URINE Analysis with normal structure of nephron and physical examination and chemical examination

Urine analysis

1. URINE ANALYSIS

2. FORMATION OF URINE URINE – water + ELECTROLYTES + WASTE PRODUCTS filtered out of the blood system. Formation – 3 regulated processes FILTRATION – GLOMERULI , BOWMAN’S CAPSULE REABSORPTION SECRETION PCT , LOOP OF HENLE , DCT , COLLECTING TUBULES GLOMERULAR FILTRATION RATE – NORMAL : 125 ML/MIN OR 180 L/DAY

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. Urine Collection:

5. Collection Methods Midstream specimen Clean-catch specimen :for bacteriological studies 24 HR or Timed specimen : for protein and hormone analysis Catheter specimen : Used for routine and bacteriological study in bedridden patients. Infant : Either by attaching a clean plastic bag around baby’s genitalia and leaving it there for some time or suprapubic puncture Ideally urine should be examined within 2 hrs of voiding, if delayed, should be preserved.

6. Composition

7. PHYSICAL EXAMINATION

8. Volume Total volume can be evaluated only from 24-hour urine sample Normal individual 24-Hour urinary output: 600 to 2000 ml out of which about 400 ml is produced during night

9. Increase in Urine volume Decrease in Urine volume < 4 00 ml/ 24 hours- Oliguria <100 ml/ 24 hours or complete cessation- Anuria >2000 ml/ 24 hours- Polyuria >500 ml during night- Nocturia Specific gravity- <1.018 Cause s 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 Causes Oliguria: High grade febrile states Acute glomerulonephritis (decreased glomerular filtration) Congestive cardiac failure or dehydration (Renal hypoperfusion) Anuria: Acute tubular necrosis Complete urinary obstruction

10. COLOUR Colors Conditions Colorless Dilute urine (diabetes mellitus, diabetes insipidus, overhydration) Red Hematuria , Hemoglobinuria , Porphyria, Myoglobinuria Dark brown or 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

11. Appearance-Clarity Normal, freshly voided urine is clear in appearance. Cause Appearance Diagnosis 1. Amorphous phosphates White and cloudy on standing in Disappear on addition of a drop of   alkaline urine dilute acetic acid 2. Amorphous urates Pink and cloudy in acid urine Dissolve on warming 3. Pus cells Varying grades of turbidity Microscopy 4. Bacteria Uniformly cloudy; do not settle at the bottom following centrifugation Microscopy, Nitrite test

12. Odour Some abnormal odors with associated conditions are: • Fruity : Ketoacidosis , starvation Mousy or musty: Phenylketonuria Fishy: Urinary tract infection with Proteus , Rancid - Tyrosinaemia . Ammoniacal : Urinary tract infection with Escherichia coli , old standing urine. Foul : Urinary tract infection Sulfurous : Cystinuria Burnt sugar - MSUD

13. Specific Gravity (SG) Normal SG of random sample of urine is 1.003 to 1.030 Range of 24 hour sample in Normal adults with adequate fluid intake- 1.015- 1.025. SG of normal urine is mainly related to urea and sodium . SG increases as solute concentration increases and decreases when temperature rises (since volume expands with rise in temperature). Causes of increase in SG of urine are diabetes mellitus (glycosuria), nephrotic syndrome (proteinuria), fever , and dehydration . Causes of decrease in SG of urine are diabetes insipidus (SG consistently between 1.002-1.003), chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating ability of tubules ) and compulsive water drinking

14. CORRECTION: For every 3⁰C increase: add 0.001 For every 3⁰C decrease: subtract 0.001 CORRECTED SPECIFIC GRAVITY= Observed specific gravity+ (Room temp-15) x 0.001 3

15. Methods of measuring Specific Gravity C. Refractometer SG can be precisely determined by a refractometer, which measures the refractive index of the total soluble solids Higher the concentration of total dissolved solids, higher the refractive index . Extent of refraction of a beam of light passed through urine is a measure of solute concentration, and thus of SG Requires only 1-2 drops of urine Result is read from a scale or from digital display Urinometer - This method is based on the principle of buoyancy (i.e. the ability of a fluid to exert an upward thrust on a body placed in it).

16. pH Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic). Urine pH depends on diet, acid base balance, water balance, and renal tubular function Acidic urine is found in ketosis (diabetes mellitus, starvation, fever), urinary tract infection by Escherichia coli , and high protein diet Alkaline urine may result from urinary tract infection by bacteria that split urea to ammonia ( Proteus or Pseudomonas ), severe vomiting , vegetarian diet, old ammoniacal urine sample and chronic renal failure.

