining than in Rom anowsky preparations.
,
761
760. Dual esterase reaction in HCL. Two CE- positive nc ut ro phils. a TJ.L ce ll with localized BE po sitivity. and an He with gra nular positivit y tending to sho w a cresccntic localization at eithe r side of the nucleu s.
76 1. Alk aline phosphatase reaction in neutroph ils in HCL. Th e score is almos t invari abl y high as in this case whe re most neutrophils sho w + + + to + + + + pos itivity.
---
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762
•
762-776 . An example of atyp ical HCL (HC L variant), with high leucocytosis, presence of monocytes. normal LA P score and chronic course. 762. Rom anowsky stain. showing more central nuclei . mo re co nspicuo us nucleoli. and coarse r cyto plasmic projections than usual in HCs. 763. SB sta in. sho wing thr ee negative HCs. two positive neutrophil s and two monocytes with var iable scatte red positiv e granu les. 764. PAS sta in showing the usual HC pattern - quit e unlike that of. for exa mple. e l l o r PL L. 765. Acid phosphatase sho wing a mo re ma rkedly po lar d istribu tion than in typica l HCL. 766. Dual este rase in atypical HCL. Two CE-posi tive ncutrc phils. o ne heavily vacuolated and with only a few positive gra nules . two BE-positive vacuolated mon ocytes and three nucleolated HCs. one showing polar BE positivity .
767
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767-772 . Various sections of hone marrow trephin e biopsies from patients with Hef. ami marrow infiltration. Marrow involvem ent in HC L may he irregular and patchy, but where it occurs the lie distribut ion is diffu se rather than foc al.
•
767. A low-power view of a bo ne marrow sec tio n heavily infiltrated with B eL. Th e overall ccllular ity is around 70'Yo with substantial redu ction in the proport ion of fat spaces; the cell populati on ca n be seen to have two main compon ent s. HCs with relatively light-staining nuclei and ampl e cytoplasm . and erythroblasts with smalle r size and den sely stained nuclei. Onc o r two megakaryocytcs can be discerne d , but there is a notable abse nce of granulocytcs. in keeping with the mark ed pe riphe ral leucope nia almost invariably found in I-I C L.
•
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; A Giem sa-st ain ed section fro m the sam e biopsy as in 767. demonstrating more striking ly the wide spacing of HC nuclei and the co nde nsat ion o f peripheral cytomic mate rial. including hairy proce sses. at th e cell te rfaces, prod ucing the so-ca lled ' ha lo' effect. - 9. PAS reaction on a thin sectio n from the sa me pr pa ra tion as in 767 and 768. sho wing cyto plas mic itivity in the HCs. with a re cognizably mixed diffuse gra nular patte rn closely rese mbling that sec n in .-ear prepa rations (cf. 757) . There is a positive mast towards the to p centre .
,
•
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770
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• • 770 and 771. T hick and thin sectio ns. respective ly, from par affin- a nd plastic-em bedded bone ma rro w tre phin e biopsy prep ar at ion s taken from a pati en t with Be l d uring th e course o f remi ssion-induction treatment with alpha-in terferon . In bot h sect ions, cha racte ristic HCs are now in a minority, thoug h readily recog nizable; no rma l haemopoietic mar row elements predomina te, with erythroblasts most numerous bu t with megakar yocytes and eve n occas ional gran ulocytcs present. In 770 a tissue mast cell is see n at the lower right and a plasma ce ll at the upper left ncar a mitotic figure . In th is pai r of slides the thin section offers little additional info rma tio n . 772. A further example of a thin section from a marrow biopsy in HCL . this time from a patient with a co nco mita nt toxoplasma infection. giving rise to a marked immunoblastic reaction with numerous transformed lyrnphocytes in the pe riphe ral blood. and both lymph oplasrnacytoid and plasma cells in the bone marrow . In the field shown here these reactive immune cells predominate. with smaller nu mbe rs 'of scatte red HCs recognizable by thei r paler nuclei and larger size.
246
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• 77~7 75 . Sections ofbon e marrow trephine biopsy fro m patients with p eriph eral Tscell lymp ho m a and marrow infiltration . Th is type of lymphoma is m orphologically variable, composed of T cells ranging f rom sm all m ature /ymphocy tes to large im munoblasts, and classifiable as
of either low or high-grade severity. They show a high incidence of bone marrow involveme nt, mostly diffuse but occasionally with a randomly scattered foca l distri-
bution. The histological structure of infiltrates mimics that of the parent lymphoma , with an inflammatory element and prominent vascularity, difficult to distinguish f rom pleomo rphic Hodgkin 's disease or various reactive stales such as A I L , connective tissue diseases. or im m une deficiency syndrom es. including A IDS.
n 3. A very low-power view of a large focal infiltrate . • 'ith relative ly norm al mar row above the upper trabecular process. To the right. the margin of the focus is DOt sharp but me rges into a zone of diffuse infiltration. , 4. A high-power field fro m a seco nd case of polyIDOrp hic peripheral T-cell lymp ho ma , showing diffuse . tration with large lymphoma ce lls. so metimes . ucleated and superficially rese mbli ng Reedern berg ce lls. polymorphs with de nse twisted nuclei. aod elonga ted vascula r endothe lial cells. ep ithelioid . -te s and occas ional fibrob lasts. A PAS stai n o n the same biopsy material as in 774 out the PAS-pos itive vascular endo thelial ce lls scattered epithelioid histiocytes.
•
• 775
•
776
776-7 88. Circulating abnormal neap/astir T cells from cases of Sezary 's syndrome (SS) . This disorder is a variant of mycosis f ungoides (M F) with leukaemic involvement of blood and bone marrow , as well as the sk in infiltration and tile subcutaneous fo ci of rumour cells and reactive elements kno wn as Pautrier's microabscesses, fo und in AI F. The T cells of ss are usually CD3+. CD-I+. and have helper f unction , although occasionally CD3+. CD8+. suppressor ma rkers and fu nction may be fo und. 776. Rom anowsky stain: the conspicuous nuclear con volutions show n on electron microscopy (777) of
the same ce lls arc barely detecta ble as nuclear creasing by light microsco py. 777. El\1prepa ration from the same case The circulating Sezarv cells show a 'ce reb rifo rm' nuclea r section, with striking invaginntions. Glycogen gra nules are con spicuous in the cytoplasm, 778. PAS reaction: most cells in this case are negative, but occasionally fine o r moderat ely coarse gra nular positivity is present. Nucleoli are well show n in some of the neoplastic cells, as is the creased or cleft appearance of some nuclei . T he PAS reaction in Seza ry ce lls is quite variable, some cases having much mo re mar ked gra nular positivity.
2 ~8
77 9
779. Rom anowsky sta in o n a bu ffy coat prepar ation from pe riphe ral bloo d of anot her patien t with SS. showing six Scza ry cells with typical high nucle arcytoplasmic rati o and deepl y ind ent ed o r clef t nuclei.
78 1
more co nsp icuo us tha n in th e previou s case illustrat ed in 776 . One of these cells shows marked cyto plasmic baso philia. a fea ture present in only a small mino rity o f Sczary cells. The field in 779 also conta ins five normal lymphocytcs. an eosi no phil po lymo rph and 1\\'0 monocytcs .
7g0. 5 B stain on the sa me preparation as in 779. revealing sudanoph ilia in only one cell. an eosinophi l. while the rem aining mixture o f the larger Sezary ce lls and smaller Iymphocytcs sho ws negativ e reactio ns. Agai n. o nc of the Sczary cells shows de ep cytoplasmic baso philia. 1. PAS reaction o n the same buffy coat preparation sho ws coarse blocks and/o r variable gra nules of PAS · positive glycogen in most of the Sezary cells. togeth er with the usual dense positivit y in a neut roph il polymorph and a less stro ng . finely gra nular and diffuse . reac tio n in a myelocyte and a stab cell. Th e rem aining lympho. tes in this field a rc negat ive .
249
782
782 . Rom anowsky sta in on a buffy coat prep ar ati on from another patient with SS, showing a furth er exa mple o f the ran ge of cytological appearances in th is diso rder . Exce pt for th e single neu troph il po lymo rp h at the left , all the cells in this field are neop lastic Sezary cells. mostly with mor e basophilic cytoplasm than in the previous illustr atio ns. but again with a gene rally high nuclear-cytopl asmi c ra tio . The ce ntral ce ll has inde ntat ion s of the nuclear o utline and th e cell imm ed iately below it has a dar k line of nucl ear folding across the upper pa rt of the nucleus. 783 . The PAS reaction in this case agai n shows strong posltivity in the Sezary cells. with coarse granules and blocks in many of the m. There are four no rmally positive ne utrophil polymo rp hs and part of a fifth, and also a nega tive mon ocyte with folded cyto plasmic edge at bottom left and a wea kly pos itive myelocyte at the top right. 784 . Acid phosphatase reaction in this case sho ws moderately stro ng positivity in two monocytes and a few scattered granules in th e polymor ph s, while the predominating Sezary cells most ly show weak granular reactions wit h only occas iona lly the pa ra nuclea r disposition characterist ic of ma ny T cells . Thi s wea k sca ttered granule pattern witho ut par an uclear do ts is the picture most often encountered in SS. 785. This dual esterase stain in the sa me case sho ws no rm al reac tio ns in monocytes. neu tro phil s and an eosinophil, but nega tive reaction s in the Seza ry cells.
250
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.
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.
786
788
786. Anothe r variant of ma ligna nt ce ll cytology in SS. In this field , from a Rom an owsky-stain ed buffy coat smear. all th e ce lls, apa rt from a single stab cell above the ce ntre. are e ither no rma l Iymphocytcs or Sczary. The co ntras t in density of nuclear chromatin is striking, and in this case the deep infolding of the nuclear membrane can be better envisaged than in 782. perhaps because the blood sample was kept in anticoagulant for some hours before the buffy coat preparation was made . a procedure which tends to exaggerate nuclear membra ne irregularities . 787. A high-power view of two Seza ry cells from a fresh peripher al bloo d smear, show ing well-ma rked nuclear infoldin g but also the ex iste nce of multiple ma ll nucleoli, especially in th e lower cell. a feature which is no t usually cons picuous in Seza ry cells . 88. A lower-power view of the same peripheral blood sample as in 787. showi ng the usual finding in SS of a positive cell-s u rface me mbrane reaction in the neoplastic T cells to an immu nocytochemical A PAAP stain with the MAh CD4.
251
7
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789. A nest of mature plasma cells from the marrow o f a patient with cat-scratch disease, showing some reactive plasmacytosis. Th e vacuolation and tendency to buddin g and fragmentation o f the cytoplas m may ind icate en hanced activity. Th er e is a central macroph age nucleu s with filmy ill-defined surro unding cytoplasm. a Usha ped residual me gakary ocyte nucleu s with scattere d platelets bel ow it at th e lower left , and a fragmen t co ntai ning free iron at th e bo rde r o f th e plasma cell to the uppe r left o f th e gro up. 790 . A nothe r exa mple o f reactive mature plas ma cells in the bo ne mar row of a patient with rheumatoid art hr itis . wher e po lyclona l plasm acytosis is co mmo nly associated with th e usual hype rgammaglobul inaem ia . 79 1. A low-powe r view of a sec tion of bone ma rro w trephine biopsy from an elde rly pat ient with a chro nic infective process and reactive plasmacytosis. Overall cellularity is low. with man y fat spaces. b ut among the residual hae mopoietic ma rro w cells sca ttered plasma cells with their ecce ntric nuclei are freque ntly to be see n.
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792-797 . A series of paired fields from consecutively stained bo ne marrow sm t'ar prep arations to demonstrate the presence of free stainable iron in plasma cells. In each case the first illustration of the pair (792, 794 and 796) shows a Romano wsky -stained field, and the second (793, 795 and 797) the same field after decolorization and restaining by the Prussian blue reaction. Th e presence of free iron in plasma cells may be a result of transfer of this mat erial from macrophages (two macrophage nuclei afe present in 794 and 795) via cytoplasm ic bridges. rather than a consequence of direct phagocytic action . but it appears 10 be most com monly associated with alcoholism . although it may also be seen in infective hep atitis, as in tire present case. The iron has been sh own bv ultrastructural stud ies to be located in m em branebo und lysosomal vesicles and not in mitochondria. thus diffe ring fr om the erythroblastic accumulations in sideroblustic anaemia.
97
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798, A group of plasma cells with mo re immature features than those in 789 and 790, from a pati ent with mult iple myel om a . 799 . Variabilit y in the size . maturity and gene ra l staining characteristics of plasma cells, in the marrow of a patient with multiple myelom a . 800 . Bone marrow trephin e biopsy in myeloma . showing the predominance of neopl astic plasm a cells of va ria ble size and nuclear den sity. with co nside rab le cyto plasmic fragmentat ion . and' wit h eros ion and cellular infiltrati on of the tr abecular bo ne at o ne poi nt.
254
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803
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SOl. Circulati ng plas ma ce lls from a case of plasm a cell leukae mia with predomina ntly mat ure non -nucleolatcd
cells. Note absence ofrouleaux formation . 802. Acid phosphatase reaction in the same case: there is strong localized positivity, but considerably less inte nse th an usually scen in typical myelom a cells of bone marrow (see 825 and 826).
803. Dual esterase reaction : the same case shows only vcry weak BE positivity in the plasma cells in co mpa riso n with th e stro nger reaction for BE or CE or both enzymes common in typical myelo ma cells of bone marrow (see 827). 80·1. A re ma rkab le exa mple of inte rnuclear brid ging in a case of myelo ma . Most myeloma cells had two o r three linked nuclei.
255
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865. Plasma cells in myeloma. showing a remarkable tendency to cytoplasmic disruption. 806. A multinucleated plasma cell in myeloma.
807 and 808. The develop ment of flaming cells. The smooth eosinophil component which makes up the
'flaming' character in certain plasma cells is usually first seen at the cell periphery. It OrIC'" contrasts sharply with intense basophilia in the (emaining cytoplasm',
256
• •
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• 8 12
809. A pai r o f fully de veloped flaming cells. HIO and 811. Th e development o f thcsaurocytcs -Iar gc plasma cells with d ark . so mewha t pycnoti c nuclei and extensive fibrillary cytoplasm. some times having the appeara nce of division into compa rtments . These 'storage cells' usually have 'flaming' characteristics in their rema ining cyto plasm.
812 and 813. Large nuclea r inclusions. PAS-positive . occasio nally sce n in plasma cells in myeloma (a nd in o ther co nditions). The ir significance is un known ; they are less likely tha n cyto plasm ic inclusions to represent secre tio n or synt hesis prod ucts .
813
257
814
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814 a nd 815. Plasm a cells in myelom a , sho wing an accumu lation of sphe rica l inclu sion s, bluish in co lour . pro babl y represe nting an abnormal concent ration of immunog lob ulin precurso r. Ce lls may become full of the se bod ies (called Russell bo dies by some authorit ies) and arc then so me times referred to as Mo tt cells .
816 and 817. An other type of sphe rical inclusion . eosi no phil-sta ining and PAS-positive , found mu ch less often in smears of plasm a cells. Thi s type of inclusion is also given th e nam e Ru ssell body. perhap s with more historic al justifica tion. 818 . A n un usua l form of locul ar degene ration in the cyto plasm o f a pla sma cell from myel om a.
819. Azurop hilic rod s (rese mbling Aucr rods, but negative to SR . pe roxidas e and PA S sta ining) are not rare in plasma cells, but in th is unusual myelom atous case the majority of the plasm a cells co nta ined many such rods .
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820. Three myeloma cells, two with 'flaming' ce ll feat ur es a nd th e third with mu ltipl e small vacuoles ofte n co nta ining azurophilic materia l. po ssibly irregu lar deposits of defec tive paraprotein within autop hagic vacuoles. 821 and 822 . An even mor e unu su al case of myeloma . whe re nearl y all the plasma cells we re d istor ted by lar ge crystalline inclusion s . having so me resem blan ce to
Charcot-Leyden crystals . and presumably represen ting the prod uct of diso rde re d synthe tic ac tivity in the ce lls. T he c~ 1I cytoplasm sho wed wea k PA S positivity. bu t (he inclu sions were negat ive (822) .
823 and 824. PAS reac tions in plasma cells fro m case s of mu ltiple myelom a. Reaction s ra nge fro m nega tive to we akly positive . with occa siona l gran ules against a we a ke r diffu se ba ckground of faint positive tinge . T he sphe rical bluish incl usions illustrated in 814 a nd 815 a re PAS-negat ive .
H2~
259
825
825. The o ne striking cytoche mical characterist ic of plasma ce lls. no rmal and pat hologi ca l. is their co nsistently strong ad d phosp hatase reaction . illustrated in th is general view of a bone marrow smea r from a P..uie nr with myelo ma . 826. A seco nd exam ple of acid phosphatase reacti on in myelom a cells . T he mo re sharply parti culate deposit arises from the use of naphthol AS-S I phosph oric acid as substrate , compared with u-naphthyl acid phosphate . as used for 825.
.
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827. Dual este rase reaction in a marrow smear from a pat ient with myelom a . Th e cells contain scatte red CE pcsi ti vny in this case . but no BE as is so me time!'> fo und . ."
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829
•
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Section of hone marrow trephine biopsy from a t wit h myeloma . s ho wing typica lly variable _)', with differences in ce ll size a nd in nuclear tin co nde nsa tio n. and with con spicuous pale red Krawc lear inclusions in sever al o f th e myelom a ce lls
nerally more primitive nuclear patt ern s. Th ese pr umably th e sa me t ype of inclusion as sho wn in : _ 813. Anothe r typica l ex ample of myel oma ce ll cy to logy
in histo logical sections o f bone marr ow. Th ere sional hinucle ated cells and som e var iabil ity in ty. bu t in ge neral the cell nuclei arc mor e den sely tic wit h more co nspicuo us clock-face marki ngs III
28 .
Iyelomato us prolif eration is frequ entl y nodul ar ribu tio n. and this section from ano the r bo ne tr ephine biopsy sho ws the mar gin of a foca l mye loma cell gro wth. at the uppe r righ t. .........._ resid ual nor mal haem opo ietic mar row. with stic. gra nulocytic an d megak aryocytic co rnall visible . at the lower left . Ov er all ccllulariry ar ea is relative ly low . with num erous fat spaces.
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831-833. Transformati on of myeloma to AML. 831. Myeloma marrow with chiefly mature welldifferentiat ed myeloma ce lls. but with occasion al blast cells of myc lomonocytic cytology. 832. A higher-pow er view to co ntrast the ra nge o f myeloma cell morph ology with two nucleolated myeloid hlast cells. R33. Marrow smea r fro m th e sa me patien t at a later stage of transformation, sho wing predo minance of acute myclo monocytic leuk aemi a (A MML) blast cells with scanty residu al plasma ce lls and ra re late r grenu locyt e stages .
262
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A bo ne mar row smear from a patient with macrolinac mia. Lymphocy tcs , mostly with disrupting or mal cytoplasm, pr edomin at e. Th ere is also a tissue ce ll prese nt.
.0: . Another exa mple o f bon e marrow cyto logy in 8IICfOglo bulinaemia. Th e red ce lls show cons picuo us ux formatio n, the lymphocytcs predom inate and scanty cytoplasm. and there is a plasma cell prese nt ell as a ve ry den sely gra nu lar tissue mast cell. An alive name for this last cell. ' baso phil ball cell". here be descriptively ap t.
Another fie ld from the sa me preparation . wit h lym phocytcs but a disrupted tissue mast cel l.
The neopJastic cells in this field from the bo ne _ ....,.. of another case sho w occasional ' Iymphocytoid' features. Phagocytic RE cells arc also _"'PKu O US.
263
83H. PAS stain in macroglobulinaemia: there is scattered granular PAS positivity in the cytoplasm of two R E cells. hut the lymphocytes and plasmacytoid cells in this disease show little or no reaction .
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839. Dual esterase stain in macroglobulin aemia . showing several tissue mast cells with coa rse CE positivity in a marrow fleck. The neoplastic lymphoi d cells a nd RE cells in the fleck show only weak scatte red gra nular pos itivity to both BE and CE . 840. Free-iron stain on bone marr ow smear in macroglobulinaemia. The e xcessive amount of free iron. both scatte red and in macroph ages. is evide nt. 841. Romanowsky stain of marrow from macroglobulinaemia . showing a macroph age heavily laden
with iron . 842. A similar cell to that in 8-11, stained with the Prussian blue stain for free iron .
