[First report on assessment of the status of engraftment after allogeneic hematopoietic stem cell transplantation by using denaturing high-performance liquid chromatography].

Monitoring engraftment of donor cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is supposed to be important for the early diagnosis of graft failure or relapse of malignancy. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeats (STR) as markers are attractive because they are sensitive and can be performed rapidly. The intent of this study was to test a novel approach for assessment of donor engraftment using denaturing high-performance liquid chromatography (DHPLC) combined with STR-PCR. The feasibility of this assay and the accuracy of semi-quantitative results were tested in-vitro by using serial DNA mixtures from unrelated individuals. The results showed that dilution experiments of the mock chimerism sample revealed a clear correlation between the percentage of donor or recipient DNA and the proportion of allele peak areas, with the limit of detection for a minor DNA percentage being 5%. Discrimination between donor and recipient was possible in all patients analyzed (n = 51) except for 5 patients whose pre-transplant samples were not available and identical twins in one case. STR results were the same as values obtained by capillary electrophoresis combined with fluorescence labeling multiply PCR. Results were also compared with data obtained with FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors or with PCR-detectable disease-specific gene products analysis. The results of the microsatellite analysis correlated well with the corresponding clinical findings. Full donor chimerism (FDC) were detected in all patients; decreasing values of donor chimerism were detected concomitantly with the appearance of relapse of disease in 3 patients. Samples from eight patients receiving HLA mismatched-haploidentical transplants from related donors together with cord blood transplants from unrelated donors were analyzed by this method. The results showed all 8 patients achieved FDC derived from related donors. It is concluded that this novel approach allows a rapid, sensitive, economical, auto-mated and non-isotopic STR-PCR testing, thus provides a reliable alternative for assessment of the status of engraftment after allo-HSCT.