17. CHEMICAL Examination

18. CAUSES OF PROTEINURIA PROTEINS Normally, kidneys excrete scant amount of protein in urine (up to 150 mg/24 hours) Proteinuria refers to protein excretion in urine greater than 150 mg/24 hours in adults

19. Tests for Detection of Proteinuria 1.Heat and acetic acid test (Boiling test) : This test is based on the principle that proteins get precipitated when boiled in an acidic solution On adding 2 drops acetic acid ,if precipitation remains=Proteins+ If precipitate becomes clear=Phosphates+ Method: 1 2 3

20. 2.Heller’s Nitric Acid Ring Test: Principle- Concn HNO 3 causes denaturation and hence precipitation of proteins. 0.5ml HNO3 + 0.5ml Urine along the sides of the TT—Ring at the junction if protein+

21. 3.Sulphosalicylic acid test ( CONFIRMATORY) (Principle-ACID PRECIPITATION) Addition of sulphosalicylic acid(3-5%) to the urine causes formation of a white precipitate if proteins are present 4.Reagent strip test :(Principle- COLORIMETRIC TEST) The reagent area of the strip is coated with an indicator and buffered to an acid pH which changes color in the presence of proteins. The principle is known as “protein error of indicators”-meaning that one color appears if protein is present and another color if protein is absent.

22. Indications for quantitative estimation of proteins in urine are: 1.Diagnosis of nephrotic syndrome 2. Detection of microalbuminuria or early diabetic nephropathy 3.To follow response to therapy in renal disease mg/24 hr < 30 30-300 >300 Condition Normal Microalbuminuria Overt albuminuria Proteinuria >1500 mg/ 24 hours indicates glomerular disease ; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria ; in tubular, hemodynamic and post renal diseases , proteinuria is usually < 1500 mg/ 24 hours earliest sign of renal damage in diabetes mellitus (diabetic nephropathy).

23. Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine Bence Jones Proteinuria Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. Excess production of these light chains occurs in plasma cell dyscrasias like multiple myeloma and primary amyloidosis. Detected using Urine Protein Electrophoresis.

24. Glucose Causes of Glycosuria 1. Glycosuria with hyperglycemia : • Endocrine diseases: diabetes mellitus, acromegaly, Cushing’s syndrome, hyperthyroidism, pancreatic disease • Non-endocrine diseases: central nervous system diseases, liver disorders • Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides • Alimentary glycosuria (Lag-storage glycosuria) 2.Glycosuria without hyperglycemia • Renal glycosuria • Fanconi’s syndrome • Toxic renal tubular damage.

25. Tests for Detection of Glucose in Urine Urine Reagent Strip Test is specific for Glucose PRINCIPLE: The result is reported in grades as follows (Fig. 1.10): Nil: no change from blue color Trace: Green without precipitate 1+ (approx. 0.5 grams/dl): Green with precipitate 2+ (approx. 1.0 grams/dl): Brown precipitate 3+ (approx. 1.5 grams/dl: Yellow-orange precipitate 4+ (> 2.0 grams/dl): Brick- red precipitate BENEDICT’S TEST

26. Ketones Excretion of ketone bodies ( acetoacetic acid, β- hydroxybutyric acid, and acetone ) in urine is called as ketonuria Causes of Ketonuria : 1)Diabetic Ketoacidosis : Seen more commonly 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

27. Methods of testing: Reagent Strip method Nitroprusside Tablet test Gerhardt ferric chloride test (Only for acetoacetic acid) Test tube nitroprusside method of Rothera Principles of Rothera’s test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid

28. Bile Pigment (Bilirubin) Urine test Hemolytic Hepatocellular Obstructive jaundice jaundice jaundice 1. Bilirubin Absent Present Present 2. Urobilinogen Increased Increased Absent Methods for detection of bilirubin in urine are foam test , Gmelin’s test , Lugol iodine test , Fouchet’s test , Ictotest tablet test, and reagent strip test . FOUCHET’S TEST

29. UROBILINOGEN Tests for Detection of Urobilinogen in Urine Reagent Strip method(SPECIFIC) Ehlrich’s Aldehyde method Reagent Strip method

30. Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection) Nitrite test If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella , etc .) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests Leucocyte esterase test detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done

31. BLOOD, Hb , Myoglobin & HEMOSIDERIN Chemical tests are positive in hematuria , hemoglobinuria , and myoglobinuria : 1) Benzidine test 2) Orthotoluidine test 3) Reagent strip test Parameter Hematuria Hemoglobinuria Myoglobinuria 1. Urine color Normal, smoky, red, Pink, red, or Red or brown   or brown brown   2. Plasma color Normal Pink Normal 3. Urine test based on Positive Positive Positive peroxidase activity 4. Urine microscopy Many red cells Occasional red cell Occasional red cell 5. Serum haptoglobin Normal Low Normal 6. Serum creatine kinase Normal Normal Markedly increased

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