Examination of urine

Urine examination step by step, Normal finding, time of collection, physical chemical examination, cast, crysteals in urine

1. Examination of Urine by Dr Sweta Biswas Das M.B.B.S., D.G.O. 1 st Year PGT Pathology R G. Kar MC&H, Kolkata 1 Moderator: Prof (Dr.) Tushar Kanti Das, HOD

2. Composition of normal urine (24 hrs) in adults Sl No Parameters Values Sl No Parameters Values 1 Volume 600-2000 ml 10 Urea nitrogen 12-20 gm 2 Specific gravity 1.003-1.030 11 Uric Acid 250-750 mg 3 Osmolality 300-900 mOsm /kg 12 Sodium 40-200 mEq 4 pH 4.6-8.0 13 Potassium 25-125 mEq 5 Glucose < 0.5 gm 14 Chloride 110-250 mEq 6 Proteins < 150 mg 15 Calcium (low calcium diet) 50-150 mg 7 Urobilinogen 0.5-4.0 gm 16 Formiminoglutamic acid ( FIGlu ) < 3 mg 8 Prophobilinogen 0.2 mg 17 Red cells, epithelial cells and white blood cells ≤ 1-2 per high power field 9 Creatinine M: 14-26 mg/kg F: 11-20 mg/kg 2

3. Indication of urine analysis Suspected renal diseases Glomerulonephritis Nephrotic Syndrome Pyelonephritis Renal failure Urine tract infection diagnosis Metabolic disorders i.e. Diabetes mellitus Differential diagnosis of jaundice Detection of Plasma cell dyscrasias Diagnosis of pregnancy 3

4. Collection of urine: Time of collection (1/3) Single specimen First morning voiding Most concentrated Acidic pH Formed element preserved like casts, cells Used for Routine examination Fasting Glucose Protein Nitrite Pregnancy test Microscopic analysis of cellular elements Orthostatic proteinuria Bacteriological analysis 4

5. Collection of urine: Time of collection (2/3) Single specimen The Random Specimen Can be collected at any point of time Routine examination Post-prandial specimen Collected two hours after a meal in the afternoon Insulin therapy monitoring in Diabetes Mellitus Urobilinogen 5

6. Collection of urine: Time of collection (3/3) 24 –hour-specimen First urine discarded in morning Clean 2 litres bottle with cap- used Whole day and night urine collected Next day first urine in morning also collected Preserved at 4-6 ◦ C during collection After collection immediate transportation to lab Thoroughly mixed and a part of whole sample used for quantitative estimation of Protein Hormones 6

7. Collection of urine: Methods of collection Midstream specimen Used for all types of examination Collected after voiding initial half of urine Clean-catch specimen Used for bacteriological culture Urethral opening- cleaned with soap and water Collected after voiding initial half of urine Catheter specimen Used for bacteriological culture For bed ridden and obstructed urinary tract patient Infants Aspiration done above the symphysis pubis 7

8. Changes occur in room temperature after prolonged standing Increase in pH- production of ammonia Formation of crystal- calcium and phosphate precipitation Loss of ketone bodies- volatile Decrease of glucose- glycolysis by bacteria and cells Oxidation of bilirubin to biliverdin Oxidation of urobilinogen to urobilin Bacterial proliferation Disintegration of cellular elements 8

9. Preservation of urine sample Test to be done within 2 hrs Can be kept at 4-6 ◦ C for maximum 8 hrs Routine analysis- preservatives avoided Preservative used in 24 hrs sample Hydrochloric acid- Adrenaline, Nor-adrenaline, vanillyl mandelic acid, steroids Toluene- measurement of chemicals Boric acid- general preservative Thymol- inhibit bacteria and fungi Formalin- formed elements 9

10. Physical examination of urine 10

11. Physical examination: appearance Appearance Diagnosis Cause White and cloudy on standing in alkaline urine Disappear on addition of a drop of dilute acetic acid Amorphous phosphates Pink and cloudy in acid urine Dissolve on warming Amorphous urates Varying grades of turbidity Microscopy Pus cells Uniformly cloudy, do not settle at the bottom following centrifugation Microscopy, nitrite test Bacteria 11

