pathology

1. Standardization of
Urinalysis

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.

7. Specimen Collection
Supra-pubic Needle Aspiration

8. − Macroscopic Examination
− Chemical Analysis
− Microscopic Examination
− Culture
− Cytological Examination
Types of Analysis

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

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

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

12. 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)

13. 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.

14. Chemical Analysis

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

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

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

18. 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.
Uses and Limitations of Urine Glucose Detection

19. 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 CuSO4

20. ++
+
trace
400 600 800 1000200
UrinalysisGlucoseResult
Blood Glucose (mg/dL)
Urine versus Blood Glucose
Negative

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

22. 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

23. 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

24. 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

25. 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

26. 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

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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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)

35. 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

36. 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

37. 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

38. Reading time in a nutshell

39. 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

40. 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

41. 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

42. Microscopic Examination
RBCs

43. Microscopic Examination
WBCs

44. Microscopic Examination
Squamous Cells

45. Microscopic Examination
Tubular Epithelial Cells

46. Microscopic Examination
Transitional Cells

47. Microscopic Examination
Transitional Cells

48. Microscopic Examination
Oval Fat Body

49. 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

50. Microscopic Examination
Bacteria

51. Microscopic Examination
Yeasts

52. 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

53. Microscopic Examination
Casts

54. Microscopic Examination
RBCs Cast - Histology

55. Microscopic Examination
RBCs Cast

56. Microscopic Examination
RBCs Cast - Histology

57. Microscopic Examination
WBCs Cast

58. Microscopic Examination
Tubular Epith. Cast

59. Microscopic Examination
Tubular Epith. Cast

60. Microscopic Examination
Granular Cast

61. Microscopic Examination
Hyaline Cast

62. Microscopic Examination
Waxy Cast

63. Microscopic Examination
Fatty Cast

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

65. Microscopic Examination
Crystals in acidic urine

66. Microscopic Examination
Crystals in alkaline urine

67. Microscopic Examination
Calcium Oxalate Crystals

68. Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape

69. Microscopic Examination
Triple Phosphate Crystals

70. Microscopic Examination
Urate Crystals

71. Microscopic Examination
Leucine Crystals

72. Microscopic Examination
Cystine Crystals

73. Microscopic Examination
Ammonium Biurate Crystals

74. Microscopic Examination
Cholesterol Crystals

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

76. List of instruments

77. 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.

78.  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

79. Priciple

80. Iris Diagnostics Division
iChem®100

83. 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

87. video

88. Cytological Examination
Staining:
 Papanicolau
 Wright’s
 Immunoperoxidase
 Immunofluorescence

89. Cytology: Normal

90. Cytology: Normal

91. Cytology: Reactive

92. Cytology: Reactive

93. Cytology: Polyoma (Decoy Cell)

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

95. Cytology: TCC Low Grade

96. Cytology: TCC Low Grade

97. Cytology: TCC High Grade

98. Cytology: TCC High Grade

99. Cytology: Squamous Cell Ca.

100. Cytology: Renal Cell Ca.

101. Cytology: Prostatic Carcinoma

102. Thank you