Urine analysis in pathology clinical

pathology

1. Standardization of Urinalysis Dr. Appy Moderator- Dr. Manisha T.

2. Types of Specimen Collection

3. Specimen Collection for routine urinalysis First morning voiding (most concentrated) Record collection time Type of specimen (e.g. “ clean catch ” ) Analyzed within 2 hours of collection Free of debris or vaginal secretions

4. Collection of urine is started in the morning and subsequent samples are collected till the next morning. Preservatives used: Refrigeration at 4 degrees Toluene- 1 ml / 50ml of urine. Preserves chemical constituents. SPECIMEN COLLECTION FOR 24 HOURS URINALYSIS

5. Thymol – 1% of thymol, gives false positive for proteins Acid- HCl, sulfuric acid and boric acid can be used. Formalin- 6-8 drops of 40% formalin per 100 ml of urine. Preserves RBCs and Pus cells, gives false positive test for sugar.

6. Specimen Collection Supra-pubic Needle Aspiration

7. Macroscopic Examination Chemical Analysis Microscopic Examination Culture Cytological Examination Types of Analysis

8. Macroscopic Examination Volume : normal – 700-2500ml/24 hours Polyuria- >2500ml Excess intake Diabetes insipidus Diabetes mellitus

9. Oliguria: <500ml Less intake Dehydration Renal ischaemia Anuria: <150ml Renal stones Renal or prostatic tumors Severe renal ischaemia

10. Appearance normal-clear Cloudy U.T.I. calculi dye Milky lipiduria Chyluria Turbid Crystals infection

11. Macroscopic Examination Odor: Ammonia-like: (Urea-splitting bacteria) Foul, offensive: Old specimen, pus or inflammation Sweet: Glucose Fruity: Ketones Maple syrup-like: Maple Syrup Urine Disease Color: Colorless Diluted urine Deep Yellow Concentrated Urine, Riboflavin Yellow-Green Bilirubin / Biliverdin Red Blood / Hemoglobin Brownish-red Acidified Blood (Actute GN) Brownish-black Homogentisic acid (Melanin)

12. Foam If a normal urine specimen is shaken or agitated sufficiently, a white foam can be forced to develop at its surface that readily dissipates on standing. Moderate to large amounts of protein (albumin) in urine cause a stable white foam to be produced when the urine is poured or agitated. When bilirubin is present in sufficient amounts, the foam if present will be characteristically yellow.

13. Chemical Analysis

14. Conventional tests Benedicts test Heat coagulation test Rothera ’ s test Hay ’ s test Ehrlich ’ s test Fouchet ’ s test Benzidine test

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

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

17. 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 CuSO 4 test for reducing sugars. Uses and Limitations of Urine Glucose Detection

18. Sugar Disease (s) - Galactose Galactosemias - Fructose Fructosuria, Fructose Intolerance, etc. - Lactose Lactase Deficiency - Pentoses Essential Pentosuria - Maltose Non-pathogenic * NOT Sucrose because it is not a reducing sugar Detection of Reducing Sugars* by CuSO 4

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

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

21. Significance - Increased direct bilirubin (correlates with urobilinogen and serum bilirubin) Limitations - Interference: prolonged exposure of sample to light - Only measures direct bilirubin--will not pick up indirect bilirubin Other Tests - Ictotest (more sensitive tablet version of same assay) - Serum test for total and direct bilirubin is more informative Uses and Limitations of Urine Bilirrubin Detection

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

23. 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 version of same assay) - Serum glucose measurement to confirm DKA Uses and Limitations of Urine Ketone Detection

24. 1.000 1.005 1.010 1.015 1.020 1.025 1.030 Chemical Principle Read up to 2 minutes RR: 1.003-1.035 The Urine Dipstick: Specific Gravity pKa change of an polyelectrolyte; indirect measure of density

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

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

27. 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 Uses and Limitations of Urine Blood Detection

28. 5.0 6.0 6.5 7.0 7.5 8.0 8.5 The Urine Dipstick: pH 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) Chemical Principle Read up to 2 minutes R.R.: 4.5-8.0

