Physical and chemical examination of urine

Presentation on Important aspects of Urine examination

1. Physical and Chemical examination of Urine Dr. Himil Parikh First year resident GCS Medical college, Ahmedabad Guided by- Dr. Deepak Joshi Dr. Kalpen Patel

2. Introduction Urine can be used to look for physical and biochemical abnormalities. To screen for and/or to aid in diagnosis   of conditions such as a urinary tract infections, kidney disorders, liver problems, diabetes or other metabolic conditions, to name a few. For example: Persistent amounts of albumin and other proteins in the urine (proteinuria) indicate kidney damage.

3. Indications Suspected RENAL DISEASES like glomerulonephritis, nephrotic syndrome, pyelonephritis, acute and chronic renal failure. Detection of URINARY TRACT INFECTION. Detection and monitoring of METABOLIC DISORDERS like Diabetes mellitus. Differential diagnosis of JAUNDICE. Detection and monitoring of PLASMA CELL DYSCRASIAS. Diagnosis of PREGNANCY.

4. Collection of Urine Improper collection---- may invalidate the results Containers for collection of urine should be wide mouthed, clean and dry. Time of Collection Single specimen 24 hour specimen First voided specimen Random Postprandial specimen

5. First voided specimen: Most concentrated and has acidic pH, so formed elements (cells and casts) are well preserved. It is preferred for urinalysis. Random specimen: Single specimen collected at any time of day and is sufficient for routine urine examination (though not preferred , it is the most frequently received specimen ) Post- prandial specimen: (collected 2 hours after a meal in the afternoon) Sometimes requested for estimation of glucose or of urobilinogen.

6. Collection for Routine Urinalysis: About 15 ml of midstream sample (especially in female patients) is cleanly collected. Collection for Bacterial Culture: Collect clean catch sample. 24- hour specimen: Quantitative estimation of proteins, hormones and electrolytes

7. Collection Methods Midstream specimen Clean-catch specimen Timed collection Catheter specimen Used for routine and bacteriological study in bedridden patients. Infants Either by attaching a clean plastic bag around baby’s genitalia and leaving it there for some time or suprapubic puncture

8. Collection and Transport Guidelines All urine collection and/or transport containers should be: Clean and leak proof. Break-resistant Material of container should not interfere and Container should not be re-used. Capacity of at least 50 ml (routine) and at least 3 litre (24-hour sample) Amber coloured containers for light sensitive analytes

9. Light sensitive analytes Bilirubin Phorphobilinogen Porphyrins Urobilinogen

10. Storage of urine Ideally the specimen should be examined within 2 hours of voiding. If delay is expected, it should be refrigerated at 4-6° Celsius for up to 8 hours.

11. Effect of Storage on urine: Increase in pH: Due to production of ammonia from urea by urease producing bacteria. Formation of crystals: Precipitation of phosphate and calcium. Loss of ketone bodies : since they are volatile. Decrease in glucose: Due to glycolysis and utilization of glucose by cells and bacteria. Oxidation of bilirubin to biliverdin : false-negative test for bilirubin Oxidation of urobilinogen to urobilin: false-negative test for urobilinogen. Bacterial proliferation Disintegration of cellular elements (Especially in alkaline and hypotonic urine.

12. Preservation of Urine Sample Not recommended for routine analysis as they interfere with reagent strip techniques and chemical test for protein. Preservatives for 24-hour urine sample: Hydrochloric acid: Used when detecting adrenaline, nor-adrenaline, vanillylmandelic acid (VMA) and steroids. Toluene: It forms a thin layer and hence physical barrier against bacteria and air. Boric acid: General preservative (sample can be kept for 24 hours without refrigeration) Thymol: Inhibits bacteria and fungi . Formalin: Excellent for preservation of formed elements.

13. Urinalysis The basic (routine) urinalysis consists of four parts: Specimen evaluation Physical examination Chemical examination Microscopy of urine sediment/ Sediment examination

14. Specimen Evaluation Urinalysis

15. Preanalytical Assessment Before proceeding for examination, specimen must be evaluated in terms of its acceptability. Minimum Labeling Requirements: Patient’s full name, Date and Time of collection Intactness: There shouldn’t be any leakage, spillage and damage to container. Timing of collection: First voided morning urine is the best for Routine analysis. Preferences: If multiple investigations are to be done from a single specimen, bacteriologic examination should be performed first . Hence, volume of urine should be noted properly.

