Urine -Physical and Chemical Examination and Reagent Strips

Reference - Henry Clinical Pathology , Kawthalkar Clinical Pathology

1. Physical and Chemical Examination Of Urine Presented by– Dr. Pritika Nehra

2. URINE EXAMINATION is an important lab test as many different diseases can display abnormalities in the urine . Basic urinalysis consists of gross examination of the urine, as well as a dipstick analysis for blood, white blood cells, sugar, and other substances. A microscopic analysis of urine may be necessary in many cases. This is done to detect cellular elements, casts, and crystals.

3. RENAL DISEASES . URINARY TRACT INFECTION METABOLIC DISORDERS like DM,DI. JAUNDICE. PLASMA CELL DYSCRASIAS . Diagnosis of PREGNANCY . GENETIC Abnormalities – Cystinuria , Phenylketonuria Indications of Urine Examination

4. Specimen Evaluation Gross / Physical Examination Chemical Screening Sediment Examination Components of Basic Urinalysis

5. 1) PROPER LABELING – Patient’s full name, Date and Time of collection Specimen Evaluation

6. 2) PROPER SPECIMEN – Methods of urine collection Midstream Clean Catch Specimen Catheter Specimen Plastic Bag Specimen- infants Supra pubic aspiration Three glass Specimen - prostate infection

7. Collection of urine for A) Routine examination containers should be: wide mouthed , Clean and leak proof. Break-resistant , Disposable , clear material Capacity of 50ml(routine) and 3lt. (24hr sample) Amber coloured containers for light sensitiveAnalytes 15 ml of midstream sample B) Culture examination Midstream clean catch sample Plated within 2hrs of collection (if refrigerated – 24hrs) No preservatives If single specimen is received for multiple tests then bacteriological examination should be done first.

8. C) Proper Preservative REFRIGERATION (mc) Ideally the specimen should be examined within 2 hoursof voiding. If delay is expected, can be refrigerated for maximum of 24hrs 4-6°C refrigeration upto 8hrs is best general preservation method

9. Preservatives for testing of Steroids HCl + copper sulphate VMA HCl Porphyrin Sodium carbonate CHEMICAL PRESERVATIVES – Formalin 3 drop/100 ml ( formed elements) Toluene 2 ml/100 ml (chemical substances) Boric acid 0.5gm/60 ml (general preservative) Thymol 1crystal/100m (sediments ) Conc. HCl (Adrenaline, nor-A, VMA, steroids)

10. D) Visible Signs Of Contamination Effect of Storage on urine: 1.Decrease in clarity , foul smeling odour Increase in pH. 2. Formation of crystals 3. Loss of ketone bodies 4. Decrease in glucose 5. Oxidation of bilirubin to biliverdin 6. Oxidation of urobilinogen to urobilin 7. Increase in nitrites 8. Disintegration of cellular elements

11. Physical Examination Of Urine

12. COLOR Normally, urine is AMBER coloured and CLEAR

13. COLOR PATHOLOGICAL NON PATHOLOGICAL Pink to Red to Red -Brown Red blood cells Haemoglobin Myoglobin Porphyrins Beets( anthocynin ) Methyldopa YELLOW TO ORANGE Bilirubin Urobilin Concentrated urine Rifampicin Acriflavine Carrots

14. COLOR PATHOLOGICAL NON PATHOLOGICAL Brown to Black Methaemoglobin Rhabdomyolysis Homogentisic acid Melanin Iron compounds Levodopa Chloroquine Metronidazole Blue to Green Biliverdin Pseudomonas Infection Food dyes Additives Amitryptiline Cimetidine Methylene blue Vit B complex

15. CLARITY

16. Cloudy urine Dissolves on acidification Dissolves on warming (60 °C) Does not dissolve PHOSPHATE AMMONIUM URATE CARBONATE URIC ACID URATES PATHOLOGICAL WBC BACTERIA CHYLURIA LIPIDURIA

17. ODOUR Freshly voided urine- Typical aromatic odour (Volatile organic acids) On standing- Conversion of urea  ammonia– Faint ammoniacal odour

