"Abnormal Constituents of Urine" practical

"Abnormal Constituents of Urine" practical

1. Abnormal constituents of Urine Amit Jha Lecturer UCMS, Bhairahawa

2. Urine Water (95%) & Solids (5%)] Urinary out put: 1-1.5 L per day. Almost all substances found in urine are also find in blood. may also contain cells, casts, crystals, mucus & bacteria.

3. Urine: Provides information about functioning & abnormalities of kidneys & urinary tract Help in diagnosis of various systemic diseases [+nce or –nce of several substances in urine]

4. Normal constituents of urine Creatinine 0.8 – 1.8 gm/L Uric Acid 0.5 gm/L Urea 25-30 gm/L Ca ++ 0.2 gM /L PO 4 3- 1.7gm/L K + 1.7 gm/L Na + 3.5 gm/L Cl - 6-16 gm/L

5. Preservation For routine analysis, urine is best examined fresh. Bacterial growth will ruin a specimen if analysis is delayed for >3 hrs . Refrigeration : best way to preserve if analysis is delayed. Refrigeration for >24hrs isn’t recommended .

6. Chemicals used Toluene Thymol [for sugar estimation] Formalin Boric acid Camphor Toluol [for acetone estimation] Chloroform

7. Changes occur in non preserved specimen Urea → NH 3 ↑es pH ↓ Glucose d/t bacterial utilization ↓ KB d/t volatilization ↓ bilirubin d/t exposure to light ↑ bacterial number ↑ turbidity bacteria & amorphous Disintegration of RBCs casts ↑ nitrite d/t bacterial reduction of nitrate Changes in color d/t oxidation or reduction of metabolite

8. URINE ANALYSIS Physical Chemical Microscopic

9. MACROSCOPIC EXAMINATION OF URINE Color Clarity Odor Volume Specific gravity pH

10. Color : Normal urine is of amber color due to +nce of urochrome ( urobillin & urobilinogen) in urine. Colourless : Dilute urine Dark: Concentrated urine. Colorless Dark High fluid intake Use of diuretic DM DI Alcohol Low fluid intake Excessive sweating Dehydration (burns, fever)

11. Abnormal colour of urine 11 Cloudy Excess PO4, Urates , Pus cells, Bacterial contamination Red Frank Hematuria , hemoglobinuria , Myoglobinuria , Intake of Pyridium , Phenolphthalein Ingestion of Beet root, Black berries Deep yellow Obstructive Jaundice , Ingestion of Vitamin B complex Greenish Obst . Jaundice [excess Billirubin or billiverdin ] Phenol poisoning Blue Methylene Blue poisoning Brown black Hemorrhage in bleeding, Acidic urine, Porphyria Black Alkaptonuria Milky +nce of Chyle Cola Nephritic syndrome

12. Clarity (Transparency) Normal urine clear or transparent Any turbidity will indicate +nce of either of the following: WBCs (pus). RBCs Epithelial cells Bacteria Casts Crystals Lymph Semen Phosphate

13. ODOR Normal fresh urine Faint aromatic odor d/t +nce of volatile acids Standing for long time Ammoniac odor Bacterial action of pus (UTI) Offensive odor Ketoacidosis Fruity odor Phenylketonurea Mousy odor

14. VOLUME Adult 600 – 2500 ml /24hr 0.5-1ml /kg/hr ~ 1.5L/24hr Children 200–400ml/24hr 4ml/kg/ hr Oligouria ↓ in urine flow [< 400 ml] Polyuria ↑ in urine flow [> 2500 ml] Anuria <100ml/day Nocturia ↑ urination during night

15. Causes of anuria : Severe Renal Defect Loss of urine formation mechanism. Due to +nce of stone or tumor. Post transfusion hemolytic reaction. Incompatibility between donor`s & receiver's blood → hemolysis → excess Hb causes blockage of renal tubules → acute renal failure.