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R43. A high- powe r view of a sectio n of hone marr ow trep hine hiopsy from a patient with Iymphoplasmacytoid immunocyto ma and macroglo bulin aemia. providi ng a clea r demonstration of the mixed lymphocytic. plasmaeyrie. and inte rmediate cytology of the predom inant infiltrating cells. Several binucleated plasma ce lls can he see n. 844. A smear of bone marrow aspir ate from the same case as in 843. sho wing the much superior cyto logical detail of such pre parations. There is a central norm oblast. but all the re maining cells in this field are of the Iymphoplasmacytoid ser ies . with a good seq ue nce of intermediate developmental appearances. from Iymphocyte to plasma ce ll. 845 A low po wer view of a section of bone marrow trephine biopsy from a case of Waldenstrom 's macroglobulinaemia with lymph nod e invo lvement . where lymph node biopsy reveal ed the histological features of Iymphoplasrnacytoid immunocytom a. The field sho ws the edge of a neoplastic nodule . with a marked 'cosine philic' reaction in the immedia tely surrounding residual marrow. here seen at the up per part of the field. Th e tumo ur cells appear to have gene rally un iform cytology at lo w po wer.
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846 A higher power view of the same specime n as in 845. Th e infiltratin g tumour cells are now see n to range fro m the predo mina nt mature- looking lymphocytic. ce ntrocytic or plasmacytoid cells. to much less matu re larger cells with pale-staining nuclei and occasional nucleo li. 847 and 848. Thin sections fro m plastic-embedded trephine biopsy mat erial taken from the bon e mar ro w of another patie nt with macroglo bulin aemia . The lowpowe r view (847) illustrates diffuse invo lvement of residual marrow with lymp hoplasmacytoid cells and furthe r 'eosinophilic' mast cells. and also a conspicuo us scatte red background deposi t of amo rphous eos ino phil deposit. probably represe nting amyloid mat erial. T he higher magnificatio n of 848 reveals qu ite clea rly the Iympho plasmacytoid nat ure of the infiltrate and the presen ce of various normal myelo id precursors, and a lso confirms the prese nce of num erous tissue mast cells.
•
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849
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849-851. Sections from a hone marro w trephine biopsy tak e n from anot her patient with macro globulinaemia . 1149 sta ined by H&E . and 850 and 851 by the PAS reaction to show various examples of Dut chcr body formatio n in the neapl astic Iyrnphcplasmacyto id cells. Th ese are PAS-positive nuclear inclusion bod ies prohab ly derived from cyto plasmic im ·aginations . Similar. but ge nera lly much smaller. int ra nuclear inclusions occur in myeloma and less often in reactive plasma cells. but the lar ge inclusio ns know n as Dutcher bod ies arc see n especially in macroglobulin aemi a. where they may be so lar ge . as in 849 , as to ca use fragment ati on of the nucleus. A PAS-positive body stretching the nuclear membrane and occupying most of the cell is shown in 850. while in 851 a who le se ries of these bodies , at vario us stages of deve lopm ent . appears in the lower pa rt of the field . Th ere is a single PAS · positive polymorph near the top.
H51
Part 4 Miscellaneous cells from bone marrow or blood smears , reticulo-endothelial cells , osteoclasts and osteoblasts , foreign cells and parasites
Reticulo-endoth elial ( RE) cells (re ticulum ce lls. histiocytes. macrophagcs) are co mmon in the bo ne mar row and have bee n illustr ated several times previously in this boo k. Th ey take up foreign particl es. free iro n. fat globules, specific gra nules from disrupted granulocytes, and o the r cell fragments. and therefore ofte n co ntai n pha gocytosed mat er ial. Their cyto plasm appea rs fragile and is readily broken up in the smea ring process o r str etched out between neighbourin g cells - so that the cytoplasmic outlines may be difficult to recognize. T hey may co ntain various inclusions - sea-blue histiocyte material, pscudo-Gaucher cell bire fringent lipid or blue crystals and gre y-green crystals. In ce rta in lipid sto rage disea ses the y appear grossly swollen with ahno rmal fibrillary or globular deposits of lipids. The nature of this lipid inclusion materi al in the two main forms of lipidosis. Ga ucher's disease and NiemannPick's disease . is illustr at ed in this section and discussed in the acco mpanying captions. Glycogen stor age disease is also associa ted with the accumulatio n of inclusion materi al. parti cularl y in rnacrophages. but 10 a lesser exte nt in man y other hae mic ce lls. In chro nic infective or reactive states involving the bone marrow. rangin g from tuberculosis to sarco idosis. macrophage proliferation may lead to forei gn-bod y giant-ce ll (or Langhans' ce ll) forma tion . with appea rances similar to those encountered in the lymph nod e imprints illustrated in 1098 and 1099. Anothe r group of gra nuloma to us states frequ en tly involving the bon e marrow a rc those of the ' histiocytosis X: family. including Hand-Sch uller-Chri snnu syndrome. eosi nop hilic gran uloma and l.cttcrc r-Siwc disease. A ll arc thou ght to involve proliferation of Langerhans' cells. pro minent in the skin but probably
arising in the bon e marrow. and wit h mixed cytol ogical features of macr op hagcs and dend ritic RE cells. The Leishman-D ono van bodies . the protozoal par asites of Leishmani asis. appea r most prominently in RE macrophagcs. Malignant histioc ytosis is a ra re neopla stic state of the RE cell and even rarer is histiocytic leuk aemia. Examples of each ar e illustrated in this sectio n. Apart fro m RE cells . most of the other ce lls illustrated here ar c less commonly encountered. hut they have highly cha racteristic cyto logical features. and o nce these features are appreciated the cells arc unlikely to be co nfused with normal or abn ormal varian ts of more common cell lines in the bone marrow or blood. A non-malignant form of histioc ytic proliferation with enhanced phagoc ytosis. arising in var iou s oppo rt unistic virus infection s. especially in immun ocomp rom ised subjects . is virus -associated haem ophagocytic synd rome (VAHS); examples of this co ndi tion in trephine and aspira tio n marrow biopsies are illust rated here. Several different examples of tum o ur cells invadin g the marrow a re shown. Alth ou gh an isolated tumour cell may rarel y be iden tified as such in the abse nce of more typical cell clumps, the feature which allows identification in most instances is the occ urrence of ce ll nest s. frequently partially syncytial. of cells not belon ging to any hae mic serie s. The identifi cation cannot often go beyond ' metastasizing tumour ce lls', but comparison with the histology. cytology and cytochemistry of the range of illustrative metastascs shown in %(~I024 may provide a suggestive indicat ion of likely origin . Apart from neuroblastoma. medullobl astoma and chemodectoma all relatively rare tum ours with characteristic cytology found in yo ung people the meta sta ses most often found in marrow aspirat es o r trephin e biop sies from o lder -c
269
patient s. usually with lcucoery throblastic anaemia. are from primary malignant tumou rs of lung. breast, kidney or thyroid. and examples of all these are shown here. Certain of the more common parasites which may be seen in smears of blood or bone marrow are also illustrated. They are found mostly in tropical or sub-
tropical zones, but their appe arance should be recognized by all haematologists: although what may actually be seen unde r the microscope (as in the photo graphs her e) does not always provide detail comparahle with the diagrams and painti ngs in parasitology texts.
152
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852 . A ph agocytic re ticulo-e ndot helial ( RE) ce ll co nta ining variou s part icles of cellu lar deb ris. These cells ar c see n o nly rarely in the peripheral blood. altho ugh they may occas iona lly be found in buffy coat smears in some chro nic infective sta tes, as in the mo nocytemacrophage seq ue nce in 855-857 . but th ey are common in bo ne marr ow smea rs. as here. where they are gene ra lly to be found towar ds th e tail of the smear or in marrow flecks. Their th in flattened lep tochromatic nuclei, with fine chromat in markings and usually inconspicuo us nucleoli. and thei r extensive fragile cytoplasm. usua lly with inde finite mar gins. as shown here . a rc characteristic. 853 . T his is prohahly an RE cell with phagocytosed eos inophil granules from a d isrupted myelocyte . T he alte rna tive poss ihility is that it represents a pa rtia lly smeared degen er at ing eos inophil myelocyte . 854 . A gro up of R E cells with poorly o utlined filmy cytoplasm, from the bone ma rrow in a case of pernicious anaemia .
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855. A gro up of three cells of the monocyte-macrophage system in buffy coa t of peri pheral blood . from a patie nt with bacteria l endocarditis. The middle cell is clearly a macrop hage. whe reas the contiguous mononuclear cells are still of vacuola ted monocytic cyto logy. 856. A similar macroph age in the buffy coat of this patie nt, stained to show the free-iro n content.
857. Another example of the monocyte-macroph age seque nce in peri pheral blood . The re are Iymphocytes at top and boitom of th is field. a nd six neut roph il polymorph s. with three cells showing s t agc~ in the monocyte to macro phage tra nsformation , all having phagocytic vacuoles and the uppermost showing the most macrophagic cytology .
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859. SB stai n: strong positi vity. probably surroundi ng phagoc ytose d materi al . including free-iron pa rticles . Th ere a re scatte red S8 posit ive gra nules in a mon ocyte . 860. PAS reaction: three RE ce lls. on e with scatte red PAS -positive granules and the other two with weak PAS reaction but chiefly scattered free-iron particles spreading across the field diagonally.
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862. Acid phospha tase reaction : three strongly positive RE cells. contai ning phagocy tosed cell remnant s and large particl es of free iron heavily coated with acid phospha tase . 863. Dual esterase reaction: the phagocytic RE cell shows the typica l very strongly positive BE reaction .
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1167
a case of congenital dyscrythropoietic anaem ia (CDA) type I. R E cells co ntaining such blue crystals. and
pseudo-Ga ucher cells. RE cells with crumpled swollen cytoplasm. are found not uncommonly in C DA .
R65 and 866. Increased visibility a nd clear birefringence
of these crystals, with reversal of refringence on 9
867 and 868. A not he r Ga uchcr-like cell with d uplicate unde r polarized light showing refringence. Th is specimen was from a case of CML, a co nd itio n in which the pseudo-Gauche r cell phenomen on is also not infrequent. 868
275
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870 and 871. An SBH. stained consecutively by Lcishma n and free-iron stain s. to demonst rate that the gra nular material is chiefly negative for iro n. 872 and 873. An iron-laden macroph age with som e SC3blue material similarly stained consecutively for CCJrn parison. In this case much of the heavier gra nularity co nta ins free iron .
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276
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874. A, thin section of marrow trephine biopsy from a case of MDS with numerous pscudo-Gaucher cells. histiocytes showing distended granular cytoplasm . A ltho ugh the H& E stain does not furt her identify the nat ure of the gra nules . these are the same cells as in 875 and 876 . where the gra nules of man y of them are shown to be sea-blue by the use of Rom anowsk y staining.
877
•
875 and 876. Romanowsky-stained bon e mar row sme a rs from the same case as illustrat ed in 874 , sho wing SBHs . with neighbouring ea rly myeloid cells having 'h e defective gra nularity co mmonly found in the myelodysplastic sta tes. In 875 there are three . and in 876 five. histiocyte or macrophage nuclei visible in the respective sea-blue cell nests. 878
877 and 878. Another example of a pseudo-Gauchcr cell. in this case from the bone mar row of a child with AML. emerging into remission o n che motherapy . Th e appearance of the striated and chunky cyto plas mic material is see n un der no rma l microscopic illumin at io n and the n under polari zed light. Th e inclusion mat erial is stro ngly birefringent.
277
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879. G rey-gree n crystal s in a disrupte d RE cell. Th ese inclusions occur chiefly in myeloid leuk acm ias and arc not biref ringent. 880. Anot her exa mple of the non -birefrin gent type of 'grey-green crystal in a macroph age from the bone marrow of a pat ient with A ML. Th e flat nucleu s and spread ing cytoplasm of the macro phage are typical. and the ce ll conta ins othe r phagocytosed mater ial , probably chiefly iron. in addition to the grey-green crysta l. 881. A genuine Gauche r cell in the bone marrow of a patie nt with Ga ucher's disease , showing typical coa rse o nio n-skin lipid inclusion mat erial.
278
882
I
882. Two further Ga ucher cells in the bone marrow one typical cell and one heavily vacuolated. 883. Less typical Ga uche r cells from the same specimen . The cytoplasm is mor e granular and less fibrillary. 884 and 885. Ga ucher cells under polarized light: the birefringence is shown in the same cells with the polarize r turned to a 9()O angle .
885
886-889 . Marrow trephine sectio ns and aspirate smear fro m ano ther patient with G auc her's disease . In the lowpo wer field in 886, the nodul ar ap peara nce results from the pe rsistence of normal hae mopoieti c marrow , with little infiltrat ion in the vicinity of the bony trabeculum at the upper left. but elsewhere the Gaucher cell infiltrate was very extensive in this case , Th e characte ristic histo logical appeara nce of these cells in marrow sections stained with H&E is well sho wn in the higher-power view of 887, with their ample and swollen cyto plasm. diste nded with fibrill ar mate rial. In the reticulin silver sta in of 888 a scatt erin g of interl acing ret iculin fibrils, somewhat increased ove r normal. is revea led. while the accumulated cytoplasmic materi al has a mo re gran ular appe arance in this stain a nd there is eve n a suggestion of weak argyrophilia in the cytoplasm of some hlstioc ytcs. Th e smear prepa ratio n in 889, with the usu al Romanowsky stain , shows the same Gaucher cells as in the preced ing sections. here with a mixed fibrillary and granular or foa my appearance. T he striking and highly charac teristic appea ra nce of Gaucher cells is d ue to the accumulation within expanded Iysosomes o f the lipid glucosy l ccramide. resulting from deficiency of the enzyme glucocc rebrosidasc .
890
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891
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890. R E cells laden with foamy deposits of abnormal lipid iri the hon e marr ow from a patient with NicmannPick disea se. Th e lipid in these cells is sphi ngomyelin. 891. Abnorm al cells. probably RE macroph ages. with variable reticulated cytoplas mic conte nt of abnormal stor age material. fro m the marrow of a pati ent with a
'histiocytosis' of the eosinophil granuloma-HandSchuller-Christian disease group . These granul om atous conditions. including also Letterer-Si we disease. are someti mes grouped toget her under the name ' Histiocytosis X' . They appear to share an origin from Langerhans' cells which prob ably arise in the bone marrow, typically express CO l and HLA -DR antige ns. and which arc characterized by a mixture of feat ures of macrophages and immun o-stimulator y de ndritic ce lls.
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893 and 894. Lo w- and higher-power views. respectively . of another example of fihrosing granulo ma with eosinop hilia in a section of bone marrow trephine biopsy, from a patient with Hodgkin's disease (H D). The granulo mas were high ly localized in the otherwise normal marrow shown a t th e lower left part of the field in 893. Th e histiocy tic prolifer at ion and the eosin oph ilic rea ctio n arc clearl y eviden t in the high-power field of 894. Granulo mas of this so rt arc not diagnostic of bone marrow involvem ent - and thus of Stage IV d isease - but are suggestive. 895. A nothe r example of histiocytic proliferation in the ho ne marrow. with diffuse infiltr ation througho ut the who le section. from a pati ent with a chronic myelod ysplastic state. with refracto ry anaemi a , some incre ase in blasts and persiste nt mod eratel y severe thr ombocytepenia . The histiocy tes appear swollen . with distende d cytoplas m,
282
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896-898. Furt her fields from the same tr eph ine biops y of hon e marrow as shown in 895 , with two high-power ileitis from an II& E sta in and o ne from a G iemsa-stai ned prepa ration. Th e histiocyte cyto plasm her e shows so me granularity and there is evide nce of phagocytic activity. especially in 897. where there arc also sever al mult inucleated hisriocytes. In the Gicmsa stain a few of the histiocy tes show a suggest ion of sea-blue inclusio n materi al. As scc n previo usly. this appearance is not co nfined to a single spec ific lysosom al enzyme deficiency as in the ma in her editar y lipid sto rage diseases. but appears to occur most often as a manifestation of myelodysplasia and perhaps accompanying increased ce ll dest ruct ion invo lving especially the gra nulocyte and th rom bocyte lines .
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glycogen storage disease. type 2. Th is is another example of a lysosom al sto rage disease arising from the heredit ary defici ency of a single enzyme. in this instance.
alpha-La-glucosidase . There are commonly vacuolcs prese nt in pe riphera l blood lyrnphocyres in this disease. but similar vacuo lcs occ ur in many ot her disorde rs and the y arc o nly d iagnosticall y helpful in directing atte ntion to the need for o ther cytoc hemical and biochemical investigations and bo ne marro w st udies . In this disease. the PAS reaction gives stro ng pos itivity of vacuolcs and cytoplasmic gra nules of a very cha racte ristic kind . In 899 a Rom anowsky sta in of a bo ne ma rro w fleck shows coarse semi-vac uo lated chu nks of inclusion material in a macrophage. while in 900 a PAS stain on the same cell reveals typically st rong positivity of the chunky inclusions of glycogen-contai ning material. A more crowded field . including glycogen- laden megakaryocyt cs . is illustrated in 901. also a PAS stain. where the megakaryoc ytes have co nspicuous glycogen inclusion bodies. The Iymphocyte with coarse PA S positivity seen in 902 is from a buffy coat preparation . which first raised the possibil ity of glycoge n storage disease as a diagnosis in this case . In 903 and 9()..1. both from the marrow aspirate , the intensely positive glycogen storage material is shown in a neutrophil polymorph and in an eosinoph il.
284
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905
906
907
905-921. Cytology and cytochemistry of bon e marrow smears· from very uncommon cases of histiocytic leuk aemia or lcukuemic histiocytosis. The cells involved sho w many fea tures ofactivated macrop huges. including large size and a m ark ed tendency to sp read over the slide surface, very conspicuous forma tion of cytoplasmic pinocytotic vacuoles and peripheral pseudopodia , with an obvious intense phagocytic activity . Cytochemical/y, the reactions differ somewhat fro m thOJC of normal marrow macrophages, with general sudanophobia, un· expcctedty strong PA S staining. weak acid and alkaline phosphatases, and almost negative butyrate esterase. These pa tterns may reflect a neoplastic functional defect or, possibly, enzy me exhaustion following increased phagocytic activity .
908
905. Ma rro w cells fro m an un usual case of histiocytic leukae mia , sho wing gene ral cyto logical similarities to the ce lls de picted in 891. Phagocytic inclusions are conspicuous.
909
906and 907. Further field s fro m the marrow of the same case of histiocytic leuk aemia showing cells of primitive cyt ology. but with scattered vacuo les, occasional multiple nuclei and freq uent giant nucleoli. 908 and 909. A nother example of this unus ual type of histiocytic leukaemia with blast cells again showing very large size. around 30-40 microns in diameter, irregu lar and so metimes fragmenting cytop lasmic borders, and coarse vacuo lation with occasio nal erythrophagocytosis.
91
910
912
910 and 9 11. SB reaction shows the se primitive cells to be negative in bo th cases . Th er e is a posit ive neutrophil in eac h of the illustr ate d fields . 912 and 913. Th e PAS reaction shows rem ark ahl y stro ng positivit y in virtually eve ry cell in eac h case . It is clea r that man y. alth ough not all. of the vacuo les co ntain glycogen. while the inte nsity of the stai n is emphas ized by the relatively weak . tho ugh normal , reac tion in the neutrophil in 913.
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916 and 917. Du al es te rase reaction in smear s fro m eac h o f the two cases o f histiocytic leukaem ia illustrated o n the pre ceding page. Th e blast ce lls are almos t entire ly nega tive . with no mo re th an a very weak tinge of buty rate esterase (BE) positivity and a few fine chloroaceta le esterase (Cfi) gra nules in 916. Both fields co ntai n norm ally reacting Cfi-pos itive neu tr oph ils and Bfi-po sitive rnacrophages.
920
91S-920. Free- iro n stains showing a po sitive reaction in some of the primitive histiocytic cells . The posirivity is likely to be the result of ingested erythroid cells, and it is noteworthy th at the single Fe-posi tive blast cell amo ng eight which ar e ne gative in 920 also co ntains other cellular debris and has clearl y bee n especially active in phagocytosis. 921 921. Alkaline phos pha tase cytochemistry o n a hone marrow smear from the seco nd of the two cases shows a weak tinge of positivity in seven blast ce lls. a co nside rably st ro nge r reaction in an eighth . and a neutrophil po lymo rph with mod erate cyto plas mic reactio n . Again . the obvious phagocy tic activity of the mo re strongly posi tive blast ce ll mak es it see m likely th at at least part of the alkali ne phosph atase activ ity shown in the histiocytic blast cells is seco ndary to ingestion of positive neutrophil cyto plasm. although part ma y be native to th ese unusual leukae mic cells.
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9U . A furth er ex ampl e of increased phagocy tic activity in malignant hist iocytosis. Th e whole clu mp of er yth roblasts. showing faint diffuse PAS positivit y. is within the cytoplasm of a spread RE cell with nucleus to the left of the clump .