18. Volume Volume of only the 24 hour specimen of urine is to be measured . Average 24hr urinary output in adults is 600 to 2000ml Called as Amount Causes Polyuria >2000ml in 24 hours Diabetes mellitus, Diabetes insipidus Chronic renal failure Diuretic therapy 2. Oliguria < 500 ml in 24 hours Fever Dehydration Congestive heart failure Acute glomerulonephritis Anuria < 100 ml or NO output in 24 hours Complete urinary tract obstruction Acute tubular necrosis Acute glomerulonephritis

19. Ratio of weight of a volume of urine to the weight of the same volume of distilled water at a constant temperature. Main contributors - UREA (20%) and NaCl (25%) 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. Specific Gravity

20. Specific gravity of distilled water= 1.000 Normal sp.gr. of urine Random sample 1.003 to 1.030 ( at least > 1.023) 24 hr sample with normal fluid intake 1.016 to 1.022 After a standard water load < 1.007 Abnormal sp.gr. of urine HYPO- STHENURIA Consistently < 1.007 DI, GN, pyelonephritis ISO- STHENURIA Fixed at 1.010 Severe Renal Damage HYPER-STHENURIA Higher than normal Fever, dehydration, CHF, adrenal insufficiency, DM, nephrotic syn.

21. Methods of Specific Gravity measurement INDIRECT METHODS 1. Reagent strip method 2. Urinometer DIRECT METHODS 3. Refractometer 4. Falling drop method M/C EMPLOYED

22. Reagent test strips These measure the ion concentration in urine Contents of a reagent strip pad- (1) polyelectrolyte (2) indicator substance (3) buffer 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 .

23. The strips have colour pads with increment of reading of 0.005 Advantages : 1. This method is not affected by the high amounts of protein and sugar and so there is no need for the correction of value. 2. 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.

24. 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: 1. Only a few drops of urine are required 2. The results obtained are quite accurate DISADVANTAGES : 1. Valid only for Urine 2. Damaged by heat above 150° F and by immersion of eyepiece and focusing ring in water 3. Requires daily calibration for accurate results

25. 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 . Falling Drop Method

26. Osmolality Indicates the number of particles of solute per unit of solution Normal fluid intake- 500 to 850 mOsm /kg water. 800 to 1400 mOsm/kg water in dehydration, After a period of dehydration, the osmolality of the urine should be three to four times that of the plasma 40 to 80 mOsm/kg water during water diuresis . 285 mosm /kg in terminal renal failure Measured by freezing point depression using osmometer . A solution containing 1osmol or 1000 mOsm/kg water depresses the freezing point 1.86° C below the freezing point of water

27. Chemical Examination Of Urine

28. REAGENT STRIPS Primary method used for the chemical examination of urine. Reagent strips are dip- and -read strips for in vitro diagnostic use only for testing various contents in urine. It is measured by comparison of test paper attached to plastic strip with color of chart blocks printed on vial label.

29. Testing Test urine as soon as possible after receipt. Test a well-mixed, unspun urine sample. Urine samples must be at room temperature before testing. Do not use reagent strips in the presence of volatile acids or alkaline fumes Dip reagent strip into urine briefly—no longer than 1 second. Follow exact timing recommendations for each chemical test. Hold reagent strip close to the color chart, and read under good lighting. Know sources of error, sensitivity, and specificity of each test on the reagent strip. Think! Make correlations between patient history and individual test, and then follow through.

30. URO-DIP CHECK 300 By ERBA

31. 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

32. Normal pH is 4.6 to 8.0 ( avg -6) Methods: 1. Reagent strip method 2. Litmus paper test 3. pH meter Urine pH 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 Chronic renal failure

33. • 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. Reagent strip method 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

34. Litmus paper test : pH meter : Other Methods:

35. Normal- Up to 150 mg/24 hours , average protein concentration varying from 2 to 10 mg/dl Proteins from plasma (albumin) and Proteins from UT ( Tamm horsfall protein , IgA , Tubular epithelial cells , WBC’s, desquamated cells . 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 PROTEIN

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

37. (> 4 gm/day) Nephrotic syndrome (loss of albumin , globulin) Acute and rapidy progressive glomerulonephritis Chronic glomerulonephritis Diabetic nephropathy, severe Renal amyloidosis Lupus nephritis Toxemia of pregnancy End Stage Renal Disease Miscellaneous – malaria,malignant hypertension,heavy metals,drugs Heavy Proteinuria

38. 1. Glomerular Proteinuria - increased permeability of glomerular capillary wall SELECTIVE - excretion of LMW proteins NON SELECTIVE - HMW proteins (γ globulins) are also excreted 2 . Tubular Proteinuria - Qualitative Categories Of Proteinuria LMW proteins like microglobulin , RBP, lysozyme , and free immunoglobulin light are excreted in urine while albumin excretion is minimal. Eg - acute and chronic pyelonephritis , heavy metal poisoning, TB of kidney, interstitial nephritis, cystinosis , Fanconi syndrome and rejection of kidney transplant.

