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Quantitative determination of bone marrow transplant engraftment using
fluorescent polymerase chain reaction primers for human identity markers
SJ Scharf, AG Smith, JA Hansen, C McFarland and HA Erlich
Department of Molecular Genetics, Roche Molecular Systems, Inc, Alameda, CA
94501, USA.
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.
Volume 85,
Issue 7,
pp. 1954-1963,
04/01/1995
Copyright © 1995 by The American Society of Hematology
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