Standardization of Urinalysis

SPECIMEN COLLECTION FOR ROUTINE URINALYSIS

– 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

Collection of urine is started in the

morning and subsequent samples are

collected till the next morning.

Preservatives used:

Refrigeration at 4 degrees

Toluene- 1 ml / 50ml of urine. Preserves

chemical constituents.

SPECIMEN COLLECTION FOR 24 HOURS URINALYSIS

Thymol – 1% of thymol, gives false positive for proteins

Acid- HCl, sulfuric acid and boric acid can be used.

Formalin- 6-8 drops of 40% formalin per 100 ml

of urine. Preserves RBCs and Pus cells, gives

false positive test for sugar.

Specimen Collection

Supra-pubic Needle Aspiration

− Macroscopic Examination

− Chemical Analysis

− Microscopic Examination

− Culture

− Cytological Examination

Types of Analysis

Macroscopic Examination

Volume : normal – 700-2500ml/24 hours

Polyuria- >2500ml

Excess intake

Diabetes insipidus

Diabetes mellitus

Oliguria: <500ml

Less intake

Dehydration

Renal ischaemia

Anuria: <150ml

Renal stones

Renal or prostatic tumors

Severe renal ischaemia

Appearance normal-clear

Cloudy

U.T.I.

calculi

dye

Milky

lipiduria

Chyluria

Turbid

Crystals

infection

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)

Foam If a normal urine specimen is shaken or

agitated sufficiently, a white foam can be forced to develop at its surface that readily dissipates on standing.

Moderate to large amounts of protein (albumin) in urine cause a stable white foam to be produced when the urine is poured or agitated.

When bilirubin is present in sufficient amounts, the foam if present will be characteristically yellow.

Chemical Analysis

Conventional tests

Benedicts test

Heat coagulation test

Rothera’s test

Hay’s test

Ehrlich’s test

Fouchet’s test

Benzidine test

Chemical AnalysisUrine Dipstick

Glucose

Bilirubin

Ketones

Specific Gravity

Blood

pH

Protein

Urobilinogen

Nitrite

Leukocyte Esterase

Negative

Trace (100 mg/dL)

+ (250 mg/dL)

++ (500 mg/dL)

+++ (1000 mg/dL)

++++ (2000+ mg/dL)

The Urine Dipstick:Glucose

Glucose + 2 H2O + O2 --->Gluconic Acid + 2 H2O2

Glucose Oxidase

3 H2O2 + KI ---> KIO3 + 3 H2O

Horseradish Peroxidase

Chemical Principle

Read at 30 seconds

RR: Negative

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.

Uses and Limitations of Urine Glucose Detection

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

Detection of Reducing Sugars* by CuSO4

++

+

trace

400 600 800 1000200

Urin

aly

sis

Glu

co

se R

esu

lt

Blood Glucose (mg/dL)

Urine versus Blood Glucose

Negative

Negative

+ (weak)

++ (moderate)

+++ (strong)

The Urine Dipstick:Bilirubin

Bilirubin + Diazo salt ---------> AzobilirubinAcidic

Chemical Principle

Read at 30 seconds

RR: Negative

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

Uses and Limitations of Urine Bilirrubin Detection

Negative

Trace (5 mg/dL)

+ (15 mg/dL)

++ (40 mg/dL)

+++ (80 mg/dL)

++++ (160+ mg/dL)

The Urine Dipstick:Ketones

Acetoacetic Acid + Nitroprusside

------> Colored Complex

Chemical Principle

Read at 40 seconds

RR: Negative

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 version of same assay)

- Serum glucose measurement to confirm DKA

Uses and Limitations of Urine Ketone Detection

1.000

1.005

1.010

1.015

1.020

1.025

1.030

Chemical Principle

Read up to 2 minutes

RR: 1.003-1.035

The Urine Dipstick:Specific Gravity

pKa change of anpolyelectrolyte;indirect measureof density

Significance

- Diabetes insipidus

Limitations

- Interference: alkaline urine

- Does not measure non-ionized solutes (e.g. glucose)

Other Tests

- Refractometry

- Hydrometer

- Osmolality measurement

Uses and Limitations of Urine Specific Gravity

Negative

Trace (non-hemolyzed)

Moderate (non-hemolyzed)

Trace (hemolyzed)

+ (weak)

++ (moderate)

+++ (strong)

The Urine Dipstick:Blood

Diisopropylbenzene dihydroperoxide +

Tetramethylbenzidine

------------> Colored ComplexHeme

Chemical Principle

Lysing agent to lyse red blood cells

Read at 60 seconds

RR: Negative

Analytic Sensitivity: 10 RBCs

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

Uses and Limitations of Urine Blood Detection

5.0

6.0

6.5

7.0

7.5

8.0

8.5

The Urine Dipstick: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)