16. Physical Examination Urinalysis

17. Physical examination It includes: Volume Appearance Odor Specific gravity

18. Volume Total volume can be evaluated only from 24-hour urine sample. Main determinant of urine volume is water intake. Normal individual: 24-Hour urinary output: 600 to 2000 ml out of which about 400 ml is produced during night (Reference: Todd and Stanford) Exceptions: Pregnancy- diurnal variation may be reversed Young children- 3-4 times more urine than adults (ml/per body weight)

19. Increase in Urine volume Decrease in Urine volume >2000 ml/ 24 hours- Polyuria >500 ml during night- Nocturia Specific gravity- <1.018 <500 ml/ 24 hours- Oliguria <100 ml/ 24 hours or complete cessation- Anuria Causes Causes Diabetes mellitus (Osmotic diuresis) Diabetes insipidus (Failure to secrete ADH) Chronic renal failure (Loss of concentrating ability of renal tubules) Diuretic therapy Polydypsia Caffeine/ alcohol intake Oliguria : High grade febrile states Acute glomerulonephritis (decreased glomerular filtration) Congestive cardiac failure or dehydration (Renal hypoperfusion) Anuria : Acute tubular necrosis Complete urinary obstruction

20. Appearance Colour Clarity Normally, urine is AMBER coloured and CLEAR Colour Pathological Non pathological White Chyle Pus Phosphates Admixture of semen (in males) Yellow to Orange Bilirubin Urobilin Concentrated urine Vit - B Complex therapy Rifampicin Acriflavine Pyridium Carrots Senna Pink to Red Haemoglobin Myoglobin Porphyrins Red blood cells Beets(anthocynin) Aminopyrine Methyldopa Food color

21. Colour Pathological Nonpathological Red to Brown to Purple Porphobilinogen Uroporphyrin Brown to Black Homogenistic acid Melanin Methaemoglobin (Black-water fever) Phenol Porphyrins Iron compounds Chloroquine Levodopa Metronidazole Quinine Blue to Green Biliverdin Pseudomonas infection Methylene blue Vit B complex Phenyl salicylate Amitryptiline

22. Black water fever Complication of Malaria Massive hemolysis Mostly autoimmune due to interaction of Plasmodium and Quinine

23. Urine Colour Chart

24. Clarity Non- Pathologic Turbidity due to precipitation of crystals or non-pathologic salts known as AMORPHOUS In alkaline urine In acidic urine Precipitation of- Phosphate Ammonium urate Carbonate Redissolves on acidification Precipitation of- Uric acid Urates Redissolves on warming at 60°

25. Pathologic Leukocytes Bacteria Chyluria Lipiduria Cloudy urine (just like phosphate containing urine) which doesn’t dissolve on acidification Uniform opalescence which will remain even after acidification or centrifugation. Milky Urine - contains lymph due to rupture of lymph vessels into urinary tract E.g. Filariasis Pseudochyluria occurs with use of paraffin based vaginal creams in treatment of Candida infection Milky Urine - Seen with Nephrotic syndrome (contains TGLs and cholesterol) Also in sustained skeletal trauma with fractures to major long bone Smoky urine - RBCs Hazy urine - Mucous

26. Ether extraction Extracted Not Extracted Chyluria Pseudochyluria Lipiduria Triglycerides Yes No Differentiating Chyluria from Lipiduria

27. Odour Freshly voided urine- Typical aromatic odour (Volatile organic acids) On standing- Conversion of urea  ammonia by urease producing bacteria – Faint ammoniacal odour Odour Cause Fruity Ketonuria Mousy Phenylketonuria Fishy UTI by Proteus, Tyrosinemia Ammoniacal UTI with E. coli, Old standing urine Foul Urinary Tract Infection (UTI)

28. Odour Cause Maple syrup Maple syrup urine disease (MSUD) Rancid Tyrosinemia Cabbage Methionine malabsorption Sweaty feet Isovaleric acidemia and glutaric acidemia Rotten eggs Cystinuria

29. Specific Gravity Also known as Relative mass density Ratio of weight of a volume of urine to the weight of the same volume of distilled water at a constant temperature. Main contributors to specific gravity of urine are urea and sodium Measures the concentrating and diluting power of kidney. Concentrating ability of kidney is one of the first function to be lost as a result of tubular damage. Normal range of random specimen -1.003-1.035 Range of 24 hour sample in Normal adults with adequate fluid intake - 1.015- 1.025.

30. Specific gravity and Osmolality are not the same! Specific Gravity: Indicates relative proportions of dissolved solid components to total volume of the specimen i.e. DENSITY of urine Larger particles like glucose and protein tend to increase the Sp.Gr . more than smaller electrolytes Osmolality: Indicates number of particles of solute per unit of solution No difference occurs in the results with different sized particles i.e. glucose and protein OR smaller electrolytes * In certain circumstances, measurement of Osmolality is preferred over measurement of Specific gravity.