29. Significance - Acidic (less than 4.5): metabolic acidosis, high-protein diet - Alkaline (greater than 8.0): renal tubular acidosis (>5.5) Limitations - Interference: bacterial overgrowth (alkaline or acidic), “ run over effect ” effect of protein pad on pH indicator pad Other Tests - Titrable acidity - Blood gases to determine acid-base status Uses and Limitations of Urine pH Detection

30. Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase Buffers from the protein area of the strip (pH 3.0) spill over to the pH area of the strip and make the pH of the sample appear more acidic than it really is. pH Run Over Effect

31. Negative Trace + (30 mg/dL) ++ (100 mg/dL) +++ (300 mg/dL) ++++ (2000 mg/dL) The Urine Dipstick: Protein Chemical Principle H H H H H H Pr Pr Pr Pr Pr Pr “ Protein Error of Indicators Method ” Pr Pr Pr Pr Pr Pr Tetrabromphenol Blue (buffered to pH 3.0) H + H + H + H + H + H + Read at 60 seconds RR: Negative

32. Physiological Renal - Severe muscular exertion - Glomerulonephritis - Pregnancy - Nephrotic syndrome - Orthostatic proteinuria - Renal tumor or infection Pre-Renal Post-Renal - Fever - Cystitis - Renal hypoxia - Urethritis or prostatitis - Hypertension - Contamination with vaginal secretions Causes of Proteinuria

33. Primary - Lipoid nephrosis (severe) - Membranous glomerulonephritis - Membranoproliferative glomerulonephritis Secondary - Diabetes mellitus (Kimmelsteil-Wilson lesions) - Systemic lupus erythematosus - Amyloidosis and other infiltrative diseases - Renal vein thrombosis Nephrotic Syndrome (> 3.5 g/dL in 24 h)

34. Significance - Proteinuria and the nephrotic syndrome. Limitations - Interference: highly alkaline urine. - Much more sensitive to albumin than other proteins (e.g., immunoglobulin light chains). Other Tests - Sulfosalicylic acid (SSA) turbidity test. - Urine protein electrophoresis (UPEP) Uses and Limitations of Urine Protein Detection

35. 0.2 mg/dL 1 mg/dL 2 mg/dL 4 mg/dL 8 mg/dL The Urine Dipstick: Urobilinogen Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex (Ehrlich ’ s Reagent) Chemical Principle Read at 60 seconds RR: 0.02-1.0 mg/dL

36. Significance - High: increased hepatic processing of bilirubin - Low: bile obstruction Limitations - Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin) - Cannot detect low levels of urobilinogen Other Tests - Serum total and direct bilirubin Uses and Limitations of Urobilinogen Detection

37. Reading time in a nutshell

38. Preservation - Cells and casts begin to disintegrate in 1 - 3 hrs. at room temp. - Refrigeration for up to 48 hours (little loss of cells). Specimen concentration - Ten to twenty-fold concentration by centrifugation. Types of microscopy - Phase contrast microscopy - Polarized microscopy - Bright field microscopy with special staining (e.g., Sternheimer-Malbin stain) Microscopic Examination General Aspects

39. Microscopic Examination Per High Power Field (HPF) (400x) > 3 erythrocytes > 5 leukocytes > 2 renal tubular cells > 10 bacteria Per Low Power Field (LPF) (200x) > 3 hyaline casts or > 1 granular cast > 10 squamous cells (indicative of contaminated specimen) Any other cast (RBCs, WBCs) Presence of: Fungal hyphae or yeast, parasite, viral inclusions Pathological crystals (cystine, leucine, tyrosine) Large number of uric acid or calcium oxalate crystals Abnormal Findings

40. Erythrocytes - “ Dysmorphic ” vs. “ normal ” (> 10 per HPF) Leukocytes - Neutrophils (glitter cells) More than 1 per 3 HPF - Eosinophils Hansel test (special stain) Epithelial Cells - Squamous cells Indicate level of contamination - Renal tubular epithelial cells Few are normal - Transitional epithelial cells Few are normal - Oval fat bodies Abnormal, indicate Nephrosis Microscopic Examination Cells

