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Viva Questions & Answers: Urine Examination in Pathology

PART 1 - GENERAL URINE EXAMINATION

Q1. Which sample of urine is best for routine examination? A: Early morning, mid-stream urine sample (first void). It is concentrated, has consistent pH, and cellular elements are better preserved.

Q2. What are the three parts of routine urine examination? A:

Physical examination (colour, appearance, volume, odour, specific gravity)
Chemical examination (pH, protein, sugar, ketone bodies, bile salts/pigments, blood)
Microscopic examination (casts, cells, crystals, organisms)

Q3. What is the normal volume of urine excreted in 24 hours? A: 1000-1500 mL/day (average 1200 mL).

Q4. Define oliguria, anuria, polyuria, and nocturia. A:

Oliguria: Urine output < 400 mL/day
Anuria: Urine output < 100 mL/day (no urine)
Polyuria: Urine output > 2500 mL/day
Nocturia: Excessive urination at night

Q5. What is the normal colour of urine and what causes variations? A: Normal urine is pale yellow to amber (due to urochrome pigment).

Dark yellow/amber: dehydration, fever
Red/pink: hematuria, hemoglobinuria, myoglobinuria, beetroot ingestion, rifampicin
Dark brown/black: alkaptonuria, melanuria
Green: biliverdin, Pseudomonas infection
Pale/colourless: diabetes insipidus, overhydration, chronic renal failure

Q6. What is the normal specific gravity of urine? A: 1.003-1.030 (typically 1.015-1.025).

Increased in: dehydration, excessive sweating, acute nephritis, glycosuria, albuminuria
Decreased in: diabetes insipidus, chronic nephritis, polyuria

Q7. How is specific gravity measured? A:

Urinometer: Float the instrument in urine; read the meniscus. Temperature correction: add 0.001 for every 3°C above calibration temperature; subtract 0.001 for every 3°C below.
Refractometer: Measures refractive index. Needs only 1-2 drops; no temperature correction needed. More accurate.

Q8. What is the normal pH of urine? A: 4.5 to 8.0 (usually slightly acidic, ~6.0).

Acidic urine: starvation, diabetes, high-protein diet, diarrhea, dehydration
Alkaline urine: vegetarian diet, urinary tract infection (urea-splitting bacteria), renal tubular acidosis, prolonged standing (urea → ammonia)

Q9. What are the changes that occur in standing urine at room temperature? A:

Increase in pH (alkalinization)
Turbidity due to crystal/bacterial growth
Loss of ketone bodies (volatile)
Oxidation of bilirubin to biliverdin
Oxidation of urobilinogen to urobilin
Bacterial multiplication
Decrease in glucose (bacterial consumption)
Disintegration of cellular elements (casts, RBCs, WBCs)

Q10. How is urine preserved? A:

Refrigeration at 4-6°C for up to 8 hours
Toluene: 1 mL per 50 mL urine - forms surface layer, preserves chemical constituents
Formalin: 6-8 drops of 40% formalin per 100 mL - preserves RBCs and pus cells (false positive for sugars)
Thymol: 1% solution (false positive for proteins)
Boric acid / HCl / H2SO4: for specific chemical tests

Q11. What is the significance of urine odour? A:

Normal: slightly aromatic
Fruity/acetone: ketonuria (diabetes mellitus, starvation)
Mousy/musty: phenylketonuria (PKU)
Maple syrup: maple syrup urine disease (MSUD)
Foul/ammonia: bacterial decomposition/UTI

PART 2 - BENEDICT'S TEST (Test for Reducing Sugar/Glucose)

Q12. What is Benedict's test? A: A qualitative test for detecting reducing sugars (especially glucose) in urine.

Q13. What is the principle of Benedict's test? A: Glucose (a reducing sugar) reduces the cupric ions (Cu²⁺) in Benedict's reagent (alkaline copper sulfate solution containing sodium citrate and sodium carbonate) to cuprous oxide (Cu₂O), which forms a coloured precipitate. The reaction is:

Cu²⁺ (blue) + Reducing sugar → Cu₂O (coloured precipitate) + oxidized sugar

Q14. What is the composition of Benedict's reagent? A: Copper sulfate (CuSO₄), sodium citrate, and sodium carbonate in water (alkaline solution).

Q15. What is the procedure for Benedict's test? A: Take 5 mL of Benedict's reagent in a test tube. Add 8 drops of urine. Boil for 1-2 minutes (or place in boiling water bath for 5 minutes). Observe the colour change.

