Transcript Urinalysis

Urinalysis
Course: IDPT 5005
School of Medicine, UCDHSC
Francisco G. La Rosa, MD
[email protected]
Associate Professor, Department of Pathology
University of Colorado Denver Health Sciences Programs, Denver, Colorado 80045
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
Clean Catch
Specimen Collection
Supra-pubic Needle Aspiration
Types of Analysis
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Macroscopic Examination
Chemical Analysis (Urine Dipstick)
Microscopic Examination
Culture (not covered in this lecture)
Cytological Examination
Macroscopic Examination
Odor:
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−
−
Ammonia-like:
Foul, offensive:
Sweet:
Fruity:
Maple syrup-like:
(Urea-splitting bacteria)
Old specimen, pus or inflammation
Glucose
Ketones
Maple Syrup Urine Disease
Color:
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Colorless
Deep Yellow
Yellow-Green
Red
Brownish-red
Brownish-black
Diluted urine
Concentrated Urine, Riboflavin
Bilirubin / Biliverdin
Blood / Hemoglobin
Acidified Blood (Actute GN)
Homogentisic acid (Melanin)
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.
Chemical Analysis
Chemical Analysis
Urine Dipstick
Glucose
Bilirubin
Ketones
Specific Gravity
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
The Urine Dipstick:
Glucose
Chemical Principle
Negative
Glucose Oxidase
Trace (100 mg/dL)
+ (250 mg/dL)
Glucose + 2 H2O + O2 --->
Gluconic Acid + 2 H2O2
++ (500 mg/dL)
Horseradish Peroxidase
+++ (1000 mg/dL)
++++ (2000+ mg/dL)
3 H2O2 + KI ---> KIO3 + 3 H2O
Read at 30 seconds
RR: Negative
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.
Detection of Reducing Sugars* by CuSO4
Sugar
- Galactose
- Fructose
Intolerance, etc.
- Lactose
- Pentoses
- Maltose
Disease(s)
Galactosemias
Fructosuria, Fructose
Lactase Deficiency
Essential Pentosuria
Non-pathogenic
* NOT Sucrose because it is not a reducing sugar
Urinalysis Glucose Result
Urine versus Blood Glucose
++
+
trace
Negative
200
400
600
800
Blood Glucose (mg/dL)
1000
Microscopic Examination
General Aspects
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
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
Microscopic Examination
Cells
Erythrocytes
- “Dysmorphic” vs. “normal”
(> 10 per HPF)
Leukocytes
- Neutrophils (glitter cells)
- Eosinophils
More than 1 per 3 HPF
Hansel test (special stain)
Epithelial Cells
- Squamous cells
- Renal tubular epithelial cells
- Transitional epithelial cells
Indicate level of contamination
Few are normal
Few are normal
- Oval fat bodies
Abnormal, indicate Nephrosis
Microscopic Examination
RBCs
Microscopic Examination
RBCs
Microscopic Examination
WBCs
Microscopic Examination
Squamous Cells
Microscopic Examination
Tubular Epithelial Cells
Microscopic Examination
Transitional Cells
Microscopic Examination
Transitional Cells
Microscopic Examination
Oval Fat Body
Microscopic Examination
LE Cell
Microscopic Examination
Bacteria & Yeasts
Bacteria
- Bacteriuria
Yeasts
- Candidiasis
Viruses
- CMV inclusions
More than 10 per HPF
Most likely a contaminant
but should correlate with
clinical picture.
Probable viral cystitis.
Microscopic Examination
Bacteria
Microscopic Examination
Yeasts
Microscopic Examination
Yeasts
Microscopic Examination
Cytomegalovirus
Microscopic Examination
Casts
Erythrocyte Casts:
Glomerular diseases
Leukocyte Casts:
Pyuria, glomerular disease
Degenerating Casts:
- Granular casts
- Hyaline casts
- Waxy casts
- Fatty casts
(oval fat body casts)
Nonspecific (Tamm-Horsfall protein)
Nonspecific (Tamm-Horsfall protein)
Nonspecific
Nephrotic syndrome
Microscopic Examination
Casts
Microscopic Examination
RBCs Cast - Histology
Microscopic Examination
RBCs Cast
Microscopic Examination
RBCs Cast - Histology
Microscopic Examination
WBCs Cast
Microscopic Examination
Tubular Epith. Cast
Microscopic Examination
Tubular Epith. Cast
Microscopic Examination
Granular Cast
Microscopic Examination
Hyaline Cast
Microscopic Examination
Waxy Cast
Microscopic Examination
Fatty Cast
Significance of Cellular Casts
Erythrocyte Casts
Leukocyte Casts
Bacterial Casts
Single Erythrocytes
Single Leukocytes
Single Bacteria
Verrier-Jones & Asscher, 1991.
