CM Handout

CLINICAL MICROSCOPY ANALYSIS OF URINE AND OTHER BODY FLUIDS CLINICAL MICROSCOPY

Roderick D. Balce, RMT

ANALYSIS OF URINE, OTHER BODY FLUIDS, AND MISCELLANEOUS SPECIMENS Roderick D. Balce

Centro Escolar University MEDICAL TECHNOLOGY 1

CLINICAL MICROSCOPY CONTENT

ANALYSIS OF URINE AND OTHER BODY FLUIDS

1. Introduction to Urinalysis 2. Quality Assurance 3. Physical Examination of Urine 4. Chemical Examination of Urine 5. Microscopic Examination of Urine 6. Urinalysis Automation 7. Renal Structure, Functions, and Diseases 8. Renal Calculi 9. Urine Screening Tests

2 4 6 7 10 12 13 16 17


10. Pregnancy Tests 11. Cerebrospinal Fluid 12. Semen 13. Synovial Fluid 14. Serous Fluids 15. Amniotic Fluid 16. Feces 17. Sputum and BAL 18. Gastric Fluid

19 20 22 24 26 28 29 30 31

1. Introduction to Urinalysis A. History  Urinalysis – marked the beginning of laboratory medicine; included observations of color, turbidity, odor, volume, viscosity, and even sweetness

 5th century BC – Hippocrates wrote a book on uroscopy  1140 AD – color charts were developed that described the significance of 20 different colors  1627 – Thomas Bryant wrote a book about charlatans (pisse prophets) which inspired the passing of the first medical licensure law in England

 1694 – Frederik Dekkers’ discovered albuminuria by boiling urine  17th century – microscope was invented which led to the examination of urinary sediment and to the development by Thomas Addis of methods for quantitating the microscopic sediment

 1827 – Richard Bright introduced the concept of urinalysis as part of routine patient examination

B. Utilities of Analysis 1. To aid in the diagnosis of diseases 2. To screen asymptomatic populations for undetected disorders 3. To monitor the progress of disease and the effectiveness of therapy

C. Specimen Considerations 1. Composition of Urine: 95% water, 5% analytes a. Organic components – urea, creatinine, uric acid, ammonia, undetermined nitrogen, others + + 2+ b. Inorganic components – Cl , Na , K , P, Ca , phosphates, sulfates 2. Types of Urine Specimen/ Collection Techniques a. First morning – routine screening, pregnancy test, detection of orthostatic proteinuria b. Random – routine screening c. 24-hour – quantitative chemical tests, hormone studies d. 12-hour – Addis count e. Afternoon specimen (2-4 pm) – urobilinogen determination f. Fasting/Second morning – diabetic screening/monitoring g. 2-h Postprandial – diabetic monitoring h. Glucose Tolerance – accompaniment to blood samples in GTT i. Drug testing specimen – collection requires stringent protocols (COC); temperature should be within 32.5- 37.7ºC; blueing agent added to the toilet water reservoir in unwitnessed collection j. Midstream clean-catch – routine screening, bacterial culture k. Catheterization – bacterial culture l. Suprapubic aspiration – bacterial culture, cytology m. Three-glass collection – diagnosis of prostatic infection 2

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3. Specimen Preservation a. Methods of Preservation Preservatives Refrigeration Phenol Toluene Thymol Formalin Sodium fluoride Boric acid Saccomanno Fixative

Comments Prevents bacterial growth for at least 24 hours; preserves organized sediments; maintains an acid pH up to about 8 hours; and does not interfere with chemical tests Precipitates amorphous materials increasing the specific gravity Does not interfere with routine tests; causes an odor change Does not interfere with routine tests; floats on surface of specimens and clings to pipettes and testing materials Preserves glucose and sediments well; interferes with acid precipitation tests for protein Excellent sediment preservative; acts as a reducing agent, interfering with chemical tests for glucose, blood, LE, and copper reduction Prevents glycolysis; is a good preservative for drug analyses; inhibits reagent strip tests for glucose, blood, and leukocytes Preserves protein and formed elements well; does not interfere with routine analyses other than pH; interferes with drug and hormone analyses Preserves cellular elements; for cytology studies

b. Changes in Unpreserved Urine Parameter Color Odor pH Nitrite Bacteria Clarity Glucose Ketones Bilirubin Urobilinogen Cells and casts

Change Modified/ darkened Increased

Decreased

Cause Oxidation or reduction of metabolites Bacterial multiplication or breakdown of urea to ammonia Bacterial breakdown of urea to ammonia/ loss of CO2 Multiplication of nitrate-reducing bacteria Multiplication Bacterial growth and precipitation of amorphous material Glycolysis and bacterial use Volatilization and bacterial metabolism Photooxidation to biliverdin Oxidation to urobilin Disintegration in dilute alkaline urine

4. Urine Volume (24-hour) a. Average daily output: 1,200-1,500 mL b. Variations 1) Polyuria – abnormal increase in urine output  Clinical significance: diabetes mellitus, diabetes insipidus 2) Oliguria – abnormal decrease in urine output  Clinical significance: dehydration, renal insufficiency, poorly compensated heart disease, calculi formation, kidney tumors 3) Anuria/Anuresis – total suppression of urine production  Clinical significance: severe acute nephritis, Hg poisoning, obstructive uropathy, kidney failure 4) Nocturia – excretion of more than 500 mL urine at night 5) Diuresis – transitory increase in urine volume

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2. Quality Assurance A. Pre-analytical Aspects of Quality Assurance 1. Specimen Collection a. Appropriate specimen container (capacity, need for a sterile or opaque container) b. Minimum labeling requirements (patient’s name, date and time of collection) c. Specimen type and volume required for each test 2. Specimen Receipt or Rejection Criteria for Specimen Rejection  Unlabeled or mislabeled containers  Request form incomplete or lacking  Nonmatching labels and requisition forms  Visibly contaminated specimens (feces or toilet paper)

    

Containers with contaminated exteriors Inappropriate specimen type Specimens of insufficient quantity Improperly transported specimens Incorrect urine preservative

3. Specimen Processing  Immediate processing (within 2 hours for routine UA) to improve specimen TAT and prevent changes in specimen integrity  For timed specimens: adequate mixing, volume measurement, aliquoting

B. Analytical Aspects of Quality Assurance 1. Reagents  Deionized water for reagent preparation: check pH and purity every week and bacterial count every month  Reagent strips: store in opaque container with dessicant at room temperature; check with positive and negative controls every 24 hours (+ control: ± 1 color block, - control: water not acceptable) 2. Equipment Routine performance checks, calibration, and preventive maintenance schedules Frequency Checks performed Daily or weekly Clean rotor, trunnions, and interior with suitable disinfectant. Every 3 months Check speed and timer. Microscopes Daily Clean and adjust if necessary (e.g. Kohler illumination, phase ring) Annually Preventive maintenance and cleaning. Reagent strip Daily Calibrate reflectance meter with standard reagent strip. readers Daily or periodically Clean mechanical parts and optics. Refractometers Every shift or daily Calibrate with distilled water (1.000) and at least one standard of known SG e.g. 3% NaCl (1.015), 5% NaCl (1.022), 7% NaCl (1.035), or 9% sucrose (1.034). Acceptable tolerance: target ± 0.001 Equipment Centrifuges

3. Microscopy Guidelines for Standardizing Microscopic Examination of Urine Sediment a. Volume of urine examined 10, 12, or 15 mL b. Speed of centrifugation 400 g c. Length of centrifugation 5 minutes d. Sediment preparation 0.5 or 1 mL left after decantation 20 µL or 0.02 mL e. Volume of sediment examined f. Sediment examination At least 10 LPFs and 10 HPFs 4

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4. Quality Control a. Internal QC  Two levels of commercial controls must be run and recorded at the beginning of each shift and if reagents are changed, an instrument malfunction has occurred, or if test results are questionable  QC data must be retained for 2 years and should be reviewed daily and monthly to detect errors. b. External QC/Proficiency testing  Analysis of lyophilized or ready-to-use specimens sent by a regulatory agency

C. Post-analytical Aspects of Quality Assurance 1. Critical or Panic Values  Pathologic urine crystals, strongly positive test for glucose and ketones, presence of a reducing substance other than glucose and ascorbic acid in an infant’s urine 2. Reporting and Interpretation of Results  Reporting format must be standardized and reference values must be included in the results form Sample standardized urine microscopic reporting format Quantitate an average of 10 fields both under low and high power. Do not quantitate budding yeast, mycelial elements, trichomonas, mucus threads, or sperm, but do note their presence. Epithelial cells: Rare (0–5), Few (5–20), Moderate (20–100), Many (>100) per LPF Casts: 0–2, 2–5, 5–10, >10 per LPF RBCs: 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 per HPF WBCs:0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 per HPF Crystals: Rare (0–2), Few (2–5), Moderate (5–20), Many (>20) per LPF or HPF Bacteria: Rare (0–10), Few (10–50), Moderate (50–200), Many (>200) per HPF

D. Approaches to Quality Management 1. Total Quality Management  focuses on teams, processes, statistics, and services that meet or exceed customer expectations  strives to continually look for ways to reduce errors (defect prevention) by empowering employees to assist in solving problems and understand their integral role within the system (universal responsibility) 2. Continuous Quality Improvement  strives to continually improve practices and not just meet established quality standards  patients are the ultimate customers of CQI but also include health-care providers, personnel in other departments, and the patient’s family and friends 3. Six Sigma  hands-on process with the single mantra of improvement (improved performance, improved quality, improved customer and employee satisfaction)  based on statistics and quantitative measurements which involve the determination of the number of defects per million opportunities (DPMO) and the reduction of the same to near zero