Q16. How do you interpret the results of Benedict's test?

Colour of Precipitate	Grading	Approximate Glucose (g%)
Blue (no change)	Negative	0
Green precipitate	+	0.5%
Yellow/green precipitate	++	1.0%
Orange precipitate	+++	1.5%
Brick red precipitate	++++	2.0% or more

Q17. What is glycosuria? What are its causes? A: Presence of glucose in urine.

Diabetes mellitus (most common)
Renal glycosuria: Normal blood glucose but low renal threshold (<180 mg/dL)
Hyperadrenalism (Cushing's syndrome)
Hyperthyroidism
Pregnancy (gestational)
Alimentary glycosuria: after excessive carbohydrate ingestion
Stress glycosuria: after head injury, stroke

Q18. What is the renal threshold for glucose? A: Normally ~180 mg/dL. When blood glucose exceeds this level, glucose appears in urine.

Q19. Which sugars other than glucose can give a positive Benedict's test? A: All reducing sugars - lactose, fructose, galactose, pentose, maltose. (Sucrose is a non-reducing sugar and gives a negative result.)

Q20. What are the causes of a false positive Benedict's test? A:

Lactose in urine (lactosuria - normal in pregnancy/lactation)
Drugs: cephalosporins, penicillin (large doses), nalidixic acid, salicylates, ascorbic acid (Vitamin C), formalin (if used as preservative)
Galactose (galactosemia), fructose, homogentisic acid (alkaptonuria)

Q21. What is the difference between Benedict's test and glucose oxidase test (dipstick)? A:

Benedict's detects ALL reducing sugars (non-specific)
Glucose oxidase (dipstick) is specific ONLY for glucose
Drugs like cephalosporins give false positive with Benedict's but NOT with glucose oxidase strip

PART 3 - HEAT AND ACETIC ACID TEST (Test for Protein/Albumin)

Q22. What is the Heat and Acetic Acid test? A: A qualitative test for detecting protein (mainly albumin) in urine.

Q23. What is the principle of the Heat and Acetic Acid test? A: Proteins are denatured and precipitated on heating. Acetic acid is added to:

Maintain an acidic pH (proteins precipitate best at their isoelectric point, near pH 4-5)
Dissolve phosphates and carbonates that may form a false white precipitate on heating
Distinguish protein precipitate (persists/increases with acid) from phosphate/carbonate precipitate (dissolves with acid)

Q24. What is the procedure for the Heat and Acetic Acid test? A:

Fill a test tube 2/3 with urine
Tilt the tube and heat only the UPPER part over a flame until it boils
Observe for turbidity/precipitate in the upper part (compare with the lower unheated portion)
If turbidity appears, add 2-3 drops of 5% acetic acid
Observe again

Q25. Why is only the upper part of the test tube heated? A: The lower unheated portion acts as a control. By comparing the heated upper part with the clear lower part, any turbidity in the heated portion can be confirmed as protein. This avoids confusion with naturally turbid urine.

Q26. How do you interpret the Heat and Acetic Acid test?

Observation	Interpretation
No turbidity	Negative (no protein)
Turbidity appears on heating, dissolves on adding acetic acid	Phosphates/carbonates (false positive - not protein)
Turbidity appears on heating, persists/increases with acetic acid	POSITIVE - Protein present
Turbidity appears on heating, dissolves on heating (not precipitate)	May be radiographic contrast media

Q27. What are the causes of proteinuria? A:

Pre-renal: fever, cardiac failure, severe anaemia, hypertension, myeloma
Renal (glomerular): glomerulonephritis, nephrotic syndrome, diabetic nephropathy
Renal (tubular): tubular damage, Fanconi syndrome
Post-renal: UTI, renal tuberculosis, bladder carcinoma, urethritis
Orthostatic/Postural proteinuria: appears when standing, disappears when lying down (benign)

Q28. Which proteins are detected by the heat and acetic acid test? A: Mainly albumin, also globulins. However, Bence Jones proteins (light chains in myeloma) have a unique behavior - they precipitate on heating to 40-60°C and RE-DISSOLVE on boiling at 100°C.

Q29. What is Bence Jones proteinuria and its significance? A: Bence Jones proteins are immunoglobulin light chains (free kappa or lambda chains) excreted in urine. Seen in multiple myeloma, Waldenström macroglobulinemia, and amyloidosis. They precipitate at 40-60°C and redissolve at 100°C (pathognomonic behavior).

