Examination of Urine

1. Examination of Urine Dr/Mohamed MahmoudNourEldein PhD Biochemistry Assistant Professor of Biochemistry Faculty of Medicine Umm AL-Qura University

2. Urinary System

3. Introduction • Urine is formed in the kidneys, is a product of ultrafiltration of plasma by the renal glomeruli which is a network of arteriolar capillaries, each glomeruli is surrounded by Bowman’s capsule (a double epithelial sac) like a rounded funnel which leads to the tube.

4. Purpose • Urine contains important metabolic information • Urine is cheap, simple, & readily available • General evaluation of health • Diagnosis of disease or disorders of the kidneys or urinary tract • Diagnosis of other systemic disease that affect kidney function • Monitoring of patients with diabetes • Screening for drug abuse (eg. Sulfonamide or aminoglycosides)

5. Collection of urine specimens • The first voided morning urine (most concentrated) - qualitative • Random urine (routine) • 24hrs sample- quantitative • Mid-stream clean catch (MSCC) (for urine culture)- UTI • Post prandial sample-D.M Attention • Need to be examined within 1 hour

6. Clean Catch

8. 24 hour urine sample • For quantitative estimation of proteins • For estimation of vanillyl mandelic acid, 5-hydroxyindole acetic acid, metanephrines • For detection of AFB in urine • For detection of microalbuminuria

9. Types of Analysis 1- Macroscopic Examination Physical characteristics : color, odor, turbidity, volume, & specific gravity • Chemical Analysis (Urine Dipstick) : pH, glucose, protein, ketones, pus (WBC’s & bacteria), RBC’s, hemoglobin, bile 2- Microscopic Examination of urine sediment: crystals, cells, etc.

10. Physical examination • Volume • Color • Odour • Reaction or urinary pH • Specific gravity

11. Urinary volume • The average daily urine output 1200 - 1500 mL (1.2 - 1.5 L) • The normal daily range of urine output 600 - 2000 mL (0.6 - 2.0 L) • Polyuria- >2000ml • Oliguria- <400ml • Anuria-complete cessation of urine(<200ml) • Nocturia-excretion of urine by an adult of >500ml with a specific gravity of <1.018 at night (characteristic of chronic glomerulonephritis)

12. Causes of polyuria > 2000ml • Diabetes mellitus • Diabetes insipidus • Polycystic kidney • Chronic renal failure • Diuretics • Intravenous saline/glucose

13. Oliguria <400ml • Dehydration-vomiting, diarrhea, excessive sweating • Renal ischemia • Acute tubular necrosis • Obstruction to the urinary tract • Acute renal failure

14. Urine Color and Clarity • Urine color and clarity can indicate what substances may be present in urine. • Confirmation of suspected substances is obtained during the chemical and microsopic examination.

15. Urine Color • Normal urine color ranges from pale yellow to deep amber — the result of a pigment called urochrome • Most changes in urine color are harmless and temporary and may be due to: • Certain foods – beets may turn urine red • Dyes in foods/drinks • Supplements – vitamins • Prescription drugs

16. Abnormal colors of urine & possible causes • Unusual urine color can indicate an infection or serious illness . • Colourless- dilution, diabetes mellitus, diabetes insipidus, diuretics • Milky- genitourinary tract infection • Orange-fever, excessive sweating, bilirubin • Red-beetroot ingestion,haematuria • Brown/ black- alkaptunuria, melanin • Green - bile, Pseudomonas bacteria

17. Examples of Urine Color

18. Urine Clarity • Urine clarity refers to how clear the urine is. • Terms used: clear, transparent, slightly cloudy, cloudy, or turbid. • “Normal” urine can be clear or cloudy. • The clarity of the urine is not as important as the substance that is causing the urine to be cloudy. • Turbidity - “cloudiness” due to particulate matter suspended in urine

19. Urine Clarity • Substances that cause cloudiness but that are not considered unhealthy include: • mucous, • sperm and prostatic fluid, • cells from the skin, • normal urine crystals, and • contaminants (like body lotions and powders). • Other substances that can make urine cloudy (such as red blood cells, white blood cells, or bacteria) indicate a condition that requires attention.

20. Examples of Urine Clarity

21. Odour • Normal= aromatic due to the volatile fatty acids • Standing (“old”) urine takes on an ammonia odor due to urea-splitting bacterial • Foul, offensive: Old specimen, pus or inflammation • Sweet: Glucose • Fruity: Ketones

22. Chemical Analysis

23. Urine dipsticks (Reagent Strips) • Urine dipstick are plastic strips on which are attached to a series of chemically impregnated absorbent pads, each pad contain certain chemicals that react with substance in the urine producing a color change in pad, this color change is compared with a series of known standards.

24. Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase Chemical Analysis Urine Dipstick

25. Reagent Strips

26. Procedure • Reagent strips are used only once and discarded. • Testing • Perform within 1 hour after collection • Allow refrigerated specimens to return to room temperature. • Dip strip briefly, but completely into well mixed, room temperature urine sample. • Withdraw strip. • Blot briefly on its side. • Keep the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

27. Procedure • Instruments are available which detect color changes electronically and prints out results

28. Handling and Storage of Strips Handling and Storage Keep strips in original container Do not touch reagent pad areas Reagents and strips must be stored properly to retain activity Protect from moisture and volatile fumes Stored at room temperature Use before expiration date

29. Sources of Error • Timing - Failure to observe color changes at appropriate time intervals may cause inaccurate results. • Lighting - Observe color changes and color charts under good lighting. • QC - Reagent strips should be tested with positive controls on each day of use to ensure proper reactivity. • Sample - Proper collection and storage of urine is necessary to insure preservation of chemical.

30. Sources of Error • Testing cold specimens - would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing • Inadequate mixing of specimen - could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping • Over-dipping of reagent strip - will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

31. Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) The Urine Dipstick: Glucose Chemical Principle Glucose Oxidase Glucose + 2 H2O+ O2---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

32. Uses and Limitations of Urine Glucose Detection Significance • Diabetes mellitus. • Renal glycosuria. Limitations • Interference: reducing agents, ketones. • Only measures glucose and not other sugars. • Renal threshold must be passed in order for glucose to spill into the urine. Other Tests • CuSO4 test for reducing sugars.

33. Urine versus Blood Glucose ++ + Urinalysis Glucose Result trace Negative 200 400 600 800 1000 Blood Glucose (mg/dL)

34. Negative + (weak) ++ (moderate) +++ (strong) The Urine Dipstick: Bilirrubin Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

35. Bilirubin • Bilirubin is a byproduct of the breakdown of hemoglobin. • Normally contains no bilirubin. • Presence may be an indication of liver disease, bile duct obstruction or hepatitis. • Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.

36. Ketones • Ketones are excreted when the body metabolizes fats incompletely (ketonuria)

37. Negative Trace (5 mg/dL) + (15 mg/dL) ++ (40 mg/dL) +++ (80 mg/dL) ++++ (160+ mg/dL) The Urine Dipstick: Ketones Chemical Principle Acetoacetic Acid + Nitroprusside ------> Colored Complex Read at 40 seconds RR: Negative

38. Uses and Limitations of Urine Ketone Detection Significance - Diabetic ketoacidosis - Prolonged fasting Limitations - Interference: expired reagents (degradation with exposure to moisture in air) - Only measures acetoacetate not other ketone bodies (such as in rebound ketosis). Other Tests - Ketostix (more sensitive tablet version of same assay) - Serum glucose measurement to confirm DKA

39. Specific gravity • Depends on the concentration of various solutes in the urine. • Measured by-urinometer - refractometer - dipsticks • Specific gravity reflects kidney's ability to concentrate. • Want concentrated urine for accurate testing, best is first morning sample. • Low – specimen not concentrated, kidney disease. • High – first morning, certain drugs

40. Urinometer • Take 2/3 of urinometer container with urine • Allow the urinometer to float into the urine • Read the graduation at the lowest level of urinary meniscus • Correction of temperature & albumin is a must. • Urinometer is calibrated at 15or 200c So for every 3oc increase/decrease add/subtract 0.001 For 1gm/dl of albumin add0.001

42. 1.000 1.005 1.010 1.015 1.020 1.025 1.030 The Urine Dipstick: Specific Gravity Chemical Principle X+ + Polymethyl vinyl ether / maleic anhydride ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromthymol Blue indicator to form a colored complex. Read up to 2 minutes RR: 1.003-1.035

43. Uses and Limitations of Urine Specific Gravity Significance - Diabetes insipidus Limitations - Interference: alkaline urine - Does not measure non-ionized solutes (e.g. glucose) Other Tests - Refractometry - Hydrometer - Osmolality measurement (typically used with water deprivation test)

44. High specific gravity(hyperosthenuria) • Normal-1.016-1.022 • Causes All causes of oliguria Glycosuria

45. Low specific gravity(hyposthenuria) • All causes of polyuria except glycosuria • Fixed specific gravity (isosthenuria)=1.010 Seen in chronic renal disease when kidney has lost the ability to concentrate or dilute

46. Blood • Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys. • False positive readings most often due to contamination with menstrual blood.

47. The Urine Dipstick: Blood Negative Chemical Principle Trace (non-hemolyzed) Lysing agent to lyse red blood cells Moderate (non-hemolyzed) Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine ------------> Colored Complex Trace (hemolyzed) Heme + (weak) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs ++ (moderate) +++ (strong)

48. Uses and Limitations of Urine Blood Detection Significance - Hematuria (nephritis, trauma, etc) - Hemoglobinuria (hemolysis, etc) - Myoglobinuria (rhabdomyolysis, etc) Limitations - Interference: reducing agents, microbial peroxidases - Cannot distinguish between the above disease processes Other Tests - Urine microscopic examination - Urine cytology

49. Urinary pH/ reaction • Reaction reflects ability of kidney to maintain normal hydrogen ion concentration in plasma & ECF • Normal= 4.6-8 • Tested by- 1.litmus paper 2. pH paper 3. dipsticks

50. 5.0 6.0 6.5 7.0 7.5 8.0 8.5 The Urine Dipstick: pH Chemical Principle H+ interacts with: Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high pH), to form a colored complexes(dual indicator system) Read up to 2 minutes R.R.: 4.5-8.0