Principle: Bilirubin

Diazo Rxn

*Bilirubin coupled with diazonium salt in acid medium and forms Azobilirubin

*color change from tan->beige->pink

Expected value of Bilirubin should be

Negative

Specificty for Bilirubin

Bilirubin

False positives for bilirubin caused by..

*Drug induced color changes, such as phenazopyridine, indican-indoxyl sulfate.

*Large amounts of chlorpromazine metabolites.

*Metabolites or etodolac

*A yellow orange to red color with indican

False Negatives of Bilirubin caused by...

*Ascorbic acid >25mg/dL

*high nitrite concentrations(as seen in UTImay decrease sensitivity)

,* improper storage, resulting in, oxidation or hydrolysis to nonreactive biliverdin and free bilirubin; light exposure. 

Principle for Urobilinogen

*For chemstrip and rapignost strips azocoupling reaction of urobilinogen with diazonium salt in an acid medium to form an azo dye. Color changes from light pink to dark pink are observed.

*For use with Multistix strips modified Ehrlichs reaction is done. Urobilinogen present reacts with Ehrlich’s reagent (p-dimethyaminobenzaldehyde) in concentrated hydrochloric acid to form a red compound (aldehyde). Color changes from light orange-pink to dark pink are observed.

False positive of Urobiligen caused by..

* any other Ehrlichs reactive substance

*atypical colors caused by sulfonamides, p-aminobenzoic acid, p-aminosalicyclic acid,

*substances that induce color mask results, such as drugs, and beet ingestions, 

False negatives results for urobilinogen caused by

*Formalin, a urine preservative.

*Improper storage, resulting in oxidation to urobilin.

Principle for Specific Gravity

Ionic solutes present in the urine cause protons to be released from a polyelectrolyte. As the protons are released, the pH decreases and produces a color change of the bromthymol blue indicator from the blue-green to yellow-green. 

Specificity of Specific Gravity

ionic solutes

Princple of Nitrite testt

*Based on Griess test involving diazo reaction

*Nitrite react with aromatic amine in an acid medium to produce diazonium salt.

*diazonium salt then coupled with another aromatic ring (quinoline) to give azo dye (pink or red) 

Falso positve for nitrites caused by

*Highly colored substances that mask results, such as drugs, beet ingestion.

*Improper storage with bacterial proliferation. 

*conversion of nitrate to nitrite as result of bacterial contamination of specimen

False negative test for nitrites caused by..

Ascorbic acid intergerence

*not enough time in bladder to convert nitrate to nitrite

*insufficient dietary nitrate for bacteria to convert to nitrite (starvation, fasting, or intravenous feeding)

Principle of Leukocyte test

*diazo reaction

*strip contains ester that is hydrolized by leukocyte esterase to form its alcohol and acid

* the aromatic ring is then coupled with diazonium salt to form azo dye (purple color)

Chem strips= indoxyl ester

Multstix=pyyrole amino acid ester

Specificity of Leukocyte chem strip test

False positive of leukocytes caused by

*highly colored substances that mask results, such as drugs, beet ingestion.

*Vaginal contamination of urine.

*strong oxidzing agents such as chlrorine, bleach, and urinary preservatives such as formalin

False negative of leukocyte test caused by

*Lymphocytes present are not detected,

*increased glucose >3g/dL

*high specific gravity,

*Drugs such as cephalexink cephalothin, tetracycline, and gentamicin

*oxalic acid (metabolites of asborbic acid)

*High levels of albumin >500 mg/dL

Why is confirmatory testing used?

 What is Protein-Sulfosalicylic Acid (SSA)?

Confirmatory test for protein

  Used to confirm the presence of protein when reactions indicating a trace or more are obtained or when reagent strip values are in doubt.

Principle of SSA test?

Specificity of SSA

What are the SSA results?

1+ SSA test result means...

turbid, but can see lines and read print through tube (15-30 mg/dL) 

2+ SSA test results mean...

cannot read through but can see lines (40-100 mg/dL) 

3+ SSA test results means..

cannot see through, fine granules may be present (150-350 mg/dL) 

4+ SSA test result means

flocculent precipitate or gelled tube (greater than 500 mg/dL

What is the SSA sensitvity?
 

5 – 10 mg of protein/dL

What are the SSA false reactions?

False reactions:

  Positive = Radiographic dyes; Penicillins

  Negative = highly buffered urine; alkaline urine

Protein Precipitation by Heat Principle?

Principle for Glucose-Copper Reduction Test

Glucose-Copper Reduction Test 

False postive test by...

Ketones-Nitroprusside test (Acetest) sensitivity

Ketone Nitroprusside table test False reactions

0.05 – 0.1 mg/dL

&conjugated bilirubin

Bilirubin Diazo tablet test false reactions

What is cytometry

definition of Fluorescent flow cytometry- 

*Reflectance Photometry: quantifies the intensity of the colored product generated by the reaction.

*Light emitting diode (LED) emits a specific wavelength of light onto the test strip

*Colored product absorbs the light, the darker the test pad the less reflected light.

*A detector captures the reflected light and converts to an electronic signal that results in a result.

Cell population identified by...