Q30. What drugs/substances can cause false positive in the heat and acetic acid test? A:

Thymol (used as preservative)
Radiographic contrast media (X-ray dye) - but the precipitate dissolves with heat
Penicillin (large doses)
Highly concentrated urine

PART 4 - ROTHERA'S TEST (Test for Ketone Bodies)

Q31. What is Rothera's test? A: A qualitative test for detecting ketone bodies (acetone and acetoacetic acid) in urine.

Q32. What are ketone bodies? Name them. A: Ketone bodies are intermediary metabolites of fat metabolism:

Acetoacetic acid (acetoacetate) - major ketone
Beta-hydroxybutyric acid (3-hydroxybutyrate) - major in DKA
Acetone (from spontaneous decarboxylation of acetoacetate)

Q33. What is the principle of Rothera's test? A: Acetone and acetoacetic acid react with sodium nitroprusside [Na₂Fe(CN)₅NO] in the presence of concentrated ammonia (alkali) to form a purple/permanganate-coloured ring at the junction of the two layers. This is known as the Legal reaction or nitroprusside reaction.

Note: Beta-hydroxybutyric acid does NOT react with nitroprusside (major limitation).

Q34. What is Rothera's mixture (reagent)? A: A mixture of ammonium sulfate and sodium nitroprusside (ground together in a mortar). Some protocols use crystals separately.

Q35. What is the procedure for Rothera's test? A:

Take 5 mL of urine in a test tube
Saturate it with Rothera's mixture (ammonium sulfate + sodium nitroprusside powder)
Gently add 1-2 mL of concentrated ammonia solution down the side of the tube
Allow to stand (do not mix)
Observe for a coloured ring at the junction of the two layers

Q36. How do you interpret Rothera's test?

Observation	Result
No ring	Negative
Faint pink ring	Trace
Purple/permanganate-coloured ring at junction	POSITIVE - Acetone/acetoacetic acid present

Q37. What is ketonuria? What are its causes? A: Presence of ketone bodies in urine (>1 mg/24 hr is abnormal).

Diabetic ketoacidosis (DKA) - most important
Starvation and prolonged fasting
Vomiting and diarrhoea
High-fat, low-carbohydrate diet
Eclampsia of pregnancy
Acute febrile illnesses in children
Thyrotoxicosis
Glycogen storage diseases

Q38. Why does Rothera's test not detect beta-hydroxybutyric acid? A: Beta-hydroxybutyrate lacks a keto group (it's a hydroxyl acid), so it does NOT react with nitroprusside. In severe DKA, beta-hydroxybutyrate predominates, so Rothera's test may be weakly positive or even negative despite significant ketosis.

Q39. What is the normal level of ketone bodies in urine? A: Up to 1 mg/24 hours (not normally detectable by routine tests).

Q40. What is ketosis vs ketonemia vs ketonuria? A:

Ketosis: Accumulation of ketone bodies in body fluids
Ketonemia: Excess ketone bodies in blood (normal: < 1 mg/dL; in DKA: > 3 mmol/L)
Ketonuria: Excess ketone bodies excreted in urine

PART 5 - HAY'S SULPHUR TEST (Test for Bile Salts)

Q41. What is Hay's Sulphur test? A: A simple qualitative test for detecting bile salts in urine.

Q42. What is the principle of Hay's Sulphur test? A: Bile salts act as surface-active agents (emulsifying agents/detergents). They lower the surface tension of urine. Sulphur powder, which normally floats on the surface of water due to surface tension, sinks to the bottom when bile salts are present (because surface tension is reduced). In normal urine (no bile salts), sulphur powder floats.

Q43. What is the procedure for Hay's Sulphur test? A:

Take 3 mL of urine (test) in one test tube
Take 3 mL of distilled water (control) in another test tube
Sprinkle a pinch of sulphur powder gently on the surface of both

Q44. How do you interpret Hay's Sulphur test?

Observation	Result
Sulphur powder floats (like in control)	Negative - No bile salts
Sulphur powder sinks to bottom	POSITIVE - Bile salts present

Q45. What are bile salts? Name them. A: Bile salts are sodium and potassium salts of bile acids conjugated with glycine or taurine:

Sodium glycocholate
Sodium taurocholate
Sodium glycodeoxycholate
Sodium taurodeoxycholate

Q46. What are the causes of bile salts in urine (choluria)? A: Bile salts appear in urine in obstructive jaundice (cholestasis):

Cholelithiasis (gallstones)
Carcinoma of head of pancreas
Cholangiocarcinoma
Viral hepatitis (hepatocellular jaundice with intrahepatic cholestasis)
Primary biliary cholangitis
Drug-induced cholestasis

Q47. In which type of jaundice are bile salts present/absent in urine?

