An eight-colour flow cytometric method for the detection of reference values of lymphocyte subsets in selected healthy donors.

Determination of immunoregulatory cells in peripheral blood is important in the management of disease or in the therapeutic approaches that involve alterations in lymphocyte subpopulations. The aims of the present study were (1) to develop a standard multiparametric flow cytometric method for phenotypic detection and enumeration of lymphocyte subsets so as to reduce the variability in both sample preparation methodology and flow cytometric operations; (2) to furnish reference values of lymphocytes by using a selected healthy population; and (3) to examine the influence of age and sex on the distribution of lymphocytes expressing surface markers. Eighty healthy donors were analysed, and ten-parameter, eight-colour analytical procedure was performed. We furnished a panel to detect and to enumerate lymphocyte subpopulations by a multiparametric flow cytometric method to set the reference values to a selected healthy population. These values showed statistically but not clinically significant differences in T lymphocyte subsets and natural killer cells. Furthermore, significant age-related correlations in T lymphocyte and natural killer cells were observed. Lastly, males and females in relation to age showed a significant different trend in T and B lymphocyte subsets. We confirmed that this study provides a rapid and accurate method for the detection and quantification of lymphocyte subsets that could be utilized in the clinical settings. The definition of reference values in the healthy selected population could be helpful also to better define the disease status and to evaluate the treatment efficacy during clinical trials.

The plasma levels of soluble HLA-G molecules correlate directly with CD34+ cell concentration and HLA-G 14bp insertion/insertion polymorphism in cord blood donors.

BACKGROUND: Cord blood provides haematopoietic stem cells for allogeneic transplantation and, thanks to the naivety of its immune system, has several advantages over other sources of stem cells. In the transplantation setting, the presence of immunosuppressive human leucocyte antigen (HLA)-G molecules has been advocated to prevent both rejection and Graft-versus-Host disease. HLA-G is physiologically expressed throughout pregnancy and is contained in cord blood at birth. Moreover, it has recently been reported that not only cord blood mesenchymal cells, but also CD34+ cell progenies produce soluble HLA-G (sHLA-G). We tried to identify the largest producer of sHLA-G among 85 healthy cord blood donors at Pavia Cord Blood Bank, correlating the sHLA-G concentration with the HLA-G 14bp insertion/deletion (INS/DEL) genotype and CD34+ cell concentration. MATERIALS AND METHODS: We measured sHLA-G levels in 36 cord blood plasma stored at -20 °C for 2 months and 49 cord blood plasma stored at -196 °C for 4-6 years, by enzyme-linked immunosorbent assay. All cord blood donors were genotyped for the HLA-G 14bp INS/DEL polymorphism by polymerase chain reaction. For each cord blood unit, we measured the cell concentration by flow cytometry. RESULTS: We did not find differences in sHLA-G levels between cord blood plasma aliquots stored for 4-6 years at -196 °C and cord blood plasma aliquots stored for 2 months at -20 °C. We observed a higher sHLA-G concentration in cord blood plasma donors who carried the HLA-G 14bp INS/INS genotype and had higher CD34+ cell concentrations (P=0.006). DISCUSSION: This is the first report showing that the best cord blood stem cell donor is also the best sHLA-G producer, particularly if genetically characterized by the HLA-G 14bp INS/INS genotype. If the therapeutic role of sHLA-G molecules were to be finally established in the transplantation setting, our data suggest that cord blood plasma donors can provide a safe source of allogeneic sHLA-G immunosuppressive molecules ready for transfusion.