Qantitative Analysis Of Lineage-specific Chimerism Using Real-time PCR And Single Nucleotide Polymorphisms

【Objeetive】To establish a novel post—transplant assay approach for quantitative assessment of mixed hematopoietic chimerism using real—time polymerase chain reaction(PCR)of single nucleotide polymorphisms(SNPs),and analyse the application of real—time SNP—PCR method for patients treated by allogeneic HSCT.【Method】For the purpose of developing an chimerism analysis method for Chinese patients which underwent the hematopoietic stem cell transplantation,we searched the dbSNP database and gotseven SNPs to development an informative panel.Sub—elected CD3+T lymphocytes using anti—CD3 immunomagnetic beads reagents by magnetic activated cell separator。Establish a SNP—based quantitative method using real—time PCR,and then analyse three clinical samples received the allogeneic stem cell transplantation.【Result】1.Positive selection of CD3+T cells using the MACS magnetic cell sorting systemCD3+T lymphocytes were successfully selected using the MACS magnetic cell sorting system,and the sorting purity was greater than 96%.2.Selection of the SNP panelWe searched and got seven SNPs from dbSN P database.These seven SNPs include three type of base substitution,in which 3 of A/G,3 of C/T,l of T/G type.The selection guideline is that the allele frequence of each SNP should be between 0.3～0.8 and all SNPs should distributed on the different chromosomes.For generalizing the detection method,we made the efforts to cover the various base substitution type.3.Establish a SNP—based quantitative method using real—time PCR.UV spectrophotometer measured the concentration of sample DNA.In order to detect the donor/recipient chimerism we produced a C allele rs4245 locus of standard curve.The negative and positive template were validated by repeated genotyping.Then we made the gradient concentration ranged from 100 to 0.01%.After amplifying this set of templates with real-time PCR,the operation software output standard curve:y=-3.5976x+38.327.The amplification efficiency was O.897 and R2was 0.9969.Mean Ct value of each concentration was 22.11,25.15,29.20,33.06 and 36.36.It showed that the linear relationship of this standard curve was fine.The sensitivity this standard curve reached was not lower than 0.1%.and the amplification effect of the standard sample template is also good.4.Analysis of the clinical samplesThe purity of CD3+T lymphocytes selected by immunomagnetic bead separation using flow cytometry analysis was 97%.Amplify the chimera samples(donor genotype:TT,receptor genotype:CC),and we got CT mean value obtained for the 21.23 into the above—mentioned standard curve rs4245.According to the output,as well as total DNA calculate chimeric degrees for 16.23%.【Conclution】The MACS magnetic cell sorting system can be carried out quickly and efficiently separating blood cells,.We established the quantitative detection method by preparation of the artificial chimerism,and this method made a greater improvement in detection sensitivity,accuracy and stability than traditional methods.