Type of Jaundice	Bile Salts in Urine	Bile Pigments in Urine
Obstructive (cholestatic)	Present	Present
Hepatocellular	Present (variable)	Present
Haemolytic (pre-hepatic)	Absent	Absent

Q48. What is the role of bile salts as emulsifying agents? A: Bile salts emulsify dietary fats in the intestine, increasing their surface area for digestion by lipase. They have a hydrophilic and hydrophobic end (amphipathic), making them effective detergents.

Q49. How is Hay's Sulphur test different from Fouchet's test? A:

Hay's Sulphur test: Tests for bile SALTS (detects surface tension reduction)
Fouchet's test: Tests for bile PIGMENTS (bilirubin) - uses BaCl₂ to precipitate bilirubin, then Fouchet's reagent (FeCl₃ + trichloroacetic acid) turns it green/bluish-green

QUICK SUMMARY TABLE

Test	Detects	Principle	Positive Result
Benedict's test	Reducing sugars (glucose)	Cu²⁺ reduced to Cu₂O by reducing sugar	Coloured precipitate (green → brick red)
Heat & Acetic acid test	Protein (albumin)	Heat denaturation + acid precipitation	White turbidity persisting/increasing with acetic acid
Rothera's test	Ketone bodies (acetone, acetoacetate)	Nitroprusside reaction in alkaline medium	Purple/permanganate ring at junction
Hay's Sulphur test	Bile salts	Reduction of surface tension by bile salts	Sulphur powder sinks

Sources: Henry's Clinical Diagnosis and Management by Laboratory Methods (Elsevier); TESTS FOR ABNORMAL CONSTITUENTS IN URINE, ZMC Dahod; ilovepathology.com viva questions

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Viva Questions & Answers: CBC (Complete Blood Count) and PBS (Peripheral Blood Smear)

PART 1 - COMPLETE BLOOD COUNT (CBC / HEMOGRAM)

Q1. What is CBC (Complete Blood Count)? A: CBC is a panel of blood tests that evaluates the cellular components of blood. It includes:

RBC count
Hemoglobin (Hb)
Hematocrit / Packed Cell Volume (PCV)
RBC indices (MCV, MCH, MCHC, RDW)
WBC count and differential count
Platelet count and MPV

Q2. What anticoagulant is used for CBC? Why? A: EDTA (Ethylenediaminetetraacetic acid) - dipotassium salt (K₂EDTA) in the purple/lavender-topped tube.

It chelates calcium ions, preventing coagulation
Preserves cell morphology best for blood film preparation
Does not cause cell swelling (unlike citrate/oxalate)
Ratio: 1.5-2 mg EDTA per mL of blood

Q3. What are the normal values for CBC?

Parameter	Male	Female
Hemoglobin	13-17 g/dL	12-15 g/dL
RBC count	4.5-6.2 million/µL	4.0-5.4 million/µL
PCV/Hematocrit	40-54%	36-47%
MCV	80-100 fL	80-100 fL
MCH	27-31 pg	27-31 pg
MCHC	32-36 g/dL	32-36 g/dL
RDW	12-15%	12-15%
WBC	4,000-11,000/µL	4,000-11,000/µL
Platelet count	1.5-4.5 lakh/µL	1.5-4.5 lakh/µL

Q4. What is hemoglobin? What is its normal value in a newborn? A: Hemoglobin is a conjugated protein (globin + haem) that carries oxygen. In newborns, Hb is 14-20 g/dL (higher due to HbF and physiological polycythemia).

Q5. Define anemia. How is it classified by WHO? A: Anemia = reduction in hemoglobin concentration below normal for age and sex. WHO criteria for anemia:

Men: Hb < 13 g/dL
Non-pregnant women: Hb < 12 g/dL
Pregnant women: Hb < 11 g/dL
Children 6 months-5 years: Hb < 11 g/dL

Q6. What is PCV (Packed Cell Volume) / Hematocrit? A: PCV is the fraction of blood volume occupied by RBCs, expressed as a percentage. It is measured by centrifuging blood in a microhematocrit tube and comparing RBC column height to total blood column height.