16. Causes of polyuria : ↑ed fluid intake ↑ed salt & protein intake Addison’s disease Intravenous saline or glucose Chronic glomerulonephritis Diuretics intake Psychogenic polydipsia DM DI Causes of Oliguria : Water deprivation Dehydration Prolonged vomiting Diarrohea Excessive sweating Acute renal failure Hypotension Renal Ischemia Obstruction [ Calculi,Tumor , Prostatic hypertrophy]

17. pH One of imp. functions of kidney is pH regulation. Blood pH: 7.4 & urine pH: ~ 6.0 (4.6 – 8.0) [due to secretion of H + & reabsorption of HCO 3 - ] Urine pH ≥ 9, indicate that urine is stand for a long time & must be rejected. Acidic urine Alkaline urine Acidosis Alkalosis DKA UTI [Proteus] Starvation RTA Dehydration Vegetarian diet Diarrhea E. coli infection Muscular fatigue

18. Clinical significance of pH Determine existence of acid base disorder. Precipitation of crystals to from stone requires specific pH for each type. Hence , pH control may inhibit formation of these stones. Crystals in acidic urine Crystals in alkaline urine Ca oxalate Ca carbonate Uric acid Ca phosphate Mg Phosphate

19. Specific gravity Normal: 1.015-1.025. Theoretical extremes: 1.003 to 1.032. Contamination during collection & storage gives false value. Sp. gravity is ↓ed in Excessive water intake DI Chronic glomerulonephritis All cases of polyuria [ exc ept DM] Sp. gravity is ↑ed in DM [Glycosuria] Nephrosis [ Albuminuria ] All cases of oliguria Hematuria Hemoglobinuria Execessive sweating

20. Low fixed specific gravity Due to loss of concentrating ability by damaged tubule, sp. gravity of urine is fixed at 1.010. Found in: Chronic glomerulonephritis -end stage kidney ADH def. Polycystic kidney Chronic pyelonephritis

21. Chemical examination of Urine Sugar Blood Porphyrin Protein Mucin 5-HIAA KB Bile salt Hb Bile pigment

22. Urine examination for +nce of Sugar Glycosuria is defined as presence of sugar in urine in a amount that can be detected by chemical methods. Reducing subst. found in urine: Sugar Non-sugar Glucose [DM, Endocrine disorder] CHCl 3 , Formaldehyde [preservative] Lactose [Pregnancy, Lactation] Homogentistic acid Fructose Ascorbic acid

23. Hyperglycemic glycosuria Blood glucose > Renal threshold for glucose → glycosuria Occurs in Endocrinal disorder DM Cushing’s syndrome Hyperpituitarism Hyperadrenalism

24. Alimentary Glycosuria High glucose intake at once for > 1 week ↓ ↓ed tolerance of body for glucose ↓ Glycosuria

25. Renal glycosuria Defect in renal tubule ↓ Subsequent lowered renal threshold for glucose ↓ G lycosuria Occurs in: RTA Heavy metal poisoning Fanconi’s Syndrome

26. Benedict’s Test General test for Reducing sugars Reagent’s composition: 26 CuSO 4 17.3gm Provide Cu ++ Na 2 CO 3 100gm Provide alkaline medium Na-Citrate 173gm Cu ++ chelating agent [slowly releases Cu ++ ] Dist. water 1000ml

27. Benedict’s Test Copper reduction test in alkaline medium Principle : Reducing sugars under alkaline medium, tautomerise to form enediols (powerful reducing agent), which reduces Cu ++ to Cu + . CuSO 4 → Cu ++ + SO 4 -- Cu ++ + Na-citrate → Cu-Na-citrate complex Reducing sugar → Enediol Enediol + Cu ++ → Cu + + sugar acids Cu + + OH - → CuOH 2 CuOH → Cu 2 O (↓) 27

28. Procedure 5mL of Benedict’s reagent was taken in a test tube. 8 drops of urine was added. Mixed well. Boiled for 2 min Cooled & color was observed. Observation Inference Sample A Sample B

29. Benedict’s Test 29 Blue color -ve Green colour Trace < 0.5gm% Green PPT + 0.5-1.0 gm% Green to yellow PPT ++ 1.0 – 1.5 gm% Yellow to red PPT +++ 1.5-2.0 gm% Brick Red PPT ++++ >2.0 gm% Final color formed is dependent on amount of reducing sugars +nt in given sample, thus benedict’s test is known as Semi-quantitative test.

