Application of MALDI-TOF MS in clinical difficult blood group typing / 中国输血杂志

【Objective】 To explore the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in the genotyping of difficult blood typing samples, and to provide evidence for clinical blood transfusion. 【Methods】 Three ambiguous blood group samples, submitted to Shanghai Blood Center by Shanghai regional hospitals, were studied, of which Sample1 included the proband and his parents. Serological methods were used to perform blood group typing, direct antibody test, unexpected antibody screening and identification test. Blood group genotyping was performed by using the MALDI-TOF MS detection systeme stablished in our laboratory. Sanger sequencing was used to confirm gene mutation sites, and serological or flow methods were used to verify specific samples′ phenotype. 【Results】 Serological results indicated the existence of antibodies against high frequency antigens in sample 1 (including proband and her mother), 2 and 3. The genotyping results of MALDI-TOF MS showed that the proband of sample 1 was Di(a+ b+ ), her father was Di(a-b+ ), her mother was Di(a+ b-), sample 2 was p, and sample 3 was Jr(a-). Sequencing results of three samples were consistent with mass spectrometry typing results. Serological results showed that sample 2 had a p phenotype. The flow cytometry results suggested that sample 3 had a Jr(a-) phenotype. 【Conclusion】 For the first time, we applied MALDI-TOF MS technology to blood type genotyping of ambiguous clinical samples in China. Compared with other genotyping methods such as PCR-SSP, MALDI-TOF MS has the advantages of rapid detection, high throughput and high specificity, which would contribute to identification of difficult blood typing samples in the future, as well as rare blood group screening.

The role of miR-492 in the regulation of OK blood group antigen expression on red blood cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells.</p><p><b>METHODS</b>Two 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis.</p><p><b>RESULTS</b>The sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A.</p><p><b>CONCLUSION</b>miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.</p>

Development of a multiplex polymerase chain reaction system for screening low-frequency blood group antigens K and Ytb / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>A multiplex PCR system for screening rare blood group antigens K and Yt(b) was constructed to study the distribution of the two blood groups in a Uygur population in Xinjiang, China.</p><p><b>METHODS</b>Sequence-specific primers (SSP) were designed based on single nucleotide polymorphism sites of KEL and ACHE alleles encoding the two blood group antigens. The system was designed for simultaneously detecting the two antigens by optimizing the PCR reaction. Three hundred and sixty-two randomly selected healthy individuals were screened. Products of PCR were further analyzed for heterozygosity.</p><p><b>RESULTS</b>The system was set up successfully. No KK sample was identified and 9 K+ k+ , 41 Yt (a+ b+ ), 4 Yt (a- b+ ) were found among the 362 samples.</p><p><b>CONCLUSION</b>The established PCR-SSP based multiple PCR system is efficient to screen the rare blood group antigens K and Yt(b). The information of rare blood donors obtained from the screening can be used to improve the capability of compatible transfusion.</p>

Screening of rare blood group Lu(a-b-) phenotype and study of its molecular basis in ethnic Han Chinese from Shanghai region / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the frequency of rare blood group Lu(a-b-) phenotype in a population from Shanghai region, and to explore the molecular basis of Lu(a-b-) by detecting the Lu and Lu relative mediator gene EKLF/KLF1.</p><p><b>METHODS</b>Donors from Shanghai region were screened for Lutheran blood group by monoclonal anti-Lub using serological methods. Individuals with Lu(b-) were determined Lua, P1 and i antigens. Fifteen exons of the LU gene and 3 exons of the EKLF/KLF1 gene for the identified Lu(a-b-) samples were amplified and sequenced.</p><p><b>RESULTS</b>Ten Lu(a-b-) donors were obtained from 44 331 donors from Shanghai region. No homozygous or heterozygous mutations were found in the LU gene, whilst 7 mutations in EKLF/KLF1 gene were identified in the 10 samples.</p><p><b>CONCLUSION</b>The frequency of rare Lu(a-b-) blood group in Shanghai was approximately 0.02%, and all the individuals had an In(Lu) phenotype. The molecular basis of such samples may be related to mutations in the EKLF/KLF1 gene.</p>

Application of multiplex PCR for the screening of genotyping system for the rare blood groups Fy(a-), s-,k-,Di(b-) and Js(b-) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen rare blood groups Fy(a-), s-, k-, Di(b-) and Js(b-) in an ethnic Zhuang population.</p><p><b>METHODS</b>Sequence-specific primers were designed based on single nucleotide polymorphism (SNP) sites of blood group antigens Fy(b) and s. A specific multiplex PCR system I was established. Multiplex PCR system II was applied to detect alleles antigens Di(b), k, Js(b)1910 and Js(b) 2019 at the same time. The two systems was were used to screen for rare blood group antigens in 4490 randomly selected healthy donors of Guangxi Zhuang ethnic origin.</p><p><b>RESULTS</b>We successfully made the multiplex PCR system I. We detected the rare blood group antigens using the two PCR system. There are five Fy(a-), three s(-), two Di(b-) in 4490 Guangxi zhuang random samples. The multiplex PCR system I has achieved good accuracy and stability. With multiplex PCR systems I and II, 4490 samples were screened. Five Fy(a-), three s(-) and two Di(b-) samples were discovered.</p><p><b>CONCLUSION</b>Multiplex PCR is an effective methods, which can be used for high throughput screening of rare blood groups. The rare blood types of Guangxi Zhuang ethnic origin obtained through the screening can provide valuable information for compatible blood transfusion. Through screening we obtained precious rare blood type materials which can be used to improve the capability of compatible infusion and reduce the transfusion reactions.</p>