Rapid Review of Hematology.1E.2014.pdf

Ramadas Nayak

MBBS MD

Professor Department of Pathology Kasturba Medical College Manipal University Mangalore, Karnataka, India [email protected]

Sharada Rai MBBS MD Associate Professor Department of Pathology Kasturba Medical College Manipal University Mangalore, Karnataka, India [email protected]

Foreword

AR Raghupathy

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD New Delhi • London • Philadelphia • Panama

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Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2014, Jaypee Brothers Medical Publishers All rights reserved. No part of this book may be reproduced in any form or by any means without the prior permission of the publisher. Inquiries for bulk sales may be solicited at: [email protected] This book has been published in good faith that the contents provided by the authors contained herein are srcinal, and is intended for educational purposes only. While every effort is made to ensure accuracy of information, the publisher and the authors specically disclaim any damage, liability, or loss incurred, directly or indirectly, from the use or application of any of the contents of this work. If not specically stated, all gures and tables are courtesy of the authors. Where appropriate, the readers should consult with a specialist or contact the manufacturer of the drug or device. Rapid Review of Hematology First Edition:

2014

ISBN 978-93-5090-961-4 Printed at

Dedicated to Students who inspired us, patients who provided the knowledge, our parents and family members who encouraged and supported us.

Foreword DEPARTMENT OF PATHOLOGY

BANGALORE MEDICAL COLLEGE VICTORIA HOSPITAL COMPLEX BENGALURU 560 002 Phone: 670 1150 Ext.: 314, 315, 316, 317

It gives me great pleasure to write a short foreword for this new book on Rapid Review of Hematology. Tis is a well-written concise but precise and student-friendly text that will be highly valuable to medical students. It will help in revising and reinforcing the fundamental concepts in hematology. It is very well organized with optional and correct usage of good pictures, schematic diagrams and flow charts. Every essential topic has been discussed giving opt importance and stress on salient features. Each statement mentioned in the text is well written as it carries the required essential points. In short, this book provides within one volume a user-friendly reviewof the basic essential concepts in hematology. It will be of great help to not only second year MBBS students, but also for students preparing for entrance examinations, and students of allied sciences. Tis book will certainly serve as a valuable gift and a valuable addition to the students’ library and the user will definitely appreciate the content and presentation of the information in this book. In conclusion, I am sure, this book brought out by Dr Ramadas Nayak and Dr Sharada Rai will be a very useful compendium for second year MBBS students, the students preparing for entrance examinations, and students of allied health sciences. I hope the reader of this new book will get as much pleasure and knowledge as I did.

AR Raghupathy MBBS MD PGDHHM (IGNOU) Professor and Head Department of Pathology Bangalore Medical College and Research Institute Bengaluru, Karnataka, India

Preface Hematology is one of the rapidly expanding fields of medicine and emerging as a clinical specialty in its own right. Hematology is difficult to teach at the undergraduate level, as it is a part of the curriculum in Pathology, during which undergraduate students do not have enough exposure to diseases of blood. Tis results in less attention to hematological diseases at undergraduate level. After many years of teaching undergraduates, we found that undergraduate students either neglect hematology or find it difficult to understand the subject. It is a nightmare for many students especially during examinations. Tere are many hematology textbooks, but undergraduates face difficulties to refresh their knowledge of hematology during examinations. Tis encouraged us to write a book to fill the niche, to provide basic information to an undergraduate in a nutshell. With this view in mind, Rapid Review of Hematology is intended for the undergraduates from medical, dental and paramedical fields. Most students are fundamentally “visually oriented”. As the saying “one picture worth thousand words”, it encouraged us to provide many illustrations (e.g. etiopathogenesis, clinical presentation, complications, peripheral blood smear and other relevant laboratory tests).

Organization Tis book is organized into four sections namely disorders of red cells, disorders of white cells, disorders of hemostasis and clinical scenario. Te final section deals with common clinical scenario encountered during theory examination.

How to use this book We recommend that this book not to be used as a hematology textbook rather than a supplement to “Essentials in Hematology and Clinical Pathology” (Authored by Dr Ramadas Nayak, Dr Sharada Rai and Dr Astha Gupta). Te concepts of hematology have been oversimplified in this book, but all the information, the student will ever need to know, have been provided. Te readers are requested to give more emphasis on word in bold letters that represents the key words to be remembered. Te peripheral smear and bone marrow findings have been highlighted in colored background. Boxes have been provided at the sides of main text. Tese include some of the key points as well as commonly expected questions during examinations. Tis book can serve as a source of rapid review of hematology.

Ramadas Nayak Sharada Rai

Acknowledgments •

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Our sincere thanks to Ms Prathiba Bhat for her untiring efforts, patience and excellent support in creating many illustrations for this book. Acknowledgments are also due to Dr Astha Gupta (Consultant Pathologist, New Delhi, India), Dr Rakshatha (KS Hegde Medical college, Mangalore, Karnataka, India), Ms Rekha Nayak, Ms Rashmitha Nayak, and Mr Ramnath Kini for their contribution in the preparation of the manuscript. Our sincere thanks to Dr AR Raghupathy, Professor and Head, Department of Pathology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India, for his support and guidance. We are grateful to Dr K Ramnarayan, Vice Chancellor of Manipal University, Manipal, Karnataka, India, and Dr M Venkatraya Prabhu, Dean, Kasturba Medical College, Mangalore, Manipal University, Karnataka, India, for their encouragement. We are grateful to all our friends, undergraduate and postgraduate students who have inspired and supported us. We wholeheartedly thank Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Managing Director), Mr arun Duneja (Director-Publishing), Ms Chetna Malhotra Vohra (Sr Manager, Business Development) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for publishing the book in the same format as wanted well in time. We acknowledge the wonderful work done by Ms Sunita Katla (Publishing Manager), Ms Samina Khan (PA to Director), Mr KK Raman (Production Manager), Mr Rajesh Sharma (Production Coordinator), Ms Seema Dogra (Cover Designer), Mr Sarvesh Kumar Singh (Proofreader), Mr Rajesh Ghurkundi (Graphic Designer), and Mr Raj Kumar (DP Operator) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.

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We thank especially Mr Venugopal V and Mr Vasudev H of M/s Jaypee Brothers Medical Publishers (P) Ltd, Bengaluru Branch, Karnataka, India, for taking this book to every corner of Karnataka.

Contents Section 1:

Disorders of Red Cells

1. Anemias of ImpairedRed Cell Production

3

Anemia 3 Red cell indices 4 Iron deficiency anemia 5 Megaloblastic anemia 8 Pernicious anemia 11 Aplastic anemia 13

2. HemolyticAnemias Due to Red Cell Membraneand Enzyme Defects Hemolytic anemia 16 Hereditary spherocytosis 17 Glucose-6-phosphate dehydrogenase deficiency

16

20

3. ThalassemiaSyndrome

22

Classification of hereditary defects in hemoglobin 22 Thalassemia syndrome 22 b-thalassemia 22 b-thalassemia major 23 b-thalassemia minor/trait 27 a-thalassemia 28

4. Sickle Cell Disease

29

Sickle cell disease 29 Sickle cell anemia 29 Sickle cell trait 34

Other Anemias

36 Immunohemolytic anemias 36 Hemolytic disease of the newborn 36 Antiglobulin (Coombs) test 39 Autoimmune hemolytic anemia 40 Fragmentation syndrome 41 Paroxysmal nocturnal hemoglobinuria 41 Anemias of blood loss 41 Sideroblastic anemias 42

xiv

Rapid Review of Hematology

Section 2:

Disorders of White Cells

6. Quantitativeand QualitativeDisordersof Leukocytes

45

Normal differential leukocyte count (DLC) 45 Quantitative disorders of leukocytes 45 Qualitative disorders of leukocytes 50 Infectious mononucleosis (Glandular fever) 51

Acute Leukemia

52 Acute leukemia 52 Acute lymphoblastic leukemia/lymphoma

55

Acute myelogenous leukemia 57 Myeloid sarcoma 59

8. MyelodysplasticSyndromes

60

Myelodysplastic syndromes 60

9. Myeloproliferative Neoplasms Myeloproliferative neoplasms (MPN) Polycythemia or erythrocytosis 63 Polycythemia vera 63 Essential thrombocythemia 65 Primary myelofibrosis 66

62 62

10. ChronicMyelogenousLeukemia

68

Chronic myelogenous leukemia 68 Natural history of chronic myeloid leukemia 70

11. ChronicLymphocyticLeukemia/SmallLymphocyticLymphoma

73

Chronic lymphocytic leukemia 73 Hairy cell leukemia 75

12. Plasma Cell Neoplasms Plasma cell myeloma (multiple myeloma) 76 Plasmacytoma 80 Immunoglobulin deposition disease 80 Monoclonal gammopathy of uncertain significance (MGUS)

76

80

3. LymphoidNeoplasms

81

Classification of lymphoid neoplasms 81 Follicular lymphoma (FL) 82 Diffuse large B cell lymphoma (DLBCL) 83 Burkitt lymphoma (BL) 83 Mature T cell and NK cell neoplasms 85

4. Hodgkin Lymphomas Definition Classification87 87 Morphology of neoplastic cells 88 Classical Hodgkin lymphoma 88 Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) Etiology and pathogenesis of Hodgkin lymphoma 93

87

92

Contents

Laboratory findings 93 Staging of Hodgkin lymphoma 94 Differences between Hodgkin lymphoma and non-Hodgkin lymphoma 94

15. LangerhansCell Histiocytosis/HistiocytosisX

95

Morphology 95 Laboratory findings 95

Section 3:

Disorders of Hemostasis

16. Disordersof Primary Hemostasis

99

Normal hemostasis 99 Classification of hemostatic disorders 99 Bleeding disorders caused by vessel wall abnormalities 99 Bleeding disorders due to abnormalities of platelet 100 Thrombocytopenia 100 Immune thrombocytopenic purpura 102 Thrombocytosis 104 Qualitative platelet disorders 104

17. BleedingDisorders:Due to Abnormalitiesof Coagulation/ClottingFactor Classification of coagulation disorders 105 Hereditary coagulation disorders 106 Hemophilia 106 Hemophilia A (Factor VIII deficiency) 106 Hemophilia B (Christmas disease, factor IX deficiency) von Willebrand disease (vWD) 108 Acquired coagulation disorders 109 Disseminated intravascular coagulation 110

105

108

18. ThromboticDisorders:HypercoagulableState

113

Hypercoagulable state (Thrombophilia) 113 Inherited hypercoagulable states 114 Acquired hypercoagulable states 114

Section 4:

Clinical Scenario

ClinicalScenario

119 Symptoms and signs that suggest a blood disease 119 Patterns strongly suggestive of a blood disease 120

Appendix

127

Bibliography

133

Index

135

xv

Anemias of Impaired Red Cell Production CHAPTER 1

SECTION

Disorders of Red Cells

1

1

Anemias of Impaired Red Cell Production

1 CHAPTER

ANEMIA

Q. Define anemia.

Definition •

• •

Decrease below normal of the hemoglobin concentration (Hb)/RBC count/hematocrit (packed cell volume). Reduction of the total circulating red cell mass below normal limits. Decrease in the oxygen-carrying capacity of the blood, which leads to tissue hypoxia.

WHO criteria for anemia: adult males Hb <13 g/dL and adult female Hb <12 g/dL.

Anemia may be absolute (decreased RBC mass), orrelative (associated with a higher plasma Grading of anemia: volume). Anemia is conventionally used for absolute anemia. mild (Hb 9.1–10.5 g/dL), moderate (Hb 6.0–9.0 g/dL) and severe (Hb < 6.0 g/dL).

Classification of Anemia 1. Morphological classification (able 1.1): it is based on: a. Red cell size (normocytic, microcytic, or macrocytic), and b. Degree of hemoglobinization (normochromic or hypochromic).

Anemia is characterized by decreased oxygen carrying capacity of blood. Shows decreased Hb and PCV.

TABLE 1.1: Morphological classification of anemia Type of anemia SizeofRBCs

Microcytic hypochromic Smallerthannormal

Normocytic normochromic Normal

Macrocytic Largerthannormal

Central pallor in RBCs

More than 1/3

Mean corpuscular volume (MCV)

Reduced(<80fL)

Normal(82–98fL)

Increased(>100fL)

Mean corpuscular hemoglobin concentration (MCHC)

Reduced(<30g/dL)

Normal(31–36g/dL)

Normal(31–36g/dL)

Examples

Irondeficiencyanemia, thalassemia

Morphology of RBC

Normal

During blood loss, anemia of chronic diseases

Q. Classify anemia.

Normal

Classification: anemias are mainly classified based on 1) morphology and 2) etiology.

Deficiency of vitamin B12 and folic acid Spurious anemia is the term used when RBC concentration decreases due to hemodilution as seen in third semester of pregnancy.

4

SECTION 1

Disorders of Red Cells

Anemia is the expression of underlying disease and from treatment point, the cause of anemia must be identified.

2. Etiological classification: Te etiological classification of anemia is listed in able 1.2. TABLE 1.2: Etiological classification of anemia 1. IMPAIRED RED CELL PRODUCTION Disturbed Proliferation and Maturation of Erythroblasts Defective DNA synthesis – Megaloblastic anemias due to deficiency or impaired utilization of vitamin B 12 and folic acid – Anemia of renal failure due to deficiency of erythropoietin – Anemia of chronic disease due to iron sequestration and relative erythropoietin deficiency – Anemias of endocrine disorders Defective hemoglobin synthesis •

Causes of anemia: 1. Decreased RBC production 2. Increased RBC destruction (hemolysis) or 3. Blood loss.

•

– Defective heme synthesis: iron deficiency, sideroblastic anemia – Defective globin synthesis: thalassemias Marrow Replacement Primary hematopoietic neoplasms : acute leukemia, myelodysplastic syndromes Marrow Infiltration (myelophthisic anemia) Metastatic neoplasms Disturbed Proliferation and Differentiation of Stem Cells Aplastic anemia, pure red cell aplasia •

•

•

Iron deficiency anemia is the most common anemia.

2. INCREASED RED CELL DESTRUCTION (HEMOLYTIC ANEMIAS) Intrinsic (Intracorpuscular) Abnormalities Hereditary – Membrane abnormalities: spherocytosis, elliptocytosis – Enzyme deficiencies: glucose-6-phosphate dehydrogenase, pyruvate kinase – Disorders of h emoglobin synthesis ◆ Deficient globin synthesis: thalassemia syndromes ◆ Structurally abnormal globin synthesis (hemoglobinopathies): sickle cell anemia Acquired – Membrane defects: paroxysmal nocturnal hemoglobinuria Extrinsic (Extracorpuscular) Abnormalities Antibody-mediated – Isohemagglutinins: transfusion reactions, Rh disease of the newb orn – Autoantibodies: idiopathic (primary), drug-a ssociated, systemic lupus erythem atosus Mechanical trauma to RBCs: – Microangiopathic hemolytic anemia: disseminated intravascular coagulation Infections: malaria •

•

•

•

•

3. BLOOD LOSS • •

Q. Write short notes on red cell indices.

Red cell indices: MCV, MCH, MCHC and RDW.

RED CELL INDICES Red cell indices are useful in morphological characterization and diagnosis of anemias. Tey are either directly measured or automatically calculated by specialized instruments. Red cell indices include: 1. Mean Corpuscular Volume (MCV) • MCV is indicative of average volume of the RBC and is expressed in femtoliters (fL). • •

Microcytic anemia have MCV < 80 fL and macrocytic anemia have MCV> 100 fL.

Acute: trauma Chronic:lesions of gastrointestinal tract (e.g. carcinoma colon), gynecological disturbances

It is used for classification and differential diagnosis of anemias. Normal range: 82–98 fL.

MCV =

PCV × 1000 RBC count in millions

= 0.45 × 1000/5 = 90 fL

Anemias of Impaired Red Cell Production CHAPTER 1

2. Mean Corpuscular Hemoglobin (MCH) MCH < 26 pg is seen in • MCH indicates the amount of Hb (weight) per RBC and is expressed as picograms (1 pgmicrocytic anemia and -12 MCH > 33 pg is seen in = 10 g). macrocytic anemia. • It is of limited value in differential diagnosis of anemias. • Normal range: 27–32 pg MCH = Hb (in g/L)/RBC (in millions/

L) = 15

×

10/5 = 30 pg

3. Mean Corpuscular Hemoglobin Concentration (MCHC) MCHC<31 g/dL is seen in • MCHC denotes the average concentration of hemoglobin in the RBC taking volume intohypochromic RBC such as IDA and thalassemia. account. It is expressed as g/dL (earlier it was expressed as %). MCHC >36 g/dL is an • It is a better indicator of hypochromasia than MCH. indication of hyperchromic • Normal range: 31–35 g/dL. RBCs. MCHC = Hb (in g/dL)/PCV = 15/0.45 = 33 g/dL 4. Red Cell Distribution Width (RDW) RDW is useful for • RDW is a quantitative measure of anisocytosis. differentiating anemia due to iron deficiency and • Normal RDW is 11.5% to 14.5%. thalassemia. • A normal RDW indicates that RBCs are relatively uniform in size. A raised RDW indicates that red cells are heterogeneous in size and/or shape. In early iron deficiency anemia, RDW increases along with low MCV while in thalassemia trait, RDW is normal with low MCV. RDW = (Standard deviation

÷

mean cell volume) × 100

IRON DEFICIENCY ANEMIA Iron deficiency anemia (IDA) is themost common nutritional disorder.

Etiology (Table 1.3) IDA is due to deficiency of iron causing defective heme synthesis.

Q. Discuss the etiopathogenesis of iron deficiency anemia.

TABLE 1.3: Causes of iron deficiency anemia 1. Dietary deficiency/lack • • • •

Milk-fed infants Elderly with improper diet and poor dentition Low socioeconomical sections Vegetarians (contains poorly absorbable inorganic iron)

2. Impaired absorption • • •

Total/partial gastrectomy Intestinal absorption is impaired in sprue, other causes of intestinal steatorrhea and chronic diarrhea Specific items in the diet, like phytates of cereals, tannates, carbonates, oxalates, phosphates and drugs can impair iron absorption

3. Increased demand/requirement • •

• • •

In adult men and postmenopausal women, deficiency may be due to chronic gastrointestinal blood loss.

Iron is absorbed in the duodenum.

Growing infants, children and adolescents Pregnancy and lactation

4. Chronic blood loss: due to bleeding from the •

Dietary deficiency is the commonest cause of IDA.

Gastrointestinal tract (e.g. peptic ulcers, gastric carcinoma, colonic carcinoma, hemorrhoids, hookworm infestation or nonsteroidal anti-inflammatory drugs) Urinary tract (e.g. renal or bladder tumors) Genital tract (e.g. menorrhagia, uterine cancer) Respiratory tract (e.g. hemoptysis)

Infants who consume large amounts of cow's milk are susceptible to develop IDA.

5

6

SECTION 1

Disorders of Red Cells

Stages of IDA in sequence: absent of iron stores→decreased serum ferritin→decreased serum iron→increased TIBC → decreased iron saturation→ microcytic hypochromic anemia.

Pathogenesis of Iron Deficiency Anemia It is due to decreased synthesis of heme and can be divided into 3 stages. Stage 1 (Iron depletion): iron adequate to maintain normal hemoglobin level and only serum ferritin decreased. Stage 2 (Iron deficient erythropoiesis): lowering of serum iron and transferrin saturation levels without anemia (Hb, MCV and MCH within normal range). Bone marrow shows iron deficient erythropoiesis. Stage 3 (Iron deficiency anemia): low serum iron, serum ferritin and transferrin saturation. Impaired hemoglobin production. Morphologically,first reduction in the size (microcytic) and later increase in the central pallor (hypochromia) of RBCs. •

•

•

Laboratory Findings

Q. Discuss the laboratory findings in iron deficiency anemia.

Peripheral Blood

MCV, MCH and MCHC are reduced. RDW is raised.

• •

Hemoglobin and hematocrit (PCV): decreased Red cell indices: – MCV: <80 fL (normal 82–98 fL) – MCH: <25 pg (normal 27–32 pg) – MCHC: <27 g/dL(31–36 g/dL) – RDW: increased and >15%. It is earliest sign of iron deficiency (normal 11.5–14.5%).

Q. Write short notes on peripheral • Peripheral smear (Figs 1.1 and 1.2): smear findings in iron deficiency – RBCs: microcytic (small) and hypochromic (pale). Severe anemia shows ring/pessary cells. anemia. Moderate anisocytosis and poikilocytosis pencil/cigar-shaped cells. – WBCs: normal; eosinophilia in hookworm infestation. Peripheral smear shows – Platelets: normal microcytic hypochromic • RBCs. Reticulocyte count: low for the degree of anemia.

Fig. 1.1:Peripheral blood smear showing microcytic hypochromic red blood cells

Fig. 1.2:Diagrammatic appearance of peripheral blood smear with microcytic hypochromic red blood cells

Anemias of Impaired Red Cell Production CHAPTER 1

Bone Marrow • • • • • •

Cellularity: moderately hypercellular. M:E ratio: varies from 2:1 to 1:2 (normal 2:1 to 4:1). Erythropoiesis: hyperplasia and micronormoblastic maturation. Myelopoiesis: normal. Megakaryopoiesis: normal. Absence of bone marrow iron: “Gold standard” test, demonstrated by negative Prussian blue reaction.

Bone marrow shows micronormoblastic eythroid hyperplasia. Marrow iron is absent. Prussian blue reaction negative.

Serum Iron Profile (Table 1.4)

Reduced: serum iron,

TABLE 1.4: Serum iron profile in IDA

ferritin, % transferrin saturation. Increased: TIBC, TFR and red cell protoporphyrin.

Normalrange

ValueinIDA

Serumferritin

15–300µg/L

<15µg/L

Serumiron

50–150µg/dL

10–15µg/dL

Serumtransferrinsaturation

30–40%

Total plasma iron-binding capacity (TIBC)

310–340 µg/dL

Serum transferrin receptor (TFR)

0.57–2.8 µg/L

Redcellprotoporphyrin

30–50µg/dL

Observation

<15% 350–450 µg/dL 3.5–7.1 µg/L >200µg/dL

Reticulocyte Hemoglobin It is decreased and is an early feature of IDA.

Clinical Features of IDA

The earliest laboratory indicator of IDA is reduced reticulocyte hemoglobin.

Q. Mention the various clinical features of iron deficiency anemia.

Nonspecific and related to both severity and the cause of the anemia (e.g. gastrointestinal disease) Onset: insidious. Nonspecific symptoms: fatigue, palpitations, breathlessness, weakness and irritability. Pharyngeal/esophageal webs formed cause dysphagia. Patterson-Kelly or Plummer-Vinson syndrome: Patterson-Kelly or Plummer-Vinson – Microcytic hypochromic anemia syndrome: microcytic – Atrophic glossitis hypochromic anemia, – Esophageal webs atrophic glossitis and esophageal webs. Congestive heart failure in severe anemia. Central nervous system: pica-unusual craving for substances with no nutritional value like clay or chalk. Craving for ice (pagophagia)specific to iron deficiency. Pica may be the cause rather than effect of IDA. • • • •

• •

Physical Findings Diminished tissue enzymes cause characteristic epithelial changes of iron deficiency anemia. Angular stomatitis and glossitis Chronic atrophic gastritis Koilonychia (spoon nails) • • •

Koilonychia (spoon nails) is a physical finding seen in iron deficiency. First fingernails become thin and flat-platonychia, then brittle and finally spoon shaped.

7

8

SECTION 1

Disorders of Red Cells

Q. Enumerate the causes of microcytic hypochromic anemia.

Causes of Microcytic Hypochromic Anemia • • • • •

Q. Discuss the causes and pathogenesis of megaloblastic anemia.

Iron deficiency anemia Talassemia major Anemia of chronic disorders Others: alcohol, lead poisoning and drugs Sideroblastic anemia (rare cause).

MEGALOBLASTIC ANEMIA Anemias characterized by defective/impaired DNA synthesis and distinct megaloblasts in the bone marrow. Megaloblastic anemias are common among anemias due to impaired red cell production.

Vitamin B12 is present in animal products.

Etiology of Megaloblastic Anemia (Table 1.5) TABLE 1.5: Causes of megaloblastic anemia VITAMIN B 12 DEFICIENCY Deficiency of vitamin B12 and folic acid are the main causes of megaloblastic anemia.

1. Decreased Intake: inadequate diet, “pure vegetarians” (vegans) 2. Impaired Absorption Gastric: deficiency of gastric acid or pepsin or intrinsic factor – Pernicious anemia – Post-gastrectomy Intestinal – Loss of absorptive surface ◆ Malabsorption syndromes ◆ Diffuse intestinal disease, e.g. lymphoma, systemic sclerosis ◆ Ileal resection, Crohn disease – Bacterial or parasitic competition for vitamin B12 ◆ Bacterial overgrowth in blind loops and diverticula of bowel ◆ Fish tapeworm infestation 3. Increased Demand: pregnancy, hyperthyroidism, disseminated cancer •

•

FOLIC ACID DEFICIENCY Folic acid is absorbed in the jejunum.

1. Decreased Intake: inadequate diet—alcoholism, malnutrition 2. Impaired Absorption Malabsorption states: nontropical and tropical sprue Diffuse infiltrative diseases of the small intestine (e.g. lymphoma) Drugs: anticonvulsant phenytoin and oral contraceptives 3. Increased Loss: hemodialysis 4. Increased Demand: pregnancy, infancy, disseminated cancer, markedly increased hematopoiesis 5. Impaired Utilization: folic acid antagonists, such as methotrexate • • •

Deficiency of vitamin B12 and folic acid → delayed nuclear maturation → megaloblast → macrocyte.

Pathogenesis of Megaloblastic Change 1. Impaired DNA synthesis: megaloblastic anemia is commonly due to deficiency of vitamin B12 (cyanocobalamin) or folic acid. Both are required for the synthesis of DNA. a. Delayed maturation of nucleus. Te nuclear maturation lags behind the cytoplasmic maturation and results in abnormally large nucleated erythroid precursors named asmegaloblasts.

Ineffective erythropoiesis and hemolysis are responsible for anemia.

b. Cytoplasm matures normally. RBCs are larger than normal→ macrocytes. c. Affects all rapidly dividing cells of the body (including skin, GI tract, and bone marrow). 2. Ineffective erythropoiesis: megaloblast precursors undergo intramedullary destruction.

Anemias of Impaired Red Cell Production CHAPTER 1

Laboratory Findings of Megaloblastic Anemia Blood findings in vitamin B12 and/or folic acid deficiency are similar.

Q. Write short note on the laboratory findings in megaloblastic anemia.

Peripheral Blood • •

•

•

Hemoglobin and hematocrit (PCV): reduced Red cell indices – MCV: above 100 fL (normal 82–98 fL) – MCH (normal 27–32 pg) – Normal MCHC (31–36 g/dL) Peripheral smear (Figs 1.3 and 1.4): pancytopenia (decreased RBC, WBCs and platelets). – RBCs: ◆ Macrocytic and oval (egg-shaped macro-ovalocytes)-diagnostic. ◆ Most macrocytes lack the central pallor (Figs 1.3 and 1.4). ◆ Marked variation in the size and shape of red cells (anisopoikilocytosis). ◆ Evidence of dyserythropoiesis: basophilic stippling, Cabot ring and Howell Jolly bodies. – WBCs: ◆ Decreased WBC count (leukopenia). ◆ Hypersegmented neutrophils (more than five nuclear lobes): first and specific morphological sign of megaloblastic anemia. These neutrophils are also larger than normal (macropolys). – Platelets: decreased.

Reticulocyte count: normal or low.

Megaloblastic anemia Pancytopenia Macro-ovalocytes Hypersegmented neutrophils Macropolys. •

•

•

•

In megaloblastic anemia due to vitamin B12 deficiency, reticulocyte count may be normal or low and high reticulocyte count is seen on 7th day following vitamin B12 therapy.

Dimorphic Anemia • Combined vitamin B12/folic acid and iron deficiency. •

Peripheral smear shows two populations of RBCs namely: macro-ovalocytes and microcytic hypochromic (Fig. 1.5).

Fig. 1.3: Peripheral blood smear showing macro-ovalocytes (arrows) and hypersegmented neutrophil (inset )

Fig. 1.4:Diagrammatic peripheral blood smear showing macro-ovalocytes (thick arrows) and hypersegmented neutrophil (thin arrow )

9

10

SECTION 1

Disorders of Red Cells

A mixture of microcytic hypochromic and macrocytic RBCs is termed as dimorphic picture and occurs in mixed deficiency of iron and folic acid or vitamin B12.

Fig. 1.5: Diagrammatic peripheral blood smear of dimorphic anemia showing macro-ovalocytes and microcytes

Megaloblastic anemiabone marrow: Megaloblasts Giant metamyelocytes. •

•

Megaloblast are large, abnormal precursors of RBCs seen in the bone marrow of patients with megaloblastic anemia.

Bone Marrow • •

•

•

• •

Cellularity: moderately to markedly hypercellular. M: E ratio: due to marked erythroid hyperplasia, M: E ratio is reversed ranging from 1:1 to 1:6 (normal 2:1 to 4:1). Erythropoiesis: megaloblastic type (Figs 1.6 and 1.7) – Megaloblasts: large, abnormal counterparts of normal normoblasts . Megaloblast shows asynchrony of nuclear and cytoplasmic maturation. The cytoplasm shows normal hemoglobinization. – Ineffective erythropoiesis: developing megaloblasts die in marrow (intramedullary hemolysis). Myelopoiesis: – Myeloid cells adequate in number. – Granulocytic precursors display nuclear-cytoplasmic asynchrony in the form of giant metamyelocytes and band forms. Megakaryopoiesis: normal or increased in number. Bone marrow iron: moderately increased.

Te differences between normoblasts and megaloblasts are shown in able 1.6 Q. List the differences between TABLE 1.6: Differences betweennormoblast and megaloblast normoblast and megaloblast. Characteristics Normoblast Megaloblast

Megaloblasts: Nuclear maturation lags behind cytoplasmic maturation. Nuclei have open sievelike chromatin. •

•

Cellsize

Normal

Largerthancorrespondingnormoblast

Nuclearchromatin

Normal

Opensieve-like

Nuclearmaturation

Normal

Lagsbehindcytoplasmicmaturation

Mitosis

Normal

Increasedandabnormal

Maturation in bone marrow

Normal (Late > intermediate > early normoblast)

Increased proportion of more primitive erythroid cells (Late < intermediate < early megaloblast)

Evidence of dyserythropoiesis

Absent

Present (irregular nuclei, Howell Jolly bodies)

Myelopoiesis

Normal

Found in

Normal bone marrow

Showsgiantmetamyelocytes Bonemarrow of megaloblastic anemia

Anemias of Impaired Red Cell Production CHAPTER 1

Fig. 1.6:Bone marrow aspirate showing megaloblastic precursors (arrows) in varying stages of maturation (inset shows early megaloblast)

Fig. 1.7: Diagrammatic picture of bone marrow aspirate showing megaloblastic precursors (thick arrows) in varying stages of maturation

Biochemical Tests for Megaloblastic Anemia Common for both vitamin B12 and folic acid deficiency Deoxyuridine suppression Deoxyuridine suppression test : it is a sensitive measure of deficiency of 5, 10-methylene HF,test is abnormal even before the morphological which occurs in both folic acid and vitamin B12 deficiency. changes. • Serum homocysteine • Serum bilirubin: mild increase causes mild jaundice • Serum iron and ferritin • •

Plasma lactate dehydrogenase (LDH) Serum vitamin B 12/folate decreased.

Diagnostic tests for vitamin B12 deficiency • Serum vitamin B12 levels: decreased – Serum methylmalonic acid – Urinary excretion of methylmalonic acid • Schilling test for vitamin B12 absorption (Refer page 12). Specific tests for folic acid deficiency • Serum folic acid levels: decreased • FIGLU in urine: excessively excreted.

PERNICIOUS ANEMIA Pernicious anemia (PA) is an autoimmune disease due to deficiency of intrinsic factor causing

Schilling test determines the cause of vitamin B12 deficiency.

Q. Discuss the etiopathogenesis and morphology of pernicious anemia.

impaired absorption of vitamin B12 and megaloblastic anemia. Rare in India. A genetic predisposition is suspected. Age: older age—fifth to eighth decades of life Sex: females are more involved than males (F: M is 1.5: 1).

Vitamin B12 is absorbed in terminal ileum and requires IF.

11

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Disorders of Red Cells

PA: autoimmune disease Atrophic gastritis IF deficiency Autoantibodies.

