Flow Cytometric Analysis of Donor-Recipient Chimerism Using Anti-HLA Antibody after HLA-Mismatched Cord Blood Transplantation.

Background: Although umbilical cord blood has been used successfully as an alternative donor source to treat hematological disorders, cord blood transplantation (CBT) is frequently complicated by graft failure and relapse of original diseases. Since recipient-derived hematopoietic cells persist or increase under these reasons, analysis of donor-recipient mixed chimerism (MC) might be useful for their diagnosis.

Objective: Since most CBT involves human leukocyte antigen (HLA)-mismatched transplantation especially for adult patients, we have developed a flow cytometry-based method of MC analysis using anti-HLA antibodies (HLA-FACS method) to detect mismatched antigens.

Patients and methods: We analyzed MC in 14 patients after CBT using HLA-FACS method including 9 cases with weekly analysis using peripheral blood in first 4 weeks for monitoring of the engraftment and 9 cases with serial analysis using bone marrow samples for detection of the minimal residual disease (MRD).

Results 1 (monitoring of engraftment): In our first patient with acute myelogenous leukemia (AML), MC in peripheral blood could be detected at week 1 after CBT; the recipient-derived cells were mainly observed in the CD4⁺ T-cell subset. Recipient-derived cells gradually decreased and completely disappeared at week 4 after CBT. In our second patient with myelodysplastic syndrome, recipient-derived cells decreased to 1.5% of mononuclear cells at week 2 after CBT, then started to increase at week 3 and persisted with the frequency of around 10% by week 6. MC was mainly caused by recipient-derived monocytes in this patient. After tapering of cyclosporine A, a rapid decrease of recipient-derived cells was observed from week 6 to week 10. Another 7 patients showed complete donor chimerism at week 3 after transplant.

Result 2 (monitoring of MRD): In the first case, the percentage of recipient-derived cells among the CD34⁺CD45^dim fraction in bone marrow was 0.2% on day 159. On day 281, however, these cells significantly increased to 5.7%. To elucidate whether the recipient-derived CD34⁺CD45^dim cells were leukemia cells or not, we separately sorted donor- and recipient-derived CD34⁺CD45^dim cells and analyzed the leukemia-specific AML1/ETO chimeric genes by the fluorescent in situ hybridization method. Recipient-derived CD34⁺CD45^dim cells but not donor-derived ones were positive for AML1/ETO fusion signals. We have detected significant frequency of recipient cells in the CD34⁺CD45^dim fraction of bone marrow in the other patient followed by clinical diagnosis of leukemia relapse at 3 weeks later. No sign of recipient recurrence was observed in other 7 patients.

Conclusion: These results provided a proof of concept for an approach that may have utility in the early diagnosis of graft failure and relapse of leukemia by the HLA-FACS method for patients received HLA-mismatched CBT.