异基因造血干细胞移植植活状态的变性高效液相色谱检测研究的首次报告

异基因造血干细胞移植后动态检测供者嵌合状态对判断移植效果和实施临床早期干预治疗具有重要意义。在临床上已有多种方法用于植活检测，本研究用变性高效液相色谱(DHPLC)技术结合聚合酶链反应扩增短串联重复序列(STR—PCR)检测造血干细胞移植植活状态的特点及可靠性。用非亲缘个体的系列DNA混合体在体外检测此测定方法的可行性和半定量结果的准确性。结果表明，体外模拟混合嵌合体检测实验，显示扩增前供者DNA嵌合百分率与扩增后供受者DHPLC色谱峰峰面积比值呈直线相关，最小检出DNA百分比为5％。用该方法分析51例移植对的结果显示：45例均至少有2个可以区分彼此的有信息住点(另外5例无移植前受者标本，1例为同卵双生双胞胎)；39例与荧光标记多重PCR扩增STR结合毛细管电泳方法进行对照，准确性为100％；29例与性染色体FISH分析，27例与特殊基因标记的分析结果一致；所有病例均检测到完全供者细胞嵌合状态(FDC)；对3例患者观察到临床复发的同时，检测到供者嵌合体的比例明显下降，其中2例为混合嵌合体，1例转变为受者自身型。在此基础上，我们将该方法首次用于对8例人类白细胞抗原(HLA)配型不同一单倍型亲缘供者干细胞及无血缘关系脐带血混合移植患者植活情况的动态检测。结果显示，8例混合移植患者STR—PCR检测结果均为FDC，来源均为亲缘供者。结论：用DHPLC技术结合短串联重复序列具有快速、经济、无污染和自动化高等特点，用于检测异基因造血干细胞移植物植活状态是可靠的。

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.