urinalysis

URINALYSISSpecimen 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

Prepared by Amjad khan

Macroscopic Examination

Chemical Analysis (Urine Dipstick)

Microscopic Examination

Culture (not covered in this lecture)

Cytological 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-GreenBilirubin / Biliverdin

− Red Blood / Hemoglobin

− Brownish-red Acidified Blood (Actute GN)

− Brownish-black Homogentisic acid (Melanin)


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

Urine Dipstick

The Urine Dipstick:


Glucose

Chemical Principle

Glucose Oxidase

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 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

The Urine Dipstick:

Bilirrubin

Chemical Principle

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

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

Chemical Principle

Acetoacetic Acid + Nitroprusside

------> Colored Complex

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

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)

+ (weak)

++ (moderate)

+++ (strong)

Hemolyzed

Blood

Chemical Principle

Lysing agent to lyse red blood cells

Diisopropylbenzene dihydroperoxide +

Tetramethylbenzidine

------------> 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

Chemical Principle

Methyl Red (at high concentration; low pH) and

H+ interacts with:

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

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:


Protein

Chemical Principle

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

Chemical Principle


Urobilinogen + Diethylaminobenzaldehyde

(Ehrlich’s 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

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

Esterases

------------> 3-hydroxy-5-phenyl pyrrole

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


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)


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


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


RBCs


WBCs

Squamous Cells

Tubular Epithelial Cells

Transitional Cells


Oval Fat Body

LE Cell


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.

Bacteria

Yeasts

Cytomegalovirus


Casts


Erythrocyte Casts:Glomerular diseases

Leukocyte Casts: Pyuria, glomerular disease

Degenerating Casts:

- Granular casts Nonspecific (Tamm-Horsfall protein)

- Hyaline castsNonspecific (Tamm-Horsfall protein)

- Waxy casts Nonspecific

- Fatty casts Nephrotic syndrome

(oval fat body casts)

RBCs Cast

RBCs Cast - Histology

RBCs Cast - Histology


WBCs Cast

Tubular Epith. Cast

Granular Cast Hyaline Cast

Waxy Cast


Fatty Cast

Significance of Cellular Casts


Crystals


- Urate

Ammonium biurate

Uric acid

- Triple Phosphate

- Calcium Oxalate

- Amino Acids

Cystine

Leucine

Tyrosine

- Sulfonamide

Calcium Oxalate Crystals

Triple Phosphate Crystals

Urate Crystals


Leucine Crystals

Cystine Crystals

Ammonium Biurate Crystals

Cholesterol Crystals


Cytological Examination

Staining:

– Papanicolau

– Wright’s

– Immunoperoxidase

– Immunofluorescence

Cytology: Normal

Cytology: Reactive

Cytology: Reactive


Cytology: Polyoma (Decoy Cell)

Cytology: Polyoma (Decoy Cell)Immunoperoxidase to SV40 ag

Cytology: TCC Low Grade

Cytology: TCC High Grade


Cytology: Squamous Cell Ca.

Cytology: Renal Cell Ca.

Cytology: Prostatic Carcinoma


Urinalysis


urinalysis

Health & Medicine

amjad khan

measures glucose

limitations interference

glucose fruity

indirect bilirubin

direct bilirubin correlates

urinalysis specimen

seconds rr