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3. Physical Examination of Urine A. Color 1. Normal – varies from almost colorless, straw or light yellow to dark yellow, yellow-orange, or amber 2. Variations a. Colorless/pale yellow – recent fluid consumption, polyuria, DM, DI b. Amber/ orange – bilirubin, acriflavine, phenazopyridine, nitrofurantoin, phenindione c. Yellow-green – oxidation of bilirubin to biliverdin d. Blue/green – Pseudomonas infection, amitriptyline, methocarbamol, clorets, indican, methylene blue e. Pink/red – intact RBCs, hemoglobin, myoglobin, porphyrins, beets, menstrual contamination f. Brown/black – methemoglobin, homogentisic acid, melanin, argyrol, methyldopa, levodopa, metronidazole

B. Odor 1. Normal – faint aromatic due to volatile acids; becomes ammoniacal as the specimen stands 2. Variations a. Ammoniacal (freshly voided) – UTI g. Mousy – PKU b. Rancid – tyrosinuria h. Sweaty feet – isovaleric acidemia c. Maple syrup/ caramel-like – MSUD i. Rotting fish – trimethyl aminuria d. Sulfur odor – cystine disorders j. Fecaloid – recto-vesicular fistula e. Fruity/ sweet – diabetes ketoacidosis k. Cabbage/ hops – methionine malabsorption f. Mercaptan – asparagus, garlic, and eggs l. Bleach – contamination

C. Transparency 1. Normal: 2. Variations:

Clear – no visible particulates, transparent Hazy – few particulates, print easily seen through urine Cloudy – many particulates, print blurred through urine Turbid – print cannot be seen through urine Milky – may precipitate or be clotted

D. Specific Gravity 1. Normal Values: depend on the patient’s degree of hydration 2. Methods a. Urinometry 1) Urinometer/Hydrometer - weighted float that is designed to sink to a level of 1.000 in distilled water; calibrated at 20°C; less accurate than other methods; requires large volume of urine 2) Corrections  Temperature – for every 3°C that the urine temperature is above or below the calibration temperature, 0.001 is respectively added to or subtracted from the reading  Protein – subtract 0.003 for every g/dL; Glucose – subtract 0.004 for every g/dL b. Refractometry 1) Refractometer/ TS meter - measures refractive index; compensated between 15°C and 38°C 2) Corrections: protein and glucose only; temperature correction not done c. Harmonic Oscillation Densitometry 1) Mass gravity meter – used by Yellow IRIS automated workstations to measure specific gravity 2) Principle: Sound waves of specific frequency are generated at one end of the tube and as the sound waves oscillate through urine, their frequency is altered by the density of the specimen.

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4. Chemical Examination of Urine A. Summary of Chemical Tests Parameter (Normal values) 1. Specific Gravity

Clinical significance

Additional comments

1. Monitoring patient’s hydration. 2. Detection of loss of renal concentrating ability 3. Diagnosis of diabetes insipidus 4. Determination of unsatisfactory specimens due to low concentration 2. pH 1. Respiratory or metabolic acidosis/ alkalosis 2. Renal tubular acidosis (Average, 6.0; 3. Renal calculi formation random, 4.54. Treatment of UTI 8.0; fasting, 5. Precipitation/ identification of crystals 5.5-6.5) 6. Determination of unsatisfactory specimen 3. Protein Degrees of proteinuria: a. Mild – < 1.0 g/day (<30 mg/dL or b. Moderate – 1.0-4.0 g/day <150 mg/day; c. Heavy – > 4.0 g/day Negative rgt Types of proteinuria: strip test) a. Pre-renal – intravascular hemolysis; muscle injury; severe infection and inflammation; multiple myeloma b. Renal (glomerular) – diabetic nephropathy, amyloidosis, glomerulonephritis, autoimmune disorders, toxic agents, hypertension, strenuous exercise, preeclampsia, dehydration, orthostatic proteinuria c. Renal (tubular) – Fanconi syndrome, toxic agents, severe viral infections d. Post-renal – lower UTI; injury or trauma; menstrual contamination; prostatic fluid; spermatozoa; vaginal secretions

4. Glucose (<15 mg/dL; Negative rgt strip test)

Types of Glucosuria: a. Hyperglycemia-associated – diabetes mellitus, endocrine disorders, pancreatic disorders, CNS disorders, disturbance in metabolism, liver disease, drugs, gestational diabetes mellitus b. Renal-associated – renal tubular dysfunction, tubular necrosis, Fanconi syndrome, osteomalacia, pregnancy 7

Causes of acid urine: emphysema, diabetes mellitus, starvation, diarrhea, dehydration, acidproducing bacteria, high protein diet, medications Causes of alkaline urine: hyperventilation, vomiting, renal tubular acidosis, urease-producing bacteria, vegetarian diet, old specimens Tests for protein: a. Heat and Acetic Acid Test  Grading: diffused cloud (+1); granular cloud (+2); distinct flocculi (+3); large flocculi (+4) b. SSA Test/ Cold Protein Precipitation `

Grade Degree of Turbidity and Conc. in mg/dL

Neg No increase in turbidity (<6) Trace Noticeable turbidity (6-30) +1 Distinct turbidity, no granulation (30-100) Turbidity with granulation, no flocculation +2 (100-200) Turbidity with granulation and flocculation +3 (200-400) +4 Clumps of protein (>400)  False (+): mucin, uric acid, penicillin, tolbutamides, radiocontrast media, sulfonamides, cephalosporins  False (-): highly buffered alkaline urine  Correlate with reagent strip results c. Tests for microalbuminuria  Micral test and Immunodip  Significant values reported as AER Tests for Glucose (Copper Reduction): a. Benedict’s test  Positive result: brick red precipitate b. Clinitest  Rgts: CuSO4, NaOH, sodium citrate, Na2CO3  False(+): other reducing sugars, ascorbic acid, drug metabolites, cephalosporins  False (-): pass-through phenomenon  Correlate with reagent strip results

CLINICAL MICROSCOPY 5. Ketones (Negative rgt strip test)


Ketone bodies: Acetone – 2% Acetoacetic acid – 20% β-hydroxybutyric acid – 78% Causes of Ketonuria: diabetes mellitus, starvation, fasting, weight reduction, strenuous exercise, malabsorption, pancreatic disorders, inborn errors of amino acid metabolism

a. Hematuria – renal calculi, glomerular disorders, pyelonephritis, tumors, trauma, (Negative rgt toxic chemicals, anticoagulant therapy, strip test) strenuous exercise, and menstruation b. Hemoglobinuria– hemolytic anemias, transfusion reactions, severe burns, malaria,strenuous exercise c. Myoglobinuria – muscular trauma, crush syndrome, prolonged coma, convulsions, muscle-wasting diseases, alcoholism, drug abuse, extensive exertion 7. Bilirubin 1. Diagnosis of hepatitis, cirrhosis, other liver disorders, and biliary obstruction (Negative rgt 2. Determination is more significant when strip test) combined with serum bilirubin and urine urobilinogen Blood Urine Urine Bilirubin Bilirubin UBG Hemolytic Inc Neg +++ Hepatic Inc +/++ Obstructive Inc/N +++ Normal 8.Urobilinogen 1. Only a small amount is normally found in urine. (< 1.0 mg/dL 2. Useful for the early detection of liver or <1.0 Ehrlich disease and for the diagnosis of hemolytic unit) disorders, hepatitis, cirrhosis, and carcinoma 3. Absence in urine may indicate biliary obstruction 6. Blood

9. Nitrite (Negative rgt strip test) 10. LE (Negative rgt strip test)


Tests for Ketones: a. Gerhart’s, Lindeman’s – diacetic acid b. Frommer’s, Walhauster’s, Lange’s, JacksonTaylor’s, Rantzmann’s, Lieben’s – acetone c. Legal’s, Rothera’s, Acetest, Ketostix – diacetic acid and acetone d. Osterberg, Hart’s test – β-hydroxybutyric acid Tests to differentiate hemoglobin and myoglobin: a. Ammonium Sulfate Method  Reagent: 2.8 g (NH4)2SO4  Hemoglobin is precipitated by (NH4)2SO4 and is not detected in the supernatant. b. Enzyme assays c. Absorption Spectrophotometry d. Immunodiffusion Technique e. Electrophoresis Tests for Bilirubin: a. Foam Shake Test b. Oxidation Test (Gmelin or Fouchet’s method)  Acidic oxidation of bilirubin into a rainbow array of colors: green (biliverdin), blue (bilicyanin), and yellow (choletelin) c. Diazotization Test (Ictotest)  Reagents: p-nitrobenzene-diazonium-ptoluene sulfonate, Na2CO3, boric acid, SSA  (+) Result: blue to purple color Tests for Urobilinogen: a. Ehrlich’s Tube Test  Reagent: p-dimethylaminobenzaldehyde  (+) Result: cherry red color b. Scwartz-Watson Differentiation Test  UBG - soluble in both chloroform and butanol  PBG - insoluble in both  ERCs- soluble only in butanol

1. Diagnosis of cystitis and pyelonephritis 2. Evaluation of antibiotic therapy 3. Monitoring of patients at high risk for UTI 4. Screening of urine culture specimens 1. Detection of bacterial and nonbacterial UTI 2. Inflammation of the urinary tract 3. Screening of urine culture specimens

NOTES: __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 8

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B. Summary of Reagent Strip Testing Regent strip test

