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Quantitative evaluation of post-bone marrow transplant engraftment status using fluorescent-labeled variable number
R A Luhm1, D B Bellissimo, A J Uzgiris
1The Diagnostic Laboratories of the Blood Center of Southeastern Wisconsin, Milwaukee, WI, USA.
Background:
The analysis of highly polymorphic variable number of tandem repeat (VNTR) loci is useful for the estimation of donor-host chimerism in bone marrow transplant recipients.
Methods And Results:
A rapid and sensitive engraftment assay has been developed in which the VNTR loci, D1S80, D17S5, D1S111, and apoB, are amplified with fluorescent-labeled (Cy5.5) oligonucleotide primers, followed by analysis using the Visible Genetics, Inc, OpenGene System. The degree of chimerism is then calculated by determining the percentage of host contribution to the total informative allele peak area. Reconstitution experiments and analysis of 383 posttransplantation DNA samples, isolated from 71 different bone marrow transplant recipients, were evaluated as part of assay development. Reconstitution studies showed assay linearity and sensitivity of at least 1%. Patient results were compared with a previous analysis in which unlabeled PCR products were quantified on silver-stained polyacrylamide gels. High concordance was observed between fluorescent analysis and silver-staining method in all 71 patients.
Conclusions:
Fluorescent analysis offers many advantages over previous methods, including faster turnaround time, decreased DNA requirements, greater resolution and/or sensitivity, and objective interpretation.