Dipstick Assay for Urinalysis

Chemical Test Principles

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Bayer Multistix Product Insert

Dipstick
Dipstick or reagent strips Strip of plastic containing small pads of reagents Each pad contains a colorimetric reagent to detect a different analyte Color generated is proportional to concentration of the analyte present in the sample.

Dipstick Assay Analytes

1. 2.

3. 4. 5.

Glucose Bilirubin Ketones Specific Gravity Blood

6. pH 7. Protein 8. Urobilinogen 9. Nitrite 10. Leukocytes

Available in commercial urine dipsticks

Test Principle - Glucose

Reagents on the dipstick: Enzymes Glucose Oxidase, Peroxidase Chromogens Potassium Iodide, Tetramethylbenzene, o-tolidene, 4Aminoantipyrene

Note: 1) This test will work only for glucose. Other sugars such as sucrose, lactose, galactose and fructose cannot be detected. 2) Other reducing sugars can be detected using the Benedicts test. However, this involves a heating step. The sensitivity of the Glucose Oxidase method is more than that of a Copper reduction test such as Benedicts test.

Test Principle - Bilirubin

Reagents on strip: 2,4-dichloroanilene diazonium salt, 2,4-dichlorobenzene diazonium salt

Note: 1) This test may give false negatives upon prolonged exposure of the urine sample to light. Bilirubin is photosensitive, and gets converted into Biliverdin upon light exposure.

Test Principle - Ketones

Reagents on strip: Sodium Nitroprusside

Note: 1) This test will not detect Acetone or hydroxybutyric acid.

Test Principle - Blood

Reagents on the strip: Diisopropylbenze dihydroperoxide, 3,3,5,5-tetramethylbenzidine

Note: 1) This test can usually show free Hb, myoglobin as well as RBCs. Upon testing, RBCs will lyse, and form a speckled pattern on the pad. 2) False positives may arise from the presence of oxidizing contaminants in the sample.

Test Principle Specific Gravity

1.000 1.005 1.010 1.015 1.020
1.025

X+ + Polymethyl vinyl ether / maleic anhydride ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromothymol Blue indicator to form a colored complex

Reagents on strip Poly(methyl vinyl ether/ maleic anhydride) , Sodium Hydroxide, Bromothymol Blue

1.030

Note: 1) These readings will not be accurate in case the urine is alkaline. 2) Test does not measure non-ionized solutes such as glucose.

Test Principle - pH
H+ ions react with Methyl Red and Bromomethyl Blue indicators to form coloured complexes. Methyl Red high conc. of H+ , low pH Bromothymol Blue low conc. of H+ , high pH Reagents on strip : Bromothymol Blue, Methyl Red

Note: 1) These reagents can indicate pHs between 5.0 and 8.5. 2) Values of pH may be affected by certain medications, or bacterial growth in the sample. 3) In a commerical dipstick, buffers from the next reagent zone for protein may also run-over into the pH zone and provide an incorrect value.

Test Principle - Protein

Based on the Protein Errors of Indicators method. The point of colour change of some indicators is different in the presence of a protein than in the absence of the protein. This is due to proton accepting behavior of proteins at a constant pH Reagents on strip: Tetrabromphenol, acidic buffer.

Note: 1) Test is sensitive to albumin, but not to globulins, Bence-Jones proteins, and muco-proteins. 2) Highly alkaline urine will lead to false positives.

Test Principle - Urobilinogen

Reagents on strip: Paradiethylaminobenzaldehyde Ehrlich Aldehyde Reaction

Note: 1) Several interfering substances such as paminosalicylic acid, sulfonamides, p-aminobenzoic acid may react with the reagent. 2) Test cannot indicate the absence of Urobilinogen.

Test Principle - Nitrite

Reagents on strip : Para-arsanilic acid, 1,2,3,4-tetrahydrobenzo (h)-quinolin-3-ol

Note: 1) Bacterial overgrowth in sample with interfere with the test. 2) Can only detect bacteria than reduce nitrate to nitrite.

Test Principle Leukocytes

Reagents on strip: Derivatised pyrrole amino acid ester/ indoxyl ester derivative, diazonium salt

Note: 1) False positives may occur in case strong oxidizing contaminants are present 2) False negative may occur if the specific gravity is high due to glucose or protein.

List of Reagents from a Product Insert

UrinCheck Healthscreen 10, Express Diagnostics, USA

References
Graffs Textbook of Routine Urinalysis & Body Fluid, Chapter 4 Urinalysis - Francisco G. La Rosa, University of Colorado UrinCheck HealthScreen 10 product insert. Practical Guide to Urine Analysis, Clinitek Product Insert