Principle

Color reaction

Reagents

Sources of interference

M: Poly(methylvinyl ether) Blue-greenmaleic anhydride, BTB F(+): High concentration of protein yellow C: Ethyleneglycol-bis F(–): Highly alkaline urine (45 s) (aminoethylether), BTB Double indicator Methyl red; Red-yellow- Runover from adjacent pads 2. pH system bromthymol blue blue (60 s) Old specimens F(+): Highly buffered alkaline urine, M: Tetrabromphenol blue pigmented specimens, chlorhexidine, Protein error of C: Tetrachlorophenol Blue-green phenazopyridine, QACs (detergents), 3. Protein indicator tetrabromosulfon(60 s) antiseptics, loss of buffer, high SG phthalein F(–):Proteins other than albumin, high salt concentration, microalbuminuria M: Glucose oxidase, M: green- F(+): Oxidizing agents, detergents Glucose peroxidase, KI brown F(–): Ascorbic acid, ketones, high SG, low 4. Glucose oxidase reaction C: Glucose oxidase, C: yellowtemperatures, improperly preserved peroxidase, TMB green (30 s) specimens F(+): Phthalein dyes, highly pigmented Sodium M: Sodium nitroprusside Purple red urine, levodopa, medications 5. Ketones nitroprusside C: Sodium nitroprusside (40 s) containing SH group reaction and glycine F(–): Improperly preserved specimens M:Diisopropylbenzene F(+): Strong oxidizing agents, bacterial Pseudodihydroperoxide TMB peroxidases, menstrual contamination Blue-green 6. Blood peroxidase C: 2,5-dimethyl 2,5F(–): High SG, crenated cells, formalin, (60 s) activity of heme dihydroperoxyhexane captopril, nitrite, ascorbic acid, unmixed TMB specimen F(+): Highly pigmented urine, indican, M: 2,4-dichloroaniline Tan, pink, phenazopyridine, metabolites of Lodine diazonium salt 7. Bilirubin Diazo reaction or violet F(–): Specimen exposure to light, C:2,6-dichlorobenzene (30 s) ascorbic acid >25 mg/dL, high diazoniumtetrafluoroborate concentration of nitrite F(+): PBG, indican, procaine, M: paradiethylaminop-aminosalicylic acid, sulfonamides, benzaldehyde Red methyldopa, chlorpromazine, pigmented 8.Urobilinogen Ehrlich reaction C: 4-methoxybenzene (60 s) urine diazonium tetrafluoroF(–): Old specimens, formalin, high borate concentration of nitrite F(+): Old specimen, highly pigmented M: p-arsanilic acid, urine tetrahydrobenzoquinolinol F(–):Nonreductase-containing bacteria, C: sulfanilamide, 3Pink 9. Nitrite Greiss reaction lack of urinary nitrate, insufficient contact hydroxy- 1,2,3,4(60 s) time between bacteria and nitrate, tetrahydrobacteria converting nitrite to nitrogen, 7,8-benzoquinoline antibiotics, ascorbic acid, high SG M:Derivatized pyerole F(+): Strong oxidizing agents, formalin, 10. Granulocytic amino acid ester, highly pigmented urine, nitrofurantoin Purple Leukocyte esterase diazonium salt F(–):  protein, glucose, oxalic acid, (120 s) esterase reaction C: Indoxylcarbonic acid ascorbic acid, gentamicin, ester, diazonium salt cephalosporins, tetracyclines 1. Specific gravity

pKa change of polyelectrolytes

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5. Microscopic Examination of Urine A. Sediment Examination Techniques 1. Standardization of Manual Microscopic Technique (refer to QA notes) 2. Sediment Stains Stain Sternheimer-Malbin Toluidine blue Lipid Stains: Oil Red O and Sudan III Hansel stain Prussian blue stain

Action/Function Delineates structure and contrasting colors of the nucleus and cytoplasm; identifies WBCs, epithelial cells, and casts Enhances nuclear detail; differentiates WBCs and RTE cells Stains triglycerides and neutral fats orange red; identifies free fat droplets and lipidcontaining cells and casts Stains granules of urinary eosinophils Stains structures containing iron; identifies hemosiderin granules in cells and casts

3. Microscopy Type Bright-field Phase-contrast Polarizing Dark-field Fluorescence DIC

Function Used for routine urinalysis Enhances visualization of elements with low refractive indices e.g. hyaline casts Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals Often used for unstained specimens; and aids in identification of T. pallidum Used to visualize naturally fluorescent microorganisms or those stained by a fluorescent dye Produces a three-dimensional microscopy-image and layer-by-layer imaging of a specimen

B. Sediment Constituents 1. Cells Red blood cells

White blood cells

Renal tubular ECs

Biconcave, anucleate discs, Granular, larger than RBCs, Vary in size and shape; 7 µm in diameter ~12 µm in diameter with eccentric nucleus Normal: 0-2/hpf Normal: <5/hpf Most clinically significant Hematuria: glomerulonephritis, Pyuria: urinary tract >2/hpf = tubular injury renal calculi, malignancy infection or inflammation Neutrophils: most common Bilirubin-laden: hepatitis Ghost cells in dilute urine Glitter cells:sparkling Hemosiderin-laden: Crenated in hypertonic urine appearance and Brownian hemolytic conditions Dysmorphic in glomerular Oval fat bodies: lipiduria, movement Eosinophils: AIN bleeding nephrotic syndrome Mononuclears: graft rejection Bubble cells: ATN Squamous ECs

Bacteria

Abundant, irregular cytoplasm and a prominent central nucleus about the size of an RBC

Gram – rods of family Enterobacteriaceae, and cocci esp. Staphylococcus and Enterococcus species

No clinical significance (except clue cells = covered with Gardnerella vaginalis coccobacilli)

Bacteriuria – indicates UTI when seen in conjunction with pyuria and a positive urine culture 10

Transitional ECs Spherical, polyhedral, and caudate; with central nucleus Abnormal morphology indicates malignancy or viral infection Singly, in pairs, or in clumps (syncytia) following invasive urologic procedures e.g. catheterization

Yeast cells

Parasites

Small, refractile oval structures; Candida albicans most common True yeast infection accompanied by WBCs; seen in diabetes, immunocompromised state and vaginal moniliasis

T. vaginalis –most common; may resemble a WBC, transitional, or RTE cell; causes nonspecific urethritis S. haematobium – inhabits the urinary bladder; ova have a characteristic terminal spine

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2. Casts (See Formation and degeneration of casts on page 32) Hyaline cast

Red blood cell cast

White blood cell cast

Orange-red under LPO; Colorless with low refractive more fragile and have a index; prototype of all casts more irregular shape Normal: 0-2/lpf Indicates Seen with other pathological glomerulonephritis and casts in cases of AGN, damage to the capillary CGN, APN, and CHF structure of the nephrons Granular cast

Granular appearance, irregular borders, multilobed nuclei Seen in pyelonephritis (+WBCs and bacteria) and interstitial nephritis (+eosinophils)

Waxy cast

Matrix contains coarse or fine granules from the disintegration of cellular casts, filtered proteins, or lysosomes secreted by RTE

RTE cell cast Consists of RTE cells incorporated into the cast matrix Advanced tubular destruction, associated with exposure to toxic agents

Broad cast

High RI; brittle consistency (appear fragmented with jagged ends and notches);homogenous, dark pink with supravital stains Extreme urine stasis, Indicates stasis of urine flow indicating CRF; final phase of cast degeneration

Renal failure cast; mostly granular and waxy formed in the collecting ducts Extreme urine stasis and destruction of the tubular walls

Fatty cast Highly refractile; matrix may contain few or many fat droplets, and intact oval fat bodies may be attached to the matrix Seen in lipiduria in conjunction with oval fat bodies and free fat droplets

3. Crystals a. Normal and Iatrogenic Crystals in Acidic Urine Uric acid Amorphous urates Acid urates

Sodium urates

Macroscopically resemble brick dust Small brown spheres; Colorless birefringent Microscopically appear may cluster in pairs needles as yellow-brown and triplets granules Soluble in alkali Soluble in alkali and heat; convert to uric acid crystals when acidified Increased in gout, Commonly seen in Rarely encountered and have little clinical leukemia, and Lesch- refrigerated significance Nyhan syndrome specimens Yellow-brown; pleomorphic; birefringent

Calcium sulfate

Hippuric acid

Long, thin colorless needles or prisms identical to calcium phosphate

Yellow-brown or colorless, needles, rhombic plates and four-sided prisms Soluble in hot water Soluble in acetic acid and alkali Rarely seen; Rare; associated with no clinical foods containing significance benzoic acid b. Normal Crystals in Alkaline Urine Amorph. phosphates Calcium phosphate Macroscopically appear milky white; Microscopically granular in appearance

Colorless, flat rectangular plates or thin prisms often in rosette formations

Radiographic dye Flat, colorless, notched rhombic plates; highly birefringent

Sulfonamide crystals

Calcium oxalate Dihydrate – colorless, octahedral envelope Monohydrate – oval or dumbbell-shaped Soluble in dilute HCl Ethylene glycol poisoning, renal Calculi Ampicillin crystals

Colorless to yellowColorless needles that brown needles, tend to form bundles sheaves of wheat,and following refrigeration rosettes

Soluble in 10% NaOH Soluble in acetone

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Associated with a Indicates inadequate hydration among markedly elevated SG patients being treated for UTI; may cause (>1.040) tubular damage if crystals form in the nephron Triple phosphate Prism shape resembling “coffin lids”; birefringent under polarized light

Ammonium biurate

Calcium carbonate

Yellow-brown spiculeSmall, colorless, covered spheres dumbbell or spherical; described as “thorny may occur in clumps apples” Soluble in acetic acid Soluble in acetic acid Soluble in dilute acetic acid and heat with evolution of gas Seen in refrigerated Common constituent Presence of urea-splitting bacteria (e.g. No clinical significance specimens of renal calculi Proteus, Pseudomonas) 11

CLINICAL MICROSCOPY c. Abnormal Crystals Cystine Colorless, hexagonal plates; may be confused with colorless uric acid crystals


Cholesterol

Leucine

Rectangular plates with a notch in one or more corners; highly birefringent

Yellow-brown spheres that demonstrate concentric circles and radial striations


Tyrosine Fine colorless to yellow needles that frequently form clumps or rosettes

Soluble in ammonia, Soluble in hot alkali or Soluble in alkali or Soluble in chloroform dilute HCl alcohol heat Cystinuria; renal calculi formation

Lipiduria (nephrotic syndrome)

Bilirubin Clumped needles or granules with the characteristic yellow color Soluble in acetic acid, HCl, NaOH, ether, chloroform

