Evaluation of cross-reactive antiplatelet antibodies in whole blood by means of flow cytometry - limitations and pitfalls
Magdalena Boncler, Cezary Watała, Jacek Golański
Med Sci Monit 1998; 4(3): BR413-418
Platelet surface membrane alloantigens are believed to be the cause of alloimmunization phenomena following blood transfusions or due to antigenic immunocompetence between mother and fetus encountered in pregnant women. Under such conditions of incompatibility, alloimmunization can result in the resistance of donor platelets following blood transfusions. The advantage of flow cytometry in determining the occurrence of platelet alloantibodies (in comparison to other methods, like MAIPA) is that it is easy to perform with small aliquots of whole blood, the obtained results can be quantitatively evaluated and stored for later comparisons, and the technique can be also used in cases when platelet counts are low, such as in thrombocytopenias. The methods principle is that more alloantigenic or donor-incompatible platelets bind with more immunoglobulins in the recipient's plasma. Blood samples were incubated with FITC-labelled F(ab')2 antibodies, directed against IgG, adsorbed on the platelet's surface. Higher incompatibility resulted in the enhanced binding of F(ab')2-FITC antibodies. The attributed FITC-derived fluorescence, reflecting the extent to which platelets were coated with specific immunoglobulins, intensified along with the increasing immunoincompetence of the recipient's platelets and donor's plasma. Control values of the fluorescence ratio (FR = RFIF(ab')2-FITC: RFIIgGFITC) fluctuated between 1.08-1.77 (min-max), which corresponded to the normal range (mean±1SD) of 1.015-1.835. In other cases, the FR values for AB0-incompatible 'pairs' fluctuated between 1.75-3.26 which corresponded to the normal range (mean±1SD) of 1.606-3.464. The increase of FR above 1.75-1.85 may suggest a higher risk for serologic incompatibility and considerably downgrade successful blood transfusions.
Keywords: platelet surface antigens, platelet-reactive antibodies, platelet serology, Flow Cytometry, Alloimmunization, alloantigenicity, Immunocompetence