39. Glomerular and tubular proteinuria . (1) selective proteinuria , (2) non-selective proteinuria , and (3) tubular proteinuria

40. 3. Overflow proteinuria : immunoglobulin light chains or Bence Jones proteins hemoglobin ( intravascular hemolysis ) myoglobin (skeletal muscle trauma) lysozyme (AML M4 or M5). 4. Hemodynamic proteinuria : high fever, hypertension , heavy exercise, congestive cardiac failure Cold exposure

41. Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in recumbent position ,due to lordotic posture that causesIVC compression between the liver and vertebral column. Post-renal proteinuria : This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter , bladder, prostate, or urethra.

42. Bence Jones Proteinuria monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. occurs in plasma cell dyscrasias . (overflow proteinuria ). When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60-70°C), and precipitate disappears, on further heating at 85-100°C (while precipitate of other proteins does not). When cooled (60-85°C), there is reappearance of precipitate of Bence Jones proteins. replaced by protein electrophoresis Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine

43. Protein screening tests Qualitative tests Reagent strip Sulphosalicylic acid test Heat Precipitation test Heller’s nitric acid test Quantitative tests Esbach’s Albuminometer Immunological method There are other Quantitave methods available but these have been found unsatisfactory.

44. Reagent strip test Principle (protein error of indicators)- Reagent area of strip is coated with an indicator (tetrabromophenol blue) and buffered to an acid pH 3 which changes color in the presence of proteins indicator undergoes color change from yellow to shades of green Sensit i ve to albumin 5-10 mg/dl Negative with Bence-Jone s protein, Hb , Mb

45. Advantage: • avoids false-positive reactions with organic iodides (e.g. Radiograph contrast and tolbutamides or other drugs). Drawbacks: • Lack of sensitivity of the reagent strip to globulins • False positive results can occur with highly pigmented urine, quaternary ammonium compounds, 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.

46. Sulfosalicylic Acid Method (Qualitative): Principle - Proteins are denatured by organic acid and precipitate out of sol. Detects albumin, Hb, myoglobin,Bence Jones protein Procedure :- A dd 2-3 drops of 3% Sulphosalicylic acid in 2ml urine, wait for 10 minutes and observe for degree of precipitation

47. Interpretation Negative : No turbidity (0.005 gm/dl) Traces : Perceptible turbidity ( 0.020 gm/dl) 1+: Distinct turbidity (0.01 - 0.05 gm/dl) 2+: Turbidity with granulations (0.05 - 0.20 gm/dl) 3+: Turbidity with flocculations (0.20 - 0.5 g/dl) 4+: Clumps of ppt (>1 gm/dl) False +ve - Tolbutamide, penicillin, sulfonamides, radiographic dyes False –ve - Alkaline, very dilute urine

48. GLUCOSE Glycosuria occurs when the blood level is >180 - 200 mg/dl Causes of Glycosuria : 1. Renal Glycosuria (due to low renal threshold) e.g. Fanconi’s Syndrome 2. Gestational Glycosuria (Reduced Renal threshold) 3. Alimentary glycosuria 4. Endocrine causes-DM, Acromegaly, Cushing syndrome, hyperthyroidism, pancreatic disease 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.