Normal: Males 40-54%, Females 36-47%
Used to calculate MCV and MCHC

Q7. What are RBC indices? Define each one. A: RBC indices are calculated values describing the size and hemoglobin content of RBCs:

MCV (Mean Corpuscular Volume) = PCV (%) × 10 / RBC count (millions/µL). Normal: 80-100 fL. Indicates average RBC size.
MCH (Mean Corpuscular Hemoglobin) = Hb (g/dL) × 10 / RBC count (millions/µL). Normal: 27-31 pg. Average weight of Hb per RBC.
MCHC (Mean Corpuscular Hemoglobin Concentration) = Hb (g/dL) × 100 / PCV (%). Normal: 32-36 g/dL. Average Hb concentration per unit volume of RBCs.
RDW (Red Cell Distribution Width): Measures variation in RBC size (anisocytosis). Normal: 12-15%. Elevated in mixed deficiency anemias, early iron deficiency.

Q8. How do you classify anemia using MCV?

MCV	Type	Causes
< 80 fL	Microcytic	Iron deficiency, thalassemia, sideroblastic anemia, lead poisoning
80-100 fL	Normocytic	Aplastic anemia, hemolytic anemia, acute blood loss, anemia of chronic disease
> 100 fL	Macrocytic	Megaloblastic (B12/folate deficiency), liver disease, hypothyroidism, alcohol

Q9. What is polycythemia? Types? A: Polycythemia = increase in RBC mass/Hb above normal.

Primary (Polycythemia vera): Myeloproliferative disorder - autonomous RBC overproduction; JAK2 mutation present
Secondary: Due to increased erythropoietin - hypoxia (high altitude, COPD, cyanotic heart disease), renal cell carcinoma
Relative/Spurious: Decreased plasma volume (dehydration, burns)

Q10. What is the normal WBC count? What is leukocytosis and leukopenia? A: Normal WBC: 4,000-11,000/µL

Leukocytosis: WBC > 11,000/µL
Leukopenia: WBC < 4,000/µL

Q11. What is the differential leucocyte count (DLC)? Normal percentages?

Cell	Normal %	Absolute Count
Neutrophils	40-70%	2,500-7,000/µL
Lymphocytes	20-40%	1,000-4,800/µL
Monocytes	2-8%	200-800/µL
Eosinophils	1-4%	100-500/µL
Basophils	0-1%	0-300/µL

Q12. What causes neutrophilia? What is a "left shift"? A: Neutrophilia (>7,000/µL): Bacterial infections, tissue necrosis (MI, burns), stress, corticosteroids, CML, myeloproliferative disorders, pregnancy.

Left shift: Appearance of immature neutrophils (band forms, metamyelocytes, myelocytes) in peripheral blood - indicates intense demand/marrow response. Seen in severe bacterial infections, sepsis, leukemia.

Q13. What causes neutropenia? A: Viral infections, typhoid, tuberculosis, aplastic anemia, SLE, chemotherapy/radiation, drug-induced (clozapine, carbimazole, phenytoin), megaloblastic anemia.

Q14. What causes lymphocytosis? A: Viral infections (EBV/infectious mononucleosis, CMV, HIV, viral hepatitis), tuberculosis, pertussis (whooping cough - very high lymphocyte count), CLL, lymphomas.

Q15. What causes eosinophilia? A: Mnemonic NAACP:

Neoplasm (Hodgkin lymphoma, eosinophilic leukemia)
Allergic conditions (asthma, hay fever, urticaria)
Adrenal cortex insufficiency (Addison's disease)
Collagen vascular diseases (PAN, SLE)
Parasitic infections (helminths - Ascaris, hookworm, filariasis)

Also: drug hypersensitivity, skin diseases (pemphigus, eczema), tropical eosinophilia.

Q16. What causes monocytosis? A: Tuberculosis, subacute bacterial endocarditis (SBE), typhoid, malaria, leishmaniasis, inflammatory bowel disease, monocytic leukemia, recovery phase of acute infections.

Q17. What is the normal platelet count? Define thrombocytopenia and thrombocytosis. A: Normal: 1.5-4.5 lakh/µL (150,000-450,000/µL)

Thrombocytopenia: Platelets < 1.5 lakh/µL
Thrombocytosis: Platelets > 4.5 lakh/µL
Critical bleeding risk: Platelets < 20,000/µL (spontaneous bleeding possible)

Q18. What are causes of thrombocytopenia? A:

Decreased production: Aplastic anemia, leukemia, megaloblastic anemia, chemotherapy, viral marrow suppression
Increased destruction: ITP (immune thrombocytopenic purpura), DIC, TTP, HUS, hypersplenism
Sequestration: Splenomegaly

Q19. What is MPV (Mean Platelet Volume)? A: MPV measures average size of platelets. Normal: 7-11 fL.

