Flow Cytometric Quantification of Human Platelet Membrane Glycoproteins in Citrated Whole Blood

Objectives: Flow cytometric quantification of human platelet membrane glycoprotein complexes (GP) Ib, GP IIb/IIIa and the membrane protein P-selectin (CD62) is an established method of diagnosing inherited platelet receptor defects. In order to overcome pre-analytical limitations of utilizing platelet rich plasma (PRP) from patients with thrombocytopenia or abnormally large platelets a method utilizing citrated whole blood (CWB) was developed and compared to the conventional PRP method.

Methods: CWB or PRP from healthy blood donors (n = 13) was added to phosphate-buffered saline and stained with fluorescein isothiocyanate (FITC)-conjugated antibodies against GP Ib, GP IIb/IIIa, GP IIb, or CD62 and a phycoerythrin (PE)-conjugated antibody against CD235a, an erythrocyte membrane protein. Platelet activation was induced by phorbol 12-myristate 13-acetat (PMA). The expression of GP Ib, GP IIb/IIIa, GP IIb and CD62 on CD235a-negative cells was quantified by flow cytometry after 15 minutes of incubation.

Results: Flow cytometric analysis was feasible in normal CWB samples of 45 µl volume. CWB and PRP samples showed no differences of mean (± standard deviation) fluorescence intensity (GP Ib 81.2 ± 11.1 vs. 96.9 ± 5.0, GP IIb/IIIa 277.0 ± 26.4 vs. 269.3 ± 20.9, GP IIb/IIIa with PMA 387.6 ± 31.4 vs. 372.3 ± 53.3, GP IIb 32.5 ± 9.7 vs. 35.9 ± 8.6, GP IIb with PMA 43.4 ± 11.9 vs. 45.1 ± 15.7, CD62 1.7 ± 0.4 vs. 2.2 ± 0.7, CD62 with PMA 48.5 ± 7.4 vs. 37.3 ± 4.2, respectively). The ratio of the fluorescence signal with PMA to that without PMA showed an increase in CWB in comparable proportions to PRP, with a ratio of 30.6 ± 8.2 vs. 18.6 ± 5.0 measured for CD62. GP IIb/IIIa (1.4 ± 0.1 vs. 1.4 ± 0.1), and GP IIb (1.4 ± 0.2 vs. 1.2 ± 0.1) remained unaffected by PMA in both CWB and PRP.

Conclusions: Flow cytometric quantification of platelet membrane proteins in CWB is comparable to the conventional method using PRP. The novel method is robust and reliable, spares time-consuming preparation of PRP, and requires smaller amounts of blood. Flow cytometric differentiation between giant platelets and erythrocytes is feasible and allows identification of rare inherited platelet membrane disorders.