Armistead Paul M, Mohseni Mehrdad, Gerwin Roslyn, Walsh Emily C, Iravani Masoud, Chahardouli Bahram, Rostami Shahrbano, Zhang Wandi, Neuberg Donna, Rioux John, Ghavamzadeh Ardeshir, Ritz Jerome, Antin Joseph H, Wu Catherine J
Department of Medical Oncology, M.D. Anderson Cancer Center, Houston, TX, USA.
Exp Hematol. 2008 Sep;36(9):1205-15. doi: 10.1016/j.exphem.2008.04.004. Epub 2008 Jun 11.
We aimed to create a molecular assay to monitor erythroid (red blood cell [RBC]) engraftment in any patient following allogeneic hematopoietic stem cell transplantation, independent of disease-specific mutations.
We identified 10 common single nucleotide polymorphisms (SNPs), expressed by genes encoding RBC antigens and structural proteins. These SNPs were polymerase chain reaction-amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies validated that each SNP can quantitatively measure donor/recipient DNA and RNA.
We directly genotyped 23 patients who underwent hematopoietic stem cell transplantation and their human leukocyte antigen-matched donors and found a median of three informative SNPs (i.e., discordant between donor and recipient) per pair. By using the informative RBC SNPs to quantify donor-derived RBC transcripts, we compared rates of RBC engraftment in 13 patients with hemoglobinopathies vs donor mononuclear cell (white blood cell [WBC]) engraftment. Consistent with known ineffective erythropoiesis associated with hemoglobinopathies, we detected up to threefold greater RBC-specific compared to overall WBC engraftment in five of eight patients who were mixed chimeras by transplant day 30. The remaining three of eight who received ABH-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment that was related to persistence of host-derived anti-isohemagglutinin antibodies.
This RNA-based assay can be used to monitor RBC-specific engraftment regardless of a patient's specific hemoglobin mutation or even diagnosis. We propose that panels of expressed SNPs informative for other cell lineages can be created to comprehensively assess the impact of novel stem cell-based therapies on lineage-specific engraftment.
我们旨在创建一种分子检测方法，以监测异基因造血干细胞移植后任何患者的红系（红细胞[RBC]）植入情况，而不依赖于疾病特异性突变。
我们鉴定了10个常见的单核苷酸多态性（SNP），它们由编码RBC抗原和结构蛋白的基因表达。这些SNP通过聚合酶链反应从外周血提取的总RNA中扩增，外周血中含有有核红系祖细胞。混合研究验证了每个SNP都能定量测量供体/受体DNA和RNA。
我们对23例接受造血干细胞移植的患者及其人类白细胞抗原匹配的供体进行了直接基因分型，发现每对中平均有3个信息性SNP（即供体和受体之间不一致）。通过使用信息性RBC SNP来量化供体来源的RBC转录本，我们比较了13例血红蛋白病患者的RBC植入率与供体单个核细胞（白细胞[WBC]）植入率。与已知的与血红蛋白病相关的无效红细胞生成一致，在移植后30天为混合嵌合体的8例患者中的5例中，我们检测到RBC特异性植入比总体WBC植入高多达三倍。接受ABH不相容移植物的其余8例中的3例，与WBC植入相比，RBC植入至少低0.5倍，这与宿主来源的抗异血凝素抗体的持续存在有关。
这种基于RNA的检测方法可用于监测RBC特异性植入，而不管患者的特定血红蛋白突变甚至诊断如何。我们建议可以创建对其他细胞谱系有信息性的表达SNP面板，以全面评估新型干细胞疗法对谱系特异性植入的影响。
