Urinalysis

Course: IDPT 5005

School of Medicine, UCDHSC

Francisco G. La Rosa, MD
Francisco.LaRosa@uchsc.edu
Assistant Professor, Department of Pathology
University of Colorado at Denver Health Science Center, Denver, Colorado
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

Macroscopic Examination
Chemical Analysis (Urine Dipstick)
Microscopic Examination
Culture (not covered in this lecture)
Cytological Examination
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)
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)
Glucose + 2 H2O + O2 --->
+ (250 mg/dL) Gluconic Acid + 2 H2O2

++ (500 mg/dL) Horseradish Peroxidase

3 H2O2 + KI ---> KIO3 + 3 H2O
+++ (1000 mg/dL)
Read at 30 seconds
++++ (2000+ mg/dL)
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 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

Urine versus Blood Glucose
Urinalysis Glucose Result

++

trace

Negative
200 400 600 800 1000
Blood Glucose (mg/dL)
The Urine Dipstick:
Bilirrubin

Negative Chemical Principle

+ (weak) Acidic Azobilirubin

Bilirubin + Diazo salt --------->
++ (moderate)
Read at 30 seconds
+++ (strong) 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) Read at 40 seconds

RR: Negative
++++ (160+ mg/dL)
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
Chemical Principle
1.005
X+ + Polymethyl vinyl ether / maleic anhydride
1.010 --------------->
X+-Polymethyl vinyl ether / maleic anhydride + H+
1.015
H+ interacts with a Bromthymol Blue indicator to
1.020 form a colored complex.

1.025 Read up to 2 minutes

RR: 1.003-1.035
1.030
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:
Blood

Negative
Chemical Principle
Trace (non-hemolyzed)
Lysing agent to lyse red blood cells

Moderate (non-hemolyzed)
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
Trace (hemolyzed)
Heme
------------> Colored Complex
+ (weak)

++ (moderate) Read at 60 seconds

RR: Negative
Analytic Sensitivity: 10 RBCs
+++ (strong)
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 Chemical Principle

H+ interacts with:
6.5
Methyl Red (at high concentration; low pH) and
Bromthymol Blue (at low concentration; high
7.0
pH), to form a colored complexes
(dual indicator system)
7.5

8.0 Read up to 2 minutes

R.R.: 4.5-8.0
8.5
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 Buffers from the protein area of
the strip (pH 3.0) spill over to the
Blood pH area of the strip and make the
pH pH of the sample appear more
acidic than it really is.
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
The Urine Dipstick:
Protein

Chemical Principle
Protein Error of Indicators Method
Negative Pr
H Pr
H Pr
Trace H
Pr Pr
+ (30 mg/dL) H H
H Pr
++ (100 mg/dL) Tetrabromphenol Blue +
H+ H H
+
(buffered to pH 3.0)
H + +
+++ (300 mg/dL) Pr Pr H+ H
Pr
Pr Pr
++++ (2000 mg/dL) Pr
Read at 60 seconds
RR: Negative
Causes of Proteinuria

Functional 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
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 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

The Urine Dipstick:
Urobilinogen

0.2 mg/dL Chemical Principle

1 mg/dL Urobilinogen + Diethylaminobenzaldehyde

(Ehrlichs Reagent)
2 mg/dL -------> Colored Complex

4 mg/dL
Read at 60 seconds
8 mg/dL 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

Acidic
Negative Nitrite + p-arsenilic acid -------> Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
Positive
----------> 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)
Read at 2 minutes
+++ (strong) 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
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) 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
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 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
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 Nonspecific (Tamm-Horsfall protein)
- Hyaline casts Nonspecific (Tamm-Horsfall protein)
- Waxy casts Nonspecific
- Fatty casts Nephrotic syndrome
(oval fat body casts)
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
Wrights
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
Urinalysis

Disease Diagnosis
 Diluted urine, request a voided urine in the morning
Case 1 If persisting low SG, possible diabetes insipida
A microscopic may give negative results

Glucose Negative

Bilirubin Negative
A 35-year old man undergoing routine pre
employment drug screening.
Ketones Negative
Physical characteristics: Clear.
S.G. 1.001 Microscopic: Not performed.
Drugs Identified: None.
Blood Negative
Questions:
pH 5.5

Protein Negative - What is your differential diagnosis?