Etiopathogenesis

•

•

•

• •

•

An autoimmune disease due to destruction of gastric mucosa. Stomach shows damage to parietal cells, dense infiltration by lymphocytes and plasma cells → chronic atrophic gastritis → failure of production of intrinsic factor. Presence of autoantibodies: two major types of autoantibodies— – Anti-intrinsic factor (IF) antibody ◆ ype I (blocking) antibody: blocks the binding of vitamin B12 to IF. Present in 50–75% of the cases. ◆ ype II (binding) antibody: attaches to the IF–vitamin B12 complex and prevent its binding to receptors in the ileum. Present in about 40% of patients. – Parietal cell (Type III) antibody: neither specific for PA nor other autoimmune disorders. It is found in 90% of patients.

Morphology Alimentary System Atrophic gastritis may predispose to carcinoma stomach.

• •

Atrophic glossitis: tongue shiny, glazed and beefy. Stomach: – Diffuse chronic atrophic gastritisand impaired secretion of hydrochloric acid, pepsin and intrinsic factor. ◆ Histologically atrophy of the glands, with loss of both chief cells and parietal cells. ◆ Nuclei of mucosal cells look similar to that of megaloblasts. ◆ Dense infiltration by lymphocytes and plasma cells. – Intestinal metaplasia.

Central Nervous System Found in 75% of cases. • Demyelination in the dorsal and lateral tracts: subacute combined degeneration Peripheral neuropathy. •

Laboratory Findings (Fig. 1.8)

Q. Write short note on laboratory findings in pernicious anemia.

Blood, bone marrow and biochemical test findings are similar to those described earlier for megaloblastic anemias (Refer page 9 to 11).

Specific Diagnostic Tests for Pernicious Anemia Schilling test: diagnostic of PA but now very infrequently performed.

•

• • •

Schilling test for vitamin B12 absorption: abnormal – Radioactive vitamin B12 is used to assess the status of intrinsic factor (IF) and vitamin B12. – Helps in distinguishing megaloblastic anemia due to IF deficiency (pernicious anemia) from other causes of vitamin B12 deficiency. Serum antibodies to intrinsic factor a re highly specific for pernicious anemia Achlorhydria with histamine/pentagastrin stimulation. Severe deficiency of intrinsic factor.

Anemias of Impaired Red Cell Production CHAPTER 1

Pernicious anemia present with features of megaloblastic anemia due to vitamin B12 deficiency. In addition, it may show features of atrophic gastritis and achlorhydria.

PA patients sometimes have a lemon-yellow color owing to a combination of pallor and mild jaundice caused by excess breakdown of hemoglobin. Nonmegaloblastic causes of macrocytic anemia: 1. Alcohol 2. Liver disease 3. Myxedema 4. Cytotoxic drugs 5. Myeloma 6. Aplastic anemia 7. Reticulocytosis 8. Red cell aplasia.

Fig. 1.8: Clinical features and laboratory findings in pernicious anemia

Clinical Features of Megaloblastic Anemia Te clinical features of vitamin B12 deficiency anemia and pernicious anemia are: • Onset: insidious and progresses slowly. • Classic triad of presentation: weakness, sore throat and paresthesias. • ongue: painful red “beefy” tongue. • Neurological manifestations: – Bilateral peripheral neuropathy:glove and sock distribution ofnumbness or paresthesia – Demyelination of spinal cord: subacute combined demyelination/degeneration of dorsal and lateral tracts—ataxia , uncoordinated gait, impairment of vibration and position sense. • Atherosclerosis: serum homocysteine level is raised and is a risk factor for atherosclerosis and thrombosis.

APLASTIC ANEMIA Hematopoietic stem cell (HSC) disorder characterized by: • Pancytopenia (anemia, neutropenia and thrombocytopenia) •

With markedly hypocellular bone marrow(less than 30% cellularity).

Etiology Te most common causes associated with aplastic anemia are shown in able 1.7.

Q. Mention the various clinical features of megaloblastic anemia.

Folate deficiency anemia presents with features of megaloblastic anemia due to vitamin B12. Unlike with vitamin B12 deficiency, neurological symptoms does not occur.

Q. Write short notes on aplastic anemia.

13

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Disorders of Red Cells

6 “I” s of the causes of aplastic anemia: 1. Idiopathic 2. Ingestion of drugs and chemicals 3. Idiosyncratic 4. Irradiation 5. Infections and 6. Inherited.

TABLE 1.7: Common causes of aplastic anemia 1. ACQUIRED Idiopathic Acquired defects in stem cell Immune mediated Secondary Chemical Agents Cytotoxic drugs: alkylating agents, antimetabolites Inorganic arsenicals Idiosyncratic Chloramphenicol • •

•

•

•

•

Benzene Chloramphenicol

•

•

Phenylbutazone

•

•

Penicillamine Carbamazepine Gold salts Organic arsenicals Methylphenylethyl hydantoin Physical Agents: whole-body irradiation Viral Infections: hepatitis virus, Epstein-Barr virus, cytomegalovirus , herpes zoster ( Varicella zoster) , HIV •

•

•

2. INHERITED:fanconi anemia, telomerase defects

Pathogenesis: Direct damage to the hematopoietic stem cells and progenitor cells. Immune-mediated destruction. Primary stem cell abnormality—inherited defect in the stem cells.

Pathogenesis (Fig. 1.9)

•

•

•

Fig. 1.9:Pathogenesis of aplastic anemia

Clinical Features • •

Any age of both sexes Insidious – Progressive weakness, pallor and dyspnea due to anemia – Frequent (mucocutaneous bacterial infections) or fatal infections due to neutropenia

Anemias of Impaired Red Cell Production CHAPTER 1

– Bleeding manifestations in the form of petechiae, bruises and ecchymoses due to thrombocytopenia.

Laboratory Findings Peripheral Blood • • •

•

Hemoglobin PCV Reticulocyte count: markedly decreased. Peripheral smear: pancytopenia, i.e. decreased red cells, neutrophils and platelets. – RBCs: normocytic normochromic anemia – WBCs: total leukocyte count decreased. Neutrophils markedly diminished and neutropenia is a reflection of the severity of aplasia. Initial stages, lymphocytes normal in number as the disease progresses their count decreases. – Platelets: count is decreased.

Bone Marrow •

Marrow aplasia—best appreciated in a bone marrow (trephine) biopsy – Cellularity: marked hypocellularity. – Hematopoiesis: paucity of all erythroid, myeloid and megakaryocytic precursors. – Other cells: lymphocytes and plasma cells are prominent.

No Splenomegaly Diagnosis: diagnosis is made with peripheral bloodand bone marrow biopsy findings.

Differential Diagnosis •

Should be distinguished from other causes of pancytopenia (able 1.8)

TABLE 1.8: Causes of pancytopenia Decreased bone marrow function •

• •

Aplastic anemia – Idiopathic – Secondary – Inherited Myelodysplastic syndromes Bone marrow infiltration with – Leukemia – Lymphoma – Myeloma – Tumors (carcinoma) – Granulomatous diseases (e.g. tuberculosis, sarcoidosis)

•

• • •

Nutritional deficiencies: – Megaloblastic anemia (vitamin B 12 and folic acid deficiency) Paroxysmal nocturnal hemoglobinuria Myelofibrosis (rare) Hemophagocytic syndrome

Increased peripheral destruction •

Reticulocyte count is markedly low in aplastic anemia and is characteristic feature.

Hypersplenism

Prognosis: unpredictable.

Bone marrow elements are replaced by fat and aspiration usually yields dry tap.

Absence of splenomegaly and in its presence the diagnosis of aplastic anemia should not be made.

15

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2

Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects

CHAPTER

Q. Define and classify hemolytic anemia.

Normal lifespan of red cell is about 120 days. In hemolytic anemias RBC survival time is considerably shortened.

HEMOLYTIC ANEMIA Definition Hemolytic anemias are due toincrease in the rate of red cell destruction (hemolysis).

Classification of Hemolytic Anemias (Table 2.1) Depending on: • •

•

Location of hemolysis: intravascular and extravascular Source of defect causing hemolysis: intracorpuscular defect and extracorpuscular defect Mode of onset: hereditary and acquired disorders.

TABLE 2.1: Classification and causes of hemolytic anemia Breakdown of normal RBCs occurs in the macrophages of the bone marrow, liver and spleen.

Intrinsic (intracorpuscular) abnormalities

Extrinsic (extracorpuscular) abnormalities

Hereditary RBC membrane abnormalities – Membrane skeletal abnormalities: spherocytosis, elliptocytosis – Membrane lipids: abetalipoproteinemia Enzyme deficiencies – Enzymes of hexose monophosphate shunt: glucose-6-phosphate dehydrogenase – Glycolytic enzymes: pyruvate kinase Disorders of hemoglobin synthesis – Deficient globin synthesis: thalassemia syndromes – Structurally abnormal globin synthesis (hemoglobinopathies): sickle cell anemia

Antibody-mediated Isohemagglutinis: Rh disease of the new-born, transfusion reactions Autoantibodies: idiopathic (primary), drugassociated, systemic lupus erythematosus Mechanical trauma to RBCs Microangiopathic hemolytic anemia: disseminated intravascular coagulation Defective cardiac valves Infections:malaria Drugs, chemicals and toxins

•

•

Decreased red cell survival does not always cause anemia as there is a compensatory increase in red cell production by the bone marrow.

•

Acquired Membrane defects: paroxysmal nocturnal hemoglobinuria •

•

•

•

•

• • •

Drugs: oxidant drugs, primaquine, dapsone, etc. Chemicals: naphthalene, nitrites, nitrates, etc. Toxins: snake venom, lead poisoning, clostridial sepsis

Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2

Location of Hemolysis

Q. List the differences between extravascular hemolysis and

It may be intravascular and/or extravascular. Te differences between these two types areintravascular hemolysis. listed in able 2.2. TABLE 2.2: Differences between extravascular andintravascular hemolysis Characteristics

Extravascularhemolysis

Intravascularhemolysis

Site of hemolysis

RE system (spleen, bone marrow)

Within circulation

Splenomegaly

Usual

Uncommon

Moderately raised Normal Not seen

Mildly raised Decreased Positive

Urine Hemoglobinuria Hemosiderinuria •

Absent Absent

Present Present

Examples

Thalassemia, sickle cell anemia

G6PD deficiency, PNH

In most hemolytic anemias red cell destruction is extravascular.

Laboratory findings • • •

Serum bilirubin-unconjugated Serum haptoglobin Hemoglobinemia

•

HEREDITARY SPHEROCYTOSIS Hereditary spherocytosis (HS) is a rareinherited hemolytic anemia resulting from thedefect Q. Describe the etiopathogenesis in the red cell membrane. of hereditary spherocytosis. Normal structure of RBC membrane is depicted in Figure 2.1.

Etiopathogenesis • •

HS, is due to defect in the RBC membrane protein.

Autosomal dominant disorder The common mutations RBC membrane protein defect caused by variousmutations. Most common mutations involve ankyrin, band 3, spectrin or band protein involve ankyrin, band 3, spectrin, or band protein 4.2. 4.2.

Mechanism of Hemolysis in HS (Fig. 2.2) •

•

HS: intrinsic defect of RBC membrane-extravascular

Young HS RBCs are normal in shape. But as they age, they undergo loss of membrane hemolysis. fragments in the circulation. Tese smallRBCs assume a spherical shape(spherocytes). Spherocytes are rigid, inflexible and less deformable. Tey get trapped in the spleen leading to premature destructionof spherocytes.

Fig. 2.1: Structure of the red cell membrane

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Disorders of Red Cells

Fig. 2.2: Mechanism of hemolysis in hereditary spherocytosis

Laboratory Findings

Q. Write short notes on laboratory findings in HS.

Peripheral Blood

In hereditary spherocytosis MCHC is > 35 g/dL.

Spherocytes and reticulocytosis are observed in the peripheral blood.

• •

•

Peripheral smear: very important for diagnosis (Figs 2.3 and 2.4). – RBCs:

◆ Spherocytes are most distinctive but not pathognomonic. Spherocytes are small, darkstaining (hyperchromic)RBCs without any central pallor. ◆ Polychromatophilia due to reticulocytosis. – WBCs: total leukocyte count ( TLC) increased. – Platelets: normal.

Spherocytes may also be seen in autoimmune hemolytic anemia and burns.

• Bone marrow shows erythroid hyperplasia.

Hemoglobin: decreased and level depends on degree of hemolysis. Red cell indices: – MCV: reduced (normal 82–98 fL) – MCHC: raised and > 35 g/dL (normal 31–36 g/dL).

Reticulocyte count: increased (Fig. 2.5).

Bone Marrow • • • •

Cellularity: markedly hypercellular Erythropoiesis: erythroid hyperplasia Myelopoiesis: normal Megakaryopoiesis: normal.

Fig. 2.3: Peripheral blood smear with numerous spherocytes (arrows)

Fig. 2.4: Diagrammatic peripheral blood smear with numerous spherocytes (arrows)

Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2

Fig. 2.5: Smear shows reticulocyte with blue filamentous/granular material (new methylene blue stain) (arrows)

Biochemical Findings • • •

Serum bilirubin: mildly raised. Urine urobilinogen: increased. Serum haptoglobin: decreased.

Osmotic Fragility Test Osmotic fragility is increased and there is shift of the curve to the right (Fig. 2.6).

Clinical Features • • •

Age: anytime from the neonatal period to adulthood. Family history: most (75%) are inherited asautosomal dominant trait. Anemia: mild to moderate.

Fig. 2.6: Osmotic fragility test. Normal curve (blue) and increased osmotic fragility in hereditary spherocytosis

HS: osmotic fragility is increased with a shift of curve to the right.

Clinical features of intermittent jaundice, splenomegaly and spherocytes in the peripheral smear is highly suggestive of HS.

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Disorders of Red Cells

• • • •

Jaundice: intermittent attacks, precipitated by pregnancy, fatigue, or infection. Splenomegaly: moderate (500 to 1000 g). Gallstones: pigment gallstones. Aplastic crises: may be triggered by an acute parvovirus infection.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY • • • •

Hemolytic disease due to red cell enzyme defects. In G6PD deficiency, RBCs are susceptible to oxidative injury by free radicals. It is an X-linked recessive disorder and its full expression is seen only in males. Tere are different subtypes.

G6PD deficiency is an intrinsic defect and hemolysis is primarily intravascular.

Role of G6PD (Fig. 2.7)

In G6PD, RBCs exposed to oxidant stress, the hemoglobin is oxidized to methemoglobin which forms Heinz bodies in the cytoplasm of RBCs.

Sequence of Events in G6PD Deficiency

• Reduced glutathione (GSH) in the normal RBCs protects them against oxidant injury by breakdown of compounds such as H2O2 to H2O. Te housekeeping enzyme, G6PD is required for normal GSH.

In G6PD deficiency, oxidants can cause both intravascular and extravascular hemolysis. In G6PD deficiency, there is decreased synthesis of reduced glutathione. RBCs when exposed to oxidant stress (during infections, exposure to drugs or chemical, fava beans)accumulate H2O2. It damages RBC membrane causinghemolysis. Hemolyzed red cellsliberate hemoglobin. • •

•

•

Te hemoglobin is oxidized by oxidants leading to formation of methemoglobin, which forms Heinz bodies (Fig. 2.8) in the cytoplasm of RBCs.

Fig. 2.7: Role of G6PD against injury by oxidants

Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2

Fig. 2.8: Peripheral blood smear in G6PD deficiency with “bite cells” (arrows). Inset shows Heinz bodies (supravital stain)

•

Heinz bodies removed from RBC membrane by macrophages in the spleen and produceG6PD deficiency has a protective effect against bite cells. Tese bite cells areremoved viaerythrophagocytosis in the spleen. Plasmodium falciparum malaria.

Clinical Presentation G6PD deficiency manifests in several distinct clinical patterns. Usually present as acute selflimited acute intravascular hemolytic anemia following exposure to oxidative stress.

Laboratory Findings Peripheral Blood • •

•

•

Hemoglobin: decreased. Reticulocyte count: increased. Peripheral smear: – RBCs: moderate anisopoikilocytosis withpolychromatophilia, microspherocytesand bite cells (Fig. 2.8).Heinz bodies identifiedwith a supravital stainand are best seen during active hemolysis. – WBCs: mild leukocytosis. – Platelets: normal.

G6PD deficiency–oxidant damage to RBC • Bite cells • Heinz bodies.

Self-limited hemolysis: primarily theold red cells are hemolyzed , hence hemolysis is self-limited.

Urine Hemoglobinuria will be found during hemolysis and may last for about 1–6 days.

G6PD: enzyme analysis– confirmatory test.

RBC Enzyme Analysis Tests for G6PD deficiency are positive and should be assessed a few weeks after the acute hemolytic episode.

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3

Thalassemia Syndrome

CHAPTER

Q. Classify hereditary disorders of hemoglobin. The term hemoglobinopathy is usually used for a qualitative hereditary disorder of hemoglobin.

CLASSIFICATION OF HEREDITARY DEFECTS IN HEMOGLOBIN

Hemoglobin defects may be quantitative (reduced production of normal hemoglobin) or qualitative (production of abnormal hemoglobin). • Quantitative defect: genetic mutations in the globin loci (e.g. thalassemia) may quantitatively reduce the synthesis of a-globin or b-globin chain. It leads to net reduction of hemoglobin. • Qualitative defect: genetic mutations in the a-globin or b-globin locus may produce abnormal hemoglobin (e.g. sickle cell anemia). Te abnormal hemoglobin may be functionally normal, but its physical or physiologic properties differ from normal hemoglobin.

Q. Classify thalassemia syndromes.

THALASSEMIA S YNDROME • Tese are group of inherited disorders due toabnormality of globin production. • It is characterized bydecreased or absence of synthesis of either a or b-globin chain of adult hemoglobin, HbA (a2b2).

In b-Thalassemia, there is decreased/absence of synthesis of b-chains. In a-Thalassemia, there is reduced/absence of synthesis of a-chains of globin.

Classification Tey are mainly classified as: • b-Talassemia syndromes: impaired synthesis ofb-chains of globin. • a-Talassemia syndromes: impaired synthesis ofa-chains of globin. • Miscellaneous thalassemia syndromes.

b-THALASSEMIA • Autosomal recessive hereditary disorder • Diminished synthesis of b-globin chains and normal synthesis of a-chains.

Thalassemia Syndrome CHAPTER 3

Molecular Pathology

Point mutations leading to aberrant RNA splicing is

• b-globin chains are encoded by a single gene. the most common cause of • Te molecular errors over 200 genetic defects leading tob-thalassemia have been identified. b-thalassemia. • Different types of mutations in b-globin gene can occur but mainly point mutations rather than gene deletions (unlike in a-thalassemia). Te mutations result in defects in transcription, RNA splicing and modification, translation via frame shifts and nonsense codons. Mutations leading to aberrant RNA splicing are the most common cause.

Clinical and Genetic Classification (Table 3.1) TABLE 3.1: Clinical and genetic classification of b-thalassemias Clinicalsyndromes

Genotype

b-thalassemiamajor

Homozygous( b0/b0,b+/b+) or double heterozygous ( b0/b+)

•

Clinicalfeatures •

Severe form, severe anemia and transfusion dependent High level of HbF in the blood

0

+

+

+

0

+

b-thalassemiaintermedia

Variable( b /b , b /b , b /b, b /b)

Moderately severe and not transfusion dependent

b-thalassemia minor/b-thalassemia trait

Heterozygous (b0/b, b+/b)

Mild anemia and asymptomatic

b-THALASSEMIA MAJOR

•

b0 = Total absence of b-globin synthesis; b+ = Markedly reduced or diminished b-globin synthesis; b = normal b-globin synthesis.

b-thalassemia is the commonest quantitative

• It is a hereditary hemolytic anemia due to absence of synthesis of b-globin chain of disorder of hemoglobin. hemoglobin. Te synthesis of a-globin chain is not affected. • Homozygous form of b0/b0 or b+/b+ or double heterozygous b0/b+ (able 3.1) b-thalassemia major also called Mediterranean or • Most common in Mediterranean countries, parts of Africa and South East Asia. Cooley’s anemia.

• Hemolytic anemia is of severe degree.

Pathophysiology of b-thalassemia Major (Fig. 3.1) Consequence of Defective or Absent b-chains

Q. Describe the pathophysiology/ pathogenesis ofb-thalassemia major.

• Severe hemolytic anemia due to: 1. Absence of b-globin chain: results inabsence of synthesis of HbA (a2b2). Tis produces b-thalassemia major Absence of synthesis of RBCs that are poorly hemoglobinized (hypochromic) and small in size (microcytic). HbA produces severe 2. Ineffective erythropoiesis: unpaired and excess a-chains aggregate into insoluble microcytic hypochromic precipitates, which bind to and damage the membrane of erythroid precursors . Tese anemia erythroid precursors fail to mature andundergo apoptosis in the marrow. Increased synthesis of 3. Extravascular hemolysis: RBCs with a-chain inclusions are removed by macrophages of HbF. spleen (extravascular hemolysis). • Synthesis of fetal hemoglobin (HbF): the ϒ-globin chain synthesis continues even 6 months after birth and combines with a-globin leading to increased levels of HbF (a2ϒ2). Te level of HbF varies from 30% to 90%. •

•

Consequences of Ineffective Erythropoiesis • Changes in bone marrow: marked erythroid hyperplasia. • Changes in bone: – Skull X-ray: hair on end (“crew-cut”) appearance (Fig. 3.2) – ypical facies: thalassemic facies (Fig. 3.3)—prominent forehead, cheekbones and upper jaw.

b-thalassemia major Thalassemic facies Crew cut appearance on skull x-ray Splenomegaly. •

•

•

23

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Disorders of Red Cells

Fig. 3.1: Pathogenesis of β-thalassemia major and its consequence

• Extramedullary hematopoiesis: in liver and spleen → consequent hepatosplenomegaly. • Cachexia: develops in untreated patients.

b-thalassemia major Iron overload damgaes parenchymal organs due to hemosiderosis and secondary hemochromatosis.

Iron Overload and its Consequences

Failure to thrive, retarded growth, monogoloid face, and hepatosplenomegaly are clinical features of

Clinical Features

b-thalassemia major.

4–5 years of age. • Bone changes: those who survive longer develop distortion of skull and facial bones. X-ray skull shows hair on end appearance(Fig. 3.2) and face shows a characteristicthalassemic facies (Fig. 3.3). • Marked splenomegaly: up to 1500 grams due to hyperplasia and extramedullary hematopoiesis. • Extramedullary hemopoiesis: liver and lymph nodes may show extramedullary hematopoiesis.

•

• Causes of iron overload: 1. Increased absorption of dietary iron from duodenum 2. Hemolysis 3. Repeated transfusions (usual mode of treatment). • Consequences: iron overload produceshemosiderosis and secondary hemochromatosis and damages to parenchyma of organs (e.g.heart, liver and pancreas).

• Age: infants develop moderate to severe anemia 6–9 months after birth. • Growth and development: untreated/untransfused childrenfail to thrive and die within

Thalassemia Syndrome CHAPTER 3

Fig. 3.2: X-ray appearance of skull in b-thalassemia showing hair-onend appearance (Courtesy: Dr Nuthan Kamath)

Fig. 3.3: Appearance of typical thalassemic facies (Courtesy: Dr Nuthan Kamath)

• Iron overload: multiple blood transfusions may lead to iron overload and result in hemosiderosis and secondary hemochromatosis (heart, liver and pancreas).

Laboratory Findings Peripheral Blood

Q. Mention the laboratory findings inb-thalassemia major.

• Hemoglobin (ranges from 3 to 8 g/dL)and hematocrit (ranges from 8 to 23%): markedly reduced • RBC count increased/normal (in contrast to iron deficiency anemia). • Reticulocyte count increased and in the range of 5 to 15%. • Red cell indices: – MCV decreased and in the range of 45–70 fL (normal range 82–98 fL). – MCHC decreased and in the range of 22–30 g/dL (normal range 31–35 g/dL). RDW normal – MCH decreased and in the range of 20–28 pg (normal range 27–32 pg). MCV, MCH and MCHC – RDW-within normal limits (in contrast to iron deficiency anemia where it is increased). decreased. • Peripheral smear: – RBCs: ◆ Microcytic hypochromic anemia ◆ Moderate to marked anisocytosis and poikilocytosis ◆ Many target cells (Figs 3.4 and 3.5) ◆ Basophilic stippling ◆ Nucleated red cell precursors (normoblasts) in variable numbers (5–40%). – WBCs: leukocytosis with mild left shift. – Platelets: normal.

Bone Marrow • • • • • •

Cellularity: markedly hypercellular. M: E ratio: reversed to 1:1 to 1:5 depending upon the degree of erythroid hyperplasia. Erythropoiesis: normoblastic with marked er ythroid hyperplasia. Myelopoiesis: normal. Megakaryopoiesis: normal. Bone marrow iron: markedly increased due to increased dietary absorption and hemolysis.

Q. Write short note on peripheral smear findings in b-thalassemia major. b- thalassemia major: RDW normal. The peripheral blood smear shows microcytic hypochromic anemia, target cells and anisopoikilocytosis. Bone marrow in bthalassemia major shows marked normoblastic erythroid hyperplasia. Marrow iron is markedly increased.

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Disorders of Red Cells

Fig. 3.4: Peripheral blood smear in β-thalassemia showing target cells (arrows)

Fig. 3.5: Diagrammatic appearance of peripheral blood smear in -thalassemia showing target cells (short arrows) and nucleated red cells (long arrows)

Biochemical Findings • • • •

Bilirubin: increased—mainly of unconjugated type. Urine urobilinogen: increased Serum haptoglobin: markedly reduced. Serum iron status: – Serum iron, serum ferritin and transferrin saturation are markedly increased – otal iron binding capacity (IBC): reduced.

Reduced/absence of synthesis of b-chains; the excess a-chains combine with γ-chains leading to increased HbF.

Special Tests

Note:normal adult cell contains 96% HbA (a2b2), 3% HbA22(a2d2) and 1% HbF(a2 γ2).

TABLE 3.2: Hemoglobin F and A 2 percentage in thalassemia syndromes

• Fetal hemoglobin (HbF): increased to 30% to 90% (normal range 0 – 1%). • Hemoglobin electrophoresis (able 3.2): – b+ thalassemia (b+/b+ or b0/b+ genotypes): demonstrates bands of both HbA and HbF. – bo thalassemia (b0/b0 genotype): since no b-chains are formed, there is no HbA. Major hemoglobin is HbF with normal or low HbA 2. • High performance chromatography(HPLC): HbF is increased (30–90%). HPLC measures various fractions of hemoglobin (Hb) and is used for confirmation of diagnosis. • Prenatal diagnosis bymolecular analysis of DNA. • Estimation of globin chains: normally a: b ratio is 1:1. Lack of b chain alter this ratio to 5–30:1

Type

HbF

HbA2

b -Thalassemia major (homozygous)

30–90%

<3.5%

b -Thalassemia intermedia (double heterozygous)

10–30%

<3.5%

b -Thalassemia minor/trait (heterozygous)

0–5%

3.6–8%

Thalassemia Syndrome CHAPTER 3

Differences between Iron Deficiency Anemia and b-Thalassemia Major (Table 3.3) TABLE 3.3: Differences betweeniron deficiency anemiaand b-thalassemia major Character

Irondeficiencyanemia

b-thalassemia major

Etiology

Deficiency of iron

Reduced synthesis of b chain

Decreased (< 5 million/cu mm)

Increased (> 5 million/cu mm)

– Type of RBCs – Anisopoikilocytosis – Target cells

Microcytic hypochromic Mild to moderate Absent

Microcytic hypochromic Severe Present

•

Bone marrow iron

Absent

Markedlyincreased

•

Serum iron profile – Serum ferritin – Serum iron – TIBC

Reduced < 15 µg/L Reduced Increased

Increased (300 – 1000 µg/L) Increased Normal

•

Fetal hemoglobin (HbF)

Normal(0–1%)

Markedlyincreased(30–90%)

•

RDW

Increased

Normal

Laboratory findings •

RBC count

•

Peripheral smear

b-thalassemia major should be differentiated from iron deficiency anemia. Treatment with iron in b-thalassemia major worsens the iron load and its consequences.

Clinical features •

Age

•

Growthanddevelopment

•

Hepatosplenomegaly

X-ray findings

Any age Normal

Presented years 2 < of age Retarded

Absent

Present

Nil

Haironendappearance

b-thalassemia intermedia: it is a clinical entity intermediate between thalassemia trait and thalassemia major.

Abbreviations: RDW, red cell distribution width; TIBC, total iron-binding capacity.

b-THALASSEMIA MINOR/TRAIT • More common thanb-thalassemia major. • Most patients are heterozygous for thalassemic gene. • Usually asymptomatic and anemia is mild.

Laboratory Findings inb-Thalassemia Minor • Peripheral blood: microcytosis, hypochromia,basophilic stippling and target cells. • Bone marrow: mild er ythroid hyperplasia. • Hemoglobin electrophoresis: increase in HbA2 (a2d2) to 4 to 8% of the total hemoglobin (normal 2.5 ± 0.3%). HbF levels may be normal or slightly increased. NESTROF test positive • NESROF test (Naked eye single tube red cell osmotic fragility test):positive. because the microcytic – In this test, 0.02 mL of patient’s blood is added to 5 mL of 0.35% saline in a test tube. hypochromic RBCs of – After half an hour white paper with a dark black line is held behind the tube. b-thalassemia minor are – Te microcytic hypochromic RBCs of thalassemia minor are resistant to lysis thanresistant to lysis than normocytic normochromic normocytic normochromic RBCs. RBCs. – Hence, the black line on the paper is not clearly visible through the test tube compared to normal cells. • Estimation of HbA2: HPLC is used for accurate estimation. HbA 2 estimation is diagnostic and level ranges from 4% to 8%.

27

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Disorders of Red Cells

b-thalassemia trait should be differentiated from iron deficiency (Table 3.4).

TABLE 3.4: Differences betweeniron deficiency anemia and b-thalassemia minor/trait C ha ra c ter

I r o nd e fi c i e n c ya n e m i a

b-thalassemia minor

Deficiency of iron

Reduced synthesis of b chain

Microcytichypochromic

Microcytichypochromic

– Serum ferritin – Serum iron – TIBC

Reduced < 15 µg/L Reduced Increased

Normal /slightly incresaed Normal Normal

•

HbA2 level

Normal or decreased (2.5 + 0.3 %)

•

RBC count

<5million/cumm

•

RDW

Increased

Etiology Laboratory findings

a-Thalassemia: anemia due to— Lack of adequate hemoglobin Effect of excess unpaired non-a-chains (b, γ, d). •

•

•

Peripheral smear - RBCs

•

Serum iron profile

Increased (4–8 %) >5million/cumm Normal

a-THALASSEMIA • Inherited disorders characterized byreduced or absent synthesis of a-globin chains. • Autosomal recessive disorder.

Molecular Pathology In contrast to a single gene coding b-globin chain, each a-globin chain are encoded by two genes. Deletion of a-gene is the most common cause of reduced a-chain synthesis.

a-thalassemia is one of the cause of non-immune

Clinical Syndromes

hydrops fetalis.

a-chain Fournumber genes control synthesis. of a-thalassemia varies greatly depending on the of a-globin genes deletedSeverity (able 3.5). Each of the four a-globin genes normally contributes 25% of the total a-globin chains.

Immune hydrops fetalis is a hemolytic disease caused by blood group incompatibility between mother and fetus.

TABLE 3.5: Clinical syndromesassociated witha-thalassemia disorders Clinical syndrome

No. of

Clinicopathological features

a-globin deleted Silent carrier state

1

Asymptomatic

a-Thalassemia trait

2

Usually asymptomatic. Normal hemoglobin level or minimal anemia

Hemoglobin H disease

3

Moderate microcytic hypochromic anemia

Hydrops fetalis (Hb Barts)

4

Severe form, fatal and usually results in intrauterine death

Sickle Cell Disease

4 CHAPTER

SICKLE CELL DISEASE Definition Sickle cell disease (SCD) is a group of hereditary disorders of hemoglobin characterized by production of defective hemoglobin called sickle hemoglobin (HbS). On low oxygen tension or deoxygenation, HbS imparts sickle shape to RBCs. HbS is produced due toqualitative defect in hemoglobin production caused by mutation in β-globin gene.

