Flow cytometry with anti HLA-antibodies: a simple but highly sensitive method for monitoring chimerism and minimal residual disease after HLA-mismatched stem cell transplantation

Abstract

Transplantation of HLA-mismatched stem cells may allow determination of chimerism status of single cells by differential expression of HLA molecules. Monoclonal antibodies against HLA antigens can be used to determine the HLA type of sub-populations by standard flow cytometry. Blood samples from 23 patients transplanted from HLA-mismatched family donors were monitored using HLA-specific antibodies. Suitable antibodies could be found for all donor recipient pairs by using differences in HLA Bw4 and Bw6 groups or other serological antigens. Pretransplant controls of donor and recipient were used to correct for variable fluorescence intensities of the antibodies and sub-populations. Owing to the high sensitivity, cell populations with a minimum frequency of 0.1% were detectable. Flow-cytometric analysis was confirmed by chimerism analysis of immunomagnetically isolated T cells by standard PCR technique. In addition to chimerism evaluation, HLA antibodies improved the detection of leukemic cells after transplantation with aberrant phenotype. In conclusion, flow cytometry using antibodies against HLA antigens is an interesting tool for determination of chimerism and minimal residual disease after HLA-mismatched transplantation. Information about the chimerism status is given on a single-cell level and allows fast and convenient analysis of sub-populations.

Haploidentical SCT in children: an update and future perspectives

“…Frequent analyses of T cell chimerism can help to predict graft rejection, either by PCR analysis of VNTR regions or by flow cytometry of lymphocyte subsets with MoAb against HLA Ags of recipient and donor. 37,38 In case of increasing autologous signals, (1) cessation of immunosuppression and (2) if necessary, low-dose donor lymphocyte infusions are recommended to restore a complete donor chimerism. 38 Moreover, ongoing studies evaluate the role of co-transplanted MSC in the prevention of graft failure.…”

Preservation of Immune Repertoire by Selective Depletion of Haploidentical Grafts

“…The HLA disparity of donor and recipients allows a simple and detailed analysis of the engraftment status of various lymphocyte and myeloid cell populations [27]. Donor-derived cells can be easily identified using antibodies specific to the disparate HLA.…”

New strategies for haploidentical transplantation

“…In addition, the HLA disparity can also be helpful for the very early detection of impending recurrence of leukemia by flow cytometry (12 A major advantage of haploidentical donors over umbilical cord blood, a stem cell source that is also immediately available, is the availability of the donor for post-transplant cellular therapies either for prevention of relapse or the treatment of viral infections by adoptive T-cell transfer. In contrast to umbilical cord blood, where the number of stem cells is limited but important for successful transplantation (21), large numbers of haploidentical stem cells can be collected from the donor by repeated collections if necessary and these large stem cell numbers allow engraftment across the HLA-barrier (6) even after reduced toxicity conditioning regimens (22).…”

“…Chimerism analysis can easily and rapidly be done using anti-HLA antibodies conjugated to fluorochromes, as host and donor cells can be differentiated due to the existent HLA-mismatch in haploidentical transplantation (12). The detection of residual host hematopoiesis can be combined with the flow cytometric detection of minimal residual disease (84) and allows very early detection of an impending relapse and an immediate intervention for its prevention.…”