A refined method for lymphocyte isolation from the placenta

Authors

  • Silvia Miranda,

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
    2. IDEHU, University of Buenos Aires, Junín 956 4P, (1113)Buenos Aires, Argentina;
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  • Tobias Pöhlmann,

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
    2. Department of Dermatology, Friedrich-Schiller-University Jena, Germany
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  • Katharina Förster,

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
    2. Department of Dermatology, Friedrich-Schiller-University Jena, Germany
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  • Rita Neundorf,

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
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  • Andreas Kaufmann,

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
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  • Udo R. Markert

    1. Department of Gynecology and Obstetrics, Friedrich-Schiller-University Jena, Germany;
    2. Department of Dermatology, Friedrich-Schiller-University Jena, Germany
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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.

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