Kroll H, Carl B, Santoso S, Bux J, Bein G
Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Germany.
Transfus Med. 2001 Jun;11(3):211-9. doi: 10.1046/j.1365-3148.2001.00307.x.
The immunization against alloantigens present on platelets, granulocytes and red blood cells (RBCs) is responsible for various clinical syndromes. Since the molecular basis of these antigens has become clear during the last decade, genotyping is nowadays used in several laboratories. However, many DNA-based techniques still have to be evaluated. We therefore organized a workshop on the genotyping of the most relevant alloantigens on platelets and granulocytes as well as on selected RBC alleles. DNA was isolated from peripheral blood lymphocytes or from B-lymphoblastoid cell lines (B-LCL). We distributed samples for the identification of platelet (n = 7), granulocyte (n = 6) and RBC (n = 4) polymorphisms, respectively. There were 33 institutions in Germany, Austria and Switzerland, which participated in at least one part of the workshop. Twenty-four laboratories reported results on HPA-1, and 23 laboratories on HPA-2, -3, and -5 typing. In addition, five laboratories typed for HPA-4 and -6. The HNA-1a/b (NA1/NA2) alleles were identified by eight laboratories, one of which also typed for HNA-1c (SH). The most frequent genes of the ABO (A1, B, O) and Rh (D, C, c, E, e) systems were typed by 12 participating laboratories, and an additional four laboratories restricted their RBC typing to the RHD gene. The typing technique mainly used for all three cell lineages was the polymerase chain reaction with sequence-specific primers. Other techniques were restriction fragment length analysis, oligonucleotide ligation assay, enzyme-linked mini-sequence assay or direct sequence analysis. The following typing errors were observed: HPA: 15/1442 (1.0%), HNA: 4/108 (3.7%), ABO: 5/96 (5.2%) RH 1/320 (0.3%). Our workshop demonstrated the existence of a number of reliable techniques for the genotyping of blood cell alloantigens and a high standard in the participating laboratories. In addition, we could show the usefulness of B-LCL as a source of reference DNA. However, the 5.2% rate of mistyping in the ABO system demonstrated that further efforts are needed to improve the precision of the genotyping techniques. Future workshops will have to challenge methods and participants with rare variants of RBC genes to guarantee reliable genotyping, e.g. in prenatal diagnosis of fetomaternal incompatibility.
针对血小板、粒细胞和红细胞（RBC）上存在的同种异体抗原进行免疫会引发各种临床综合征。在过去十年中，由于这些抗原的分子基础已逐渐明晰，如今一些实验室开始采用基因分型技术。然而，许多基于DNA的技术仍有待评估。因此，我们组织了一次关于血小板和粒细胞上最相关同种异体抗原以及选定红细胞等位基因基因分型的研讨会。DNA从外周血淋巴细胞或B淋巴母细胞系（B-LCL）中分离得到。我们分别分发了用于鉴定血小板（n = 7）、粒细胞（n = 6）和红细胞（n = 4）多态性的样本。德国、奥地利和瑞士共有33个机构参与了该研讨会的至少一部分内容。24个实验室报告了HPA-1的检测结果，23个实验室报告了HPA-2、-3和-5的分型结果。此外，有5个实验室对HPA-4和-6进行了分型。8个实验室鉴定了HNA-1a/b（NA1/NA2）等位基因，其中1个实验室还对HNA-1c（SH）进行了分型。12个参与实验室对ABO（A1、B、O）和Rh（D、C、c、E、e）系统中最常见的基因进行了分型，另外4个实验室将其红细胞分型限制在RHD基因上。用于所有三种细胞谱系的主要分型技术是序列特异性引物聚合酶链反应。其他技术包括限制性片段长度分析、寡核苷酸连接测定、酶联微序列测定或直接序列分析。观察到以下分型错误：HPA：15/1442（1.0%），HNA：4/108（3.7%），ABO：5/96（5.2%），Rh：1/320（0.3%）。我们的研讨会表明存在多种可靠的血细胞同种异体抗原基因分型技术，且参与实验室具有较高水平。此外，我们还展示了B-LCL作为参考DNA来源的实用性。然而，ABO系统中5.2%的分型错误率表明，仍需进一步努力提高基因分型技术的准确性。未来的研讨会将必须用红细胞基因的罕见变异挑战方法和参与者，以确保可靠的基因分型，例如在母胎血型不合的产前诊断中。
