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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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