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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.
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Fig. 1
Assessing the degree of mixed chimerism in an arbitrary cell mixture using PCR amplification of a microsatellite in the class II Eb gene of the murine MHC. Donor (D, C57BL/6, H-2kb mice) and recipient (R, BALB/c, H-2kd) splenocytes were mixed in vitro in various proportions (D to R=0 to 100, 5 to 95, 10 to 90, 25 to 75, 50 to 50, 75 to 25, 90 to 10, 95 to 5, and 100 to 0) in dilution experiments. PCR amplification of the class II Eb gene and flow cytometry analysis were performed using the cell mixtures. The DNA fragments amplified from the donor and recipient were 107 and 139bp, respectively. The lanes show the results for artificial mixtures of donor and recipient splenocytes.
Fig. 2
Assessing the degree of mixed chimerism in arbitrary cell mixtures using flow cytometry. Donor (C57BL/6, H-2kb mice) and recipient (BALB/c, H-2kd) splenocytes were mixed in vitro in various proportions (D to R=99 to 1, 95 to 5, 90 to 10, 10 to 90, 5 to 95, and 1 to 99) for dilution experiments. Donor (H-2Kb) and recipient (H-2Kd) cells were distinguished during lymphoid gating by staining with fluorescein isothiocyanate-labeled anti-H-2Kb and phycoerythrin-labeled anti-H-2Kd antibodies (PharMingen, San Diego, CA), respectively. Stained cells were analyzed using CellQuest software on a FACSCalibur flow cytometer (both from Becton Dickinson, Mountain View, CA). The percentage of donor-derived cells was calculated by dividing the number of donor cells by the total net number of donor plus recipient cells that showed positive staining.
Fig. 3
Donor chimerism in peripheral blood of an allogeneic mixed chimera following MHC-mismatched non-myeloablative BMT. Engraftment/mixed chimerism at 3 weeks post-BMT was evaluated using PCR amplification of a microsatellite in the class II Eb gene. Allogeneic mixed chimeric mice (lanes 1, 2, and 4) showed both the 107-bp (donor, C57BL/6, H-2b) and 139-bp (host, BALB/c, H-2d) fragments, whereas non-transplanted recipient mice as a negative control (lane 3) showed only the 107-bp (host, BALB/c, H-2d) fragment. The relative ratios of donor-derived cells in the PB from those mice were 76, 42, 0, and 89% of all lymphocytes, respectively. The DNA-based assay developed in this study roughly correlated with the results of the flow cytometry assay.
Fig. 4
Assessing NOD/Scid mice using PCR amplification of a microsatellite in the class II Eb gene of the murine MHC. The presence of NOD/Scid-derived cells can be detected using PCR amplification of a microsatellite in the class II Eb gene of the murine MHC, although NOD/Scid mice do not express the H-2 haplotype antigen. The amplified DNA fragments of the C57BL/6 (H-2b), BALB/c (H-2d), and NOD/Scid mice were 107, 139, and 80bp, respectively.
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