288
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925-927. A se ries of fields at gene ra lly high magnifications, from thin section s of plastic-embedd ed trephine hiop sy mat erial from anot he r patient with malignant hist iocytosis. In 925 the exte nsive accumulation of haem oside rin in many histiocytes is especially noticeable; in the field chosen in 926. the re are again many haernosiderin-laden histiocytcs. but the very considerable infiltrate of primitive mon ocyte-macro phage histiocytes. with palely sta ining leptochr omat ic nuclei and conspicuo us nucleoli. is also eve n mor e clea rly evide nt than in 925. All these features ar e visible in the still higher-power view of 927. whe re the re is also an exce ptionally dr am ati c example of mu ltip le phagocytos is of red cells. Malignan t histiocytosis ma y somelimes be d ifficult to diffe re ntiate from virus-associated hacmophagocytic syndrome (928-939). especially when . as in the prese nt case . ther e is ex te nsive haemoph ago cytos is by more mature histiocytcs. eithe r deri ved fro m less differ enti at ed precursors or perhaps devel oping as a reactive co mpo ne nt . but the presence o f num erous primitive ce lls stro ngly suggests the fo rme r malignan t sta te .
289
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290
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ingestion of platelets. In 933 the macrop hage contains ma ny inges ted red cells and platelets. while the macro-phages in 9.,1.4 and 935 show phagocytosis of two early normoblasts and eight later normobl asts. res pective ly. T his type of gross and indi scriminat e ph agocytic activity is more o fte n see n in VA HS than in malignant histiocytosis. 936 and 937. Further illustr ation s of massive phage cytos is by marrow macrophagcs in a pati ent with refractory anaemia with excess blasts (RAE B) and immune supp ressio n secondary to chemotherapy. later develo ping VAH S following an opport unistic viral infectio n. The Rom an owsky stai n in 936 sho ws a cell with gross accu mulation o f gra nulocyte de bris. includ ing dark-staining nuclear re mna nts and bo th ne utrophil and eosi no phil gran ules . To the left a re several somewhat dys plastic myelocytes, a lymphocyte and an ea rly eryt hroblast. In 937 the S8 stai n o n a similar cell gro up reveals the strong sud anophilia of the ingested polymorph granules.
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cursors. The dual esterase reaction shown in 939 is unu sual , in that the mass of ingested po lymo rp hs appea rs predominan tly BE-positive rather tha n having the normal C E positivity sho wn by gra nulocytes . as man ifest for example in the ne ighbouring myelocytes in this field . Some residu al CE reac tion ca n still be made o ut. however. and it see ms likely that this e nzyme is bei ng ove rridden by high activity o f the endoge no us BE of the engulfing macrophage, 9~O
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and ~1. Low- and higher-power fields. respectively. from a trephine biopsy o f bone marr ow tak en from the same patient at a later stage o f disease . The specime n now sho ws an oedematous marrow stro ma with prolifer ati on o f fibrobl asts and development of fibrosis. perhaps seco nda ry to the earlier chemotherapy. but also with persiste nt mature cell histiocyt ic increase and the eme rge nce of some Iymphoplasmacytoi d reaction to the co nti nuing intrac table viral infection.
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952
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953 and 954. Examples of tissue mast cells in bone marro w smears. These cells have been illustra ted earlier in macrog lobulinaemia. where they are usually to be seen , but they also occur in small numbers in normal marrow and may show an increase in aplastic and hypoplastic stales. They differ from basophil polymorphs in their lack of nuclear lobulation and generally larger size. and in their absence from normal peripheral blood and freq uent presence in subc uta neous tissue . T hey do ap pea r to have their origi n in the bo ne marr ow. howeve r. and may he involved in a malignant mastocytosis which may be fran kly leu kacmi c. Like baso phils. they show the metachromatic staining of their granules with toluidine blue produced by acid mucopolysaccharides. but this reflects their high content of heparin sulphate whereas the basophil reaction is due chiefly to chondroitin sulphate. 955. PAS react ion shows st ro ng positivity in a mast cell. T his reaction gene rally resem bles that of basophil polymorphs. although it is less coa rsely gran ular . 956. Dual esterase reaction showing strong chloroacetate esterase positivity in a mast cell . The reaction here diners from that of basophtls. which is essentially negative for all the cytochemically demonstrable este rascs.
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957-961. Cyto logy and cytoc hemistry of bone marrow as pira te prep ara tion s fro m a patien t with mas t cell leuk aemi a. As ca n be see n in these illustrations, the neo plasric mast cells te nd to have relatively higher nuclea r-cytoplasmic ratio than normal mast cells . with often less de nse gra nularity and a patch y disposi tion of gra nules . The nucl eu s is usually round , but may show a degree of indentation. as in some ce lls in 959 and 961, while the cytoplasmic ou tline may be elongated and eve n spind le-shaped . as in 957 and 960. There is a particul ar tenden cy for malignant mast cells to occu r in clumps and in marrow flecks. where they may be mixed with fibro blasts, e rythrcblasts . mcga karyocytcs and gran ulocytcs, and may be difficult to recognize except at the edges . as in 958. The ce lls may show a rneta chro ma tic staining reaction no t o nly with to luidine blue but also with SR, as see n in 959 , wher e the da rk red co lour o f the mast cell gra nules co ntrasts with the black stai n o f the neutroph il myelocyte at the right and of a few sca tte red ne utrophil gra nules. The d ual este rase reaction of 960 shows stro ng C E pos jtivity, while the PA S react ion in 961 reveals a weak tinge of positi vity in most mast ce lls, with peripher al gra nular positivit y in ma ny. Of all the cytoc hemical reaction s. the most help ful in disti ngu ishing neopl astic mast cells fro m the basophils they may resemble is the este rase reaction , wher e mas t cells are stro ngly positi ve for chloroacctatc (a nd also aminoca proate) este rase , . for which bascphils a re nega tive .
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• 962-966. Examples of vascular endothelial cell clump s in the peripheral blood . The very regular nuclear structure and size and the tendency for these cells to occur in sheets or streaks along the dire ction of spreading of the film assist in recognition. Th ese ce lls arc for eign to blood, and are lifted from the intima of the vein during insertion o r withdrawa l of the needle used for collecting the blood sample. Th e nuclei in 965 both sho w conspicuous Ban bodies. indicating the presence of the inactive X-chromosom al materi al of no rmal female cells . In 966 can be seen alkaline phosph atase pos itivity in a strand of vascular endothelium. cross ing a field of negative immature marrow cells.
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9S0 and 981. Examples from two differ ent subjec ts , of ne uro blasto ma cell nest s in th e bon e marrow . T he cells have a resemblance individually to Iymphobl asts o f ac ute leukaem ia . but frequentl y sho w a whorled ar ra nge me nt in small clumps, as illustrat ed her e. 9H2 . PA S reaction on a clump of neu robl astom a ce lls.
showing a negati ve re act io n, co ntrasting with the usual co arse positivity enco untered in a pro portion of lymphoblas ts in ALL.
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983-987 . Furth er cytological and cytoche mical features of neur obla stoma cells as seen in bone marrow aspirat es. Th e high-power view of a Romanowsky-stained smea r in 983 reveals a clump of neuroblastom a cells with characte ristic fine nuclear chro matin co nta ining severa l dense hyper chromatic spo ts, but o nly small and sometimes poo rly distinguishable nucleoli. Th e cytoplasm of these cells is ofte n frag ile and easi ly disrupted . so that the cells may appear as naked nuclei. In 984 the acid phosph at ase reaction in a neuroblasto ma cell nest shows the tumo ur ce lls to have o nly very weak positivi ty. without any sign of the par anuclear concentration seen in T Iympho blasts. Mixed cell clumps with ce ntral neuroblastom a cells and peri pheral haem ic cells some times occur . whe n they may be confused with norm al mar row Decks, especially when dense . Examples are illustr ated sta ined with the du al esterase rea ction in 985 and the Prussian blue sta in for free iron in 986. In 985 there is the usual CE positivit y in granulocytes and so me BE positivity in later norrnoblasts and a megakaryocyte at the edge s of the cell clump , while the neuroblastoma cells show moderat ely strong, finely granular, BE posiiivity . Th ere is no det ect able free iro n in the tum our cell nest see n in 986. nor in the pe ripheral normobla sts . Th is contrasts with the finding of norm al qu ant ities of free iron in the marro w Deck of haemopoietic tissue from the same slide . illustrated in 987, where neuro blastoma cells, if prese nt. arc no t easily distinguished .
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993-997 . Thin sectio ns from bo ne marrow tre phine biopsies from pat ients with medull obl astom a invading the marrow . Thes e tumour cells bea r some resemb lance to ne uroblasto ma cells , but they are ge ne rally mo re stro ngly stai ning , and form sma ller ce ll clumps or chai nlike co lumns o f single cells in the bo ne marrow. and do not usually ma n ifest th e swirled or who rled arra ngeme nt co mmon in ne uroblastoma. The low-power fields of 993 and 994 show th ese cha racteristic gro wth patterns well, with resid ual no rmal marrow ele men ts in the bac kgro und and a sectioned capi llary at the left in 994 . Th e higher magnification of 995 and 996 reveals the moderately dense nuclear chromatin and tende ncy to fo rm ce ll chai ns and small syncytia l clumps . In a still higher-pow er field (997), th e nuclei o f the medu lloblasto ma cells can be see n to have the sa me type of hyperch rom atic spo ts as previou sly sho wn in neuroblasto ma ce lls.
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1002. Alka line phosph atase reaction is negati ve in the tumo ur cells (in contrast to RE cells). A positive polymor ph is present.
1003. Acid phosphatase reaction shows moderately coarse scattered granular positivity in tumour cells.
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1001. Clumpe d cells. darkly staining. and unlike any cells normally found in the marrow. allow a diagnosis of tumour ce ll metastasis to be made . This marrow smear was from a patient with disseminated carcinoma of the stomach.
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1005 and 1006. Cytological de tail of cell clumps from the same patient with gastric ca rcinoma . The tendency to syncytium formatio n is evident. 1007. A part ially syncytia l cell clump of seco ndary deposi t from carcinoma of the bronchus in a bone marrow smear .
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1008. A fur lher exa mple of bronchial ca rcino ma ce lls in ma rro w, showing almos t nothing but tum our ce lls (a nd clumped ery throcy tes). A large multinucleat ed syncyt ium is present . pe rha ps of tumo ur ce lls, but po ssibly an osteoc last. 1009. Bron chial carcino ma cells in marrow fro m a third patient, here showing a te nde ncy to rosette forma tio n.
1010- 1012. Examples of malignant cells in bone marrow asp irates fro m a patient with dissemi nated carcinoma of the breast.
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and 1018. Low- and higher-power views. respectively. of a section of marrow trephine biopsy heavily infiltrated with a fibrosing breast carcinoma . The relative infrequency of epithelial cell nests and the predominance of dense fibrosis are well shown in 1017. and the irregular cytology of the malignant cell clumps amid the surrounding collagen and fibrocytes appears in grea ter derail in 1018. 1017
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1021. A case of bo ne mar row metastasis from prostati c ca rcino ma see n in a tre phine biopsy sec tion. In this lowpower view there is evident erosion of the bo ny trabecu lae and a conspic uo us fibro tic reaction around the tumour cell nests.
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represen ts sarcomatous malignant change. Areas of exte nsive erosion and destru ction of the trabecular bone can be see n at the pe riphery. particularly at the upper edge, while most of the remaining field is occupied by whor ls of fibrosing tumour cells. 1024. A high-power view of a section of bo ne mar row trephine biopsy from a patient with Ewing's sarcoma of bone . sho wing the cyto logy and mitotic activity of the tumo ur cells. with a multinucleat ed giant tu mo ur cell at the bottom right. Middl e right of the field is a spur of normal marrow tissue penetratin g the tumou r mass.
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1029. Involvement of bo th marrow and periph eral blood in the terminal stage o f HD may produce a leukaemi a-like picture . Thi s doe s not occ ur commo nly. but most exa mples reported appe a r to have shown mon ocytoid primi tive cells. Illustrat ed here is the periphe ra l blood picture in a term inal leukaemi c tran sformation o f HO. wher e the ce lls pro babl y be long to the same lineage as R-S cell s.
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1033. A blood smear in infection with Bartonella baci lliformis. Many eryt hrocy tes con tain the orga nisms. with rod-s hape d o r somet imes cocco- bacillary fo rm. The disease pro duced by this organ ism in man , Bartonellosis. Ca rrio n's disease o r Oroya fc ..er. is associa ted with macrocytosis and hae molytic ana emia, and th is smea r shows some polychromasia. 1034. Yeast-like bod ies of blastom ycosis in a spicule of bo ne mar row . possibly und ergoi ng phagocytosis by RE cells. 1035. A n RE cell contai ning many Leishrnan-Don ovan bodies. fro m the ma rrow of a patient with kala-azar. Variou s other mar row cells. a megakaryocyte. several myelocytes and a lat c normoblast are also present.
315
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1036. Ano the r exa mple of Leis hma nia, with o rganisms within a mon ocyte and ot he rs liberated amo ng th e surrounding cells. Below the monocyte is a clump of platelets for comparison with the more sharp ly and positive ly staining Leishmania bodies.
•
1037. Furt her Leishmania parasites in a monocyte l macrophage. 1038 and 1039. Co nsecutive Ro manowsky and PAS reac tions o n the same field fro m a smear of bo ne mar row aspirate tak en fro m a pati ent with Leish ma niasis. T he cen tra l pai r o f macrophag cs contain bet wee n them a dozen parasites. which can be seen in the PAS stain of 1039 to be weakly PAS-positive. There is a necrotic Iymphocyte below the macrophages and normally PASpositive neutrophils to either side .
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316
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1040. Exa mples of leishmania Don ovan i infantum in a group o f macrophagcs from a hon e marrow aspirate taken from an infected Ethio pian child . Para sites may be sce n lying free as well as in the macrophagcs.
ICJ.II and 1CJ.I2. Co nsecutive photographs of a binucleated macrophage co ntaining numerous parasites in a marrow smea r from a patient infected with Leishmania Brazilien sis, showing the sharp birefr ingence of the parasites unde r polarized light.
1CJ.I2
1043 an d 1044. High-p ower and very high-po wer views. res pec tively , of a field from a section of bo ne marrow tre phine bio psy tak en from a pat ien t with Leishmaniasis . In 1043 th e pleom o rph ic cyto logy can be seen. with co nspicuo us increase in plasma cells and occasional hisriocytcs amo ng th e normal haem opoiet ic tissue . Th c Leishm ani a parasites are difficult to d iscern in sections . of bo ne mar row, but a gro up of extrace llular pa rasi tes can just be dist inguished in 1044. a little to th e right and above the cent re of th e field . Th e cyto logy of th e predomina nt plasma cells is also well show n at th is magnification. 1045. Toxoplasma with in the cytopl asm o f a macrophage . T wo free orga nisms are pre sent tow ard s the bottom right . and othe r single free o rganisms elsewhere. Their larger size, spindle sha pe and banded nucle us dist inguis hes the m clea rly from Leis hma nia and fro m plate lets .
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1046-1048 . Plasm odium falc iparum infec tion .
1046. Ring form trop hozoites in eryth rocytes. T he presence of seve ral rings in a single ery throcyte is strong ly suggestive of P. falc ipar um . 1047. Anot her field showing further ring forms in many red cells . There is also a mon ocyte in this field . HMS. Crescentic ga rnetocytes, diagnostic of P. [alciparu m . In falcipa rum malaria the schizo nts do not appear in the per ipher al blood and on ly the ring-shaped trophozo ites and the cresce ntic gamc tocytes are likely to be sccn in blood smears.
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1049-1052. P. malariae infectio n.
1049. Ring-form tro phozo ites in erythrocytes. 1050. A n example of the coa rse ring forms found in P. malariae infect io ns (a nd also in P. vivax and P. ovale, but no t in 1'. [a/ciparum) . 1051. Maturin g troph ozoite. 1052. Schizonts contai ning be twee n six and 12 ro und merozoi tes, togeth er with clumps of coa rse dark brow n pigme nt.
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320
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105-1
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1055
1053. Ring for ms in P. vivax infection . 105~ . Ring-form and mo re mature tr op hozoi tes, from P. o vale infection . Schuffner's dot s in the erythrocy tes are co nspicuo us. as they may also be in P. vivax infectio ns.
1055. A schizont in P. l 'i l 'OX infecti on. containing som e 20 merozoites and a quanti ty of den se bro wn pigment. 1056 . P. \-'i\lax infection . Ring-fo rm tro phozoites, a nd liberation of merozo itcs from a fully deve loped schizont.
1056
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1057
1057 and 1058. A nother exa mp le of a schizont in P. vivax infection. illustr at ing the appea rance of a Rom an owsky-stained smear under norm al illum inat ion and then under pola rized light. The pigment dots are seen to be stro ngly birefringent.
1059 and 1060. Further consecutive stains of the same schizont as shown in the previous two figures . In 1059 the slide has been restained by the Prussian blue re action for free iron . but little or no blue staining can be seen , The pigment particles do. neve rt heless, contain iron which C3n be rendered accessib le to the Prussia n blue reaction by prior trea tme nt with hydroge n perox ide . and in 1060 a blue reaction . albe it faint , can be detected afte r this sequence of expos ures.
322
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. 1063
1061. Tryp anosoma gumbiense, the most import ant species causing African trypan osomiasis. Th e kinet oplast is much smaller th an the centra l nucleus, the undul atin g membrane is broad and the contor tions of shape variable.
1062. T. brucei, present in some areas of Africa, has generally similar morph ology. 1063. T. cnai. the ca use of Chagas ' disease o r South Am er ican trypa noso miasis. has a much larger kinet oplast , a less conspicuo us undul atin g membrane , and a more fixed horseshoe configuration.
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Part 5 Imprints and sections of lymph nodes and spleen
Cyto logical study of imprin t or needl e aspirate samples of lymph nodes an d splee n co mp leme nts histo logical st udy o f biopsy specimens and is especially va luable in the diagn osis of infecti ve and reactive lymp hade nopat hy, lympho mas and so me ot her metastatic tumo urs . and . in th e case of splenic mat erial. also in lipid oses, mali gn an t histiocytosis and so me pri marily haem arological d isorders with ex tra me du llary haemopoiesis. T he no me ncla ture of lym ph nod e ce lls is confusing and several systems exist. In the illustrati on s here , certa in of the more widely used synonyms are given initially, afte r which the simple Kicl classification is used chiefly. readers bei ng expec ted to tra nslate as necessar y. T he cytological differences between lymphomas, for example, are ge ne ra lly clear in the slides de picted, eve n if se ma ntic d ispute re ma ins . Phagocytic reticu lo-endoth eli al cells (RE cells) in bo ne mar row and blood have been illustrate d pre viously in thi s volume; simila r cells arc conspicuo us in lymph nodes whe re their development fro m mo nocytes through an inte rme d iate mon ocyte/m acrophage or epit he lioid cell may be envisaged . Ce lls o f th e same family - perhap s the sa me ce lls in differ ent to pog raphical sites or at differ ent functiona l stages o f activity have be en separated by histo logists and electron microscopists int o a se ries of type s. A pa rt fro m th e actively phagocytic R E ce ll (histiocy te , macrophage , starry sky cell, histiocytic reticulum cell) (CD 11b/c+) and th e ep ithe lioid cells, fibrob lastic, dendriti c and inte rdi gitating R E or reti cu lum cells have been describ ed . Th ese latter are no t ofte n clearly distin guishable in imp rint s, perha ps beca use they te nd to rem ain in the suppo rting stru cture of the node an d do not easily come free in tou ch or smear prepara tions . Th e R E cells th at do appear in imp rints are show n in Ro man owsky preparations and also with va rious cytoche mica l and
324
immunocy toc he mica l sta ins which fre que ntly re nder them particul arl y co nspicuous . Such stains may hel p in ide ntifying the vari an ts listed above - fibro blastic R E ce lls are describe d as stro ngly positive for alkaline phosph atase bu t o nly weakly so for es terases an d acid phos p hatase; den dr itic ce lls (C D 14 + , C D23 +) , mostly fro m germina l ce ntres . are negative for phos phatases and weak in es terase ; interdigitating ce lls (C D40+ ). chiefly from extrafollicular T vcel l zones , are nega tive for alka line phos pha tase and react wea kly for acid phos pha tase and es te rase . The aut hors ' impressio n is that the se crite ria are no mor e th an ro ugh guidel ines; ce rtai nly. the cytoc he mical po sitivity o f clea rly ph agocytic RE cells varies wide ly and is probab ly mu ch influe nced by the nature of the ingested ce llular material. T he degree of variability of reaction in the cell gro up as see n in imprints is illustrated in this sect ion . We have includ ed illustrations of a wide ran ge of malign an t disor ders affecti ng lymph no des. fro m Hod gk in's d isease (H D) and non -H od gkin ' s lymp homa (N H L) . to seconda ry met astases fro m various tumours of non -haem ic cell origi n. Primar y lymph nod e tumou rs are ofte n immedia tely diagn osab le with reason able ce rtai nty from impri nts mad e at biopsy, and typi cal findings for variants of H D and N HL are pro vided . Histol ogical appeara nces in lymp h no de sections arc also incl ude d , par ticul arl y when th e struct ural informa tio n th ey pro vide is necessary for , or especially relevan t to , di agnostic classificati on . Fo r HD , the conventiona lly accepted div ision s into Iymphocyte predo mina nt, nod ular sclerosi ng , mixed cellularity and Iymp hocyte dep leted are followed , while for NH L the Kiel classificat ion is gene ra lly used . The table here sho ws the co nd itio ns included in th at class ificatio n. together with their equivalence to an int ernatio nally agree d 'working for mu lati on ' .
