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

• Chae H
• Park HH

• KMID: 2252172
• DOI: http://doi.org/10.5045/kjh.2009.44.1.42

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.

Keyword

Anti-CD41a; Clone 96.2C1; Platelet glycoprotein IIb/IIIa; EDTA; CaCl2

MeSH Terms

Antibodies
Antibodies, Monoclonal
Anticoagulants
Binding Sites
Blood Platelets
Calcium
Citric Acid
Clone Cells
Edetic Acid
Glycoproteins
Heparin
Immunophenotyping
Leukemia
Antibodies
Antibodies, Monoclonal
Anticoagulants
Calcium
Citric Acid
Edetic Acid
Glycoproteins
Heparin

Figure

• 

  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.

Reference

References

1. Neumeier D, Pauls R, Wifling I, Fateh-Moghadam A, Knedel M. Immunophenotyping of lymphocyte subpopulations using monoclonal antibodies in an analytical flow cytometer: evaluation of the method and reference values. J Clin Chem Clin Biochem. 1985; 23:765–75.

2. Behnken LJ, Heicke B, Löhr G, Maier-Lenz H, Musil J. Detection of lymphocyte subpopulations in EDTA whole blood samples using flow cytometry. Stability and normal values. Arzneimittelforschung. 1985; 35:1311–6.

3. Carrell NA, Fitzgerald LA, Steiner B, Erickson HP, Phillips DR. Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy. J Biol Chem. 1985; 260:1743–9.

4. Fitzgerald LA, Phillips DR. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem. 1985; 260:11366–74.

5. Nishiyama M, Hayashi S, Futsukaichi Y, Suehisa E, Kurata Y. Usefulness of immunoplatelet measurement using the flow cytometric method for accurate platelet counting in hematological patients with severe thrombocytopenia. Rinsho Byori. 2004; 52:103–8.

6. Serotec. Mouse antihuman CD41 antibody datasheet. WWWdocument. URL.http://www.ab-direct.com/catalog/datasheet-MCA467A647T.html.

7. Bauer JD. Methods in coagulation disorders. Bauer JD, Ackermann PG, Toro G, editors. Clinical Laboratory Methods. 9th ed.Mosby, USA: Mosby;1982. p. 256.

8. Nomura S, Nagata H, Oda K, Kokawa T, Yasunaga K. Effects of EDTA on the membrane glycoproteins IIb-IIIa complex–analysis using flow cytometry. Thromb Res. 1987; 47:47–58.

9. Fiorin F, Steffan A, Pradella P, Bizzaro N, Petenza R, De Angelis V. IgG platelet antibodies in EDTA-dependent pseudothrobocytopenia bind to platelet membrane glycoprotein IIb. Am J Clin Pathol. 1998; 110:178–83.

10. Holt C. Inorganic constituents of milk. III. The colloidal calcium phosphate of cow's milk. J Dairy Res. 1982; 49:29–38.