12. Physical examination: volume 24 hrs average urine output- 600 to 2000 ml (adult) Polyuria More than 2000 ml per day Occurs in Diabetes Mellitus, Diabetes Insipidus , Chronic renal failure, diuretic therapy. Oliguria Less than 400 ml per day Febrile states, acute glomerulonephritis, congestive cardiac failure, dehydration Anuria Less than 100 ml per day Acute tubular necrosis, acute glomerulonephritis, complete urinary tract obstruction 12

13. Physical examination: colour Different colours of urine Found in colorless Dilute urine(diabetes mellitus, diabetes insipidus, red Haematuria , hemoglobinuria, porphyria, myoglobinuria Dark brown to black Alkaptonuria, melanoma brown hemoglobinuria yellow Concentrated urine Yellow green or green biliverdin Deep yellow with yellow foam bilirubin Orange or orange brown Urobilinogen, porphobilinogen Milky white chyluria Red or orange fluorescence with UV light porphyria 13

14. Physical examination: odour Fruity: ketoacidosis, starvation, Musty: phenylketonuria Fishy: UTI ( Proteus ), tyrosinaemia Ammoniacal: UTI ( E. coli ) Foul: UTI Sulfurous: cystinuria 14

15. Physical examination : pH/reaction (1/2) Normal range: 4.6-8 Tested by Litmus paper pH indicator paper pH meter Reagent strip test 15

16. Physical examination: pH/reaction (2/2) Acidic urine: Ketosis - diabetes mellitus, starvation, fever UTI by E. coli High protein diet Alkaline urine: Strict vegetarian UTI by Proteus or Pseudomonas Sever vomiting CRF 16

17. Physical examination: Specific gravity (1/2) Depends on the concentration of various solutes in the urine Normal range :1.003 to 1.030 at 20 O C Measured by : Urinometer Refractometer Reagent strip method 17

18. Physical examination: Specific gravity (2/2) High specific gravity Diabetes mellitus( glycosuria) Nephrotic syndrome (proteinuria) Fever Dehydration Low specific gravity Diabetes insipidus Chronic renal failure 18

19. Chemical examination of urine 19

20. Chemical examination: Protein Causes of proteinuria ( 1/3) Normal value: 150 mg per day Glomerular proteinuria: due to increased permeability of glomerular capillary wall Selective (only albumin and transferrin bands seen by electrophoresis) Nonselective (pattern same as serum) 20

21. Chemical examination: Protein Causes of proteinuria ( 2/3) Tubular proteinuria: Proteinuria caused by renal tubular dysfunction. acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of kidney Overflow proteinuria: Proteinuria associated with increased production of abnormal low molecular weight proteins, Bence jones protein (plasma cell dyscrasias) Hemoglobin (intravascular haemolysis) Myoglobin (skeletal muscle trauma) Lysozyme (acute myeloid leukemia type M4 or M5) 21

22. Chemical examination: Protein Causes of proteinuria (3/3) Hemodynamic proteinuria: alteration of blood flow causes increase protein filtration High fever Hypertension Congestive cardiac failure Heavy exercise Post-renal proteinuria: Inflammation and neoplasia of Renal pelvis Ureter Bladder Prostate Urethra 22

23. Tests for Detection of Protein: Heat and acetic acid test Principle: Protein get coagulated when boiled Method: 2/3 rd of test tube filled with urine Heat upper portion of urine in inclined test tube till it boils Cloudiness appears due to coagulation Few drops of 10% acetic acid added to dissolve phosphorus –helps to detect false positivity 23

24. Tests for Detection of Protein: Reagent strip method Principle: Indicator strip coated with bromo -phenol blue indicator Buffered to pH 3.0 with citrate Protein changes ionization of dye Semi quantitative method Notes: Mainly reactive to albumin False negative- Bence Jones protein, myoglobin, haemoglobin False positive- Gross haematuria, highly alkaline urine, contaminated urine 24

25. Tests for Detection of Protein: Sulphosalicylic acid test Principle: Protein gets precipitated after adding organic acid like sulphosalicylic acid Method: 2ml of clear urine in a test tube If urine neutral or alkaline- add one drop of glacial acetic acid Add 2-3 drops of 3-5% sulphosalicylic acid Check turbidity against dark background Notes: More sensitive and reliable than boiling test False positive- gross haematuria, highly concentrate urine, radiographic dye, excess uric acid, tolbutamide, sulphonamide, salicylates, penicillins False negative- very dilute urine Can detect- albumin, myoglobin, haemoglobin, Benes Jones Protein 25

