COMPARISON OF THREE DIFFERENT TECHNIQUES FOR ISOLATION OF NEUTROPHILS FROM BLOOD AND THEIR UTILITY IN PERFORMING NITROBLUE TETRAZOLIUM TEST

Background & Objectives: Assays for neutrophils constitute an important component of screening tests in clinical immunology. There are no standard protocols for performing many of these tests and procedures vary from one laboratory to another. In addition, normal ranges for these assays in healthy Indian population have not been defined. Hence, an attempt is made to evaluate and present a simple technique for WBC isolation and NBT test. Methods: The study involved participation of 30 healthy adult volunteers. Ten of blood sample collected from each subject was subjected to three different procedures for isolation of WBCs - Ficoll-Hypaque gradient, dextran sedimentation and gelatin sedimentation methods. Cells isolated from these procedures were then used to perform NBT test. Smears were prepared, stained with Giemsa and results were expressed as % of stimulated and unstimulated cells. Results: The mean cell yield from both dextran and gelatin methods was comparable (2921.67cells vs 2806.67cells/cu mm). The cell yield from Ficoll-Hypaque method was much lower (1408.33 cells/cu mm). In NBT test, the mean readings of stimulated (61%) and unstimulated cells (18%) were almost similar in all three procedures of cell isolation. Conclusions: Comparison of procedures show that gelatin and dextran sedimentation methods yield high amount of relatively purified WBCs. The efficacy of cells isolated from all three procedures in NBT test was almost similar. The range of stimulated and unstimualted cells in the subjects were within expected levels. Gelatin sedimentation is economical, easy to perform and can be adopted to any clinical laboratory for WBC isolation