Spyridonidis Alexandros, Zeiser Robert, Wäsch Ralph, Bertz Hartmut, Finke Jürgen
Department of Hematology and Oncology, Albert Ludwigs University Medical Center, Freiburg, Germany.
Clin Transplant. 2005 Jun;19(3):350-6. doi: 10.1111/j.1399-0012.2005.00347.x.
Hematopoietic chimerism has been demonstrated to be relevant for donor cell engraftment and detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). In the light of increasing numbers of non-myeloablative aHCT as a treatment modality sensitive, rapid, and accurate chimerism monitoring techniques acquire novel relevance.
We evaluated the informativeness of five microsatellite markers in 376 donor/recipient pairs and evaluated the ability of capillary electrophoresis to detect mixed chimerism after aHCT. The sensitivity for capillary electrophoresis with respect to different markers was determined by limiting dilution assays with mixed chimerism samples containing defined amounts of cells or DNA. Furthermore, capillary electrophoresis was applied in 17 retrospectively selected patients with a mixed chimerism detected previously by gel electrophoresis, having undergone aHCT for different hematologic diseases and initially achieving a complete donor chimerism.
In 163 of 165 (98%) of all related and 210 of 211 (99%) unrelated transplants the microsatellites identified informative alleles. The sensitivity and accuracy was higher with capillary electrophoresis when compared with gel electrophoresis with three of five microsatellites. Potential pitfalls with the application of capillary electrophoresis was preferential amplification and the occurrence of stutter peaks in the representative area. Investigation of the selected patient samples demonstrated that detection of a mixed chimerism was earlier with capillary electrophoresis when compared with gel electrophoresis. The detected recipient genotype by capillary electrophoresis examination, despite a negative gel electrophoresis result, ranged from 0.7 to 7.1%.
We conclude that chimerism assessment with our five microsatellites identified informative alleles in 99% of all donor/recipient pairs and may therefore be of use when establishing an institutional chimerism testing procedure. Capillary electrophoresis displayed a high sensitivity and accuracy for detecting a mixed chimerism in vitro and in vivo.
造血嵌合体已被证明与异基因造血细胞移植（aHCT）后供体细胞植入及微小残留病的检测相关。鉴于非清髓性aHCT作为一种治疗方式的应用日益增多，灵敏、快速且准确的嵌合体监测技术具有了新的重要性。
我们评估了376对供体/受体中五个微卫星标记的信息量，并评估了毛细管电泳检测aHCT后混合嵌合体的能力。通过对含有确定数量细胞或DNA的混合嵌合体样本进行有限稀释分析，确定了毛细管电泳对不同标记的灵敏度。此外，毛细管电泳应用于17例先前通过凝胶电泳检测到混合嵌合体的回顾性选择患者，这些患者因不同血液系统疾病接受了aHCT，最初实现了完全供体嵌合体。
在所有165例相关移植中的163例（98%）和211例无关移植中的210例（99%）中，微卫星鉴定出了信息性等位基因。与凝胶电泳相比，五个微卫星中的三个在毛细管电泳时灵敏度和准确性更高。毛细管电泳应用的潜在陷阱是代表性区域的优先扩增和拖尾峰的出现。对所选患者样本的研究表明，与凝胶电泳相比，毛细管电泳检测混合嵌合体更早。尽管凝胶电泳结果为阴性，但通过毛细管电泳检测到的受体基因型范围为0.7%至7.1%。
我们得出结论，使用我们的五个微卫星进行嵌合体评估，在所有供体/受体对中的99%鉴定出了信息性等位基因，因此在建立机构嵌合体检测程序时可能有用。毛细管电泳在体外和体内检测混合嵌合体时显示出高灵敏度和准确性。