26. Test for detection of Glucose in urine BENEDICT TEST (1/2) Principal: When urine is boiled in Benedict solution, blue alkaline copper sulphate is reduced to red brown cuprous oxide if a reducing agent is present. Method: Take 5ml of Benedict’s reagent in a test tube, add 8 drops of urine. Boil the mixture. Nil: no change from the Blue color Trace: Green without precipitate 1+(approx 0.5grams/dl)Green with precipitate 2+(approx 1.0 grams /dl) Brown precipitate 3+(approx 1.5 grams /dl) Yellow orange precipitate 4+(approx >2grams /dl ) Red precipitate 26

27. Test for detection of Glucose in urine BENEDICT TEST (2/2) Detects all reducing substances like glucose, fructose ,lactose Sensitivity is 200 mg of glucose /dl 27

28. Test for detection of Glucose in urine Reagent strip method Specific for glucose Based on glucose oxidase peroxidase reaction More sensitivity(sensitivity 100 mg glucose/dl) 28

29. Causes of glycosuria Glycosuria with hyperglycaemia : Diabetes Acromegaly Cushing’s disease Hyperthyroidism Drugs like corticosteroids Glycosuria without hyperglycaemia : Renal tubular dysfunction 29

30. Ketone bodies Types Acetone Acetoacetic acid β hydroxy butyric acid They are products of fat metabolism 30

31. Test for ketone bodies Rothera’s test Principal: Acetone and acetoacetic acid react with sodium nitroprusside in the presence of alkali to produce purple color Method: Take 5ml of urine in a test tube and saturate it with ammonium sulphate, then add one crystal of sodium nitroprusside . then slowly run the liquor ammonia along the sides of the test tube. Formation of purple colored ring at junction indicates positive test Rothera’s test is sensitive to 1-5 mg /dl of acetoacetate and 10-25 mg/dl of acetone 31

32. Other tests for detection of ketone bodies Acetest tablet test Ferric chloride test (Gerhardt’s test) Reagent strip method 32

33. Causes of ketonuria Diabetes Non diabetic causes : High fever Starvation Sever vomiting Diarrhoea Glycogen storage diseases 33

34. Bilirubin Fouchet’s test Principal: Bilirubin absorbs to Barium Chloride and results in green color formation when fouchet’s reagent is added. Method: In 5 ml of urine add 2.5 ml of 10% Barium chloride and mix well .then filter to obtain the precipitate on a filter paper .To precipitate add 1 drop of Fouchet’s reagent. Developement of blue green color around the drop indicates presence of bilirubin 34

35. Other tests for bilirubin Foam test Gmelin’s test Lugol’s iodine test Reagent strips with diazo reagent 35

36. Urobilinogen Ehrlich test Method: In 5 ml of urine add 0.5 ml of Ehrlich’s reagent (HCL 20 ml ,distilled water 80 ml , para dimethylaminobenzaldehyde 2gm)Allow to stand for 5 mins . development of pink colour indicates positive test. 36

37. Causes of Urobilinogen in urine Hemolytic jaundice Early hepatitis Hepatocellular jaundice 37

38. Bile salt Hay’s surface tension test Method: In 5ml of urine sprinkle a pinch of sulphur particles.If bile salt is present sulphur particles will sink to the bottom because bile salts lower the surface tension of urine . 38

39. Blood in urine Benzidine test Principle : The peroxidase activity of haemoglobin decomposes hydrogen peroxide releasing nascent oxygen ,which in turn oxidizes benzidine to give blue colour Method : Mix 2ml of benzidine solution with 2ml of hydrogen peroxide in a test tube .Take 2ml of urine and add 2ml of mixture .A blue or green colour within 5 min indicates positive reaction. 39

40. Causes of Haematuria Disease of urinary tract Glomerular disease : Glomerulonephritis Berger’s disease Lupus nephritis Henoch-Schonlein purpura Non glomerular disease: Calculus , tumor, infection, tuberculosis, pyelonephritis , trauma, carcinoma of prostate Hematological condition: Coagulation disorders ,sickle cell disease 40

41. MICROSCOPIC EXAMINATION 41

42. Microscopic examination Qualitative technique Urine must be freshly voided Examined without excessive delay in order to prevent cellular degeneration If preservative is required ,then 1 crystal of thymol or 1 drop of formalin (40%)is added to 10 ml of urine Well mix sample of urine(10 -15ml )is centrifuged in machine for 5mins at 1500 rpm. The top part(supernatant)is discarded. A drop of urine left at the bottom of test tube (sediment)is placed on the glass slide and covered with cover slip it is examined under high power 42