High MPV: Large platelets - ITP, myeloproliferative disorders, hyperthyroidism (young/immature platelets)
Low MPV: Small platelets - aplastic anemia, chemotherapy (platelet production decreased)

Q20. What are the causes of raised ESR? A: ESR (Erythrocyte Sedimentation Rate) rises in any condition that increases plasma proteins (especially fibrinogen):

Acute/chronic infections, TB, rheumatoid arthritis, SLE, multiple myeloma, malignancy, anemia, pregnancy, myocardial infarction
Normal: Males 0-15 mm/hr (Westergren), Females 0-20 mm/hr

PART 2 - PERIPHERAL BLOOD SMEAR (PBS)

Q21. What is a peripheral blood smear? A: A PBS is a thin film of blood spread on a glass slide, stained, and examined under a microscope to study the morphology, number, and types of blood cells (RBCs, WBCs, and platelets).

Q22. What are the indications for peripheral blood smear? A:

Anemia - to classify type and find morphological cause
Leukopenia / leukocytosis - to find cause and type
Suspected leukemia or lymphoma
Thrombocytopenia / thrombocytosis
Detection of blood parasites (malaria, microfilaria)
To detect inclusion bodies in RBCs
To confirm or verify automated CBC analyzer results
To confirm manual platelet count (when clumping suspected)

Q23. What stains are used for PBS? A:

Leishman's stain - most commonly used in India (Romanowsky type)
Wright's stain - used commonly in Western countries
Giemsa stain - used for parasites (malaria), chromosomes
May-Grünwald Giemsa (MGG) - used in many labs All are Romanowsky stains (combination of eosin + methylene blue derivatives).

Q24. What is the principle of Leishman's stain? A: Leishman's stain is a polychrome stain (Romanowsky type). It contains eosin (acidic dye, stains alkaline components red/orange) and methylene blue (basic dye, stains acidic components blue/purple). Methanol acts as fixative. When diluted with buffer, the methylene blue is polychromed and produces azure dyes that stain nuclei and granules.

Nuclei, basophil granules: blue/purple
Cytoplasm of RBCs, eosinophil granules: pink/red
Neutrophil granules: lilac/violet

Q25. How is a peripheral blood smear prepared? A:

Clean a glass slide
Place a small drop of blood (from finger prick or EDTA tube) near one end
Place a spreader slide at 30-45° angle in front of the drop
Draw the spreader back to touch the blood - let it spread along the edge
Push the spreader slide forward in one smooth, quick motion
Air-dry the smear
Fix and stain with Leishman's stain

Q26. What are the characteristics of a good blood smear? A:

Gradual transition from thick to thin end
Smooth, even distribution of cells
No ridges, holes, or waves
Ends before the edge of the slide
RBCs just touching but not overlapping in the counting area
A "feathered edge" at the thin end

Q27. What is the area of the smear used for examination? A: The monolayer zone (transition zone between thick and thin areas) - where RBCs are spread individually, just touching but not overlapping. This area gives the best morphological detail.

Q28. What are the components examined in a PBS? A:

RBC morphology: size, shape, colour, inclusions
WBC: types, morphology, toxic changes, abnormal cells
Platelet: count estimate, clumping, size, giant platelets
Differential leucocyte count (100 cells counted)
Background: parasites, rouleaux, agglutination

Q29. How is WBC estimated from PBS? A: Estimate: Count WBCs in 10 high-power fields (40x objective), calculate average per field, multiply by 2,000 (factor for low-power field count method). Alternatively:

Estimated WBC = Average WBCs per HPF × 2,000

Q30. How are platelets estimated from PBS? A: Count platelets in 5-10 oil-immersion fields (100x), calculate average, then:

Estimated platelet count = Average platelets per OIF × 15,000-20,000/µL Normal: 7-20 platelets per OIF (oil immersion field)

PART 3 - RBC MORPHOLOGY IN PBS

Q31. What is anisocytosis? A: Variation in the size of RBCs. Seen in iron deficiency anemia, megaloblastic anemia, mixed anemias. Quantified by RDW on CBC.

Q32. What is poikilocytosis? A: Variation in the shape of RBCs. Seen in hemolytic anemias, iron deficiency, thalassemia, etc.