30. KETONURIA Usually found ketone bodies in human body & urine are:- β-Hydroxy butyrate  -- Acetoacetate ------  Acetone [Primary] Normal level of ketone bodies in blood: 70mg/dl Renal threshold for ketone bodies: 1mg/dl normallly excreted in urine. [<20mg/day] ↑ed KB in urine Intake of high fat & low carbohydrate diet Starvation Uncontrolled DM Prolonged vomiting

31. Rothera’s test [ Nitroprusside test] Reagents : (NH 4 ) 2 SO 4 Crystals Precipitate protein NH 3 solution Provide alkaline medium Freshly prepared 5% Na- Nitroprusside solution

32. Rothera’s test [ Nitroprusside test] Principle : Saturation of urine with (NH 4 ) 2 SO 4 leads to settling down of proteins as precipitate. KB remains at surface. In alkaline medium, KB reacts with sodium Nitroprusside to give purple/pink ring (at interface).

33. Rothera’s test [ Nitroprusside test] Procedure : 2 mL of urine was taken in a test tube. 3 drops of nitroprusside solution was added to it. 2 mL of NH 3 solution was added slowly along the side wall of tube. Observation Inference Sample A Sample B

34. Urinary protein ( proteinuria ) Tamm horse fall protein: protein normally found in urine (<30 mg/24hr). [undetectable by routine methods] Proteinuria : defined as +nce of protein in urine that can be detected by routine methods. Pre renal Renal Post renal Cardiac disease Glomerulonephritis Severe UTI Fever Nephrotic syndrome Lesions of renal pelvis Cancer Nephritic syndrome Lesions of bladder Collagen disease Carcinoma of kidney Lesions of prostate Intra-abdominal tomors Pyelonephritis Lesions of urethra Rejection of kidney allograft

35. Classification: Glomerular Non glomerular Tubular overflow

36. Glomerular Causes : Immune complex deposition AGE deposition [Diabetic Nephropathy] ↑ in glomerular permeability due to: ↑ in pore size of glomerular memb . & loss of -ve charges due to podocyte foot process retraction & basement memb . damage. >3.5gm/24hr: hallmark for diagnosis of Nephrotic syndrome

37. Tubular Low mol. Wt. Proteins are normally filtered by glomerulus & completely reabsorbed in PCT. [eg: β 2 -microglobulin, Ig - light chains, & RBP] Loss of tubular function ↓ ↓ reabsorption ↓ Tubular proteinuria Causes : Toxic agents [Heavy metal, Drugs]

38. Overflow: Caused by excess production of Ig light chains. Multiple myeloma or monoclonal gammopathy of uncertain significance (MGUS)

39. Microalbuminuria Albuminuria : well-known predictor of poor renal function in patients with type 2 DM & in essential HTN . Microalbuminuria : Urinary albumin 30 - 300 mg/24-hr urine. Overt Albuminuria ( Macroalbuminuria ) Urinary albumin excretion of ≥300 mg/24hr urine Urinary albuminuria comprises 20–70% or urinary total protein excretion

40. Bence Jones Protein Abnormal low mol. wt. globin consisting of light chains of Ig (kappa or lamda ). Characteristics feature : Precipitate at 40-60°C Precipitate dissolve at 100°C. Precipitate reappear on cooling Bence jones proteinuria occurs in Multiple myeloma.

41. Detection of proteinuria by Heat & acetic acid test Heat & acetic acid test: Principle: Native protein (Albumin) when heated at pI, due to unfolding of quarternary , tertiary & secondary structure gets denatured & thus precipitated. Phosphates in alkaline urine also gives similar results. Addition of acetic acid, confirms presence of protein if white coagulum is formed.

42. Procedure 2/3 rd of test tube was filled with urine. Upper part of tube was heated. Observed & few drops of acetic acid was added. Observation Inference Sample A Sample B

43. Heller’s test Principle : Conc. HNO 3 form acid meta protein in reaction with protein to form precipitate at the interface. Procedure 3 mL of conc. HNO 3 was taken in a test tube. 2 mL of urine was added along the side wall of tube. Observation Inference Sample A Sample B

44. Bile pigments found in urine In Normal urine [< 0.02mg%] In abnormal urine Urochrome [Chemical nature unknown] Traces of urobilin [Small amount can’t be detected] Bilirubin [in freshly voided urine] Urobilinogen Biliverdin [develops on standing urine from oxidation of bilirubin] Urobilin [decomposition product of bilirubin or urobilinogen due to action of light or action of bacteria] Significance of bilirubinuria Only conjugated bilirubin appears in urine. It occurs with even minimal degree of jaundice & may be detected before clinical jaundice is evident.