31. Increased specific gravity Diabetes mellitus Dehydration Proteinuria (Nephrotic syndrome) Glycosuria Decreased specific gravity Polydipsia Diabetes insipidus Diuretics Early stages of Chronic Kidney Disease Causes: Hyposthenuria: Consistently low specific gravity; <1.007 Hypersthenuria: Consistently high specific gravity Isosthenuria: Fixed specific gravity at 1.010 (occurs in Chronic Kidney Disease)

32. Methods of Specific Gravity measurement Reagent strip method - Most commonly employed Urinometer Refractometer Falling drop method - Least commonly performed INDIRECT METHODS DIRECT METHODS

33. Reagent Strip method Has 3 main ingredients: PRINCIPLE : pKa change of pretreated polyelectrolyte in relation to the ionic concentration of urine. Higher the concentration of urine, lower the pKa as pKa refers to the proton donating capacity of an acid and stronger the acid, lower the pKa. Polyelectrolyte Indicator substance Buffer

34. Advantages: This method is not affected by the high amounts of protein and sugar and so there is no need for the correction of value. This is a method unaffected by Temperature changes. Disadvantages: False readings may be obtained if there is a run-over from adjacent reagent area in excessively wetted strips.

35. Urinometer Direct method of assessing specific gravity of urine at room temperature which is based on the principle of BUOYANCY. ADVANTAGES: Results are directly obtained and depend on the solutes dissolved in urine. Easily performed and quick assessment of results DISADVANTAGES : Requires minimum of 15 ml for measuring specific gravity Correction of 0.001 should be made for each 3° C rise or fall in temperature. Correction for every g/dl of protein and sugar is required (0.003 for 1 g/dl of protein and 0.004 for 1 g/dl of glucose)

36. URINOMETER

37. Refractometer Indirect method related to content of dissolved solids present It is obtained by ratio of velocity of light in air to the velocity of light in a solution ADVANTAGES: DISADVANTAGES : Only a few drops of urine are required The results obtained are quite accurate Valid only for Urine Damaged by heat above 150° F and by immersion of eyepiece and focusing ring in water Requires daily calibration for accurate results

38. Falling drop method More accurate and precise than both Urinometer and Refractometer A drop of urine is allowed to fall in a specially designed column. This drop encounters two beams of light, first one starts the timer and the second one stops it. This falling time is measured electronically and expressed as specific gravity.

39. Chemical Examination Urinalysis

40. Chemical examination of urine Reagent strips are the primary method used for the chemical examination of urine. They represent multiple complex, state-of-the- art chemical reactions. Advantages of Multistix Strip Quick screening of urine chemistry Reliable, specific and sensitive Avoids use of various corrosive reagents, different type of glass wares and other laboratory material required for wet chemical testing of urine It can be performed in uncentrifuged urine and doesn’t require acidification Less labour intensive and can be automated for large laboratories. Less chance of human error

41. Reaction/ pH pH is the scale for measuring acidity or alkalinity On standing, urine becomes alkaline due to formation of ammonia from urea and loss of CO2. Hence, freshly voided urine specimen is required. Methods: Normal pH is 4.6 to 8.0 Reagent strip method Litmus paper test pH meter

42. Reagent strip method Indicators methyl red and bromothymol blue give a range of orange , green , and blue colours as the pH rises, permitting estimation of pH values to within half a unit within the range of 5 to 9. Acidic urine Alkaline urine Diabetes mellitus UTI by urea splitting organisms (Proteus and Pseudomonas) Starvation Severe vomiting Fever Vegetarian diet (Citrus fruits) UTI by E. coli Old ammoniacal urine sample High protein diet Chroni c renal failure

43. Problems: If the strips get excessively wet, acid buffer from the protein patch runs into the pH patch, causing it to become orange. Results are not precise unless the test is performed on freshly voided urine as with time, the pH increases with formation of ammonia and release of CO2. Reagent Strip

44. Other Methods: Litmus paper test: Blue litmus paper turns red , in acidic urine and Red litmus turns blue in alkaline urine. Not very precise, not preferably used pH meter: Electrode of pH meter is dipped in urine and the reading is taken. It is used when exact pH is required.

45. Uses: To evaluate Renal acidosis/alkalosis and compensatory mechanism activation in pulmonary acidosis/alkalosis. Evaluation of Tubular function (Exchange of Anions and Cations) Helps identify crystals in urine When alteration of urinary pH is required in treatment of stones Certain drugs work better at a certain pH ( For e.g .: Streptomycin is effective in UTI if urine is kept alkaline)

46. Protein in Urine Normal- Up to 150 mg/24 hours in a concentration varying from 2 to 10 mg/dl Tamm- Horsfall glycoprotein( urumucoid ) secreted by the distal tubular cells and ascending loop of Henle . It constitutes 1/3 rd or more of the total urinary protein loss. Protein has a very low maximal tubular rate of reabsorption meaning increased filtration of protein quickly saturates the reabsorptive mechanism. Hence, detection of abnormal amounts of protein- IMP indicator of Renal disease

47. Common screening tests Qualitative/ Semiquantitative Colorimetric Reagent strip test Precipitation-based testing Reagent Strip - This method takes advantage of the protein error of pH indicators. Because proteins carry a charge at physiologic pH, their presence will elicit a pH change. The reagent strip is impregnated with tetrabromphenol blue buffered to an acid pH of 3 OR tetrachlorophenol tetrabromosulfophthalein. Variable shades of green develop if protein is present in urine.

