Lecture 4: Chemical Examination of Urine

Reagent Strips

performed routinely in the laboratory as part of urinalysis because it is a simple and rapid means for performing medically significant chemical analysis of urine.

• Leukocytes esterase

• Nitrites

• Urobilinogen

• Protein

• pH

• Blood

• Specific Gravity

• Ketones

• Bilirubin

• Glucose

What are the parameters in the Reagent strips

• RBCs and WBCs

• Strip in urine for longer period

• Excess urine on the strip

• Timing of reactions

• Reagent strips and color charts are NOT interchangeable

• Refrigerated specimen

Errors cause by improper reagent strip technique

Reagent Strip Test Handling

• must be protected from deterioration

• Keep container sealed with desiccant to prevent light and moisture exposure.

• After removing a strip, immediately cover the bottle to protect remaining strips.

Specimen Preparation

Refrigerated specimens should be brought to room temperature before testing.

Qualitry Control

Check reagents with positive and negative controls every 24 hours

5.0 - 6.0 (slightly acidic)

Normal morning urine specimen

• To aid in determining the systemic acid-base disorders

• Management of urinary conditions

• Precipitation of inorganic chemicals that is dissolved in urine

• Treatment of UTI

• Dietary intake

• Improperly preserved specimen

Clinical significance for pH

• Double Indicator System

• Methyl red & Bromthymol blue

Reagent Strip Reaction of pH

• Principle

• Reagents (Indicator)

• 5.0 to 8.5 in 0.5 increments

• 5.0 to 9.0 in 1.0 increments

pH sensitivity:

• Multistix

• Chemstrip

Protein

Clinical significance is for detection of proteinuria

Albumin

Major serum protein

• Protein Error of pH Indicators

• Tetrabromophenol blue (multistix)

• Tetrabromosulfonphthalein (Chemstrip)

• 60 seconds

Protein

• Principle

• Reagents

• Reading time

• 15-30 mg/dL of albumin (Multistix)

• 6 mg/dL of albumin (Chemstrip)

Proteins sensitivity

• Prerenal proteinuria

• Renal proteinuria

• Postrenal proteinuria

3 Major categories of clinical proteinuria

Prerenal proteinuria

This affects the plasma prior to reaching the kidney. It is not an indicative of an actual renal disease. This conditions are transient.

NOT discovered / detected in routine urinalysis.

Bence Jones Proteins

This is a primary example of protenuria that is due to increased serum protein leves.

These are found in patients with multiple myeloma.

Coagulates at 40 C - 60 C, 

Dissolves at 100 C

Bence - Jones Protein Principle

• Glomerular Protenuria

• Microalbuminuria

• Orthostatic (postural) Proteinuria

• Tubular Proteinuria

• Postrenal Protenuria

Renal Protenuria

Glomerular proteinuria

Impaired selective filtration in glomerulus allows RBCs, WBCs, and increased serum protein into urine.

Strenuous exercise, dehydration, hypertension

Reversible causes of glomerular filtration

Microalbuminuria

Early indicator of diabetic nephropathy and renal complications.

Helps prevent cardiovascular risks by stabilizing blood glucose and controlling hypertension.

Orthostatic (Postural) Protenuria

• Benign protenuria related to patient posture.

• Appears after vertical posture; disappears when horizontal.

Tubular Protenuria

• Caused by disorders affecting tubular reabsorption

• Protein in urine is usually mildly elevated, unlike glomerular protenuria

Postrenal Protenuria

Related to the lower urinary tract: ureters, bladder, prostate, urethra, vagina

Sulfosalicylic Acid Precipitation Test

• Cold precipitation test for protein detection

• Standard laboratory concentration: 3% SSA

Negative

No increase in turbidity,

Less than 6mg/dL protein range

Trace

Noticeable turbidity, 6-30 mg/dL protein range

1 +

Distinct turbidity, no granulation, 30-100 mg/dL

2+

Turbidity, granulation, no flocculation, 100-200 mg/dL protein range

3+

Turbidity, granulation, flocculation, 200-400 mg/dL protein range

4+

Clumps of protein, Greater than 400 mg/dL protein range

Microalbumin Testing

Is a reagent strip for the Micral test.

Principle : Enzyme Immunoassay

Specimen: First morning urine

Heat and Acetic Acid Test

• Perform on centrifuged specimens

• Heat the upper portion of the tube

• Add 5% acetic acid

• Heat again

• Grade the degree of cloudiness.