- What would you do next to confirm your
Urobilinogen 0.2 mg/dL suspicion?
- Would you order a microscopic analysis
Nitrite Negative on this sample?
L.E. Negative
 Possible gallbladder or hepatic disease.
Case 2 No hemolytic anemia. Perform bilirubins in serum
Microscopic unlikely to provide additional info

Glucose Negative

Bilirubin +++
A 42-year old woman presents with dark urine
Ketones Negative
Physical characteristics: Red-brown.
S.G. 1.020 Microscopic: Not performed.
Blood Negative
Questions:
pH 5.5
- What is your differential diagnosis?
Protein Negative - Could this be a case of hemolytic anemia?
- How would you rule it out?
Urobilinogen 0.2 mg/dL - What tests would you order next? Why?
Nitrite - Would you order a microscopic analysis?
Negative

L.E. Negative
 Possible UTI, request culture and antibiotic sensitivity

Case 3
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
A 42-year old man presents painful urination
Bilirubin Negative

Physical characteristics: dark red, turbid

Ketones Negative
Microscopic: leukocytes = 30 per HPF
S.G. 1.030 RBCs = >100 per HPF
Bacteria = >100 per HPF
Blood +++
Questions:
pH 6.5 - What is your suspected diagnosis?
- What would you do next?
Protein Trace
- What do you make of the nitrite test?
Urobilinogen 1.0 mg/dL - How would the microscopic exam differ if
the S.G. were 1.003?
Nitrite Negative - Is this a common diagnosis for this type of
patient?
L.E. +++
 Diabetes
Case 4 May be decompensated and with ketoacidosis
Ketones should become negative after treatment

Glucose ++

Bilirubin Negative

Ketones Trace A 27-year old woman presents with severe

abdominal pain.
S.G. 1.015
Physical characteristics: clear-yellow.
Blood Negative Microscopic: Not performed.
pH 6.0
Questions:
Protein Negative - What is the most likely diagnosis?
- What do you make of the ketone result?
Urobilinogen 1.0 mg/dL - What do you expect to happen to the ketone
measurement when treatment begins?
Nitrite Negative

L.E. Negative
 Glomerulonephritis
Case 5 RBC casts reveals renal cortex involvement
RBC cast are not always present in GN

Glucose Negative
8-year old boy presents with discolored urine
Bilirubin Negative

Ketones Physical characteristics: Red, turbid.

Negative
Microscopic: erythrocytes = >100 per HPF
S.G. 1.015 (almost all dysmorphic)
Red cell casts present.
Blood +++
Questions:
pH 6.5 - What is the most likely diagnosis in this
Protein case?
+
- Does the presence of red cell casts help you
Urobilinogen 1.0 mg/dL in any way?
- If the erythrocytes were not dysmorphic
Nitrite Negative would that change your diagnosis?
L.E. Negative
 Functional proteinuria?
Case 6 Microscopic may reveal a few leukocytes
Request protein concentration in 24 h urine

Glucose Negative
22-year old man presenting for a routine
Bilirubin Negative
physical required for admission to medical
Ketones Negative school

S.G. 1.010 Physical characteristics: Yellow

Microscopic: Not performed
Blood Negative
Questions:
pH 5.0
- What is your differential diagnosis?
Protein + - Would you order a microscopic analysis on
this sample?
Urobilinogen 0.2 mg/dL - What would you do next to confirm the
diagnosis?
Nitrite Negative

L.E. Negative
 Common Findings in:
Acute Tubular Necrosis
Glucose

Bilirubin

Ketones

S.G. Decreased Microscopic:

Blood +/-
Renal tubular epithelial cells
pH
Pathological casts
Protein +/-

Urobilinogen

Nitrite

L.E.
 Common Findings in:
Acute Glomerulonephritis
Glucose

Bilirubin

Ketones
Microscopic:
S.G.

Blood Increased Erythrocytes (dysmorphic)

pH Erythrocyte casts
Protein Increased
Mixed cellular casts

Urobilinogen

Nitrite

L.E.
 Common Findings in:
Chronic Glomerulonephritis
Glucose

Bilirubin

Ketones

Decreased
Microscopic:
S.G.

Blood Increased
Pathological casts
pH (broad waxy casts, RBCs)
Protein Increased

Urobilinogen

Nitrite

L.E.
 Common Findings in:
Acute Pyelonephritis
Glucose

Bilirubin

Ketones Microscopic:
S.G.
Bacteria
Blood
Leukocytes
pH Leukocyte, granular, and
Protein Trace
waxy casts
Renal tubular epithelial
Urobilinogen
cell casts
Nitrite Positive

L.E. Positive
 Common Findings in:
Nephrotic Syndrome
Glucose

Bilirubin

Ketones

S.G. Microscopic:
Blood
Oval fat bodies
pH
Fatty casts
Protein ++++ Waxy casts
Urobilinogen

Nitrite

L.E.
 Common Findings in:
Eosinophilic Cystitis
Glucose

Bilirubin

Ketones

S.G. Microscopic:
Blood +
Numerous eosinophils
pH
(Hansels stain)
Protein NO significant casts.
Urobilinogen

Nitrite

L.E.
 Common Findings in:
Urothelial Carcinoma
Glucose

Bilirubin

Ketones

S.G. Microscopic:
Blood +
Malignant cells on
pH
urine cytology (urine
Protein sample should be submitted
separately to cytology, void
Urobilinogen
or 24 hrs.)
Nitrite

L.E.
Acknowledgment:

Dr. Brad Brimhall

Questions ?