48. Advantage: Advantage of avoiding false-positive reactions with organic iodides (e.g. Radiograph contrast and tolbutamides or other drugs) . Decreased chance of Human error Rapid results Drawbacks: Lack of sensitivity of the reagent strip to globulins False positive results can occur with highly pigmented urine, quaternary ammonium compounds, amidoamines in fabric softeners, chlorhexidine, and excessive leaching of the acid buffer of the test strip by excessive wetting. Highly buffered alkaline urine will also provide false-positive results.

49. Reporting: Most methods will detect 5 to 20 mg of albumin per decilitre Results can be read in a “plus” system as negative, trace, and 1+ to 4+

50. Protein quantification The quantification of urine can be done by obtaining 24 hour urine sample Normal Protein: Creatinine ratio values are: <0.2 g Protein per g of Creatinine in Adults and Children >2 years <0.5 g Protein per g of Creatinine in Children <2 years Quick Quantification When 24 hour sample is not available/feasible, Protein: creatinine ratio can be used to estimate renal function.

51. MINIMAL PROTEINURIA (<1 gm/day) Exercise Fever Emotional stress Chronic Pyelonephritis Nephrosclerosis Chronic interstitial nephritis Postural proteinurias Transient proteinurias MODERATE PROTEINURIA (1-4 gm/day) Nephrosclerosis Toxic nephropathies Degenerative, Malignant, Inflammatory conditions of Lower urinary tract Irritative conditions of Lower urinary tract like Calculi Pre- eclampsia Multiple myeloma

52. MARKED PROTEINURIA (> 4 gm/day) Nephrotic syndrome Acute glomerulonephritis Chronic glomerulonephritis, severe Diabetic nephropathy, severe Renal amyloidosis Lupus nephritis Toxemia of pregnancy End Stage Renal Disease

53. FUNCTIONAL PROTEINURIA Usually less than 0.5 g/day Seen with dehydration and strenuous exercise Also with Congestive heart failure, cold exposure and fever TRANSIENT PROTEINURIA Proteinuria only on certain occasions and otherwise normal Requires 6 monthly follow-up.

54. Sulfosalicylic Acid Method (Qualitative): Depends on formation of a precipitate on addition of 3% Sulfosalicylic acid for determination of the presence of protein. This method will detect about 5 to 10 mg/dl . Albumin, globulins, glycoproteins, and Bence Jones proteins are all detected. * There are Quantitave methods available like Sulfosalicylic acid method and Trichloroacetic acid method but these quantitative methods have been found unsatisfactory. Confirmatory test

55. Glucose and Other Sugars in Urine The presence of detectable amounts of glucose in urine is termed glycosuria. Glucose may appear in the urine at different blood glucose levels, and there is not always a concomitant hyperglycemia. Glycosuria usually occurs when the blood level is greater than 180 - 200 mg/dl (Reference: Henry’s Clinical Diagnosis and Management by Laboratory Methods – 23 rd edition) Causes of Glycosuria: Renal Glycosuria (due to low renal threshold – Acquired/Congenital) e.g. Fanconi’s Syndrome Gestational Glycosuria (Reduced Renal threshold) Alimentary glycosuria – On consuming high amounts of carbohydrates Others: Endocrinal disorders (e.g. Acromegaly, Cushing’s syndrome, Hyperadrenocorticism, Pheochromocytoma, Diabetes Mellitus, etc)

56. Fanconi’s syndrome Fanconi's syndrome  is a  syndrome  of inadequate  reabsorption  in the proximal  renal tubules  of the  kidney . The syndrome can be caused by various underlying congenital or acquired  diseases , by  toxicity  (for example, from  toxic heavy metals ), or by  adverse drug reactions . It results in various small  molecules  of  metabolism  being passed into the  urine  instead of being  reabsorbed  from the  tubular fluid  (for example,  glucose ,  amino acids ,  uric acid ,  phosphate , and  bicarbonate ). Most common inherited cause in pediatric age group is Cystinosis … other causes include, Expired tetracyclines (forms epitetracyclines and anhydrotetracyclines ) Multiple myeloma Wilson’s disease, etc

57. Methods of Glucose estimation Reagent strip method Copper reduction tests Normally, 50 mg of disaccharides are excreted in 24 hours. With severe Intestinal diseases like sprue or acute enteritis , this level may rise to 250 mg or more.