Q33. Describe the different abnormal RBC shapes and their significance:

RBC Shape	Description	Associated Condition
Microcyte	Small RBC (MCV < 80 fL)	Iron deficiency, thalassemia
Macrocyte	Large RBC (MCV > 100 fL)	Megaloblastic anemia, liver disease
Spherocyte	Small, round, dense, no central pallor	Hereditary spherocytosis, AIHA
Target cell (codocyte)	Bull's-eye appearance	Thalassemia, iron deficiency, obstructive jaundice, HbC
Sickle cell (drepanocyte)	Crescent/sickle shape	Sickle cell disease (HbSS)
Schistocyte	Fragmented RBC (helmet cell)	Microangiopathic hemolytic anemia, DIC, TTP, HUS, prosthetic valves
Tear drop cell (dacryocyte)	Tear drop shape	Myelofibrosis, iron deficiency anemia, thalassemia
Elliptocyte/Ovalocyte	Oval/elliptical RBC	Hereditary elliptocytosis, megaloblastic anemia, iron deficiency
Burr cell (echinocyte)	Regularly spiculated, 10-30 spicules	Uremia, artifact
Acanthocyte (spur cell)	Irregularly spiculated, 3-12 spicules	Abetalipoproteinemia, liver disease, McLeod syndrome
Stomatocyte	Mouth/slit-shaped central pallor	Hereditary stomatocytosis, liver disease, alcohol
Bite cell (keratocyte)	Bite taken out of RBC	G6PD deficiency (Heinz body removal by spleen)
Blister cell	Vacuole/blister at periphery	G6PD deficiency
Rouleaux	Stack of coins appearance	Multiple myeloma, macroglobulinemia, chronic infection

Q34. What are RBC inclusions? Name and describe them.

Inclusion	Stain	Significance
Howell-Jolly bodies	Dark purple dots (DNA remnants) on Leishman stain	Post-splenectomy, hyposplenia, megaloblastic anemia
Basophilic stippling	Blue dots (aggregated ribosomes)	Lead poisoning, thalassemia, sideroblastic anemia
Heinz bodies	Denatured Hb - requires supravital stain (crystal violet)	G6PD deficiency, unstable hemoglobins
Cabot rings	Ring/figure-of-8 shaped filaments (nuclear remnants)	Megaloblastic anemia, severe anemia
Malaria parasites	Ring forms, trophozoites, schizonts (Giemsa stain)	Plasmodium falciparum/vivax malaria
Pappenheimer bodies	Iron-containing granules - Prussian blue stain	Sideroblastic anemia, post-splenectomy

Q35. What is hypochromia? What causes it? A: RBCs with increased central pallor (pale zone > 1/3 of cell diameter), due to reduced hemoglobin content. Causes: iron deficiency anemia, thalassemia, sideroblastic anemia, anemia of chronic disease.

Q36. What is polychromasia? A: RBCs staining blue-gray due to residual RNA (reticulocytes stained with Romanowsky stain). Indicates active erythropoiesis / reticulocytosis. Seen in hemolytic anemia, hemorrhage, response to treatment of anemia.

PART 4 - WBC MORPHOLOGY IN PBS

Q37. Describe the identifying features of each WBC type:

Neutrophil:

Size: 12-15 µm
Nucleus: Multi-lobed (3-5 lobes) connected by thin filaments
Cytoplasm: Pink with fine lilac/violet granules
Function: Phagocytosis of bacteria (first line of defense)

Lymphocyte:

Size: 7-12 µm (small lymphocyte)
Nucleus: Large, round, occupies most of cell, dark purple, eccentric
Cytoplasm: Scanty, sky-blue, no granules
Function: Adaptive immunity (B cells - antibodies, T cells - cell-mediated)

Monocyte:

Size: 15-20 µm (largest WBC)
Nucleus: Kidney/horseshoe/cerebri-form shaped, folded, blue-gray
Cytoplasm: Abundant, grey-blue, with fine azurophilic granules and vacuoles
Function: Phagocytosis, antigen presentation; precursor to macrophages

Eosinophil:

Size: 12-17 µm
Nucleus: Bi-lobed (spectacles/dumbbell shaped)
Cytoplasm: Large, prominent, brick-red/orange granules
Function: Allergic reactions, parasite defense

Basophil:

Size: 10-14 µm
Nucleus: Often obscured by granules; S-shaped or bilobed
Cytoplasm: Large, dark purple/black granules (contain histamine, heparin)
Function: Allergic and inflammatory reactions

Q38. What are toxic changes in neutrophils? Their significance? A: Toxic changes indicate severe infection/systemic illness:

Toxic granulation: Heavy, dark purple granules (increased primary granules)
Döhle bodies: Blue-gray cytoplasmic inclusions (rough ER remnants)
Cytoplasmic vacuolation: Phagolysosomes
Hypersegmentation: >5 lobes - seen in megaloblastic anemia
Shift to left: Band neutrophils and immature forms

Q39. What are Auer rods? What is their significance? A: Pink/red rod-shaped crystalline cytoplasmic inclusions in myeloid blast cells. Pathognomonic of Acute Myeloid Leukemia (AML). Composed of fused primary (azurophilic) granules.

