Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers

Abstract

We have developed a quantitative, nonisotopic method using variable number tandem repeat (VNTR) and short tandem repeat (STR) markers for monitoring donor cell engraftment in marrow transplant recipients. Posttransplant DNA from the recipient is amplified with fluorescent polymerase chain reaction (PCR) primers for polymorphic markers that distinguish donor alleles from recipient alleles. The fluorescent PCR products are then separated on agarose or acrylamide gels on the Applied Biosystems 373A Sequencer (Foster City, CA). Using GeneScan 672 software (Applied Biosystems) to analyze the separated alleles, we can correlate allele peak areas to the percentage of donor or recipient DNA. We quantitate engraftment in a mixed chimeric sample by mixing pretransplant recipient and donor DNAs in a range of percentages and amplifying the mixtures to produce a standard curve. By amplifying and analyzing the posttransplant sample DNA(s), we can determine the extent of engraftment by interpolating the percent peak area of the informative allele(s) from this standard curve. This approach provides a precision of measurement ranging, depending on the marker, from 3.5% to 8.0% (percent coefficient of variation) and an accuracy of engraftment determination ranging from 97% to 99%, with a sensitivity of detection of 1% donor or recipient DNA. We retrospectively analyzed a panel of 32 patients and found seven to be informative for some degree of mixed chimerism, indicative of either residual normal host cells or leukemic relapse. An analysis of different cell lineages obtained posttransplant showed different degrees of engraftment in myeloid and T-cell populations. In summary, this method can provide an accurate, quantitative assessment of mixed chimerism in patients posttransplant. Such information may be useful in the future in guiding early implementation of additional treatment designed to circumvent graft failure or suppress relapse.