Quantitative Analysis Of NK Cell Chimerism After Hematopoietic Stem Cell Transplantation By SNP-PCR

Objective: To investigate the feasibility and efficiency of the fluo-rescence-activated cellsorting (FACS) skill, which is used to sort the natural killer (NK) cells from the periphera l bloodof the patients after allogeneic hematopoietic stem cell transpla ntation (allo-HSCT). Then toestablish a novel method, which is based on single nucleotide polymorphism (SNP) genotypingand carried out by real-time polymerase chain reaction (RT-PCR) with Cycling probe, forquantitative detection of chimerism of the NK cells in the patients after allo-HSCT. The aims ofthese detections are to explore their applica tion value, accuracy and superiority, and to observethe relationships in the early period between the formation of this chimerism and the outcome ofdisease .Methods: 7 SNP locis were screened from five different chromosome s and correspond ingCycling probes and primers were composed to identify informative markers for detectingchimerism in each donor/recipient pair before allo-HSCT. The NK cells were sorted from theperiphera l blood of 20 norma l persons (9 ma les, 11 fema les) and 4 donor/recipient pairs by FACS.DNA of NK cells samples was extracted, then the chimerism rate of each informative markerwas analyzed by Cyclea ve PCR based SNP. The accuracy and sensitivity of this new methodwere verified by a series of modeling chimerism from norma l persons.Results:①The percentage of norma l persons NK cells is (12.86±3.62) %. After cell sorting,the purity is (96.15±2.03) %, and the recovery rate is (95.08±2.16) %. The percentage of NKcells of patients after allo-HSCT is (11.01±2.08) %. After sorting, the purity is (96.22±2.16) %,and the recovery rate is (95.27±1.18) %.②At least one informative marker could be found inover 78.5% of 4 donor/recipient pairs and 10 artificia l donor/recipient pairs formed 20 norma lpersons .③The slope of the 7 time amplications of the plasmid was-2.3681, the intercept was36.5919,correla tion coefficients were more than 0.995, which was close to the theoretica l level.④Value s of the artificia l mixed chimerism measured by SNP-PCR are close to the predeterminedvalue s(P＞0.01). A linear correla tion with artificia l chimerism is over 0.99, and a sensitivity of0.01% proved reproducible.Conclusion: FACS is efficient, fast and easy in sorting the NK cells from periphera l blood,especia lly for the patients after hematopoietic stem cell transpla ntation in their early recoverystage. And the change of these NK cells'chimer ism is correla ted with their disease status. So thequantitative detection of chimerism ma y be useful to predict the disease recurrence and then toguide clinica l interventions , in order to improve the outcomes . This new assay provides a more accurate, reliable and rapid quantitative assessment of chimerism after allo-HSCT than thetraditiona l method.