Severe liver disease

4. Urinary Sediment Artifacts a. Starch granules – highly refractile spheres with a dimpled center; resemble fat droplets when polarized, producing a Maltese cross formation b. Oil droplets and air bubbles – highly refractile and may resemble RBCs c. Pollen grains – spheres with a cell wall and occasional concentric circles; large size may cause the microscopist to be out of focus d. Hair and fibers – may be mistaken for casts but are longer, more refractile, and are able to polarize light e. Fecal artifacts – from improperly collected specimens or due to the presence of a recto-vesical fistula; may appear as plant and meat fibers or as brown amorphous materials in a variety of sizes and shapes

6. Urinalysis Automation Instrument 1. Semiautomated chemistry analyzers

Tests Urine chemistries Specific gravity

Clarity Urine chemistries Specific gravity

Principle Reflectance photometry Reflectance photometry Reflectance photometry or spectrophotometry Light transmittance or light scatter Reflectance photometry Refractive index measurement

Urine sediment analysis

Flow cytometry

Color, clarity, urine chemistries

Same as no. 2

Color 2. Fully automated chemistry analyzers 3. Automated Microscopy analyzers

4. Fully automated urinalysis systems or workstations

Specific gravity Urine sediment analysis

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Refractive index measurement (old Yellow IRIS models used harmonic oscillation densitometry) Flow cytometry or intelligent microscopy system

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7. Renal Structure, Function, and Diseases A. Anatomy of the Kidney 1. Basic Structure (Refer to figures 1 and 2 on page 32) 2. Nephron – basic functional unit of the urinary system, 1-1.5 million per kidney a. Types 1) Cortical – constitute approximately 85% of the nephrons; located in the cortex of the kidney 2) Juxtamedullary – have longer Henle’s loops that extend deep into the medulla of the kidney b. Major Parts 1) Glomerulus (Renal Corpuscle) – highly specialized tuft of capillaries 2) Renal tubules a) Bowman's capsule – forms the beginning of the renal tubules b) Proximal convoluted tubule – reabsorption of water and essential substances; major site for removal of nonfiltered substances c) Loop of Henle – selective reabsorption i. Descending limb – freely permeable to water but not to solutes ii. Ascending limb – impermeable to water but permeable to solutes d) Distal convoluted tubule – reabsorption of Na and H2O continues e) Collecting duct – further concentration of the filtrate

B. Physiology of Urine Formation 1. Renal Functions a. Renal Blood Flow b. Glomerular Filtration 1) Glomerular Filtration Rate (GFR): 120 mL/min 2) Characteristics of the Glomerular Filtrate: SG of 1.010; protein- and cell-free 3) Factors Affecting Glomerular Filtration a) Cellular Structure i. Capillary Wall Membrane – made up of endothelial cells that are fenestrated ii. Basement Membrane/ Basal lamina – further restriction of large molecules and blood cells iii. Visceral Epithelium of Bowman’s capsule – podocytes with intertwining foot processes b) Glomerular pressure – 75 mmHg; results from the smaller sizes of the efferent arteriole and glomerular capillaries c) Renin-Angiotensin-Aldosterone Mechanism – responds to changes in blood pressure and plasma sodium content monitored by the juxtaglomerular apparatus c. Tubular Reabsorption 1) Active Transport – glucose, amino acids, salts, chloride, sodium 2) Passive Transport – water, urea, sodium 3) Counter Current Mechanism – maintains the osmotic gradient of the medulla d. Tubular Secretion Major Functions: 1) Elimination of waste products not filtered by the glomerulus + – – 2) Regulation of acid-base balance through secretion of H that react with HCO3 , PO4 , and NH3

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C. Evaluation of Renal Functions 1. Glomerular Filtration Tests a. Clearance Test – standard test to measure the filtering capacity of the glomeruli b. Methods 1) Urea Clearance – earliest method because of presence in all urine specimens and the existence of chemical methods of analysis 2) Radioisotope Clearance – involves injection of radionucleotides; not only determines GFR but also enables visualization of filtration in one or both kidneys 3) β-2 microglobulin Clearance – dissociates from HLA at a constant rate and is rapidly removed from the plasma; not reliable in patients with history of immunologic disorders or malignancy 4) Cystatin C Clearance – small protein (MW 13,359) produced at a constant rate by all nucleated cells, readily filtered by the glomerulus, and reabsorbed and broken down by the renal tubular cells; serum marker recommended for pediatric patients, diabetics, elderly, and critically ill patients 5) Inulin Clearance – reference method; extremely stable substance that is neither reabsorbed nor secreted by the tubules; exogenous procedure 6) Creatinine Clearance – recommended method; endogenous procedure a) Calculation 𝐔𝐕 Formula: 𝐂 = where, C=clearance in mL/min; V=urine volume in mL/min; 𝐏 U=urine creatinine in mg/dL; and P=plasma creatinine in mg/dL To adjust for body size: 𝐂 =

𝐔𝐕 𝐏

𝐱

𝟏.𝟕𝟑 𝐀

where, A=body surface area

b) Normal Values: 75 – 112 mL/min (F); 85 – 125 mL/min (M)

2. Tubular Reabsorption Tests a. Concentration Tests 1) Fishberg Test – patients are deprived of fluids for 24 hours prior to measuring the specific gravity 2) Mosenthal Test – compares the volume and SG of day and night samples 3) Osmometry – used to measure the osmolarity of serum and urine by comparing a colligative property value of the sample with that of pure water a) Normal Values: 275 – 300 mOsm (serum); 50 – 1, 400 mOsm (urine) b) Clinical Osmometers – FP and VP osmometers 4) U:S Osmolarity Ratio – important in determining whether DI is caused by decreased ADH production or lack of renal response to ADH a) should be at least 1:1 under normal random conditions b) should reach 3:1 after controlled fluid intake or ADH injection 5) Free Water Clearance – an expansion of the U:S Osmolarity Ratio; used to determine the ability of the kidneys to respond to the state of body hydration a) Calculation: CH2O = V − Cosm where, CH2O=free water clearance; V=urine volume in mL/min b) Interpretation of Results + value: excess water is excreted 0 value: no renal concentration or dilution would be taking place – value: less than the necessary amount of water is being excreted

3. Tubular Secretion and Renal Blood Flow Tests a. Methods 1) Phenolsulfonphthalein (PSP) – not currently performed due to difficulty in standardization and interpretation of results 2) Indigo Carmine Test – used by urologists as confirmatory test for unilateral kidney disease 3) Para-aminohippuric Acid Test (PAH) – used to measure effective renal plasma flow 4) Titratable Acidity and Urinary Ammonia – used to determine the ability of the kidneys to produce + an acid urine through the tubular secretion of H ions and the production and secretion of NH3 by the cells of the DCT

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D. Renal Disorders 1. Glomerular Disorders Disorder 1. Glomerulonephritis a. Acute or Poststreptococcal Glomerulonephritis b. Rapidly Progressive Glomerulonephritis c. Goodpasture’s Syndrome

2. Vasculitis a. Wegener’s Granulomatosis b. Henoch-Schonlein Purpura 3. IgA Nephropathy

4. Membranous Glomerulonephritis

5. Membranoproliferative Glomerulonephritis 6. Chronic Glomerulonephritis

7. Nephrotic Syndrome

8. Minimal Change Disease 9. Focal Segmental Glomerulosclerosis

Etiology

Urinalysis results

Other tests

Deposition of immune complexes, in conjunction with streptococcal infection, on the glomerular membrane Deposition of immune complexes from systemic immune disorders on the glomerular membrane Attachment of a cytotoxic antibody formed during viral respiratory infections to glomerular and alveolar basement membranes

Macroscopic hematuria, proteinuria, RBC casts, granular casts Macroscopic hematuria, proteinuria, RBC casts

ASO titer, antigroup A streptococcal enzymes BUN, creatinin, creatinine clearance Anti-glomerular basement membrane antibody

Binding of antineutrophilic cytoplasmic antibody to neutrophils in vessel walls Disruption of vascular integrity following viral respiratory infections Deposition of IgA on the glomerular membrane resulting from increased levels of serum IgA

Macroscopic hematuria, proteinuria, RBC casts Macroscopic hematuria, proteinuria, RBC casts Early: Macroscopic or microscopic hematuria Late: hematuria, casts, proteinuria, glucosuria Microscopic hematuria, proteinuria

ANCA

Hematuria, proteinuria

Serum complement levels

Thickening of the glomerular membrane following IgG immune complex deposition associated with systemic disorders Cellular proliferation affecting the capillary walls or the glomerular basement membrane Marked decrease in renal function resulting from glomerular damage precipitated by other renal disorders

Macroscopic hematuria, proteinuria, RBC casts

Hematuria, proteinuria, glucosuria, cellular, granular casts, waxy, and broad casts Disruption of the electrical charges that Microscopic hematuria, produce the tightly fitting podocyte heavy proteinuria, RTE barrier resulting in massive loss of cells, oval fat bodies, fat protein and lipids droplets, fatty casts Disruption of the podocytes following Heavy proteinuria, allergic reactions and immunizations transient hematuria, fat droplets Disruption of podocytes associated Proteinuria, microscopic with heroin/analgesic abuse and AIDS hematuria

FOBT Serum IgA

ANA, HBsAg, FTA-ABS

BUN, creatinine, creatinine clearance Serum albumin, cholesterol, TAG

Serum albumin, cholesterol, TAG Drug test, HIV test

2. Vesicotubulointerstitial Disorders Disorder 10. Acute Tubular Necrosis

Etiology Damage to RTE cells caused by ischemia or toxic agents

Urinalysis results Microscopic hematuria, proteinuria, RTE cells, RTE cell casts 11. Fanconi’s Syndrome Inherited in association with cystinosis Glucosuria, possible cystine and Hartnup disease or acquired crystals (exposure to toxic agents) 12. Cystitis Ascending bacterial infection of the Leukocyturia, bacteriuria, bladder microscopic hematuria, mild proteinuria, increased pH 15

Other tests Hemoglobin, hematocrit, cardiac enzymes Serum and urine electrolytes, chromatography Urine culture