Chemical Principle

Read up to 2 minutes

R.R.: 4.5-8.0

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

Uses and Limitations of Urine pH Detection

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.

pH Run Over Effect

Negative

Trace

+ (30 mg/dL)

++ (100 mg/dL)

+++ (300 mg/dL)

++++ (2000 mg/dL)

The Urine Dipstick:Protein

Chemical Principle

H

H

H

H

H

H

Pr

Pr

Pr

Pr

Pr

Pr

“Protein Error of Indicators Method”

Pr Pr

Pr

PrPr

Pr

Tetrabromphenol Blue

(buffered to pH 3.0)H+

H+

H+H+

H+ H+

Read at 60 seconds

RR: Negative

Physiological 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

Causes of Proteinuria

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

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

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)

Uses and Limitations of Urine Protein Detection

0.2 mg/dL

1 mg/dL

2 mg/dL

4 mg/dL

8 mg/dL

The Urine Dipstick:Urobilinogen

Urobilinogen + Diethylaminobenzaldehyde

-------> Colored Complex

(Ehrlich’s Reagent)

Chemical Principle

Read at 60 seconds

RR: 0.02-1.0 mg/dL

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

Uses and Limitations of Urobilinogen Detection

Reading time in a nutshell

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 ExaminationGeneral Aspects

Microscopic Examination

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

Abnormal Findings

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 ExaminationCells

Microscopic ExaminationRBCs

Microscopic ExaminationWBCs

Microscopic ExaminationSquamous Cells

Microscopic ExaminationTubular Epithelial Cells

Microscopic ExaminationTransitional Cells

Microscopic ExaminationTransitional Cells

Microscopic ExaminationOval Fat Body

Bacteria- Bacteriuria More than 10 per HPF

Yeasts- Candidiasis Most likely a contaminant

but should correlate withclinical picture.

Viruses- CMV inclusions Probable viral cystitis.

Microscopic ExaminationBacteria & Yeasts

Microscopic ExaminationBacteria

Microscopic ExaminationYeasts

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 ExaminationCasts

Microscopic ExaminationCasts

Microscopic ExaminationRBCs Cast - Histology

Microscopic ExaminationRBCs Cast

Microscopic ExaminationRBCs Cast - Histology

Microscopic ExaminationWBCs Cast

Microscopic ExaminationTubular Epith. Cast

Microscopic ExaminationTubular Epith. Cast

Microscopic ExaminationGranular Cast

Microscopic ExaminationHyaline Cast

Microscopic ExaminationWaxy Cast

Microscopic ExaminationFatty Cast

Bacterial Casts

Single Leukocytes

Leukocyte Casts

Verrier-Jones & Asscher, 1991.

Single Erythrocytes

Erythrocyte Casts

Single Bacteria

Significance of Cellular Casts

Microscopic ExaminationCrystals in acidic urine

Microscopic ExaminationCrystals in alkaline urine

Microscopic ExaminationCalcium Oxalate Crystals

Microscopic ExaminationCalcium Oxalate Crystals

Dumbbell Shape

Microscopic ExaminationTriple Phosphate Crystals

Microscopic ExaminationUrate Crystals

Microscopic ExaminationLeucine Crystals

Microscopic ExaminationCystine Crystals

Microscopic ExaminationAmmonium Biurate Crystals

Microscopic ExaminationCholesterol Crystals

Need for automation

Time saving

Easy to process

Multiple samples can be processed

Reduce the need for man power

Uniformity

Sensitive

Specific

List of instruments

Principle

Aspirates and dispenses fixed amount of urine on each pad of the strip

Reads the strip via reflectance method.

Assess color of a specimen by using four wavelengths of light to obtain the tone (light, normal, dark) and hue of a urine specimen.

Light scatter is used to determine the turbidity

Specific gravity is measured by assessing refractive index of LED-emitted light.

dual wavelength reflectance to measure the pH and chemical constituents of urine

Priciple

Iris Diagnostics Division iChem®100

Factors That Require Standardization in the Microscopic Examination Urine volume used (e.g., 10 mL, 12 mL, 15 mL)

Speed of centrifugation (400 or 450 Å~ g)

Time of centrifugation (5 minutes)

Concentration of sediment prepared (e.g., 10 : 1, 12 : 1, 15 : 1, 30 : 1)

Volume of sediment examined—determined by commercial slides used and microscope optical properties (i.e., ocular field number)

Result reporting—format, terminology, reference intervals, magnification used for assessment

video

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

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