Analysis of Chimerism Following Allogeneic Bone Marrow Transplantation by Fluorescent-in-Situ Hybridization

Abstract

Twelve patients (9 males and 3 females) with chronic myelogenous leukemia, underwent CD8+ T cell depleted allogeneic bone marrow transplantation with a sex-mismatched donor. To assess chimerism we performed fluorescent in-situ hybridization for the X and Y chromosome at different time points after BMT. Patient median age was 33 years (range, 27-48): median time to transplant was 28 months (range, 5-87). All patients received thiotepa 10 mg/kg; cyclophosphamide 120 mg/kg and 12.0 Gy of fractionated total body irradiation. CD8+ cells were depleted from the normal donor marrow with anti-CD8 murine monoclonal antibodies and immunomagnetic beads. Bone marrow aspirates were studied at >60, 60–140, 140–300, and <300 days post BMT. Hybridization was done on mononticlear cells and a median of 518 cells were counted per slide with a fluorescent microscope. The median percentage of donor cells was 99.04%. 98.21%, 98.15%, and 99.52% at >60, 60-140, 140-300, and <300 days after BMT.

Mixed chimerism was a rare occurrence after CD8 depleted allogeneic BMT and occured only at low levels. Inhibition of repopulation by host hematopoietic cells may be associated with the graft-versus-leukemia effect against CML.

Key Words:

• Chimerism
• post allogeneic bone marrow transplantation
• fluorescent-in-situ hybridization
• FISH