Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

Hemolytic disease of the newborn due to anti-Jra from a Chinese mother with one novel and one classic heterozygous mutation.

OBJECTIVE: Investigation of a Jr(a-) family samples, identification of the mutant and assessment of the differences of Jr antigen density of the Jr(a-) family members, random adult and newborn individuals' RBCs. BACKGROUND: The anti-Jra antibody is generated when a Jr(a-) individual pregnant or transfused with Jr(a+) blood unit, which can lead to mild-to-moderate hemolytic disease of the foetus and newborn (HDFN) or hemolytic transfusion reaction (HTR). Several mutations had been identified. The anti-Jra caused HDFN is not rare in East Asia, but due to the lack of antibody and molecular background, it is likely to lead missed detection. METHODS AND MATERIALS: One G4P1 woman had been detected as IAT positive during prenatal examination. Suspected as anti-Jra after the laboratory serological testing, the maternal sample was further assessed by molecular analysis. The antigen density was detected by flow cytometry after reacting with anti-Jra serum in family members and the normal individuals. RESULTS: One novel frameshift mutation c.717delC and one previously identified mutation c.706C > T in ABCG2 was identified on proband. The infant haemoglobin(Hb) and bilirubin increased significantly after exchange transfusion and the severe HDFN was relieved. Flow cytometry results showed that the Jra antigens on adult RBCs were significantly less than those on the infant. CONCLUSION: The c.717delC mutation can lead to the shortening of protein ABCG2 in the site of p.Leu307Stop, result in the loss of Jra antigen. The difference in antigen density between adult and infant RBCs may be a possible reason that leads to severe HDFN but not transfusion reaction. Breastfeeding may lead to slower recovery from HDFN.

NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes.

Autophagy and phagocytosis are two important processes that capture and digest materials found in cellular interiors and exteriors, respectively. Aged red blood cells (RBCs) are cleared by phagocytes in vivo. We focused on determining whether autophagy occurs after phagocytes swallow sunset erythrocytes, and whether the degree of autophagy is related to scavenging ability of phagocytes to erythrocytes. In addition, the ability of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to regulate erythrocyte clearance by phagocytes and its association with autophagy-related protein 16-like protein 1 (ATG16L1) are confirmed. We constructed a stable and low-NLRP3 expression THP-1 cell line using CRISPR/Cas9 technology. The analysis of erythrocyte clearance and autophagy of THP-1 cells with low NLRP3 expression showed that autophagy changes together when THP-1 engulfs aged RBCs. The occurrence of autophagy was dominated by microtubule-associated protein 1A/1B-light chain 3- (LC3-) associated phagocytosis accompanied by canonical autophagy. A negative correlation exists between the clearance of RBCs by THP-1 cells and the degree of autophagy. Downregulating the expression of NLRP3 in THP-1 cells can simultaneously inhibit the scavenging ability of THP-1 to erythrocytes and the degree of autophagy. In addition, the autophagy inhibitor bafilomycin A1 (BafA1) can enhance the phagocytosis ability of THP-1 to erythrocytes and promote the NLRP3 activation in THP-1 cells, while the autophagy inducer rapamycin inhibits the phagocytosis ability of THP-1 to RBCs and downregulates the NLRP3 activation. Results showed that autophagy and phagocytosis may be dynamic balance processes that can provide sufficient nutrition and energy to cells. Choosing NLRP3 as a target may regulate the phagocytic ability and the degree of autophagy in the meantime. These findings may be a potential strategy for regulating the clearance rate of phagocytes to aged RBCs and the secretion of proinflammatory cytokines to ensure transfusion safety.

RHD alleles contributing to serologically weak D phenotypes in China: A single-centre study over 10 years.

BACKGROUND AND OBJECTIVES: In cases of serologically weak D phenotypes, RHD genotyping may identify discrepant serotyping results and protect the patient against allogeneic immunization. This study aimed to conduct a comprehensive analysis of weak D alleles in China. MATERIALS AND METHODS: We collected samples carrying weak D antigen during a 10-year period from 2005 to 2014. The intensity and epitopes of D were analysed serologically. Genomic DNA was extracted and used for RHD sequencing and heterozygote analysis. In particular, an in vitro expression method for functional verification of the rare and novel in-frame deletion mutation was developed and then combined with homologous modelling results for analysis. RESULTS: We studied a total of 283 weak D samples from volunteer blood donors and identified 45 RHD alleles among them, 11 of which were reported for the first time. Ten (3.5%) samples surprisingly carried DEL allelic variants and as many as 40 (14.1%) carried the wild-type RHD genotype. Combination of the results of functional experiments and in silico analysis suggested that the rare in-frame deletion mutation may reduce the expression of D antigen by affecting the RhD protein structure. CONCLUSIONS: This study provides an enhanced overview of the distribution characteristics of RHD alleles in Chinese subjects with serologically weak D. An in vitro method to predict the biological significance of variant RHD alleles was also provided. We found inconsistent genotyping and phenotypic results in some samples, indicating the existence of additional regulatory mechanisms.

OK/basigin expression on red blood cells varies between blood donors and correlates with binding of recombinant Plasmodium falciparum reticulocyte-binding protein homolog 5.

BACKGROUND: Recently, basigin (BSG), which carries OK antigens on red blood cells (RBCs), was reported to be the receptor of the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRh5). BSG-PfRh5 is the only essential receptor-ligand pair in P. falciparum invasion that is known to date. However, the kind of OK/BSG polymorphism involved in the selection pressure caused by P. falciparum malaria has not been determined. STUDY DESIGN AND METHODS: Blood samples were collected to detect the expression of OK/BSG. The coding region of PfRh5 was cloned and expressed. Enzyme-linked immunosorbent assay-based erythrocyte binding assay was used to measure the recombinant PfRh5 (rPfRh5) binding of RBCs with different OK/BSG expressions. Sequencing of the BSG gene and quantification of the BSG mRNA were performed for selected samples. The candidate microRNAs (miRNAs), which might target the BSG gene, were obtained by miRNA sequencing. Dual-Luciferase reporter assay and overexpression of identified miRNAs were performed in K562 cells. RESULTS: The rPfRh5 was successfully expressed and verified. The OK/BSG expression levels varied among blood donors and were strongly associated with rPfRh5 binding. No single-nucleotide polymorphism was related to the OK/BSG expression. A potential BSG regulator, miR-501-3p, was identified by miRNA sequencing and Dual-Luciferase assay, but was not proven to regulate the expression of BSG in K562 cells. CONCLUSION: Although the mechanism of OK/BSG expression and regulation on RBCs has not been fully clarified, our findings suggest that the OK/BSG expression levels on RBCs might be related to P. falciparum invasion. Moreover, posttranscriptional regulation might play a role in controlling the OK/BSG expression.

[The role of miR-492 in the regulation of OK blood group antigen expression on red blood cells].

OBJECTIVE: To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells. METHODS: 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. RESULTS: 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. CONCLUSION: miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum.

In Dictyostelium, soluble cell adhesion molecule, DdCAD-1, regulates cell-cell interaction through an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes revealed that the potential DdCAD-1 interacting protein was between 64 and 98 kDa. To isolate and identify the anchoring protein, GST-DdCAD-1 and anchoring protein were cross-linked in vivo by chemical cross-linker and stable protein complex was isolated by co-immunoprecipitation assays. The protein cross-linked to DdCAD-1 was extracted from the gel slice and trypsinized. The peptides were subjected to analysis by mass spectrometry, which showed that the putative anchoring protein belongs to ATP-binding cassette transporter family.