Evaluation of bone marrow transplantation efficiency by competitive PCR on Y sequences.

Laboratoire de Biologie Cellulaire et Mol~culaire, Centre Hospitalier Universitaire de Poitiers, 86021 Poitiers Cedex, France Allogeneic bone marrow transplantat ion (BMT) is now a c o m m o n t rea tment for hematological diseases. Before the transplantation, the hemopoiet ic cells of the patient are eradicated. In spite of this pretransplant treatment, some apparently normal recipient hemopoiet ic cells can remain, leading to a mixed chimerism p h e n o m e n o n . (1) It is very impor tant to moni to r this chimerism to follow the kinetics of recipient cell amoun t after BMT and possibly to prevent the relapse of a residual disease. Early at temps to evaluate the mixed chimerism were based on immunological and cytogenetic methods. Conventional molecular detections of host cells included Southern blotting and RFLP analyses; their sensitivity was 1 2 % . More recently, PCR techniques were performed to assess the chimerism. (2) The in vitro amplification was used either to detect microsatellites (3) or Y chromosomal material. (4~ The latter strategy is available in one case out of four, when there is a sex mismatch with a female donor and a male recipient. The long arm of the h u m a n Y chromosome contains a highly repeated nucleotide sequence arranged in a head-totail manner . (s~ The repeated area represents 800--5000 copies on the Y chromosome. These 3.56-kb EcoRI fragments belong to the h u m a n Y chromosome-specific repeated DNA family (DYZl locus) and are composed of several hundred variants of a basic pentanucleotide (TTCCA). A fragment of this sequence overlapping the EcoRI restriction site can be detected by PCR. This me thod was already used for fetal sex determination (6'7) and may also provide a way of studying the BMT chimerism. In this paper we propose a simple, rapid, and efficient me thod to evaluate this chimerism after sex-mismatched allogeneic BMT. A prel iminary PCR detect ion system is described. If Y amplified fragments are detected in recipient blood samples, we quant i fy the remaining Y sequences by PCR with an internal exogenous standard. This exper iment is performed simultaneously on blood samples (B) and buccal epithelial cells (E) to compare hemopoiet ic and nonhemopoiet ic cells from the same patient. The B/E ratio represents the percentage of mixed chimerism. Because the analysis is repeated every 3 months after BMT, high percentages may announce a possible relapse. MATERIALS AND METHODS