Classification of NUL \\'o rkin~
Form ulation
Kiel Eq uivalent
Low grade
Low grade
A. Sm all lymphocytic. SL cons istent with CLL. Plasmacytoid B. Fo llicular, sma ll cleaved
A . Lymphocytic: C LL Lymphoplasmacytic Lymphoplasmacytoid Lymphocpithelioid T-zone lymphoma F-SC B .lC. Cc ntrocyticJ ccntroblastic fo llicular and diffuse F-M
C. Follicular, mixed small cleaved and lar ge ce ll interm ediate grade D . Follicular . larg e cell F-L
D . Ccntroblasridce ntroeyrie follirular o r diffuse E. Diffuse. small cleaved D-SC E . Cenlrocytic . diffu se F. Diffu se. mixed sm all D-M F. Ccntrocytidcentroand large cell +1blastic diffu se sclerosis/epit hc lioid cells High grade G . Diffuse lar ge cc ll D-L G . Ccntroblastic. diffuse High grade H . Immunoblastic .largclB H . Immunobl astic ce ll (plasmacyto id . (B or T ) po lymo rpho us. +1C D 30 (ki - I)+ la rge ce ll ana plastic. T o r B epithelioid cells) I. Lymphoblastic . LB I. Lymphoblastic co nvo luted convoluted T -cell non -convoluted unclassified J . Lymphoblast ic. small SNC J . Lymphoblastic . non-cleaved Burkitt Bur kill ce ll type (B -ce ll) cell -type
Descriptions o f the histology and cytol ogy of lymphoid tissu es and cells and of th e reactive and ne oplastic conditions illustrated are given in further de tail in the captions. Chromosome abnormalities frequen tly occur in NHL. These include especially t(14 ;18) and t(II ;14) in follicular lymphomas. involving the bel genes on ISq and Ilq with the H-chain locus on 14q : the t(8 ;14) of Burkitt's lymp homa and lymphoblastic lymphomas o f sim ilar cyto logy. involvi ng c-rnyc o n 8q with the sam e Hchai n locus ; and a ran ge of transloca tions in T-c ell lymp homas invo lving th e various tcl ge nes o n lOq. lip and 14q. or the c-myc gene on Sq. with the alpha T-cell receptor locus on 14q. Several examples of sections or imprints from nodes with seco ndary metastases of solid tumours are given , mostl y fro m nod e biopsies made before the primary tumo ur had been di agn osed and when lymphoma had been suspected . Since haematologists a re frequ ently involved in the interpretation of such node biopsies. an appro priate range is illustrated he re . Needle biopsy of the splee n may occasionally be diagnostica lly va luab le in co nd itions such as lipidoses and certain tropical d iseases like Leishm ani asis; a few illustrat ive examp les of the appearances of sp leen as pirates or imprints are provided . Th e occur rence o f Icukaemic or lymphomato us infiltra tio n in pleura , peritoneum or meninges, may lead to the presence. o f neoplastic ce lls in pleural. ascitic o r ce rebrospina l fluids. The neoplastic cells which may be see n in ce ntrifuge d deposits from these fluids look mu ch as th ey do in buffy coat preparations from blood o r in marro w. as illustr ated alrea dy in earlier sections of th is atlas. They are acco rd ingly not shown again here . bu t some illustrations are given of pleural lining cells . which must be differentiated fro m neoplastic ones.
Miscellaneous: Mycosis fungoides and Sezary synd rome (T< ell). extramedullary plasmacytoma (Bccell), histiocytic and non -classifiable lymphomas.
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1064
1064 . Leis hman stai n: lym ph nod e im print: showing three lar ge immun obl asts with ample baso philic cytoplasm , and ge ne ra lly p rimitive nucle i with nucl e oli more centrally than peripher ally situated; a somewhat sma ller cc ntro blas t or lar ge follicula r ce ntre ce ll, top left . with high er nucl ear-cytopl asmi c ra tio but also with cytoplasmi c basophilia a nd three nucl eoli more towar ds th e pe riphe ry of the nucl eu s ; mature Iyrnph ocytes appear above a nd be low t he ce n tro blas t nea r the low est immunob last; there is a single plasma cell near the central immunobl ast and a mon ocyt e/m acroph age with a sma ll inclusion a t the left o f the field . Th e rem a ini ng ce lls a re centrocytes o r sma ll fo llicula r centre ce lls (Fee). 1065. Le ishma n sta in: lymph nod e imprint : seve n immunoblas ts illustrate th e range o f size and cytology in this group. Th e re ma ining ce lls in thi s field are mos tly mature Iymphocytes. 1066 . Leishman sta in: lymph nod e im pr int : a co lle ction of ce lls fro m the follicu lar ce nt re a rea of a lymph no de impri nt in reactive hyperplas ia secondary to infection . Against a backgroun d of small mature Iym ph ocytes arc occasiona l small follic u la r"ce nt re ce lls (ce ntrocytcs) a nd about a dozen large FCCs or centroblasts wit h nu cleo li a nd so me cytoplas mic basophilia . In the upper right co rner are seve ral imm unoblasts. a nd to their left a histi ocyti c R E ce ll a nd a plasma celL
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1067. Leishm an sta in: lymph node imprint : field to illustr ate seven large cells involved in a sequence of probable developmental cha nges along the mono cyteepithelioid cell-macrophage (R E ce ll) line . Th e remaining cells in this field are chiefly small lymphocytes . 1068. Leishman sta in : lymph nod e imprint: an exa mp le of an epithelioid ce ll cluster in a lymph node showing a reactive hyperpl asia seco nda ry to toxoplasma infection . Around the cluster arc chiefly Iymphocytcs but with a histiocytic RE cell (starry sky cell) at the bottom and six larg e mon onucl ear cells. o ne in mitosis. at the ce ntre and lower right of the field. Th ese cells are proh abl y immunob lasts. 1069. Leishman stai n: lymph nod e imprin t: R E cells, perhaps of the int erdigitating vari et y. with little evidenc e of phagocytosis, but with neighbouring Iymphocytes , occupy much of the lower half of this field. Two are binucleated, the left with a n adja cent prob able tissue mast cell and the right with a more basoph ilic cyto plasm th an usual. The upper half shows a mixture of small Iymph ocytes with dense nuclei and follicular ce ntre ce lls o r centrocytes with more ope n nuclei . sho wing tran sition toward s centroblasts and with a single immunoblast toward s the top right corner.
3'"27
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10
1070. SS stain: lymph nod e imprint : Sudan black po sitivit y is shown in a hist iocyt ic R E cell and in three cells o f th e monocyte-m acroph age line . All lymphoid
elements are negative. 1071. SB stain: lymph node imprint : positivit y is shown in a neutrophil polymorph. Th e remainin g cells, including macrophege s. centrocytes, cent rob lasts and immunoblasts, are all S8-negative .
1072. PAS re action : lymph node imprint : thi s pr ep aration from the same nod e as in 1069 shows fine granular positivity in a few mononuclear cells o nly. One on the left is probably an RE cell of th e int erdi git ating type with granular PAS po sitivit y sprea ding out in the cyto plas mic prolongation s be tween neighbouring cells. Two oth er positive cells toward s the top of the field are . respectively. a ccntro blast and a ce ntrocyte. A ver y occasional Iymphocytc also shows detecta ble gra nular positivity.
32 '
1074
1073 . PAS reacti on : lymph node imprin t: this preparationshow s no rmall y po sitive reactions in neutrophil polymorphs but gen er ally negative reacti on s in Iymphocytes. centrocytes and ccnt robl asts. alth ou gh ther e is a single small block of PAS- positive mat eri al in o ne lymphocyte ju st be low the centre of the field . and weak diffuse o r finely gra nular reaction s in the nar row cytoplasmic rim of eac h of two larger cells. probably
1075
centroblasts, above the centre. 1074 . Alk aline phosphatase : lymph node imprin t: this prepar ation from a reactive node shows lymphocytes. ce ntrocytes. ce ntrob lasts and immunoblasts all to be negat ive for this enzyme . while a single neu trophil po lymorp h shows + + positivit y and a lar ge phagocytic RE cell is o nly very weakl y positive - despite its obvious past ingestion of various o ther cell types . probably including a neut roph il.
1075. A lkaline phosphatase : lymph node imprint: strong positivity is manifest in a phagocytic RE cell with ingested cellular remn ant s: positivel y reactin g fibrils spread o ut ove r a neighbo uring lymphocy te to give it a spurious appea ra nce of pos itivity.
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• 1076. Acid pho sphat ase: lymph nod e impr int : very strong positivity is seen in a phagocytic RE cell containing much ingested material, while at the opposite corner of the field a smaller monocyte-m acrophage shows less intense butstill striking po sitiviry. Most of the remaining cells are of the lyrnph ocyte , centrocyte, centroblast familyand show no more than occasional weak granular positivity, but betwee n the cells spreads a network of positively reacting fibrils proba bly deriv ed from the larger histiocytic RE cell or perhaps from an inconspicuous dendritic RE cell.
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1077. Acid phosphatase: lymph node imprint: several strongly positive RE cells of variable size and shape are seen against a background of Iymphocytes. These show variable scattered positivity without any clear example of T-cell type focal paranuclear reaction . There is a wea kly positive polymo rph at the lower right. 1078. Acid phosphatas e: lymph node imprint: a phagocytic or histiocytic RE cell with much ingested material (sta rry sky cell) shows relatively little acid phosphatase positivity in contrast to a more strongly positive monocyte or epithelioid cell. Lymphocytes here show mostly either negative orscattered granularreactions and there are two large mononuclear cells, with variable weak granular posi tivity. which are probably immunoblasts. At the lower right corner is a positively reacting plasma cell.
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• 1080
9
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1081
1079. Du al este rase reacti on : lymph nod e imprint: this prep ar ation shows chiefly cortical area Tu lymphocytes, with stro ng localized dots of butyra te esterase ( BE) positivity, surrounding lar ger ce lls of the monocytemacrophage system. only one of which shoe..s moderately strong BE positivity, the remainder being almost negative with only a few chloroacetate esterase (Cfi} pos itive granules . A norma lly CE-positive pol ymorph is present.
1080. Dual esterase reaction : lymph node imp rint: BE positivity of moderate-to-strong deg ree in epithelioid cells in this case. One has ingested aCE-positive neutrophil. Most lymphocytes here do not show the localized Tit type of BE positivity and are presuma bly chiefly B cells. 1081 and 1082. Dual esterase reaction : lymph node imprint: these fields from the same case of reactive hype rplasia show strong BE posirivity in RE cells probabl y of the histiocytic o r ph agoc ytic variety. wea ker reactions in occasional mon ocyte-macroph age ce lls. and loca lized do t-like positi vity in certain lymph ocytes. presumab ly of the Tit subset. A probable dendritic RE cell with lo ng intercellular processes but rather weak BE positivity is also shown in 1082.
11182
331
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1083-1096. Sections and imprints from lymph nod e biopsies taken fro m patients with toxoplasmosis . The protozoan parasite, although more easily detected in imprints, is not often identifiable in sections, bur the histological p icture is suffic iently cha racteristic to suggest the diagnosis in m ost cases.
1083. A low-power view of a lymph nod e section from a patie nt with toxopl asma infec tion. Th e histological picture is a mixed one, with d istended sinuses including a marked proliferat ion of histiocytes. and with further histiocytic hyperpl asia in the par aco rtical areas , but also with follicular hyper plasia and nume rous activated B cells appearing as immunoblasts and plasm acytoid cells. In this field the paler areas represen t gro ssly dila ted sinuses with in the co rtex . 1084. A higher -power field from the same area as shown in 1083. illust ra ting (he pa raco rt ical lymphocytes to the left and the dense accum ulatio n of reactive histiocyt es in the dil ated sinus to the right. The pa le vesicular nuclei of the mo nocytoid histiocytes and the frequ en t pre sence of ingested mat erial in their cyt opla sm are clea rly visible here . while similar large histiocytes are occasiona lly to be seen amo ng the par acor tical cells. 1085. Anot her high-po wer field fro m a different area in the sa me node. show ing the edge of a hype rplastic follicle . sharply de marcate d fro m (he para follicular lymphoid cuff. but with numerous lar ge histiocytes.
0.
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1086
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1086-1090. Imp rints from a lymph nod e biopsy ta ke n from anot her pa tient with toxoplasma infect ion. showing the cytology and cytoc hemist ry of the reactive process. The cells in the Rom anowsk y prepara tion in 1086 include a macroph age with spreading cytoplas m and seve ral mo nocytoid ce lls surro unded by a predo minance of Iymph ocytes. In the SB-sta ined field sho wn in 1087 there is a single nor mally sudanop hilic neu trophil and a met ach rom atically sta ining tissue mast cell, while all the remain ing cells. includin g immunoblasts and mon ocyto id rnacroph ages. are S8-negativ e . Th e PAS-st ained field illustrat ed at lower pow er in 1088 again has a mixed cytological con tent. with a few scatter ed PA S-po sitive neutrop hils, includi ng o ne within th e cyto plasm of a macrop hage. and vario us lympho cytcs. centrob lasts and immuno blasts, as well as the conspicuo us lar ge mon ocyt oid macro phages. Apa rt from the ncut rophil s. most cells he re appear PA S-negat ive . or with no mo re th an a weak cytoplas mic tinge of reactivity . visible in some macrop h ages. In both the du al este rase and the acid p hos phatase reaction s sho wn in 1089 and 1090 the macrop hages and their monocyti c precu rso rs are picked o ut by their positive re action s. that for BE con trasting with th e CE reaction of neutrophils in 1089. The majo rity o f th e lymphoid eleme nts ar e negat ive for bot h reactio ns. in co nfor mity with their pre do mina ntly B-cell lineage.
1089
1090
333
1091
1091
1093
109 1- 1094. Impri nts from another toxoplasma l lymph node biopsy . In 1091 there is a central mo nocytoid macrophage surrounded by imm u nob last s, whic h in turn arc surrounded by Iymphocytes and centrocytes. Th e more detailed cyto logy of these cells is shown at higher power in 1092. wit h two monocytoid macrophages to the upper right , and with eight immunoblasts around and to the left of them . plus a centroblast at mid-bottom of the field . and Iymph ocytes and centrobl asts peripher ally . In 1093 . aga inst a simila r cytological back ground , there are two mature macrophagcs conta ining inges ted mat eri al wh ich includes nuclear debris but also elliptical toxoplasma prot ozoa . The PAS sta in in 1094 shows two stro ngly PAS-positiv e neutrophils, most ly ne gat ive immu no blasts. gra nular cyto plas mic positivi ty in three centrocy tes to the left of th e field and in a ce ntrob last be low th e right-h and neutrophil. and pos itivity in a to xoplasma par asite amo ng ot he r cellul ar debri s in a macrophage at the to p right corner . 1095 and 1096. Lymph nod e imprints. from a patie nt with recurrenc e o f lymph ad en opathy te n yea rs after initial d iagn osis o f toxopl asmosis. Th e presen ce o f irnmuno blastic and mon ocyte-macroph agc prolifcratio n with num erou s mitoses. a ppare nt in 1095. and th e mul tinucleated macroph age with mu ch ingested materi al . incl ud ing a proba ble toxoplasm a o rga nism at the ext reme right o f the cyto plasm . visib le in 1096 . co nfirms persistence o r rec ur re nce of the disease .
1097 . .
•
1097-1099 . Leishman stain: lymph node imprints , sho wing chronic inflammatory changes. includin g especially the fo rmation of Langhans giant cells.
1099
1097. A gro up of epithe lio id cells with some phagocytic activity surro unde d by Iymphocy tes and centroc ytes from the chronic lymph adenitis of sarcoidosis.
1098. A foreign- body giant cell or Lang hans cell from the same conditio n. 1099. Anothe r ex ampl e of a Langha ns multinucleated giant cell from tubercu lous lymp hadenit is.
335
11 1100
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• 11 1102
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1100- 1105. Cytoc he mical features of typical Langha ns giant cells as see n in imprint s of a lymp h node biop sy fro m a fema le patient with chronic gra nulo matous disease . Th ese cells a re probably de rived fro m multiple nuclear division s witho ut cytokinesis occurring in macropha ges . The seque nce o f stains illustr ated her e includes . first . a Ro manowsky prepar ation . with flat nuclei of moder ate chro ma tin co nce ntra tion. witho ut clear nucleoli bu t with single are as o f localized chroma tin co nde nsation. usually at the pe riphe ry . eac h probab ly represe nti ng the inac tive X chromo some or Bar r body. The bluc agra nular cytoplas m is eviden tly fra gile and eas ily disru pted . In ll Ol the SR stain is esse ntially negative except for a few scatte red granules probably derived fro m an ingested neut roph il polymo rp h. T he haem ato xylin co unte rsta in used in the PAS reaction of 1102 again shows the Barr bod ies well. while the cytoplasm has weak diffuse PAS-positivity. 'The dual esterase react ion of 1103 has a modera tely stro ng gran ular positivity fo r BE in the giant cell. with aCE-pos itive neutrop hil top left. In 1104 ther e is the strong positive acid phospha tase reactivity to be expecte d in a cell of macrop hag ic o rigin. but the alkaline ph osph atase stai n in 1105 is negative .
336
11
11
Uh
1107
1106-1120. A section and a sequence afimpri nts f rom a lymph node biopsy taken fro m a patient with Leuerer Stwe syndrom e, a form of histiocytosis X . the protife rotin' mix ed granulom atous slate involvin g the nodal equivalent of epiderm al Lan gerhans cells, perhaps related 10 or derived fro m dend ritic reticulum cells.
I HIM
1106. Th is section shows an area of th e co rte x with sinus expansio n and monon uclea r histiocytic o r Langerh ans cell hyperpl asia in the lower part. and se veral multinuclea ted giant cells amo ng a prepo nderance of Iymphocytc s in the upp er part. Alt hough these giant ce lls bear some resem blance to Langha ns foreign body giant cells. they arc gene rally smaller . with fewer and oft en more cent ra lly placed nuclei . 1107 and 1108 . Imp rints from th e same node biopsy as in 1106. sho wing at low- and higher -power magnifica tion the cyto logy o f th e atypical Xchistio cytcs or Lan gerhan s cells. Th ey tend to occur in clumps. have a more irregu lar nucle us and more robu st cytoplas m than Langhan s foreign body giant cells. alth ou gh with some tenden cy to exte nd processes. Th ey ofte n appear to co ntain p hagocyt osed materi al. th ou gh less active in this respe ct th an th e co mmon histiocyte .
337
11
1109 •
1111
Ill.
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1109- 1114. A sequence ofstains ofimprints f rom lymph node biopsies taken from patients with eosinop hilic granuloma, another variant of histiocytosis X .
11
1109. Thi s Rom anowsky-stained preparation shows a nest of eosino phils surrounde d by a mixtur e of large monocytoid Langerh ans cells and Iymphocytes. 1110. A similar high-power view of the second case sho ws the mor e det ailed cytology of the Langer hans cells and their close resemb lance to monocytoid macrophages. 1111 and 111 2. Low- and higher -power field s, from SB-staincd prepa rations from the two cases, illustrating that the histiocytic cells are generally suda nophobic . 11 -
1113 and 1114. PAS stai ns fro m the two cases . In 1113 there are probably six histiocytes present. two (one of them with unstained crysta lline inclusions) sho wing strong PAS-positivity. o ne containing bro wnish gra nules of free iron . one nega tive except for a small tetrad of positive mater ial. pe rhaps a phagocytosed sta rch grain . and the two smaller histiocytes nega tive. Prob ably only the last three cells are Langerhans ce lls. the ea rlier ones being phagocy tic macroph ages. Th e cells from the second case in 1114 are mostly negative . except for weak gra nular positivity and a diffuse tinge in o ne large cell to the left of the field. These are pro babl y all Langerhans cells.