49. Methods of Glucose estimation 1. REAGENT STRIP METHOD 2. COPPER REDUCTION TEST Benedict’s Test Clinitest Tablet

50. 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. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change False neg a tives – ketones, ascorbic acid, salicylates, E.coli infection False positive – Presence of oxidizing agents

51. Copper Reduction Tests: Done to cross verify when false-negative is suspected due to ascorbic acid • excretion of sugars other than glucose is suspected (infants) Benedict’s Test Principle -In benedict reagent cupric ion present in form of copper sulphate (blue color) which is reduced by reducing substance like glucose to cuprous oxide (brown red color)

52. Defect in carbohydrate metabolism or absorption, the body compensates by metabolizing increasing amounts of fatty acids. • 3 ketone bodies (products of incomplete fat metabolism ) present in the urine are acetoacetic acid (20%), acetone (2%), and 3-hydroxybutyrate (about 78%). Causes of Ketonuria 1. Diabetic Ketoacidosis : Type 1DM 2. Nondiabetic ketonuria : Acute febrile diseases, starvation, Hyperemesis of pregnancy 3. Lactic Acidosis:Can coexist with Diabetes mellitus, Renal failure, Liver disease. KETONES

53. Methods: 1. Reagent Strip method 2. Test tube nitroprusside method of Rothera 3. Nitroprusside Tablet test 4. Gerhardt ferric chloride test (Only for acetoacetic acid) Commonly used method is the Reagent strip method, detects as low as 10 mg of acetoacetic acid. None of the tests mentioned above detects Beta- Hydroxybutyric acid

54. Principle : Based on nitroprusside reaction for ketone . • 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 . Reagent Strip method

55. Other Tests Rothera’s Test Principle -Acetone & acetoacetic acid react with sodium nitroprusside in the presence of alkali to produce purple colour ring. Method- take 5ml of urine in a test tube & saturate it with ammonium sulphate. Then add one crystal of sodium nitroprusside. Then gently add liquor ammonia along the sides of the test tube. Sensitivity - 1-5 mg/dl AAA, 10-15 mg/dl Acetone

56. • Gross hematuria : visible to the naked eye • Microscopic hematuria : 3 or >3RBCs/HPF • Reagent strip method is more sensitive than urine microscopy .Yet, it is better to confirm this by microscopy. Causes of Hematuria 1. Diseases of Urinary tract : Glomerular diseases : Glomerulonephritis , Berger’s disease, lupus, HSP Non- glomerular disease : Calculus, tumour, TB,pyelonephritis etc 2 . Hematological conditions : Coagulation disorders, SCD Presence of Red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria HEMATURIA

57. Principle : Liberation of oxygen from peroxide in the reagent strip by peroxidase like activity of hemoglobin, lysed erythrocytes or myoglobin Detect 0.3-0.5 mg/dl • 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 . Reagent Strip method

58. Causes of myoglobinuria injury to skeletal or cardiac muscle, crush injury myocardial infarction dermatomyositis , severe electric shock thermal burns. Chemical tests used for blood or hemoglobin also give positive reaction with myoglobin Ammonium sulfate solubility test is used as a screening test for myoglobinuria ( Myoglobin is soluble in 80% saturated solution of ammonium sulfate , while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria ).

59. • Breakdown product of Hemoglobin . • Normal adult urine has only 0.02 mg/dl of Bilirubin • Increased urinary Bilirubin occurs in- 1. Obstruction to bile outflow from liver 2. Hepatocellular disease with decreased excretion of conjugated Bilirubin in bile * Presence of Bilirubin in urine means presence of only CONJUGATED BILIRUBIN BILIRUBIN

60. Methods: 1. Reagent Strip method 2. Diazo tablet method 3. Wash-through tablet method • when urine is kept for a longer period of time(especially when exposed to light), bilirubin glucuronide gets converted to 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 . The diazo test reacts positively to bilirubin in amounts as low as 0.05 to 0.1 mg per decilitre.

61. Principle : The test is based on coupling reaction of bilirubin with diazonium salt in strongly acidic medium. • 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 False neg a tive- Exposure to light , ascorbic acid False positives – Coloured metabolites of drugs ( Rifampicin and chlorpromazine Reagent Strip method

62. 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 Bile salts and Bile pigments(BSBP)

63. • Conjugated bilirubin in colon is acted upon by bacteria to form: Urobilinogen which 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 Causes Of Increased Urobilinogen 1. Hepatocellular damage. 2. Congestive heart failure (liver congestion) 3. Infection such as Cholangitis 4. Hemolysis ( Urobilinogen without bilirubin UROBILINOGEN

64. Methods: 1. REAGENT STRIP METHOD 2. 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.