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13. Acute Pyelonephritis Infection of the renal tubules and interstitium related to interference of urine flow or reflux of urine from the bladder, and untreated cystitis 14.Chronic Pyelonephritis Recurrent infection of the renal tubules and interstitium caused by structural abnormalities affecting the flow of urine 15. Acute Interstitial Allergic inflammation of the renal Nephritis interstitium in response to certain medications 16. Renal Failure

May be gradual progression from the original disorder to chronic renal failure or end-stage renal disease


Leukocyturia, bacteriuria, WBC casts, bacterial casts, microscopic hematuria, proteinuria Leukocyturia, bacteriuria, WBC, bacterial, granular, waxy, and broad casts, hematuria, proteinuria Hematuria, proteinuria, leukocyturia, WBC casts

Urine and blood cultures

BUN, creatinine, creatinine clearance

Urine eosinophils, BUN, creatinine, creatinine clearance Proteinuria, renal Creatinine glycosuria, abundance of clearance, BUN, granular, waxy, and broad creatinine, casts (telescoped sediment) electrolytes

8. Renal Calculi A. Renal Lithiasis Disorder 17. Renal Lithiasis

Etiology Deposition of renal calculi or kidney stones in the calyces and pelvis of the kidney, ureters and urinary bladder

Urinalysis results Microscopic hematuria

Other tests Chemical analysis of kidney stones

B. Examination of Renal Calculi 1. Chemical Composition of Renal Calculi a. Calcium oxalate or phosphate b. Magnesium ammonium phosphate c. Uric acid d. Cystine 2. Qualitative Examination of Renal Calculi a. Physical Examination (appearance, size/weight) b. Chemical Examination 1) Pulverize the stone. 2) Ash a small portion over a hot burner. 3) Distribute the remaining pulverized stone into 7 tubes as follows: Tube 1 Uric Acid 2 Carbonate and Oxalate 3 Phosphate 4 Calcium 5 Magnesium 6 Ammonium 7 Cystine

Reagents 20% sodium carbonate, Folin reagent 10% HCl, MnO2 ammonium molybdate, 25% HNO3 10% HCl, 20% NaOH 10% HCl, 20% NaOH, p-nitrobenzene azoresorcinol 10% HCl, 20% NaOH, Nessler’s solution 28% NH4OH, 5% NaCN, 5% sodium nitroprusside 16

Positive Result Deep blue Bubble formation Yellow White cloud Blue Orange-brown Red-purple

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9. Urine Screening Tests A. Major Disorders of Metabolism Disorder

Abnormal urinary constituent

Etiology

Clinical manifestations

I. Amino Acid Disorders A. Phenylalanine – Tyrosine Disorders 1. Phenylketonuria

Phenylalanine hydroxylase deficiency

Phenylalanine, phenyllactic acid, phenylpyruvic acid

Mousy odor of urine, mental retardation

2. Tyrosinuria

Tyrosine transaminase or p-hydroxy phenylpyruvic acid oxidase deficiency; liver disease or underdevelopment of the liver

p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid

Tyrosine and leucine crystals in urine; liver and renal disease (temporary or permanent)

3. Alkaptonuria

Homogentisic acid oxidase deficiency

Homogentisic acid

Dark blue to black pigmentation of cartilage and connective tissues, liver and cardiac disorders

4. Melanuria

Overproliferation of melaninproducing cells

Melanin

Malignant melanoma

Deficiency in decarboxylases and other enzymes needed for the conversion of ketoamino acids to fatty acids

Ketoacids (α-ketoisovaleric; α-ketoisocaproic; α-keto,β-methylvaleric)

Maple syrup odor of urine, failure to thrive, mental retardation

Isovaleryl CoA in the leucine pathway

Isovalerylglycine

Sweaty feet odor of urine

Error in the metabolic pathway converting valine, isoleucine, threonine, and methionine to succinyl CoA

Propionic and methylmalonic acid

Early severe illness, vomiting, metabolic acidosis, hypoglycemia, increased serum ammonia, ketonuria

Indican

“Blue diaper syndrome” Generalized aminoaciduria (Fanconi’s syndrome)

Carcinoid tumors involving the argentaffin cells of the intestine

5-HIAA (>25 mg/24 hours)

Vascular and bronchospastic symptoms (carcinoid syndrome)

Defect in renal tubular reabsorption

Cystine, lysine, arginine, ornithine or Cystine and lysine only

Sulfur odor of urine Cystine crystals in urine Formation of urinary calculi Fanconi’s syndrome

B. Branched Chain Amino Acid Disorders 1. Maple Syrup Urine Disease (MSUD) 2. Organic Acidemias a. Isovaleric Acidemia b. Propionic and Methylmalonic acidemias

C. Tryptophan Disorders 1. Indicanuria Intestinal disorders Hartnup disease 2. Abnormal Urinary Excretion of 5-HIAA D. Cystine Disorders 1. Cystinuria

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2. Cystinosis

Inborn error of metabolism

Cystine

Deposition of cystine crystals in many areas of the body

3. Homocystinuria

Cystathionine β-synthase deficiency

Homocystine and methionine

Failure to thrive, cataracts, mental retardation, thromboembolic problems

II. Porphyrin Disorders

Inborn error of metabolism or acquired through erythrocytic and hepatic malfunctions or exposure to toxic agents

ALA, porphobilinogen, uroporphyrin, coproporphyrin

Port wine color of urine Neurologic/psychiatric symptoms Cutaneous photosensitivity

III. Mucopolysaccharide Disorders

Inborn error of metabolism

Dermatan sulfate, keratan sulfate, heparan sulfate

Mental retardation Accumulation of mucopolysaccharides in the cornea of the eyes Abnormal skeletal structure

Hypoxanthineguanine phosphoribosyl transferase deficiency

Uric acid

Severe motor defects, mental retardation, tendency toward self destruction, gout, renal calculi, “orange sand in diapers”

Galactose

Failure to thrive, liver disorders, cataracts, severe mental retardation

IV. Purine Disorders * Lesch-Nyhan Disease

V. Carbohydrate Disorders/Meliturias * Galactosuria Inborn error of metabolism (GALT, galactokinase, or UDP-galactose-4-epimerase deficiency)

C. Summary of Screening Tests for Metabolic Disorders Test

Ferric Chloride Tube Test

2,4-Dinitrophenylhydrazine Phenistix Acetest Nitrosonaphthol Cyanide nitroprusside Silver nitroprusside Clinitest Sodium nitroprusside p-nitroaniline CTAB Test Metachromatic staining Guthrie Test

Disorders Phenylketonuria and 5-HIAA Tyrosinuria Alkaptonuria Melanuria MSUD Indicanuria Phenylketonuria, MSUD, Tyrosinuria, Acidemias Phenylketonuria Tyrosinuria Melanuria MSUD, Acidemias Tyrosinuria, MSUD 5-HIAA Cystine disorders Alkaptonuria Homocystinuria Cystinosis, Melituria Melanuria Methylmalonic academia Mucopolysaccharidoses Mucopolysaccharidoses Phenylketonuria

18

Observation Blue-green Transient green Transient blue Gray-black Green-gray Violet-blue with CHCl3 Yellow Green-gray Transient green Red Purple Red Violet with HNO3 Red-purple Black Red-purple Orange-red Red Emerald green White turbidity Blue spot Growth of B. subtilis

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10. Pregnancy Testing A. Types of Pregnancy Test 1. Immunoassays a. Principle: Detection of hCG using monoclonal antibodies. b. Methods 1) Agglutination Immunoassays – direct or agglutination-inhibition 2) Competitive Radioimmunoassay a) Principle: serum hCG and the radiolabeled hCG compete for the binding of anti-hCG b) Sensitivity – 5 mIU/mL 3) EIA (Sandwich ELISA) a) Principle: Detection of hCG based on color indicator reaction mediated by an enzyme (e.g. ALP); commonly used in home-based pregnancy tests b) Sensitivity – 2-5 mIU/mL 4) Immunochromatography (lateral flow tests) a) Principle: The labeled antibody-dye conjugate in the reaction zone binds to the hCG in the specimen forming an antibody-antigen complex. This complex binds to the anti-hCG antibody in the test zone and produces a colored band when the hCG concentration is equal to or greater than 20 mIU/ml. In the absence of hCG, the reaction mixture continues flowing through the absorbent device past the test zone allowing the binding of unbound conjugates to the reagent in the control zone. b) Interpretation of Results:

2. Bioassays a. Principle: Introduction of hCG to a test animal produces characteristic changes in reproductive organs. b. Methods Test Animal Route of Injection Positive Result 1) AschheimImmature female Enlargement of the ovary; corpora Subcutaneous Zondek mouse hemorrhagica and corpora lutea 2) Friedmann/ Hyperemia of the uterus and Female virgin rabbit Free marginal ear vein Hoffman corpora hemorrhagica Subcutaneous (into the Oogenesis (Extrusion of eggs 8-12 3) Hogben Female frog dorsal lymph sac) hours after injection) Intramuscular Spermatogenesis (Release of 4) Galli-Mainini Male frog (gastrocnemius) sperm cells into the cloaca) 5) Frank-Berman Female virgin rat Subcutaneous Ovarian hyperemia 6) Kupperman Female virgin rat Intraperitoneal Ovarian hyperemia

B. Sources of Errors 1. False-positive: production of hCG in the pituitary; tumors characterized by significant amounts of hCG; ectopic pregnancy and incomplete abortion; intake of chlorpromazine, phenothiazine, and aspirin 2. False-negative: low titer or concentration of hCG; low sensitivity of test animal or assay method; use of toxic urine (bacterial contamination, increased electrolyte levels, salicylates, and barbiturates)

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11. Cerebrospinal Fluid  Imperfect ultrafiltrate of plasma produced in the choroid plexuses within the ventricles of the brain at a

rate of approx. 500 mL/day maintaining a total volume of 90-150 mL in adults or 10-60 mL in neonates.