Classification of Sickle Cell Disease (Table 4.1) TABLE 4.1: Classification of sickle cell disease Sicklecellanemia(SS) •

Homozygous state—both the β-globin chains are abnormal/defective

Sickle cell diseases are hemoglobinopathies characterized by qualitative defect in hemoglobin synthesis.

Sickle cell anemia is a homozygous state in which both β-globin chains are abnormal.

Sicklecelltrait(AS) •

Heterozygous state—one gene is defective (for HbS) and while the other gene is normal (for HbA)

Sickle cell trait: one β-globin chain is abnormal and other β-globin chain is normal.

Other sickling syndromes (Compound heterozygous) •

If both the β-globin chains have different abnormalities, (e.g. Hb SC, Hb S-β-thalassemia)—termed as compound heterozygous

SICKLE CELL ANEMIA Characteristic Features • Autosomal recessive disorder manifests early in life. • β Homozygous state (SS) caused inin the -globin gene.no HbA. • HbS constitutes more than 70%by ofamutation hemoglobin their RBCs with

Etiopathogenesis • Production of abnormal hemoglobin called sickle hemoglobin (HbS).

Sickle cell anemia: autosomal recessive disorder with extravascular hemolysis.

HbS provides protection against falciparum malaria.

Q. Discuss the etiopathogenesis of sickle cell anemia.

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Disorders of Red Cells

Fig. 4.1: Replacement of glutamic acid with valine in the sixth position of β-globin

Replacement of the glutamic acid residue by valine in 6th position of β-globin chain.

• Missense point mutation: in HbS, there issubstitution of glutamic acid by valine in the 6th position the β-globin chain of hemoglobin (Fig. 4.1). It alters the solubility or stability

of the hemoglobin and produces hemolytic anemia. • HbS is responsible for the characteristics of the disease.

Molecular Basis of Sickling (Fig. 4.2) •

During low O2 tension or deoxygenation, HbS molecules undergo aggregation and polymerization.

During low oxygen tension or deoxygenation RBCs assume sickle shape and predisposes to vessel occlusion.

RBCs in sickle cell anemia have shorter lifespan and causes hemolytic anemia.

Fig. 4.2: Pathogenesis of sickle cell anemia

Sickle Cell Disease

•

• •

•

•

CHAPTER 4

If deoxygenation continues, the aggregated HbS molecules form long needle-like fibers (or pseudocrystalline structures known as tactoids) within RBCs. Te tactoids grow in length beyond the diameter of RBCs and distort RBC shape. RBC become elongated and assumes ashape like sickle (or crescent moon or holly-leaf or boat) and predisposes to stasis and vascular occlusion. When the oxygen tension returns to normal, the sickled red cell returns to normal shape. Recurrent sickling causes red cell membrane damage and these RBCs becomeirreversibly sickled cells (ISC).

Factors Affecting Sickling (Table 4.2) TABLE 4.2: Factors affecting sickling Factors

Favorssickling

Type of other associated hemoglobins

-

HbA

-

HbF

HbC

-

Slowing of bloodstream

-

Transit time in microvasculature MCHC Intracellular pH Other factors

Hinderssickling

Increased MCHC Decreased pH

In sickle cell anemia, HbF hinders sickling.

Decreased MCHC -

Temperature above 37°C

-

Infections

-

Abbreviation:MCHC, mean corpuscular hemoglobin concentration.

Mechanism of Red Cell Damage

With repeated sickling the RBCs become irreversibly

• HbS polymerization: when HbS polymerizes, it grows beyond the RBC membraneand sickled cells (ISC) and leads

project through it.

to RBC membrane damage

• Dehydration: repeated episodes of sickling leads to increased dehydration of RBCs. Teseand hemolysis.

RBCs become more rigid and nondefromable(irreversible sickled cells). • Percentage of ISC: degree of the hemolysis correlates with the percentage ofirreversibly

sickled cells. • Impaired cation homeostasis: structural changes in the RBC membrane causes theinflux

of Ca+ ions, which activate an ion channel resulting in theefflux of K+ and H2O.

Pathogenesis of the Microvascular Occlusions Most serious clinical features are due to occlusion of microvasculature. • Deformability: sickle cells are rigid and tend to aggregate. Te aggregated sickle cells block the small blood vessels. • Factors that slow the blood flow: RBC cytoskeletal damage slow the movement of RBCs through microvascular beds. • Higher expression of adhesion molecules: sickle cells express higher levels of adhesion molecules and thus become abnormally sticky to the endothilium. • Inactivation of nitric oxide: lysed sickle cells liberate free hemoglobin, which binds and inactivates nitric oxide (NO). Tis narrows the vessels and produces microvascular stasis and sickling.

Most serious clinical features of sickle cell anemia are due to microvascular occlusion.

31

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Disorders of Red Cells

Clinical Features (Fig. 4.3) The cardinal clinical features are due to chronic hemolytic anemia, crises (recurrent painful episodes), infections and chronic organ damage.

• Presence ofHbF in the first 6 monthsof life has a protective role. • Symptoms appear after 6 monthsof age as the HbF disappears. • Infants and children present with acute problems like severe infection, acute chest

syndrome, splenic sequestrationand stroke. • Chronic hypoxia in children is responsible for generalizedimpairment of growth and

development. Adults manifest with chronic organ damage.

Chronic Hemolytic Anemia • Lifelong hemolysis (mainly and unconjugated causeschronic hemolytic , which is of moderate degree . Tisextravascular) produces raised (indirect) anemia bilirubin , and

predisposes topigment bilirubin gallstones (cholelithiasis) andcholecystitis.

Four crises encountered in sickle cell anemia: sickling crisis, hemolytic crisis, aplastic crisis and sequestration crisis.

Recurrent splenic infarction due to sickling crisis lead to autosplenectomy.

Crises Four types of crises are encountered. Tese are: 1. Sickling crisis (vaso-occlusive/pain/painful/infarctive crisis) • Most common • Blockage of microcirculationby sickled red cells produceshypoxic injury and infarction. • Bone:manifest as thehand-foot syndrome , dactylitisof the bones of the hands or feet or both. • Lung: acute chest syndrome (dangerous) . • Spleen: acute abdominal pain due to infarcts of abdominal viscera caused by occlusion of vessels. Recurrent splenic infarction results inautosplenectomy.

Infants most commonly present with dactylitis.

Most common cause of death in adults is acute chest syndrome.

Fig. 4.3: Various effects of vascular occlusion and hemolysis in sickle cell anemia

Sickle Cell Disease

CHAPTER 4

2. Hemolytic crisis • Rare type and presents withmarked increase in hemolysis. 3. Aplastic crisis • Associated with parvovirus B19. • Reticulocytopenia.

Reticulocytopenia is seen in aplastic crisis and reticulocytosis in sequestration crisis.

4. Sequestration crisis • Usually occurs in children. • Sudden trapping of bloodin spleen or liver causes rapid enlargement of the organ and drop in hematocritleading to hypovolemic shock.

Other crises encountered rarely are hypoplastic crisis and megaloblastic crisis (due to inadequate folate).

Increased Susceptibility to Infections

Susceptible to acute

• Common infections are pneumonia due to Pneumococcus, meningitis due to S. infections with encapsulated organisms. pneumoniae and osteomyelitis due to Salmonella. Increased frequency of osteomyelitis is

due to bone infarcts, which act as a nidus for infection. • Septicemia and meningitis are the most common causes of death in children.

Causes of susceptibility to infections: • Hypofunction of spleen: In children: due to congestion and poor blood flow. Common pathogens: In adults: due to multiple infarcts and resultantautosplenectomy. S. pneumonia, • Defects in the alternative complement pathway. Salmonella and Impairs opsonization of encapsulated bacteria such as pneumococci Haemophilus and influenzae.Pneumococcus.

Chronic Organ Damage Particularly seen in the spleen, bones, kidneys, heart, lungs, brain and skin. • Spleen – Children after 6 months of life present with splenomegaly (up to500 g). – After 5–6 years of age, the spleen gets fibrosed and gradually reduces in the size due to multiple infarcts. – Gradualloss of splenic function secondary to infarcts results in autosplenectomy. • Bone: osteomyelitis, particularly with Salmonella typhimurium • Extremities: skin ulcers over the lower extremities

Laboratory Findings in Sickle Cell Anemia Peripheral Blood • • • •

Hemoglobin: decreased. Hematocrit (PCV): decreased. ESR: reduced. Reticulocyte count: increased and range from 3% to 10%.

Peripheral smear • – RBCs: ◆ Normocytic normochromic to mildly hypochromic. ◆ Moderate to severe degree of anisopoikilocytosis. ◆ Characteristic cell is the sickle cell—appear as long, curved cells with pointed ends (Figs 4.4 and 4.5); may also show target cells (due to red cell dehydration) and ovalocytes. ◆ Polychromatophilia due to reticulocytosis. – WBCs: mildly increased with shift to left. – Platelets: mildly increased.

SCA: severe hemolytic anemia Sickling crisis Autosplenectomy.

Q. Laboratory findings in sickle cell anemia.

Sickle cell anemia: ESR is reduced because sickle cells do not form rouleaux.

Peripheral smear shows characteristic sickle cells number of which varies.

33

34

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Disorders of Red Cells

Fig. 4.4: Peripheral blood smear with sickle cells (arrows)

In severe cases, skull bone shows crewcut appearance in roentgenograms.

Extramedullary hematopoiesis can also develop as a compensatory mechanism.

Fig. 4.5: Diagrammatic peripheral blood smear with sickle cells (arrows)

Bone Marrow • Cellularity: hypercellular. • Erythropoiesis: compensatory normoblastic erythroid hyperplasia, which expands the marrow and causes resorption of bone and secondary new bone formation. • Myelopoiesis: normal. • Megakaryopoiesis: normal. • Iron stores: usually increased.

Serum Findings • Serum bilirubin: raised and predisposes to pigment gallstones. • Iron status: raised serum iron, serum ferritin and transferrin saturation. • Serum haptoglobin: reduced. •

Urine Urobilinogen: increased.

Diagnostic/Confirmatory Tests • Sickling test: – Sickling is induced by adding a reducing (oxygen-consuming)agent like 2% sodium

Sickle cell anemia:HbS 70–90%, HbF 10–30%, no HbA.

• • • •

metabisulfite or sodium dithionite to blood sample. – Red cells with HbS showsickled (Fig. 4.6) and holly leaf appearance. – It is diagnostic of sickle cell anemia. Hemoglobin electrophoresis: HbS is a slow moving compared to HbA and HbF. Estimation of HbF: in homozygous state constitutes about 10–30% of hemoglobin. HPLC: useful for confirmation of diagnosis. Prenatal diagnosis: by analysis of fetal DNA obtained by amniocentesis or chorionic villous biopsy, to detect the point mutations.

SICKLE CELL TRAIT Heterozygous state for the hemoglobin S mutation and shows both HbA and HbS (HbAS). One defective gene (from one parent with HbS) and while the other gene is normal.

Sickle Cell Disease

CHAPTER 4

Sickling test is a diagnostic test for sickle cell anemia.

Fig. 4.6: Sickling test. Sickled red cells (arrows) induced by reducing agent (2% sodium metabisulfite)

Pathogenesis

Sickle cell trait: Usually no anemia •

In sickle cell trait, the hemoglobin A in RBCs prevents hemoglobin S polymerization. However, No significant clinical RBCs may sickle under extreme conditions (e.g. flight at high altitude in unpressurized aircraft, features Amount of HbS varies deep sea diving). •

•

•

Clinical Features

from 25% to 40% Hb A in RBCs prevents polymerization of Hb S.

Usually asymptomatic. Normal growth and development, lifespan and life expectancy.

Laboratory Findings Peripheral Blood • Hemoglobin: normal or mildly decreased. • Peripheral smear: – RBCs: normocytic normochromic picture with very few target cells and mild degree of anisopoikilocytosis. – WBCs: normal. – Platelets: normal.

Bone Marrow Hypercellular because of a compensatory normoblastic erythroid hyperplasia.

Diagnostic Tests • Hb electrophoresis: demonstrates two bands of HbS and HbA. • Sickling test: sickling test is positive. • High-performance liquid chromatography (HPLC):useful for confirmation of diagnosis.

In sickle cell trait: HbS 40–45% and HbA 55–60%.

35

5

Other Anemias

CHAPTER

Immunohemolytic anemias are characterized by the destruction of RBCs by either allo or auto antibodies.

Immunohemolytic anemias are mainly classified as: 1. Alloimmune and

IMMUNOHEMOLYTIC ANEMIAS Anemias due to premature RBC destruction(hemolysis) mediated by antibodies that bind to RBCs. Te antibodies may be either allo or auto type.

Classification of Immunohemolytic Anemias ( Table 5.1) TABLE 5.1: Classification of immunohemolytic anemias

2. Autoimmune hemolytic anemia.

Alloimmune hemolytic anemia • •

Hemolytic disease of the newborn Hemolytic transfusion reactions: mismatched blood transfusion

Autoimmune hemolytic anemia •

Hemolytic transfusion reactions are due to ABO mismatch. The antibodies present in the recipient’s serum coat donor’s RBCs and lead to intravascular hemolysis.

•

•

Warm antibody type (IgG antibodies active at 37°C) – Primary (Idiopathic) – Secondary: autoimmune disorders (systemic lupus erythematosus), drugs, lymphomas Cold agglutinin type (IgM antibodies active at 4°C–18°C) – Acute: mycoplasmal infection, infectious mononucleosis – Chronic: idiopathic, lymphomas Cold hemolysin type (Donath-Landsteiner antibodies)

Alloimmune Hemolytic Anemia • •

Q. Write short notes on hemolytic disease of newborn. • • •

Production of antibody against foreign antigen not present on individual’s red blood cell. Allo-antibodies are present either in the serum or bound to red cells.

HEMOLYTIC DISEASE OF THE NEWBORN It is an allo-immune hemolytic anemia developing in the fetus and newborn baby. Hemolysis is extravascular. HDN develops when the IgG antibodies against blood group of fetus passes from mother to fetus through the placenta.

Other Anemias

•

CHAPTER 5

Occurs in two forms: HDN may be either due to – Rh incompatibility in which mother is Rh negative and fetus is Rh positive. Te anti-D Rh or ABO incompatibility between mother and fetal antibodies are responsible for the hemolytic anemia. RBCs. – ABO incompatibility in which mother’s blood group is O and fetus is either of A or B blood group. Eitheranti-A or anti-B antibodies cause hemolysis.

Rh Hemolytic Disease of the Newborn (Fig. 5.1) Rh hemolytic disease of the newborn is more important than due to ABO incompatibility.

Pathogenesis • •

•

HDN usually does not Occurs when mother is Rh (D antigen) negative and fetus is Rh positive. manifest during first Sensitization occurs when fetal Rh positive RBCs enter into Rh negative mothers. Rhpregnancy. Sensitization negative mother develops anti-Rh antibodies. develops during delivery Sensitization occurs only at the time of delivery or during miscarriage. So, it does not or miscarriage. manifest in the first pregnancy.

Rh HDN develops when mother is Rh-ve and fetus is Rh+ve.

Fig. 5.1: Pathogenesis of Rh hemolytic disease of the newborn

37

38

SECTION 1

Disorders of Red Cells

Hydrops fetalis is fatal condition, characterized by left and right-sided heart failure producing generalized edema and may result in death.

•

•

In subsequent pregnancy, anti-Rh antibodies from mother cross placenta and coat the Rh positive fetal red cells. Tese antibodies cause immune destruction of fetal red cells results in severe hemolytic anemia leading to jaundice of the newborn. Fetus may develop cardiac failure—hydrops fetalis(immune type).

Clinicopathological Features In Rh HDN, high levels of unconjugated bilirubin can cross blood brain barrier causing kernicterus and

• •

Infants may have jaundice at birth. When the disease is severe, the levels of unconjugated bilirubin in the blood are high and bilirubin can pass the blood brain barrier.

•

Bilirubin is deposited the is central nervous system (especially the basal of ganglia) producing neurological damageinand known as kernicterus (yellow coloration cerebellum and basal ganglia due to bilirubin deposition). It can cause death of the infant.

death of infant.

Prevention of Rh HDN: by the prophylactic removal of fetal cells entering the maternal circulation before sensitization develops, by injecting anti-D into the Rh D negative mother.

Laboratory Findings Peripheral blood Hemoglobin: decreased. Reticulocyte count: increased. • •

Peripheral smear: normocytic normochromic anemia with nucleated RBCs and polychromatophils.

•

•

Peripheral smear: – RBCs: normocytic normochromic anemia with numerous nucleated RBCs, polychromatophils and occasional spherocytes. – WBCs: normal. – Platelets: normal.

Antiglobulin test (Coombs test): antibodies in the mother and baby are detected by indirect and direct Coombs test respectively (Fig. 5.2).

In direct antiglobulin test, patient’s RBCs are used where as in indirect antiglobulin test patient’s serum is used for the test.

Fig. 5.2: Direct and indirect methods of antiglobulin test (Coombs test)

Other Anemias

CHAPTER 5

Serum findings Serum bilirubin: increased. Lactate hydrogenase (LDH): increased. Haptoglobin: decreased.

Antiglobulin test is useful for diagnosis of HDN.

ABO Hemolytic Disease of the Newborn

ABO HDN is more common but less severe. It may be

• • •

• • •

•

It is less severe. seen in first pregnancy. Te fetus may be affected in the first pregnancy of a mother with blood group O. Te IgG antibodies to A or B from maternal blood cross placenta and enter the fetal circulation. Tese anti-A or anti- B antibodies react with A and B antigenic determinants present in fetal fluids and tissues. Tis results in consumption of major portion of the maternal IgG and the small portion, which is left combines with fetal red cells causing only mild hemolysis.

ANTIGLOBULIN (COOMBS) TEST It is useful to detect the presence of incomplete antibody (IgG) and/or complement on the RBC membrane.

Q. Write short notes on Coombs (antiglobulin) test.

Principle •

• •

RBCs coated with incomplete antibody (IgG) or C3 complement does not cause agglutination of RBCs. Coombs reagent contains antibodies (antiglobulins) against human IgG/IgM/complement. If the RBCs coated by incomplete antibody or complement, are treated with Coombs reagent, the antiglobulins in the reagent willinduce agglutination of such RBCs.

Types of Antiglobulin Test (Fig. 5.2) • •

Direct (Coombs) antiglobulin test (DA) Indirect (Coombs) antiglobulin test (IA)

Direct Antiglobulin Test (Fig. 5.2) Direct antiglobulin test (DA) (direct Coombs test)detects antibodies (IgG) and/or complement coatedon the surface of patient’s RBCmembrane. Patient’s RBCs are taken in a test tube and washed three times in normal saline. Coombs (anti-globulin) reagent is added and observed for agglutination. Agglutination indicates the presence of antibody on the RBC membrane and interprets as positive DA. • • •

Uses of Direct Antiglobulin Test Hemolytic disease of the newborn (HDN), in which direct Coombs test is performed on the •

•

• •

newborn baby’s red cells from the cord blood. Autoimmune hemolytic anemia: to demonstrate in vivo attachment of antibodies to red cells. Drug induced red cell sensitization. Investigation of hemolytic transfusion reaction.

There are 2 types of antiglobulin test: direct and indirect.

Patient’s red cells are used in direct antiglobulin test.

39

40

SECTION 1

Disorders of Red Cells

•

Patient’s serum is used for indirect antiglobulin test.

In HDN, newborn baby’s RBCs from cord blood is used for direct antiglobulin test, which will be positive.

Indirect Antiglobulin Test (Fig. 5.2) Indirect antiglobulin test (IA) (indirect Coombs test)detects the presence of incomplete (IgG) antibodies and/or complement in the patient’s serum. In this test, patient’s serum is taken and “O” Rh positive cell suspension of any normal individual is added. “O” Rh positive RBCs are coated with (lgG) anti-Rh antibodies (if present)the in patient’s serum. Add Coombs (antiglobulin) reagent and examine for agglutination. Agglutination of RBCs indicates the presence of antibodies in the patient’s serum and test is reported as positive for indirect antiglobulin test. •

• • •

Patient’s serum + O Rhpositive RBC suspension + Coombs reagent → Agglutination (test positive).

Uses of Indirect Antiglobulin Test • •

The type of antibody causing autoimmune hemolytic anemia may be warm antibody or cold agglutinin or cold hemolysin.

Hemolytic disease of newborn: mother’s serum is tested to detect anti-Rh antibody. Cross-matching for blood transfusion:to detect incompatibility of recipient’s serum with donor’s cells.

AUTOIMMUNE HEMOLYTIC ANEMIA •

•

Antibodies against self-antigens on the RBC membrane cause premature destruction of RBCs. Anti-RBC antibodies can be divided into three general categories (able 5.1). Interaction of the autoantibody with the red cell antigen is dependent on the temperature, i.e. warm or cold type.

Warm AIHA: mediated by IgG autoantibodyoptimally active at 37°C.

Warm Antibody Type • • • • •

Most common type (50–70%). Idiopathic (primary) or secondary to drug exposure or predisposing disease. IgG type antibodies combine with RBC antigen at 37°C— warm antibody. Direct antiglobulin test: DA (Coombs test)positive in 90–95% cases. LE cell test: positive in SLE with secondary autoimmune hemolytic anemia (AIHA).

Cold Agglutinin Type • • •

Caused bycold agglutinins. Mediated by IgM antibodies optimally active below 30°C. Occurs as a complication of infections (e.g. infectious mononucleosis, Mycoplasma infections) and lymphoid neoplasms.

Cold Hemolysins Type (Donath-Landsteiner Antibodies) • • •

Autoantibodies directed against the P antigen system on red cells. Responsible for a rare disorder known asparoxysmal cold hemoglobinuria. Direct antiglobulin test is usually negative.

Other Anemias

CHAPTER 5

FRAGMENTATION SYNDROME Te RBCs subjected to trauma (physical or mechanical) in the circulation can undergo fragmentation and result in intravascular hemolysis leading to hemolytic anemias. Tese are known as fragmentation syndrome.

Classification According to the site of hemolysis it is classified as: Macroangiopathic (large vessels) hemolytic anemia: red cell trauma from an abnormal vascular surface (e.g. prosthetic heart valve, synthetic vascular graft). •

•

Microangiopathic hemolytic anemia (MAHA): it occurs in capillaries due to abnormal narrowing of the lumen (e.g. disseminated intravascular coagulation).

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA It is arare and is the only hemolytic anemiaacquired mutation in the hematopoietic stem cell.

PNH is an acquired disorder in which there is deficiency of GPI linked proteins, which normally protect the red cells against complement mediated lysis.

Etiology and Pathogenesis •

•

•

Acquired mutations in the phosphatidylinositol glycan-group A (PIGA) gene in the hematopoietic stem cell. PIGA gene mutation causes deficient synthesis of GPI-linked proteinsin blood cells and loss of anchor for decay-accelerating factor (DAF). Normally, DAF responsible for In PNH, RBCs are very complement degradation. sensitive to complementmediated hemolysis. RBCs are abnormally sensitive to complement-mediated intravascular hemolysis .

Clinical Features •

•

Intravascular hemolysis: hemoglobin in acidic urine is converted into acid hematin and results in dark brown urine . Trombosis: in the hepatic, portal or cerebral veins.

Laboratory Findings •

•

Ham’s acidified serum test and sucrose hemolysis test: patient’s RBCs undergo lysis when PNH: Ham’s acidified serum incubated with acidified serum (Ham test) or sugar (sucrose hemolysis test). test and sucrose hemolysis Flow cytometry: detects RBC deficient in GPI-linked proteins (CD55 and CD59) and istest +ve. useful for diagnosis of PNH.

ANEMIAS OF BLOOD LOSS Acute Blood Loss (Hemorrhage) •

•

During recovery phase of acute blood loss,

Causes loss of intravascular volume and if massive can lead tohypovolemic shock and peripheral smear show death. reticulocytosis. Bleeding may be external (e.g. open fracture, knife wound) orinternal (e.g. ruptured spleen, ruptured abdominal aneurysm).

41

42

SECTION 1

Disorders of Red Cells

•

Peripheral smear: – RBCs: normocytic normochromic anemia. Polychromasia during the recovery phase due to increased reticulocytes. – WBCs: leukocytosis. – Platelets: increased in number (thrombocytosis) during recovery phase.

Chronic Blood Loss Produces anemia when the rate of blood loss exceeds the regenerative capacity of the bone marrow or when iron reserves are depleted and results iniron deficiency anemia.

Sideroblastic anemias are rare refractory anemias which may be hereditary or acquired.

SIDEROBLASTIC ANEMIAS Rare heterogeneous group of refractory anemias characterized by: Ring sideroblasts in the bone marrow aspirate (Fig. 5.3). Dimorphic peripheral blood picture:microcytic hypochromic red cells in hereditary form and macrocytic in the acquired forms of the disease mixed with normochromic cells. Iron-containing inclusions (Pappenheimer bodies)in the RBCs. Increased serum iron concentration and markedly increased storage iron. Ineffective erythropoiesis. • •

•

•

•

It is classified as: 1. Hereditary sideroblastic anemia 2. Acquired sideroblastic anemia: idiopathic or secondary.

Fig. 5.3:Ring sideroblasts with partial perinuclear ring of iron granules

SECTION

Disorders of White Cells

2

Quantitative and Qualitative Disorders of Leukocytes

NORMAL DIFFERENTIAL LEUKOCYTE COUNT (DLC) Te normal range of DLC in an adult is presented in able 6.1.

6 CHAPTER

Differential leukocyte count (DLC) is one of the routine, useful and important investigations.

TABLE 6.1: Normal range of different leukocytes in an adult Typeofwhitebloodcell

Normalrange

Neutrophils

40–70% (2.0–7.0 × 109/L)

Lymphocytes

20–40% (1.0–3.0 × 109/L)

Monocytes

2–10% (0.2–1.0× 109/L)

Eosinophils

1–6% (0.02–0.5× 109/L)

Basophils

Less than 1% (0.02–0.1 × 109/L)

QUANTITATIVE DISORDERS OF LEUKOCYTES Leukocytosis

Q. Define leukocytosis and list

An increase in the total number ofleukocytesin the bloodmore than 11,000/cu mm(11 × 109/L). its causes. Causes: common causes of leukocytosis are shown in able 6.2. TABLE 6.2: Common causes of leukocytosis •

•

•

•

Infections – Bacterial – Viral infections (e.g. infectious mononucleosis) Leukemia – Acute – Chronic: chronic lymphocytic leukemia and chronic myeloid leukemia Leukemoid reactions Physiological – Pregnancy – Exercise

Leukocytosis is usually due to increase in the neutrophils, but may also be due to increased lymphocytes (or rarely monocytes and eosinophils).

46

SECTION 2

Disorders of White Cells

Leukopenia is the decrease in the WBC count below 4,000/cumm.

Leukopenia Total leukocyte count is less than 4,000/cu mm (4 × 109/L). Causes: common causes of leukopenia are shown in able 6.3. TABLE 6.3: Common causes of leukopenia

The causes of leukopenia include typhoid and paratyphoid fever and aplastic anemia.

•

•

Typhoid and paratyphoid Anemia – Aplastic an emia – Megaloblastic anemia

•

Hypersplenism

•

Drugs including cytotoxic drugs

•

Radiation

•

Rarely leukemia

Q. Define neutrophilia and mention its causes.

Disorders of Neutrophils

Neutrophilia: absolute neutrophil count more than 8000 cells/mm.

An absolute neutrophil count of more than 8000/cu mm (8 × 109/L). Differential count shows 9 more than 70% neutrophils and is usually accompanied by leukocytosis (15–30 × 10 /L).

Common causes of neutrophilia are infections, inflammatory conditions and tissue necrosis.

Neutrophilia (Fig. 6.1)

Causes of neutrophilia: major causes of neutrophilia are shown in able 6.4.

TABLE 6.4: Major causes of neutrophilia 1. Pathological: – Acute bacterial and fungal infections: ◆ Localized: pyogenic microorganisms causing infections, e.g. pneumonias, pyogenic meningitis, cellulitis, diphtheria, abscess, tonsillitis, etc. ◆ Generalized: septicemia, acute rheumatic fever – Acute inflammatory processes: inflammatory conditions (acute appendicitis), vasculitis – Tissue necrosis: burns, myocardial infarction, gangrene, neoplasms (tumor necrosis) – Acute stress or hypoxic states: following hemorrhage, hemolysis and surgery – Myeloproliferative neoplasms: chronic myeloid leukemia, polycythemia vera – Metabolic: uremia, acidosis, gout – Miscellaneous: eclampsia, steroid therapy 2. Physiological: – Exercise (shift from marginating pool to circulating pool), newborns, extremes of temperature, pain, emotional stress and during obstetric labor

Fig. 6.1:Peripheral smear showing neutrophilia

Quantitative and Qualitative Disorders of Leukocytes CHAPTER 6

Leukemoid Reaction

Leukemoid reaction: benign exaggerated

Benign leukocytic proliferation characterized by atotal leukocyte count of more than 25 × leukocyte proliferation to be differentiated from 109/L with immature white cells (like band forms, metamyelocytes and myelocytes). It is different from chronic myelocytic/myeloid leukemia (able 6.5).

leukemia.

TABLE 6.5: Differences between leukemoidreaction and chronic myeloid leukemia

Q. Tabulate the differences between leukemia and leukemoid reaction.

Clinical features

Leukemoidreaction

Chronicmyeloidleukemia

Features of causative disease

Splenomegaly, and bone pain are common Neutrophils in bacterial infections show toxic granules.

Peripheral blood findings WBC Total WBC count

Moderately increased, rarely exceeds Markedly increased and usually 50 × 109/L

50 × 109/L

Differential leukocyte count

Shift to the left with few immature forms. Toxic granulation seen

Shift to the left with numerous immature forms. Myelocyte and neutrophil peak

Eosinophiliaandbasophilia

Variable

Leukocyte alkaline phosphatase (LAP)

Present

Increased

Dohle bodies are small round to oval structures seen in the cytoplasm can also be observed in bacterial infections.

Decreased

RBC Anemia

Usuallyminimalorabsent

Severeandprogressive

Platelets Number

Variable

Normal or increased

Extramedullary myeloid tumors

Absent

Present

Philadelphia chromosome

Absent

Present

Neutropenia (Agranulocytosis) Reduction in the absolute neutrophil count (total WBC band forms) below 1.5 × 109/L (1500/cu mm).

In leukemoid reaction LAP score is raised and neutrophils may show toxic granulation.

Neutropenia: absolute neutrophil count below

×

% segmented neutrophils and 1500 cells/cu mm.

Etiology: the causes of neutropenia are presented in able 6.6.

Eosinophilia (Fig. 6.2) Eosinophil count of more than 450/cu mm(0.45 × 109/L). Causes of eosinophilia are presented in able 6.7.

Fig. 6.2:Peripheral smear showing eosinophilia

Eosinophilia: eosinophil count more than 450 cells/ cu mm.

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Disorders of White Cells

TABLE 6.6: Causes of neutropenia 1. Inadequate production:

Agranulocytosis: neutrophil count below 0.5 × 109/L. The patients are highly susceptible to bacterial and fungal infections.

– Suppression of stem cells : in these disorders granulocytopenia represents a component of pancytopenia ◆ Aplastic anemia ◆ Marrow infiltration ◆ Metastatic tumors ◆ Granulomatous disorders – Suppression of committed granulocytic precursors ◆ Drugs and chemicals (e.g. sulfonamides, analgesics, arsenicals) ◆ Ionizing radiation – Diseases associated with ineffective hematopoiesis

◆ Megaloblastic anemias: vitamin B12 or folate deficiency ◆ Myelodysplastic syndromes – Congenital: Kostmann syndrome (rare) – Severe infections ◆ Bacterial (e.g. typhoid, paratyphoid, septicemia) ◆ Viral (e.g. influenza, infectious mononucleosis, hepatitis, measles) ◆ Rickettsial (e.g. scrub typhus) ◆ Protozoal (e.g. malaria, kala-azar) 2. Increased destruction of neutrophils: – Immunologically mediated destruction ◆ Idiopathic ◆ Secondary ◊ Drugs ◊ Autoimmune disorders, e.g. systemic lupus erythematosus – Splenic sequestration may be associated with pancytopenia 3. Shift from the circulating pool to marginating pool: – Hemodialysis and cardiopulmonary bypass 4. Idiopathic: mechanism not known – – – –

Q. Define eosinophilia and list its causes.

Hodgkin and non-Hodgkin lymphoma Chronic lymphocytic leukemia Viral infections (HIV, hepatitis) Cyclic neutropenia

TABLE 6.7: Causes of eosinophilia 1. Allergic/atopic conditions – Asthma – Hay fever – Allergic rhinitis

– Urticaria – Drug reactions

2. Parasitic infestations (with tissue invasion) – Roundworm infestation – Filariasis Eosinophilia is seen in allergic reactions and parasitic infestations with tissue invasion.