1115
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1115-1120 . Further cytochemical stains from the first of these {WO cases, where, despite the undoub ted diagnosis of eosinop hilic granuloma. there is an intense con· comitant proliferation of phagocytic macrophages in parallel with the proliferation of Langerhans cells.
1119
• •
1115 and 1116. Th e immense amo unt of free iron demonstr able by the Prussian blue reaction in the lymph nod e macroph agcs from this pa tient is shown in 1115. Th is accumu lation is no dou bt chiefly in phagocy tic macrop hages, o ne of which, laden with iron and also much ingested materi al. includi ng numerou s red cells from which the iron is derived , is sho wn in 1116. 1117 and 1118. Differ enl fields from the same du al esterase stai n. In eac h there is a single CE -pos itive neutrop hil and various negative lyrnphocytes and centrocy tes . Three weakly BE-positive large ce lls. possibly La nge rha ns cells . are illust rated in 1117. contrastin g with the two much more coa rse ly BE-positive phagocytic macrop hages in 1118.
1120
1119 and 1120. Acid and alkaline phosphat ase stai ns, respectively. o n the lymph nod e biop sy materia l, bo th field s showing positi vity in ph agocyti c macroph ages. Lan gerh an s cells a re difficult to identify in these preparations but appea r generall y negative for both pho sph at ases.
339
1121
1121-1130. A small selection of lymph node biopsy sections f rom patients with Hodgkin 's disease (HD), to illus trate features of nodal structure relevant to diagnosis of pathological variants , not readily apparent fr om imprints.
1121 and 1122. Both these fields are taken from an exa mple o f the lymph ocyte pred omin ant va riant . where lymp hocytcs and centrocytes grea tly predominate . but wher e both binucleat ed Reed-Sternberg (R-S) cells and large mononucle ar Hodgkin's ce lls can be distinguished. These cells. with their conspicuous large single centrally placed nucleoli . are quite numerous and promin ent in 112t. b ut less frequen t thnugh ea sily eno ugh identified in 1122. Diagnosis from imprint mat eri al ma y be difficult when R· S and Hod gkin 's ce lls are few. and th er e may be confusion with lymphocytic non- Hod gkin 's lymphom a {l HL ) in the interpre ta tion of bot h imprints and secti ons.
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1124-1127. Furt her illustrations of structural pattern s of nodal and cellular architecture in NSHD . In 1124the H&E stain shows a coa rse hand of densely fibrotic mate rial separating islands of neop lastic tissue , including a predominance of lacunar cells, conspicu ous among the surrounding Iymphocytcs. In 1125 and 1126 similar fields from sectio ns stained with the Mallor y and Ponceau S methods respectively pick out the collagenous tissue with blue and red staining and the ncoplastic cell nests with nuclei stained red or brown . The higher-power view of an H&E-stained field in 1127 shows the more derailed cytology of the lacunar cells, with their nucleolated nuclei and well-defined cytoplasm contracted within the lacunar space" These cells are surro unded by lyrnphocyt es with den sely chromatic nuclei. intersperse d with larger a nd more lightly staining cent rocytes .
34 1
128
•
1128-1130. A low-po wer and two higher-power views of different areas o f an H&E-st ain ed section of a lymph nod e biop sy. from a pat ient with th e mixed ce llularity variant o f HD (MC H D) . The pictu re is a gene ra lly pleomorphic one . with . in 1128. a pseudofollicular structure prod uced by an expanding nodule of neop lastic Hod gkin's cells compressing the surround ing lymphocytes into the appea ra nce o f a sheath. but with man y scatte red Hod gkin 's and R-S cells also present amo ng the Iymphocytes. In 1129 seve ral of these large neo plastic ce lls can be d ist ingu ished. an especially conspicuous one at the lower centre. but there is also a marked eosinophilic infiltra tio n a nd so me fibro tic activity appare nt amo ng the ncopl astic tissue and the redu ced lymphocytic component. An oth er area o f th e section, see n in 1130, has a high er proport ion o f resid ua l Iym phocytes and centrocytes bu t agai n a conspicuous pro liferation of R-S and Hod gkin's cells.
11.
31
1132
1131-1133. Leishm an stain: lymph node imprints: H Dmix ed cellularity.
1133
1131 and 1132. Low- and higher -power views. respec tively, of the pleomo rphic picture commonly see n in node imprint s from the disease . especia lly in the mixed cellularity variant. Lymph ocytes, centrocytcs, occasional centroblasts and immuno blasts mingle with plasma cells. eosinop hil and, sometimes , neutrop hil polymo rphs, and monocyte-macroph agcs. There is a single large R-S cell with twisted or overlap ping double nucleu s and large dark violaceo us nucleoli in eac h figure . The extent of eosinophilia in this exa mple is unusual , but some cosinop hils can commo nly be found in most imprint preparat ion s fro m HO.
1133. An other area of the same slide . again showing lympho cytcs. centrocytes, centrob lasts and an immunoblast (top rigbt), togetb er witb a plasma cell and many eosinop hils. Ce ntrally. there is a macroph age or RE cell with , on the right . a mononuclear R-S cell or Hodgki n's cell with cha racte ristic nuclear chro matin and nucleoli.
343
• 113.3
1134-1136. Leishman stain: lymph node imprints: II D lymphocyte predominant . 1134. A low-powe r view providing a more uniformly lymphocytic and centrocyt ic backgro und. but with one binuclea ted R-S cell and severa l mononuclear Hodgkin's cells. Occasio nal monocyte-macrophage type cells can be secn. 1135. A higher-power view of the same slide in which all the cell types men tioned abo ve can be more clea rly ide ntified. 1136. A still higher magnification of an R-S cell from this specimen , to illustra te the typical reticular nuclear chromatin and the large da rk violet nucleoli. Th e surrounding cells a rc mostly lymphocytes.
11-
11311
11-10
9
1141
1137-1139. Lelshm an stain: lymp h node imprint: /ID
1142 1139. Yet anothe r area in th e sa me slide sho ws a co llection o f histiocytic R E cells. som e with ingested cell debris. which arc clearly morphologically distinct from the R-S and Hodgkin 's cells of the previous figures.
11-10-11-15. Leishman stain: lymph node imprints : variants of giant cells in liD. 1140-1142. Typically binudeated R-S cells. Th er e is also a monon uclea r Hodgkin's cell in 1142.
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characte ristic nuclear and nucleo lar pattern s . In IJ44 a pai r of these cells, one possib ly with overl apping doub le nucleus . are se parated by a pair of RE cells o f clea rly co ntrasting nuclear and cyto plasmic morphol ogy. 1145. A mitotic figure in an R-S ce ll. showing the clearly intact nucle o li surrounded by separated chromosome s.
To the left of this cell is a macrophage.
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1146. PA S reactio n: lymph nod e imprint: HD. A cle arl y binucleated R-S cell and five o ut of six mo nonuclear Hod gkin's cells. includ ing o ne in mitosis. show glob ules or blocks of PA S pos itivity . Although the giant cells of HD are mos t often PAS -negat ive . the appearance of glob ules of positivity as in this figure . o r even coarsely scat te red granular posit ivit y, is by no mean s rare .
1147. Acid phosphatase stain: lymp h node imprint: HO . A binucleated R-S ce ll shows strong posuivi ty. 1148. D ual esterase reaction: lymph nod e imprint : HD. Hod gkin's cells usua lly show little esterase positivity , as here . where weakly scatte red BE and CE granules are detectable. in sharp contrast to the stro ng BE positivity in a neighbouring giant RE cell. An eos ino phil. a CE -positive neu tro phil and variably BEpositive lyrnp hocytes co mplete th e field .
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1149 a nd 1150. Leishm a n stai n: lym ph nod e imprint : chro nic lymphaden itis . Mult inucleat ed gia nt ce lls. with flat e pithe lial-type nu clei and re la tive ly sma ll o r incon spic uo us nucleoli; no t to be mistake n for HD gia nt ce lls. 1151 . Dua l es terase reaction from th e same speci men . One of the mu ltinuclea ted giant cells shows strong BE positivity, unlike R-S ce lls which are usu ally negative .
Localized dot-like positivity is seen also in some probable T cells and there is CE positivity in several ncu trophils and mixed reaction in a plasma cell.
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1153
1152-11 57. Lymph node imprints :[ollicular lymphoma. cen trocytic-centrobiastic; mixed small and large [olliculur cen tre-cell lymphoma.
11 5~
1152. Leishman stain: lower-power view of a mixtu re of centrocytcs and centroblasts, with a scattering of mature Iymphocytcs. 1153 . A higher-power view of the same preparation. revealing large single o r occasio nally multiple nucleoli in the larg er cells - ce ntro blasts - which often show indentation o r cleavi ng of the nucleus. The two varieties of sma ll-cell. den sely pachych rom ati c Iymphocytes and more lightly sta ined cen trocytes, ca n be clearl y differentiated .
1154 . SS rea ctio n in this nod e imprint shows all the lymphoma ce lls to be negative. Th e single positively reacting ce ll is a neu trophil pol ymorph .
~9
1156
1155
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1155. PA S reaction o n the same nod e imprint shows a little positivity in cyto plasmic fragme nts , and so me tinge and fine granular react ion in lymph oma cells.
1156. Acid phosph atase reactio n o n this specimen shows variable + to + + + positivity in lymphom a cells. scatte red and not part icularly loca lized in the pa ranucl ea r zo ne . There is a st ro ngly positive ij.E cell present. 1157. Dual este rase react ion here shows CE positivity in a neutro phil po lymo rp h and in a tissue mast cell. with BE positi vity of modest degree in an RE cell. possibly dendritic. with fine processes exte nding bet ween neighbo uring lympho ma cells. Th e lymph oma ce lls arc essentially negative .
350
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1158-11 62. Lymph node sections f rom cem rocyttccensroblastic malignant lym ph oma. IlS8 and 1159. A very low-power view and a higherpower field fro m an H&E -stain ed section. In 1158 mo st of th e field is occupied by part of a nod ule of lymphoma cells. wit h com p ressed norm al pa ra follicula r tissue at th e upper and left edges. The tum our appears paler th an the predominantl y lymphocytic surr ounding tissue . Th e mixed cytology of th e follicular tumour is shown in de tai l in 1159 ; the ce m roblasts have large and pal el y staining vesicul ar nuclei wit h periphe ral nucleo li. th e cc nt roc ytcs are smaller with dar ker nuclei and inco nsp icuo us nucleoli . and th er e arc one o r two deeply stai ne d small Iymphoc ytcs. Several mito tic figures ca n be see n .
1162
1160-11 62. Immunocytochemical PAP reaction s on sec tions from the same biopsy. A negative co nt rol is see n in 1160. and a reaction with the pan -B f\.lAb C D19 in 1161 - the latter showing stro ng po sitivity in more matu re residual lymphocytes at the up pe r left and weaker posi tivity in the ce ntrocytes and ce ntroblasts co mprising the bulk o f the tumour follicl e in the lowe r right of th e field . Th e negative T cell s, scanty with in th e tumour tissue bu t more frequent in the cortical area , sta nd o ut clea rly. A rea ction with th e leu cocyt e co mmo n MAb C D~5 is see n in 1162 wher e all th e cells in the section sho w positivi ty of grea te r or less inten sity.
351
11
1163
1163-1168. Lymph node lymphoma, B-cell type.
impr ints:
1163 and 1164. Leishman stain: low- and higher power views of the neopl astic lympho blast tumour cells; they sho w roun ded nuclei with moderately fine chro matin and nucleoli varying in numb er between one and thr ee o r four . mostly no t at the nuclear mem brane . The re is moderately dee p basophilia in the narrow cytoplasmic rim . 1165. 58 stai n: ther e is a single positive neutrophil polymo rph. but the Iymphoblasts and occasio nal Iymphocytcs are all quit e negative.
352
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lymphoblastic I
11 -
1168
1166. PA S reaction: the blast cells are virtually entirely negative: there is some granular positivity in o ne lymphocyte and the usual strong reaction in the single neutrophil present.
11 67. Acid phosphatase: the blast cells are again negative while a phagocytic RE cell is strongly positive . A few positive granules are see n in a single plasma cell
above the RE cell. 1168. Dual esterase : the Iymphobl asts show fine scattered BE positivity. A neutrophil polymorph is CEpositive.
33
11
1169
1169-1174. Lym ph node imp rints: immunoblastic lymphoma, B-cell typ e, with some plasmacyto id differ entiation. 1169 and 1170. Leishman stain: low- and high-power views sho wing large immunobl astic ce lls with mod er ate amo unts of basophili c cyto plasm and leptochromat ic nuclei conta ining large but poo rly defined central nucleoli. Most immunoblasts contain globular inclusions, staining a greyish-green colour. like Russell bodies, presumably represent ing secreted immunoglobulin . Thi s feature is o nly occas ionally see n in cases of immunoblastic lympho ma . but when pre sent indicat es the secret ory Bccell natu re of the tumour. Am ong the surro unding small cells , chiefly lymp hocytes and ce ntrocytes, are som e with plasmacytic morpholo gy occas ionally containing similar Russell-body inclusion mate rial. Scatte red cytoplasmic fragments are conspicuous. especially in the high-power view. where multiple small cytop lasmic vacuoles are visible in the immu nob lasts .
1171. PAS reaction shows the immunob lasts to be essentially negati ve. as are the globular inclusion s in this insta nce , altho ugh more ofte n such inclusions sho w weak PA S positivity. A single pos itive neutrophil ca n be seen .
354
1171
1173
2
1174
1172-11 74. Imprints of lymph node biopsies from another two cases of immunob lastic lymphoma with gene rally similar cytology but without the globular inclusions seen in the previous case . In the first , the field shown in 1172 again illustrates the characteristic cytology of the immunoblasts and also the presence of lymphoplasmacytoid cells amon g the lymphocytes and centrocytes; the immunoblasts from the second case , depicted in 1173, have more irregular nuclear outlines, as may sometimes be see n. The PAS reaction on the cells from this exampl e. sho wn in 1174, is more variable in positivit y than was the case in 1171, ranging from negative to quit e strongly pos itive. with fine or coa rse granules, as at the top left and lower right . respectivel y. of this field. An occas ional block of PAS posuivity was present in the immunobl asts in this case and o ne such block is seen free from the cytopl asm in the centre of the field. Variable PAS positivit y of this kind is not uncomm on in immunoblastic lymphoma.
-.
355
11 -
11 75-1178. Lymph node imprints: hairy eel/leukaemia
tucu .
1175 . Lei shrnan stain: the spec imen is composed almost entirely of hairy cells (HCs) . Th ere is gross cyto plasmic fragme ntation but there arc virtua lly no nor mal lymph node ele ments . A single Iymphocyte can he seen near the top left-hand corne r.
11 76. PA S reaction: there is diffuse or finely gra nular positivity in the background . which is large ly made up of the fragmen ted cytoplasm of HCs. 1177. Acid phosp hatase: most cells show moder ately stro ng granular positivity. and ther e is also conside rable backgro und scatte red positivity from cyto plasmic fragmenta tion. O nly four norm al lyrnphocytes can be de tecte d . 117H. Dual esterase: there is strong BE positivi ty in a histiocytic RE cell. and weaker reac tion in the cytoplasmic rim of many HCs and in the scattered fragme nts of disru pted He cyto plasm.
356
•
I
-
79
1180
118 1
11 79-1187 . Sections and imprints of lym ph node biops ies fro m patients with peripheral T-cell lymp huma s (Len nen :s lymp homa) . Tslyrnphoblastic lymph oma s. lik e T-A L L , which they closely resemb le and into which they usually und ergo transformation. ere regarded as being derived from precursor T cells. but anothe r group of lymphomas with diffu se histological distribution and variable clinical course r eacl with ma ture T-cell markers, often with CD.J. and do not possess Td T. Their mo, pho logy is pleom orphic, with the lymph om atous cells either mature T cells or T immunoblasts or. more com m only. a heterogeneous m ixture. but with a m arked reactive com po nent of epithelioid histiocytes and increased vascularity , The neap/astir T cells may sho w atypical surface antigen expression , som etimes failing to react with the pan · T marker CD 7, fo r example, but their clonal origin can be confirmed by the demonstration of specific TCR gene rearrangement in Southern blots. 1179-11 8 1. Successively increasi ng magnifications of areas from a section of lymph node showing the character istic features of T-cell lymphom a ; the increased vascularity and conspicuous epit helioid cell reaction among the ma lignant Iymphocytes are manifest in 1179 and 1180. while de tails of the Teccll and epithelioid-cell cyto logy a re revealed in 1181.
357
11 ,
182
1182 and 1183. Low- and higher-power views, respectively, of a lymph node section from another case of peripheral T-celllymphoma, again illustrating the mixed but here chiefly large-cell immunoblastic neoplastic cytology, the marked vascularity and the epithelioid histiocytes, although the last component is less conspicuous in this case than in the previous one . In 1183 there is a multinucleated giant cell, probably of Langhans type and derived from the reactive epithelioid histio-
11
cytes, a finding quite common in this kindof lymphoma . 1184. An immunoperoxidase reaction against the MAb CD4 , on a section from the same biopsy, showing the presence of the helper phenotypic antigen on the malignant T Iymphocytes and immunoblasts. The larger epithelioid cells and the vascular endothelial cells are negative.
.
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35
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85
1187
1185-1187. Romanowsky, PAS and acid phosph atase stains on imprints made from a lymph node biopsy from a similar case of periph eral T-cell (Lennert's) lymphoma . In imprints . the feature o f increased vascularity cannot, of course, be demonstrated, and the
epithelioid histiocytic proliferation is also less easy to
appreciate than in sections, becausethe epithelioidcells tend to adhere to the cut surface o f the node and no t to
be adequately represe nted in the imprint. The mixed neoplastic T-cell cyto logy . with a range from mature lymphocytes to immunoblasts, is apparent in 1185, however . with a gene rally negative PAS reaction exce pt for a single block in o ne immunobl ast (shown in 1186) - but with the strong localized paranuclear acid
phosphatase positivity typically found in T cells (as seen in 1187).
•
359
11
•
• 11
• •
•
1188-1 191. Sections and an imprint fr om an example of true histiocytic lymphoma ( TH L ), a localized variant of the generalized system ic disease malignant histiocytosis. Diagnosis usually requires cytochemical or immunocytochemical confirmation of the cell lineage, with acetate or butyrate esterase and acid ph osphatase staining. or with positive reactions la CD14, since the neoplastic histiocyres in THL often show little ph agocytic activity.
1188-1190. Sections of lymph node biops y from a localized nodal enlargement without evid en ce of generalized disease . showing predominance of histiocytes, demonstrable as such by cytochemic al stainin g for the mon ocyte-macrophage en zyme alpha- naphthyl acetate esterase . as shown at low- and higher-power magnification in 1188 and 1189, res pective ly. In 1190 an acid phosph atase sta in o n anoth er sectio n fro m the same material shows the strong positivity cha racte ristic of histiocytes . 1191. A high-power field from an imprint preparation fro m the same nod e biops y. showing an unu sual example of multiple phagocytosis. Th e large r macrophage (histiocyte) contai ns two ingested cells. one probably a ccntroblast and the ot her an uniden tifiable cell in metaphase of mitosis. while the smaller macrophage below contai ns seve ral fragments of ce llular debris.
360
1191
1193
92
1194
1192-1194. A lymph nod e imprint : Leishm an sta in: myelom a. Th e fields illustr ate variable size and staining characteristics of ' infiltratin g plasma cells. occas ional mar ked multinuclea rity . and the gene ral contrast between the plasma cell eleme nts and the backgro und centrocytcs , with their lighter nuclei and clear cyto plasm. In 1194. there is an example of a mitotic figure in a myelom a cell with probable polyploidy. while ano the r more norm al mitotic figure can be see n in a neighbouring centrob last.
361
11
1195- 1197. A lymph node biopsy : imp rint preparations. Leishman stain. Node infiltration by testicular teratoma. 1195. Three large tumo ur cells among normal and reactive lymph node cells.
1196 and 1197. Cha racte ristic tumou r cell clumps: the cytological structure of these teratoma cells is quite
distinct from any normal or lymphomatous component to be found in lymph nodes .
362
11
1199
198
1200
1198-1202 . Lymph ov arian carcinoma.
nod e
imp rints:
1201
metastasizing
1198and 1199. Two fields from the same Romanowskystained preparation. to illustrate the striking morph ology of the ovarian tumourcells. with variability in size and degree of cytoplasmic baso philia and large deeply staining nucleoli. The cells are unlike any encountered in either normal or lymphomatous lymph nodes. 12~1202 . The cytochemical stains shown here - the PAS reaction in 1200, an acid phosphatase stain in 1201 , and an alkaline phosphatase stain in 1202 - all show strong positive reactions in the tumour cell cytoplasm, again unlike any cells of haemic origin.