Q40. What are smudge/smear cells (basket cells)? A: Degenerated, fragile lymphocytes that rupture during smear preparation, leaving bare nuclei with smeared chromatin. Characteristic of Chronic Lymphocytic Leukemia (CLL).

Q41. What are atypical lymphocytes (reactive lymphocytes)? A: Large, activated lymphocytes with abundant basophilic cytoplasm, irregular nucleus. Characteristic of infectious mononucleosis (EBV infection). Also called Downey cells or virocytes.

PART 5 - PLATELET EXAMINATION IN PBS

Q42. How do platelets appear on PBS? A: Small (2-4 µm), anucleate, pale blue/colorless cells with pink/purple granules. Normally 7-20 per oil-immersion field.

Q43. What are giant platelets? Their significance? A: Platelets as large as or larger than RBCs. Seen in:

ITP (immune thrombocytopenic purpura)
Bernard-Soulier syndrome
Myeloproliferative disorders
May-Hegglin anomaly

Q44. What is platelet satellitism? A: Platelets adhering to neutrophils in a ring pattern. An in vitro phenomenon occurring in EDTA anticoagulated blood - can cause spurious thrombocytopenia on automated counters. Correct by using citrate tube.

PART 6 - CLINICAL INTERPRETATION

Q45. What PBS findings suggest iron deficiency anemia? A: Microcytic hypochromic anemia - small pale RBCs, anisocytosis, poikilocytosis, pencil cells (elongated RBCs), target cells. WBC and platelets usually normal (platelets may be elevated - reactive thrombocytosis).

Q46. What PBS findings suggest megaloblastic anemia? A: Macrocytic anemia - oval macrocytes (ovalomacrocytes), hypersegmented neutrophils (>5 lobes in >5% of neutrophils), Howell-Jolly bodies. Pancytopenia in severe cases.

Q47. What PBS findings suggest thalassemia? A: Microcytic hypochromic anemia with marked anisocytosis and poikilocytosis, target cells, tear drop cells, nucleated RBCs (normoblasts), basophilic stippling.

Q48. What PBS findings suggest sickle cell disease? A: Sickle cells (drepanocytes), target cells, Howell-Jolly bodies (functional asplenia), nucleated RBCs, reticulocytosis, polychromasia.

Q49. What PBS findings suggest CML (Chronic Myeloid Leukemia)? A: Leukocytosis with full spectrum of myeloid cells (myelocytes, metamyelocytes, band forms, neutrophils, eosinophilia, basophilia), thrombocytosis, mild anemia. Philadelphia chromosome (BCR-ABL) confirms.

Q50. What PBS findings suggest malaria? A: Ring forms (early trophozoites) in RBCs on Giemsa-stained smear. In P. falciparum: multiple rings per cell, banana-shaped gametocytes, no enlarged RBCs. In P. vivax: enlarged stippled RBCs (Schüffner's dots), amoeboid trophozoites.

QUICK REFERENCE SUMMARY

Test	Normal Value	Raised in	Decreased in
Hb (male)	13-17 g/dL	Polycythemia	Anemia
Hb (female)	12-15 g/dL	Dehydration	Blood loss
WBC	4,000-11,000/µL	Infection, leukemia	Viral illness, aplasia
Platelets	1.5-4.5 lakh/µL	Reactive, MPD	ITP, DIC, aplasia
MCV	80-100 fL	B12/folate def	Iron def, thalassemia
Neutrophils	40-70%	Bacterial infection	Viral, drugs
Eosinophils	1-4%	Allergy, parasites	Cushing's, stress
Lymphocytes	20-40%	Viral, CLL	HIV, steroid

Sources: Henry's Clinical Diagnosis and Management by Laboratory Methods (Elsevier); Cleveland Clinic CBC reference; Peripheral Smear Viva Answers - IGMCRI (Dr. Manjula / Zaphyrianz batch); StatPearls - Normal and Abnormal CBC with Differential

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