– Hookworm infestation

3. Fungal infections(e.g. coccidioidomycosis) 4. Skin diseases – Dermatitis (eczema) – Scabies

– Pemphigus – Dermatitis herpetiformis

5. Hematological diseases – Chronic myeloid leukemia – Hodgkin lymphoma – Eosinophilic leukemia

– Polycythemia – Acute myelomonocytic leukemia

6. Miscellaneous – Tropical eosinophilia – Löeffler’s syndrome – Eosinophilic granuloma

– Pulmonary eosinophilia – Hypereosinophilic syndrome

Quantitative and Qualitative Disorders of Leukocytes CHAPTER 6

Basophilia Normally basophils (Fig. 6.3) are less than 1% of WBCs in peripheral blood. Causes of basophilia include chronic myeloid leukemia, immediate hypersensitivity reactions, mastocytosis, etc.

Monocytosis (Table 6.8) More than 10% of differential count or an absolute monocyte (Fig. 6.4) count exceeding 500/ cu mm (0.5 × 109/L).

Fig. 6.3:Diagrammatic appearance of basophil

TABLE 6.8: Causes of monocytosis 1. Infections – – – – –

Bacterial: tuberculosis, bacterial endocarditis, brucellosis Protozoal: malaria, kala-azar Spirochetal:syp hilis Rickettsial: typhus, rocky mountain fever Recovery phase of neutropenia and acute infections

2. Inflammatory diseases – Inflammatory bowel disease: ulcerative colitis, Crohn disease – Autoimmune diseases: systemic lupus erythem atosus, rheumatoid arthritis – Sarcoidosis

Fig. 6.4:Diagrammatic appearance of monocyte

3. Hematologic malignancies – – – –

Acute monocytic, myelomonocytic and myelocytic leukemias Chronic myelomonocytic leuk emia Hodgkin lymphoma Multiple myeloma

Lymphocytosis 9

Lymphocyte (Fig. 6.5) count more than 4,000/cu mm (4 × 10 /L) in adults and more than 8,000/cumm (8 × 109/L) in child. Common causes of lymphocytosis are given in the able 6.9

TABLE 6.9: Causes of lymphocytosis 1. Acute infections – Viral infections: infectious mononucleosis, mumps, measles, chickenpox, infectious hepatitis – Toxoplasmosis

Lymphocytosis: lymphocyte count more than 4,000/cu mm in adults and more than 8,000/cumm (8 × 109/L) in child. Dengue fever is caused by flavi virus transmitted by freshwater mosquito (Aedes egypti). Peripheral smear shows transformed lymphocytes and thrombocytopenia.

2. Chronic infections/inflammatory diseases – – – –

Tuberculosis Syphilis Brucellosis Inflammatory bowel disease: Crohn disease and ulcerative colitis

3. Hematologic malignancies – Acute lymphoblastic leukemia – Chronic lymphocytic leukemia – Non-Hodgkin lymphoma with spill over – Adult T cell leukemia/lymphoma – Hairy cell leukemia

Fig. 6.5:Diagrammatic appearance of lymphocyte

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Disorders of White Cells

Lymphocytopenia Lymphocyte count below 1,500/cu mm (1.5 (3 × 109/L) in children.

×

109/L) in adults and below 3000/cu mm

Some of the important causes of lymphocytopenia are listed in the able 6.10. TABLE 6.10: Causes of lymphocytopenia 1. Increased destruction – Corticosteroids – Cytotoxic dru gs – Radiation 2. Decreased production – Aplastic anemia – Advanced malignancy: Hodgkin lymphoma – Infections: AIDS, miliary tuberculosis 3. Increased loss via GI tract – Obstruction to intestinal lymphatic drainage (e.g. tumor) – Congestive heart failure

QUALITATIVE DISORDERS OF LEUKOCYTES Qualitative disorders of leukocytes are rare familial disorders that manifest as morphologic changes in the leukocytes (Fig. 6.6).

Chediak-Higashi anomaly is associated with increased susceptibility to pyogenic infections.

CGD is associated with impaired phagocytosis and killing of organisms. Fig. 6.6:Various quantitative disorders of leukocytes

Quantitative and Qualitative Disorders of Leukocytes CHAPTER 6

INFECTIOUS MONONUCLEOSIS (GLANDULAR FEVER) Acute, benign, self-limiting lymphoproliferative disordercaused by Epstein-Barr virus (EBV). • Incubation period: 4 to 8 weeks. • Mode of transmission: oropharyngeal secretions (kissing), hence the nickname kissing disease.

EBV infects B cells but the peripheral blood shows CD8 + T cells, which appear as atypical lymphocytes.

Pathogenesis • • • •

EBV infects B lymphocytes by binding to CD21 (CR2) receptor. Viral infection begins in the submucosal lymphoid tissues of nasopharynx and oropharynx. Virus remains dormant inside the B cells. B cells are“immortalized” and are capable ofproliferation indefinitely. Lesions caused by EBV:

Clinical Features •

•

Age: young adults among upper socioeconomic classes in developed nations and children

of low socioeconomic status. Signs and symptoms: classical triad – Fever – Pharyngitis (sore throat) – Lymphadenopathy.

Laboratory Findings • •

•

Total leukocytes count increased (12,000 to 25,000 cells/cu mm): absolute lymphocytosis. Atypical lymphocytosis (mononuclear cells): these are CD8 + subset (cytotoxic) of T cells and not the virus-infected B cells. Serological tests – Demonstration of heterophile antibodies ◆ Paul Bunnell test is characteristically positive. ◆ Monospot test is a sensitive slide test. – Demonstration specific antibodies against EBV antigens: ◆ Antibody against viral capsid antigens (anti-VCA). ◆ Antibodies to Epstein-Barr nuclear antigen (EBNA).

1. Infectious mononucleosis 2. Burkitt lymphoma 3. Nasopharyngeal carcinoma 4. Hodgkin lymphoma 5. X-linked lymphoproliferataive disorders, and 6. Body cavity lymphoma.

Q. Mention the laboratory findings in infectious mononucleosis.

Demonstration of specific antibodies to EBV is the most specific test for infectious mononucleosis.

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7

Acute Leukemia

CHAPTER

ACUTE LEUKEMIA Q. Define and classify leukemia.

Definition

Acute leukemia is a malignant disease of the bone marrow stem cell and its c haracteristic features are: Normally blast cells are less • Bone marrow: diffuse replacement with proliferating neoplastic blast cells that fail to mature. Blast cells more than 20%(WHO criteria) of the nucleated cells in the marrow. than 5% of nucleated cells in the marrow. • Peripheral blood: abnormal numbers and forms of immature white blood cells. Aleukemic/subleukemic leukemia is characterized by very few/no blasts in the peripheral blood. Leukemia: malignant disease of bone marrow stem cell, arises in the marrow and spreads.

Acute leukemia are mainly divided into two groups namely acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML).

Etiology and Pathogenesis Risk Factors: risk

factors (able 7.1) may cause mutations in the proto-oncogenes and tumor suppressor genes. TABLE 7.1: Risk factors for acute leukemia ENVIRONMENTAL FACTORS • •

Ionizing radiation Drugs: – Alkylating agents—nitrogen mustard, chlorambucil, etc. – AML occurs in myeloma patients treated with melphalan – Leukemia follows chemotherapy of lung and ovarian cancer

•

Chemicals:benzene (used in paint industry, plastic glues, etc.) GENETIC DISORDERS Example: Down syndrome (ALL or AML), Fanconi anemia (AML), ataxia telangiectasia (ALL, NHL) ACQUIRED DISORDERS • •

PNH and aplastic anemia may transform into acute leukemia AML may develop de novo or secondary to myelodysplastic syndrome (MDS)

Acute Leukemia CHAPTER 7

Classification

Q. Classify acute leukemia.

raditional classification depending on microscopic appearance of the involved cell and the course of leukemias is presented in able 7.2. TABLE 7.2: Traditional classification ofleukemia Acute leukemia – Acute myelogenous/myeloblastic/myelocytic/myeloid leukemia (AML) – Acute lymphoblastic/lymphocytic leukemia (ALL)

•

Chronic leukemia – Chronic myeloid leukemia (CML)

•

– Chronic lymphocytic leukemia (CLL)

FAB Classification of Acute Leukemias •

•

FAB criteria for the

First French, American and British (FAB) classification (1976) was based on the diagnosis of acute leukemia: bone marrow 1. morphological and 2. cytochemical characteristics of blast cells. should show a blast count Revised FAB classification(able 7.3): it includes of 30% or more. 1. M: Morphology and cytochemistry of blast cells 2. I: Immunophenotyping 3. C: Cytogenetics 4. M: Molecular genetics.

TABLE 7.3: Revised French,American and British (FAB) classification of acuteleukemias Acute Lymphoid Leukemia L1

Small homogenous cells with inconspicuous nucleoli

L2

Large cells with variable size and 1–2 nucleoli

L3

Large, homogeneous cells with finely stippled chromatin and prominent nucleoli. Cytoplasm is basophilic and vacuolated

Acute Myeloid Leukemia M0

Minimally differentiated AML

M1

AML without maturation

M2

AML with maturation

M3

Promyelocytic leukemia

M4

Myelomonocytic leukemia

M5

Monocytic leukemia

M6

Erythroleukemia

M7

Megakaryocytic leukemia

WHO Classification (2008) of Acute Leukemia (Table 7.4)

WHO classification of ALL: two main categories namely, 1. Precursor-B lymphoblastic leukemia/lymphoma, and 2. Precursor T lymphoblastic leukemia/lymphoma.

TABLE 7.4: WHO classification (2008) ofacute lymphoblastic and myeloid leukemia A. Acute Lymphoblastic Leukemia I. Precursor-B lymphoblastic leukemia/lymphoma II. Precursor-T lymphoblastic leukemia/lymphoma B. Acute Myeloid Leukemia I. AML with recurrent genetic abnormalities ◆ AML with t(8;21)(q22;q22);RUNX1-RUNX1T1 ◆ AML with inv(16)(p13;1q22);CBFB-MYH11 Contd...

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Disorders of White Cells

Contd...

WHO classification of AML: based on clinical, morphological, immunophenotypic and genetic features.

Minimum blast cells in bone marrow should be more than 20%.

◆ APL with t(15;17)(q22;q12); PML-RARA ◆ AML with t(9;11)(p22;q23); MLLT3-MLL II. AML with MDS-related changes III. Therapy-related myeloid neoplasms IV. AML not otherwise specified ◆ AML minimally differentiated ◆ AML without maturation ◆ AML with maturation ◆ Acute myelomonocytic leukemia ◆ Acute monoblastic and monocytic leukemia ◆ Acute erythroid leukemia megakaryoblastic leukemia ◆ Acutesarcoma V. Myeloid VI. Myeloid proliferation related to Down syndrome

Q. List the differences between Abbreviations: AML, Acute myeloid leukemia; APL, Acute promyelocytic leukemia; MDS, Myelodysplastic syndrome myeloblast and lymphoblast. It is important to differentiate between lymphoblast and myeloblast because of difference in treatment and prognosis of AML and ALL.

Differences between Myeloblast and Lymphoblast (Table 7.5) TABLE 7.5: Differences between myeloblast and lymphoblast based on morphology and cytochemistry

Myeloblast: comparison with lymphoblast has 4 Ms M: more in size M: more nucleoli (3–5) M: moderate cytoplasm M: myeloperoxidase +ve

Size

Auer rod :+.

Cytoplasmicgranules Auer rod

Lymphoblast (Figs 7.1 and 7.3)

Myeloblast (Figs 7.2, 7.4 and 7.5)

2–3 times the size of lymphocyte

3–5 times the size of lymphocyte

Cytoplasmic characters Amount Color

Scanty (less cytoplasm than myeloblast) Scanty to moderate (more cytoplasm than lymphoblast) Blue

Gray

Agranular Negative

Mayhavecytoplasmicgranules Positive

Nuclear characters Nuclearchromatin Nucleoli N:C ratio Accompanying cells

Uniform,coarse

Uniform,fine

Inconspicuousor1to2 High

3to5,prominent High

Lymphocytes

Promyelocytes,myelocytes,meta myelocytes, band forms and neutrophils

Cytochemistry Myeloperoxidase Sudan Black Fig. 7.1:Diagrammatic appearance of lymphoblast

Fig. 7.2:Diagrammatic appearance of myeloblast

PAS Nonspecificesterase

Fig. 7.3:Periodic acid Schiff (PAS) stain showing lymphoblast with block positivity

Negative

Positive

Negative

Positive

Block positivity

Negative

Negative

PositiveinM4andM5

Fig. 7.4:Myeloblast stained positively with myeloperoxidase (MOP)

Fig. 7.5:Myeloblast stained positively with Sudan Black

Acute Leukemia CHAPTER 7

ACUTE LYMPHOBLASTIC LEUKEMIA/LYMPHOMA •

• •

Acute Lymphoblastic Leukemia/Lymphoma (ALL) is a group of neoplasms consisting of lymphoblasts. Lymphoblast is immature, precursor B (pre-B) or  (pre-) lymphocyte. WHO classification (able 7.4): – Precursor B cells ALL (about 85%) seen in childhood and present as acute leukemias. – Precursor T cells ALL (15%) present in adolescent males as lymphomas, often with involvement of mediastinum (thymus).

Molecular Pathogenesis •

Differentiating malignant pre-B and pre-T lymphoblasts on morphology is difficult.

Requires immunophenotyping for

Chromosomal abnormalities are found in about 90% of ALLs. subclassification of ALL. – Numerical abnormality: hyperploidy (>50 chromosomes) and hypoploidy. – Structural abnormality: balanced chromosomal translocations (e.g. Philadelphia chromosome). ◆ Most -ALLs have mutations in NOCH1 gene. ◆ Most B-ALLs have mutations in genes PAX5, E2A and EBF or a balanced translocation T-ALL has worse prognosis compared to B-ALL. t (12; 21) involving the genes EL and AML1.

Classification of Acute Lymphoblastic Leukemia (Tables 7.3, 7.4 and 7.6) TABLE 7.6: Characteristics of FAB subtypes of acute lymphoid leukemias (ALL) FAB type Cell size

L

1

Small cell size

L2 Largeheterogeneous cell population

L3 Large, homogeneous cell population

Morphologically, as per the FAB classification lymphoblast are classified as L1, L2 and L3.

Nuclear characteristics Shape

Regular

Irregular,cleftingand indentation common

Regular, oval or round

Chromatin

Condensed

Dispersedchromatin

Finelystippled

Nucleolus

Smalland inconspicuous

Visible, 1–2 in number

Usually prominent

Scanty

Variable, often abundant

Moderately abundant

ALL-L1 has better prognosis than ALL-L3.

Cytoplasmic characteristics Amount Cytoplasmicbasophilia

Slighttomoderate

Cytoplasmicvacuolation

Absent

Variable Variable

ALL-L3 is a leukemic counterpart of Burkitt lymphoma.

Strong ProminentandoilredO stain positive

Clinical Features Age: most common hematological malignancy of children. Most common between 1 and 5 ALL is the most common leukemia in children and years of age and between 30 and 40 years. Sex: slight male preponderance. Onset: abrupt.

is usually associated with lymphadenopathy.

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Disorders of White Cells

Symptoms are due to bone marrow infiltration by blasts. Bone marrow failure anemia neutropenia thrombocytopenia. •

•

•

Symptoms: • Bone marrow failure: – Anemia: causes fatigue, weakness. – Neutropenia: infections by bacteria or opportunistic fungi. Develop sore throat and respiratory infections. – Trombocytopenia: bleeding into the skin and mucosa in the form of purpura or ecchymoses. – Bone pain and sternal t enderness. • Extramedullary infiltration: – Lymphadenopathy: 75% of patients, usually involvecervical lymph nodes. – Bone pain and tenderness. – Hepatosplenomegaly: splenomegaly is more common than hepatomegaly. – Mediastinal thymic mass: more common in -ALL. • CNS involvement: spread into the meninges causesleukemic meningitis ALL (pre-B). • Testicular involvement (ALL).

Laboratory Findings

Q. Write short note on laboratory/ peripheral smear findings in acute Peripheral lymphoblastic leukemia. •

Subleukemic leukemia: total WBC count lower than 4 × 109/L and peripheral blood shows very few blasts. Aleukemic leukemia: total white cell count is low 9

(< 4 × 10 /L) with no blasts in the peripheral blood. Lymphoblasts should be differentiated from myeloblasts (see Table 8.3).

• •

•

Blood

Total WBC count : markedly raised ranging from 20× 109/L to 200 × 109/L Platelet count: reduced (thrombocytopenia). Hemoglobin: decreased and may be as low as 3 g/dL. Peripheral smear (Figs 7.6 and 7.7): – RBCs: normocytic normochromic anemia. – WBCs: total count markedly increased and 20% or more lymphoblasts . ◆ Morphology of lymphoblasts: ◊ Larger than small lymphocyte ◊ High N:C ratio ◊ Nucleus with condensed chromatin and nucleoli are either absent or inconspicuous ◊ Scant to moderate agranular basophilic cytoplasm. – Platelets: thrombocytopenia.

Fig. 7.6: Peripheral blood smears in acute lymphoblastic leukemia showing lymphoblasts (arrows). Inset shows lymphoblast with block positivity with PAS stain

Fig. 7.7: Diagrammatic peripheral blood smear in acute lymphoblastic leukemia showing lymphoblasts (arrows)

Acute Leukemia CHAPTER 7

Cytochemistry of Lymphoblasts • • •

PAS: cytoplasmic aggregates ofPAS positive (Figs 7.3 and 7.6) material(block positivity). Myeloperoxidase (MPO) negative. Sudan black B negative.

Lymphoblast: cytoplasm shows block positivity with PAS stain.

Bone Marrow • • • •

Cellularity: markedly hypercellular due to proliferation of blasts. Erythropoiesis and myelopoiesis: reduced. Megakaryopoiesis: megakaryocytes gradually decrease. Blasts: constitute 20–100% of the marrow cells.

Immunophenotyping Terminal-deoxynucleotidyl-transferase (TdT) + in pre-Band pre-T lymphoblasts. • Immature B cells + positive for pan B cell marker CD19 and CD10 (CALLA—common ALLDistinction between antigen). precursor B and T cell ALL requires lineage-specific • Precursor  ALL cells are positive for CD2, CD5 and CD8. markers.

Biochemical Findings •

•

Serum uric acid: raised due to destruction of leukemic cells during chemotherapy leading to hyperuricemia. LDH: raised, because of increased turnover of leukemic cells.

CSF Examination o know/rule out CNS involvement. Prognosis: prognostic features of ALL are presented in able 7.7. TABLE 7.7: Prognostic factors in ALL Unfavorableprognosis

Favorableprognosis

Age

Below2yearsandabove10years (adolescence or adulthood)

Between 2 to 10 years

Sex

Males

Females

Total WBC count

High (more than 50,000 cells/cu mm)

Low

Meningeal involvement

Present

Absent

Cytogenetic abnormalities

t(9;22) (the Philadelphia chromosome)

Hyperploidy, trisomy of chromosomes 4, 7 and 10 and t(12;21)

Time required for clearing blasts from blood

More than 1 week

Less than 1 week

Presence of Philadelphia chromosome in ALL: prognosis unfavorable.

Prognosis is far better in ALL than AML. 95% of children develop complete remission. 75 to 85% are cured with current chemotherapy.

ACUTE MYELOGENOUS LEUKEMIA Definition: neoplasm of hematopoietic progenitors characterized by proliferation resulting in accumulation of immature myeloblasts in the marrow. Classification of acute myelogenous leukemia (AML): refer ables 7.3 and 7.4.

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Disorders of White Cells

AML synonyms: acute myeloid/myeloblastic/ myelocytic leukemia.

Molecular Pathogenesis •

•

Many recurrent genetic abnormalities can disrupt genes encoding transcription factors involved in normal myeloid differentiation. Mutated tyrosine kinase activation is a common.

Clinical Features AML: develop at any age. Usually 15–60 years of age.

Symptoms are due to anemia, neutropenia and thrombocytopenia.

Acute promyelocytic leukemia (AML-M3) may be associated with widespread bleeding due to DIC.

Age: AML may develop at any age, but is more common in adults. Onset: acute leukemias areabrupt in onset. Symptoms: related to depressed marrow function. • Bone marrow failure: – Anemia: fatigue and weakness. – Neutropenia: life-threatening infections by bacteria or opportunistic fungi. – Trombocytopenia: bleeding, patient may also develop disseminated intravascular coagulation (DIC) in AML M3 and primary fibrinolysis. – Bone pain and tenderness • Extramedullary infiltration – Gingival hypertrophy (M4 and M5) and infiltration of skin (leukemia cutis). – Hepatosplenomegaly: usually more than in ALL.

Laboratory Findings Q. Write short note on laboratory/Peripheral Blood peripheral smear findings in AML. • Total WBC Count : markedly raised •

Subleukemic leukemia: total WBC count lower than 4 × 109/L and peripheral blood shows very few blasts.

•

Aleukemic leukemia: total white cell count is low (< 4 × 109/L) with no blasts in the peripheral blood. AML: Auer rods in the cytoplasm of myeloblasts, seen in AML; not in CML.

Myeloblasts stain positively with myeloperoxidase and Sudan black B.

ranging from 20× 109/L to 100 × 109/L. Hemoglobin: decreased and ranges from 5 to 9 g/dL. Peripheral smear (Figs 7.8 and 7.9): – RBCs: normocytic normochromic type of anemia. – WBCs: total WBC count markedly increased. ◆ Differential count: more than 20% myeloid blasts. May show more than one type of blast or blasts with hybrid features. ◆ Morphology of myeloblasts ◊ 3 to 5 times larger than the diameter of a small lymphocyte. ◊ High N:C ratio. ◊ Fine nuclear chromatin with 2-4 variably prominent nucleoli. ◊ More cytoplasm than lymphoblasts—azurophilic, peroxidase-positive granules. ◊ Presence of Auer rods is definitive evidence of myeloid differentiation. ◆ Auer rods are azurophilic needle-like peroxidase-positive structures in the cytosol of myeloblasts (M2 and M3 subtype). – Platelets: moderate to severe thrombocytopenia and causes bleeding from skin and mucosa.

Cytochemistry of Myeloblasts (Figs 7.10 and 7.11) •

Stain positively with myeloperoxidase (MPO) and Sudan black B.

•

Monoblasts stain with nonspecific esterases. Both in subleukemic and aleukemic leukemia bone marrow contains blasts more than 20%.

Bone Marrow • • •

•

Cellularity: markedly hypercellular. Erythropoiesis: markedly suppressed. Myelopoiesis: suppression of myeloid maturation and myeloblasts constitute more than 20% of marrow cells. Megakaryopoiesis: gradually decreased.

Acute Leukemia CHAPTER 7

Fig. 7.8: Peripheral smear in AML with myeloblasts. Inset shows myeloblast with Auer rod

Fig. 7.9: Diagrammatic peripheral blood smear in AML with myeloblasts. One myeloblast with two Auer rods (arrow)

Fig. 7.10:Myeloblast stained positively with myeloperoxidase

Fig. 7.11:Myeloblast stained positively with Sudan black

Immunophenotyping Diagnosis of AML is confirmed by using stains for myeloid specific antigens.

AML prognosis: Fulminant course and has worse prognosis than ALL. Cytogenetic markers are major determinants of prognosis. •

•

Cytogenetics Very important in the WHO classification of AML (able 7.4).

MYELOID SARCOMA

Myeloid sarcoma synonym: Extramedullary myeloid tumor/granulocytic sarcoma/chloroma.

umor mass consisting of myeloid blasts with or without maturation occurring at extra– medullary sites. Myeloid sarcoma is • On sectioning: tumor is green (hence the term chloroma) frequent in skin, lymph node, GI tract, bone, soft • Microscopically myeloblasts with or without features of promyelocytic or neutrophilic tissue and testis. maturation.

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8

Myelodysplastic Syndromes

CHAPTER

MYELODYSPLASTIC SYNDROMES MDS: cytopenias with hypercellular bone marrow. About 30% progress to AML.

Myelodysplastic Syndromes (MDS) are a heterogeneous group ofacquired clonal stem cell disorders affecting stem cells. MDS is characterized by: • Progressive cytopenias • Dysplasia in one or more cell lines • Ineffective hematopoiesis • Risk of development of AML.

Classification • •

Idiopathic or primary MDS Secondary/therapy-related MDS (t-MDS): complication of previous cytotoxic drug or radiation therapy.

WHO classification of myelodysplastic syndromes is presented in Table 8.1.

Clinical Features • • • •

Elderly above 60 years Slightly more common in males Symptoms are due to cytopenias About 10% to 40% of MDS patients progress to AML.

Laboratory Findings •

Peripheral smear: cytopenias in the peripheral blood – RBCs: mild to moderate degree of macrocytic or dimorphic anemia. – WBCs: normal or low total leukocyte count. – Platelets: variable thrombocytopenia, large hypogranular or giant platelets.

Myelodysplastic Syndromes CHAPTER 8

TABLE 8.1: WHO classification of myelodysplastic syndromes Disease

Peripheralbloodpicture

Bonemarrowfeatures

1. Refractory cytopenia with Unicytopenia or bicytopenia 1 unilineage dysplasia (RCUD): refractory anemia (RA), refractory neutropenia (RN), refractory thrombocytopenia (RT)

Unilineage dysplasia in > 10% of the cells in one myeloid lineage < 5% blasts < 15% ring sideroblasts

2. Refractory anemia with ring sideroblasts (RARS)

Anemia No blasts

Dyserythropoiesis >15% ring sideroblasts <5% blasts

3. Refractory cytopenia with multilineage dysplasia (RCMD)

Bi/pancytopenia Rare blast

Dysplasia in > 10% of cells in 2 myeloid lineages (neutrophil

No rods <1 ×Auer 109/L monocytes

and/or erythroid and/or megakaryocytes) < 5% blasts No Auer rods ± 15% ring sideroblasts

4. Refractory anemia with excess blasts-1 (RAEB-1)

< 5% blasts2 Bi/pancytopenia No Auer rods <1 × 109/L monocytes

5–9% blasts Unilineage or multilineage dysplasia No Auer rods

5. Refractory anemia with excess blsts-2 (RAEB-2)

5–19% blasts Cytopenia Auer rods ± 3 <1 × 109/L monocytes

10–19% blasts Unilineage or multilineage dysplasia Auer rods ± 3

6. MDS unclassified (MDS-U)

< 1% blasts Cytopenia only

< 5% blasts Unequivocal dysplasia in less than 10% of cells in one or more myeloid cell lines when accompanied by cytogenetic abnormalities considered as presumptive evidence for diagnosis of MDS

7. MDS with isolated del (5q)

No or rare blasts Anemia Platelets increased or normal

< 5% blasts Increased to normal megakaryocytes with hypolobated nuclei. Isolated 5q deletion. No Auer rods

1. Cases with pancytopenia should be classified as MDS-U 2. If marrow blasts < 5% with 2–4% myeloblasts in blood = RAEB-1. Cases with RCUD and RCMD with 1% myeloblasts in blood should be classified as MDS-U 3. Cases with Auer rods and < 5% myeloblasts in blood and 10% in marrow = RAEB 2

Bone Marrow Dysplasia of all non-lymphoid lineages (erythroid, granulocytic, monocytic and megakaryocytic) associated with cytopenias. • Cellularity: hypercellular. • Erythropoiesis: dysplastic changes in erythroid precursors with megaloblastoid change and presence of ringed sideroblasts in iron stain. • Myelopoiesis: hyperplasia with dysgranulopoiesis. • Megakaryopoiesis: dysmegakaryopoiesis—pawn ball megakaryocytes. • Iron stores: increased with ring sideroblasts. Ineffective hematopoiesis

Bone Marrow Trephine Biopsy Abnormal localization of immature precursors (ALIP) in (refractory anemia with excess blasts (RAEB).

Bone marrow in MDS: pawn ball megakaryocytes, dysgranulopoiesis, erythroid precursors with megaloblastoid change and presence of ringed sideroblasts.

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Myeloproliferative Neoplasms

CHAPTER

MYELOPROLIFERATIVE NEOPLASMS (MPN) MPN peaks in the 5th to 7th decade. All MPN show splenomegaly.

Definition: clonal hematopoietic stem cell disorders characterized byproliferation of one or more of the myeloid lineages(erythroid, granulocytic, megakaryocytic and mast cells). • Splenomegaly and hepatomegaly due to sequestration of excess hematopoietic cells or proliferation of abnormal hematopoietic cells.

WHO Classification of MPN It is presented in able 9.1. TABLE 9.1: WHO (2008) classification ofmyeloproliferative neoplasm (MPN) WHO (2008) Myeloproliferative neoplasms Chronic myelogenous leukemia, BCR-ABL-1 positive Chronic neutrophilic leukemia Polycythemia vera—JAK2 V617F or exon 12 mutation Primary myelofibrosis—JAK2 or MPL mutation Essential thrombocythemia Platelet count > 450 × 109/L JAK2 mutation • •

Chronic eosinophilic leukemia, NOS No BCR-ABL1, PDGFRA, PDGFRB or FGFR1 translocation •

Mastocytosis—KIT mutation Myeloproliferative neoplasm, unclassifiable

Pathogenesis Presence of mutated, constitutively activated tyrosine kinases leads to proliferation of hematopoietic stem cells and results in hypercellular marrow.

Myeloproliferative Neoplasms CHAPTER 9

POLYCYTHEMIA OR ERYTHROCYTOSIS Polycythemia is characterized byincrease in the RBC mass, usually with a corresponding increase in hemoglobin level. Pathophysiologic classification of polycythemia is given in able 9.2. TABLE 9.2: Pathophysiologic classification of polycythemia ABSOLUTE Primary (low erythropoietin level) Polycythemia vera Secondary (high er ythropoietin level) Physiologically appropriate – Compensatory – Lung disease – Living in high-altitude – Cyanotic heart disease (Tetralogy of Fallot) Physiologically inappropriate (with increased erythropoietin) – Paraneoplastic: erythropoietin-secreting tumors (e.g. renal cell carcinoma, uterine leiomyoma, hepatocellular carcinoma) •

•

Increase in red cells can be absolute or relative.

•

RELATIVE Reduced plasma volume Hemoconcentration (dehydration due to diarrhea, vomiting) Gaisböck’s syndrome (spurious polycythemia) • •

POLYCYTHEMIA VERA Definition: polycythemia vera (PV) is an acquired myeloproliferative neoplasm arising from malignant transformation ofhematopoietic stem cell. • It is characterized bytrilineage (erythroid, granulocytic, and megakaryocytic) hyperplasia in the bone marrow. • It leads touncontrolled production of red cells, granulocytes and platelets (panmyelosis) and leads to erythrocytosis (polycythemia) and or granulocytosis and thrombocytosis.

Q. Write short notes on polycythemia vera.

Molecular Pathogenesis (Figs 9.1 and 9.2) •

• • •

Normally, a tyrosine kinase protein called JAK2 (Janus 2 kinase gene), is activated followingJAK2 mutation is diagnostic of polycythemia binding of the growth hormone erythropoietin. vera. JAK2 then activates a signaling pathway causing cells to replicate. Tis process is strictly regulated by various feedback pathways. Polycythemia vera (PV) is due to mutation in tyrosine kinaseJAK 2 V617F, which causes PV: erythropoietin is proliferation of not only erythroid lineage but also granulocytic and megakaryocytic lineage. decreased.

Clinical Features 1. Insidious. 2. Late middle age (median age at onset is 60 years). 3. Plethora and cyanosis, headache, dizziness andvisual problems result from vascular PV: most symptoms are disturbances in the brain and retina. due to the increased red 4. Trombotic episodes: e.g.deep venous thrombosis,myocardial infarction, thrombosisof cell mass and hematocrit. hepatic veins (producing Budd-Chiari syndrome).

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Fig. 9.1:Normal signaling by JAK2

Fig. 9.2:In polycythemia vera, the presence of a mutant version of JAK2 results in dysregulated downstream signaling in the absence of erythropoietin

Phases PV develops into acute myelogenous leukemia in 2% to 5%.

Tere are three phases of Polycythemia vera • Proliferative phase: erythroid proliferation and increased red cell mass. • Spent phase: in 10%, excessive proliferation of erythroid cells ceases with stable or decreased RBC mass. • Myelofibrosis: about 10% progress to myelofibrosis.