1202
363
12O-a
103
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1203-1208. Lymph node imprints : infiltration with carcinoma lung. 1203. Rom anowsky staining revea ls replacemen t of lymphoid tissue by tu mour cells with fragile and generally poo rly defined cytoplasm, and with smoo th structureless nuclei, occasionally mul tiple . T he re are widespread scatt ered vacuoles . possibly of cytoplasmic orig in. 1204. SB stain shows the lung carcinoma cells to be sudanopho bic. There is a single SB-positive neutrophil in this field , but the rema ining cells are probably all tumou r ce lls.
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1205. Th e PAS reaction here shows a group of tumour cells with mod erately stro ng granular cytoplasmic posinvi ty . surrounded by normal lymphoid cells with darker nuclea r stai ning a nd negative PAS reaction . 1206. Th e dua l esterase sta in in this field reveals CE positivity in a neut roph il and moderatel y stro ng BE positivity in the larger , more lept ochromatic tumo ur ce lls. with generally negativ e reactions in the occas ional small da rk lympho id cells. 1207. Th e acid phosph ata se reaction in this view show s a strongly pos itive macrophage. but no more than a weak scattered gra nular reaction in the tumour cells with little evide nce of focal localization . 1208. The alkaline phospha tase rea ction is positive in several of the large r neopla stic cells.
120"
"
121
1209and 1210. Lym ph node im prints: cervical node with secondary gastric carcifl om a. Low- and higher -power views, respectiv ely, of a Romanowsky-stained preparation, showing a clump of tumour cells with large size, high nuclear-cytoplasmic ratio , irreg ularly cleft and indented nuclei with several poorl y defined nucleoli. and dee ply basophilic cytoplasm. There are two or three degenerating necrobiotic carcino ma cell remna nts a nd two normal neu trophils in each field. 1211. Neuroblastoma cells in a lym ph node imprint. The field shown of th is Romanowsky-stained pre paration contains only two residual Iymphocytes. their dar kly sta ined nuclei sta nding out in contrast to the paler generally leptochro matic nucle i of the preponderating ne urob lastoma cells. Alt hough in this area of the lymph node imprint the whorled clump ing of neur oblastoma cell nests is not apparent. the cells show very consp icuously the feat ure of dense chro matin spots. sometimes called chromoccntres or karyocentres. within many of the nuclei. cha racteristic especially of this type of tumour and see n in few others except the related medulloblastoma.
1211
,
365
120
1111
1212--1215. A clump a/ breast carcinoma cells in a lymph
1214
node imprint. 1212. In this Romanowsky stain the breast carcinoma cells appear as a largely syncytial clump with over 70 nuclei, many overlapping. and some showing earl y necrobiotic changes. with an opening and reticula tion of the nuclear chro matin . Most nuclei have a smooth and flat appearance , with occasional purple nucleoli visible. A diagnosis of breast carcinoma secondary could not be made from this specimen. I2B-1215. Cytochemical reactions ... to the PAS stain in 1213, to acid phosphatase in 1214, and to dual esterases in 1215- all show strong positivity. in the case of the esterase reaction to BE. It is interesting to note that each of these cytochemica lly stained cell clumps shows more clearly defined cytoplasmic outlines and a less syncytial appea rance than in the Romanowsky stain, perhaps because the cytochemical reaction products tend to emphasize the periph ery of the cytoplasm, as is notably the case in the PAS reaction.
1215
12t
1218
1216-1219. Sections of spleen f rom patients with HCL. In this disease the spleen is generally moderately
enlarged, with diffuse infiltration with HCs. The splenic fo llicles, Malpighian bodies, and the whole of the white pulp are much atrop hied and often no longer visible,
while the red pulp shows Q characteristic combination of heavy infiltration with HO and areas of marked pseudovascular dilatation engorged with red cells, giving an app earance resemblin g haemangioma . The pseudosinuses are actually lined chiefly by tartrate-resistant acid
phosphatase (TRA P)-positive HCs rather than vascular endoth elial cells.
1216 and 1217. Low- and higher-power fields, respectively, from a thin section, showin g an area o f diffuse infiltration of sple nic co nical tissue with HCs . The typical halated appea rance of the HCs. with their centra lly placed nuclei, irregularly marked nucle ar
chromatin, andample cytoplasm against an eosinophilic background of red ce lls, is well shown . 1218. A low-power view of a thicker secti on from the spleen of another case of HCL. more strongly staining with H&E , showing the pseud osinuses eng orged with
red cells in the upper half of the field and the predo minance of HCs in the less vascular lower half.
367
• 219
1220
1219. A higher -power view of par t of the same field as that shown in 1218. where the pscudosinuses with their evident lining of HCs, and the characteristic nuclearand cytoplasmic patterns of these ce lls. are shown in more detail. 1220 and 1221. A splenic imprint: He L: Leishman stain .
1220. Typical appearances of intact HCs in spleen imprint. The nuclear pattern and moderate amount of
greyish cytoplasm suggest the diagnosis. although hairy processes cannot be distinguished. A single RE cell is prese nt. but no monocytes, no granulocytes and almost
no normal small lymphocytes. 1221. A spleen imprint from anot he r case of HCL. with rather more variable nuclear patternsand a tendency to cytoplasmic process formation and fragmentation . A few nonnallymphocytes and neutrophil polymorphs can be seen and there is a characteristic background of red cells from the congested pulp.
1221
1213
....
1 22~
1222-1229. Splenic imprints : Gaucher's disease. Since one of the most striking clinical features of Gaucher's disease is splenomegaly , and the diagnos tic cells may not be found in the bone marrow aspirates or trephin e biopsies taken in the course of investigating the accom panying anaemia or cytopenias. this is one of the fe w haematological conditions fo r w hich needle biopsy ofthe spleen may still be diagnos tically valuable. 1222 and 1223. Leishm an stain: various Ga uche r cells show the granul ar . fibrillar and onion-skin patt erns of cytoplasmic lipid inclusion mate rial.
1224. 5B stai n: two granulocytes show suda nophilia but the two Gaucher cells are essentiall y negat ive.
369
• 12'
•
1225 and 1226. PAS reaction: a consecutive pair of photographs o f the same field containing two Ga uche r cells; the first shows PAS positivity of diffuse or finely granular disposition and the seco nd, under polarized light, shows the refri ngence of the fibrill ar inclusion material. The brightly refringent PAS·pqsitive 'hot cross bun' structure at the bottom is a starch granule, from
surgical glove-powder contamination. 1227. Acid phosp hatase: the Ga ucher cells show strong positiviry.
370
•
28
1229
.
,
..
1228. Dua l esterase: two neutrophil polymorphs show normal CE positivity, while the group of Gaucher ce lls show moderately strong BE positivity .
1230
1229. Prussian blue reaction : the Gaucher ce ll in the ce ntre of the field shows free iron staining of varied intensity in much of the cytoplasm.
1230 and 1231. Anothe r dupl icate pair of phot ographs of a field showing starch-powder contamination, similar to that app eari ng in 1225 and 1226. In this instance. the contamination is very much heavier and is seen in a lymph node imprint from a patient with follicular (centroblastic-centrocytic) lymphoma.
1231
371
11
1232
1232. A splenic puncture smear: Leishman stain. A conspicuous cluster of serosa l cells, picked up as the needle traverses the pcritoneallining cells of the spleen surface. The ir flat epithelial nuclear structure and lanceolate cytoplasm arc very charac teristic. 1233 and 1234. Lcishman stain : splenic puncture smear. Malignant histiocytosis (histiocytic medullary reticulosis), showing gross phagocytosis of red cells by malignant histiocytic RE cells. In 1233there is a mitotic figure in one such RE cell, which also contains the remnants of some six or seven erythrocytes, while a second histiocytic RE cell contains within its cytoplasm some 20 erythrocytes. In 1234 a similar cell contains more than 30 erythrocytes .
312
11.:
1235
,
•
•
.,.... •• I
•
\
r •
•
1235. PAS reac tion on the same prep aration as the previous two figures. Th e malignant histiocytic RE cell illustrated here contains four erythroblasts and shows PAS positivity in the surrounding cytoplasm.
1236 and 1237. Prussia n blue reaction on the same preparation at low and higher magnification to show the accumulation of free iron in the malignant histiocytes consequent upon the ingestion and breakdown of erythrocytes.
•
• \
•
,
•
• •
1~
• 1238
123
123&-1243. Submaxillary salivary gland imprint: biopsy of a regional lymph node included this material, which is illustrat ed here /0 aid identification ofsimilar unexp ected cytological biopsy findings. 1238. Leishman stain: clump o f ce lls from a spread and
disrupted salivary-gland acinus. 1239. Leishman stain: a more co mpact clump of acinar
cells. 1240. SB stain: several clumps of secretory epithelial
acinar ce lls show strong sudanophilia.
374
•
I' •
12-1
IUI
•
• •
•
• • • 1241. PA S rea ctio n: further acinar cell clumps showing stro ng PA S positivity of the mucinous secret ory content. Th e very dense PAS· positive materi al near the cell clumps is sta rch from surgical gloves.
1242. Acid phosphat ase prepa ration showing strong positivity. Th e acco mpanying lymphocyte s give some impression of the gia nt size of these salivary gland cells. 1243. Dual esterase : the acina r columnar epithe lial cells show scatte red BE positivity.
•
1:2.
. ----
2~6
124'
•
:' 1244-1247. Impr int preparations from a post-auricular nodular swelling occ urring in a patient with 1(8;21) AML in haematological remission. A local leuka emic recurrence was susp ected , but biopsy revealed only the cerum inous gland cells illustrated here . 1244. Rom anowsky stain of this imprint material shows typical ceruminous glandular epithelium , with the cells containing multipl e dark blue staining granules of secretion. 1245. An acid phosphatase stain shows these cells to be extremely rich in cytoplasmic enzyme. 1246 and 1247. Consecutively stained fields from this imprint material, 1246 bein g a de color ized Le ishman prep ar ation rest ained with the Prussian blue reaction for free iron, and 1247 the same field reph oto graph ed after the slide had been furth er stained by the PAS reaction .
12-18
• ~.
• 12~9
•
1248. A pleural effusion from a patient with HD : Leishman stain : the specimen shows pleural lining epithelial cells, sometimes binucleated (not to be confused with Hodgkin's cells) and a mixed exudate of ne utrophil polymorphs and Iymphocyte s. 1249. A high-power view of a PAS rea ction on pleural exudate cells. Th e large cells with stro ng and coa rsely granular PAS positivity are pleural lining cells. Neutrophil polymorp hs and an occasiona l lymphocyte show the normal expected positiviry.
376
•
Appendix: staining techniques ROM ANOW SK Y STA INS Purifi ed rea gen t meth od IHeyl! R~agents:
(a) Azur e Bvthiocyanate (He yl): 1.5 g. in 200 ml. dimeth yl sulphoxide (DMSO) . (b) Eos in Y (G) (Heyl): 0 .5 g. in 300 ml. methanol. (c) Stock. stai n : Mix: (a) and (b) togeth er. Store in da rk. (d) Buffer : 2.38 g. HEPES in 1 I. distilled water (O.OI M); pH adjus ted 10 6.8 with IN NaGH . Store at + 4°C. (e) Wor king stain: Add 3 ml. stock stain to 41.5 mI. buffer plus 2.5 ml. DMSO. Fresh working slain should be made up dail y. Ted nique: 1. Fix air-dried smea rs for 5 minute s with stock stain. 2. Rinse briefly in distilled water . 3. Cover or immerse in Co plin jar with working stain for 2535 minutes (for blood or bone marrow smea rs, respect ively) . 4. Rinse in distilled water for 2 minutes a nd blot dry . Lelshman
\ 1ay-G rii nwald-Giemsa Reagents: (a) May-Griinwald sta in: Prepare 0.3% so lution of powder in methanol by grinding with pestle and mortar . Filter after 2-3 days. Befor e use dilute 1:1 with buffer solution (phos pha te buffer. pH 7.2) . Diluted so lution should be discarded afte r o ne da y. (b)Gie msa : Add 0.6 g. Gie msa powder to 50 ml. methanol. Sha ke to dissolve. Add 25 mt. glycerine. Filter after 2-3 days . Befor e use dilute 10 ml. stoc k solution with 90 ml . of phosph at e buffer (pH 7.2). Technique: Air-dri ed smea rs of blood or bo ne marrow are used . Coplin jars a re used throughou t. I. Fix 15 minutes in metha nol. 2. Tr a nsfer without blotting to diluted May-Griinwald solulion : 15 minutes . 3. Drain o ff stain o n filter paper without blotting a nd tra nsfer to diluted G iemsa so lutio n for 30 minutes. 4. Tr an sfer to phosphate buffer (pH 7.2) and agitate for 10- 20 seco nds . 5. Rem ove and blot dry.
Reagents: (a) Leishman sta in (dry powder ): 0.15 g. in 100 ml. met hanol. (b) Phosphate buffer pH 7.2 . Techn ique: 1. Fix.air-dried smears in und iluted Leishman for 3 minutes. 2. Di lutel:2 with phospha te buffer for 8-10 minutes. 3. Wash in distilled wate r or phosphate buffer . 4. BIoI dry . N.B. 8-/0 minutes staining fo r periph eral blood sm ears is quite sufficie nt. but bone marrow specimens require 10-20 minutes dep ending on the cellutarity .
-
FREE IRO :-lSTA IN
SUDAN BLACK B
(after MacFadu an and Da vis, 1947)
(aftn Sheehan and Storey. 1947)
1. Fix. air-d ried smea rs in formalin vapour for 30 minut es. 2. Wash in distilled water for 2 minutes. 3. Im merse in Co plin jar co ntaining equal parts 2% potas sium fer rocy anide (Prussian blue) a nd 2% dil ution of pure (37%) hydr ochloric ac id for I hou r . 4. Rinse with distilled water . 5. Counterstain with 0 . 1% nucle ar fast red made up in 5% aluminium sulpha te solutio n for 30 minu tes.
Reag,nls: (a) Suda n black B (SB) (Gurr) : 0.3 g. in 100 ml. abso lute etha nol. (b) Buffer : Disso lve 16 g. crys talline phe no l in 30 ml. absolute et ha nol. Add to 100. ml. distilled wat er in which 0.3 g. hydrated disodium hyd rogen phosph ate (Na2HPO..+ 12H 20 ) has been dissolved . (c) Work ing stai n: add 40 ml. buffe r to 60 ml. S8 so lution a nd filte r by suctio n. Kee ps 2- 3 months. Store in refriger ator .
Staining jars must be iro n-free .
Technique: - I. Fix a ir-d ried smea rs in formalin vapour for ~IO minutes. 2. Wash briefly in distilled water a nd blot dry . L fmmerse in work ing sta in for 1 hour. 4. Wash off with 70% e thanol. 5. Co unte rstain with Leishman o r MGG . -40% wlv fo rm alde hy de saturated filter pap " in bottom of Copiin jar.
377
PAS REACTION
PE ROXIDASE
imodified f rom McMonus. /946)
Mod ified Graha m-Knetl technique
Rtagt lflS:
(a) Peri odi c acid sol ution: Dissolve 5 g. pe riod ic acid crystals in 500 ml. dist illed water . Sto re in dark bot tle. Keep s for 3 months .
(b) Basic fuchsin: Dissolve 5 g. basic fuchsin in 500 ml. hot distilled wate r. Filt er when coo l. Saturate with SOl gas by bubbling for 1 hour. Shak e with 2 g. activated cha rcoa l in conical flask for few minut es unt il j ust clear a nd filter imme diately through Wha tm an No . I filter into a dark bottle. Charcoal extraction should be done in fume cupboa rd . Solution kee ps for 3--6 mo nths de pending o n how
often used. Technique: 1. Fix air-dried sme ars for 10 minutes in IQ ml . 40'Yo forma lin and 90 ml. e tha nol so lutio n. 2. Wash bri efly in tap wat er . 3. Treat with periodic ac id so lutio n for 10 minut es . 4. Wash in distilled water a nd blot dry . 5. Imm er se in Schiff's basic fuchsin in Co plin ja r fo r 30 minutes. ( Return fuchsin solu tio n to stoc k bo ttle imm ed iatel y after use. ) 6. Wash in tap wate r for 5-10 minut es . 7. Counterstain with aqueous haematoxylin for 10- 15 minutes. Co ntro l smears ar e exposed to saliva ry digestion for 30 minutes between stages 2 a nd 3.
ALKAI.ISE PH OSI'll,\TASE
Rt agtnLf: (a) Stock propanediol buffer . 0.2M: Dissolve 10.5 g. 2amino-I-methyl propane-I I :3)-diol in 5(X) ml. distilled water . Store at 4°C; discard after 3 months. (b) Working buffer . 0.05M: Mix 25 ml. stock buffer with 5 ml. O. IN HQ and make up to 100 mt. with distilled water . (e) Subst rate. to be made up freshl y immediately before use : Sod ium alpha-na phthyl phosp hate: 35 mg. Fa..t Garnet GB C salt: 35 mg. Working buffer : 35 mt. (d ) Methyl green : 2% in distilled water . freed from co ntamination with met hyl violet by extraction with chloroform and kept free by storage at room temperature in co ntinuo us cont act with chloroform . Tuh niqut: 1. Fix air-dried smears in 10% formalin in absolute methanol for 30 seco nds (use sto pwatch) at Cl-5°C. 2. Pour fres hly pr epared substrate directl y on to slides and incubate for 5- 10 minut es at room temperature. Substrate must be used with in 5 minutes of preparation . 3. Rinse slides in tap water for 10-15 seco nds . 4 . Counterst ain with meth yl green for 1ll-15 seco nds . Good posi tive co ntrols are provid ed by slides from pol ycythae mia. infection. hairy ce ll leukaemia o r Hodgkin's disease .
I. Fix air-d ried smears for 30 seconds (use stopwatch ) in 10 ml. -to% form alin a nd 90 ml. ethanol sol ution at room temperature . 2. Wash with tap water for 10 seco nds and blot dry . 3. Dissolve app rox . 250 mg. of hen zidine or o-t otidine " in 6 ml. eth anol a nd dilute with 4 ml. distilled wate r- Add 0 .02 ml. hydrogen peroxide (20 vol. ). When solution i co mplet e. pour o nto slide without filtration a nd a llow to react for 7 minutes . 4. Wash wi th tap water for 10 seco nds a nd allow to dry in ai r . 5. Counters tai n with Lcishm an , diluted immediate lv with buffer, or use stand ard Ma y-Grunwald-Gi em sa tech niq ue for co unte rsta ining . • N .B. Both these compounds are carcinogenic and should be handled with care. Diaminohenzidin e ((lAB ) method for peroxid ase R~agtn ts:
(a) Fixative : Buffer ed fonnol ace to ne (B FA) - Na 2HPO.. 40 mg. , KH 2PO.. 200 mg. , ace to ne 90 ml. . co nce ntrat ed form alin 50 ml., distilled water 60 ml. Stor e a t 4°C a nd use co ld for fixatio n. (b) Stock phosphat e buffer : Dulbecco A , pH 7.3 - NaCl-tO g.. KCl 1 g. . anhydrous Na2H PO .. 5.75 g. , KH 2PO .. I g. . distilled water I 1. (c) Working buffer: Dilut e I I. stoc k buffer with 4 1. distilled water. (d) Subs tra te : 3.3 'd ia mino benzi dine (D A B) . (c ) H yd ro gen pe rox ide ( 1(x) vol.). (f) Working incubatio n solutio n: Dissolve 30 mg. DAB in 60 mt. wo rking bu ffer and add 120 1. of lOO va t. HlO l ; use immedi at ely. (g) Car azzi's aqueo us haematoxylin : Disso lve 75 g. potassium aluminium sulphate in 121) ml. warm dist illed water ; add 1.5 g. haematoxylin powder dissolved in 3(X) ml. glycerol by grinding with pestle and mortar ; dissolve 0.3 g. sod ium iod ate in a littl e wat er a nd add grad ually. TtC'hniqut: I. Fix air-dried smea rs in BFA fo r 45 seco nds . 2. Rinse with distilled water and drain dry . 3. Incu bat e in working substrat e so lution for 10 minutes. 4. Rinse with distilled water. 5. Counterstain with Ca razzi' s (or ot her wat er -soluble ) haematoxylin for I minute . 6. Rinse with distilled wat er and a ir-d ry.
AC ID PHOSPHATASE
Reagenu: (a) Naphthol AS·B I phosphoric acid : 10 mg . (b ) Fast garn et GB C salt (G urr) : 10 mg . (c) Walpolc's acetate buffer , O.IM, pH 5.0: 50 ml. (d ) Filter freshly prepared substrate into Coplin jar . TeC'h niq u~:
1. 2. 3. 4. 5.