41. Microscopic Examination RBCs

42. Microscopic Examination WBCs

43. Microscopic Examination Squamous Cells

44. Microscopic Examination Tubular Epithelial Cells

45. Microscopic Examination Transitional Cells

46. Microscopic Examination Transitional Cells

47. Microscopic Examination Oval Fat Body

48. Bacteria - Bacteriuria More than 10 per HPF Yeasts - Candidiasis Most likely a contaminant but should correlate with clinical picture. Viruses - CMV inclusions Probable viral cystitis. Microscopic Examination Bacteria & Yeasts

49. Microscopic Examination Bacteria

50. Microscopic Examination Yeasts

51. Erythrocyte Casts: Glomerular diseases Leukocyte Casts: Pyuria, glomerular disease Degenerating Casts: - Granular casts Nonspecific (Tamm-Horsfall protein) - Hyaline casts Nonspecific (Tamm-Horsfall protein) - Waxy casts Nonspecific - Fatty casts Nephrotic syndrome (oval fat body casts) Microscopic Examination Casts

52. Microscopic Examination Casts

53. Microscopic Examination RBCs Cast - Histology

54. Microscopic Examination RBCs Cast

55. Microscopic Examination RBCs Cast - Histology

56. Microscopic Examination WBCs Cast

57. Microscopic Examination Tubular Epith. Cast

58. Microscopic Examination Tubular Epith. Cast

59. Microscopic Examination Granular Cast

60. Microscopic Examination Hyaline Cast

61. Microscopic Examination Waxy Cast

62. Microscopic Examination Fatty Cast

63. Bacterial Casts Single Leukocytes Leukocyte Casts Verrier-Jones & Asscher, 1991. Single Erythrocytes Erythrocyte Casts Single Bacteria Significance of Cellular Casts

64. Microscopic Examination Crystals in acidic urine

65. Microscopic Examination Crystals in alkaline urine

66. Microscopic Examination Calcium Oxalate Crystals

67. Microscopic Examination Calcium Oxalate Crystals Dumbbell Shape

68. Microscopic Examination Triple Phosphate Crystals

69. Microscopic Examination Urate Crystals

70. Microscopic Examination Leucine Crystals

71. Microscopic Examination Cystine Crystals

72. Microscopic Examination Ammonium Biurate Crystals

73. Microscopic Examination Cholesterol Crystals

74. Need for automation Time saving Easy to process Multiple samples can be processed Reduce the need for man power Uniformity Sensitive Specific

75. List of instruments

76. Principle Aspirates and dispenses fixed amount of urine on each pad of the strip Reads the strip via reflectance method. Assess color of a specimen by using four wavelengths of light to obtain the tone (light, normal, dark) and hue of a urine specimen.

77. Light scatter is used to determine the turbidity Specific gravity is measured by assessing refractive index of LED-emitted light. dual wavelength reflectance to measure the pH and chemical constituents of urine

78. Priciple

79. Iris Diagnostics Division iChem®100

80. Factors That Require Standardization in the Microscopic Examination Urine volume used (e.g., 10 mL, 12 mL, 15 mL) Speed of centrifugation (400 or 450 Å~ g) Time of centrifugation (5 minutes) Concentration of sediment prepared (e.g., 10 : 1, 12 : 1, 15 : 1, 30 : 1) Volume of sediment examined—determined by commercial slides used and microscope optical properties (i.e., ocular field number) Result reporting—format, terminology, reference intervals, magnification used for assessment

81. video

82. Cytological Examination Staining: Papanicolau Wright ’ s Immunoperoxidase Immunofluorescence

83. Cytology: Normal

84. Cytology: Normal

85. Cytology: Reactive

86. Cytology: Reactive

87. Cytology: Polyoma (Decoy Cell)

88. Cytology: Polyoma (Decoy Cell) Immunoperoxidase to SV40 ag

89. Cytology: TCC Low Grade

90. Cytology: TCC Low Grade

91. Cytology: TCC High Grade

92. Cytology: TCC High Grade

93. Cytology: Squamous Cell Ca.

94. Cytology: Renal Cell Ca.

95. Cytology: Prostatic Carcinoma

96. Thank you