1. Urinalysis Francisco G. La Rosa, MD Course: IDPT 5005
School of Medicine, UCDHSC
Francisco G. La Rosa, MD
Associate Professor, Department of Pathology
University of Colorado Denver Health Sciences Programs, Denver, Colorado 80045

2. Specimen Collection 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

3. Clean Catch

4. Specimen Collection
Supra-pubic Needle Aspiration

5. Types of Analysis Macroscopic Examination
Chemical Analysis (Urine Dipstick)
Microscopic Examination
Culture (not covered in this lecture)
Cytological Examination

6. 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)

7. Macroscopic Examination
Turbidity:
Typically cells or crystals.
Cellular elements and bacteria will clear by centrifugation.
Crystals dissolved by a variety of methods (acid or base).
Microscopic examination will determine which is present.

8. Chemical Analysis

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

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

11. 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.

12. Detection of Reducing Sugars* by CuSO4
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

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

14. Microscopic Examination
General Aspects
Preservation
- Cells and casts begin to disintegrate in 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)

15. Microscopic Examination
Abnormal Findings
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

16. Microscopic Examination
Cells
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

17. Microscopic Examination
RBCs

18. Microscopic Examination
RBCs

19. Microscopic Examination
WBCs

20. Microscopic Examination
Squamous Cells

21. Tubular Epithelial Cells
Microscopic Examination
Tubular Epithelial Cells

22. Microscopic Examination
Transitional Cells

23. Microscopic Examination
Transitional Cells

24. Microscopic Examination
Oval Fat Body

25. Microscopic Examination
LE Cell

26. Microscopic Examination
Bacteria & Yeasts
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.

27. Microscopic Examination
Bacteria

28. Microscopic Examination
Yeasts

29. Microscopic Examination
Yeasts

30. Microscopic Examination
Cytomegalovirus

31. Microscopic Examination
Casts
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)

32. Microscopic Examination
Casts

33. Microscopic Examination
RBCs Cast - Histology

34. Microscopic Examination
RBCs Cast

35. Microscopic Examination
RBCs Cast - Histology

36. Microscopic Examination
WBCs Cast

37. Microscopic Examination
Tubular Epith. Cast

38. Microscopic Examination
Tubular Epith. Cast

39. Microscopic Examination
Granular Cast

40. Microscopic Examination
Hyaline Cast

41. Microscopic Examination
Waxy Cast

42. Microscopic Examination
Fatty Cast

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

44. Microscopic Examination
Crystals
- Urate
Ammonium biurate
Uric acid
- Triple Phosphate
- Calcium Oxalate
- Amino Acids
Cystine
Leucine
Tyrosine
- Sulfonamide

45. Calcium Oxalate Crystals
Microscopic Examination
Calcium Oxalate Crystals

46. Calcium Oxalate Crystals
Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape

47. Triple Phosphate Crystals
Microscopic Examination
Triple Phosphate Crystals

48. Microscopic Examination
Urate Crystals

49. Microscopic Examination
Leucine Crystals

50. Microscopic Examination
Cystine Crystals

51. Ammonium Biurate Crystals
Microscopic Examination
Ammonium Biurate Crystals

52. Microscopic Examination
Cholesterol Crystals

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

54. Cytology: Normal

55. Cytology: Normal

56. Cytology: Reactive

57. Cytology: Reactive

58. Cytology: Polyoma (Decoy Cell)

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

60. Cytology: TCC Low Grade

61. Cytology: TCC Low Grade

62. Cytology: TCC High Grade

63. Cytology: TCC High Grade

66. Cytology: Squamous Cell Ca.

67. Cytology: Renal Cell Ca.

68. Cytology: Prostatic Carcinoma

69. Questions ?

70. Disclaimer:
The images and texts presented in this slide show are solely for educational purposes and not intended for commercial or pecuniary benefit. The images have been obtained from Dr. La Rosa’s personal collection, from text books used during the teaching of this chapter, and from published articles and educational works. Reproduction of these images can be done only for educational use.
Reference: USA Copyright Law, Section 110, “Limitations on exclusive rights: Exemption of certain performances and displays”).
[Download] the USA Copyright Law version, October 2007.