A. Specimen Considerations 1. Specimen collection a. Lumbar puncture b. Other methods: cisternal puncture, lateral cervical puncture or through ventricular cannulas 2. Volume collected: up to 20 mL Order of draw, tests, and manner of storage Tube Tests Storage 1 Chemistry and immunology Frozen 2 Microbiology Room temperature 3 Cell count and differential Refrigerated 4 Additional tests Depends on test to be performed

B. Gross Examination 1. Appearance a. Normal: colorless and crystal clear b. Variations: 1) Hazy, cloudy, turbid, milky or purulent – WBCs and microorganisms, proteins in disorders that affect blood-brain barrier, and production of IgG within the CNS 2) Oily – radiographic contrast media 3) Clotted – proteins in disorders affecting blood-brain barrier; traumatic tap 4) Bloody – traumatic tap or subarachnoid hemorrhage Distribution of blood Supernatant Clot formation Erythrophagocytosis D dimer test

Traumatic tap Uneven Clear and colorless Present Absent Negative

Hemorrhage Even Xanthochromic Absent Present Positive

5) Xanthochromic – indicates presence of RBC degradation products or other pigments Supernatant color Pink Yellow

Orange Yellow-green Brown

Associated diseases/disorders RBC lysis/hemoglobin breakdown products RBC lysis/hemoglobin breakdown products Hyperbilirubinemia CSF protein > 150 mg/dL (1.5 g/L) RBC lysis/hemoglobin breakdown products Hypervitaminosis A (carotenoids) Hyperbilirubinemia (biliverdin) Meningeal metastatic melanoma

2. Viscosity a. Normal: similar to that of water b. Clinical significance of viscous CSF: metastatic mucin-producing adenocarcinomas, cryptococcal meningitis, liquid nucleus pulposus 20

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C. Chemical Examination 1. Total protein a. Normal values: 15 – 45 mg/dL or less than 1% of the plasma level b. Methods: 1) Turbidimetry – based on TCA or SSA and sodium sulfate protein precipitation; uses benzethonium or benzalkonium chloride as precipitating agents in automated methods and micromethods 2) Dye-Binding techniques – uses Coomassie brilliant blue or Ponceau S 3) Colorimetric-spectrophotometric methods – based on Lowry or Biuret method 4) Immunoassays – used to measure the amount of specific proteins e.g. MBP 5) Electrophoresis – detection of oligoclonal bands in the gamma region c. Protein fractions  Prealbumin, albumin, transferring, α-globulins (haptoglobin, ceruloplasmin, α2-macroglobulin), gamma globulins (IgG, IgA), tau protein (carbohydrate-deficient transferrin fraction)  Not present in significant amounts: fibrinogen, IgM, β-lipoprotein 2. Albumin and IgG Measurements a. CSF/Serum albumin index– reflects the integrity or permeability of the blood-brain barrier; values < 9 = intact BBB 𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑛𝑖𝑛 (𝑚𝑔/𝑑𝐿) 𝐶𝑆𝐹/𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 𝑖𝑛𝑑𝑒𝑥 = 𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿) b. CSF IgG index – calculated to measure IgG synthesis within the CNS values > 0.70 = IgG production within the CNS 𝐼𝑔𝐺 𝑖𝑛𝑑𝑒𝑥 =

𝐶𝑆𝐹 𝐼𝑔𝐺 (𝑚𝑔/𝑑𝐿)/𝑠𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 (𝑔/𝑑𝐿) 𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑚𝑔/𝑑𝐿)/𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔/𝑑𝐿)

3. Glucose a. Normal values: 50–80 mg/dL b. Important consideration: Blood sample should be drawn about 2 hours prior to spinal tap to allow time for equilibration between the blood and fluid glucose. 4. Lactate a. Normal values: <25 mg/dL b. Important consideration: Xanthochromic or hemolyzed fluid will produce falsely elevated results Chemical results for the differential diagnosis of meningitis Bacterial Tubercular Fungal Viral Protein

Markedly elevated

Glucose

Markedly decreased (usually ≤40 mg/dL) > 35 mg/dL

Lactate

Moderate to markedly elevated Decreased (may be <45 mg/dL) > 25 mg/dL

Moderate to markedly elevated Normal to decreased > 25 mg/dL

Moderately elevated Normal Normal

5. Glutamine a. Normal values: 8 to 18 mg/dL b. Clinical significance of elevated levels: liver disorders that result in increased blood and CSF ammonia, Reye’s Syndrome, coma of unknown origin, and disturbance of consciousness 6. Enzymes a. CK-BB isoenzyme – increases about 6 hours following ischemic or anoxic insult b. LD – increased in patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid hemorrhage c. ADA – increased in tubercular meningitis (abundant in T lymphocytes) d. Lysozyme – significantly increased in patients with both bacterial and tubercular meningitis

D. Microscopic Examination 1. Total cell count  Dilution (using normal saline), counting, and calculation of the number per µL are done using the same procedure as WBC count; Fuchs-Rosenthal–type chamber may be used 21

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2. WBC count a. Normal values: <5/µL (adult), <30/µL (neonates) b. Recommended Dilutions: Clarity

Dilution

Slightly hazy Hazy Slightly cloudy Slightly bloody Cloudy, bloody, turbid

1:10 1:20 1:100 1:200 1:10,000

Amount of sample

30 µL 1 mL of 1:100 dilution

Amount of Diluent (3% acetic acid) 270 µL 570 µL 2, 970 µL 5, 970 µL 9 mL

c. Calculation: 𝑊𝐵𝐶/µ𝐿 =

𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑁𝑜. 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑎𝑐𝑕 𝑠𝑞𝑢𝑎𝑟𝑒

3. Differential Cell Count Predominant cells seen in CSF Type of Cell Clinical Significance Lymphocytes and Monocytes Normal (70:30 in adults) Neutrophils Bacterial meningitis, cerebral hemorrhage Erythrophages Intracranial hemorrhage Blast forms Leukemias and lymphomas Plasma cells Multiple sclerosis, lymphocyte reactions Ependymal and choroidal cells Diagnostic procedures Malignant cells Metastatic carcinomas, primary CNS carcinoma Eosinophils Intracranial shunt malfunctions nRBCs Bone marrow contamination during spinal tap

E. Microbiologic and Serologic Examination 1. 2. 3. 4. 5. 6. 7. 8.

Gram stain and culture Limulus lysate test Bacterial agglutination tests (immunochromatographic assay, latex agglutination) Nucleic acid amplification tests (PCR and 16S rRNA sequencing) Acid-fast stain or fluorescent antibody stains and culture DOT ELISA India ink or nigrosin stain Latex agglutination test (detection of cryptococcal antigen)

12. Semen  Composition: spermatozoa 5%, seminal fluid 60%–70%, prostatic fluid 20%–30%, and bulbourethral

secretions 5%

A. Specimen Considerations 1. Specimen collection a. Masturbation – recommended method b. Condom method – Silastic or nonlubricant-containing rubber or polyurethane condoms c. Vaginal vault aspiration d. Coitus interruptus 22

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2. Important considerations a. Patient preparation – sexual abstinence of 2 to 5 days; must empty bladder before collection b. Specimen container – prewarmed sterile glass or plastic containers c. Transport – kept at 37 °C, delivered to the laboratory within 1 hour of collection d. Fertility testing – 2-3 samples tested at 2-week intervals; 2 abnormal samples considered significant

B. Gross Examination Parameter Findings 1. Appearance Gray-white, translucent Yellow Red/ rust color Turbid 2. Volume 2–5 mL <2 mL >5 mL 3. Liquefaction 30 to 60 min >2 hours 4. Viscosity Pours in droplets Clumped, stringy, or gel-like 5. pH 7.2 to 8.0 >8.0 <7.2

Clinical significance Normal Pyospermia, contamination, prolonged abstinence, medications Bleeding Infection Normal Improper functioning of one of the semen-producing organs Prolonged abstinence Normal Deficiency in prostatic enzymes Normal Deficiency in prostatic enzymes Normal Infection within the reproductive tract Increased prostatic fluid

C. Microscopic Examination 1. Sperm concentration and count a. Recommended procedure:  Dilution: 1:20 using sodium bicarbonate in formalin, saline, or distilled water  Both sides of a Neubauer counting chamber are loaded and counted.  Calculation: When using the above procedure and counting in the typical WBC squares, Sperm/mL = sperm counted (average of 2 sides) x 100,000 When using the above procedure and counting in the typical RBC squares, Sperm/mL = sperm counted (average of 2 sides) x 1,000,000 To solve for total sperm count, Sperm/ejaculate = sperm/mL x specimen volume b. Normal values: >20 million/mL (concentration); >40 million/ejaculate (count) c. Clinical significance of abnormal counts: azoospermia, necrospermia, oligospermia 2. Sperm motility a. Recommended procedure:  Performed on well mixed, undiluted, liquefied semen within 1 hour of collection  Speed and direction are both evaluated using approximately 20 hpfs Grade Interpretation 4 Rapid, straight line motility 3 Slower speed, some lateral movement 2 Slow forward progression, noticeable lateral movement 1 No forward progression 0 No movement b. Normal % motility: >50% within 1 hr, quality >2.0 c. Clinical significance of abnormal motility: midpiece and tail abnormalities 3. Sperm morphology a. Recommended procedure:  Evaluation is done on a thin smear stained using Wright’s, Giemsa, or Papanicolaou (best stain)  200 sperm are evaluated under OIO for abnormalities in the head, midpiece, and tail  Strict criteria: oval-shaped head (approx. 5 x 3 µm) tail approx. 45 µm long acrosomal cap normal in size no big cytoplasmic droplet  % Normal forms: >14% (strict criteria), >30% (routine criteria) 23

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D. Additional Testing Additional tests for abnormal semen analysis Routine semenalysis findings

Additional test

Decreased motility with normal count Decreased count

Eosin-nigrosin stain Fructose level determination MAR, Immunobead test, Decreased motility with clumping Agglutination with male serum Normal analysis with continued infertility Agglutination with female serum Test Hamster egg penetration Cervical mucus penetration Hypo-osmotic swelling test In vitro acrosome reaction