Fix ai r-dried smea rs in forma lin vapour for 4 minut es . Wash briefly in ta p wat er and blot d ry . Incubate in subs trate sol ution a t 3TC for 1-1 '12 ho urs. Wash briefl y in tap water . Co unterstain with aqueous haematoxylin for 11l- 15 minut es. For assessment of tartrate resistance add HXJ mg. U + ) tartaric acid to the substrare.
• DUAL ESTERASE
Reagents: (a) Chlo ro acc tate substrate so lutio n : use at o nce O.JM phosphate buffer (pH 8 .0) : 10 ml. Alpha-naphthol A S-D chloroaceta te : 0.25 mg. (O.7 x 1O--4 M ) in n.l ml. acetone . Fast blu e BB salt : 15 mg. (Sx lO-·l M). (b) But yrat e substra tc solution: use at once O.IM phosph ate buffer (pH 8.0): III ml. Alpha -naphthyl butyrat e : 0.5 mg. (2.33x IO-" M) .
Fast garnet GB C salt: 3 mg. (9 x l~M ). For testin g fluoride inhibition , add NaF 1.5 mg.lml. to buff er .
Techniq ue: 1. Fix air-d rie d smea rs in form alin vapour for 4 m inutes . 2. W ash briefly in di stilled wat er a nd blot dr y. 3. Incubate in freshly prepared chloroacctate substrate solution fo r 5---15minutes at room temperature . 4. Wash briefly in d istilled water and blot dr y. 5. Incubate in freshly prepared butyrate substratc solution for 15-30 minutes, at room temperature and away from light. 6. Wash briefly in distilled wate r. 7. Counterstain in aqu eou s haematoxylin for 5 minut es. 8. Wash in distilled water . blot dry and examine .
IMM UNO CYTOCH EMI CAL M ETH ODS
Immun oper oxidase - indirect PAP (peroxidase-antiperoxidase) method Reagents: (a ) Specific primar y antibodies. e .g. ra bbit ant i-human Ig anti sera . specific for the different heavy and light chains; mou se monoclon al antibod ies. (b ) Appropriate seco ndary antibodies. e.g. swine anti-rabbit immun oglobulin ; goa t anti-mo use immuno globulin. (c) Appropriate rabbit or mou se antipe roxidase- pe rc xidase co mplex. (d) DAB pe roxidase cytoc hemical reag ent s. A solution of 5 mg. DA B in to ml. tr is-salin e solution plus 1 drop of 100 vol. hydroge n perox ide is freshly prepared and filter ed befo re use. Technique: I . Fix air-dried smears in methanol for 10 minutes to block endogenous peroxidase . 2. Add primary antibody and incub ate for 15-30 minut es. A titrat ion effect may be achieved by using con centration s ranging from 1 in 20 to 1 in SW . 3. Wash in tris-saline solution for 5 minute s. 4 . Add seco ndary antibody at 1 in 20 to 1 in I(X) for 15-30 minu tes. 5. Wash as in 3. 6. Add PA P oomplex 1 in 20 to 1 in 40 for 15-30 minut es. 7. Wash as in 3. 8 . Ad d DA B substrate and incubate for 10 minut es. 9. Co unters tain with aqueous haem atox ylin 1 minute . rinse and blot dry .
Immunoalkaline phosphatase - indirect alkaline phosphatase-anti-alkaline phospha tase (A PAA Pj methnd Reagents: (a ) Primar y antibodies - any mur ine monoclon al . for example. (b) Appropriate seco ndary an tibodies - anti -mou se immunoglobulin in this case . (c) APAAP complex, con sisting of alkaline pho sphatase and mou se monoclonal antibody specific for alkaline phosphatase . (d ) Incubation mediu m for alkaline phosphata se . 2 mg. of naphthol AS~BI (or AS-MX) pho sphate are dissolved in 0.2 ml. of N ,N-dimeth ylfonnamide , to which 9.8 ml. O.lM T ris-H Cl buffer, pH 8.2. are added . Immediately before use , add 10 mg. Fast Red TR salt (or ano the r suita ble diazonium sa lt) and filter directly on to slides .
When necessary, endogenous alkaline phosph atase activit y ean be inhibited by addin g 0.24 mg.lml. (l mmol./I.) levam isole to buffer. Technique fo r smears and imprints: 1. Fix air-dried smears in buffered formalin-acetone (Na zHP0 4 20 mg. , KH 2 P0 4 100 mg.• aceto ne 45 ml. . co ncentrated formalin 25 ml. , distilled water 30 ml.) for 30 seconds at room temperatu re. wash briefly in distilled water and allow to dr y. 2. Immerse in 'Iris-buffered saline (TBS ) for 10 minutes, then tip off excess. 3. Add primar y mouse rno noclonal antibody and incubate in moist chamber for 30 minutes at room temp erature. Optimal di lution of MAb in TBS has to be selected for individual MAbs . 4. Wash slides in TBS with three changes. 3 minute s each. 5 . Add anti-mouse Ig , diluted 1:25 in TBS. for 15-30 minutes. 6. Wash as in 4 abo ve . 7. Add APAAP co mplex , diluted 1:50 . in TB S, for 15-30 minutes. 8. Wash as in 4 a bove . Intensity of final staining may be increa sed by repeating steps 5-8 . with reduced incub ation times. 9. Add freshly prepared alkalin e phosphatase incub at ion medium for 15-30 minutes. 10. Wash-briefly in tap water . rinse in distilled water and allow to dry . . 11. Counterstain with haematoxylin for 2 minute s. 12. Mount slides in an aqueous medium such as glycerine jell)' orinDPX.
379
• Ch loramphe nicol. effect in eryrhro blas ts 75 Chromos omes (see Leukaem ia . Lymphom a) N umbers in light type refe r to page Chronic le uka e mia (see Le ukae mia) re pictu to bold in ose th , xt te numbers in Congenit al uyserythr opcietic an aemia and ca ption numbers. (C D A) 7l1-l18 Cottage loaf ce lls (see Rcd ce lls) A bbrev iati on s 380 Crenation (see Red ce lls) Aca mhocyres (st't' Red ce lls) Crescent form ation (see Red ce lls) Acid phosphatase reaction (see a/so Crys tals ind ivid ua l ce lls and disorders ) - in plasma ce lls 819. 821 . 822 - tec hni que 37H - in RE ce lls 864-8M. 879 Acute leukaemia (st't" leukaem ia) Dohle bodies . 69 , 390-393 A du lt T-cellleu kaem iallympho ma (A TL ) Do wn's syndro me 192 - tran sient mye loprolifer ative d isorder in A lde r's a nomaly {Alde r-Re illy a nomaly) 70. 566-568 389 Drep an oc ytes (see Re d ce lls) Alk a line phosphat ase re actio n (set" aim D rum stick a ppendage s 259. 26 1- 263 indi vidual ce lls and disorders ) Dual esterase reaction (see also individua l - technique 378 cells and d isorders) Amyloid materia l in macroglo bulinaemia - techn ique 379 1147 Echinocy tes (set' Red cells) Anaemia Elliptocy tosis (see Red cells ) - ap lastic and hypo pla stic l-l , 24-27 Endothel ial cells . vascu lar. in blood 962- co nge nita l d ysery th ro poi e tic (C D A) 14, 966 7l1-l18 Eosinoph il (set' Myelocyt e . Polymorp hs) - dimorphi c 8, 9 Eos inophilia 70 - hae mo lyt ic 13. 19-2 2, 129. 135-137, 15K- in ALL 62 1 ~2-I 160 - in A ML 292-JO.1 - - foetal 37 - familial 2H9-29 1 - iron deficienc y 15. 1~IOH . 111. 132. 133 - reactive 283. 8-15 - le ucoe ryt hro blasnc 15 . 131 Eos inophilic granuloma 1109-1120 - macrono rmobla...ti c 11.31- 37 Eosinoph ilic sta ining. diffuse. in myelocyt es - megalobl astic 12. 39-69 560-562 - - gran uloc ytes in 12. 69.73.74. 257. 265 E pithe lioid ce lls 32.... 1097 (see aim - pe rnicious (see Me ga lo blast ic ) Mo nocy te-macr o phage system) - sickle cell 13. 15-1. 155 Ery rh raemic mye losis a nd ery throleuka emia - side ro blasric 15. 112- 116 1... . 89-105 An gioimmu noblasuc lymphade no pa thy ... acid phosphatase in 103. 10-1 (A IL ) 6I\1-M.l - dual esterase in 105 A nisocytos is (see Red ce lls) - PAS re act io n in 1JK-102 A rtefacts - pe roxidase reactio n in 96 - buccal m UCO!\aI ce lls in a marrow smear - Prussian blue stai n in 118 970 - Su da n black stain in 97 - crescent ce lls (set' Red cell s ) Erythroblasts 11 - droplet co ntaminati on ofhlood smear - ab normal 11, 19-69.72 . 73.75-11 8 967.968 - in CDA . 78-88 - Ferrata stages of degenera tion 969 - giant . in eryrhraem ic myelosis 90-9-1. IIN) - pseudo- elli ptocy tosis (set' Red ce lls) - no rm al (see Normobl asts) 11. I-I S - se baceous skin cell in a ma rrow smea r 972 - PAS reaction in 36. 37. 84. 98-102. IUS. - starch granule contamin ation 1225. 1226. 118. 1-19. .....9 . -153 1230. 1231 Es te rase (see Du al es terase reactio n) Auer rods . cytology and cytochem istry o f Fa t cells. stro ma l. in ma rro w smear 973. 199. 200. 202. 203. 205. 208-225.2 29.231. 974 237-239.2-I1 .2-I2 .~2-I7 . 25 1 .-I 1 5 .41 7 Flamin g ce lls KlI7-809 B ce lls (set' also Le ukae mia . chronic Foe tal haemoglo bi n 18 lymphocy tic a nd prolymphocytic. a nd Foe tal haemolyt ic d isease . PAS rea ct io n in Lym pho ma . non-Hod gki n's) 7.190. 6-13. erythrobl asts 37 647 Foll icu la r ce ntre ce lls (see Lym ph node Bartone lla baci llifo rm is infe ctio n 1033 imprints and sections. Lymph o ma ) Basophils (see Mye locyt e . Po lymorphs ) Gauche r 's d isease 269. 881-889. 1222-1 229 Basop hil sti ppling (see Red ce lls) G lycogen (set' PA S rea ction in specific Birefringe nce (see G a uc he r 's d isease . condition s) Re ticu lo-endo the lia l cells . Starch - inclusion bodies in megakary ocytes 465. gra nu les ) -169.470 Bla stomycosis 10M - sto rage disease 269 . 149.899-9 04 Brilliant cresy l blue sta in 71. 127. 129. 161. GranuI0C )1es.67. IM-W-I (seealso 162 Met am yelocyte . M yel o blast . M yelocyte . Bu rkitt 's lymph om a . marrow invasion in Po lymo rph s . Pro myelocy tcs. Sta b ce lls) 7~747 Grumpre cht's shadows 676 Burr cells Isee Red cells) H ae mo glo bin-F. Klei ha ue r reaction for 18 Ca bo t ri ngs 156. 157 Haem o glo bm -H inclu sion s 162 Centroblasrs a nd centrccyres (see Lymph H aem o po iesis . cu rrent concepts . activator s node imprints and sections. Lymphom a) and inhibitors 7.8 Ce ru mino us gland cells in post-auri cular H airy ce lls (see Leukae mia . hai ry ce ll) nod e biop sy lz.u-I2-I 7 H and-Schu ller -Christi an dise ase 269 . 891 Chedia k- Higashi-S teinbrinck anomaly 70. Helmet ce lls (set' Red cells) 398 He inz bodies 160 Ch e modecto ma ce lls in marro w IJlJK-IOO3
Index
Hisriocyt es, sea blue 869-873. 898 (see also RE ce lls) Histioc ytic medullar y reticulosis ( set' M alignan t histioc ytosis) H istioc vtosis-X 269 . 891 , 89 2. uos-r 120 Hod gkin's dise ase (H D) 32-1 - lymph node sections and imprints in 11211I.l8 - ma rrow granulom a in 893 . 89-1 - marrow infilt ra tio n in 1025-1028. 1 0~'O1032 - Reed-Ste rnberg cell le ukae m ia in IU29 Ho we ll- l o lly bodies ..... 47. S I , 62. 1-16. 1-17 - ge nesis of .56 H ypoc hro m ia (see Re d cells) Immunob lasts ae-t, 649-660. 1~1 066 . 107-1. 1078 (set' also Lympho m a. nonHodgkins. imm uno blastic ) - acid phosphat ase in 1078 - a lka line phospha tase in 1074 - PA S re action in 656 - pe rox idase reaction in 657 - Sudan black re act io n in 1071 . Im munocyroc he mical met hod s (see indiv idu al ce lls an d d isorders) - technique s 379 Inclusio n bodies - in immu no blasts 1169-1171 - in leucocyte s 396. 397 - in plasma cells 81 2-81 7. 821 . 822 - in macro glo bulinae mia 849-S..lil Infect ion (see Leucocytos is of infection ) Infectiou s mononuc leosis . Iymphocy tes in 6-19-651 Iron , free (see also Pru ssian blue stain) I.... 15. 109-118. - ...purious sudanoph ilia on 977, 978 Kala-a zar 1035-1 ...... Kle iha uer re action 18 Langerha ns cells 269 . 891. 892. 11(~1120 Langhans ce lls 269. 10'l8. 1099. 1100-1105 La rge granular lym phocytosi s (LGL) 192. 643. ... LE ce lls 384 . 385 Leishman stain 3n Le ishman-D onovan bodies 269. 1035--10+1 Leis hmaniasis (see Ka la-aza r ) Lep tccytes (see Red cells) Leuere r-Siwe syndrome (see Histiocyt osis-
sn
X)
Le ucocytcs (see G ra nulocytes . Lymphcc yrcs , Mon oc yte . Normal blood leucocyte s. No rmal bo ne marrow cells) Le ucocytosi s of in fect ion - ba cte rial - - alkal ine phos phatase in 273 - - Dohle bodies in 390-393 - - immu noblasrs in 65-1.657. 658 - ... to xic gran ules in 390...392. 658 - viral a nd mycoplas mal - - imm uno blasts in 649-653. 655, 659. 660 Le ucopenia 69 Le ukaemia - acu te lymp ho blastic (A LL) 190. 595-639 - - acid phos phatase in 605. 618, 623 - - d ual esterase in 606 . 619. 620. 63 2 - - cytogene tics of 190 - - eos ino ph ilia in 621-62-1 - - FA B class ifica tion of 190 - - - L1 . L2 . L3 va ria nts illus tra ted 596. 597.610. 612-617. 627 - - imm unocy toch emi stry in 607-609. 628. 62" - - immunol ogica l classifica tion of ... - - nul l. C- A LL. pre-B . B- a nd Tvari ants illustr at ed 607-610. 613--617. 625-<>29.633-<>39 - - normoblastic hyperplas ia in 6.'6
381
....
- - PAS reacnon in 600 . 60 1. 603. 6l).S . 615 . - - ptrox)dao;,r reaction in 59'9. 6JO - - Sudan blacL. stai n in 598. 611. 614. 626.
iJ l - acute mi xed lympboclas nc-myeloblastic phenotype 19 1. 6JO-632 - acu te myeloi d (AML) 67 - - acid phosphatase in 2U6. 243. 255. 422. ' $1 - - cyt o ge ne tics of (set' also specific defects below) 68 - - d ua l este rase in 193. 207. 224.225 .229. 2.a.a--2-I7. 256. 303 . JO..&. 3 13• .s2J . 42-1. 427. .s42• .s43. 446. 455. 502. 510 - - erythro- I ~ , 89-105 - - erythromyeloi d 450-455 - - erythromyetomonocytic ....7-449 - - FA R classificat io n of 68 - - immunocytochemistry in I'M. 198 - - megakarycblastic .$82--495 - - monoblastic/monocvnc 410-4.0 - - myeloblastic 190-2 15 - - myetomonocyuc 4-$-I.--U6 - - PAS reaction in 192. 197.20.1.205. 22122.\. 228. ~U2 . 2..10.&. 301 • •'02. 312 . 419-421 • .s26 .~39-JJI .""9 • .s53.4S7 . 492--495.501 . 519.532 - - peroxidase re action in 200. 2 16. 217. 415• .s16. 5 11-513 - - promy elocy nc ( A PL) wit h I ( 15;17) 226256 - - Suda n blac k ..rain in I 'll . 196.202.203. 218.219.227.237-239.253. 299.300. 311. 411.413.414.41 7.4.\7.438.445. -148. 452.486.491.499.500. 514-518. 530.53 1 - - Type lfType 11 lineage a nd
differentiation classification 68 -
-
-
- with basoph ilia J09-3 13 - with defec ts of 5q o r 7q 521-523 - with inv o r ins 3;3 488-195 - with t(6 ;q ) 524-532 - with t(8 ;2 1) 208--225 - with triso m y IS 496-502 - wit h cq- 5Ol-520 - wit h Ilq23 ab normalities ~3 - with 16q abnormalities 292-J04 chronic lymphocytic (CLL) 191 . 676-700 - aci d phosphatase in 681 - alkali ne phospbatase in 6IU - d ual esterase in 682. 683 - gra nular (G LL) 192,643,64-4 - immu nocy toch e m istry in 685. 686 - ma rro w histo logy in 687-692 - ma ligna nt tra nsformati on - Richt er's svndrome 69l-700 - "PA S re act io n in 679. 680 - smear ce lls in 676-678 - T-cetl va ria nt 686 chronic mye loid (C M L) 68. 320-JSJ - acid phosp hata se in .M7. 361 . 369 - alka line ph osphat ase in 332-3.M - basopbils in 305, 320. 321. 33 7, 371-37.1 - bla stic crisis (se!' mal igna nt progr essio n be low) - defective ne utro phil gra nu lari ty in .\25 - du al este rase reaction in 348. 362. 370. 3lI2
- - ju ve nile form o f 68 . 375-383 - - liver infiltration in J28 - - malignant progression to - - - lympho blast-type cris is .\63-370 - - - mye loblast-type crisis 349-362 - - - myelofibr osiv megakaryocyuc/ myeloprolifera tive state .\.\1J..-.3.sH - - ma rro w histology in 326. 327• .\39-JoU. 349--351 • .l6.\-J6S - - PA S rea ction in .3-16••160. 368. 373
3 2
-
- pe ro xidase rea ction in 335 - Ph chro moso me in 332 - skin infiltrati on in 329-331 - Sud an black stain in 336-338. 359. 367 chro nic myelomonocytic (C M ML) .wH. J09 . 588-59" - emerging re mission in acu te fo rms , cytologica l fea tures of 536-547 - hairy ce ll (HCL) 191. 7" 8--772 - - acid phosphatase in 75H. 759. 765. 1177 - - a typical variant 762-766 - - azu ro phil inclusions in 751 - - bo ne marrow secti ons in 767-712 - - du al este rase in 760. 766. 1178 - - lymph node impri nts in 1175-11 78 - - ne ut rophil alka line phosph atase in 76 1 - - PAS reaction in 752 . 757. 764. 769. 1176 - - peroxidase rea ction in 657 - - phagocytosis by hair y ce lls 755 - - ribosome-lame lla co mplex in 7.as - - sple nic sec tion a nd imp rints in 12161221 - - Sudan black sta in in 756. 763 - - vacuol ated cyt oplasm in 750. 755 - histiocv nc 269 , 905-921 - - acid phosphat a se in 9 1" . 915 - - alka line phosphat ase in 921 - - d ua l est erase in 916. 9 11 - - fre e iro n in 918--920 - - PA S reaction in 9 12. 913 - - Sudan black sta in in 910. 911 - mast ce ll 957-%1 - plasma cell ( PC L) KOI~13 - prolymphocytic (PLL) 191, 66J--675 - - acid phosphatase in 670. 672. 674 - - B-eell varietv of 664 . 666 . 669. 67" . 675 - - d ual esterase in 671. 673. 675 - - PAS reac tion in 6tIS. 669 - - Sudan black stain in 667 - - T-cel l variety of 665. 66H. 670-673 Leukaem ic reticuloe ndot hel iosis (L RE ) (see le ukaemia , hairy ce ll ( HCL» Lymph node im prints a nd sections 3H - cyto lo gy a nd cytochemistry of 1064-11 78 - - in ca rcino ma - - - br e ast 1212-1215 - - - gastric 1209. 1210 - - - lung I lOl-l 208 - - - ovary 1198-1202 - - in chronic lymphadenit is 11.s9-1151 - - in HC L 1175-1178 - - in HD - - - Iymphocy te pred o min ant 1121. 1122. I IJ.l- I 136 - - - mixed cellularity 1128--1133 - - - nodula r sclerosing 11~1127 . 11371139 - - - Reed-Sternberg cells in 1131-1 1.&7 - - in myeloma 1192-1194 - - in neuroblastom a 1211 - - in non -H od gkin 's lym phoma (NH L) (s!'t'tJb o Lym phoma) 1152-1171 - - in re acti ve hyperplasia 1064-IOH2 - - in sarcoidosis 1097. 1098 - - in secondary testi cul ar terat om a 11951197 - - in tox oplasmosis 1083-1096 - - in tubre rculo us lym phadenitis 1099 Lymphoblasts 190, 595-639 (see Leukaemia, acute lymphoblastic (A LL) . Lympboma . fympboblaenc) Lymp hocytes 7, 190, 6.atJ-6.I8 - azurophil gra nu les in 642 - B-cell fea tures 643. 647 - bin uclcated 641 - granu les in A lde r 's a no ma ly 389 - reactiv e {see- lmmunoblasts) - T -ce ll fea tures
- - T -r-s cppressor ce lls 6-12--6-$.4. 6-16. 647 - - T- ~-hel per ce JJ s ll4.l--6.s8 Lym phoma 192, 32..a - Hod gkin's (st't' Hodgkin's disrObe) - non-lIodgkin's (NHL) - - acid phosph atase in 721. 1156. 1167 - - a lkalin e phosphat ase in 725 - - Bu rkitt 's , marrow inv asion in 74-1-7..a7 - - centroblastic 702. 70S, 711. 7 12. 7~7J3 - - ce ntroblastic-cenrrocytic 72l-725. 11521162 - - ce ntrocvr ic 70 1, 70.1, 7 13- 7 15 - - c1as...ifi~tion o f - - - Kid 32..a, 325 - - - Wo r king formul ation 32..a , 325 - - cytogene tics in 325 - - dual esterase in 709. 722. 1157. 1168 - - foJlicular ce nt re ce ll 701. 72l-725. 11521157 - - histioc ytic , t rue (T H L) II~I 191 - - immunoblastic 703. 706-7 10. 1169-11 74 - - Le nnert's 773-775. 1179-11 87 - - lym pho blastic 716-720. 7.w. 741 - - - B-eelltype 7.w. 741. 74-1-7,,7.11 6l1168 - - - T-eell type 716-722 - - PA S re actio n in 70-1-706. 720. 724 . 747. II SS. 1166. 1171 - - Sudan black Sla in in 746. 1154. 1165 - - T-cell . peri pheral 77l-775. 1179-1187 Macroc ytosis (see Red ce lls) Macro glob ulinae m ia 192, ~51 - dual est e ra se in 839 - Durcher bodies in 8-19-851 - iron-laden macrophages in 84fl...S.t3 - mast cell s in 8.14-837. 839 - PAS reaction in 838 . 8..~. 851 Macronormoblasu 11, 31-35. 37 - PAS re actio n in 37 Macrophages (see RE ce lls) Malari a l pa rasit es 1046-1060 Malignant histioc ytos is 269, 922-927. 1233-
.237 M arr o..... histo lo gy in tre ph ine biopsy sections in
- Al l - - at relapse 637-639 - - during remi ssion ind uctio n 6JJ-6J5
- A Ml - - in assessing remis..io n ind uct ion and re lap ses 551-559 - - multilineage 5J4, 535 - - - secondary to M DS 533 - A MonL 428--433 - ang icimm unobtastic lymphadenopathy (A l l ) 661-663
- A Pl :z.l8-251 -
aplastic a nd hy poplastic a na e mia 2.&-27 artefactua l globules in 979 ca rci no ma - breast 1014-1018 - kidn ey 1022 - prost at e 1021 - thyroi d 1020
- Cl l - - foca l a nd d iffuse infiltra tive patterns 687-692 - - transfo rm a tio n to Rich ter's synd ro me 698--700
- CML326. 327. ...1 - - mal ignant progre ssion of 339-.lU• .M9357• •\6l-365• .a8J - Ewings sa rcoma 1024 - fibrosa rco ma 1023 - Gauche r 's d isease 886--SS 9 - hae mol ytic an aemia 19. 22 - HC L 480 . 767-772 - - fat -lad en macr ophages in 975. Q76
- histiocytosis-X 892 - H od gki n's d isease 283. 893. 894 .10,:40IO~U
- kala-azar 1l}.lJ. IO-U - leucocvtosis . infective a nd reactive 279-
283 ' - macroglobulinaemia SoU...S51 - malignant hi...ti0C)1OSis 92$-927 - M DS 122-124. 167. 168.569-571.575-
58..'