Glucose

most frequently performed chemical analysis on urine.

occurs in patients with hyperglycemia.

Around 6 months

At what stage of pregnancy is urine glucose testing important for detecting gestational diabetes?

They block insulin, causing insulin resistance and hyperglycemia

How do placental hormones affect insulin in gestational diabetes?

Glucose crosses the placenta, insulin does not, leading the fetal pancreas to produce insulin and store excess glucose as fat.

Why is glucose in maternal blood a risk for the fetus?

Macrosomia

What condition can result in the baby due to excess glucose in gestational diabetes?

To detect mothers at risk for gestational diabetes, preventing complications for both mother and baby.

Why is urine glucose testing important for preganant women?

• Double Sequential Enzyme Reaction

• Glucose oxidase, Peroxidase, Potassium iodide (Multistix), Tetramethylbenzidine (Chemstrip)

• 30 seconds

Glucose

• Principle

• Reagents

• Reading Time

• 75 - 125 mg/dL (Multistix)

• 40 mg/dL (Chemstrip)

Glucose sensitivity

Benedict’s Test / Copper reduction Test

It was one of the earliest chemical tests that was performed in urine.

Relies on the ability of glucose and other substances to reduce the copper sulfate to cuprous oxide.

Blue

Negative color of Benedict’s Test

Traces of reducing sugar

Green or yellow precipitate in Benedict’s Tet

Moderat

Orange red precipitate in Benedict’s Test

Large aggregates of reducing sugar

Red color in Benedict’s test

“Pass Through" Phenomenon

This happens in the presence of high glucose levels; it may be reported as negative

200 mg/dL

Benedict’s test sensitivity

• Sodium Nitroprusside Reaction

• Sodium Nitroprusside; Glycine

Ketones

• Principle

• Reagents

• Acetone - 2%

• Acetoacetic acid - 20%

• Beta-hydroxybutyrate - 78%

What are intermediate products of fat metabolism and give the percentage of each?

• Diabetic acidosis

• Strenuous exercise

• Insulin dosage monitoring

• Vomiting

• Starvation Inborn errors of amino acid

• Malabsorption/Pancreatic disorders

• Inborn errors of amino acid metabolism

Give the Chemical Significance of Urine Ketones

• 5-10 mg/dL acetoacetic acid (Multistix)

• 9 mg/dL acetoacetic acid; 70 mg/dL acetone (Chemstrip)

Ketones sensitivity

Blood

It is present in the urine either in the form of Hematuria or Hemoglobinuria

Hematuria

Cloudy Red Urine

Hemoglobinuria

Clear Red Urine

Myoglobinuria 

Is present when there is rapid destruction of skeletal muscles in which the Myoglobin appears in the urine either red or brown urine.

• Pseudoperoxidase activity of hemoglobin

• Sodium nitroprusside; Glycine (Chemstrip)

Blood

• Principle

• Reagents

• 5 to 20 RBCs/mL, 0.015 to 0.062 mg/dL hemoglobin (Multistix)

• 5 RBCs/mL, Hemoglobin corresponding to 10 RBCs/mL (Chemstrip)

Blood Sensitivity

Bilirubin

An early indicator of liver disease.

It is a degradation product of hemoglobin

destroyed after 120 days in the spleen and liver by phagocytic cells of the reticuloendothelial system

What happens when an RBC reaches the end of its lifespan?

Hemoglobin is broken down into;

• Iron —> reused by the body

• Protein —> reused

• Protoporphyrin —> converted into bilirubin

What happens to hemoglobin after RBC destruction?

It binds to albumin because it is water-insoluble , allowing it to travel to the liver.

How is unconjugated bilirubin transported in the blood?

No, because it is albumin-bound and water-insoluble

Can the kidneys excrete unconjugated bilirubin?

The liver converts it into conjugated bilirubin using glucuronyl transferase + glucuronic acid, making it water-soluble

How does the liver process unconjugated bilirubin?

It usually passes from the liver —> bile duct —> intestine and does not appear in urine

What happens to conjugated bilirubin?

Intestinal bacteria reduce it to urobilinogen, which is oxidized and excreted as stercobilin and urobilin in feces.

How is bilirubin metabolized in the intestine?

Bilirubinuria

The presence of bilirubin in urine. It occurs in hepatic jaundice when conjugated bilirubin leaks into circulation.

