Hamostaseologie 2019; 39(S 01): S1-S92
DOI: 10.1055/s-0039-1680250
Poster
P11 Thrombocytopenia and Dysfunction
Georg Thieme Verlag KG Stuttgart · New York

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

C. Berens
1   Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
,
H. Rühl
1   Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
,
J. Müller
1   Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
,
J. Oldenburg
1   Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
,
B. Pötzsch
1   Institute for Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
› Author Affiliations
Further Information

Publication History

Publication Date:
13 February 2019 (online)

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    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.


     

    No conflict of interest has been declared by the author(s).