Microscopic Examination
Crystals
- Urate
Ammonium biurate
Uric acid
- Triple Phosphate
- Calcium Oxalate
- Amino Acids
Cystine
Leucine
Tyrosine
- Sulfonamide
Microscopic Examination
Calcium Oxalate Crystals
Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape
Microscopic Examination
Triple Phosphate Crystals
Microscopic Examination
Urate Crystals
Microscopic Examination
Leucine Crystals
Microscopic Examination
Cystine Crystals
Microscopic Examination
Ammonium Biurate Crystals
Microscopic Examination
Cholesterol Crystals
Cytological Examination
Staining:
– Papanicolau
– Wright’s
– Immunoperoxidase
– Immunofluorescence
Cytology: Normal
Cytology: Normal
Cytology: Reactive
Cytology: Reactive
Cytology: Polyoma (Decoy Cell)
Cytology: Polyoma (Decoy Cell)
Immunoperoxidase to SV40 ag
Cytology: TCC Low Grade
Cytology: TCC Low Grade
Cytology: TCC High Grade
Cytology: TCC High Grade
Cytology: Squamous Cell Ca.
Cytology: Renal Cell Ca.
Cytology: Prostatic Carcinoma
Questions ?
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.
Chemical Analysis
Urine Dipstick
Glucose
Bilirubin
Ketones
Specific Gravity
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
The Urine Dipstick:
Glucose
Chemical Principle
Negative
Glucose Oxidase
Trace (100 mg/dL)
+ (250 mg/dL)
Glucose + 2 H2O + O2 --->
Gluconic Acid + 2 H2O2
++ (500 mg/dL)
Horseradish Peroxidase
+++ (1000 mg/dL)
++++ (2000+ mg/dL)
3 H2O2 + KI ---> KIO3 + 3 H2O
Read at 30 seconds
RR: Negative
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.
Detection of Reducing Sugars* by CuSO4
Sugar
- Galactose
- Fructose
Intolerance, etc.
- Lactose
- Pentoses
- Maltose
Disease(s)
Galactosemias
Fructosuria, Fructose
Lactase Deficiency
Essential Pentosuria
Non-pathogenic
* NOT Sucrose because it is not a reducing sugar
Urinalysis Glucose Result
Urine versus Blood Glucose
++
+
trace
Negative
200
400
600
800
Blood Glucose (mg/dL)
1000
The Urine Dipstick:
Bilirrubin
Negative
Chemical Principle
+ (weak)
Acidic Azobilirubin
Bilirubin + Diazo salt --------->
++ (moderate)
+++ (strong)
Read at 30 seconds
RR: Negative
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
The Urine Dipstick:
Ketones
Negative
Chemical Principle
Trace (5 mg/dL)
+ (15 mg/dL)
Acetoacetic Acid + Nitroprusside
------> Colored Complex
++ (40 mg/dL)
+++ (80 mg/dL)
++++ (160+ mg/dL)
Read at 40 seconds
RR: Negative
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
The Urine Dipstick:
Specific Gravity
1.000
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: 1.003-1.035
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)
The Urine Dipstick:
Negative
Trace (non-hemolyzed)
Moderate (non-hemolyzed)
Trace (hemolyzed)
+ (weak)
++ (moderate)
+++ (strong)
Blood
Chemical Principle
Lysing agent to lyse red blood cells
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
Heme
------------>
Colored Complex
Read at 60 seconds
RR: Negative
Analytic Sensitivity: 10 RBCs
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
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.: 4.5-8.0
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
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.
The Urine Dipstick:
Negative
Trace
+ (30 mg/dL)
Protein
Chemical Principle
“Protein Error of Indicators Method”
Pr
H
Pr
Pr
H
H
H
H
Pr
Pr
Pr
H
+
Tetrabromphenol Blue
+
++ (100 mg/dL)
H+ H H
(buffered to pH 3.0)
+
+
H
+
H
H
+++ (300 mg/dL)
Pr Pr
Pr
Pr Pr
++++ (2000 mg/dL)
Pr
Read at 60 seconds
RR: Negative
Causes of Proteinuria
Functional
- Severe muscular exertion
- Pregnancy
- Orthostatic proteinuria
Pre-Renal
- Fever
- Renal hypoxia
- Hypertension
Renal
- Glomerulonephritis
- Nephrotic syndrome
- Renal tumor or infection
Post-Renal
- Cystitis
- Urethritis or prostatitis
- Contamination with vaginal
secretions
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
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
Proteins in “Normal” Urine
Protein
% of Total
Daily Maximum
Albumin
Tamm-Horsfall
Immunoglobulins
Secretory IgA
Other
40%
40%
12%
3%
5%
60 mg
60 mg
24 mg
6 mg
10 mg
TOTAL
100%
150 mg
The Urine Dipstick:
0.2 mg/dL
1 mg/dL
Urobilinogen
Chemical Principle
Urobilinogen + Diethylaminobenzaldehyde
(Ehrlich’s Reagent)
2 mg/dL
-------> Colored Complex
4 mg/dL
8 mg/dL
Read at 60 seconds
RR: 0.02-1.0 mg/dL
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
The Urine Dipstick:
Nitrite
Chemical Principle
Negative
Positive
Acidic
Nitrite + p-arsenilic acid -------> Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
----------> Colored Complex
Read at 60 seconds
RR: Negative
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
The Urine Dipstick:
Leukocyte Esterase
Chemical Principle
Derivatized pyrrole amino acid ester
Negative
Esterases
------------> 3-hydroxy-5-phenyl pyrrole
Trace
+ (weak)
3-hydroxy-5-phenyl pyrrole + diazo salt
-------------> Colored Complex
++ (moderate)
+++ (strong)
Read at 2 minutes
RR: Negative
Analytic Sensitivity: 3-5 WBCs
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
Urinalysis
Disease Diagnosis
Case 1
Diluted urine, request a voided urine in the morning
If persisting low SG, possible diabetes insipida
A microscopic may give negative results
Glucose
Negative
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
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?
