A Novel Flow Cytometric Method for the Simultaneous Detection of Antibodies Against Platelet, Lymphocyte, And...

Platelet or neutrophil antibodies (Ab) detected by an indirect method need to be further investigated to confirm that they are HLA (lymphocyte)-specific. This can only be determined by the assessment of lymphocytes from the same donor. We have developed an improved flow cytometric method for the simultaneous detection of Abs against three distinct cell types (platelets, lymphocytes, and neutrophils) in a single suspension.

We prepared white blood cell (WBC) suspensions using an overlay procedure. To enhance the retrieval of the three cell types during flow cytometry, the acquisition time was divided into two periods, platelet phase and WBC phase, and a different live acquisition gate was applied to each phase. The sample/control ratio of mean fluorescence intensity (MFI) was measured for each cell type.

The number of cells in the starting cell suspension and the retrieved cell numbers of the three cell types were sufficient to detect their cognate antibodies. There was no significant difference between the MFI ratios observed in the current protocol and those from the conventional method (P > 0.05), and positive correlations were found (P < 0.05) for all three cell types. Each positive control serum with a specific set of Abs showed the typical comprehensive reaction pattern to the three cell types. Based on such patterns, 47% (14/30) of the patient sera that reacted with platelets were demonstrated to be due to anti-HLA Abs without platelet Abs.

Our method could enhance both interpretation accuracy of the diagnostic test results and laboratory efficiency. As an increased reaction with platelets is commonly due to anti-HLA Abs, the reactivity of the lymphocytes from the same donor should also be tested, which can be routinely done by using our protocol. © 2016 International Clinical Cytometry Society.