Laboratory Findings Peripheral Blood (Fig. 9.3) • •

Polycythemia vera is a chronic myeloproliferative neoplasm with RBC count of more than 6 million/ cu mm.

• • •

•

Hemoglobin: increased and are more than 18.5 g/dL in men and 16.5 g/dL in women. Hematocrit: increased and about 60%. Red cell count: increased and usually about 6 million/cu mm (6 × 1012/L). White cell count: normal or increased. Platelet count: normal or increased. Peripheral smear: – RBCs: show normocytic normochromic picture. – WBCs: ◆ Mild to moderate leukocytosis ◆ Neutrophils are morphologically normal ◆ Basophils often increased ◆ NAP (LAP) score is increased to 150–300 (Normal 40–100). – Platelets: abnormally large and functionally defective.

Myeloproliferative Neoplasms CHAPTER 9

PV: hematocrit is increased and > 60%.

Fig. 9.3:Normal hematocrit in comparison with anemia and polycythemia vera

Bone Marrow •

Hypercellular due to hyperplasia of all elements (trilineage hyperplasia/panmyelosis) namely erythroid, myeloid and megakaryocytic series with prominence of erythroid precursors in the bone marrow.

Bone Marrow Biopsy Shows increased reticulin fibers and fibrosis as the disease progresses.

Other Findings • •

• • •

Extramedullary hematopoiesis in the liver and spleen that causeshepatosplenomegaly. Arterial oxygen saturation (pO2): normal (75–100 mm Hg) and is useful fordifferentiating it from secondary polycythemia. Erythropoietin levels: decreased in contrast to secondary polycythemia. Serum vitamin B12 and uric acid: increased indicating increased cell turnover. JAK2 V617F mutation: it can be demonstrated.

ESSENTIAL THROMBOCYTHEMIA

In PV arterial oxygen saturation (pO2) is normal (>92%) whereas in secondary polycythemia it is <90%.

ET synonym: primary (essential/idiopathic)

Definition: chronic myeloproliferative neoplasm (MPN) primarily of megakaryocytic thrombocytosis. lineage. It is characterized byincreased megakaryopoiesis and thrombocytosis (more than 450 × 109/L).

Etiology •

ET: mutation of JAK2 gene Thrombocytosis with a

Most due to point mutations in JAK2 gene and constitutive activation of JAK2, and count of > 450 × 109/L. thrombopoietin-independent proliferation of megakaryocytes.

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ET: throbbing and burning sensation of hands and feet due to blocking of arterioles by aggregates of platelets is known as erythromelalgia.

Clinical Features • • •

Age: 50–60 years Trombosis and hemorrhage Erythromelalgia: one of the characteristic features.

Laboratory Findings •

Peripheral smear: – RBCs: normocytic normochromic. – WBCs: mild leukocytosis. – Platelets: ◆ Increased number (thrombocytosis)> 600,000/cu mm. ◆ Variation in size and shape -abnormally large platelets are common.

Megakaryocytic hyperplasia and abnormal (giant) platelets are characteristic features.

Bone Marrow • • • •

ET course: indolent.

Cellularity: mild to marked hypercellularity. Erythropoiesis: normal or mild hyperplasia. Myelopoiesis: normal or mild hyperplasia. Megakaryopoiesis: markedly increased in number with abnormally large megakaryocytes (giant megakaryocytes).

Extramedullary hematopoiesis: mild hepatosplenomegaly.

PRIMARY MYELOFIBROSIS Myelofibrosis: mutation in JAK2 gene.

Clonal MPN characterized by a proliferation of predominantly megakaryocytes and granulocytes in the bone marrow. Fully developed disease results in reactive marrow fibrosis and replaces hematopoietic cells leading to cytopenias and extensive extramedullary hematopoiesis.

Molecular Pathogenesis Most show JAK2 mutations.

Massive splenomegaly due to extramedullary hemopoiesis.

Clinical Features • • •

Age: above 60 years of age. Progressive anemia. Splenomegaly.

Myeloproliferative Neoplasms CHAPTER 9

Laboratory Findings •

Peripheral smears: – RBCs: moderate to severe degree of normochromic normocytic anemia accompanied by leukoerythroblastosis. Tear drop-shaped red cells (dacryocytes), probably due to damage in the fibrotic marrow can also be found. – WBCs: total white cell count is usually normal or reduced, but can be markedly elevated 80 to 100 × 109/L in early stages of the disease. – Platelets: they may be abnormally large. The platelet count is usually normal or elevated, but as the disease progresses the count decreases.

Bone Marrow •

• •

Cellularity: in early stages, it is often hypercellular due to increase in maturing cells of all lineages. In later stages, it is replaced by fibrosis and becomes hypocellular and diffusely fibrotic resulting in a dry tap. Erythroid and granulocytic precursors: these are morphologically normal. Megakaryocytes: these are large, dysplastic and abnormally clustered.

Bone Marrow Biopsy Stages: two stages have been recognized. 1. Prefibrotic (cellular) stage: hypercellular bone marrow. Megakaryocytes increased markedly abnormal.

Extramedullary hematopoiesis in spleen and liver produces hepatosplenomegaly. Course: variable.

Primary myelofibrosis: bone marrow fibrosis leads to cytopenias.

Bone marrow biopsy is essential for the diagnosis of myelofibrosis as aspirate and results in a dry tap late in the course of the disease.

2. Fibrotic stage: fibrosis distorts the marrowand prematurely releases nucleatederythroid and early granulocyte progenitors(leukoerythroblastosis). Reticulin stain demonstrates the increase in reticulin fibers (fibrosis).

Primary myelofibrosis: peripheral smear shows leukoerythroblastosis and tear drop cells.

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Chronic Myelogenous Leukemia

CHAPTER

CML synonyms: chronic myelocytic/myeloid/ granulocytic leukemia. CML is an acquired MPN of pluripotent hematopoietic stem cell.

CHRONIC MYELOGENOUS LEUKEMIA Definition Chronic myelogenous leukemia (CML) is one of the myeloproliferative neoplasm (MPN) of pluripotent hematopoietic stem cell characterized by overproduction of cells of the myeloid series which results in marked splenomegaly and leukocytosis. Distinguished from other myeloproliferative neoplasms by the presence of: 1. Chimeric fusion BCR-ABL gene. 2. Philadelphia (Ph)chromosome inmore than 90% of cases.

Etiology and Pathogenesis Risk factor:exposure to

Q. Write short notes on Philadelphia chromosome.

ionizing radiation and benzene.

Molecular Pathogenesis Philadelphia (Ph) Chromosome (Fig. 10.1)

Philadelphia (Ph) chromosome is a shortened chromosome 22 and is due to balanced reciprocal translocation between chromosome 9 and 22-t (9; 22).

•

•

Acquired chromosomal abnormality in all proliferating hematopoietic stem cells (erythroid, myeloid, monocytic and megakaryocytic precursors). Balanced reciprocal translocation between long arm of chromosome 9 and 22, i.e. t (9; 22) (q 34; q 11.2). It increases the length of chromosome 9 and shortening of 22. Tis shortened chromosome 22 is known as Philadelphia chromosome (Fig. 10.1).

BCR-ABL Fusion Gene (Fig. 10.2) Translocation results in a BCR-ABL fusion gene, which produces neoplastic proliferation.

• •

ABL proto-oncogene from chromosome 9joins the BCR on chromosome 22. It produces a new chimeric (fusion) gene called BCR-ABL, thus converting ABL protooncogene into oncogene. Te product of the fusion gene plays a central role in the development of CML

Chronic Myelogenous Leukemia CHAPTER 10

CML: translocation results in the head-to-tail fusion of the breakpoint cluster region (BCR) gene on chromosome 22 with the ABL (named after the abelson murine leukemia virus) gene located on chromosome 9.

Fig. 10.1:Balanced reciprocal translocation between long arm of chromosome 9 and chromosome 22 resulting in shortened chromosome 22 known as Philadelphia chromosome

Fig. 10.2:Fusion of ABL gene from chromosome 9 with BCR on chromosome 22 and its consequences

•

Te product of this oncogene i.e., oncoprotein (e.g. p210) causes cell division and inhibition of apoptosis.

Clinical Features • • •

Age: usually occurs between 40 to 60 years of age. Sex: males slightly more affected than females. Onset: insidious.

Symptoms: •

Nonspecific symptoms: fatigue, weakness, weight loss, anorexia.

CML: usually occurs between 40 and 60 years of age.

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CML: moderate to massive splenomegaly.

•

•

CML has three phases: chronic stable, accelerated and blast phase.

Fullness of abdomen due to splenomegaly (caused by leukemic infiltration and extramedullary hematopoiesis). Splenomegaly is moderate to severe and is characteristic feature in majority (80–90%) of patients. Hepatomegaly: mild or moderate seen in 60–70% of cases.

NATURAL HISTORY OF CHRONIC MYELOID LEUKEMIA Tree different phases: 1. chronic phase, 2. accelerated phase and 3. blastic phase.

Chronic/Stable/Indolent Phase (CP) • • •

Most are diagnosed in this phase. Lasts for 2 to 6 years. If not treated, progresses gradually to accelerated phase or abruptly to blastic phase.

Q. Write short notes on laboratory findings /peripheral smear in CML.

Laboratory Findings

CML: neutrophilia with the whole spectrum of mature myeloid precursors.

Peripheral blood Hemoglobin: usually less than 11 g/dL •

•

CML is characterized by anemia, extreme leukocytosis, granulocytic immaturity, basophilia,

Peripheral smear: – RBCs: normocytic normochromic anemia – WBCs: ◆ Marked leukocytosis (12–600 × 109/L) total leukocyte count usually exceeds 100 × 109/L (1,00,000/cu mm). ◆ Shift to left (shift to immaturity)— granulocytes at all stages of development (neutrophils, metamyelocytes, myelocytes, promyelocytes and an occasional myeloblasts). Predominant cells are neutrophils and myelocytes. Blasts are usually less than 10% of the circulating WBCs (Figs 10.3 and 10.4). Basophilia and eosinophilia. Decreased NAP/LAP score: NAP score in CML is decreased below 20 (normal score range is 40–100). Helpful in differentiating CML from leukemoid reaction (see Table 7.5). – Platelets: platelets range from normal (150–450 × 10 9/L) to greater than 1000 × 109/L. Up to 50% have thrombocytosis.

thrombocytosis.

◆ ◆ ◆ ◆

The preponderance of myelocyte is called as myelocyte bulge.

In CML LAP (NAP) is markedly reduced.

Bone Marrow • • • •

• •

Cellularity: markedly hypercellular due to myeloid hyperplasia. M: E ratio: often exceeds 20:1. Erythropoiesis: diminished erythropoiesis as disease progresses. Myelopoiesis: marked hyperplasia. Blast cells usually less than 10%. Basophils, eosinophils and their precursors are usually found. Megakaryopoiesis: megakaryocytes are either normal or increased. Dwarf megakaryocytes. Sea-blue histiocytes (Gaucher-like cells/pseudo Gaucher cells) are seen.

Biochemical findings: Serum uric acid raised Serum LDH raised. • •

Philadelphia chromosome and BCR-ABL fusion gene: demonstratedeither by chromosomal

analysis orfluorescent in situ hybridization (FISH) or PCR based tests.

Chronic Myelogenous Leukemia CHAPTER 10

Fig. 10.3:Peripheral blood picture in chronic/stable phase of chronic myeloid leukemia

Fig. 10.4:Diagrammatic peripheral blood picture in chronic/stable phase of chronic myeloid leukemia

Accelerated Phase (AP) (Figs 10.5 and 10.6) • • • •

× • •

CML: accelerated phase is more aggressive and

More aggressiveand lasts for few months. myeloblasts range from Myeloblasts: 10–19% in the blood or bone marrow. 10% to 19%. Striking basophilia (20% or more). Persistent thrombocytopenia (less than 100 × 109/L) unrelated to therapy or persistent 9

thrombocytosis proliferati (more than 10 /L) by therapy. Megakaryocyte on1000 in sheets oruncontrolled clusters in association with fibrosis. Persistent or increasing splenomegalyunresponsive to therapy.

Fig. 10.5:Peripheral blood picture in accelerated phase of chronic myeloid leukemia showing numerous blasts (10–19%) and striking basophilia

Fig. 10.6:Diagrammatic peripheral blood picture in accelerated phase of chronic myeloid leukemia showing numerous blasts (10–19%) and striking basophilia

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Blast Phase/Crisis (BP) CML blast crisis: blasts 20% or more, myeloblast (no Auer rods) or lymphoblasts. Prognosis: poor with accelerated phase or blast crisis.

Blood picture resembles acute leukemiaand has poor prognosis. •

Peripheral smear (Figs 10.7 and 10.8): – Blasts 20% or more. May be either myeloblast (70% cases) or lymphoblast (30% cases). Myeloblast does not contain Auer rods. – Thrombocytopenia causes bleeding episodes.

Fig. 10.7:Peripheral blood picture in blast phase of chronic myeloid leukemia showing numerous blasts (20% or more) and striking basophilia

Fig. 10.8:Diagrammatic peripheral blood picture in blast phase of chronic myeloid leukemia showing numerous blasts (20% or more) and striking basophilia

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

11 CHAPTER

CHRONIC LYMPHOCYTIC LEUKEMIA Definition

Q. Write short notes on chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) is a tumor composed of monomorphic small B lymphocytes in the peripheral blood, bone marrow and lymphoid organs (spleen and lymph nodes). Both CLL and SLL is a single entity with different presentations. Small lymphocytic lymphoma (SLL) is tissue equivalent of chronic lymphocytic leukemia CLL/SLL are tumors derived (CLL). from B lymphocytes. CLL/SLL tumor cells coexpress CD5 and CD23. • •

•

Etiology and Pathogenesis • •

Environmental factors: suggested but none proved. Hereditary factors: families with higher risk of CLL or other lymphoid neoplasms.

Cytogenetic Abnormalities Common mutations are deletions of 13q14.3, 11q22-23, and 17p13. About 20% of CLL show trisomy 12.

Clinical Features • • •

Age: between 50–60 years of age. Sex: more in males than in females (2:1). Symptoms: – Asymptomatic in about 25–30% – Nonspecific symptoms: fatigue, loss of weight and anorexia – Generalized lymphadenopathy – Immunological defects either as immune deficiency or autoimmunity.

CLL patients may be asymptomatic or present with generalized lymphadenopathy.

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Laboratory Findings CLL: absolute lymphocyte count is more than 5 × 109/L. It is the characteristic feature. CLL: lymphocytosis with smudge cells in the peripheral smear. Smudge cells are fragile leukemic cells produced due to rupture while making the

Peripheral Blood • •

•

Peripheral smear (Figs 11.1 and 11.2): – RBCs: normocytic normochromic anemia. – WBCs: ◆ Differential leukocyte count shows lymphocytosisand constitutesmore than 50%of the white cells. ◆ Lymphocytes mature type—small with scant cytoplasm, nuclei round with clumped coarse chromatin (”soccer ball”/block-type chromatin). Nucleoli absent. ◆ Smudge cells or basket cells (fragile leukemic cells). – Platelets: initially normal count and later may be decreased.

peripheral smear.

Lymphocytes constitute more than 30% of the nucleated cells of the bone marrow cells—diagnostic feature of CLL.

Hemoglobin: decreased and usually below 13 g/dL. Total leukocyte count is increased (20–50 × 109/L).

Bone Marrow • • • • •

Cellularity: hypercellular marrow due to infiltration by mature lymphocytes. Erythropoiesis: normal. Myelopoiesis: normal. Megakaryopoiesis: normal. as the disease advances neoplastic lymphocytes replace the normal erythroid, myeloid Lymphocytic infiltrate: and megakaryocytic series in thebone marrow resulting in anemia, neutropenia and thrombocytope nia.

Immunophenotype umor cells express the pan-B cell markers CD19 and CD20. CD5+ and CD23+ are distinctly positive in CLL.

Lymph Node • •

Show loss of normal architecture Diffuse infiltration by monomorphic, small, round lymphocytes

Fig. 11.1:Peripheral blood smears in chronic lymphocytic leukemia showing numerous small lymphocytes (long arrows) and few smudge cells (short arrow)

Fig. 11.2:Diagrammatic peripheral blood smears in chronic lymphocytic leukemia showing numerous small lymphocytes (long arrows) and few smudge cells (short arrows)

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma CHAPTER 11

• •

Lymphocytes have nuclei with coarse chromatin and scanty cytoplasm CLL/SLL: lymph node with Small, nodular aggregates of medium to large-sized lymphocytes known proliferation as centers proliferation centers are or pseudo-follicles or growth centers and when found are pathognomonic for CLL/SLL. pathognomonic.

Course and prognosis: median survival rate is 4 to 6 years. Tey may progress to B cell prolymphocytic transformation or into diffuse large B cell lymphoma (Richter syndrome).

HAIRY CELL LEUKEMIA Definition

Hairy cells have hair-like cytoplasmic projections.

Uncommon neoplasm of small mature B cells having abundant cytoplasm with fine hairlike cytoplasmic projections (hence the namehairy cell leukemia) when viewed under the phase-contrast microscope.

Laboratory Findings Peripheral Blood • • •

•

Hemoglobin: decreased. Total leukocyte count: decreased (leukopenia). Platelet count: decreased (less than 50 × 109/L)

HCL: it is B cell neoplasm and involves peripheral blood, bone marrow, spleen and liver and usually seen in old age.

Peripheral smear: pancytopenia – RBCs: normocytic normochromic. – WBCs: leukopenia with few hairy cells (Fig. 11.3). – Platelets: reduced.

Bone Marrow Aspiration • • •

Dry tap Hairy cells may be seen in the marrow Moderate to marked reduction in myeloid, erythroid and megakaryocytic cell lines.

Fig. 11.3:Hairy cell

Bone marrow trephine biopsy:neoplastic cells have“fried egg” or “honeycomb” appearance. HCL: bone marrow biopsyhairy cells have fried egg Reticulin stain shows marked increase of thin reticulin fibers surrounding neoplastic cells. appearance.

Spleen • •

Enlarged due to leukemic infiltrate Sinuses lined by hairy cells and grossly impart a beefy red appearance.

Immunophenotype and Molecular Characteristics is the most specific marker of hairy cell leukemia.

Tartrate resistant acid phosphatase (TRAP) positivity in the cytoplasm is a characteristic feature of HCL.

Clinical Features

HCL: only leukemia without lymphadenopathy.

Express theCD20, CD22, CD11c and CD25 (the IL-2 receptorα-chain) positivity. Annexin A 1

• • • •

Affects middle-aged to elderly men. Male-to-female ratio of 5:1. Massive splenomegaly. Pancytopenia.

HCL prognosis: indolent course and prognosis is excellent.

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Plasma Cell Neoplasms

CHAPTER

Plasma cell neoplasms are of B cell srcin in which single clone of plasma cells proliferate.

INTRODUCTION Definition Plasma cell neoplasms are group ofB cell neoplasmsassociated with theproliferation of single clone (monoclonal)of immunoglobulin-secreting plasma cells (also known as dyscrasias).

Plasma cell neoplasms: tumor cells secrete single type of complete or fragment of immunoglobulins.

Characteristics of Plasma Cell Neoplasms Monoclonal cellssecrete complete singleies. type of immunoglobulin (Ig) or Ig fragment. neoplastic Hence, are plasma known asmonoclonal gammopath Serum: single Ig proteins detected as monoclonal spike [M protein (M for myeloma)]on electrophoresis. Urine: excess of free light chainsis excreted in the urine as Bence-Jones (BJ) proteins. •

•

Classification of Plasma Cell Neoplasms (Table 12.1) TABLE 12.1: Classification of plasma cell neoplasms (WHO 2008) •

Plasma cell myeloma

•

Plasmacytoma

•

Immunoglobulin deposition diseases

•

Monoclonal gammopathy of undetermined significance (MGUS)

•

Osteosclerotic myeloma (POEMS syndrome)

PLASMA CELL MYELOMA (MULTIPLE MYELOMA) Definition Multiple myeloma is a multifocal malignant tumor of plasma cell and arises in the bone marrow.

Plasma cell myeloma is amalignant, multifocal plasma cell neoplasm of the bone marrow associated with M-protein in the serum and/or urine. Most common monoclonal gammopathy. Presents asmultiple tumor masses throughout theskeletal system. • •

Plasma Cell Neoplasms CHAPTER 12

Etiology

Plasma cell neoplasms arise from post-germinal center B cells.

Risk Factors Genetic predisposition Exposure to ionizing radiation Chronic antigenic stimulation associated with chronic infections (HIV and chronic osteomyelitis) and chronic inflammatory disorders (e.g. rheumatoid arthritis) Exposure to chemicals like benzene, herbicides and insecticides.

• • •

•

Laboratory Findings

Q. Write short notes on the

Peripheral Blood

laboratory diagnosis of multiple myeloma.

Hemoglobin: decreased and ranges from 6 to 10 g/dL.

•

•

Peripheral smear: – RBCs: normocytic normochromic anemia, red blood cells show rouleaux formation (Fig. 12.1) due to increased immunoglobulins. – WBCs: normal. – Platelets: normal.

MM: hypergammaglo– bulinemia is responsible for high ESR and rouleaux formation seen in peri– pheral smear.

ESR: high and is due to high gamma globulin (immunoglobulin) and rouleaux formation. Bleeding time: increased.

• •

Bone Marrow •

•

• • •

Cellularity: hypercellular due to myeloma plasma (myeloma) cells (neoplastic plasma cells) (Figs 12.2 and 12.3). Myeloma plasma cells: more than 30% are diagnostic. – Myeloma plasma cells are neoplastic plasma cells (Fig. 12.2), which are large oval cells having abundant pale blue cytoplasm. – The nucleus is round to oval, eccentric and shows perinuclear clearing/hof. – The nuclear chromatin appears like a clock-face/spoke wheel. – These cells are usually uninucleated or may show binucleation. – Other cells can also be seen in myeloma (Fig. 12.4). Erythropoiesis: diminished and is normoblastic. Myelopoiesis: normal. Megakaryopoiesis: normal.

Fig. 12.1:RBCs showing rouleaux formation in the peripheral blood

Bone marrow in MM: hypercellular, and contains more than 30% neoplastic plasma cell. Myeloma plasma cells are commonly called as myeloma cells.

Fig. 12.2:Bone marrow aspirate in multiple myeloma. With numerous myeloma plasma cells. Inset shows flame cell (left lower corner) and mott cell (right uppercorner)

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Serum Findings •

• •

•

Serum β2 microglobulin: prognostic marker and high values signify poor prognosis. Hypercalcemia Renal function tests: blood urea, serum creatinine and uric acid levels are raised with renal involvement. Serum albumin: decreases in advance stages of the disease.

Electrophoretic on Serum and Urine (Figs 12.5 Studies and 12.6) • •

Fig. 12.3: Diagrammatic appearance of bone marrow in multiple myeloma showing plasmablasts (short arrow) and plasma cells (long arrow)

•

Monoclonal spikesin 80% to 90% of cases. Raised monoclonal immunoglobulins in the blood. Immunoglobulin may be IgG (most common)/IgD/ IgA/IgE type. Light chains or Bence Jones (BJ) proteinsin the urine may be seen in 60% to 80% of cases. BJ protein may be of κ or λ type of light chain.

Fig. 12.5:Serum electrophoresis showing normal pattern

Fig. 12.4:Diagrammatic appearance of the various cells that can be seen in bone marrow in multiple myeloma

Fig. 12.6:Serum electrophoresis showing monoclonal immunoglobulin (“M Band”) in multiple myeloma

Plasma Cell Neoplasms CHAPTER 12

Morphology of Organs Involved • •

Bone: destructive -punched-out lytic lesions (Fig. 12.7). Renal lesions: – Myeloma kidney: light-chain cast of BJ proteindamages renal tubules. – Amyloidosis of the AL type and leads tonephrotic syndrome. ◆ Hypercalcemia leads to nephrocalcinosis ◆ Prone to acute and chronic pyelonephritis ◆ Renal failure.

MM: IgG is the most common immunoglobulin secreted.

MM: Monoclonal gammopathy peak 50 •

Clinical Manifestations (Fig. 12.8) Onset: insidious.

Age and sex: affects old age between 50 and 60 years with slight male preponderance.

•

•

to 60 years Multiple lytic lesions in bones Hypercalcemia.

MM: involved bone shows multiple punched out lytic lesions.

Fig. 12.7:Skull X-ray showing multiple punched out lytic lesions

Fig. 12.8:Clinical features and laboratory findings in myeloma

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MM: renal failure and sepsis are common causes of death.

Te clinical features of multiple myeloma are 1. Due to tumor cells causing bone lesions: Resorption of bone: this results in pathologic fractures, chronic bone pain and tenderness. Compression: lesion in the vertebra may compress the spinal cord nerve root. Hypercalcemia. Pallor: due to anemia and result in weakness and fatigue. •

•

MM: higher levels of serum β2 microglobulin are associated with poor prognosis.

• •

2. Production of M-proteins (increased immunoglobulins): Bleeding tendency Coagulation abnormalities • • •

MM: prognosis— progressive course with poor prognosis.

Extra-osseous plasmacytoma is usually found in the upper respiratory tract, especially in the nasal cavity and sinuses, nasopharynx and larynx.

Amyloidosis of the AL type. 3. Humoral immune deficiency: humoral immune deficiency predisposes to recurrent bacterial infections. 4. Renal disease: renal insufficiency, infections or nephrotic syndrome.

PLASMACYTOMA Localized proliferation forms a single discrete plasma cell tumor in bone (usually) or soft tissue. Solitary plasmacytoma of bone (osseous plasmacytoma) Extra-osseous (extramedullary) plasmacytoma • •

IMMUNOGLOBULIN DEPOSITION DISEASE Primary Amyloidosis Plasma cell neoplasm secretes abnormal immunoglobulin light chains, which may get deposited in tissues and form a β-pleated sheet structure (AL amyloid).

MONOCLONAL GAMMOPATHY OF UNCERTAIN SIGNIFICANCE (MGUS) •

MGUS: prognosis—most of the patients remain stable.

• • •

Presence of serum M protein concentration lower than 3 g/dL. Bone marrow clonal plasma cells less than 10% in an asymptomatic patient. Etiology: may represent an early stage of myeloma development. Clinical manifestations: asymptomatic.

Lymphoid Neoplasms

13 CHAPTER

CLASSIFICATION OF LYMPHOID NEOPLASMS (TABLE 13.1) TABLE 13.1: WHO classification ofthe lymphoid neoplasms(2008) I. PRECURSOR LYMPHOID NEOPLASMS B lymphoblastic leukemia/lymphoma T lymphoblastic leukemia/lymphoma

Majority (80 to 85%) of lymphoid neoplasms are of B cell srcin and remaining of T cell/NK cell type.

II. MATURE B CELL NEOPLASMS Chronic lymphocytic leukemia/small lymphocytic lymphoma B cell prolymphocytic leukemia Splenic B cell marginal zone lymphoma Hairy cell leukemia Lymphoplasmacytic lymphoma Heavy chain disease Plasma cell neoplasm Follicular lymphoma Mantle cell lymphoma Diffuse large B cell lymphoma Burkitt lymphoma III. MATURE T AND NK CELL NEOPLASMS T cell prolymphocytic leukemia T cell large granular lymphocytic leukemia Mycosis fungoides Sézary syndrome Peripheral T cell lymphoma, NOS Angioimmunoblastic T cell lymphoma Anaplastic large cell lymphoma Adult T cell NK/T leukemia/lymphoma Extranodal cell lymphoma, nasal type IV. HODGKIN LYMPHOMA Classical Hodgkin lymphoma – Nodular sclerosis classical Hodgkin lymphoma – Mixed cellularity classical Hodgkin lymphoma – Lymphocyte-rich classical Hodgkin lymphoma – Lymphocyte depleted classical Hodgkin lymphoma Nodular lymphocyte predominance Hodgkin lymphoma

Lymphoid neoplasms: most resemble some stage of B or T cell differentiation.

Lymphoid neoplasms: second most common malignant tumor in HIV.

Lymphoid neoplasms: about 1/3rd arise from extranodal sites.

T-cell lymphoblastic lymphoma or Burkitt lymphoma usually seen in childhood.

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WHO classification of lymphoid neoplasms depends on clinicopathological and immunological profile (able 13.2) and has clinical and therapeutic importance. TABLE 13.2: Cell type and its antigens detected by monoclonal antibodies Celltype

Antigendetected

T cell

CD1, CD3, CD4, CD5, CD8

Bcell

CD10,CD19,CD20,CD21,CD23,CD79a

Monocyteormacrophage

CD11c,CD13,CD14,CD15,CD33,CD64

cell NK

CD16, CD56

Stemcellandprogenitorcell

CD34

All leukocytes

CD45 (LCA)

Abbreviations: CD, cluster designation; NK, natural k iller; LCA, leukocyte common antigen

Q. Write short notes on follicular lymphoma.

FOLLICULAR LYMPHOMA (FL) Composed of follicle center (germinal center) B cells of lymphoid follicles (centrocytes and centroblasts).

Morphology FL: arises from follicle center B cells.

Gross • •

FL: centrocytes and centroblasts form poorly defined follicles.

FL: grade ranges from 1 to 3. Grade 1 with less than 5 centroblasts/hpf and grade 3 with more than 15/hpf.

Involves lymph nodes, spleen and bone marrow. Architecture of lymph node is lost; frequently infiltrate the perinodal tissue (Fig. 13.1).

Microscopy • •

Follicular (nodular) growth pattern,neoplastic follicles are poorly defined (Fig. 13.2). Two types of B cells. – Centrocytes (small cleaved cells) ◆ Cleaved nuclei ◆ Inconspicuous nucleoli. – Centroblasts (large non-cleaved cells) ◆ Round or oval nuclei with open nuclear (vesicular) chromatin ◆ Multiple (1 to 3) nucleoli. ◆ Usually 3 times the size of lymphocyte.

Fig. 13.2:Follicular lymphoma shows nodular Fig. 13.1:Diagrammatic appearanceof follicularlymaggregates of malignant lymphoid cells phoma. Neoplastic follicles are seen in both the cortex and medulla and infiltration of the perinodal tissue

Lymphoid Neoplasms CHAPTER 13

Immunophenotype: expresses CD19, CD20 (pan-B cell markers),CD10 (CALLA), surface immunoglobulin and BCL2 protein. Cytogenetics and molecular genetics: t (14; 18) (q32:q21), with IgH and BCL2 as partner genes and leads to constitutive overexpression of BCL2protein.

Clinical Features • •

Peak in sixth and seventh decades. Generalized lymphadenopathy.

DIFFUSE LARGE B CELL LYMPHOMA (DLBCL) Heterogeneous groupof aggressive, neoplasm of large B cell with diffuse growth pattern.

FL: peripheral blood smear may show lymphocytosis [less than 20 × 109/L (20,000/cu mm)].

FL: bone marrow involved in 85%.

FL: prognosis indolent.

DLBCL: aggressive, diffuse large B cell neoplasm.

Constitutes about 20 to 30% of NHL and 60% to 70% of aggressive lymphoid neoplasms.

Microscopy • •

Loss of lymph node architecture with diffuse growth pattern. Neoplastic cells: – Large round or oval cells,4 to 5 times of a small lymphocyte. – Moderate pale or basophilic cytoplasm – Nucleus equals or larger than thenucleus of a macrophage with different appearances.

DLBCL may involve lymph nodes or extranodal sites.

Immunophenotype • • •

Express pan-B cell markers such asCD19, CD20, CD22 and CD79a. Also express germinal center markerslike CD10 and BCL6. Negative for TdT.

Cytogenetics and Molecular Profile • •

Translocation of BCL2 gene: t (14; 18) translocation Mutations of the BCL6 gene.

Clinical Features • • •

More common between65 and 70 years of age. Slight male preponderance. Rapidly enlarging mass at a single or multiplenodal or extranodal sites.

BURKITT LYMPHOMA (BL) •

•

DLBCL: prognosis aggressive and rapidly fatal if untreated.

Q. Write short notes on Burkitt

Highly aggressiveB cell neoplasm, often presents asextranodal lymphoma or as an acute lymphoma. leukemia. Composed of medium-sized, monomorphic lymphoid cellswith basophilic vacuolated cytoplasm.

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Clinical Variants •

BL: aggressive B cell lymphoma, 3 clinical variants. Endemic: involves jaw and associated with EBV.