Case 2
Possible gallbladder or hepatic disease.
No hemolytic anemia. Perform bilirubins in serum
Microscopic unlikely to provide additional info
Glucose
Negative
Bilirubin
+++
Ketones
A 42-year old woman presents with “dark urine”
Negative
S.G.
1.020
Blood
Negative
pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative
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?
Case 3
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
Glucose
Negative
Bilirubin
Negative
Ketones
Negative
S.G.
1.030
Blood
+++
pH
6.5
Protein
Trace
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
+++
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?
Case 4
Diabetes
May be decompensated and with ketoacidosis
Ketones should become negative after treatment
Glucose
++
Bilirubin
Negative
Ketones
Trace
S.G.
1.015
Blood
Negative
pH
6.0
Protein
Negative
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
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?
Glomerulonephritis
RBC casts reveals renal cortex involvement
RBC cast are not always present in GN
Case 5
Glucose
Negative
Bilirubin
Negative
Ketones
Negative
S.G.
1.015
Blood
+++
pH
6.5
Protein
+
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
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?
“Functional” proteinuria?
Microscopic may reveal a few leukocytes
Request protein concentration in 24 h urine
Case 6
Glucose
Negative
Bilirubin
Negative
Ketones
Negative
S.G.
1.010
Blood
Negative
pH
5.0
Protein
+
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
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?
Common Findings in:
Acute Tubular Necrosis
Glucose
Bilirubin
Ketones
S.G.
Decreased
Blood
+/-
pH
Protein
Urobilinogen
Nitrite
L.E.
+/-
Microscopic:
• Renal tubular epithelial cells
• Pathological casts
Common Findings in:
Acute Glomerulonephritis
Glucose
Bilirubin
Ketones
Microscopic:
S.G.
Blood
Increased
pH
Protein
Urobilinogen
Nitrite
L.E.
Increased
• Erythrocytes (dysmorphic)
• Erythrocyte casts
• Mixed cellular casts
Common Findings in:
Chronic Glomerulonephritis
Glucose
Bilirubin
Ketones
S.G.
Decreased
Blood
Increased
pH
Protein
Urobilinogen
Nitrite
L.E.
Increased
Microscopic:
• Pathological casts
(broad waxy casts, RBCs)
Common Findings in:
Acute Pyelonephritis
Glucose
Bilirubin
Microscopic:
Ketones
S.G.
Blood
pH
Protein
Trace
Urobilinogen
Nitrite
Positive
L.E.
Positive
• Bacteria
• Leukocytes
• Leukocyte, granular, and
waxy casts
• Renal tubular epithelial
cell casts
Common Findings in:
Nephrotic Syndrome
Glucose
Bilirubin
Ketones
Microscopic:
S.G.
Blood
pH
Protein
Urobilinogen
Nitrite
L.E.
++++
• Oval fat bodies
• Fatty casts
• Waxy casts
Common Findings in:
Eosinophilic Cystitis
Glucose
Bilirubin
Ketones
Microscopic:
S.G.
Blood
pH
Protein
Urobilinogen
Nitrite
L.E.
+
• Numerous eosinophils
(Hansel’s stain)
• NO significant casts.
Common Findings in:
Urothelial Carcinoma
Glucose
Bilirubin
Ketones
Microscopic:
S.G.
Blood
pH
Protein
Urobilinogen
Nitrite
L.E.
+
• Malignant cells on
urine cytology (urine
sample should be submitted
separately to cytology, void
or 24 hrs.)
Acknowledgment:
Dr. Brad Brimhall