45. Fouchet’s test Reagents: 10% BaCl 2 Fouchet’s reagent [FeCl 3 in TCA] Principle : BaCl 2 react with sulphate radicals in urine to form BaSO 4 . Bile pigment gets adhered toBaSO 4 . Bilirubin (yellow) is oxidised to biliverdin (green) with FeCl 3 in presence of TCA.

46. Fouchet’s test Procedure: 5 mL of urine was taken in a test tubr . 5 mL of BaCl 2 & pinch of MgSO 4 was added to it. Filter & dry the filter paper. Few drops of fouchet’s reagent was added to filter paper & was dried. Observation Inference Sample A Sample B

47. Bile salts found in urine Na- taurocholate Na- glycocholate Bile salt appear in urine in obstructive jaundice.

48. Hay’s surface tension test [ Sulphor test] Reagent : Sulphor powder Principle : Presence of bile salts ↓ es surface tension of urine allowing sulphor powder to sink. False +ve hay’s test False -ve hay’s test CHCl 3 Thymol Turpentine Excess urobilin in urine

49. Hay’s surface tension test [ Sulphor test] Procedure : 4mL of urine was taken in a test tube. A pinch of sulphor powder was sprinkled on the surface of urine. Observation Inference Sample A Sample B

50. Determination of bile pigment & bile salt in urine is useful in differential diagnosis of Jaundice. Pre Hepatic Hepatic Post Hepatic Bile salt -nt +nt +nt Bile pigment -nt Trace to 4+ 2+ to 4+ Urobilinogen 3+ to 4+ 2+ +nt/-nt

51. Benzidine test Capable of detecting even minute amount of blood. Detect free Hb/Mb. Glacial acetic acid: Ruptures RBC Act as solvent for Benzidine powder. Reagents‘: Saturated solution of benzidine 3% H 2 O 2

52. Benzidine test Principle: Heme has peroxidase like property causing H 2 O 2 to release nascent oxygen that reacts with benzidine solution to give greenish/ bluish color. Faint green Trace Green + Greenish blue ++ Blue +++ Deep Blue ++++

53. Benzidine test Since urine is free of Hb & Mb. So, a positive test should be followed by exact cause & origin of abnormal findings. Benzidine test is +ve in: Hematuria Hemoglobinuria Myoglobinuria

54. Hematuria : > 10 intact RBC /HPF in urine Renal Post renal General disease Disease of neighbouring organ affecting kidney Neoplasm Causes of ureter Neoplasm Calculus Ureterocele Malaria Carcinoma of vagina TB of kidney Causes in bladder Neoplasm TB Calculus Purpura Carcinoma of uterus Acute glomerulonephritis Causes of Prostate Benign hyperplasia Prostatic neoplasm Prostatic TB Scurvy Carcinoma of rectum Nephritic syndrome Embolism of kidney from SABE Acute appendicitis Pyelonephritis Hemophilia Acute inflammation of fallopian tube Pyonephrosis Malignant HTN of kidney

55. Hemoglobinuria Blood Hb > Hb binding capacity of haptoglobin ↓ Hb filtered ↓ Hb appears in urine [ Hemoglobinuria ] Occurs in : Malaria Septicemia [hemolytic streptococcal infection] Sickle cell anemia Thallasemia Incompatible blood transfusion Effect of chemicals on RBC [ Sulphonamide , Phenylhydrazine , Arsenic, etc]

56. Myoglobinuria Injury to cardiac/ skeletal muscle ↓ Mb released ↓ Excreted via urine Mb: toxic to kidney [high concentration may lead to Acute renal failure. MI Infarction of large skeletal muscle Muscle damage [Injury, Electric shock, Heat stroke] Trauma