ObjectiveTo study the molecular genetic mechanism of para-bombay phenotype in two individuals.MethodsThe proband was a female.When the proband donated blood,because the forward blood group wasn't coincident with her reverse blood group,the blood and saliva specimen from proband and her family members were sent to Guangzhou Blood Center for further identification.Routine serological techniques were used to determine proband's and her family members' blood group and ABH antigen in saliva.The coding regions of FUT1 and FUT2 gene,exon 6 and exon 7 of ABO gene were amplified by polymerase chain reaction using proband's and her family members' genomic DNA.All amplified products were analyzed after being directly sequenced.The two-base deletion regions of FUT1 gene were certified by cloning and haplotype sequencing.Results Proband's and her little brother's blood group were identified as para-bombay while other family members' blood group were normal.Two-base deletion heterozygous mutations of FUT1 gene were found in proband and her brother,AG deletion at position 547-552 and TT deletion at position 880-882,which caused a reading frame shift and a premature stop eodon.Meanwhile,880-882del TT heterozygous mutation was found in proband's grandfather and her father and 547-552del AG heterozygous mutation was found in proband's mother and her little sister.ResultsOf cloning and haplotype sequencing certified that these two-base deletion mutations occurred at 547-548 and 881-882 position respectively.Three new mutations were found in FUT2 gene,390C ＞ T,418A ＞ T and 749G ＞ A,which could cause the change of amino acid at position 140Ile ＞ Phe and 250Arg ＞ Gln.Conclusions Two-base deletion heterozygous mutations in different positions in FUT1 gene were found in 2 individuals,which maybe the molecular genetic mechanism of para-bombay phenotype.Heterozygous deletion mutation in one-strand DNA wouldn't change the ABO blood group.Three new mutations were also found in FUT2 gene.( Chin J Lab Med,2012,35:815-819)

OBJECTIVETo report a rare case of hemolytic disease of the newborn (HDN) with kernicterus caused by anti-Di(a) diagnosed using high-throughput genotyping multiplex ligation-dependent probe amplification (MLPA).

METHODSConventional serological methods were used to detect the antibodies related with HDN. The genotypes of more than 40 red blood cell antigens for the newborn and her parents were obtained using the high-throughput MLPA assay. The antibody titers were tested using a standard serological method.

RESULTSThe unknown antibody against the low-frequency antigens was predicted based on the primary serological tests. The genotyping results for more than 40 red blood cell antigens of the newborn and her parents showed incompatible antigens of MNS and Diego blood group system, indicating the existence of anti-N or anti-Di(a). Further serological tests confirmed anti-Di(a) existence in the plasma of the newborn and her mother. The titer of anti-Di(a) in the mother's plasma was 1:32.

CONCLUSIONSevere HDN including kernicterus can result from anti-Di(a). High-throughput genotyping MLPA assay can help type some rare antigens in complicated cases. The reagent red cell panels including Di(a)-positive cells are necessary in routine antibody screening test in Chinese population.

Blood Group Incompatibility ; genetics ; Erythroblastosis, Fetal ; diagnosis ; immunology ; Exchange Transfusion, Whole Blood ; Female ; Genotype ; Humans ; Infant, Newborn ; Nucleic Acid Amplification Techniques ; methods ; Rh-Hr Blood-Group System ; genetics ; immunology ; Rho(D) Immune Globulin ; genetics ; immunology

OBJECTIVETo investigate the frequency of anti-Mur and Mur antigen among blood donors in Guangzhou to provide evidence for guiding clinical transfusion and prenatal screening.

METHODSDG Gel Coombs cards were used to screen active anti-Mur at 37 degrees celsius; from 2725 blood donors. Multiplex ligation-dependent probe amplification (MLPA) was used to genotype Mur antigen from 91 blood donors, and human anti-Mur serum was used to verify the phenotypes deduced from the genotypes.

RESULTSThe frequency of anti-Mur and genotyped Mur antigen was 0.04% (1/2725) and 6.59% (6/91), respectively, and the phenotyping results were consistent with the genotyping results.

CONCLUSIONThe blood donors in Guangzhou show a low frequency of anti-Mur and a relatively high frequency of Mur antigen. Genotyping using MLPA allows Mur antigen genotyping when commercial anti-Mur is not available.

Blood Donors ; Blood Group Antigens ; genetics ; immunology ; China ; Genotype ; Genotyping Techniques ; Humans ; Multiplex Polymerase Chain Reaction ; Phenotype

Heart failure is a serious and end-stage status of various heart diseases, characterized by comparatively high rate of readmission and mortality, and has become an important public health issue. The risk of readmission and mortality following discharge of an index hospitalization are key indicators to evaluate the quality of medical care among patients with acute heart failure. Therefore, it is important to carry out risk prediction research for patients with acute heart failure, quantify the disease risk, perform risk stratification, optimize clinical decision-making, elevate patients' quality of life and prognosis, and comprehensively improve the medical quality of acute heart failure. During the past 20 years, foreign researchers have developed dozens of models to predict the risk of acute heart failure readmission and mortality, and Chinese researchers have also developed up to 10 models applicable to the Chinese population. However, there is no recommended risk prediction model for acute heart failure in current clinical guidelines across China. In this report, we aim to introduce the major models for predicting the risk of acute heart failure readmission and mortality from home and abroad, focus on putting forward limitations of established models, and initiating potential directions for future studies from the following aspects: integrate multi-source data, mine emerging biomarkers, establish polygenic risk scores, optimize machine learning methods, promote flexible adjustment, and broaden approaches that applicable for various scenarios. Accordingly, this study will help facilitate domestic research in predicting the risk of readmission and mortality among patients hospitalized for acute heart failure.