Test Neutral α-glucosidase Zinc Citric acid Acid phosphatase

Clinical significance of abnormal result Necrospermia Lack of support medium Male antisperm antibodies Female antisperm antibodies

Sperm function tests Function evaluated Ovum penetration Ability to penetrate partner’s midcycle cervical mucus Membrane integrity and sperm viability Ability to produce enzymes essential for ovum penetration Chemical tests Normal values Significance of decreased values ≥20 mU/ejaculate Disorder of the epididymis ≥2.4 µmol/ejaculate ≥52 µmol/ejaculate Lack of prostatic fluid ≥200 Units/ejaculate

13. Synovial Fluid  Imperfect ultrafiltrate of plasma less than 3.5 mL in volume containing hyaluronic acid and a small

amount of protein produced by the synoviocytes

A. Specimen Considerations Specimen collection a. Arthrocentesis – volume usually collected is about 25 mL b. Tubes and tests performed 1) sterile heparinized tube – Gram stain and culture 2) sodium heparin or liquid EDTA tube – cell counts 3) nonanticoagulated tube – chemical and serologic tests 4) sodium fluoride tube – glucose analysis

B. Gross Examination 1. Color a. Normal: colorless to pale yellow b. Variations: green tinge (septic arthritis), deep yellow (inflammatory and noninflammatory disorders), red-brown (pathologic hemarthrosis) 2. Clarity a. Normal: clear b. Variations: turbid (leukocytes, fibrin, rice bodies, metal and plastic particles from patients with joint prostheses, or cartilage fragments in osteoarthritis), milky and opalescent (crystals), ground pepper appearance (pigmented cartilage fragments resulting from a metabolic disorder) 24

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3. Viscosity a. Falling drop – ability to form a string 4–6 cm long b. Ropes/Mucin clot test – 2% to 5% acetic acid is added to the fluid and the formation of clot is observed; results are reported as good (solid clot), fair (soft clot), low (friable clot), and poor (no clot).

C. Chemical Examination Test Glucose Total protein Lactate Uric acid

Clinical significance of synovial fluid chemistry results Normal values Clinical significance 70-110 mg/dL Decreased in inflammatory or septic arthritis < 3 g/dL Increased in inflammatory and hemorrhagic arthritis < 30 mg/dL Increased in septic arthritis 2–8 mg/dL Increased in gouty arthritis

Finding

Group I Noninflammatory

Viscosity Glucose WBCs/µL PMNs (%)

Good Normal <1000 <30

Differential diagnosis of joint disorders Group II Inflammatory Group III Septic Immunologic Crystal-induced Poor Low Variable Decreased Decreased Decreased 2000–75000 Up to 100000 50000–100000 >50 <70 >75

Group IV Hemorrhagic Low Normal Equal to blood <50

D. Microscopic Examination 1. WBC count a. Normal values: < 200 cells/µL b. Dilutions: done only on turbid or bloody fluids using 0.3% saline or 1% saponin in saline c. Clinical significance of elevated counts: crystal-induced arthritis, chronic inflammatory arthritis, and septic arthritis 2. Differential count * Incubation of the fluid with hyaluronidase and cytocentrifugation are recommended. Cell/Inclusion Neutrophil Lymphocyte Macrophage Synovial lining cell LE cell Reiter cell RA cell (ragocyte) Cartilage cells Rice bodies Fat droplets Hemosiderin

Cells and inclusions seen in synovial fluid Comments/Clinical significance Normally < 25% of the differential; increased in septic and crystal-induced arthritis Normally 15% of the differential; increased in nonseptic inflammation Normall seen; increased in viral infections Normally seen; similar to macrophage but may be multinucleated resembling a mesothelial cell Neutrophil containing characteristic ingested round body; seen in lupus erythematosus Vacuolated macrophage with ingested neutrophils; seen in Reiter syndrome Neutrophil with dark cytoplasmic granules containing immune complexes; seen in RA Large, multinucleated cells; seen in osteoarthritis Macroscopically resemble polished rice; microscopically show collagen and fibrin; seen in tuberculosis, septic and rheumatoid arthritis Refractile globules stained with Sudan dyes; seen in traumatic injury and chronic inflammation Inclusion within clusters of synovial cells; seen in pigmented villonodular synovitis

3. Crystal examination a. Compensated polarized light – used to demonstrate crystal polarization by placing a red compensator between the crystal and the analyzer b. Betamethasone acetate corticosteroid – control slide for the polarization properties of MSU c. Negative birefringence – yellow color under a compensated polarized light produced by a crystal when it is aligned to the slow vibration of the compensated polarized light d. Positive birefringence – blue color produced by a crystal when it is aligned to the slow vibration of the compensated polarized light 25

CLINICAL MICROSCOPY



Characteristics of synovial fluid crystals Crystal Shape Birefringence Monosodium urate Needles Negative Calcium pyrophosphate Rhombic square, rods Positive Cholesterol Notched, rhombic plates Negative Corticosteroid Flat, variable-shaped plates Positive and negative Calcium oxalate Envelopes Negative Calcium phosphate (apatite) Small particles None

Significance Gout Pseudogout Chronic inflammation Injections Renal dialysis Osteoarthritis

D. Microbiologic and Serologic Examination 1. Septic arthritis: Gram stain and culture, PCR 2. Immunologic arthritis: Test for RF, ANA, and antibodies to B. burgdorferi; CRP and complement levels

14. Serous Fluids  Ultrafiltrates of plasma contained within the pleural, pericardial, and peritoneal cavities with no

additional material from membrane cells.

A. Specimen Considerations 1. Collection techniques: thoracentesis, pericardiocentesis, paracentesis 2. Volume collected: > 100 mL 3. Collection tubes: a. EDTA – cell counts and differential b. sterile heparinized – microbiology and cytology procedures c. nonanticoagulated or heparinized – chemistry tests

B. Differentiation of Transudates and Exudates Clarity Color Spontaneous clotting pH Specific gravity Glucose Total protein LDH Pleural fluid cholesterol PF:serum cholesterol ratio PF:serum bilirubin ratio Fluid:serum protein ratio Fluid:serum LD ratio Serum-ascites albumin gradient WBC count

Transudate Clear Colorless to pale yellow No Alkaline < 1.015 As in plasma level < 3 g/dL < 200 IU/L < 45 mg/dL < 0.3 < 0.6 < 0.5 < 0.6 > 1.1 < 1000/µL

C. Gross Examination 1. Appearance a. Normal: clear and colorless to pale yellow 26

Exudate Depends on condition Depends on condition Possible Acidic > 1.015 Lower than plasma level > 3 g/dL > 200 IU/L > 45 mg/dL > 0.3 > 0.6 > 0.5 > 0.6 < 1.1 > 1000/µL

CLINICAL MICROSCOPY



b. Variations: 1) Turbid, white – microbial infection 2) Bloody or milky Clinical significance of bloody and milky effusions Bloody Hemothorax Hemorrhagic exudates Distribution of blood Uneven/streaked Even Hematocrit >50% of blood hematocrit <50% of blood hematocrit Milky Chylous Pseudochylous Appearance Milky/white Milky/green tinge Leukocytes Predominantly lymphocytes Mixed cells Cholesterol crystals Absent Present Triglycerides >110 mg/dL <50 mg/dL Sudan III staining Strongly positive Negative/weakly positive Clinical significance of other abnormal gross presentations Serous fluid Appearance Disease Pleural Brown Rupture of amoebic liver abscess Black Aspergillus infection Viscous Malignant mesothelioma Pericardial Grossly bloody Cardiac puncture, anticoagulant medications Peritoneal Green Gallbladder, pancreatic disorders

D. Chemical Examination Serous fluid Pleural

Pericardial Peritoneal

Clinical significance of other chemistry tests Test Significance Glucose Rheumatoid inflammation and purulent infection Lactate Bacterial infection Triglyceride Chylous effusions pH Pneumonia; esophageal rupture Adenosine deaminase Tuberculosis and malignancy Amylase Pancreatitis, esophageal rupture, and malignancy Adenosine deaminase Tubercular pericarditis Glucose Tubercular peritonitis and malignancy Amylase Pancreatitis and gastrointestinal perforation Alkaline phosphatase Gastrointestinal perforation BUN and creatinine Ruptured or punctured bladder Adenosine deaminase Tubercular peritonitis

D. Microscopic Examination Differential count: routinely performed to examine WBCs and demonstrate malignant cells Clinical significance of cells seen in serous fluids Serous fluid Test Significance Pleural Mesothelial cells Decreased in tuberculosis Plasma cells Increased in tuberculosis WBC >500 cells/µL Peritoneal Bacterial peritonitis, cirrhosis RBC >100,000/µL Intraabdominal bleeding (blunt trauma injury) Absolute granulocyte count >250 cells/µL Bacterial peritonitis

E. Microbiologic and Serologic Examination 1. 2. 3. 4. 5.

Gram staining and culture PCR Acid-fast staining Tumor markers and cytologic examination Measurement of RF/SLE titer and complement levels 27

CLINICAL MICROSCOPY



15. Amniotic Fluid st

rd

 Volume: approximately 35 mL during the 1 trimester, peaks during the 3 trimester (approx. 1 L)

Polyhydramnios – results from failure of the fetus to begin swallowing; indicates fetal distress Oligohydramnios – due to increased fetal swallowing, urinary tract deformities, and membrane leakage

A. Specimen Considerations 1. Specimen collection th a. Amniocentesis– transabdominal or transvaginal; performed after the 14 week of gestation b. Volume collected: 30 mL 2. Specimen handling a. Fluid for FLM tests – transported in ice and refrigerated up to 72 hours prior to testing; filtration or lowspeed centrifugation is recommended to prevent loss of phospholipids b. Fluid for cytogenetic test – incubated at 37°C prior to analysis to prolong the life of the cells c. Fluid for chemical testing – separated from cellular elements and debris ASAP d. Fluid for bilirubin analysis – placed in amber bottles