58. Reagent Strip method Principle: Based on a specific glucose oxidase and peroxidase method, a double sequential enzyme reaction ; reagent strips differ only in the chromogen used. Chemistry: Glucose + O2 Gluconic acid + H2O2 H2O2 + Chromogen Oxidized chromogen + H2O Glucose Oxidase Peroxidase *Multistix- potassium iodide chromogen - Color changes from blue to brown at 30 seconds.

59. Drawbacks: False-positive: If strongly oxidizing cleaning agents are present in the urine container. Low specific gravity may falsely elevate results. False-negative: Use of Sodium fluoride as a preservative High specific gravity Occasionally by Ascorbic acid

60. Cross-verification Copper Reduction Tests: If false-negative is suspected due to ascorbic acid, should be confirmed with Copper reduction test In case of neonates and infants where excretion of sugars other than glucose is suspected, copper reduction test is mandatory. In those instances in which the copper method is positive and the glucose oxidase method is negative, glycosuria is ruled out. Out of all Copper Reduction Tests, Benedict’s test is the most sensitive test. Other tests include: 5-drop and 2-drop Clinitest tablet methods

61. Other sugars in urine of neonates Lactose Xylose Arabinose Galactose

62. Ketones in Urine Defect in carbohydrate metabolism or absorption or an inadequate amount of carbohydrate in the diet- the body compensates by metabolizing increasing amounts of fatty acids. In ketonuria, the three ketone bodies present in the urine are acetoacetic (diacetic) acid (20%), acetone (2%), and 3-hydroxybutyrate (about 78%). Acetoacetic acid Acetone Acetoacetic acid 3-Hydroxybutyrate - CO2 +2H -2H (Irreversible) (Reversible)

63. Causes of Ketonuria 1. Diabetic Ketoacidosis: Seen more commonly and more importantly with Type 1 Diabetes mellitus. 2. Nondiabetic ketonuria: Acute febrile diseases in infants and children, Hypoalimentation (e.g. starvation), Hyperemesis of pregnancy, in cachexia and following anesthesia. 3. Lactic Acidosis: Can coexist with Diabetes mellitus, Renal failure, Liver disease.

64. Methods: Reagent Strip method Nitroprusside Tablet test Gerhardt ferric chloride test (Only for acetoacetic acid) Test tube nitroprusside method of Rothera Commonly used method is the Reagent strip method as it is more sensitive in detecting as low as 10 mg of acetoacetic acid. (Less sensitive to Acetone) None of the tests mentioned above detects Beta- Hydroxybutyric acid

65. Reagent Strip method Principle : Based on nitroprusside reaction for ketone. The multistix strip contain buffers and sodium nitroprusside which reacts with acetoacetic acid producing a pink maroon colour in 15 seconds. It is less sensitive to Acetone. Reagent strip without alkali react to acetoacetate not acetone. The chemo strip reagent strip contain sodium nitroprusside buffer and glycine which reacts with acetoacetic acid in presence of alkaline medium to form violet dye. Sensitivity : 10 mg/dl of acetoacetic acid and 70 mg/dl of acetone.

66. Positive result is indicated by change in colour from beige to violet within 60 seconds. It is not sensitive to gamma hydroxybutyric acid. With large results like 3+, urine may be diluted and reassessed. Drawbacks False positive is seen after the use of phthaleins or in presence of extremely large amount of phenyl ketones , l-dopa and 8-hydroxyquinolone metabolites False negative results occur because of loss of reagent . Reaction may also not occur if specimen handling is improper which causes acetone loss due to its volatile characteristic or consumption by bacteria.

67. Other test Rothera’s Test Saturate about 5ml of urine with ammonium sulphate. Add few crystals of sodium nitroprusside and shake well. Add liquor ammonia through the sides of the test tube. Formation of a purple ring at the junction indicate a positive test. * Not a sensitive test and hence gives False-negative even in presence of Ketones in urine.

68. Determination of Bilirubin Bilirubin is breakdown product of Hemoglobin which occurs in Reticuloendothelial cells of Spleen, Liver and Bone marrow. The unconjugated Bilirubin thus formed is transported to liver for conjugation with glucuronic acid to form Bilirubin glucuronide. Normal adult urine has only 0.02 mg/dl of Bilirubin Increased urinary Bilirubin occurs in- Obstruction to bile outflow from liver Hepatocellular disease with decreased excretion of conjugated Bilirubin in bile * Presence of Bilirubin in urine means presence of only CONJUGATED BILIRUBIN

