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: Foul, offensive: Sweet: Fruity: Maple syrup-like:

(Urea-splitting bacteria) Old specimen, pus or inflammation Glucose Ketones Maple Syrup Urine Disease

Color:

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) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) Glucose + 2 H2O + O2 ---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 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

Urine versus Blood Glucose

Urinalysis Glucose Result

++

trace

Negative

200

400

600

800

1000

Blood Glucose (mg/dL)

The Urine Dipstick:

Bilirrubin

Negative
+ (weak) ++ (moderate) +++ (strong)

Chemical Principle

Acidic Azobilirubin Bilirubin + Diazo salt --------->

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:

Negative

Ketones

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

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:

1.000
1.005 1.010 1.015 1.020 1.025 1.030

Specific Gravity

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:

5.0 6.0 6.5 7.0 7.5 8.0 8.5 Chemical Principle

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) 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 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.

Leukocyte Esterase

The Urine Dipstick:

Protein

Negative
Trace + (30 mg/dL)

Chemical Principle Protein Error of Indicators Method Pr H Pr Pr H H H

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

Pr

Pr

Causes of Proteinuria
Functional
- Severe muscular exertion - Pregnancy - Orthostatic proteinuria

Renal
- Glomerulonephritis - Nephrotic syndrome - Renal tumor or infection

Pre-Renal
- Fever - Renal hypoxia - Hypertension

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 Albumin Tamm-Horsfall Immunoglobulins Secretory IgA Other TOTAL % of Total 40% 40% 12% 3% 5% 100% Daily Maximum 60 mg 60 mg 24 mg 6 mg 10 mg 150 mg

The Urine Dipstick:

Urobilinogen

0.2 mg/dL 1 mg/dL

2 mg/dL 4 mg/dL 8 mg/dL

Chemical Principle
Urobilinogen + Diethylaminobenzaldehyde
(Ehrlichs Reagent)

-------> Colored Complex

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

Nitrite + p-arsenilic acid -------> Diazo compound

Diazo compound + Tetrahydrobenzoquinolinol ----------> Colored Complex Read at 60 seconds RR: Negative

Acidic

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) ++ (moderate) +++ (strong)

3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex 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

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
- Oval fat bodies

Indicate level of contamination Few are normal Few are normal

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 Yeasts

- Bacteriuria

More than 10 per HPF

- Candidiasis

Viruses

Most likely a contaminant but should correlate with clinical picture. Probable viral cystitis.

- CMV inclusions

Microscopic Examination
Bacteria

Microscopic Examination
Yeasts

Microscopic Examination
Yeasts

Microscopic Examination
Cytomegalovirus

Microscopic Examination
Casts Erythrocyte Casts: Leukocyte Casts:

Glomerular diseases 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 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

Case 1
Glucose

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

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

Negative
Negative 1.001 Negative 5.5 Negative 0.2 mg/dL Negative 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
Glucose

Possible gallbladder or hepatic disease. No hemolytic anemia. Perform bilirubins in serum Microscopic unlikely to provide additional info
Negative +++ Negative 1.020

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

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?

Negative
5.5

Negative 0.2 mg/dL

Negative Negative

Case 3
Glucose

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
Negative

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

A 42-year old man presents painful urination

Negative Negative 1.030 +++ 6.5 Trace 1.0 mg/dL Negative +++

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
Glucose

Diabetes May be decompensated and with ketoacidosis Ketones should become negative after treatment
++ Negative Trace 1.015

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

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?

Negative
6.0

Negative 1.0 mg/dL

Negative Negative

Case 5
Glucose

Glomerulonephritis RBC casts reveals renal cortex involvement RBC cast are not always present in GN

Negative

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

Negative
Negative 1.015 +++ 6.5 + 1.0 mg/dL

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?

Negative
Negative

Case 6
Glucose

Functional proteinuria? Microscopic may reveal a few leukocytes Request protein concentration in 24 h urine
Negative

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E.

Negative
Negative 1.010 Negative 5.0 + 0.2 mg/dL Negative 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. Blood pH Protein Urobilinogen Nitrite L.E. +/Decreased +/-

Microscopic: Renal tubular epithelial cells Pathological casts

Common Findings in: Acute Glomerulonephritis

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. Increased Increased

Microscopic:
Erythrocytes (dysmorphic) Erythrocyte casts Mixed cellular casts

Common Findings in: Chronic Glomerulonephritis

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. Decreased Increased

Microscopic:
Pathological casts (broad waxy casts, RBCs)

Increased

Common Findings in: Acute Pyelonephritis

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. Positive

Microscopic: Bacteria Leukocytes Leukocyte, granular, and waxy casts Renal tubular epithelial cell casts

Trace

Positive

Common Findings in: Nephrotic Syndrome

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. ++++

Microscopic:
Oval fat bodies Fatty casts Waxy casts

Common Findings in: Eosinophilic Cystitis

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. +

Microscopic:
Numerous eosinophils (Hansels stain) NO significant casts.

Common Findings in: Urothelial Carcinoma

Glucose

Bilirubin
Ketones S.G. Blood pH Protein Urobilinogen Nitrite L.E. +

Microscopic:

Malignant cells on
urine cytology (urine
sample should be submitted separately to cytology, void or 24 hrs.)

Acknowledgment: Dr. Brad Brimhall

Questions ?