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

Hyojin Chae; Hun Hee Park

doi:10.5045/kjh.2009.44.1.42

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Chae and Park: 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

Original Article

Korean J Hematol 2009;44(1):42-46.

Published online: 20 March 2009

DOI: https://doi.org/10.5045/kjh.2009.44.1.42

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

Hyojin Chae, M.D., Hun Hee Park

Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Korea

Correspondence to：Hun Hee Park, M.D. Department of Laboratory Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea 505, Banpo-dong, Seocho-gu, Seoul, Korea Tel: ＋82-2-2258-1655, Fax: ＋82-2-2258-1719 E-mail: phh5240@hanmail.net

Received 2 September 2008 Revised 2 February 2009 Accepted 10 February 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Anti-CD41a, Clone 96.2C1, Platelet glycoprotein IIb/IIIa, EDTA, CaCl₂

References

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Fig. 1.

Scattergram (A) and histograms of normal platelets after staining with FITC labeled anti-CD41a, clone 96.2C1 mono-clonal antibody. EDTA-anticoagulated platelets (B) show low fluorescence consistent with inhibition of antibody binding, but citrated same platelets (C) and EDTA-anticoagulated platelets after staining with CD41a, clone 96.2C1 monoclonal antibody plus CaCl₂ and heparin show high fluorescence (D). It means the inhibitory effect of EDTA is completely neutralized by addition of heparin and CaCl₂.

Table 1.

MESF values of 5 clones of monoclonal antibodies against platelet glycoprotein IIb/IIIa with different anticoagulants

	Anticoagulant	EDTA	Heparin	Citrate
Antibody clone	Anticoagulant	Mean±SD	Mean±SD	Mean±SD
CD41a	96.2C1∗	2,092±2,182	77,529±21,110	91,531±21,830
CD41a	HIP8	45,993±3,397	48,655±4,517	49,487±4,216
CD41b	HIP2	2,367±129	2,210±205	2,364±469
CD41	PM6/248	40,908±10,948	35,155±6,118	38,595±7,941
CD61	VI-PL2	7,248±311	6,490±337	6,906±264
Negative control		307±5	304±5	307±7

^∗ Clone 96.2C1 showed significant difference among anticoagulants (one way ANOVA test, P＜0.001). Abbreviations: MESF, mean equivalent soluble fluorochrome.

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