Because hemolysis produces unconjugated bilirubin, which is not water-soluble and cannot be excreted by the kisneys.

Why doesn’t hemolytic jaundice produce bilirubinuria?

• Hepatitis

• Cirrhosis

• Biliary obstruction

What liver-related disorders can cause urine ketones?

• Diazo Reaction

• 2,4-dichloroaniline diazonium salt (Multistix)

• 2,6-dichlorobenzene diazonium salt (Chemstrip)

Bilirubin

• Principle

• Reagents

• 0.4-0.8 mg/dL bilirubin (Multistix)

• 0.5 mg/dL bilirubin (Chemstrip)

Bilirubin sensitivity

ICTO Test

It is the confirmatory test of bilirubin

Blue to Purple

Result for ICTO test when bilirubin is present

• Modified Ehrlich’s Reaction

• para-dimethylaminobenzaldehyde ; 4-methyloxybenzine-diazonium-tetrafluorobate

Urobilinogen

• Principle

• Reagents

• 0.2 mg/dL , urobilinogen (Multistix)

• 0.4 mg/dL, urobilinogen (Chemstrip)

Urobilinogen sensitivity

Watson-Schwartz Differentiation Test

A classic test for differentiating between urobilinogen, porphobilinogen, and Ehrlich-reactive compounds.

Colorless urine in the upper layer and red layer in the chloroform (bottom layer)

How does urobilinogen appear in the Watson-Schwartz test with chloroform?

Red in the upper butanol layer, colorless in the lower urine layer

How does urobilinogen appear in the Watson-Schwartz test with butanol?

Yes

Is urobilinogen soluble in chloroform and butanol?

Insoluble in both; stays in the urine layer

How does porphobilinogen behave in chloroform and butanol during the Watson-Schwartz test?

Not extracted by chloroform, but extracted by butanol

How do Ehrlich-reactive compounds behave in the Watson-Schwartz test?

• Urine (Top layer) : Colorless

• Chloroform (Bottom Layer) : Red

In the Watson-Schwartz test, how does urobilinogen appear with chloroform extraction?

Urine (Top layer): Red

Chloroform (Bottom Layer) : Colorless

In the Watson-Schwartz test, how does other Ehrlich-reactive substances appear with chloroform extraction?

Urine (Top Layer): Red

Chloroform (Bottom Layer): Colorless

In the Watson-Schwartz test, how does porphobilinogen appear with chloroform extraction?

• Butanol (Top layer) : Red

• Urine (Bottom Layer) : Colorless

In Watson-Schwartz test, how does urobilinogen appear with butanol extraction?

• Butanol (Top Layer) : Red

• Urine (Bottom Layer) : Colorless

In Watson-Schwartz test, how do other Ehrlich-reactive substances appear with butanol extraction?

• Butanol (Top Layer) : Colorless

• Urine (Bottom Layer) : Red

In Watson-Schwartz test, how does porphobilinogen appear with butanol extraction?

Hoesch Screening Test for Porphobilinogen

It is a rapid screening or monitoring tests for urinary porphobilinogen.

The top of the solution is observed for the appearance of a red color.

• rapid test for UTI

• Cystitis

• Pyelonephritis (untreated cystitis)

Clinical Significance of Nitrite

• Greiss Reaction

• Para-arsanilic acid / Sulfanilamide; Tetrahydrobenzoquinolin

• 60 seconds

Nitrite

• Principle

• Reagents

• Reading time

pink azodye

Positive result for Nitrite

100,000 organisms per mL

Nitrite sensitivity

First morning specimen from urine in the bladder for 4 hours

Specimen for Nitrite

Urinary Tract Infection

Increased urinary leukocyte is an indicator of?

Bacteria (Nitrite Positive)

Positive result of Leukocyte is most frequently accompanied by the presence of?

• Leukocyte esterase

• Indoxylcarbonic acid ester

• 120 seconds

Leukocytes

• Principle

• acid ester impregnated on the reagent pad

• Reading Time

purple azodye

Resulting color reaction for Leukocyte

• pKa change of polyelectrolyte

• Poly (methyl vinyl ether/maleic anhydride) bromthymol blue (Multistix) ;

Ethylene glycol diaminoethyl ether tetraacetic

acid, bromthymol blue

• 45 seconds

Specific Gravity

• Principle

• Reagents

• Reading time

1.000 - 1.030

Specific Gravity sensitivity