A flow cytometric method for the detection of alkaline phosphatase expression on the membrane of neutrophils

Abstract

Objective To establish a flow cytometric method for the detection of alkaline phosphatase (ALP) expression on the membrane of neutrophils (mNAP). Methods EDTA- K2 anticoagulant venous bloods were collected.Expression of mNAP in peripheral blood was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-ALP monoclonal antibody.BD QuantiBRITE PE was used to generate a calibration curve for PE fluorescence and ratios of PE to anti-ALP antibody and detect the bound ALP antibodies per cell (antibodies bound per cell,AB/c).Preanalytical handling including anticoagulants (EDTA- K2,citrate,and heparin),storage temperature,storage time,and plasma ALP were optimized and measured the precision.The expression levels of mNAP from 481 healthy controls were measured to establish a clinical reference range.The mNAP levels of 84 patients with severe infection and local infection,39 patients with virus infection were determined by this method. Results For preanalytical handling,application of PBS washing can effectively eliminate the interference of plasma ALP.The mNAP levels were not influenced by different anticoagulants and storage conditions (stored for 12 h either at room temperature or 4 ℃).This method had preferable reproducibility (CV in batch were 2.01%-3.33%,average 2.67%; CV between batch were 5.80%-6.00%,average 5.90%).The median (quartiles) of mNAP in health controls were 1 758 (1 378-2 310) AB / c for men and 1 897 (1 369-3 249) AB / c for women.There was no significant difference between genders (U=27 140，P=0.243 8).The clinical reference ranges (2.5 percentile to 97.5 percentile) of mALP was 920.5-3 493.0AB / c.The expression levels of mNAP of patients with bacterial infections (13 532,9 756-16 869 AB / c ) were significantly higher than those of patients with virus infection( 1 143,536-2 012 AB / c) and healthy controls (1879,1399-2497 AB / c)( H=221.5,P<0.01). Conclusion BD QuantiBRITE PE kit can be used to standardize flow cytometer settings and quantitatively detect molecules per cell.The flow cytometric method for detection of mNAP has important clinical application for differentiating bacterial and virus infection.（Chin J Lab Med,2014,37:623-627） Key words: Neutrophils; Cell membrane; Alkaline phosphatase; Flow cytometry; Infection