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

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

73. 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.

74. Detection of Reducing Sugars* by CuSO4
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

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

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

77. Uses and Limitations of Urine Bilirrubin Detection
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

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

79. 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

80. The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005
1.010
1.015
1.020
1.025
1.030
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:

81. 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)

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

83. 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

84. Methyl Red (at high concentration; low pH) and
The Urine Dipstick: pH
5.0
6.0
6.5
7.0
7.5
8.0
8.5
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.:

85. Uses and Limitations of Urine pH Detection
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

86. pH Run Over Effect 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.

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

88. Causes of Proteinuria Functional Renal Pre-Renal Post-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

89. Nephrotic Syndrome (> 3.5 g/dL in 24 h)
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

90. Uses and Limitations of Urine Protein Detection
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)
- Bence Jones protein

91. Proteins in “Normal” Urine
Protein % of Total Daily Maximum
Albumin 40% 60 mg
Tamm-Horsfall 40% 60 mg
Immunoglobulins 12% 24 mg
Secretory IgA 3% 6 mg
Other 5% 10 mg
TOTAL 100% 150 mg

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

93. Uses and Limitations of Urobilinogen Detection
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

94. The Urine Dipstick: Nitrite Chemical Principle Negative Positive
Acidic
Negative
Positive
Nitrite + p-arsenilic acid > Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
> Colored Complex
Read at 60 seconds
RR: Negative

95. Uses and Limitations of Nitrite Detection
Significance
- Gram negative bacteriuria
Limitations
- Interference: bacterial overgrowth
- Only able to detect bacteria that reduce nitrate to nitrite
Other Tests
- Correlate with leukocyte esterase and
- Urine microscopic examination (bacteria)
- Urine culture

96. The Urine Dipstick: Leukocyte Esterase Chemical Principle
Derivatized pyrrole amino acid ester
> 3-hydroxy-5-phenyl pyrrole
Negative
Trace
+ (weak)
++ (moderate)
+++ (strong)
Esterases
3-hydroxy-5-phenyl pyrrole + diazo salt
> Colored Complex
Read at 2 minutes
RR: Negative
Analytic Sensitivity: 3-5 WBCs

97. Significance Limitations Other Tests
Uses and Limitations of Leukocyte Esterase Detection
Significance
- Pyuria
- Acute inflammation
- Renal calculus
Limitations
- Interference: oxidizing agents, menstrual contamination
Other Tests
- Urine microscopic examination (WBCs and bacteria)
- Urine culture

98. Urinalysis
Disease Diagnosis

99. Diluted urine, request a voided urine in the morning If persisting low SG, possible diabetes insipida A microscopic may give negative results
Case 1
Glucose
Negative
A 35-year old man undergoing routine pre employment drug screening.
Physical characteristics: Clear.
Microscopic: Not performed.
Drugs Identified: None.
Questions:
- What is your differential diagnosis?
- What would you do next to confirm your suspicion?
- Would you order a microscopic analysis on this sample?
Bilirubin
Negative
Ketones
Negative
S.G.
1.001
Blood
Negative pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative

100. Possible gallbladder or hepatic disease. No hemolytic anemia
Possible gallbladder or hepatic disease. No hemolytic anemia. Perform bilirubins in serum Microscopic unlikely to provide additional info
Case 2
Glucose
Negative
Bilirubin
+++
A 42-year old woman presents with “dark urine”
Physical characteristics: Red-brown.
Microscopic: Not performed.
Questions:
- What is your differential diagnosis?
- Could this be a case of hemolytic anemia?
- How would you rule it out?
- What tests would you order next? Why?
- Would you order a microscopic analysis?
Ketones
Negative
S.G.
1.020
Blood
Negative pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative

101. Possible UTI, request culture and antibiotic sensitivity Negative Nitrite test: Gram positive bacteria Lower SG may show less number of cells and bacteria Un-common diagnosis in this type of patient
Case 3
Glucose
Negative
A 42-year old man presents painful urination
Physical characteristics: dark red, turbid
Microscopic: leukocytes = 30 per HPF
RBCs = >100 per HPF
Bacteria = >100 per HPF
Questions:
- What is your suspected diagnosis?
- What would you do next?
- What do you make of the nitrite test?
- How would the microscopic exam differ if the S.G. were 1.003?
- Is this a common diagnosis for this type of patient?
Bilirubin
Negative
Ketones
Negative
S.G.
1.030
Blood
+++ pH
6.5
Protein
Trace
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
+++