69. Methods: Reagent Strip method Diazo tablet method Wash-through tablet method The reagent strip is much less reactive to free bilirubin than is the tablet test so that a difference in results becomes more apparent as the urine ages. The above point means that when urine is kept for a longer period of time(especially when exposed to light), bilirubin glucuronide gets converted free bilirubin which isn’t picked up accurately on the Reagent strip but is seen with the Tablet test Hence, Tablet tests are confirmatory tests

70. Principle : The test is based on coupling reaction of bilirubin with diazonium salt in strongly acidic medium . Multistix reagent strip: If bilirubin is present color changes from cream buff to tan which is read at 20 seconds .This uses the diazonium salt- diazotized 2,4-dichloroaniline. Detects 0.8 mg/dl of bilirubin Chemo strip reagent: Contains 6-dichlorobenzene diazonium tetraflouroborate as salt which changes color from pink to violet at 30 -60 seconds. Detects 0.5 mg/dl of bilirubin Reagent Strip method

71. Drawbacks: Urine must be fresh because bilirubin glucuronide in urine quickly hydrolyses to less reactive free bilirubin . False negative: Seen when specimen stands too long due to oxidation of bilirubin. Large amount of ascorbic acid and nitrite can also lower bilirubin results False positive: Rifampicin and large amount of chlorpromazine metabolites may cause false positive * Salicylates and urobilinogen do not interfere with the reaction .

72. Confirmatory Bilirubin Test: Diazo tablet method: The reagent strip test is much less reactive to free bilirubin than is the tablet test, so that a difference in results becomes more apparent as the urine ages. The diazo test reacts positively to bilirubin in amounts as low as 0.05 to 0.1 mg per decilitre. No purple reaction is seen with urobilin.

73. Bile salts and Bile pigments(BSBP) Bile Salts (Hay’s Sulphur powder test): Bile salts have the property of decreasing surface tension. Hence, sulphur powder sprinkled to urine will settle down if bile salts are present in urine. Bile Pigments (Fouchet’s Test): Bile pigments adhere to the precipitates of barium sulphate. On addition of Fouchet’s reagent, ferric chloride in the presence of trichloroacetic acid oxidizes yellow bilirubin to green biliverdin.

74. Determination of Urobilinogen Conjugated bilirubin reaches eventually to duodenum from liver but without getting absorbed, passes on to colon where it is acted upon by bacteria to form: Urobilinogen Mesobilirubinogen Stercobilinogen Out of all these, only small amount of urobilinogen is excreted in the urine after the enterohepatic circulation is complete. Normal output of urobilinogen in urine is 0.5 to 2.5 mg/24 hours

75. Causes of increased urobilinogen excretion: Hepatocellular damage due to viral hepatitis, drugs or toxic substances or in some cases cirrhosis. Congestive heart failure (Due to liver congestion, reexcretion into bile is impaired). Infection such as Cholangitis associated with obstruction (Urobilinogen with bilirubin) Hemolysis (Urobilinogen without bilirubin)- It can be one of the indications for malaria infestation.

76. Methods: Reagent Strip method Ehlrich’s Aldehyde method For quantitative comparative purposes in the same patient, a 2-hour test is used in which urine is collected from 2pm to 4pm after lunch. This period coincides with heightened excretion of urobilinogen , as the pH of the urine is more nearly neutral. Other 2-hour periods may be tested for comparison

77. Principle: The test is based on modified Ehrlich’s Aldehyde reaction .The test area is impregnated with p- dimethyl aminobenzaldehyde in conjunction with color ethanol which reacts with urobilinogen in strongly acidic media to form yellow shades of red brown (Multistix). This is not specific for urobilinogen . Other porphobilinogen, indole, sterol can also give same color development. Chemo strip reagent : Test area is impregnated with 4- methoxybenezene tetra flourate .This is specific for urobilinogen. Reagent Strip method

78. Drawbacks: False negative: The test can be negative as urobilinogen is quite labile and will convert to urobilin which is non-reactive therefore only fresh sample is acceptable. If formalin is present it will give false negative result False positives: Test can also be altered by p- aminosalicyclic acid, sulphonamide and p- aminobenzoic acid. Bilirubin if present will give green color.

79. Indirect Tests for Urinary Tract Infection Two most commonly utilized testing modalities for indirect assessment of bacteriuria and leukocyturia are: Reagent strip nitrite Reagent strip Leukocyte esterase Microscopic urinalysis : Rapid confirmatory test for the presence of leukocytes and bacteria Gold standard: Bacteriologic culture

80. Nitrite Detects 70% positive results when compared to cultures E-coli only: 93% agreement with cultures Most common organisms causing positive nitrite test: Escherichia coli Klebsiella Enterobacter Proteus Staphylococcus Pseudomonas

81. Reagent Strip method Principle: This is based on conversion of nitrate in urine to nitrite by the action of bacteria like Proteus species, E.coli, Enterobacter, and Klebsiella.                                             Nitrite+ p- arsinilic acid Diazonium complex This diazonium compound then couples with 1,2,3,4, tetra hydrobenzoquinolin-3- ol to produce pink color . This indicates- 10 5.  Or more organisms per ml. * (Bacteriuria is >1,00,000 bacteria per ml of urine) Acidic pH

82. This method detects the presence of 0.075 gm of nitrite/dl. Drawbacks: False positive is seen in stored sample due to contaminants and post collection bacterial proliferation; and medication (phenazopyridine) False negative result is seen due to ascorbic acid, urobilinogen or low pH, lack of dietary nitrites.