•

•

BL: medium sized B cells. Starry sky pattern.

Endemic (African) Burkitt lymphoma (BL): – Occurs in Africa, affects children and adolescents. – Associated with Epstein-Barr virus infection and malaria. – Usually involves thejaw and present as a mandibular mass. Sporadic (nonendemic) BL : – Occurs in children or young adults. – Abdominal mass and involves ileocecum and peritoneum. Immunodeficiency-associated (HIV) BL : – Involves lymph nodes and bone marrow.

Microscopy • • •

•

•

Burkitt lymphomas, irrespective of the categories, arehistologically similar. Lymph node showsloss of architecture. Involved tissues showdiffuse infiltrate of monotonous medium-sized lymphoid cells (Figs 13.3 and 13.4). Appearance of neoplastic lymphoid cells: – Medium-sized cells. – Round or oval nucleihaving clumpedcoarse chromatin with several (2 to 5) nucleoli. – Moderate amount of deeply basophilic cytoplasm, multiple, small, round lipoid (clear) vacuoles which stain positive with oil red O. – Numerous mitotic figures. Starry sky pattern: tumor cells undergo apoptosis and nuclear remnants of these apoptotic cells are phagocytosed and cleared by benign macrophages. Tese macrophages in the background of lymphoid cells creates“starry sky” appearance (Figs 13.3 and 13.4).

Fig. 13.3: Burkitt lymphoma composed of medium-sized lymphoid cells admixed with benign macrophages giving a “starry sky” appearance

Fig. 13.4: Diagrammatic appearance of Burkitt lymphoma composed of medium-sized lymphoid cells admixed with benign macrophages giving a “starry sky” appearance

Lymphoid Neoplasms CHAPTER 13

Immunophenotype • • • •

Express surface IgM, monotypic κ or λ light chain. Positive for common B cell antigens (CD19, CD20, and CD22). Positive for CD10 and BCL6. BCL2 negative.

Cytogenetic and Molecular Genetic Features (Fig. 13.5) Translocations of c-MYCgene MYC (c-MYC) is aproto-oncogene-on chromosome 8. • • •

• •

BL: translocation of c-MYC gene.

Most common translocation t (8:14) (q24; q32). Translocations of c-MYC gene, converts proto-oncogene into MYC oncogene, which leads to overexpression of MYC protein (oncoprotein). Tis causes uncontrolled cell BL: prognosis—very aggressive but responds proliferation and stimulation of apoptosis. well chemotherapy. Mutations inactivate p53. Poor prognostic factors: – Involvement of blood, bone marrow and central nervous system. – Bulk of the disease-unresected tumor of more than 10 cm in diameter. – High serum LDH levels. – Presence of residual disease after excision.

Fig. 13.5:Chromosomal translocation and activated MYC oncogene in Burkitt lymphoma

MATURE T CELL AND NK CELL NEOPLASMS

Peripheral T Cell Lymphoma (PTCL), NOS Mainly involves lymph node.

Peripheral T cell tumors constitute less than 15% of non-Hodgkin lymphomas. NK cell tumors very rare.

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PTCL: clinical features Fifth to seventh decade. Generalized lymphadenopathy. •

Microscopy •

•

• •

PTCL: prognosis—highly aggressive with a poor response to therapy.

Immunophenotype • •

Mycosis fungoides and Sézary syndrome: T cell neoplasms with skin involvement.

Immunophenotype: Express pan-T-CD2+, CD3+ and CD5.

Sézary cells are neoplastic T cells with cerebriform nuclei.

Lack d (expressed by immature  cells). Express pan- cell-CD2, C3, CD5 and eitherα β or γ δ  cell receptors (CR).

Mycosis Fungoides • • •

Mycosis fungoides has three stages: 1. Patch stage 2. Plaque stage 3. Tumor stage.

Lymph node with effacement of the normal architecture. Paracortical or diffuse infiltration by neoplastic  cells. Neoplastic T cells – Small, intermediate to large cells with sparse or abundant; clear, eosinophilic or basophilic. – Vesicular or hyperchromatic nuclei, prominent nucleoli.

Cutaneous T cell lymphoma Lymphoid cells withirregular nuclear outlines Limited to skin.

Age: most are adults or elderly.

Microscopy • • •

Epidermis (epidermotropism) and upper dermisis infiltrated by neoplastic  cells. Groups of neoplastic cells in the epidermis—Pautrier’s microabscess. umor cells haveconvoluted (cerebriform) nuclear contours.

Sézary Syndrome Rare disease and is defined by the triad namely: 1. Widespread exfoliative erythroderma

Immunophenotype: tumor cells express-CD2+, CD3+ and CD5+.

2. Generalized lymphadenopathy 3. Presence of characteristic Sézary cells in the skin, lymph nodes and peripheral blood. Prognosis: aggressive disease and most die of opportunistic infections.

Hodgkin Lymphomas

14 CHAPTER

DEFINITION HL: Malignant lymphoid neoplasms with following characteristics: Minority (1–3%) of specific neoplastic cells (Hodgkin cells andReed-Sternberg cells). Majority background ofreactive non-neoplastic cells. Usually involveslymph nodes. Majority occurs in young adults.

Hodgkin lymphoma synonym: Hodgkin disease.

• • • •

CLASSIFICATION (TABLE 14.1)

Q. Classify Hodgkin lymphoma.

Hodgkin lymphoma (HL) is broadly divided intotwo types, which differ in clinical features, behavior, morphology and immunophenotype.

Cell of Origin and Immunophenotype •

•

Classical Hodgkin lymphoma – Cell of srcin: germinal center or post-germinal center B cell – Immunophenotype: CD15 and CD 30 positive Nodular lymphocyte predominant Hodgkin lymphoma – Cell of srcin: germinal center B cell at the centroblastic stage of differentiation. – Immunophenotype: CD15 and CD30 negative.

TABLE 14.1: WHO classification (2008)of Hodgkin lymphoma •

Classical Hodgkin lymphoma (CHL) – – – –

•

Nodular sclerosis classical Hodgkin lymphoma (NSCHL) Mixed cellularity classical Hodgkin lymphoma (MCCHL) Lymphocyte-rich classical Hodgkin lymphoma (LRCHL) Lymphocyte depleted classical Hodgkin lymphoma (LDCHL)

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)

HL is mainly divided into: 1. Classical and 2. Nodular lymphocytic Hodgkin lymphoma.

Classical HL: CD15 + and CD30 +, NLPHL: CD15-,CD 30-, CD20+, and CD 45+.

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MORPHOLOGY OF NEOPLASTIC CELLS

Q. Write short note on RS cell and its variants.

Reed-Sternberg (RS) Cells are neoplastic cells (Fig. 14.1) pathognomonic of Hodgkin lymphoma. Appearance and description of diagnostic Reed-Sternberg cells and its variants are shown in Figure 14.2. Various types of cells found in Hodgkin lymphoma are listed in Table 14.2. TABLE 14.2: Types of cells found in Hodgkin lymphoma

HL: majority are nonneoplastic cells and minority are neoplastic cells.

Non-neoplasticcells •

•

•

Neoplasticcells

Reactive lymphocytes Macrophages/histiocytes

•

•

Reed-Sternberg cells (classical) Variants

– Granulocytes – Eosinophils – Neutrophils

– Mononuclear – Lacunar – Mummified

Plasma cells

– Anaplastic/pleomorphic – Lymphocyte predominant (LP) cell/popcorn

CLASSICAL HODGKIN LYMPHOMA Classical Hodgkin lymphoma (CHL) account for95% of Hodgkin lymphomas and has4 subtypes.

Q. Write short note on nodular sclerosis HL.

Nodular Sclerosis Classical Hodgkin Lymphoma (NSCHL)

Nodular sclerosis is the

Subtype of CHL characterized by collagen bands that surroundnodules and have lacunar cell variant of Reed-Sternberg cells. Most common: 40% to 70% of cases. Most between 20 and 30 years of age with equal frequency in males and females. Rarely associated with EBV. Involves mediastinal lymph nodes.

most common subtype of CHL. Lacunar cells are commonly seen.

• • • •

Fig. 14.1:Microscopic appearance of Hodgkin lymphoma showing RS cells (short arrow and inset) and Hodgkin cells (long arrow) within the background of mixed population of reactive cells

Hodgkin Lymphomas CHAPTER 14

Classical RS cell is binucleated with owl-eyed nuclei having mirror image appearance.

Lacunar cell has clear cytoplasm and seen in nodular sclerosis CHL.

LP/popcorn cell is seen in nodular lymphocyte predominant HL.

Fig. 14.2:Diagrammatic appearances and characteristic features of Reed-Sternberg cells and its variants

Microscopy of NSCHL (Fig. 14.3) • •

• •

Loss of lymph node architecture. Sclerosis and nodules: broad collagen bands (sclerosis) divide the lymphoid tissue into nodules of varying sizes and shapes. Presence of lacunar cell. Background: small T lymphocytes, eosinophils, plasma cells, and macrophages.

Immunophenotype • •

RS cells areCD15+ and CD30+; CD45- and T cell markers negative. EBV negative.

NSCHL Nodules separated by broad bands of collagen CD15+,CD30+, EBV-ve and CD 45-ve. •

•

NSCHL Prognosis: better than other types of CHL, with a cure rate of 80% to 85%.

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Fig. 14.3:Nodular sclerosis classical Hodgkin lymphoma with nodules separated by bands of collagen. Also seen are lacunar cells and RS cells in each nodule within the background of lymphocytes, eosinophils, plasma cells and macrophages

Q. Write short note on Mixed cellularity HL.

Mixed Cellularity Classical Hodgkin Lymphoma (MCCHL) • • • •

MCCHL: scattered classical RS cells and mixed inflammatory background, CD15+, CD30+ and EBV+.

Fig. 14.4:Mixed cellularity classical Hodgkin lymphoma with classical RS cells, Hodgkin cells in the background of mixed cellular population consisting of lymphocytes, eosinophils, plasma cells and macrophages

•

Second common subtype: 20% to 25% of cases More common in males Strongly associated with EBV Older age, with systemic symptoms (such as night sweats and weight loss) andadvanced tumor stage Involves peripheral lymph nodes.

Microscopy of MCCHL (Fig. 14.4) • •

MCCHL: prognosis—very good.

•

Lymph node architecture obliterated Plenty of Reed-Sternberg cells and Hodgkin cells Back ground: small lymphocytes, eosinophils (sometimes numerous), neutrophils, plasma cells and benign macrophages (histiocytes).

Immunophenotype: RS cells areCD15+, CD30+ and EBV+ (about 70%).

Lymphocyte-rich Classical Hodgkin Lymphoma (LRCHL) • • • •

Subtype of classical Hodgkin lymphoma withscattered Hodgkin and RS cells. Uncommon—about 5% of classical HL. More in elderly patients, associated with EBV in 40% of cases. Involves peripheral lymph nodes.

Hodgkin Lymphomas CHAPTER 14

Microscopy of LRCHL (Fig. 14.5) •

• •

LRCHL: Uncommon Few RS cells Abundant lymphocytes CD15+, CD30+, CD45and CD20-. •

Growth patterns: may show two patterns – Nodular—common – Diffuse—rare Only few Reed-Sternberg cells and Hodgkincells Background: abundant reactive small lymphocytes.

•

•

•

Immunophenotype: CD45–, CD20–, CD15+ and CD30+.

LRCHL: prognosis—good to excellent prognosis.

Lymph ocyte-depleted Classical Hodgkin Lymphoma (LDCHL) Subtype of classical Hodgkin lymphomarich in Hodgkin and RS cells in a background LDCHL: Rarest depleted in non-neoplastic lymphocytes. Paucity of lymphocytes Rarest—less than 5% of cases Plenty of RS cells Predominantly in older, HIV-positive patients, often EBV-associated (over 90%) CD15+, CD30+; majority Predominantly retroperitoneal lymph nodes, abdominal organs and bone marrow. are EBV+. •

•

•

•

•

•

•

Microscopy of LDCHL (Fig. 14.6) • • •

Paucity of lymphocytes. Plenty of RS cells or their anaplastic/pleomorphic variants. Histological types – Reticular: numerous Hodgkin and RS cells with depletion of lymphocytes. – Diffuse sclerosis/fibrosis: hypocellular infiltrate containing bizarre RS cells with fine fibrosis. LDCHL: prognosis—

Immunophenotype: RS cells areCD15+, CD30+; majority are EBV+.

Fig. 14.5:Lymphocyte-rich classical Hodgkin lymphoma. One RS cell is seen in a background of many small lymphocytes and few histiocytes

outcome less favorable than with other subtypes.

Fig. 14.6:Lymphocyte-depleted classical Hodgkin lymphoma with the pleomorphic variant of RS cells surrounded by fibrous tissue

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NODULAR LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA (NLPHL) • • • •

NLPHL: Uncommon Abundant lymphocytes LP cells No Hodgkin/RS cells CD20+, CD 45+ and CD15 -, C30- and EB -ve. •

Microscopy of NLPHL (Fig. 14.7) •

•

•

•

•

•

Uncommon—5% of all Hodgkin lymphomas. Not associated with EBV. Majority males, usually30–50 year of age. Involves mainlycervical or axillary lymph nodes.

•

•

Loss of lymph node architecture. Nodular and/or diffuse infiltrate ofabundant small lymphocytes with histiocytes and scattered LP cells. Lymphocyte predominant cells (LP cells)/"popcorn" cells (Fig. 14.2): – Specific to NLPHL. – Large with relatively abundant, pale cytoplasm. – Single large delicatemultilobulated nucleus or folded nuclei resembling bubbly outlines of popcorn kernels. – One or moreinconspicuous nucleoli. Hodgkin and RS cells are not found.

Immunophenotype: LP cell areCD20+, CD 45+ and CD15–, C30– and EBV–ve.Express BCL6.

NLPHL: prognosis—more likely to recur than the classical subtypes, but the prognosis is very good.

Fig. 14.7:Nodular lymphocyte predominant Hodgkin lymphoma with ‘popcorn’ cells in a background of reactive lymphocytes and few macrophages

Hodgkin Lymphomas CHAPTER 14

ETIOLOGY AND PATHOGENESIS OF HODGKIN LYMPHOMA • • •

EBV: previous EBV infection (infectious mononucleosis) ↑ risk of HL. Genetic factors: HLA-B18 higher in HL. Immune status: HL more frequent in immunocompromised patients and autoimmune diseases (e.g. rheumatoid arthritis).

Pathogenesis (Fig. 14.8) •

•

•

:

EBV and HL HL is associated with EBV infection. Activation of nuclear factor (NF-κB) common event in classical HL → rescue germinal center B cells from apoptosis → produces Reed-Sternberg cells. Accumulation of reactive cells in response to cytokines (such as IL-5, IL-6 and TGF-β) and chemokines secreted by Reed-Sternberg cells.

LABORATORY FINDINGS •

Peripheral smear: – RBCs: normocytic normochromic anemia. – WBCs: leukocytosis occurs in 1/3rd of the patients. Eosinophilia is frequent. – Platelets: normal or increased.

ESR: raised.

Bone marrow: involved in the later stages.

Fine Needle Aspiration Cytology (FNAC) RS cells/its variants against a background of inflammatory cells (depending on the subtype).

Clinical features: Painless enlargement of lymph nodes. Systemic/constitutional symptoms: fever, night sweats and weight loss. •

•

Fig. 14.8:Pathogenetic mechanism and interaction of various cell types in Hodgkin lymphoma

HL: Pel-Ebstein fever is characterized by alternating bouts of fever followed by remissions.

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Spread • •

Mainly by contiguity First nodal disease → then splenic disease, hepatic disease → and finally marrow involvement and extranodal disease.

STAGING OF HODGKIN LYMPHOMA TABLE 14.3 TABLE 14.3: Clinical staging of Hodgkin lymphomas (Cotswold revision ofAnn Arbor staging classification) Stage

Definition

I

Involvement of a single lymph node region or lymphoid structure (e.g. spleen, Waldeyer ring, thymus)

II

Involvement of two or more lymph node regions on the same side of the diaphragm (the mediastinum is a single site; hilar lymph nodes are lateralized); the number of anatomic sites should be indicated by suffix (e.g. II3)

III

Involvement of lymph node regions or structures on both sides of the diaphragm III1 With or without splenic, hilar, celiac or por tal nodes III2 With para-aortic, iliac or mesenteric nodes

IV

Involvement of extranodal site(s) beyond those designated E

E—involvement of a single extranodal site, or contiguous or proximal to known nodal site of disease

DIFFERENCES BETWEEN HODGKIN LYMPHOMA AN D NON HODGKIN LYMPHOMA HL differs from NHL in several respects and their main differences are shown in Table 14.4. Q. List the differences between TABLE 14.4: Differences between Hodgkin and non-Hodgkin lymphomas HL and NHL. Sl.No. Characteristics Hodgkinlymphoma Non-Hodgkinlymphoma

HL: extranodal involvement uncommon.

1.

Siteofinvolvement

Arisesinasinglenodeor chain of nodes (cervical, mediastinal, para-aortic)

Mainly involves multiple peripheral nodes

2.

Patternofspread

Orderlyspreadbycontiguity in a predictable fashion

Noncontiguous spread in an unpredictable fashion

3.

Mesenteric nodes and Waldeyer ring

Rarely involved

4.

Extranodalinvolvement

Uncommon

5.

Characteristic of neoplastic cells

Neoplastic cells—Hodgkin or Reed-Sternberg cells form minor tumor cell mass (1–5%)

Commonly involved Common Neoplastic cells form the major tumor cell mass

Langerhans Cell Histiocytosis/ Histiocytosis X

15 CHAPTER

INTRODUCTION • • •

Histiocytic and dendritic cell neoplasms Clonal proliferative disorder arising from Langerhans cells Langerhans cell histiocytosis (LCH) spectrum ranges from unifocal to multifocal and unisystem to multisystem disease.

MORPHOLOGY •

Light microscopy: the characteristic feature is proliferations ofLangerhans cells. – Tese are large cells 10–15 μm in diameter, moderate slightly eosinophiliccytoplasm folded, indented, grooved orlobulated nucleus having finechromatin. – Background: mixed background of eosinophils, histiocytes (mononuclear and Langerhans cell contains

multinuclear), neutrophils and small lymphocytes. •

•

LABORATORY FINDINGS • •

pathognomonic Birbeck

Electron microscopy: Langerhans cell containspathognomonic Birbeck granules—tennis granules. racket-like shape, with a zipper-like appearance . Immunological markers: express CD1a, langerin and S-100 protein.

Peripheral blood: pancytopenia (anemia, neutropenia and thrombocytopenia). Bone marrow: extensive infiltration by histiocytes.

Prognosis: depends on the age at presentation, extent of disease and rate of progression. Groups: depending on the site involved and distribution of lesion, LCH can be divided into

three groups (able 15.1).

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TABLE 15.1: Types of Langerhans cellhistiocytosis Terminology/site

Involvedtissue/organ

Eosinophilic granuloma Localized to a single site/solitary (unifocal)

Bone and adjacent soft issue (skull, femur, vertebra, pelvic bones and ribs). Less commonly lymph nodes

Hand-Schüller-Christian disease Usually bone and soft issue Multiple sites within a single system (multifocal unisystem) Letterer-Siwe disease Disseminated and multisystemic disease (multifocal multisystem

disease)

Skin, bone, liver, spleen and bone marrow

Clinicalfeatures

Usually seen in older children or adults. Presents with lytic bone lesion Usually seen in young children Multiple destructive bone lesions with adjacent soft tissue masses Usually seen in infants. Present with fever, cytopenias, skin and bone lesions and hepatosplenomegaly

SECTION

Disorders of Hemostasis

3

Disorders of Primary Hemostasis

16 CHAPTER

NORMAL HEMOSTASIS • •

Hemostasis is thebody’s response to vascular damage/injury. Includes several sequences of events at the site of vascular injury. Tey are as follows:

Primary Hemostatic Plug Platelet adhere to subendothelial structures at the site of injury. Te platelets change their shape and release granule contents. Te released contents causeplatelet aggregation and

Platelet sequence in hemostasis: platelet adhesion → release of granule contents→ platelet aggregation → primary (temporary) hemostatic plug → activation of coagulation system → fibrin → secondary (permanent) hemostatic plug.

form primary hemostatic plug.

Secondary Hemostatic Plug Exposure of tissue factor at the site of vascular injury activates the extrinsic coagulation system. Te fibrin formed develops into a secondary hemostatic plug.

CLASSIFICATION OF H EMOSTATIC DISORDERS (TABLE 16.1) 1. Bleeding disorders (hemorrhagic disorders/hemorrhagic diathesis): bleeding disorders have an abnormal tendency to bleed (hemorrhage) due to failure of hemostasis. 2. Trombotic disorders:they cause thrombus formation.

BLEEDING DISORDERS CAUSED BY VESSEL WALL ABNORMALITIES Vascular purpura (nonthrombocytopenic purpura) is group of disorders of blood vessels that results in bleeding. Tey should be distinguished from bleeding disorders due to abnormalities of platelets.

Q. Classify bleeding disorders.

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Hemostatic disorders are broadly classified as bleeding disorders and thrombotic disorders.

TABLE 16.1: Classification of disorders of hemostasis 1. Bleeding disorders

Bleeding disorders may be due to Diseases of blood vessels Platelet disorders Coagulation disorders.

– Disorders of primary hemostasis ◆ Vessel wall abnormalities ◊ Congenital, e.g. Ehlers-Danlos syndrome ◊ Acquired, e.g. Henoch-Schönlein purpura ◆ Platelet abnormalities ◊ Quantitative: thrombocytopenia (e.g. ITP, drug-induced, congenital) ◊ Qualitative: platelet function disorders - Inherited, e.g. Glanzmann thrombasthenia, Wiskott-Aldrich syndrome, Bernard Soulier syndrome - Acquired, e.g. Uremia, drugs

Vascular purpuras are also known as nonthrombocytopenic purpuras.

– Disorders of coagulation system (disorders of secondary hemostasis) ◆ Congenital:hemophilia A, B; von Willebrand disease; other coagulation factor deficiencies [XI, VII, II, V, X] ◆ Acquired:vitamin K deficiency, liver disease, disseminated intravascular coagulation

•

•

•

2. Thrombotic disorders –

Inherited

◆ Deficiency of antithrombotic factors: antithrombin III deficiency, protein C deficiency, protein S deficiency ◆ Increased prothrombotic factors: activated protein C (APC) resistance (Factor V mutation/factor V Leiden) ◆ Prothrombin (G20210A mutation) –

Acquired:fibrinolytic system defects

Classification of bleeding disorders caused by vessel wall abnormalities are presented in able 16.2. TABLE 16.2: Classification of bleeding disorders caused by vessel wall abnormalities Acquired disorders Senile purpura is due to vessel instability.

Henoch-Schönlein purpura is characterized by hypersensitivity vasculitis and palpable purpura.

1. Due to decreased amount of connective tissue – Senile purpura – Scurvy – Cushing syndrome and steroid therapy 2. Due to vasculitis – Henoch-Schönlein purpura – Infections – Drug reactions 3. Associated with plasma cell neoplasms – Amyloidosis 4. Miscellaneous – Simple easy bruising Congenital/inherited disorders – Hereditary hemorrhagic telangiectasia – Ehlers-Danlos syndrome – Marfan syndrome

BLEEDING DISORDERS DUE TO ABNORMALITIES OF PLATELET

Classification of Platelet Disorders (Table 16.3)

THROMBOCYTOPENIA •

Decrease in theplatelet count below the lower limit of 150,000/cu mm (150 × 109/L).

Disorders of Primary Hemostasis

CHAPTER 16

TABLE 16.3: Classification of platelet disorders Quantitative platelet disorders •

Thrombocytopenia

– Increased destruction – Sequestration •

– Decreased production – Dilutional

Thrombocytosis

Qualitative platelet disorders •

Hereditary

•

– Defective adhesion of platelets – Defective platelet aggregation Acquired

– Disorders of platelet secretion

Clinical Features of Thrombocytopenia • •

•

Cutaneous bleeding appears as pinpoint hemorrhages (petechiae) and ecchymoses. Mucosal bleeding. Intracranial bleed (subarachnoid and intracerebral hemorrhage) rare but serious.

Severity of Bleeding • • •

Post-traumatic bleeding—when the plateletcount is 20,000 to 50,000/cu mm Spontaneous bleeding—when the platelet count falls below 20,000/cu mm Intracranial bleeding—when platelet count is <10,000/cu mm.

Petechiae are pinpoint hemorrhages seen only with thrombocytopenia.

Intracranial bleeding occurs-when platelet count is <10,000/cu mm.

Causes of Thrombocytopenia (Table 16.4) TABLE 16.4: Causes of thrombocytopenia 1. Increased platelet destruction – Immune mediated ◆ Autoimmune ◊ Primary: immune thrombocytopenic purpura (acute and chronic) ◊ Secondary: systemic lupus erythematosus, B cell lymphoid neoplasms ◆ Alloimmune:post-transfusion or pregnancy ◆ Drug-induced:quinidine, heparin, sulfa compounds ◆ Infections:HIV infection, infectious mononucleosis, cytomegalovirus – Non-immune mediated ◆ Disseminated intravascular coagulation ◆ Thrombotic thrombocytopenic purpura, hemolytic uremic syndrome ◆ Mechanical destruction: prosthetic heart valves, malignant hypertension ◆ Microangiopathic hemolytic anemias 2. Decreased production of platelets – Generalized primary diseases of bone marrow: aplastic anemia (congenital and acquired) – Bone marrow invasion/infiltration: leukemia, disseminated cancer – Selective impairment of platelet production ◆ Drug-induced: alcohol, thiazides, cytotoxic drugs ◆ Infections: measles, human immunodeficiency virus (HIV ) – Ineffective megakaryopoiesis ◆ Megaloblastic anemia ◆ Myelodysplastic syndromes 3. Sequestration – Hypersplenism 4. Dilutional

ITP is the most common form of thrombocytopenia.

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IMMUNE THROMBOCYTOPENIC PURPURA • •

Most common form of thrombocytopenia. Due to increased destruction of platelets by immune mechanisms—mainly autoimmune mechanism.

Types of Immune Thrombocytopenic Purpura (ITP) Acute Immune Thrombocytopenic Purpura Acute ITP is seen mainly in children between 2 to 4 years.

•

Acute ITP: autoimmune disease, sudden onset, shorter duration and usually resolves within 6 months.

•

• •

•

Self-limited disease. Children: 2 to 4 years and seen equally in both sexes. Presents 1 to 3 weeks after viral (measles, rubella, EBV) infection. Platelet destruction by antiplatelet autoantibodies. Platelet count is decreased, sometimes even below 10,000/cu mm (10 × 109/L).

Clinical Features Sudden onset. Petechiae, gum bleeding, epistaxis and mild fever. Usually resolve spontaneously within 6 months. Excellent prognosis. • • • •

Chronic ITP: autoimmune disease and the antibodies are directed against glycoprotein IIb/III a of platelets.

Chronic Immune Thrombocytopenic Purpura •

• •

Persistent thrombocytopenia for more than 6 to 12 months Indolent, females are more affected than males (F:M=3:1). More common and usually seen in adults (20 to 40 years).

Pathogenesis of ITP (Fig. 16.1) Autoimmune disorder characterized by formation of antiplatelet antibodies, directed against membrane glycoproteins (most often IIb-IIIa or Ib-IX of platelets). Antiplatelet antibodies in about 80% of patients and are of the IgG type. Antiplatelet antibodies act as opsonins and are recognized by IgG Fc receptors present on mononuclear phagocytes of RE system (mainly spleen) and are destroyed there resulting in thrombocytopenia. Splenectomy causes marked improvementin 75% to 80% of patients. •

•

•

Spleen is the major site of destruction of platelets and important site of autoantibody synthesis.

ITP: splenomegaly and lymphadenopathy are uncommon and in their presence one should consider the diagnosis other than ITP.

•

Clinical Features More common infemales (F:M ratio is 3:1). Age between 20 and 40 years. Clinical features are due to thrombocytopenia: skin bleeding, mucosal bleeding, menorrhagia in females, etc. • • •

Fig. 16.1:Pathogenesis of idiopathic thrombocytopenic purpura

Disorders of Primary Hemostasis

Laboratory Findings

Q. Write short notes on laboratory findings in ITP.

Peripheral Blood • •

•

CHAPTER 16

Platelet count: markedly reduced—below 80,000/cu mm (80 × 109/L). Hemoglobin: ranges from 7 to 12 g/dL. Peripheral smear – Platelets: markedly reduced (thrombocytopenia) and abnormally large sized platelets (megathrombocytes/giant platelets). – RBCs: chronic blood loss (e.g. menorrhagia) due to ITP may lead to microcytic hypochromic anemia.

ITP: platelets markedly reduced below 80,000/ cu mm.

– WBCs: normal.

Bone Marrow • •

•

• •

• • • • •

Cellularity: hypercellular. Megakaryopoiesis: – Moderate increase in number (Fig. 16.2) of both immature and mature forms of megakaryocytes. – Immature megakaryocytes predominate-large nonlobulated single nuclei and basophilic cytoplasm (Fig. 16.3).

Erythropoiesis: – Prolonged bleeding may cause anemia leading to normoblastic erythroid hyperplasia. – Constant bleeding leads to iron deficiency and micronormoblastic erythroid hyperplasia. Myelopoiesis: normal. Storage iron: severe and chronic bleeding causes iron deficiency with reduced iron stores.

Bleeding time (B): prolonged, but P and P are normal. ourniquet test: positive. Clotting time (C): normal. ests for platelet autoantibodies: may be positive. Spleen: normal size.

Bone marrow in chronic ITP shows megakaryocytic hyperplasia with immature megakaryocytes.

ITP: bone marrow— decreased megakaryocytes—against the diagnosis of ITP.

ITP: bleeding time prolonged PT and APTT normal.

1

2

3 Fig. 16.2:Bone marrow in ITP showing moderate increase in number of megakaryocytes (arrows)

Fig. 16.3:Diagrammatic appearance of megakaryocytes in different stages of maturation (1-immature to 3-mature)

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THROMBOCYTOSIS Platelet count more than 4,50,000/cu mm is known as thrombocytosis. Causes: various causes of thrombocytosis are shown in able 16.5. TABLE 16.5: Causes of thrombocytosis Idiopathic/primary (autonomous production) • • •

Essential thrombocytosis Polycythemia vera Chronic myeloid leukemia

Secondary (reactive thrombocytosis) • • • •

Iron deficiency Malignancy Following hemorrhage Following splenectomy

QUALITATIVE PLATELET DISORDERS Classification of platelet functional (qualitative) disorders are presented in Figure 16.4 and able 16.6.

Fig. 16.4:Functional disorders of platelet

TABLE 16.6: Classification of platelet functional (qualitative) disorders A. Hereditary Aspirin blocks the cyclooxygenase enzyme of platelets and prevents aggregation of platelets.

1. Disorders of platelet adhesion: Bernard-Soulier syndrome 2. Disorders of platelet secretion: storage pool deficiency 3. Disorders of platelet aggregation: Glanzmann thrombasthenia B. Acquired 1. Drugs: aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), dipyridamole, sulfinpyrazone 2. Renal failure: uremia 3. Hematologic malignancies: myeloproliferative neoplasms and myelodysplastic syndromes

Bleeding Disorders: Due to Abnormalities of Coagulation/Clotting Factor

17

INTRODUCTION Bleeding due to coagulation disorders must be distinguished from those due to platelet/ vascular disorders (able 17.1). TABLE 17.1: Distinguishing patterns of bleeding in platelet/vascular and coagulation disorders Characteristics

Platelet/vasculardisorders

Coagulationdisorders

Onset

Spontaneousanddevelops immediately after trauma/surgery

Delayed bleeding after trauma/ surgery

Type of lesion

Petechiae, ecchymoses

Sites

Skin,mucousmembrane Common from nose, mouth, gastrointestinal and genitourinary tracts

Hematomas Deeptissues

•

Mucous membrane

Uncommon except from gastrointestinal or genitourinary tract

•

Intothejoint

Absent

Commoninseverefactordeficiencies

•

Intothemuscle

Followingtrauma

Spontaneous

CLASSIFICATION OF COAGULATION DISORDERS (TABLE 17.2) TABLE 17.2: Classification of coagulation disorders A. Hereditary coagulation disorders 1. Hemophilia A 3. von Willebrand disease

2. Hemophilia B 4. Others

B. Acquired (secondary) coagulation disorders 1. Vitamin K deficiency 3. Others

2. Liver disease

CHAPTER

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Disorders of Hemostasis

HEREDITARY COAGULATION DISORDERS Usually, due to deficiency of single coagulation factor.

vWF is synthesized by endothelial cells and megakaryocytes.

