125-P

Abstract

Aim Engraftment monitoring is crucial step for patients after Hematopoietic Stem Cell Transplantation (HSCT). STR-PCR is currently the most widely used technique, but the interpretation is difficult due to the existence of stutter bands, amplicons containing one less repeat than the normal allele, especially in double-cord or multiple donor tranplants. The idea of using pooled umbilical cord blood (CB) as an universal donor for marrow reconstitution is not new. The aim of this study was to test if qPCR method is able to detect multiple chimera generated from 6 different DNA samples. Methods We generated several DNA mixes with known ratio (50, 5, 1 and 0.1%) to mimic HSCT postransplant situation using 5 different donors, each at an equal concentration. STR-PCR (ABI) and qPCR (Celera) methods were compared using modified calculations. We asked if biallelic indels were enough to distinguish 6 individual DNA. Was qPCR sensitive enough to detect lower amounts of DNA (1,5 and 0.1%) and how easy it was to quantify each individual DNA. Results Screening: Average number of informative STR vary 3-7 for each DNA (n = 16 loci). Only 3/6 DNA had unique Indel (n = 34 loci) markers. We decided to use combination of Indel markers; 4 Indels were unique for 4 different pair. Quantification: Both STR-PCR and qPCR were able to estimate each component, however, in case of STR-PCR stutter bands were completely excluded from the calculation, otherwise it was impossible to analyze. 50% of DNA was detected as 57-58% using STR-PCR or qPCR. 10% of each donor was detected with the range of 6-15% (STR) and 7-11% (qPCR). However, qPCR was able to detect 0.1; 1 and 5% of one DNA, in which case STR results were 3,6%; 4,4% and 9.3% resp. Conclusions As expected, STR-PCR is more informative but less sensitive than qPCR. Interpretation of these data could be adapted and used in cases of multiple donors. Pooled CB in culture should be used as donor sample to test the same hypothesis.