EDTA Inhibits the Binding of Clone 96.2C1, an Anti-CD41a Monoclonal Antibody, to the Platelets and Addition of Heparin and CaCl2 to the Antibody Neutralizes the EDTA-induced Inhibitory Effect

ABSTRACT

BACKGROUND: The binding of some monoclonal antibodies platelet glycoprotein (GP) IIb/IIIa, which is frequently used for flow cytometric immnophenotyping, is known to be inhibited by EDTA. To select the ideal antibodies to be included in the 'Acute Leukemia Panel' for immunophenotyping of acute leukemia, we compared the inhibitory effect of EDTA on the binding of 5 different clones of monoclonal antibodies to platelet GP IIb/IIIa. We also discovered a simple method to neutralize this inhibitory effect. METHODS: Flow cytometric measurement of the number of platelet GP IIb/IIIa binding sites with different anticoagulants was performed using a panel of 5 clones of monoclonal antibodies against CD41 (clone PM6/248), CD41a (clone 96.2C1 & clone HIP8), CD41b (clone HIP2) and CD61 (clone VI-PL2), and the results are expressed as the mean equivalent soluble fluorochrome (MESF) values. RESULTS: The MESF value of the EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody showed a significantly lower value than the MESF of platelets anticoagulated with heparin or citrate (P<0.001). The inhibitory effect of EDTA on the binding of anti-CD41a, clone 96.2C1 antibody to the platelets was neutralized by addition of heparin and CaCl2. The mean MESF value of EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody was significantly increased by the addition of heparin and CaCl2 (P=0.0001). CONCLUSION: The false-negative results of the binding of anti-CD41a, clone 96.2C1 antibody to the platelets seem to be due to the calcium chelating property of EDTA, and the addition of CaCl2 and heparin could be used as an easy compensatory measure for the inhibitory effect of EDTA on other antibodies as well.