A new approach of rapidly isolating lymphocytes from whole blood with immune magnetic beads for organ transplant flow cytometry cross-matching

Abstract

Objective To compare immunomagnetic bead and density gradient centrifugation (Ficoll) for the separation of peripheral blood mononuclear cells (PBMC) in flow crossmatching. Method Hemocytometer was used to count and compare the numbers of WBC, PLT, RBC isolated. For quality and quantity analysis of isolated T, B and NK cells in PBMC, flow cytometry was used to detect lymphocyte surface markers by specific fluorescent antibodies. The donor PBMC was used to detect antibodies in the recipient serum. Anti-human CD3-PE and anti-human CD19-APC monoclonal antibody were used for identification of T and B cells. Statistical analysis was performed using variance analysis andt test. Result The number of PBMC Macsseparation is 0.42 times the Ficoll separation of PBMC, but the proportion of lymphocytes (99.2±0.08) ﹪higher than that of PBMC isolated lymphocyte ratio (82.5±5.27) ﹪ (t= 9.91,P< 0.01) ,and two methods of separating PBMC RBC respectively (0.001 ± 0.001) × 106/μl and (0.02 ± 0.009) ×106/μl (t= 6.64,P< 0.001) ; the number of platelets were (1.00 ± 0.05) × 103/μl and (196.00 ± 4.21) × 103/μl had statistical significance difference (t= 146.46,P< 0.01) . Flow cytometry showed that CD3+T cells in PBMCs isolated from immunomagnetic beads were (8.41 ± 0.87) ﹪, CD3-CD56+NK cell (9.35 ± 0.67) ﹪and (2.47 ± 0.07) ﹪of CD3+CD56+ NKT cells. Ficoll isolated PBMCs contain (37.36 ± 3.27) ﹪CD3+T cells, (5.79 ± 0.94) ﹪CD19+B cells and (6.60 ± 0.91) ﹪CD56+NK cells, and the differences were statistically significant (t= 24.64, 6.470, 7.70, 51.31,P< 0.01) . In the cross matching test of T and B lymphocytes, quantitative read T and B lymphocytes and the serum antibody binding were tested. As density gradient centrifugation in lymphocyte mixed with platelets, so the flow cytometry results will be mixed with false negative, but the immune magnetic beads separation does not appear false negative results. It is proved that immunomagnetic separation PBMC can be used in clinical cross matching test. Conclusion PBMC isolated with immunomagnetic bead could yielded 99﹪pure lymophictes from whole blood with less contamination of RBC and platelets and may be used for T & B cell crossmatching in organ transplantation Key words: Peripheral blood; Monocytes; Centrifugation, density gradient; Immunomagnetic bead separation; Histocompatibility testing