A Novel Method for Flow Cytometric Quantification of Human Platelet Membrane Glycoproteins in Citrated Whole Blood

Introduction: Diagnosis of platelet receptor defects can be confirmed by flow cytometry using a panel of monoclonal antibodies. The conventional method utilizing platelet rich plasma (PRP) has many limitations such as time-consuming preparation, requirement of a large sample volume or limitations in samples from patients with thrombocytopenia or abnormally large platelets. In order to overcome these difficulties a novel method was developed using a second antibody directed against an erythrocyte protein which allows differentiation between platelets and erythrocytes in citrated whole blood (CWB).

Methods: CWB or PRP from healthy blood donors and outclinic patients without platelet disorder (n= 18) as well as from two patients with Glanzmann thrombasthenia (GT) was stained with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD42b (GP Ib), CD41a (GP IIb/IIIa), or CD62. CWB was stained in addition with a phycoerythrin (PE)-conjugated antibody against CD235a, a sialoglycoprotein expressed by erythroid precursors and erythrocytes. Platelet activation was induced by addition of 2 µM phorbol 12-myristate 13-acetat (PMA). The expression of CD42b, CD41a and CD62 on CD235a-negative cells was quantified by flow cytometry with and without PMA stimulation.

Results: CWB and PRP samples showed no differences of mean (± standard deviation) fluorescence intensity (MFI)(GP Ib 74.78 ± 21.39 vs. 85.67 ± 26.03, GP IIb/IIIa 250.33 ± 59.08 vs. 234.50 ± 56.96, GP IIb/IIIa with PMA 349.50 ± 76.87 vs. 328.67 ± 72.92, GP IIb 26.10 ± 6.17 vs. 29.15 ± 8.29, GP IIb with PMA 35.58 ± 8.86 vs. 37.50 ± 8.98, CD62 1.91 ± 0.49 vs. 2.49 ± 1.14, CD62 with PMA 43.88 ± 10.89 vs. 37.78 ± 7.20, respectively). The increase of the fluorescence signal with PMA vs. without PMA was similar in CWB and PRP with a percentage increase of 141.17 ± 10.97 vs. 142.74 ± 15.08 % for GP IIb/IIIa, 136.76 ± 16.65 vs. 131.83 ± 16.64 % for GP IIb and 2432.14 ± 785.49 vs. 1701.37 ± 474.72 % for CD62. Patients with GT showed MFI values for GP IIb/IIIa and GPIIb expression less than 5 % in comparison to that of a healthy control.

Conclusion: Flow cytometric analysis of platelet membrane proteins utilizing CWB is comparable to the conventional PRP method. Using an antibody against CD235a allows distinct differentiation between platelets and erythrocytes in CWB. This new method requires smaller amounts of blood, spares time-consuming preparation of PRP and is feasible to identify rare inherited platelet membrane disorders.