43. Red Blood cells Normally no RBC found Small, smooth, yellowish, anucleate biconcave disks 7 µ in diameter- isomorphic Found Fresh urine 9-10 µ in diameter- swollen Found in hypotonic urine Smaller diameter with spikey surface - crenated hypertonic urine Variable in size and shape- dysmorphic Glomerulonephritis 43

44. White Blood cells Spherical. Granular with visible nuclei 10-15 µ in size distorted, smaller, and have fewer granules- degenerative WBC Infection – seen in clumps 44

45. Epithelial cells Small, polyhedral, columner , or oval have granular cytoplasm, eccentric nucleus seen. Found in acute tubular necrosis , pyelonephritis , viral infection, 45

46. Urinary casts Cylindrical aggregations of particles Form in the distal renal tubules and collecting ducts Composed of a precipitation of Tamm- Horsfall protein 46

47. Types of urinary casts Acellular casts Hyaline casts Granular casts Waxy casts Fatty casts Broad casts Cellular casts Red cell casts White cell casts Epithelial cell casts 47

48. Hyaline casts The most common type of casts. They are cylindrical with parallel sides and blunt rounded end, colourless , homogenous, transparent. Seen in fever, strenous exercise, glomerular proteinuria 48

49. Granular casts They are cylindrical structure with coarse or fine granules Seen in acute glomerulonephritis and pyelonephritis 49

50. Waxy casts They have homogenous smooth glassy appearance ,cracked or serrated margins and irregular broken off ends. Seen in end stage renal failure 50

51. Fatty casts They are cylindrical structures filled with highly refractile fat globules in Tamm Horsfall protein matrix Seen in nephrotic syndrome 51

52. Broad casts Form in dilated distal tubules Seen in chronic renal failure and sever renal tubular obstruction Associated with poor prognosis 52

53. Crystals in urine 53

54. Normal crystals in urine Crystals present in acidic urine Uric acid crystals : These are variable in shape and are yellow brown in colour and soluble in alkali and insoluble in acid. Found in Gout and leukemia. Calcium oxalate crystals : These are colourless envelope-shaped .commonly found in diets rich in tomatoes, cabbages. Large number are seen in Ethylene glycol poisoning. Amorphous urates :These are urate salts of potassium, magnesium, or calcium in acid urine Crystals present in alkaline urine Calcium carbonate crystals: small colorless, grouped in pair Ammonium phosphate crystals: yellow brown ,cactus like, called as thornapple Phosphates: Triple phosphates: colorless ,3-6 sided prisms with oblique surfaces at the end. Calcium hydrogen phosphate: colorless and variable shape Amorphous phosphate :colorless small granules. 54

55. Normal crystals found in urine 55 Calcium oxalate Triple phosphate Uric acid Calcium carbonate

56. Abnormal crystals in urine Cysteine crystals: colorless clear hexagonal, highly refractile plates, soluble in hydrochloric acid. Seen in Cystenuria . Cholesterol crystals : colorless refractile , flat rectangular plates with notched corner. Seen in Nephrotic syndrome and hypercholesterolemia. Bilirubin crystal: brown and variable shapes. Seen in obstructive liver disease. Tyrosine crystals : clusters of fine colorless or yellow needles. Seen in liver disease and Tyrosinemia . Leucine crystals: refractile , yellow or brown spheres with radial or concentric striation. Seen in urine along with tyrosine in sever liver disease(cirrhosis) 56

57. Abnormal crystals found in urine 57 Cysteine Cholesterol Bilirubin Leucine

58. References Burtis CA, Ashwood ER ( Eds ). Tietz fundamentals of clinical chemistry (5th Ed). Philadelphia; WB Saunders Company, 2001. Henry JB (Ed): Clinical diagnosis and management by laboratory methods. (20th Ed). Philadelphia; WB Saunders Company, 2001. Kawthalkar Shirish M, Essential of Clinical Pathology, Jaypee Brothers Medical Publishers (P) Ltd, ISBN: 978-93-80704-19-7, Pg no: 3-29 , India, 2010. World Health Organization. Manual of basic techniques for a health laboratory (2nd Ed). Geneva; World Health Organization, 2003. 58

59. Thank you 59