Abstract
OBJECTIVES: Analysis of placental lymphocytesfrom different compartments is crucial for many questions in reproductive immunology. A separation of lymphocytes after tissue disaggregations leads to a mixture of maternal and fetal lymphocytes, which are difficult to distinguish by simple methods. HLA‐A2 is expressed by 29% (Romanians) – 45% (Spanish) of a Caucasian population and might be a marker for this approach.METHODS/RESULTS: Extravillous blood is obtained by rinsing a placenta lobus with a special pump and tube system. Such blood is usually contaminated with fetal cells. Lymphocytes are separated by a common density gradient. Lymphocytes from rinsed tissue are isolated by mechanical and enzymatical disaggregation followed by a density gradient. By using this method, fetal and maternal lymphocytes are generally widely mixed. Anti‐HLA‐A2 monoclonal antibodies are produced by a murine hybridoma cell line. In positive individuals 29–53% of lymphocytes were HLA‐A2 positive. In approximately 25% of pregnancies a discordance of HLA‐A2 expression between the mother and her fetus was found. When lymphocytes are isolated from placentae of such combinations, at least HLA‐A2 positive cells can be easily detected by flow cytometry. All positive cells derive from the same individual, whereas the origin of negative cells cannot be determined. By application of a secondary microbeads labelled antimurin antibody, anti‐HLA‐A2 can be also used for magnet activated cell separation and used for enrichment of cells of one single origin.CONCLUSION: In c. 25% of pregnancies the use of anti‐HLA‐A2 antibodies allows a distinction of maternal and fetal cells and their respective enrichment.
Published Version
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