83. Leukocyte Esterase Esterase activity- Marker for human neutrophils . Because neutrophils and other cells are labile in urine, leukocyte esterase activity can be indicative of remnants of cells that are not visible microscopically. Positive leukocyte esterase- Significant number of Neutrophils either intact or lysed. It has a high negative predictive value.

84. Reagent Strip method Neutrophilic esterases catalyze the hydrolysis of esters to produce their respective alcohols and acids. Multistix: Utilizes 3-hydroxy-5- phenyl- pyyrole -N- tosyl -l- alanine ester as a substrate, which reacts in the presence of leukocyte esterase to form pyrrole alcohol. The alcohol then reacts with a diazonium salt to produce a purple colour . The intensity of the colour produced is proportional to the amount of enzyme present , which is related to the number of neutrophils present.

85. False negative: Seen in urine having- High specific gravity, High glucose concentration, Drugs like gentamycin and high protein content False positive: Contamination of urine with Vaginal fluids Trichomonas and eosinophils provide another source of esterases and hence false positive results.

86. Blood, Hemoglobin, Hemosiderin, and Myoglobin in Urine Hematuria: Presence of abnormal number of red blood cells in urine. Hemoglobinuria: Presence of free hemoglobin in urine. Myoglobinuria: Presence of myoglobin in urine. For e.g. musculoskeletal trauma

87. Hematuria Gross hematuria: Presence of increased number of red blood cells so much so that it is visible to the naked eye Microscopic hematuria: 3 or >3 red blood cells per high-powered field As it is important to identify red blood cells in urine and due to the lysis of red blood cells in urine, some studies suggest that Reagent strip method is more sensitive than urine microscopy to identify hematuria. Yet, it is better to confirm this by microscopy of a fresh urine specimen * Alkaline pH and Low specific gravity can cause in-vitro lysis of Red Blood Cells.

88. Causes of Hematuria Diseases of Urinary tract: Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus nephritis, Henoch-Schonlein purpura. Non-glomerular disease: Calculus, tumour, infection, tuberculosis, pyelonephritis, hydronephrosis, trauma, after strenuous exercise. Hematological conditions: Coagulation disorders, Sickle cell disease. * Presence of Red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria

89. Blood/ Heme proteins Reagent Strip method Principle: This method is based on the liberation of oxygen from peroxide in reagent strip by peroxidase like activity of haem in free haemoglobin of lysed RBC or myoglobin . Intact RBC are lysed on strip causing haemoglobin to react . The reagent area is impregnated with buffered mixture of organic peroxide and chromogen tetra methyl benzidine. Haemoglobin catalyses the oxidation of chromogen to produce green colour which is read at 60 seconds.  If green spot is present then intact RBCs are present If uniform green colour is present then it indicates the presence of haemoglobin or myoglobin.

90. Sensitivity is up to 0.05-3 Hb/dl of urine False positive: Contamination of urine by menstrual blood in females. In presence of oxidizing agents ( hypochlorite bacterial content, microbial peroxidase associated with UTI) False negative: Presence of ascorbic acid and formalin . Presence of nitrites in large amount will delay the reaction. * Confirmation of Hematuria can be done through Microscopy of Urine sediment

91. GFR Glomerular filtration rate (GFR) is generally considered the best overall indicator of the level of kidney function (Smith, 1951). Inulin clearance is widely regarded as the gold standard for measuring glomerular filtration rate. Simpler or more practical method is creatinine clearance test. Two approaches Endogenous Exogenous Creatinine clearance Inulin clearance

92. Creatinine is the most widely used marker of GFR for several reasons. It is an endogenous substance with a fairly constant rate of production. Creatinine is not bound to plasma proteins , and therefore is filtered freely by the glomerulus. It is not reabsorbed by the renal tubules Only a small amount is secreted by the tubules.

93. Estimated GFR (eGFR) In the Modification of Diet in Renal Disease (MDRD) study , Levey and colleagues measured GFR by I 125 -iodothalamate to derive a formula for estimating glomerular filtration rate using six variables – age, sex, serum urea nitrogen, serum creatinine and serum albumin concentration. This equation was simplified to include age, sex, race and serum creatinine as follows: GFR = 175 ×Cr × age ×1.212 (for black) × 0.742 (for women) −1.154 −0.203 Due to major limitation in accurate urine collection for measurement of creatinine clearance, mathematical formulas have now been developed to assess the GFR.

