Practical Evaluation of Engraftment and Mixed Chimerism Using PCR Amplification of a Microsatellite in the Class II Eb Gene in Murine MHC-mismatched, Nonmyeloablative Bone Marrow Transplantation

Abstract

Background: Although engraftment following murine allogeneic bone marrow transplantation (BMT) is most commonly confirmed by H2 typing using flow cytometry, recipient mice can be seriously injured during peripheral blood (PB) sampling. Therefore, we developed an alternative DNA-based assay that does not require the large volume of PB necessary for flow cytometry. Methods: A minute volume of PB from the tail vein was used to evaluate the engraftment by PCR amplification of a microsatellite in the class II Eb gene. Dilution experiments were performed to evaluate the sensitivity of this assay for detecting donor cells in mixed cell populations compared with flow cytometry analysis. Results: Early engraftment and mixed chimerism were confirmed, based on the length variation of the microsatellite in the class II Eb gene. The degree of donor chimerism in the donor-recipient cell mixture could be estimated semiquantitatively in a dilution experiment. The sensitivity of this assay by the naked eye approached 10% of the degree of donor chimerism. Conclusion: PCR amplification of a microsatellite in the class II Eb gene can be a useful alternative to flow cytometry for evaluating early engraftment and mixed chimerism following murine nonmyeloablative BMT.