Factor VIII-vWF Complex •

vWF may be located in the plasma and subendothelial

•

tissue.

•

Three common hereditary disorders are: 1. Hemophilia A (deficiency of factor VIII) 2. Hemophilia B (deficiency of factor IX) 3. von Willebrand disease (deficiency of vWF).

Factor VIII-vWF complex has two components: – Plasma factor VIII – von Willebrand factor. vWF protects factor VIII and important for its stability. Subendothelial vWF promotes platelet adhesion. Whenever there is vascular endothelial injury, plasma vWF gets adsorbed to exposed subendothelial matrix and augments adhesion of platelets.

HEMOPHILIA •

•

•

Hemophilia A and B are similar in both clinical and pathological features, the difference being in the deficient factor. Both are sex-linked recessive disordersresulting in inherited deficiency of the clotting factor or synthesis of a defective clotting factor. Males are affected and females are carriers.

HEMOPHILIA A (FACTOR VIII DEFICIENCY) • • •

•

Common hereditary X-linkedrecessive disease. About 30% of hemophiliacs may be due to acquired mutations. Reduced amount or activity of factor VIII is associated with life-threatening bleeding Bleeding is due to both inadequate coagulation and inappropriate clot removal (fibrinolysis).

Mode of Inheritance (Fig. 17.1) Hemophilia A: X-linked recessive disorder.

•

• •

•

•

X-linked recessive disease. Genes for factor VIII are located on the long arm of the X-chromosome. Does not manifest when there is a normal copy of X-chromosome. Males with a defective/mutant factor VIII gene (hemophiliac gene) on their single X chromosome (XH) suffer from hemophilia. Heterozygous females are carriersand do not express the full clinical disease because of the paired normal X-chromosome. However, females with two copies of the defective X H chromosome may rarely suffer from hemophilia.

Molecular Genetics Causative mutations include deletions, inversions, point mutations and insertions.

Clinical Features Clinical severity depends on the level of factor VIII activity with normal range expressed as percentage (able 17.3). Severe cases have less than 1% residual factor VIII activity.

Bleeding Disorders: Due to Abnormalities of Coagulation/Clotting Factor CHAPTER 17

Hemophilia A: males are suffers and females are carriers.

Fig. 17.1:Mode of inheritance in hemophilia

TABLE 17.3: Factor VIII level and clinical severity in hemophilia A Clinicalseverity

LeveloffactorVIIIactivityin percentage

Mild

Morethan6

Clinical features

Normal range for factor VIII: 45–158 IU/dL.

Inthemildestform,itmaybe unnoticed. Bleeding develops after trauma only

Moderate

2–5

Severe

Lessthan1

Bleedingaftertrauma,including dental and other surgical trauma Easy bruising Frequentandspontaneous hemorrhage into joints (hemarthrosis) and soft tissues

Common clinical presentations include: Frequent and spontaneoushemorrhage into the joints-hemarthrosis. Hemorrhage intosoft tissues. Prolonged bleeding following trauma • • •

Laboratory Findings Bleeding time: normal • • • • •

Clotting time: prolonged, but is not a sensitive test Platelet count: normal Prothrombin time: normal Activated partial thromboplastin time (AP): increased (normal 30–40 seconds)

Hemophilia A: percentage of level of factor VIII activity correlates with severity of disease. Hemophilia A: common presentation • Hemarthrosis • Hemorrhage into soft tissues.

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Hemophilia A: decreased: factor VIII Increased: APTT and clotting time.

• • • •

Factor VIII assay: essential for the diagnosisand to assess the levels and severity of disease Fibrinogen assay: normal FDP: negative Detection of carriers: by DNA markers – o detect female carriers – Prenatal diagnosis of affected fetuses.

Complications Causes of death • Intracranial hemorrhage • Prolonged bleeding.

Due to Hemophilia •

•

Treatment • Factor VIII concentrate • Recombinant factor VIII.

Due to Therapy •

•

•

Hemophilia B: • X-linked recessive disorder • Mutation in factor IX • Deficiency of factor IX.

Viral hepatitis: hepatitis B, C and D in patients who received multiple transfusions of FFP/cryoprecipitate. AIDS: in individuals who received fresh frozen plasma (FFP) or cryoprecipitate, when screening tests for HIV were not available. Factor VIII inhibitors: makes further management difficult.

HEMOPHILIA B (CHRISTMAS DISEASE, FACTOR IX DEFICIENCY) • • • •

Hemophilia B: clinical features • Usually milder than hemophilia A. • Hemarthrosis is the common presentation.

Deforming arthritis and contractures: this is due to repeated bleeding into the joints. Organization and fibrosis of intramuscular hematomas→ contractures of involved muscles. Anemia: excessive, spontaneous or repeated bleeding leads to anemia.

Clinically indistinguishable from hemophilia A X-linked recessive disorder Variable clinical severity Assay of factor IX should be done to diagnose Christmas disease (named after the first patient).

Laboratory Findings Similar to hemophilia A. Bleeding time: normal Clotting time: prolonged Platelet count: normal Prothrombin time: normal Activated partial thromboplastin time (AP): increased (normal 30–40 seconds) Factor IX assay:factor IX is decreased. • • •

Hemophilia B: decreased: factor IX increased: APTT and clotting time.

vWF: causes platelet adhesion and prevents degradation of Factor VIII in plasma. Platelet adehsion molecule is synthesized in the Weibel-Palade bodies in endothelial cells.

• • •

VON WILLEBRAND DISEASE (vWD) • • •

Most common inherited bleeding disorders Most cases areautosomal dominant disorders Variable clinical picture with more than 20 variants.

Bleeding Disorders: Due to Abnormalities of Coagulation/Clotting Factor CHAPTER 17

Categories Grouped into two major categories: Quantitative deficiency of vWF: decreased circulating vWF – ype 1- autosomal dominant, mild disorder and form about 75% of all cases – ype 3-autosomal recessive, severe disorder and least common type. Qualitative defects in vWF: – ype 2-autosomal dominant, accounts for 25% with several subtypes. •

vWD: autosomal dominant disorders caused by mutations in vWF.

•

Clinical Features • •

•

Most cases are ofmild bleeding Common symptoms – Spontaneous bleeding from mucous membranes (e.g. epistaxis) – Excessive bleeding from wounds or menorrhagia In severe cases, similar to hemophilia A.

Laboratory Findings • • • • • • • •

Platelet count: normal Bleeding time: prolonged Clotting time: prolonged ourniquet test (Hess test): positivedue to defect in platelet adhesion AP: prolonged AP P: normal vWF assay: plasma level of active vWF isdecreased Platelet function test: defective ristocetin induced platelet aggregation test is diagnostic

vWD: increased bleeding time, clotting time and prolonged APTT. Plasma vWF is decreased. Defective ristocetin induced platelet aggregation test is diagnostic.

of vWF. Laboratory tests in hereditary disorders are summarized in able 17.4. TABLE 17.4: Summary of laboratory tests in hereditary coagulation disorders HemophiliaA Bleeding time

N

APTT Factor VIII Factor IX vWF

HemophiliaB N

Increased Decreased N

Increased N

Increased Low or Normal

Decreased N

vonWillebranddisease Increased

N

Hemophilia A, B and vWD: prothrombin time, thrombin time and platelet count are normal. APTT increased in all the three.

N Decreased

Abbreviation: N, normal

ACQUIRED COAGULATION DISORDERS Coagulation Factor Abnormalities

Vitamin K dependent coagulation factors: II, VII, IX and X.

Usually characterized by multiple clotting abnormalities Vitamin K deficiency: in neonates, low levels of vitamin K levels may produce life-threatening hemorrhage during the first week of life known as hemorrhagic disease of the newborn. Liver disease: liver synthesizes all the clotting factors and severe liver disease is associated with a hemorrhagic diathesis. Other causes: disseminated intravascular coagulation that involves deficiency of several coagulation factors. •

•

•

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Disorders of Hemostasis

DISSEMINATED INTRAVASCULAR COAGULATION Widespread disorder with combination of thrombosis and hemorrhage.

Etiology Develops as a secondary complication of wide variety of disorders (able 17.5). DIC: • Sepsis, major trauma, obstetric complications and certain cancers are the common triggers.

TABLE 17.5: Major disorders associated with disseminated intravascular coagulation Infections • •

Gram-negative bacterial sepsis Fungi, viruses, Rocky Mountain spotted fever, malaria

•

Meningococcemia and other bacteria

Obstetric Complications • • •

Retained dead fetus Abruptio placentae Toxemia and pre-eclampsia

• •

Septic abortion Amniotic fluid embolism

Neoplasms • •

Carcinomas of pancreas, prostate, lung and stomach Acute promyelocytic leukemia

Massive Tissue Injury • •

Traumatic Fat embolism

• •

Burns Surgery

Vascular Disorders •

Aortic aneurysm, giant hemangioma

Immunologic Reactions •

Transfusion reactions

•

Transplant rejection

Respiratory Distress Syndrome Miscellaneous Snakebite, liver disease, acute intravascular hemolysis, shock, heat stroke, hypersensitivity, vasculitis •

DIC: widespread thrombohemorrhagic disorder secondary to wide variety of disorders.

Pathogenesis (Fig. 17.2) Disseminated intravascular coagulation (DIC) is a disorder that showscombination of 1. thrombosis and 2. hemorrhage.

1. Thrombi/Clot Formation Mechanism of thrombi formation:

A. Initiation of thrombotic process: two major mechanisms initiate the thrombotic process of DIC namely entry of thromboplastic (procoagulant) substances into the circulation and widespreadendothelial injury. Entry of thromboplastic (procoagulant) substances into the circulation: source of thromboplastic/procoagulant substance in majority istissue factor, which activates. Widespread endothelial injury: endothelial injuries expose the thrombogenic subendothelial matrix. •

•

Bleeding Disorders: Due to Abnormalities of Coagulation/Clotting Factor CHAPTER 17

Fig. 17.2:Pathogenesis of thrombosis, ischemic tissue necrosis and bleeding in disseminated intravascular coagulation

B. Development of thrombi: DIC: Both procoagulant substances (tissue factor) and endothelial injury activate coagulation • Consumption of coagulation factors system resulting in fibrin-plateletthrombi formation in the microvasculature. • Widespread thrombosis During this process there is consumption of clotting factors, fibrin and platelets. Hence, in small blood vessels. it is also referred to asconsumptive coagulopathy or defibrination syndrome. •

•

C. Consequences of thrombi formation: widespread deposition of fibrin-thrombi within the microcirculation leads to: Ischemic necrosis: microvascular thrombi producesmicro-infarcts or large areas of infarction and multiorgan failure. •

•

Microangiopathic hemolytic anemia: RBCs trapped in the intravascular fibrinthrombi deposits undergo fragmentation. Tese RBCs appear as schistocytes in blood smears; but, frank hemolytic anemia is unusual in DIC.

2. Hemorrhagic Diathesis A. Causes of hemorrhagic/bleeding diathesis: Consumption of platelets Consumption of coagulation factors Activation of fibrinolytic system. • • •

B. Mechanism of hemorrhagic diathesis fibrin-thrombi activate secondary fibrinolytic system and generateplasmin. Te plasmin cleaves fibrinogen and fibrin and generates fibrin split products (FSPs) [or fibrin degradation products (FDP)]. FSPs are potent anticoagulant and antiplatelet effect and produces hemorrhagic diathesis. Prognosis • Depends on the underlying disorder.

Clinical Features Serious, often fatal, clinical condition • •

• Mortality is high in severe cases.

Signs and symptoms are related to: Treatment – Hemorrhagic diathesis/bleeding:most common, manifest as ecchymoses, petechiae or • Removal of the bleeding from mucous membranes or at the sites of venipuncture. underlying cause – Microvascular thrombi:tissue hypoxia and infarction of the organ leading to multiorgan • Replacement of clotting factors and platelets. failure.

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Disorders of Hemostasis

Laboratory Findings in DIC DIC laboratory findings: • Increased: APTT, PT, BT, D-dimer • Decreased: platelets, fibrinogen.

Screening Assays •

• • •

Coagulation abnormalities – AP: increased as a result of consumption and inhibition of the function of clotting factors. – Prothrombin time: increased. – Trombin time (): increased because of decreased fibrinogen. – Fibrinogen: decreased. Bleeding time: increased due to decreased platelet count. Platelet count: decreased due to utilization of platelets in microthrombi. Peripheral smear: microangiopathic hemolytic anemia with schistocytes.

Confirmatory Tests • •

DIC: D-dimer test is specific diagnostic test.

•

Fibrinolysis abnormalities FDP (fibrin degradation/split products): secondary fibrinolysis results in generation of FDPs, which can be measured by latex agglutination D-dimer test: it is specific for diagnosing DIC .

Thrombotic Disorders: Hypercoagulable State

18 CHAPTER

HYPERCOAGULABLE STATE (THROMBOPHILIA) Group of inherited or acquired conditions that are associated with increased tendency or risk to develop thrombosis.

Causes of Hypercoagulability State (Table 18.1) TABLE 18.1: Major causes of hypercoagulable state A. Inherited (genetic/primary) Deficiency of antithrombotic (anticoagulant) factors 1. Antithrombin III deficiency 2. Protein C deficiency 3. Protein S deficiency Increased prothrombotic factors 1. Activated protein C (APC) resistance (Factor V mutation/ factor Va/ factor V Leiden) 2. Excessive levels of prothrombin (prothrombin G20210A mutation) 3. High levels of factors VII, XI, IX, VIII; von Willebrand factor; fibrinogen B. Secondary (acquired) 1. Antiphospholipid antibody syndrome 2. Venous stasis ◆ Prolonged immobilization ◆ Congestive cardiac failure ◆ Prolonged bed rest 3. Increased platelet activation ◆ Hematological disorders: myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, myelofibrosis), paroxysmal nocturnal hemoglobinuria ◆ Cancer ◆ Nephrotic syndrome ◆ Atrial fibrillation ◆ Myocardial infarction ◆ Heparin-associated thrombocytopenia ◆ Thrombotic thrombocytopenic purpura (TTP) 4. Increased hepatic synthesis of coagulation factors and reduced anticoagulant synthesis ◆ Oral contraceptive pill ◆ Hyperestrogenic states (pregnancy and postpartum) 5. Release of procoagulant substances: disseminated cancers 6. Tissue injury: surgery, fracture, extensive burns 7. Reduced endothelial PGI2: old age 8. Endothelial injury: homocysteinemia 9. Fibrinolytic system defects 10. Unknown: smoking, obesity, sickle cell anemia

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Disorders of Hemostasis

INHERITED HYPERCOAGULABLE STATES Clinical Presentation • • • •

Trombosis develops at young age (less than 45 years) Recurrent thromboembolism Family history of thromboembolic episodes Trombosis develops in the venous system and atunusual anatomical sites like visceral veins.

Deficiency of Antithrombotic Factors Antithrombin (AT) III Deficiency • • •

Autosomal dominant disorder Deficiency of antithrombin—either quantitative or qualitative Risk of a thrombosis—20% to 80%.

Protein C and S Deficiency •

•

Normally, activated proteins C (APC) and protein S act as a complex, which degrades activated factors V and VIIl. When there is deficiency of these proteins, the activated factor V and VIII arenot neutralized. Tis leads to activation of the clotting system and formation of thrombus.

Increased Prothrombotic Factors Factor V Leiden/Leiden

Activated Protein C (APC) Resistance (Factor V Leiden)

mutation characterized by factor Visvariant.

• •

Factor V Leiden is resistant to inhibition by activated protein C (APC). It is associated with familial thrombophilia.

•

•

Most common genetic disorder associated with familial thrombophilia. Activated proteins C (APC) and protein S complex inhibits activated factor normal V and VIII. Te variant clotting factors cannot be degraded. Point mutation in the factor V gene synthesis of a factor V variant. Tis variant is known as factor V Leiden/Leiden mutation. Factor V variant has normal procoagulant activity but isresistant to inhibition by activated protein C (APC).

ACQUIRED HYPERCOAGULABLE STATES Causes Causes of the acquired hypercoagulable states are listed in able 18.1.

Antiphospholipid Antibody Syndrome (APLA/APS) •

•

•

Presence antiphospholipid antibodies (APAs) in the plasma are associated with hypercoagulable state. Antiphospholipid antibody reacts with plasma proteins, which are bound to phospholipids (Fig. 18.1). wo important antiphospholipid antibodies: lupus anticoagulant antibody and anti-β2 glycoprotein antibody. 1. Lupus anticoagulant antibody: prolongs the phospholipid-dependent coagulation tests in vitro (e.g. prolongation of AP).

Thrombotic Disorders: Hypercoagulable State CHAPTER 18

2. Antibodies against the phospholipid–β2-glycoprotein complex: it also bind to cardiolipin antigen used in the serological test for syphilis.

Antiphospholipid antibodies includes lupus anticoagulant antibody and anti-β2 glycoprotein antibody.

Types • •

Primary antiphospholipid syndrome: no predisposing cause. Secondary antiphospholipid syndrome: association with autoimmune diseases, like systemic lupus erythematosus, hence known as lupus anticoagulant syndrome.

Clinical Features •

•

•

Antiphospholipid antiFig. 18.1: body against plasma proteins

Hypercoagulable state: commonest acquired hematologic bound to phospholipids Triad of thrombosis, recurrent spontaneous cause of recurrent thromboembolic events. Repeated spontaneous abortions: normally, tissue plasminogen activator (t-PA) is neces- abortions and immune thrombocytopenia sary for the invasion of uterine blood vessels by placental trophoblastic tissue. Recurrentmay be the presenting spontaneous abortions develop due to antibody-mediated inhibition of t-PA activity. clinical features of antiphospholipid Immune thrombocytopenia. syndrome.

Laboratory Tests Coagulation tests AP: prolonged Factor VIII levels: normal Prothrombin time: normal Trombin time: normal • • • • •

Fibrinogen level: normal.

Confirmatory test est for lupus anticoagulant: – Dilute Russell’s viper venom test (DRVV): Russell’s viper venom (RVV) activates factor X leading to fibrin clot. Lupus anticoagulant prolongs clotting time by binding to RVV and preventing the action of RVV. Antibodies against the phospholipid–β2-glycoprotein complex: – Detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). •

•

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Clinical Scenario

4

19

Clinical Scenario

CHAPTER

INTRODUCTION Medical undergraduates in pathology are frequently required to analyze clinical-oriented cases. Tese cases are provided with symptoms and signs and laboratory findings. Students are expected to often diagnose these cases with history alone. Some of the classical scenarios in hematology, which are frequently asked in pathology examination, are presented in this chapter. First, symptoms, signs and general characteristics of blood diseases are given. Next common patterns of clinical features observed in hematology are discussed, which will help in suggesting the diagnosis. Tis is followed by clinical scenarios and their interpretations.

SYMPTOMS AN D S IGN S THAT SUGGEST A BLOOD DISEASE (TABLE 19.1) TABLE 19.1: Symptoms and signs that favor a blood disease Symptomsandsigns •

•

Tiredness, weakness, malaise, lightheadedness and easy fatigability Dyspnea (breathlessness) on mild exertion relieved by lying flat

•

Pallor

•

Tachycardia, palpitation and systolic murmur

•

Pica (craving for ice or soil)

•

Spoon-shaped nails (koilonychia)

•

During pregnancy anemia may develop due to combination of iron deficiency and/or folate deficiency.

Suggestiveofblooddisease

•

Cheilosis (fissures at the corners of the mouth) Dysphagia

•

Painful red “beefy” tongue (glossitis)

•

Pain or weakness in legs

•

Peripheral neuropathy

•

Psychosis

Anemia Pallor: appreciated better in the conjunctiva, mucous membrane of tongue and nail beds.

Iron deficiency anemia

Pica is a craving for certain substances with no nutritional value like clay or chalk.

Vitamin B12 deficiency

Vitamin B12 -peripheral neuropathy: glove and sock distribution of tingling and numbness or paresthesia in the fingers and toes. Contd...

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SECTION 4

Clinical Scenario

Contd... •

Passage of dark urine (? red cells, ? hemoglobin)

Hemolytic anemia—intravascular hemolysis

•

Jaundice/recurrentjaundice

Hemolytica nemia,m egaloblastica nemia(lemonyellow jaundice)

•

Enlargedspleen

Chronichemolyticanemias,malaria,leukemia, lymphoma

•

Massive splenomegaly causes abdominal

Splenic infarction producing severe abdominal pain

Sickle cell anemia

•

Leg ulcers

•

Frontal bossing—thalassemic facies

Chronic hemolytic anemia, e.g. thalassemia major

Massive splenomegaly (greater than 20 cm in size)

Chronic myelocytic leukemia, myelofibrosis, kala-azar

•

discomfort.

PATTERNS STRONGLY SUGGESTIVE OF A BLOOD DISEASE Various patterns that favor a blood disease are mentioned below.

Pattern 1: Iron Deficiency Anemia Craving for ice (pagophagia), is believed to be the most specific to iron deficiency. Cheilosis and are koilonychia (spoon shaped or flattened) are characteristically and specifically seen in advanced cases of IDA.

1. History strongly indicative of iron deficiency (IDA): a pale patient with tiredness, weakness, malaise, a sore tongue (glossitis), pica (craving for ice)and spoon-shaped nails (koilonychia). Signs and symptoms areusually due to both anemia and the underlying cause of anemia. 2. Diagnosis of IDA would be strengthened in a patient who has underlying cause of anemia such as gastrointestinal (e.g. carcinoma colon, hemorrhoids) or gynecologic disease (e.g. menorrhagia/excessive menstrual blood flow), malnutrition, pregnancy, and malabsorption. 3. IDA: diagnosis is typically based on laboratory results. Microcytic hypochromic anemia, low MCV, MCH, MCHC, reduced serum ferritinand other serum profile observed in IDA (refer able 1.4).

Inference (case No. 1): spoon shaped nails and pica indicates IDA.

Case No. 1

Inference (case No. 2): spoon shaped nails and cheilosis indicates IDA. Menorrhagia is the cause.

Case No. 2

Inference (case No. 3): cause of IDA is bleeding from GI tract.

History: A 28-year-pregnant lady comes to hospital with complains of weakness, easy fatigability and breathlessness of 4 months duration. She is a laborer of low economic status and used to eat clay. On examination, she is pale and had spoon shaped (koilonychia) nails.

History: A 20-year-old girl complains of weakness, easy fatigability and breathlessness of 6 months duration. She also complains of heavy menstrual bleeding every month. On examination, she is pale and had spoon shaped (flattened) brittle nails and cheilosis.

Case No. 3

History: A 73-year-old male complains of increasing weakness, malaise, and easy fatigability for the past 10 months. On examination, he is pale without hepatosplenomegaly. He also complains of black tarry stool (Stool for occult blood was positive).

Clinical Scenario

Laboratory findings for cases 1, 2, 3: Hb 9.1 g/dL, hematocrit 27.3%, RBC count 3 million/cumm, WBC count 6,700/cumm and platelet count 4,20,000/cumm. MCV 70 fL, MCH 26 pg, MCHC 28 g/dL and RDW 18.

Cause of IDA: the causes for iron deficiency: Case 1 is probably due toinadequate intake (due to low socioeconomic status) or increased demand of iron during pregnancy. Case 2 is caused bychronic blood loss, due to excessive menstrual flow. Case 3 is due to chronic blood loss through gastrointestinal tract. Te black tarry stool indicates that the patient has occult bleeding probably from GI tract malignancy.

CHAPTER 19

Laboratory findings for cases 1 to 3: The decreased hemoglobin, RBC count, MCV, MCH and MCHC favors iron deficiency anemia.

•

• •

Pattern 2: Megaloblastic Anemia •

•

•

Increased RDW helps in differentiating IDA from thalassemia (refer Table 3.3).

Unlike with B12 deficiency, folic acid deficiency is not

History strongly indicative of megaloblastic anemia (vitamin B12 deficiency and/or associated with significant pernicious anemia): middle-aged, pale patient with prematurely gray hair, severe glossitis neuropathy. and stomatitis and a smooth painful tongue, significant neuropathy and difficulty in walking, uncoordinated gait, impairment of vibration sense and position sense with delirium/dementia. Patients with megaloblastic anemia due to folic acid/folate deficiency present with symptoms of anemia or of the underlying cause. Peripheral smear with macro-ovalocytes, hypersegmented neutrophils, raised MCV, bone marrow with megaloblasts are features ofmegaloblastic anemia.

Case No. 4 History: A 34-year-male, pure vegetarian, executive complains of anorexia, premature graying of hair, weight loss and tingling and numbness in fingers and toes. On examination, he is pale and anemic. The tongue is shiny and has glazed appearance (glossitis).

Inference (case No. 4): in a pure vegetarian, tingling and numbness, glossitis, raised MCV point towards vitamin B12 deficiency.

Laboratory findings: Hb 9 g/dL, WBC count 3,800/cumm, platelet count 60,000/cumm, RBC count 2.5 million/cumm. MCV is 104 fL , MCH 32 pg and MCHC is 32 g/dL.

Case No. 5 History: A 45-year-old female complains of tiredness, mild breathlessness while climbing steps and tingling and numbness. On examination, she is pale, has raspberry-red tongue, and shows numbness and paresthesia. Biochemical investigation reveals normal serum iron profile.

Laboratory findings: Hb 8.2 g/dL, WBC count 4,800/cumm, platelet count 1,20,000/cumm, MCV is 108 fL , MCH 33 pg and MCHC is 32 g/dL. Peripheral smear and bone marrow examination confirms the clinical diagnosis. Patient is further subjected to gastric biopsy and shows atrophic gastritis. Other test including serological tests is done.

Case No. 6 History: A 50-year-old male complains of loss of appetite, increasing fatigue tingling and numbness of bothtongue lower limbs and red. difficulty in walking for the past 10 months. On physical examination, he is pale and was beefy

Laboratory findings: Hb 9.2 g/dL, hematocrit 27.9%, MCV 132 fL, platelet count 242,000/cumm, and WBC count 7590/cumm. Peripheral smear shows macro-ovalocytes and hypersegmented neutrophils.

Inference (case No. 5): raised MCV, history of tingling and numbness, raspberry tongue point to megaloblastic anemia due to vitamin B12 deficiency. The evidence of atrophic gastritis points to pernicious anemia.

Inference (case No. 6): characteristic history with increased MCV, macro-ovalocytes and hypersegmented neutrophils in the peripheral smear points to megaloblastic anemia.

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Clinical Scenario

HS may present during anytime from the neonatal period to adulthood. When there is a family history, it is usually easy to suspect the diagnosis.

Pattern 3: Hereditary Spherocytosis •

•

History strongly indicative of chronic extravascular hemolysis, usually but not always hereditary spherocytosis: normal patient presenting withpallor, mild jaundice, anenlarged spleen (classic triad) and a family history of a relative who had been ‘‘cured’’ of the same problem by splenectomy. Also family history ofpigment gallstonesin a young person. Peripheral smear withspherocytes and raised osmotic fragility test are features of HS.

Case No. 7 Inference (cases No. 7 and 8): mild jaundice, anemia and splenomegaly constitute a classical triad, and along with family history/pigment gallstones favor hereditary spherocytosis.

History: A 24-year-women complains of chronic fatigue since childhood and mild icterus. On examination she is anemic, jaundiced and has moderate splenomegaly. Her mother had similar complaints and has

Positive osmotic test fragility confirms the diagnosis of HS.

Laboratory findings for cases 7 and 8:Hb 11.1 g/dL, RBC count 3.6 million/cumm. WBC count 6,800/cumm and platelet count 1,75,000/cumm. MCV is 78 fL, MCH is 32 pg and MCHC is 38 g/dL. In a laboratory test, her RBCs lyse at a higher concentration of saline (osmotic fragility test) compared to normal patients.

multiple pigment gallstones.

Case No. 8 History: A 15-year-old girl presents with weakness and fatigue. On examination, her sclera shows mild jaundice and mild splenomegaly. Family history indicated about her father h aving recurrent gallstones.

Pattern 4: Thalassemia Major •

•

History strongly indicative of thalassemia major: short stature, failure to thrive, severe pallor, jaundice,maxillary over-growth (bossing), frontal bossing, sallow complexion and splenomegaly. Laboratory finding of microcytic hypochromic anemiawith many target cells, reduced MCV,MCH and MCHC, increased serum iron normal RDWare features of thalassemia major.

Inference (case No. 9): failure to thrive, severe pallor, jaundice, splenomegaly, microcytic hypochromic anemia with target cells, reduced MCV and normal RDW points to the diagnosis of β-thalassemia major. Increased iron stores and raised serum ferritin is against the diagnosis of IDA (refer Table 3.3).

Case No. 9 History: An 11-month-old male child was brought to the pediatric outpatient by his parents and complained that the child was failing to thrive. On examination, jaundice, pallor and a palpable spleen was detected. Laboratory findings: Hb 7.8 g/dL, hematocrit 23.4%, MCV 66 fL, platelet count 1,75,000/cumm, and WBC count 8,200/cumm. His serum ferritin was 3250 ng/mL. Peripheral examination showed microcytic hypochromic anemia with many target cells. A bone marrow aspiration performed and reveals a myeloid: erythroid ratio of 1:4, and increased iron stores.

Pattern 5: Sickle Cell Anemia •

Inference (case No. 10): the recurrent episodes of pain abdomen and backache points to a vaso-occlusive crisis in sickle cell anemia.

History strongly indicative of sickle cell anemia: recurrent episodes of vaso-occlusion in connective and musculoskeletal structures anywhere in the body producing ischemia and manifesting as acute pain, tenderness and fever. Child may show generalized impairment of growth and development.Adults manifest with chronic organ damage.

Clinical Scenario

•

CHAPTER 19

Presence of sickle cells in the peripheral smear, positive sickling test, along with demonstration of HbS byhemoglobin electrophoresis and HPLC is diagnostic of sickle cell anemia.

Case No. 10 History: A 10-year-old boy complains of severe pain in the chest, abdomen and bones. On enquiry, his mother reveals that he had several episodes of severe abdominal and back pain since early childhood. Physical examination shows anemia, jaundice and leg ulcer.

Presence of sickle cells in the peripheral smear and presence of more than 70% Hb S on hemoglobin electrophoresis is confirmative.

Laboratory findings: Hb 11.0 g/dL, RBC count 3.2 million/cumm, WBC count 8,800/cumm and platelet count 1,95,000/cumm. Peripheral smear examination, hemoglobin electrophoresis and HPLC confirms the diagnosis.

Pattern 6: Immune Thrombocytopenic Purpura (ITP) •

•

The severity/nature of bleeding depends on the

History strongly indicative of acute immune thrombocytopenic purpura: sudden platelet count. development of bruising, petechiae and muco-cutaneous bleeding in a child following a bacterial or nonspecific viral (upper respiratory or gastrointestinal)infection without features of acute leukemia (such as anemia and bone tenderness). Laboratory findings of markedly reduced platelet count and megakaryocytic hyperplasia with immature megakaryocytes. In the bone marrow favors the diagnosis ofacute immune thrombocytopenic purpura.

Case No. 11 History: A 14-year-male comes to the OPD with petechial rashes of 2 days duration. He has no history of bleeding in past. He had an attack of viral infection 3 weeks before this complaint.

Inference (case No. 11): petechial rashes of short duration in a child, low platelet count is due to acute idiopathic thrombocytopenic purpura.

Laboratory findings: Hb 14.5 g/dL, hematocrit 45%, RBC count 5.1and million/cumm. WBCand count cumm and platelet count 75,000/cumm . MCV is 85 fL, MCH is 32 pg MCHC is 31 g/dL RDW7,200/ is 14.

Pattern 7: Hemophilia •

•

History strongly indicative of one of the hemophilias (A or B): a male child presenting with a serious bleeding disorder, particularly withhemarthrosis. Decreased factor VIII/IX and increased APTT is diagnostic of one of the hemophilias (A or B).

Case No. 12 History: An 18-year-male complains of knee joint swelling after minor trauma. On examination, the joint appears tense, red and swollen. Family history revealed similar complaints by his uncle.

Case No. 13 History: A 6-year-old boy gives a history of easy bruising and episodes of passing blood in urine since infancy. On examination, many ecchymoses are noted in the skin of lower limbs. Family history reveals similar complaints in members of the family involving only males and history of hemarthrosis in few of them.