•
- - hi...uocvtosi ... in 895-898 - - pse udo-Gaucher ce lls in 874 - medullobla...toma 993-997 - megalob lastic a nae mia 39~ 40 - myelofibrosis 30. 339. 340. 559. 940. 941 - mvelo ma SOO. 828-830 - n~uroblastoma 9AA-992 -N HL - - Bccell. Burkitt ce ll type 742. 743 - - cen troblastic 712 - - cen troc ytic 7 13-715 - - cemrocyuccentrob lastic 734-736 - - immu nobtastic j tu, 711 - - lymp hocytic-ce ntrocytic 737-739 - - T -cetl . convol uted lymphoblastic 719 - - T-cell. periphera l 773-775 - normal 166 -- polycythaemia 23. 28-.\0 - thrombocvtbaem ia 482 -- thrombocytopenia . autoimmune .lS. 472. 473 -- VAHS 928-931 . 940. 941 Mast cells 953-961 - d ual esterase in 960 - in macroglobulinaemia 8J.&-.837. 839 - PAS reaction in 961 - Suda n black sta in in 9$9 May-Grun wald Giemsa sta in 377 May-Hegglin anomaly 70. 394. 395 Med ullobl asto ma 993-997 Megak ary o bla sts 67 . •\41-348. 460. 484. 4X5 Megakaryoc yt es 67. 456-462. 465--195 -- in e me rg ing remi ssion o f ac ute leukaemia
536-53_ - fragments in periphe ral blood
345-~\4X .
462 - glycogen inclu..ion bodies in .ui5. 469. 470 - ph agccyrosi... by 4774 79 Me gaka ryoc yt ic myelosis 339-.348 . 482-195 Megalobla...ts 11. 39--{,9. 72. 73 - gigantic .$X - ' inte rmed iate ' or ' transit io na l" 63-69 - nuclear extrusion in 60. 61 - rosette formal ion in 53. ~ Met amvelocvte 67. 17B-172. 178-180 - eos ino phil'.284 - gia nt 69. 545 M icrocytosis (st't" Red cells) Micronormoblasts 12. 106-108. III - PA S positivity in 1118 - Pru .....ian blue reac tion in I II Monoblast 67 ( "f(,f' leukaemia . AML. mon oc yric/mono bla stic . myelcmon ocytic) Monocyte 67 . 174. 177.405--107 (St't' leukae m ia. A ML. monoc ytic/ mo no bla...tic. mvelomo nocv tic) ... ca rbo n particles' in 399 . - gra nu le.. in 4415-1( 17 ... vacuoles in 406
Monocyte-macrophages 324 ... in lymph node impri nts 1067. 1070. 1079. 1131-1135 - in peripheral blood from bacterial e ndocarditis 855-S57 Monocvtosis . rea ctive 70 Men c~lI.. 814. HI5 Multinucle a t ed giant cells in chronic Iymphade .uis 1149-1151
M velo blast 67. 169. 170. 173-175. 178 (set' leukaemia. A ML. myeloblastic) Mye locy te 67. 169-IU. 178- 180. 185. 188. 199 (see leukaemia . C M L) - basophi l 305 - eosinophil .284 - giant 257 Myelodys plastic synd ro me (M D S) 69 . 7-9. 119-121.560-565.569-59-1 (seealso Leukaemia . C M M L. Refract ory anaem ia (RA). with sideroblasts ( RA S o r RSA ). with excess blas ts ( RA E B)) Mvelofibrosis - ~arrow histology in se, 339 . J.W. 559. 9-10.941 - red cellsi n 131 M yelogranular d ysp lasia 560-562 Mvelo ma 192.798-833 (.fet' also Plasm a
Cells) - ac ute leu kaemic tran sformation o f 83 1-
lU3 - lymp h nod e imprints in 1192-11 94 a-Naphth yl acetate es te rase in megakaryccytes 476 a-Naphthyl butyrate es terase (see Dual este rase) a -Naphthyl chloroacetat e esterase (see Dual esterase ) Neurobla..tom a cell.. 9~1J92. 1211 Normal blood leu cocytes -- acid phosphatase in 318 . 6.U . 645 - dual esterase in 189. 319 . ~ - PAS react io n in 186.314 - peroxidase rea ction in 184.316 - Sudan black Sla in in 181-183.315 Normal bone marrow cell s 166 (see also indi vidual ce ll type s ) - acid phosph at ase in 187 - d ua l este rase in 188 - PA S re action in 185 - peroxidase re action in 179 - Pru ssian blu e sta in in 110 - Su dan black sta in in 180 No rmoblasts It . 1- 15 - in iro n deficiency a nae mia 106-108 - - PA S po... it ivit y in 108 - nests around central macrophage 11...15 -- - alkali ne phosphatase in 13 - - Prussian blu e sta in in 14. 15 Nucle ar bridging - in CDA 78. 79 - in myel oma 804 Nucle~r twinning - in A PL 227. 233-235. 240 - 'in myel oma 804 - in neutrophil potymorphs 264 Osteoblast.. 943-952 - acid phosphatase in 948. 949 - alka line phos phat ase in 946. 947 - PAS re actio n in 945 Os teoclast 942. 950-952 Pap pen he imer body 147 PA S (periodic acid -Sch iff) reactio n (reindiv id ual ce lls and d isorders ) - tech niq ue 378 Pelg er -H uc t anomaly 70. 387 • •'88 Pe ro xidase reac tio n ( "H't ' individua l ce lls and disorders) - techn ique 378 Pe ye r' s patches 1911 Ph (Ph ilad elphia) chro moso me 322 Plasma cell ... 19(1. 789-&33 - acid phosphatase in 802. 82..". 826 - in A MonL "12 - azu ro phil rods in 819 - in ca t-scra tch disease 789 - comparison wit h normoblasts 10 - crysta lline inclusion... in 821. 822
... cytopla...mic disruption in 805 - dua l est er ase in 803. 827 -- flaming a ppearance of cytopla... m in 8071lO'I.820 - inclusions in vacuoles in 820 - iron . free stai na ble in 792-797 - locular degenerati on in 818 ... multiple nuclei in 806 - in mveloma 798-83~' - nucl e ar bridging in 804 - nucl ear inclusions in 812. H13. 818 - PAS re action in 813. 817. 822-S24 ... in plasma cell leukaemia 801-803 - Russell bodies in 814-817 ... the ..a urocv te varian ts of 81U. 811 Pla s modi um - falcipa rum 1046-1048 - malari a e 1049- 1052 -- ovale 1054' - viva x 1053. 105.5-1lI60 Plat elets 70. 456-459. " 62-1 66 ... gianl 463 ... phagocytosis of 464 Pleural effusion ... cytology of pleura l linin g ce lls 1248 - - PA S reaction in 1249 Poikilocytosis (see Red cell s) Pol ychromasia (see Red cell s ) Polycythaemia . primary and secondary. I~ . lS-JO Polym orpbs 67 - ag ra nula r 212. 214. 21~. 268. 269. 274. 32.." - basopbil Ixt , 183. 184. IK6. 269. 30.5-308. 31J-316.321.337.3.'8 .371...373 .~24-532 ... in emerging rem issio n of ac ute leuk aemi a !-46,547 - eosi nophil 181. IS6. 181- .'ta-l. 31J-JI6. 1131-1133 - multi-lobed in pern icious a naemia 73. 7·1265 - neutrophil 73. 74. 176.181...184 , 186. 187. 189.201. 204. 207. 258-2Mt . 262-265 . 26H. 271-273. 284. 306 . 31J-316. ~'H(~399 .
....
- - gia nt gran ules in 398 - - phagocytosis of plat ele ts by 46-1 - - tox ic gra nules in 266. 267. 390-392 Pompe's d isease (see G lycogen . sto rage disease ) Pre leukaemic sta tes (set' ~1 D S ) Proerythroblasts 11. I-S. 22. 41. 42. 62. 7.5rt ... gia nt 76. 77 - vacuolation in 75 Prolympbocytes 190 (set' aim leuk aemi a . PLL) Promooocvtes 67 (see also Leuk aemi a. AML. monoblasticrmonoc vticj Pro mve locvtes . 67 . 169-171 : 176- 178. 187. 25i(st'f' ~lso le ukaemia . A ML. A PL) - coarsely gran ular in emerging remission of acute leukaemia 543. $44 ... eo ...inophil 290 ... gia nt 257 Pr u...sia n blue sta in ... in C D A 85 ... in Gaucher's disca...e 1189 ... in haemolvuc a naemia 159 - in hi...tiOC'o"iic leukaem ia 9 18. 919 - in iron deficiencv ana e mia 111 - in macroglobulinaemia SW . 842 - in mal igna nt bistiocytosi... 923. 12..'\6. 1237 - in marrow fleck!" 109- 112 - in monocyte-macropbages 856 - in non -ringed siderobla..ts 117 - in normoblast-macropha ge cell nests 14. 15
- in plasma ce lls 792-7'17
383
- in RE cetb 14. 15. 109. 110. 87 1. 873 - in ringed side roblasts 11.1-116 - in sea-blue histiocvte, 871 , 873 - in sideroblasnc anaemia Ill. 11.1-116, 118 - technique 3n Pse udo-elliptocy tosis (see Red ce lls) Pseuco-Ga ucner ce lls 864-868. 87.1-87f, Pse uoo-P e tger - Huet phen o men on 215. JX8 Pyruvate-kinase (P K) def iciency - renculocvt osis in 129 Re d ce lls' - aca m hocyt es or spur ce us 143. 163. 164 - anisocytosis 62. 72. 73. 130, I.B. 13-1. 137, I~
-
basophi l stippli ng 72. 126. 148 bu rr ce lls 13. 141 co-ocy ses (set' tar get ce lls) cot tage loaf shape defect 142 crenation }·n. 142 c re sce nt formation 140 dacryoc ytes 131.165 d repanocytes 13. 155 echi noc yte s 13. 141 elliptocytes 13. 138 helme t ce lls 152. 153 hypoc hromia 132-13-1. 150 leptocytes JSO' M3~TOCVIO'> i s 62. 72-74. 146, 153 rmcrcc yrosis 132 ·Jrma I 16. 17 ,>olkllocyt osis 12. 13. 62. 72. 73. 131. 133. 13J. ISO. 152. 154 - · '·ar· ,lro p 131. 165 - pu .. chro-nasia 70. 125. 137 - pseudo-clhptecytosis 13. 139 - -ouleau- I·U . 145 - schis tocyt osis 143. 153. 163. 164 - vc; ling Is.! . 155 - - rder tic 158. 159 - sp her oc ytcs 13. 135-137. 153 - SpUT' cells 13 . 143, 163. 164 "pu, nik ce lls 163. 164 - stomatocvt , , 13, 128. 158 - target ct.li<; j3 . 1$0. IS'. 153. 154. 163. 164 Reed- Sterub -rg cells - ac«t ph-spbatase in 1147 - d ual e- ' erase in 1148 - Ieu kae mic invol vement 1029 - :n lymp h nod e im pri nt s and ..ec tio ns 1121114H
- ill marrow 1025-1029 - PA S re action in 1146 Re fract o rv anae mia - with excess blasts ( RAE BI SfiO-565. 574. 577- 587 - with sideroblasts ( RAS or RSA ) Il2- U4 Re n culocyt e s 11.12.70. 71. 1~129, 161 Reticulo-en dot he lial ( RE) ce lls 269 . 852924 (seealso Leu kae mia . histiocytic, M acro ph ages . M alignant histiocy tos is. Mo nocyt e-macroph ages)
- acid phosphatase in 862. 1076-1 078. 11$6. 1167 - a lk.aline pbosphatase in 13. 861. 1074.
1075 -
in blastomvcosis 103-1 blue crys tals in 864-866 ce llular de bris in 852-854. 858 dual esterase in 863. I07 9-WS.2. 1148. 1157 - in emergi ng remission of acu te leukaemia 540-542 - fat -laden ?7J/!, .v : - free -iron stain in 1:1. IS. IU'J. 110.871. 873.923 - in Gauc he r's d isease 881-889.1222-1227 - - bir efringen ce of 884. K85. 1226 - grey -gree n crys tals in 879 - in Ha nd- Scb uller -Chn stian d ise ase 891 - interdigitating 1069. 1072 - in kala-azar 1035-100U - in lym ph nod e impri nts a nd sections 6 16. 1066-1070.1072-1082.1133,1139.1148, 1156, 1167 - multinuclea ted 858 .: - in Nie rna nn- Pick disease 890 " ", - PA S rea ction in 860 , 92-1. 1072 - as pseudo-Gaucber ce lls sti7. '168. 87~ 1I78 - - birefringe nce of 868 - see -blue ma terial in 86473 - in sp lee n imprint s 1220. 1222-1227 - Sud an bla ck sta in in 859. 1070 - tox oplasma in 1045 Ried er ce lls 596 Romanow sk v stai ns 377 - purified re agent method (Heyl} 377 Ro ule au x {see Red ce lls ) Rus..o:.ell bodies 814-817. 1169-11 Sa liva ry glan d impri nt 1238-12-13 Sa rcoidosis. Iymph nod e imp rints.1097. 1098 Schistocv tosis (see Red ce lls) ": ~ '-' Sea- blue histioc ytes 869-873 Sebaceous skin cell . in mar row sme ..r 972 Serosal cells (see sple nic imprints) Sezary's syndrome 191. 776-788 Sick le ce lls (see Red cells) Sideroblasts 12. 114-118 Siderocyt es (see Red ce lls) Smear ce lls. in e LL 676-678 Splenic aspirates . imprint s a nd sectio ns - cyto logy an d cytoc he mis try of 1220-
, ....
,.
384
516
1237 - in G aucher's disease 1222-1229 - in HeL 1216-1221 - in malignant histiocytosis 1233-1 237 - serosal ce lls in 1231 Spu r ce lls (set' Re d ce lls) Sputnik ce lls (see Red ce lls) Stab ce lls 169-171 , 175. 178. 187.258. 266
6 16- 0 7
9S
Star ch gran ules. in smears a nd imprints 122..~12 Z7. 1230. 1231 - birefringence of 1226. 1231 - PAS re act io n in 12~ Stem cell s 1R hae mopoiesis 8. 67 Suda n blac k stain (see indi vidual cells and d isorders) - techniqu e 377 T ce lls - helper 6-U-6.a8 - sup presso r n.&2-64-l. 646. 647 Tart ce ll JM Terato ma . testi cul a r , in lymph nod e 11951197 Th a lassaemi a . re d cells in 150. 151. 153 Th e sa uroc ytes 810 , 811 Th romboc ytopenia 70 - megakaryocytes in 70. 467-a73 Thrombocvtosi.. a nd th rombocvthaerma 70. '477-482 ' . Toxoplasma HMS. 1068. 1083-1 0% Try pa nosoma - bruce i l{){i~ - cruzi 1063 - gambiense 1061 Tubercu losis . lymp h nod e imp ri nt in 1099 Tumour ce lls in marrow 269 - bre as t carcinoma 1010- 1018 - bronchial carcinoma 1007-1009. JOl9 - chemodectoma (paraganglioma) 99~ HMI3 - Ewing 's sarcoma of bo ne 10 24 .- fibrosa~rcoma 1023 - gas tric ca rcino ma 1004-1006 - hyperne phro ma 1022 - med ulloblastoma 993-997 - neuroblastoma ~992 ".pros icca rcinom a 1021 - thy 1020 Uraemia. red cell cha nges in 143 Vacu ol ation - in bla st ce lls ... - of AL L 602.603, 613 - - of A M L 226-228. 30'1-313. 489. 509 .
/ 6:..6: 95"
.{
- in Bur kin lymp homa cells 744-747 - in e . yt hro blasts . as ch loramphenicol or alcohol effect 75 - in hairy cells 750. 755 - in irnmunoblasts 65H - in mon cc ytes aee . 649 - in mon ocyt e-m acro phage s 855-H57 Vascul ar endothe lial cells . in blood smear 962-966 - lkelinephos phatase in 966 ,infect io n (set' Imm uno bla sts) rocytes (see Imm unoblasts ) Vi ru s-associ at e d haemo ph agocyti c svnd ro me 269. 92H.-939 Wald en strtlm 's ma cro globulina em ia (see M acro glo bu linaemia )
