Lopez Genghis H, Mcbean Rhiannon S, Wilson Brett, Irwin Darryl L, Liew Yew-Wah, Hyland Catherine A, Flower Robert L
Research and Development Division, Australian Red Cross Blood Service, Queensland, Australia.
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Queensland, Australia.
Blood Transfus. 2015 Jan;13(1):78-85. doi: 10.2450/2014.0336-13. Epub 2014 Jun 5.
The Indian blood group antigens, In(a) and In(b), are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The In(b) antigen is a high frequency antigen present in all populations, while the frequency of the antithetical In(a) ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the INA and INB alleles in a blood donor cohort.
Donor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques.
In this study group, 149 INB homozygous and 2 INA/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate INA and INB alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both In(a) and In(b) antigens were present as predicted.
Genotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the INA and INB alleles were discriminated on the basis of their melting properties. The In(a) frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance.
印度血型抗原In(a)和In(b)在输血医学中具有临床意义。然而，用于鉴定这些抗原的抗血清很难获得。In(b)抗原是一种在所有人群中都存在的高频抗原，而其相对的In(a)抗原频率在白种人中为0.1%，在中东人群中高达11%。这种抗原多态性由CD44中的单核苷酸多态性（SNP）252G>C编码。本研究的目的是建立并比较两种基因分型方法，以测定献血者队列中INA和INB等位基因的频率。
通过一种新型实时聚合酶链反应（PCR）高分辨率熔解曲线（HRM）分析和一种定制的基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）检测法对151份献血者血液样本进行基因分型。对携带罕见IN*A等位基因的样本进一步采用核苷酸测序、红细胞凝集和流式细胞术技术进行研究。
在该研究组中，检测到149份INB纯合样本和2份INA/B杂合样本，HRM和MALDI-TOF MS方法之间的一致性为100%。对于PCR HRM，仅扩增子熔解无法区分INA和INB等位基因（3类SNP），然而，引入未标记探针（UP）提高了检测的分辨率。测序证实这两份非纯合样本为IN*A/B杂合，通过红细胞凝集进行表型分析，流式细胞术证实In(a)和In(b)抗原均如预期存在。
基因分型有助于保存罕见抗血清以预测血型抗原表型。在PCR UP-HRM中，INA和INB等位基因根据其熔解特性进行区分。在这个选定的献血人群中，In(a)频率为1.3%。应用此类基因分型方法有助于识别具有潜在临床意义的罕见血型表型的献血者。
