Objective: To establish a method for the genotyping of fetal M blood group antigen by extracting cell-free fetal DNA (cff-DNA) from maternal plasma, so as to guide the management of M antigen-negative pregnant women with IgG anti-M antibody during pregnancy. Methods: A realtime fluorescent quantitative PCR (realtime PCR) method was established. The specificity and sensitivity of the method were validated by dilution of genomic DNA. Subsequently, a total of 12 M antigen-negative pregnant women were enrolled. The cff-DNA was extracted from maternal plasma, and fetal M antigen genotyping was performed by realtime PCR. Fetuses were classified as M-positive or M-negative according to the presence or absence of amplification curve. The accuracy of the method was validated by comparing fetal M antigen genotyping results with the serological results using the cord or peripheral blood of the neonate at birth. Results: Among the 12 M antigen-negative pregnant women, anti-M was detected in five cases, of which four cases had IgG anti-M, and one case had fetal anemia. The results of fetal M antigen genotyping showed that 9 cases were M-positive (9/12, 75%) and 3 cases were M-negative (3/12, 25%). Serological results of blood samples collected after birth from four M-positive fetuses and one M-negative fetus were consistent with the genotyping results. Conclusion: We have, for the first time, established a non-invasive prenatal genotyping method for fetal M antigen using maternal plasma cff-DNA, and preliminarily demonstrated the feasibility of this method.

Objective: To conduct screening for rare blood types within important blood group systems for the Chinese population, such as Rh, Duffy, Kidd, P1Pk, Diego, and MNS, in the Guangzhou region, and to establish a corresponding rare blood type database and physical repository. Methods: The saline medium microplate method was used to screen blood donors with the ccDEE phenotype combined with either Jk(a-) or Jk(b-). The polybrene microplate method was employed to screen for donors with Fy(a-), s(-), Lu(b-), Di(b-), k(-), and p phenotypes. The urea lysis microplate method was applied to screen for the Jk(a-b-) phenotype. A high-resolution melting (HRM) curve method was established for screening some donors with the Di(b-) phenotype. Subsequently, expanded phenotyping of antigens in the Rh, Kidd, MNS, Duffy, P1Pk, Lewis, Kell, and Lutheran blood group systems was performed on identified rare blood type donors using monoclonal antibodies. The test results are entered into the Rare Blood Type Bank Management System of the Guangzhou Blood Center, enabling functions such as confirmation reminders and cryopreservation storage when the donor donates again. Red blood cells of rare blood types are processed into frozen red blood cells for long-term storage. Results: Among voluntary blood donors, 16 cases of the ccDEE combined with Jk(a-) phenotype were identified (0.221 7%, 16/7 216); 10 cases of the ccDEE combined with Jk(b-) phenotype (0.138 6%, 10/7 216); 78 cases of the Fy(a-) phenotype (0.169 5%, 78/46 012); 39 cases of the Lu(b-) phenotype (0.138 2%, 39/28 214); 31 cases of the s(-) phenotype (0.081 8%, 31/37 913); 22 cases of the Di(b-) phenotype (0.029 9%, 22/73 691); 30 cases of the Jk(a-b-) phenotype (0.010 1%, 30/298 250); and 1 case of the k(-) phenotype (0.001 3%, 1/77 382), which was further identified as KELnull phenotype (K0). No p phenotype donors were identified (0/88 528). A total of 228 units of frozen red blood cells were prepared. The screening results were compared and analyzed with rare blood type data from other regions. Conclusion: This study, through a combination of different screening methods, significantly improved the efficiency of rare blood type screening while remaining cost-effective. By conducting large-scale screening and performing data informatization processing, a database and physical repository of rare blood types in the Guangzhou region were successfully established. This provides a strong guarantee for the timely supply of blood to patients with difficult-to-match and rare blood types in the region, effectively enhances the level of transfusion safety in the region, and offers a practical paradigm for constructing a comprehensive blood transfusion support system.

[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.

【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 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.

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 antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. 【Methods】 ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. 【Results】 The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. 【Conclusion】 In this case, the development of anti-Jr^a in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

【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 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.