Summary auto-generated
The nitroblue-tetrazolium (NBT) test, introduced to detect bacterial infections by measuring phagocyte-mediated dye reduction, has produced inconsistent results across different laboratories. This study standardized key technical variables affecting both standard and endotoxin-stimulated NBT tests using blood samples from hospitalized patients. Researchers found that heparin concentrations should not exceed 20 units/ml, as higher concentrations increased NBT indices. Delays in test performance, particularly at room temperature, yielded false-positive results; samples should be tested within 90 minutes of collection or stored at 4°C for no more than 6 hours. The reagent composition significantly influenced results: phosphate-buffered saline at pH 7.2 with 0.1% NBT dye (without glucose) produced optimal outcomes, while acidic pH values caused cell damage and false positives. For endotoxin-stimulated tests, incubating blood with 20 μg/ml lipopolysaccharide for 15 minutes at 37°C before the standard NBT procedure produced reliable, consistent increases in reduction indices. The stimulated test enabled discrimination between normal leucocytes and those with impaired function.
Key findings
- Heparin anticoagulant concentration must be limited to 20 units/ml of blood to prevent elevated NBT indices and false results
- Blood samples should be tested within 90 minutes of collection or stored at 4°C for no more than 6 hours to avoid false-positive results from spontaneous leucocyte degradation
- Optimal NBT reagent composition is 0.1% NBT in phosphate-buffered saline at pH 7.2 without added glucose; deviations in pH and buffer type cause leucocyte clumping and cell damage
- Endotoxin-stimulated NBT testing using 20 μg/ml bacterial lipopolysaccharide for 15 minutes at 37°C produces reliable increases in neutrophil reduction indices and helps detect phagocytic dysfunction
- Strict standardization of incubation conditions, timing, temperature, and reagent composition is essential for reproducible and comparable NBT test results across laboratories
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Abstract
Optimal conditions for the NBT-reduction test have been sought. Increasing heparin concentrations up to 100 units per ml and a delay in performance of the test, especially when blood specimens are kept at room temperature, resulted in higher values for the NBT index, which then sometimes exceeded the upper limit of normal in healthy people and in uninfected patients. The effect of pH, composition of the buffer, and dye concentration was also investigated. Phosphate-buffered saline pH 7-2 containing 0-1% NBT dye, without glucose, gave the most reliable results. In endotoxin-stimulated NBT tests, the following procedure is recommended: incubation of 0-1 ml whole blood with lyophilised endotoxin 20 mug per ml. for 15 min. in a 37 degree C water bath, followed by the standard test with a 0-2% NBT solution. By this technique, the leucocyte reaction to various types of lipopolysaccharides was of the same order of magnitude. Drug therapy having an effect on blood components lowered this reaction, whatever the source of endotoxin used as stimulant. The importance of NBT-reduction tests is discussed. Standard conditions of test performance are strictly requisite if comparable results are to be obtained and if data not corresponding with the apparent clinical and other laboratory findings are to be evaluated correctly. The stimulated NBT test, performed in parallel with the standard test, is useful in the interpretation of abnormal results and in the detection of factors with a temporary or permanent effect on the phagocytic activity of pmn leucocytes.