B. Gross Examination Appearance Colorless Blood-streaked Yellow Dark green Dark red-brown

Clinical significance of amniotic fluid appearance Significance Normal (may show slight to moderate turbidity) Traumatic tap, abdominal trauma, intra-amniotic hemorrhage Hemolytic disease of the newborn Meconium Fetal death

C. Tests 1. Tests for Fetal Lung Maturity Test

Method/Principle

Thin-layer chromatography Agglutination 2. Amniostat-FLM immunoassay 3. Foam Stability Index Modified foam-shake 4. Microviscosity Fluorescence- polarization 5. Lamellar body count Resistance pulse counting 6. OD at 650 nm Spectrophotometry 1. L/S ratio

Comments

Normal values

Sphingomyelin used as internal standard; greatly ≥2.0 affected by blood and meconium contamination Uses antisera specific to phosphatidylglycerol; not Positive affected by blood and meconium contamination 95% ethanol used as anti-foaming agent ≥47 Albumin used as internal standard ≥55 mg/g Uses the platelet channel of hematology analyzers ≥32,000/mL Requires centrifugation at 2000 g for 10 min ≥0.150

2. Tests for Fetal Distress Test 1. Bilirubin ΔA450 2. AFP

Method/Principle

3. AChE

Spectrophotometry

Spectrophotometry Immunoassay

Comments ΔA450 plotted on a Liley graph to determine seveity of HDN; Hgb and meconium interfere Screening test for NTDs Confirmatory test for NTDs; greatly affected by blood contamination

28

Normal values <0.025 <2.0 MoM Undetectable

CLINICAL MICROSCOPY



16. Feces A. Gross Examination Significance of stool macroscopic characteristics Color Clinical significance Brown Normal (stercobilin, urobilin) Black Upper GI bleeding, iron therapy, charcoal, bismuth Red Lower GI bleeding, beets and food coloring, rifampin Pale yellow, white, or gray Bile-duct obstruction, barium sulfate Green Biliverdin, oral antibiotics, green vegetables Appearance Clinical significance Soft to well-formed Normal Watery Diarrhea Small, hard Constipation Bulky, frothy Bile-duct obstruction, pancreatic disorders Ribbon-like, slender Intestinal constriction Mucoid, blood-streaked Colitis, dysentery, malignancy, constipation

B. Chemical Examination Test FOBT Van de Kamer Apt test

Method/principle Pseudoperoxidase activity of hemoglobin Fecal fat extraction and titration of fatty acids Differentiation between fetal and maternal blood

Trypsin

Gelatin hydrolysis

Carbohydrates

Copper reduction

Fecal chemistry tests Reagent Result Guaiac

Blue color

NaOH (titrant)

1-6 g/day >6 g/day Pink NaOH Yellow-brown Gelatin on an Clearing x-ray film Absence of clearing Clinitest Orange-red (0.5 g/dL)

Significance GI bleeding Normal Steatorrhea Presence of HbF Presence of HbA Normal Pancreatic insufficiency Carbohydrate intolerance

D. Microscopic Examination Test Muscle fibers

Qualitative fecal fats Fecal neutrophils

Significance of stool microscopic findings Method/Principle Significance Examination of eosin-stained >10 undigested muscle fibers smear to visualize muscle indicate pancreatic fiber striations insufficiency Examination of direct smear 60 large orange-red droplets stained with Sudan III indicates malabsorption Examination of smear heated 100 orange-red droplets (6–75 with acetic acid and Sudan III µm) indicates malabsorption Microscopic count of 3/hpf indicates invasive neutrophils in stained smear condition

Other comments Only fibers with visible vertical and horizontal striations are counted Detects neutral fats only Represents total fat content 70% sensitive

*Lactoferrin latex immunoassay – detects secondary granule component of neutrophils; remains sensitive in refrigerated and frozen specimens 29

CLINICAL MICROSCOPY



17. Sputum and BAL A. Specimen Considerations Specimen collection a. Expectoration – first morning; may require induction (10% NaCl, acetylcysteine, sterile distilled water) b. Bronchoalveolar lavage – infusion of saline through a bronchoscope followed by aspiration c. Throat swab/ Cough plate or cough swab method d. Endotracheal aspiration

B. Gross Examination of Sputum 1. Color a. Yellow/yellow-green – presence of pus (pulmonary TB, chronic bronchitis) b. Bright green – jaundice, caseous pneumonia, Pseudomonas infection, rupture of liver abscess c. Red/bright red – recent hemorrhage (acute cardiac or pulmonary infarction, neoplasm invasion) d. Rust-colored – decomposed hemoglobin (lobar or pneumococcal pneumonia, pulmonary gangrene) e. Brown – congestive heart failure f. Olive green/grass green – chronic cancer g. Black – dust particles, carbon or charcoal, heavy smokers, anthracosis 2. Macroscopic Structures a. Cheesy masses – fragments of necrotic pulmonary tissue that range in size from pinpoint to pea-size; seen in pulmonary gangrene, pulmonary TB, and lung abscess b. Dittrich’s plugs – yellowish or gray caseous materials about the size of a pinhead that give a foul odor when crushed; seen in bronchiectasis, putrid bronchitis, and bronchial asthma c. Pneumoliths/Broncholiths/Lung stones – small white or gray fragments from the calcification of infected and necrotic tissue within the bronchial cavity; seen in chronic PTB and histoplasmosis d. Bronchial casts – white or gray branching tree-like casts from the bronchioles; seen in lobar pneumonia and fibrinous bronchitis e. Mycetomas – rounded masses of fungal elements seen in Aspergillus infection

C. Microscopic Examination of Sputum Microscopic Structures a. Curschmann’s spirals – spirally twisted mucoid strands frequently coiled into little balls b. Elastic fibers – refractile fibers shed off during the cougning out process; indicates destructive disease c. Crystals 1) Charcot-Leyden – hexagonal, needle-like or bipyramidal crystals; seen in bronchial asthma 2) Hematoidin – rhombic-shaped crystals; seen in pulmonary infarction and lung abscess 3) Cholesterol – notched plates; seen in lung abscess d. Cells and Inclusions 1) Creola bodies – bronchial epithelial cells with vacuolated cytoplasm and ciliated borders 2) Carbon-laden or dust cells – contain black granules; seen in pneumoconioses 3) Heart failure cells/siderophages – hemosiderin-laden cells seen in CHF and alveolar hemorrhage

D. Microscopic Examination of Bronchoalveolar Lavage 1. Cell count  Appropriate dilution for WBC and RBC counts using the Unopette system  Both sides of the hemocytometer are counted and the average of the two sides is calculated using the standard Neubauer formula 2. Differential count  slides are prepared by cytocentrifugation; 500 to 1000 cells (or at least 300) are counted and classified 30

CLINICAL MICROSCOPY



Cells and inclusions seen in BAL specimens Cells/inclusions Clinical significance Interstitial lung disease, drug reactions, pulmonary Lymphocytes lymphoma, and nonbacterial infections Cigarette smokers, bronchopneumonia, toxin exposure, Neutrophils and diffuse alveolar damage Asthma, drug-induced lung disease, parasitic infections, Eosinophils hypersensitivity, pneumonitis, and eosinophilic pneumonia Erythrocytes, erythrophages, siderophages Alveolar hemorrhage Bronchial epithelial cells Normal; more numerous in bronchial wash specimens Sulfur granules Actinomyces infection Langerhans cells Cigarette smokers, Langerhans cell histiocytosis Fat droplets/ lipid-laden macrophages Fat embolism Dust particle inclusions Pneumoconioses or asbestos exposure

E. Microbiologic Examination 1. 2. 3. 4.

Bacterial pathogens: M. tuberculosis, L. pneumophila, M. pneumoniae, Actinomyces spp. Fungal pathogens: Pneumocystis jiroveci, Cryptococcus neoformans, Histoplasma capsulatum Parasites: P. westermani, S. stercoralis, E. histolytica, E. granulosus Viruses: Influenza A and B, respiratory syncytial virus

18. Gastric Fluid A. Specimen Considerations Specimen collection a. Evacuation/gastric tubes: 1) Rehfuss’ tube – has a metal tip; swallowed by gravity; for both gastric and duodenal fluid collection 2) Levine’s tube – has the smallest diameter; inserted through the nose b. Stimulants: 1) Test meals – poor gastric stimulants (e.g. Ewald’s, Boa’s, Reigel’s, or Alcohol test meal) 2) Histamine – exerts unpleasant systemic effects on blood vessels and smooth muscles 3) Histalog/Betazole – histamine isomer with preferential effect on gastrin secretion 4) Insulin (hypoglycemia test) – used to determine completeness of vagotomy 5) Pentagastrin – stimulant of choice resembling gastrin; more rapid response than Histalog

B. Gross Examination 1. Appearance 2. Volume

a. Normal: colorless or pale gray and transluscent b. Variations: green (old bile), yellow (fresh bile), red (blood), coffee brown (old blood) a. Normal: 20-50 mL after a test meal; 45-150 mL after chemical stimulation b. Increased volume: hypomotility, pyloric obstruction, Zollinger-Ellison syndrome c. Decreased volume: gastric hypermotility

C. Chemical Examination 1. Gastric Acidity a. Total Acidity: 40-70 mEq/L b. Free HCl: 20-40 mEq/L 2. Clinical Significance a. Hyperchlorhydria – increased free HCl seen in peptic ulcer b. Hypochlorhydria – decreased free HCl seen in chronic gastritis, gastric ulcer, and stomach CA c. Achlorhydria – absence of free HCl seen in pernicious anemia, advanced gastric ulcer, and pellagra 31

CLINICAL MICROSCOPY



FIGURES

Fig 1. Sagittal section of the right kidney

Fig 2. Parts of a nephron

Fig 3. Formation and degeneration of casts 32

CM Handout

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