1. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT
Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
2. CHEMICAL TESTS
These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
Reagent Strip Test
Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
Causes of false-positive tests:
- Contamination of urine by menstrual blood in females
- Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
Causes of false-negative tests:
- Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
- Use of formalin as a preservative for urine
Evaluation of positive chemical test for blood is shown in Figure 828.2.