Lineage-Specific Chimerism Monitoring Using Flow Cytometry with Anti HLA-Antibodies in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction: Haploidentical hematopoietic stem-cell transplantation (HHSCT) is an alternative transplant strategy for patients who lack a suitable HLA-matched donor. Determining the degree of engraftment can be critical to establishing the success of a HHSCT. Short tandem repeats-PCR (STR-PCR) based chimerism analysis is widely used, but most has been performed by testing total leukocytes. Analysis of chimerism in selected lineage-specific populations could provide more important information. However, STR-PCR of isolated cells is labor intensive and time consuming. In HHSCT settings, HLA antigens were used as markers to establish the presence of chimerism. Flow cytometry can be used to evaluate chimeric status in HHSCT based on disparity of HLA antigens between donor and recipient. In this study, we aimed to observe the dynamics of changes in cell subpopulations and engraftment after HHSCT with flow cytometric lineage-specific chimerism analysis (FCM-LSCA).

Methods: Peripheral blood samples of total sixty-nine pediatric patients who underwent HHSCT in Asan Medical Center were collected between October 2011 and June 2014. Diagnoses were aplastic anemia (n=27), acute myeloid leukemia (n=21), acute lymphoblastic leukemia (n=10), non-Hodgkin’s lymphoma (n=7), non-malignant hematologic disorders (n=4). Four patients received CD34-selected graft, 26 patients received CD3-depleted graft, and 39 patients received TCRαß-depleted graft. Patients who experienced graft loss or disease relapse were six. FCM-LSCA performed using antibodies for HLA antigens and cluster of differentiation (CD) antigens. We selected a panel of specific HLA antibodies to target HLA serotypes based on the allele distribution of HLA in Korean population. FCM-LSCA of T cell and NK cell was done according to regularly scheduled protocol from the start of stem cell infusion (PID0).

Results: Fifty-two patients (75% of total patients) showed suitable anti HLA-antibodies for FCM-LSCA. The proportion of donor NK cells increased sharply and reached 100% of lymphocytes at PID 2-3 in the second or third HHSCT. In comparison, donor NK cells reached 100% of lymphocytes approximately 3 days after the first successful HHSCT. The proportion of donor NK cells reached 100% but decreased and reached 0% at PID 14-21 in failure of engraftment. CD56 dim/CD57 positive NK cells among CD56 positive cells reached 50% at PID 3 and remained over 50% until PID 22 and slowly decreased to 40% at PID 35. CD56 dim/CD57 negative cells had highest expression at PID 18 and decreased slowly. CD56 bright/CD16 negative cells reached peak proportion at PID 28, comprising 20% of CD56 positive cells. Among the gated lymphocytes, the median percentage of donor T cells at PID 10 of patients who experienced graft failure was significantly lower than that of patients who achieved sustained engraftment [5.5% versus 88.7%, P=0.000]. During scheduled monitoring, we detected mixed chimerism of T cell with increased recipient cells in two acute leukemia patients. Emergent bone marrow examination performed and diagnosed relapse of leukemia. Two of aplastic anemia patients showed complete donor chimerism on STR-PCR (more than 95% donor cells), but showed persistent mixed chimerism on T-cell LSCA (22.9% and 25.8% of recipient cells, respectively).

Conclusions: Serial monitoring of FCM-LSCA will give a better understanding of regenerating hematopoiesis after transplantation as well as engraftment or rejection kinetics. In practical consideration, preliminary evaluation of anti-HLA antibody reactivity in donor and recipients will be essential. Especially, our study strongly suggests that monitoring FCM-LSCA is useful to enable early diagnosis and effective treatment of impending graft rejection or recurrence of original disease in HHSCT.