94. Estimated GFR results are reported as ml/min/1.73m 2   A normal eGFR for adults is greater than 90 ml/min/1.73m 2 , according to the National Kidney Foundation An eGFR below 60 ml/min/1.73m 2  suggests that some kidney damage has occurred KIDNEY DAMAGE STAGE DESCRIPTION ESTIMATED GFR (ML/MIN/1.73M 2 ) 1 Normal or minimal kidney damage with normal GFR 90+ 2 Mild decrease in GFR 60-89 3 Moderate decrease in GFR 30-59 4 Severe decrease in GFR 15-29 5 Kidney failure <15

95. Albumin to Creatinine Ratio The urine albumin test or albumin/creatinine ratio (ACR) is used to screen people with chronic conditions e.g.   diabetes and high blood pressure (hypertension) - increased risk of developing kidney disease. The American Diabetes Association has stated a preference for the ACR for screening for albuminuria indicating early kidney disease. Result Interpretation < 3 mg/ mmol Normal 3.0 - 30.0 mg/ mmol Moderately increased >30.0 mg/ mmol Severely increased Interpretation of ACR: * Including nephrotic syndrome (urine ACR usually >220 mg/ mmol )

96. ACR is the Albumin Creatinine Ratio - and is the preferred test for detection of small amounts of albumin (protein) in the urine. A new finding of urine ACR of between 3.0 and 70.0 mg/ mmol should be confirmed with a first morning urine sample to establish consistent finding and exclude transient elevation due to intercurrent illness. If the urine ACR is  >70 mg/ mmol there is no need to repeat to confirm the result. * Regard a confirmed ACR of 3.0 mg/ mmol or more as clinically important proteinuria.

97. Special Tests on urine 1) Porphyrins Porphyrins require special mention as on the Reagent strip test of urobilinogen, porphyrins give positive reaction and it has to be differentiated from urobilinogen. The porphyrias are a group of diseases resulting from defects in the synthesis of heme. The Watson- Schwartz test is used to separate causes of a positive Ehrlich-reacting test and to give an indication of large amounts of urobilinogen or the presence of porphobilinogen.

98. Watson-Schwartz test Watson Schwartz test is based on solubility differences between urobilinogen and porphobilinogen. Urobilinogen can be extracted by chloroform and/or butanol , whereas porphobilinogen will remain in an aqueous phase. A positive result for porphobilinogen in the Watson-Schwartz test can be further confirmed by the Hoesch test , because the former may show false-positive results for porphobilinogen as a result of drugs such as methyldopa. Fluorescence screening procedure for porphyrin is another method using Wood’s lamp to diagnose Porphyrias.

99. Watson- Schwartz Test for Porphobilinogen To confirm: Hoesch test

100. 2) Melanin No simple specific test is available for melanuria Tests based on nonspecific colour reactions produced with ferric chloride, Ehrlich’s aldehyde reagent, and nitroferricyanide can be performed. A column cation-exchange chromatographic method allows detection of melanin metabolites in urine. 3) 5-Hydroxyindoleacetic Acid It is a metabolite of Serotonin (5-Hydroxytryptophan) found in urine. The metabolite can be analyzed directly by using High Performance Liquid Chromatography (HPLC).

101. 4) Bence Jones Proteinuria Bence Jones proteinuria is associated with multiple myeloma, macroglobulinemia, and malignant lymphomas. Electrophoresis and immunofixation electrophoresis methods are the best detection and quantification methods, along with the immunoassay measurement of free light chains 5) Microalbuminuria Microalbuminuria is the presence of albumin in urine above the normal level but below the detectable range of conventional urine dipstick methods. (Range: 20-200 mg/L – Ref: Todd and Stanford) Immunologic test systems and dye-binding chemical test strips are available for accurate diagnosis Important for monitoring Early diabetic nephropathy

102. 6) Aminoacidurias Excretion of one or more amino acids in the urine may be due to a block in a major metabolic pathway (overflow type) or a deficiency in renal tubular function (renal type). Phenylketonuria: Phenylketonuria is an autosomal recessive inherited disorder in which there is absence of the enzyme phenylalanine hydroxylase. Alkaptonuria: The enzyme homogentisic acid oxidase is deficient, and homogentisic acid is excreted in urine in large quantities. Screening Phenistix reagent strips Confirmatory Ion-exchange high performance liquid chromatography Screening Ferric chloride and silver nitrate tests Confirmatory Paper or thin-layer chromatography; capillary electrophoresis