102. Diabetes May be decompensated and with ketoacidosis Ketones should become negative after treatment
Case 4
Glucose ++
Bilirubin
Negative
A 27-year old woman presents with severe abdominal pain.
Physical characteristics: clear-yellow.
Microscopic: Not performed.
Questions:
- What is the most likely diagnosis?
- What do you make of the ketone result?
- What do you expect to happen to the ketone measurement when treatment begins?
Ketones
Trace
S.G.
1.015
Blood
Negative pH
6.0
Protein
Negative
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
Negative

103. Glomerulonephritis RBC casts reveals renal cortex involvement RBC cast are not always present in GN
Case 5
Glucose
Negative
8-year old boy presents with discolored urine
Physical characteristics: Red, turbid.
Microscopic: erythrocytes = >100 per HPF (almost all dysmorphic)
Red cell casts present.
Questions:
- What is the most likely diagnosis in this case?
- Does the presence of red cell casts help you in any way?
- If the erythrocytes were not dysmorphic would that change your diagnosis?
Bilirubin
Negative
Ketones
Negative
S.G.
1.015
Blood
+++ pH
6.5
Protein +
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
Negative

104. “Functional” proteinuria
“Functional” proteinuria? Microscopic may reveal a few leukocytes Request protein concentration in 24 h urine
Case 6
Glucose
Negative
22-year old man presenting for a routine physical required for admission to medical school
Physical characteristics: Yellow
Microscopic: Not performed
Questions:
- What is your differential diagnosis?
- Would you order a microscopic analysis on this sample?
- What would you do next to confirm the diagnosis?
Bilirubin
Negative
Ketones
Negative
S.G.
1.010
Blood
Negative pH
5.0
Protein +
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative

105. Common Findings in: Acute Tubular Necrosis
Glucose
Bilirubin
Ketones
S.G.
Decreased
Microscopic:
Renal tubular epithelial cells
Pathological casts
Blood
+ / - pH
Protein
+ / -
Urobilinogen
Nitrite
L.E.

106. Common Findings in: Acute Glomerulonephritis
Glucose
Bilirubin
Ketones
Microscopic:
Erythrocytes (dysmorphic)
Erythrocyte casts
Mixed cellular casts
S.G.
Blood
Increased pH
Protein
Increased
Urobilinogen
Nitrite
L.E.

107. Common Findings in: Chronic Glomerulonephritis
Glucose
Bilirubin
Ketones
Microscopic:
Pathological casts (broad waxy casts, RBCs)
S.G.
Decreased
Blood
Increased pH
Protein
Increased
Urobilinogen
Nitrite
L.E.

108. Common Findings in: Acute Pyelonephritis
Glucose
Bilirubin
Microscopic:
Bacteria
Leukocytes
Leukocyte, granular, and waxy casts
Renal tubular epithelial cell casts
Ketones
S.G.
Blood pH
Protein
Trace
Urobilinogen
Nitrite
Positive
L.E.
Positive

109. Common Findings in: Nephrotic Syndrome
Glucose
Bilirubin
Ketones
Microscopic:
Oval fat bodies
Fatty casts
Waxy casts
S.G.
Blood pH
Protein
++++
Urobilinogen
Nitrite
L.E.

110. Common Findings in: Eosinophilic Cystitis
Glucose
Bilirubin
Ketones
Microscopic:
Numerous eosinophils (Hansel’s stain)
NO significant casts.
S.G.
Blood + pH
Protein
Urobilinogen
Nitrite
L.E.

111. Common Findings in: Urothelial Carcinoma
Glucose
Bilirubin
Ketones
Microscopic:
Malignant cells on urine cytology (urine sample should be submitted separately to cytology, void or 24 hrs.)
S.G.
Blood + pH
Protein
Urobilinogen
Nitrite
L.E.

112. Acknowledgment: Dr. Brad Brimhall