Inference (cases No. 12 and 13): the development of ecchymosis, family history with disease affecting males, hemarthrosis, normal platelet count and prolonged APTT favor hemophilia A/B.

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Laboratory findings for cases 11 and 12: Hb 13 g/dL, hematocrit 36%, RBC count 4.4 million/cumm, WBC count 8,200/cumm and platelet count of 2,35,000/cumm. MCV is 84 fL, MCH 32 g and MCHC is 33 g/dL. Bleeding time, clotting time and prothrombin time are within normal limits. APTT is prolonged.

ALL: pallor, fatigue, bleeding, fever, and infection are due to peripheral blood cytopenias resulting from bone marrow failure.

Pattern 8: Acute Lymphoblastic Leukemia (ALL) •

•

History strongly indicativeacute lymphoblastic leukemia: child (1 to 6 years of age)/adult (30 to 40 years) presenting withrapid onset (few weeks) of pallor, fatigue, bleeding, fever, and infection. Presence of lymphadenopathy, hepato- or splenomegaly, CNS disease, testicular enlargement. Laboratory findings of marked raised WBC count, presence of more than 20% lymphoblasts in the blood and bone marrow, blasts + with PAS (block positivity) and d + are features of acute lymphoblastic leukemia.

Inference (cases No. 14 to 16): presence of anemia, thrombocytopenia and leukocytosis with presence of PAS positive blasts in the peripheral smear and bone marrow in a child is in favor of acute lymphoblastic leukemia.

Case No. 14

Bone pain is due to expansion of the marrow caused by proliferation of blasts.

Case No. 16

History: A 6-year-old boy presents with fatigue, bone pain, weakness and low-grade fever of 1-week duration. Physical examination shows anemia, sternal bone tenderness and lymphadenopathy.

Case No. 15 History: A 5-year-old boy complains of sudden onset of fever, tiredness and pallor. On examination, there are many enlarged cervical lymph nodes, hepatosplenomegaly, bone tenderness, and petechial hemorrhages on the skin.

History: A 4-year-old boy is becoming increasingly lethargic for the past 1 month. On examination, he is having fever, ecchymoses on the sk in of his lower legs and shoulder, generalized lymphadenopathy and tenderness on palpation of long bones. Laboratory findings of cases 14 to 16: Hb 8 g/dL, hematocrit 24%, WBC 18,200 cells/cumm and platelet count of 90,000/cumm. Serum LDH is markedly increased. Peripheral blood smear shows abnormal cells, which are PAS positive. A bone marrow examination shows 100% cellularity with replacement by primitive cells. These abnormal primitive cells have scanty cytoplasm and large nuclei with indistinct nucleoli. Following blood smear examination, the child is admitted to the medical oncology ward.

Pattern 9: Acute Myeloblastic Leukemia (AML) •

•

History strongly indicative acute myeloblastic leukemia: adult presenting with rapid onset (few weeks) of pallor, fatigue, bleeding, fever, and infection. Laboratory findings of marked raised WBC count, presence ofmore than 20% myeloblasts in the blood and bone marrow, presence of Auer rods (not seen in all subtypes), blasts + with MPO and Sudan Black B are features ofacute myeloblastic leukemia.

Clinical Scenario

Case No. 17 History: A 45-year-old man presents with fatigue, episodes of epistaxis, bleeding from gums, and low grade fever of 1 week duration. Physical examination reveals temperature of 37.4°C and hepatosplenomegaly.

CHAPTER 19

Inference (case No. 17): the high WBC count with the blasts and Auer rods are very characteristic for acute myelogenous leukemia.

Laboratory findings: Hb 7 g/dL, WBC count 51,000/cumm and platelet count 80,000/cumm. Peripheral blood smear shows abnormal large cells with Auer rods.

Pattern 10: Chronic Myelogenous Leukemia (CML) •

•

History strongly indicative of chronic myelogenous leukemia: patient in fifth and sixth decades of life presenting with gradual development offatigue, weakness, weight loss, anorexia and fullness of abdomen, early satiety and left upper quadrant pain or mass (due to splenomegaly). Laboratory finding of very high WBC count with myeloid precursors, neutrophils and myelocyte peak, low LAP and presence of Ph chromosome is diagnostic of chronic myelogenous leukemia.

Case No. 18 History: A 50-year-old male comes to the hospital with complaints of generalized weakness, weight loss, easy fatigability, abdominal discomfort and dragging sensation in the left hypochondrium for the last 8 months. On examination, he is pale and has marked splenomegaly. Laboratory findings: Hb 11.9 g/dL, hematocrit 36%, WBC count 1,20,000/cumm, platelet count 4,12,000/ cumm. Peripheral smear examination shows characteristic blood picture with myeloid precursors, neutrophils and myelocyte peak confirms the clinical diagnosis. The leukocyte alkaline phosphatase (LAP) score is low . Chromosomal analysis shows t(9:22) positivity. He is admitted to the oncology

Inference (case No. 18): massive splenomegaly with very high WBC count, peripheral smear with myeloid precursors, neutrophils and myelocyte peak t (9:22) positivity and low LAP score are features of chronic myelogenous leukemia (chronic phase).

department for treatment.

Pattern 11: Chronic Lymphocytic Leukemia (CLL) •

•

History strongly indicative of chronic lymphocytic leukemia: marked splenomegaly, generalized lymphadenopathy (any nodes can be involved), involvement ofspleen in an elderly patient with fatigue, loss ofweight and anorexia. Peripheral blood withlymphocytosis which constitute more than 50% of the white cells and smudge cells; bone marrow with more than 30% lymphocytes of the bone marrow cells is diagnostic of chronic lymphocytic leukemia.

Case No. 19 History: A 60-year-old male complains of increasing fatigue and shortness of breath with minimal exercise for the last 6 months. He has noted some abdominal discomfort over the past month. On examination, he has non-tender cervical lymphadenopathy. The liver is enlarged, smooth and palpable just below right costal margin. The spleen is palpated 3 cm below left costal margin.

Inference (case No. 19): older patient, abdominal discomfort (splenomegaly) nontender lymphadenopathy, hepatosplenomegaly, high WBC count with lymphocytosis and smudge cells in the peripheral smear are features of chronic lymphocytic leukemia.

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Laboratory findings: WBC count 22,000/ cumm with 78% lymphocytes on DLC. Hb 10.1 g/dL, hematocrit 33%, platelet count 2,32,000/cumm. Peripheral smear shows many smudge cells.

Pattern 12: Multiple Myeloma •

•

Inference (case No. 20): patient over 50 years of age with bone pain, pallor, lytic lesions in bone, peripheral smear with marked rouleaux formation and markedly raised ESR suggest multiple myeloma.

History strongly indicative of multiple myeloma: Patient over 50 years of age presenting with severe bone pain, pallor, and renal failure. Laboratory findings shows nonspecific findings such as raised ESR, rouleaux formation of RBC in the peripheral smear andlytic lesions in the bone. Bone marrow with more than 30% myeloma cells and M spike on electrophoresisconfirms the diagnosis of multiple myeloma.

Case No. 20 History: A 61-year-old man has dull, constant back pain for 3 months. On physical examination, he is pale. A plain film radiograph of the spine and s kull reveals several 1 to 2 cm lytic lesions of the vertebral bodies. Laboratory findings: Peripheral smear shows marked rouleaux formation, ESR is 110 mm at 1st hour. Bone marrow aspiration, urine and serum electrophoresis showed characteristic findings.

Pattern 13: Hodgkin Lymphoma (HL) •

•

Inference (case No. 21): non-tender lympadenoapthy, lowgrade fever, weight loss, night sweats and presence of characteristic Reed-Sternberg cells is diagnostic of Hodgkin lymphoma.

History strongly indicative of Hodgkin lymphoma: weight loss, fever, night sweats, lassitude, pruritis, and enlarged cervical nodes. Sometimes accompanied by pain in one or more sites almost immediately after ingestion of alcohol. Extranodal involvement is very rare. Histologically/cytologicallypresence of RS cells is diagnostic of Hodgkin lymphoma.

Case No. 21 History: A 33-year-old female complains of low-grade fevers, 6 kg weight loss, night sweats, and generalized malaise for the past 3 months. On physical examination, she has non-tender right cervical and supraclavicular lymphadenopathy. Investigation: Fine needle aspiration followed by a biopsy of cervical lymph node is performed. On microscopic examination, the lymph node showed loss of architecture and characteristic cell that confirmed the clinical diagnosis. He was admitted to the oncology ward for further management.

Bibliography 1. Beck N. Diagnostic Hematology. London,Springer-Verlag.2009. 2. Colledge NR, Walker BR, Ralston SH. Davidson’s Principles and Practice of Medicine, 21st edn. Edinburgh, Churchill Livingstone. 2010. 3. Colman RW, Marder VJ, Clowes AW, George JN, Goldhaber SZ. Hemostasis and Trombosis: Basic Principles and Clinical Practice, 5th edn. Philadelphia, Lippincott, Williams and Wilkins. 2006. 4. Goldman L, Ausiello D. Cecil Medicine, 23rd edn. Philadelphia, WB Saunders. 2007. 5. Goljan EF. Rapid Review ofPathology, 3rd edn. Philadelphia, WBSaunders. 2008. 6. Hoffbrand AV, Catovsky D, uddenham EGD. Postgraduate Hematology, 5th edn. Massachuset ts, Blackwell Publishing. 2005. 7. Hoffman R, Benz EJ, Shattil SJ, Furie B, Silberstein LE, McGlave P, Heslop HE. Hoffman’s Hematology: Basic Principles and Practice, 5th ed. Edinburgh, Churchill Livingstone. 2008. 8. Hsi ED. Hematopathology, 2nd edn. Philadelphia, WB Saunders. 2012. 9. Knowles DM. Neoplastic Hematopathlogy, 2nd edn. Philadelphia, Lippincott Williams and Wilkins. 2001. 10. Kumar V, Abbas AK, Fausto N, Aster JC. Robbins and CotranPathologic Basis of Disease, 8th edn. Philadelphia, WB Saunders. 2009. 11. Kumar V, Abbas AK, Aster JC. Robbins Basic Pathology, 9thedn. Philadelphia, WBSaunders. 2013. 12. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology, 10th edn. London, Churchill Livingstone. 2006. 13. Lichtman MA, Kipps J, Kaushansky K, Beutler E, Seligsohn U,Prchal J. Williams Hematology, 7th edn. USA, McGraw-Hill Companies. 2006. 14. Longo DL. Harrison’s Hematology and Oncology. USA, McGraw-Hill Companies. 2010. 15. Longo DL, Kasper DL, Jameson JL, Fauci AS, Hauser SL, Loscalzo J. Harrison’s Principles of Internal Medicine, 18th edn. USA, McGraw-Hill Companies. 2012. 16. McPherson RA, Pincus MR. Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd edn. Philadelphia, WB Saunders. 2011. 17. Rodak BF, Fritsma GA, Doig K. Hematology: Clinical Principles and Applications, 3rd edn. Philadelphia, WB Saunders. 2007. 18. Rubin R, Strayer DS. Rubin’s Pathology: Clinicopathologic Foundation of Medicine, 6th edn. Philadelphia, Lippincott Williams and Wilkins. 2012. 19. Stevens A, Lowe J. Pathology, 2nd edn.Edinburgh, Mosby. 2000. 20. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al., WHO Classification of umours of Haematopoietic and Lymphoid issues. IARC, Lyon. 2008. 21. Underwood JCE, Cross SS. General and Systemic Pathology, 5th edn. Edinburgh, Churchill, Livingstone. 2009.

Appendix

Different of unit, hematopoiesis. Abbreviation: BFU, bursastages forming CFU, colony forming unit

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Stages of erythropoiesis

Variations in color of RBCs and associated conditions

Appendix

Variations in shape of RBCs and associated conditions

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Inclusions in RBCs and associated conditions

Rouleaux formation and associated conditions

Appendix

Variations in size of RBCs and associated conditions

Stages of megakaryopoiesis

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Hemoparasites 1. Malarial parasites: Most common are Plasmodium vivax and falciparum 2. Microfilaria 3. Trypanosomes 4. Leishmania donovani 5. Babesiosis.

Stages of myelopoiesis

HEMOPARASITES

ABCD

Peripheral blood smear showing microfilaria

Peripheral blood smear A. Plasmodium vivax ring form, B and C Plasmodium vivax schizont and D. Plasmodium falciparum gametocyte

Index Page numbers followed byf refer to figure andt refer to table

A

Abdominal discomfort 125 Abnormal localization of immature precursors 61 Abnormalities of coagulation/clotting factor 105 globin production 22 platelet 100 Abnormally large megakaryocytes 66 ABO hemolytic disease of newborn 39 incompatibility 37 Achlorhydria 12 Acquired clonal stem cell disorders 60 coagulation disorders 105, 109 disorders 52, 100 hypercoagulable states 114 mutations 41 myeloproliferative neoplasm 63 Activated partial thromboplastintime 107 protein C resistance 114 Acute abdominal pain 32 bacterial and fungal infections 46 blood loss 41 chest syndrome 32 erythroid leukemia 54 immune thrombocytopenic purpura 102, 123 leukemia 52, 52 t, 53, 55, 72 lymphoblastic leukemia 49, 52, 53, 55, 56f, 124 lymphoma 55 lymphoid leukemias 55 t megakaryoblastic leukemia monoblastic leukemia 54 54 monocytic leukemia 49, 54 myeloblastic leukemia 52, 53, 124 myelocytic leukemia 49, 53 myelogenous leukemia 53, 57 myeloid leukemia 53, 54 myelomonocytic leukemia 48, 49, 54

promyelocytic leukemia 54 Adult  cell leukemia 49, 81 lymphoma 49, 81 Agranulocytosis 47 Aleukemic leukemia 52 Alimentary system 12 Allergic rhinitis 48 Alloimmune hemolytic anemia 36 Amyloidosis 79, 80 Anaplastic large cell lymphoma 81 Anemia 3, 19, 36, 65f of blood loss 41 of chronic disorders 8 of impaired red cell production 3 Angioimmunoblastic  cell lymphoma 81 Angular stomatitis and glossitis 7 Anisopoikilocytosis 33

Autosomal dominant disorder 17, 108 trait 19 recessive hereditary disorder 22 Autosplenectomy 33

Antiglobulin test 38, 39 Anti-intrinsic factor antibody 12 Antiphospholipid antibody syndrome 114 Antiplatelet autoantibodies 102 Antithrombin III deficiency 114 Aplastic anemia 13, 15, 46, 48, 50 crises 20, 33 Appearance of neoplastic lymphoidcells 84 typical thalassemic facies 25 f Arterial oxygen saturation 65 Asthma 48 Asynchrony of nuclear and cytoplasmic maturation 10 Atherosclerosis 13 Atrophic gastritis 121 glossitis 7, 12 Atrophy of glands 12 Atypical lymphocytosis 51 Autoimmune diseases 49 disorder 102 hemolytic anemia 36, 39, 40

Bleeding disorders 99, 100, 105 tendency 80 time 103, 107 Blockage of microcirculation 32 Blood brain barrier 38 loss 4 Bone 79 marrow 7, 10, 15, 18, 25, 27, 34, 35, 52, 57, 65, 66, 67, 77, 95, 103 aspiration 75 biopsy 65, 67 failure 56, 58 iron 27 trephine biopsy 61 pain 56 and tenderness 56 Brucellosis 49 Budd-Chiari syndrome 63 Burkitt lymphoma 81, 83, 84f, 85f

B B cell neoplasms 76 prolymphocytic leukemia 81 Basophilia 49 Bence Jones proteins 76, 78 Benign leukocytic proliferation 47 Bernard-Soulier syndrome 100, 104 Biochemical tests for megaloblastic anemia 11 Black tarry stool 120

C Carcinoma 15 Causes of aplastic anemia 14t

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eosinophilia 48 t hemolytic anemia 16 t hemorrhagic/bleeding diathesis 111 hypercoagulability state 113 iron deficiency anemia 5t overload 24 leukocytosis 45 t leukopenia 46 t lymphocytopenia 50 t lymphocytosis 49 t megaloblastic anemia 8 t microcytic hypochromic anemia 8 monocytosis 49 t neutropenia 48 t neutrophilia 46 pancytopenia 15 t thrombocytopenia 101, 101 t thrombocytosis 104 t Central nervous system 7, 12, 38 Cervical lymphnodes 56 Chickenpox 49 Christmas disease 108 Chronic antigenic stimulation 77 atrophic gastritis 7, 12 blood loss 5, 42, 121 eosinophilic leukemia 62 hemolytic anemia 32 hypoxia 32 immune thrombocytopenic purpura 102 infections 49 leukemia 53 lymphocytic leukemia 45, 48, 49, 53, 73, 74f, 81, 125 myelogenous leukemia 62, 68, 125 myeloid leukemia 45, 47 t, 48, 53, 70, 72f, 104 myelomonocytic leukemia 49 myeloproliferative neoplasm 65 neutrophilic leukemia 62 organ damage 33 Classical Hodgkin lymphoma 81, 87, 88 Classification of acute myelogenous leukemia 57 anemia 3, 4 t bleeding disorders 100, 100 t coagulation disorders 105, 105 t disorders of hemostasis 100 t hemolytic anemias 16 hemostatic disorders 99 hereditary defects in hemoglobin 22 immunohemolytic anemias 36, 36 t

lymphoid neoplasms 81 plasma cell neoplasms 76, 76 t platelet disorders 100, 101t functional disorders 104 t sickle cell disease 29, 29 t Clonal hematopoietic stem celldisorders 62 proliferative disorder 95 Combined vitamin B12 9 Common hereditary X-linked recessive disease 106 Complement-mediated intravascular hemolysis 41 Confirmatory test 112, 115 Congestive heartfailure 7, 50 Consequences of ineffective erythropoiesis 23 thrombi formation 111 Coombs test 38, 38f, 39 Crohn disease 49 Cutaneous  cell lymphoma 86 Cyclic neutropenia 48 Cytochemistry of lymphoblasts 57 myeloblasts 58

D Defective DNA synthesis synthesis 54 heme hemoglobin synthesis 4 Deficiency of antithrombotic factors 113, 114 intrinsic factor 11 Degeneration of dorsal and lateral tracts 13 Degree of hemoglobinization 3 Dehydration 31 Delayed maturationof nucleus 8 Demonstration of heterophile antibodies 51 Demyelination of spinalcord 13 Deoxyuridine suppression test 11 Dermatitis 48 herpetiformis 48 Development ofthrombi 111 Diffuse chronic atrophic gastritis 12 large B cell lymphoma 81, 83 Dilute Russell’sviper venom test 115 Dimorphic anemia 9 peripheral blood picture 42

Direct antiglobulin test 39, 40 Disorders of neutrophils 46 platelet adhesion 104 aggregation 104 secretion 104 primary hemostasis 99, 100 white cells 43 Disseminated intravascular coagulation 110, 110t, 111f Distinct megaloblasts inbone marrow 8 Donath-Landsteiner antibodies 40 Down syndrome 54 Dry tap 75 Dwarf megakaryocytes 70 Dysmegakaryopoiesis 61 Dysplasia 60

E Eczema 48 Ehlers-Danlos syndrome 100 Endemic Burkittlymphoma 84 Endothelial injury 110 Enlarged cervical lymphnodes 124 Eosinophilia 47 Eosinophilic granuloma 48, 96 leukemia 48 Epstein-Barr virus 51 Erythrocytosis 63 Erythroleukemia 53 Erythromelalgia 66 Erythrophagocytosis 21 Erythropoiesis 18, 66, 77 Esophageal webs 7 Extramedullary hematopoiesis 24, 65-67 hemopoiesis 24 infiltration 56, 58 myeloid tumors 47 Extravascular hemolysis 23

F Fab classification of acute leukemias53 Fetal hemoglobin 26, 27 Fever 51 Fibrinolysis abnormalities 112 Fibrotic stage 67 Filariasis 48 Fine needle aspirationcytology 93 Fluorescent insitu hybridization 70 Folate deficiency 48

Index

Folic acid 8 and iron deficiency 9 deficiency 8, 11, 15 Follicular lymphoma 81, 82, 82f Fragmentation syndrome 41 Functional disordersof platelet 104f Fungal infections 48

G Gallstones 20 Gaucher-like cells 70 Giant megakaryocytes 66 metamyelocytes 10 Glandular fever 51 Glanzmann thrombasthenia 100, 104 Glucose-6-phosphate dehydrogenase deficiency 20 Gold standardtest 7 Granulomatous diseases 15 disorders 48 Gum bleeding 102

H Haemophilus influenzae 33 Hairy cell 75 leukemia 49, 75, 81 Hand-Schüller-Christian disease 96 Hay fever 48 Heavy chain disease 81 menstrual bleeding 120 Heinz bodies 21, 21f Hematologic malignancies 49, 104 Hematopoietic stemcell 63 Hemoglobin 6, 9, 21, 25, 58 concentration 3 electrophoresis 26, 27, 34 Hemolytic anemia 4, 16, 23 Hemolytic crisis 33 disease of newborn 36, 39 Hemophagocytic syndrome 15 Hemophilia 106 A 106

B 108 Hemorrhage 41 Hemorrhagic bleeding 111 diathesis 99, 111 disorders 99 Henoch-Schönlein purpura 100

Hereditary coagulation disorders 105, 106, 109t disorders 29 hemolytic anemia 23 spherocytosis 17, 122 High-performance liquid chromatography 35 Hodgkin cells 87, 90, 91 lymphoma 48-50,81, 87, 88 t, 90f, 93, 93f, 94, 94t, 126 Hookworm infestation 48 Humoral immune deficiency 80 Hydrops fetalis 38 Hypercoagulable state 113, 115 Hypereosinophilic syndrome 48 Hypersegmented neutrophil 9f Hypocellular bonemarrow 13 Hypochromia 6 Hypofunction ofspleen 33 Hypovolemic shock 33, 41

I Immune thrombocytopenia 115 thrombocytopenic purpura 102 Immunoglobulin deposition disease 76, 80 Immunohemolytic anemias 36 Impaired absorption 5, 8 DNA synthesis 8 red cell production 4 Inactivates nitricoxide 31 Inactivation of nitric oxide 31 Incubation period 51 Indirect antiglobulin test 39, 40 Infectious hepatitis 49 mononucleosis 45, 49 Inflammatory bowel disease 49 diseases 49 Inherited hypercoagulablestates 114 Intestinal metaplasia 12 Intravascular hemolysis 41 Iron deficiency 104, 120 anemia 5, 6, 8, 27, 28 t, 42, 120 deficient erythropoiesis 6 depletion 6 overload 24 Irreversibly sickled cells 31 Ischemic necrosis 111

J Jaundice 20

K Koilonychia 7 nails 120 Kostmann syndrome 48

L Lacunar cell. 89 Langerhans cell 95 histiocytosis 95 LE cell test 40 Letterer-Siwe disease 96 Leukemia 15, 55 Leukemic meningitis 56 Leukemoid reaction 47 Leukocyte alkaline phosphatase 47 common antigen 82 Leukocytosis 45, 66 Leukoerythroblastosis 67 Leukopenia 46 Life-threatening bleeding 106 Location of hemolysis 16, 17 Löeffler’s syndrome 48 Loss of architecture 84 intravascular volume 41 lymph node architecture 89 membrane fragments in circulation 17 Lupus anticoagulant antibody 114 Lymph nodes 74, 82, 86, 87 Lymphoblast 54 Lymphocyte depleted classical Hodgkin lymphoma 81, 87, 91 predominant cells 92 rich classical Hodgkin lymphoma 81, 87, 90 Lymphocytopenia 50 Lymphocytosis 49 Lymphoid neoplasms 81 Lymphoma 15, 55 Lymphoplasmacytic lymphoma 81

M Macroangiopathic hemolyticanemia 41 Malignant disease of bone marrowstem cell 52 lymphoid cells 82f neoplasms 87

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Mantle cell lymphoma 81 Marrow aplasia 15 infiltration 4, 48 Massive splenomegaly 75 Mature B cell neoplasms 81  and NK cell neoplasms 81  cell and NK cell neoplasms 85 Mean corpuscular hemoglobin 5

Mononuclear cells 51 Morphological classification ofanemia 3t Morphology of myeloblasts 58 neoplastic cells 88 Mucosal bleeding 102 Multiple myeloma 49, 76, 78f, 125 tumor masses 76 Mumps 49 Mutated tyrosinekinase activation 58 Mycosis fungoides 81, 86

O

concentration 5, 31 volume 4 Measles 49 Mediastinal lymph nodes 88 thymic mass 56 Medium-sized cells 84 Megakaryocytic hyperplasia 123 leukemia 53 Megakaryopoiesis 18, 66, 77 Megaloblastic anemia 8, 9, 11, 13, 15, 46, 48, 121 precursors 11 f Menorrhagia 102 Metastatic tumors 48 Microangiopathic hemolyticanemia 41, 111

Myelodysplastic syndromes 15, 48, 54, 60, 104 Myelofibrosis 15, 64 Myeloid hyperplasia 70 proliferation 54 sarcoma 54, 59 Myeloma 15 kidney 79 plasma cells 77 Myelomonocytic leukemia 53 Myelophthisic anemia 4 Myelopoiesis 18, 66, 77 Myeloproliferative neoplasms 62, 68, 104

Microcytic hypochromic anemia 7, 25, 120 red blood cells 6 f Micronormoblastic maturation 7 Microscopically myeloblasts 59 Microvascular thrombi 111 Mild erythroid hyperplasia 27 Miliary tuberculosis 50 Missense pointmutation 30 Mixed cellularity classical Hodgkin lymphoma 81, 87, 90 Mode of inheritance 106 onset 16 transmission 51 Monoclonal gammopathy of uncertain significance 80

follicles 82  cells 86 Nephrotic syndrome 79 Neutropenia 47 Neutrophilia 46 Nodular lymphocyte predominant Hodgkin lymphoma 87, 92 sclerosis classical Hodgkin lymphoma 81, 87, 88 Non-Hodgkin lymphoma 48, 49, 94, 94t Non-neoplastic cells 87, 88 Non-steroidal anti-inflammatory drugs 104 Non-tender cervical lymphadenopathy 125 Nonthrombocytopenic purpura 99 Normochromic normocyticanemia 67 Normocytic normochromic anemia

Pathogenesis of iron deficiency anemia 6 megaloblastic change 8 Rh hemolytic disease of newborn 37 f sickle cell anemia 30 f thrombosis 111 f Pathognomonic Birbeck granules 95 of Hodgkin lymphoma 88 Pathophysiologic classification of polycythemia 63t Patterson-Kelly syndrome 7 Paul Bunnell test 51 Pemphigus 48 Periodic acid Schiff stain 54f Peripheral blood smear 6f, 9f, 56 lymph nodes 90  cell lymphoma 81, 85 Pernicious anemia 11, 12 Persistent thrombocytopenia 71 Pharyngitis 51 Philadelphia chromosome 47, 68, 69f, 70 Pigment gallstones 122 Plasma cell 89 myeloma 76 neoplasm 76, 81 lactate dehydrogenase 11 Plasmacytoma 76, 80 Plasmodium falciparum gametocyte 132 Platelet count 64, 107 function disorders 100 Plummer-Vinson syndrome 7

undetermined significance 76 spikes 78 Monocytic leukemia 53 Monocytosis 49 Monomorphic lymphoidcells 83

38, 42, 70 Nucleated redcell precursors 25 Nucleus of macrophage 83 Numbness of both lower limbs 121 Nutritional deficiencies 15

Poems syndrome 76 Polychromatophilia 21, 33 Polycythemia 48, 63 vera 62, 63, 64 f, 65f, 104 Precursor lymphoidneoplasms 81

N Neoplastic cells 88

Osmotic fragility test 19, 19f, 122 Osteosclerotic myeloma 76

P Pancytopenia 13, 75 Pappenheimer bodies 42 Parietal cellantibody 12 Paroxysmal cold hemoglobinuria 40 nocturnal hemoglobinuria 15, 41

Index

Premature destruction ofspherocytes 17 RBC destruction 36 Primary amyloidosis 80 antiphospholipid syndrome 115 hemostatic plug 99 myelofibrosis 62, 66 Produces hemolytic anemia 30 Progressive cytopenias 60 Proliferating hematopoietic stemcells 68 Promyelocytic leukemia 53 Protein C andS deficiency 114 Prothrombin time 107 Pseudo Gaucher cells 70 Pulmonary eosinophilia 48

Q Qualitative disorders ofleukocytes 50 platelet disorders 101, 104 Quantitative and qualitative disorders of leukocytes 45 deficiency of vWF 109 disorders of leukocytes 45 platelet disorders 101

R

Reticulocyte count 6, 25, 15, 18, 21, 33, 38 hemoglobin 7 Rh hemolytic disease of newborn 37 incompatibility 37 Rheumatoid arthritis 49 Ring sideroblasts 42 Rouleaux formation 131 Roundworm infestation 48 Russell’s viper venom 115

Sickling crisis 32 test 34, 35, 35 f Sideroblastic anemia 8, 42 Skin bleeding 102 diseases 48 Small lymphocytic lymphoma 73, 81  lymphocytes 89 Sore throat 51

S

Spherocytes and raised osmotic fragility test 122 Splenic B cell marginal zone lymphoma 81 Spoon shapednails 120 Stages of erythropoiesis 128 Hodgkin lymphomas 94 t megakaryopoiesis 129 myelopoiesis 132 Starry sky appearance 84f Striking basophilia 71 Structure of redcell membrane 17f Subacute combineddemyelination 13 Subleukemic leukemia 52 Substitution ofglutamic acid 30 Sudden trappingof blood 33 Suppression ofstem cells 48

Salmonella typhimurium 33 Sarcoidosis 15, 49 Scabies 48 Schilling test 11, 12 Secondary antiphospholipid syndrome 115 hemostatic plug 99 Sequestration crisis 33 Serum albumin 78 bilirubin 11, 34 ferritin 7, 26 folic acid levels 11 haptoglobin 26, 34 homocysteine 11 iron 7, 26

Raspberry-red tongue 121 RBC enzyme analysis 21 Reactive marrowfibrosis 66 Recurrent splenic infarction 32 Red cell count 64 distribution width 5 indices 43, 6, 9, 18, 25 protoporphyrin 7 size 3 Reed-Sternberg cells 87, 88, 89f, 90, 91 Refractory anemia with excess blasts 61 ring sideroblasts 61 cytopenia 61 with multilineage dysplasia 61 Renal

and ferritin 11 status 26 transferrin receptor 7 saturation 7 vitamin B 12 11 and uric acid 65 Severe anemia 7 hemolytic anemia 23, 38 infections 48 Severity of bleeding 101 Sex-linked recessive disorders 106 Sézary cells 86 syndrome 81, 86 Sickle cell

Synthesis of fetal hemoglobin 23 Syphilis 49 Systemic lupus erythematosus 49

disease 80 failure 79, 104 function tests 78 Repeated spontaneousabortions 115 Respiratory distresssyndrome 110

anemia 29, 32f, 33, 122 disease 29 trait 34 hemoglobin 29 Sickled red cells 35f

plasma iron-binding capacity 7 WBC count 58 ourniquet test 103 oxoplasmosis 49 raditional classification ofleukemia 53t

T  cell large granular lymphocytic leukemia 81 prolymphocytic leukemia 81  lymphoblastic leukemia/lymphoma 81 arget cells 122 Talassemia syndrome 22 Trombocytopenia 100, 101 Trombocytosis 101, 104 Trombotic disorders 99, 100, 113 otal iron binding capacity 26 leukocyte count 46, 47, 51, 70

139

140

Rapid Review of Hematology

ropical eosinophilia 48 uberculosis 15, 49 umors 15 ypes of antiglobulin test 39 immune thrombocytopenic purpura 102 Langerhans cell histiocytosis 96 t white blood cell 45

U Ulcerative colitis 49 Unconjugated bilirubin 38 Urine urobilinogen 26, 34 Urticaria 48 Uses of direct antiglobulintest 39 indirect antiglobulin test 40

V Vascular disorders 110 purpura 99 Vessel wall abnormalities 99 Viral hepatitis 108 infections 45, 48, 49 Vitamin B12 8, 15, 48 absorption 12 deficiency 8, 11 K deficiency 105 von Willebrand disease 105, 108

W White cell count 64

WHO classification of acute leukemia 53 lymphoblastic and myeloid leukemia 53t Hodgkin lymphoma 87 t lymphoid neoplasms 81 t MPN 62 myelodysplastic syndromes 61 t myeloproliferative neoplasm 62 t Widespread exfoliative erythroderma 86 Wiskott-Aldrich syndrome 100