【Objective】 To identify the blood group epitope of a D variant individual and analyze its molecular characteristics. 【Methods】 The saline test and indirect antiglobulin test (IAT) were used to identify the RhD serologically. The anti-human globulin gel card was used for direct antiglobulin test (DAT). RhD epitopes were detected using the epitope detection kit (D-Screen). RhCE antigens were typed using Rh typing Card. The RHD gene zygomorphism was further analyzed by PCR-RFLP. Ten exons of RHD gene were amplified by PCR and analyzed by direct sequencing. 【Results】 DAT test was negative, and the serological results showed weak expression of RhD, which was D variant. The RhD epitope test results showed that the red blood cells of this patient had a weak agglutination with 4 monoclonal anti-D against epD6.4, epD6.1, epD2.1, and epD5.4 (w+ to 2+ ), and reacted negatively with other epitope antibodies. RhCE antigen typing was Ccee; The RHD gene zygomorphism result was D+ /D-, the sequencing of RHD exons revealed that the first exon carried c. 41C>T (p.Pro14Leu) missense mutation, and its genotype was RHD^*01W.136/01N.01. 【Conclusion】 This D variant is the first weak D type 136 reported in the Chinese population, and its phenotype is weak partial D.

【Objective】 To explore the identification method and characteristics of anti-IFC against Cromer blood group. 【Methods】 ABO blood group was identified by tube method, and Rh blood group was identified by Rh typing card. Irregular antibody screening and antibody identification were carried out using microtube column agglutination technology(MCAT). The serum of the patient reacted with panel cells treated with antitrypsin, trypsin and bromelain respectively to determine the specificity of the antibody. The serum was inhibited with pure CD55 protein for antibody identification. The DAF, the regulatory gene of Cromer blood group system, was amplified and sequenced. The expression of CD55 on cell membrane was analyzed by flow cytometry. 【Results】 The patient′s blood type was B, CcDEe. The patient′s serum reacted with all the untreated panel red cells, bromelain-treated red cells, trpsin-treated red cells, and DTT treated red cells.It was negative with chymotypsin-treated cells and could be neutralized by CD55 protein. No mutation was found by DAF sequencing. The expression of CD55 on patient′s cell membrane was deficient. 【Conclusion】 This high frequency antibody was identified as anti-IFC. The transient depression in CD55 protein may due to the patient′s GI system abnormalities.

【Objective】 To identify the specificity of alloantibody against high-frequency antigens in one case suffering with severe hemolytic diseases of the fetus and newborn (HDFN) and to screen for matching blood for transfusion. 【Methods】 The HDFN test and the antibody serological identification tests in the mother were performed. Several common high frequency antigens of maternal red blood cells (RBCs) were determined. IgG subtype coated on the RBCs of the newborn was determined. The phagocytic efficiency of the antibody was tested using the monocyte phagocytosis of sensitized erythrocyte by flow cytometry in vitro. Sanger sequencing of DI gene was performed in the mother, father and mother’s brother. The diluted maternal plasma was used for large scale screening of matching blood using IAT in Coomb’s gel card. 【Results】 Di(b-) phenotype was identified in the mother of the newborn and anti-Di^b (titer: 512) related HDN was detected in the newborn. IgG1 and IgG2 subtypes of anti-Di^b were detected and the rate of monocyte phagocytosis was 88.83%(74.7/84.09). The compatible blood was not detected in the maternal relatives. Subsequently, the newborn received the matching RBCs of two Di(b-) donors identified from 5 520 blood donors and discharged from the hospital. We screened out 17 Di(b-) donors out of 51 334 blood donors, indicating that the distribution frequency of Di(b-) among blood donors in Guangzhou was about 0.033% (17/51 334). 【Conclusion】 By serology and molecular biology methods, the newborn was identified with HDFN caused by anti-Di^b, and an effective large-scale screening method for Di (b -) rare blood types was established to find matching blood, which supported the establishment of rare Di(b-) blood database.

【Objective】 To establish a high-throughput detection method for ABCG2^*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. 【Methods】 The specific primers were designed and synthesized, the sample carrying homozygous ABCG2^*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2^*376T alleles were sequenced to confirm the accuracy of the HRM method. 【Results】 A HRM method, which can detect ABCG2^*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. 【Conclusion】 The HRM method can be used to accurately screen and type ABCG2^*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

【Objective】 To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. 【Methods】 Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. 【Results】 The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. 【Conclusion】 This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

[Abstract] [Objective] To further identify the RhD phenotype and RHD genotype in the individual who have RhD negative phenotype in the primary screening, and to analyze the effect of c. 801+2T>G mutation on RhD phenotype by minigene splicing assay. [Methods] The serologic test was performed for RhD phenotype identification and absorption-elution test was performed by using monoclonal anti-D. Sanger sequencing was used to analyze the sequence of RHD genes and the newly identified splicing site mutations of RHD genes were used to construct pSplicePOLR2G micro gene expression plasmids. By using an in vitro micro gene splicing system, the mRNA splicing results were detected and analyzed using agarose and capillary electrophoresis to predict their impact on RhD phenotype. [Results] The serological test results showed that the patient's blood type was RhD-negative, but the anti-D absorption-elution test was positive, indicating a Del phenotype. The rare genotype RHD^*(1227A/801+2G) was identified in this individual. The c. 801+2T>G was a novel mutation at 5'-splice site of intron 5. The minigene splicing assay showed that c. 801+2T>G resulted in a complete skipping of RHD exon 5 in the mature transcript, forming a transcript without exon 5. [Conclusion] An individual carrying a novel mutation c. 801+2T>G in the RHD gene was found to exhibit a Del phenotype, but also carry the Asian Del allele c. 1227G>A. It was speculated that the c. 801+2T>G mutation caused RhD negative or Del phenotype based on the results of minigene splicing assay in vitro.