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Immunology & Immunological Disorders Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
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Cytotoxicity of EDTA Used in Biological Samples: Effect on Some Breast-milk Studies


Contact Person: Michael O. Ogundele (mogundel@yahoo.com)


Discussion and Conclusion

The present study has described some of the toxic effects of EDTA on the cellular elements of human breast-milk and its effect on some standard biological assays. The ability of EDTA to disrupt milk fat globule membrane, thereby releasing some of the embedded proteins, and the enclosed lipid, might explain most of the observed findings. EDTA caused a significant fall in the pH of breast milk samples partly reversed by eliminating the milk fat through high-speed centrifugation. This could be ascribed to the detergent effect of EDTA, in disrupting the milk fat globule membranes (MFGM) which encapsulate the milk lipids. The released lipids being relatively more acidic than the aqueous phase of the milk. Similar differences were observed in the level of measurable total protein content of various HBM samples, with and without EDTA.

An evidence of direct cytotoxicity of EDTA on human breast-milk cells was found by comparing the total cell count of freshly collected milk samples containing EDTA as an inhibitor of complement activation and the control samples containing PBS. This cell loss by EDTA toxicity is comparable to a similar loss during breast-milk storage at 4C for up to 12 hours or longer.

A further related observation about the influence of EDTA on biological samples include a slightly brownish discoloration of many plasma samples collected in our laboratories for various C fragment ELISA assays, from whole blood containing high concentration of EDTA (up to a final concentration of 20mM). This discoloration probably provides an evidence of toxic-induced haemolysis that has taken place in the blood samples before the separation of the plasma. The observation of the role of divalent cations in supporting the cytotoxic effects of EDTA, suggests a means by which this effect in biological samples could be prevented or kept to the barest minimum, if applicable. It also suggests that extreme care should be exercised whenever EDTA or any of its derivatives are employed in biological samples, considering the ubiquitous nature of these cations in various body secretions. The role of EDTA in the CH50 whole complement assay, when used either in the reaction or stopping buffer, demonstrates how easily the results of a laboratory analysis can be falsified, in any situation where living cells are involved. The need for exercising great caution in the employment of this agent in clinical procedures has also been stressed, because of its toxicity on mammalian cells (14). In conclusion, it is conceivable that EDTA levels of at least 10mM might cause toxic haemolysis when used as a blood anticoagulant, or for other purposes in biological samples, in the presence of divalent cations. Such conditions may, therefore, not be suitable for assays which depend on measurements of haemoglobin, methaemoglobin, or cellular components.

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Ogundele, M.O.; (1998). Cytotoxicity of EDTA Used in Biological Samples: Effect on Some Breast-milk Studies. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/immunology/ogundele0175/index.html
© 1998 Author(s) Hold Copyright