Objective To analyze the correlation between the expression of soluble glycoprotein A(GPA)in plasma of healthy blood donors and anti-M and anti-"Mia"antibodies.Methods Plasma from healthy donors from February 9,2022 to February 15,2023 was collected:irregular antibody-negative NN type(group Ⅰ,n=118)and MM type(group Ⅱ,n=51),anti-M antibody positive NN type(group Ⅲ,n=145)and anti-"Mia"antibody positive companion type(group Ⅳ,n= 87),the GPA content in plasma of different individuals in 4 groups was detected,and the difference in GPA expression was analyzed by t-test.Results The average plasma GPA contents in groupsⅠ,Ⅱ,Ⅲ and Ⅳ were 9.941±0.252,10.97±0.256,5.139±0.129 and 4.28±0.139ng/ml,respectively.The average GPA content of groups Ⅰ and Ⅱ was higher,and the average GPA content of groups Ⅲ and Ⅳ was lower,and the differences were statistically significant(all P＜0.01).Conclusion The GPA content in plasma of healthy donors with anti-M and anti-"Mia"antibodies was significantly lower than that of the antibody-negative group.The results of this study lay a foundation for further investigation of whether GPA in plasma has the ability to neutralize anti-M and anti-"Mia"antibodies,improve disease diagnosis and safe blood transfusion.

Objective:To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-).Methods:Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 ( n=1 568) and a pedigree with difficult cross-matching ( n=3) were selected as the study subjects. Serological methods were used for proband′s blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband′s RHD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors. Results:The proband′s ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband′s genotype was ABGG201N.01/ABGG201N.01 [homozygous c. 376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰. Conclusion:A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.

【Objective】 To investigate the family inheritance of α-Thalassemla gene and the risk of severe anemia in neonates caused by cold IgG anti-M. 【Methods】 ABO, Rh, MN blood groups and the specificity of unexpected antibody were identified by blood group serology. The IgG subtype and antibody titer of anti-M antibody were detected. The etiology of neonatal hemolytic disease was identified by three tests and α-Thalassemla gene diagnosis. 【Results】 Family investigation showed that father was B, CCDee, MN with no α-Thalassemla gene detected; Mother B, CcDee, NN, carrying α-Thalassemla gene; both the proband and his brother were B, CCDee, MN, carrying α-Thalassemla gene. Cold IgG anti-M was present in plasma of both the mother and the proband. The titer of the mother was 128 and that of the proband was 64. The subtype of IgG anti-M was IgG1 and IgG3. The direct anti-globulin test, release test and free test of the proband and his brother were negative, and the diagnosis was severe anemia and hemolysis caused by α-Thalassemla combined with cold IgG anti-M. 【Conclusion】 The direct antiglobulin test of neonatal hemolytic disease caused by IgG anti-M can be negative or weakly positive, and α-Thalassemla gene could be hereditary in families. The presence of α-Thalassemla gene can cause anemia, hemolysis and splenomegalysis in neonates, which could be aggravated when accompanied by cold-type IgG anti-M. In the presence of high-valency IgG antibody in plasma, blood exchange combined with transfusion can improve the curative effect.

【Objective】 To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. 【Methods】 Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. 【Results】 Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. 【Conclusion】 The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.

【Objective】 To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure. 【Methods】 The 9 blood samples with rare RhD variants were collected from 210 644 blood donors of Shenzhen Blood Center. Regular serological assaying was used for determination of Rh type for the 9 samples. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and to screen the antibodies. D-screen reagent was sued to analyze the RhD epitopes of the samples. RHD zygosity testing of the samples was detected by PCR-SSP. The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced. The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN, SIFT, PolyPhen-2 and MutationTaster software. Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD. 【Results】 A total of 9 individuals with rare RhD variants were identified as follows: RHD^*weak D type 25, RHD^*weak D type 50, RHD^*weak D type 95, RHD^*weak D type 12, RHD^*weak D type 128 and four novel RHD alleles. The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples. 【Conclusion】 Five rare and four novel RHD alleles have been identified. Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles. Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.

Lung cancer is the most frequent cancer and the leading cause of cancer death all around the world. Anti-programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) therapies have significantly improved the outcomes of non-small cell lung cancer (NSCLC) patients in recent years. However, the objective response rate in non-screened patients is only about 20%. It is very important to screen out the potential patients suitable for immunotherapy. Immunohistochemical staining of tumor tissue biopsies with PD-L1 antibodies can predict the therapeutic response to immunotherapy to some extent, but it still has some limitations. Recently some clinical studies have shown that PD-L1 expression in circulating tumor cells (CTC-PD-L1) is a potential independent biomarker and may provide important information for immunotherapy in NSCLC. This article will review technology for CTC-PD-L1 detection and the predictive value of CTC-PD-L1 for immunotherapy in NSCLC and review the latest clinical research progress.

【Objective】 To analyze the blood samples sent by hospitals in Shenzhen to solve ABO cross-match incompatibility during 2011 to 2020, so as to find corresponding solutions to improve the efficacy of blood transfusion. 【Methods】 The clinical data of 1 770 cases of cross-match incompatibility in our laboratory from January 2011 to December 2020 were collected and reviewed. The causes of cross-match incompatibility were analyzed, the types of unexpected antibodies were determined. The overall incidence of antibodies was evaluated by statistical method of classified variables. The safety of blood transfusion was safeguarded by ABO homotype plus cross-matching compatibility. 【Results】 1) The 1 770 samples, presenting cross-matching incompatibility, involved 956 patients. The average number of cross-matching per patient from 2011 to 2015 was 1.32(307/232), which increased from 1.27(103/81) in 2016 to 2.23(286/128) in 2018, and remained stable in 2019 and 2020. 2) Among 956 patients, auto-and/or allo-antibody in plasma were yielded in 90.38%(864/956), including auto-antibody plus alloantibody in 42.26%(404/956), solo auto-antibody in 20.71%(198/956) and solo allo-antibody in 27.41%(262/956). Up to 20 kinds of specific allo-antibodies were detected, belonging to 8 blood groups. Among them, 70.82%(551/778) were Rh blood group, such as anti-E(37.15%)>anti-c(20.95%)>anti-C(5.27%)=anti-e(5.27%)>anti-D(2.19%), followed by MNS [11.40%(112/778)], Kidd [5.66%(44/778)], Leiws [3.21%(25/778)], Duffy [1.80%(14/778)], Diego [1.03%(8/778)], P1 [0.39%(3/778)] and H [0.26%(2/778)]. 3) 86%(37/43) of multiple transfusion recipients, aged below 20 years old, were thalassemia, and 1-4 kinds of allo- and/or auto-antibody were yielded. 【Conclusion】 The cross-matching incompatibility were mainly caused by allo- and/or auto-antibodies, which may be induced by blood transfusion, pregnancy or autoimmune diseases such as autoimmune hemolytic anemia.Those suspicious blood samples in clinical should be sent to blood group reference laboratory for further determination, in order to ensure the safety and efficacy of blood transfusion.

【Objective】 To analyze the polymorphism of erythrocyte membrane blood group glycoprotein A (GPA) related gene GYPA in high and low endemic population for clonidia sinensis infection, aimed at investigating the correlation between erythrocyte transmembrane glycoproteins and clonorchis sinensis infection. 【Methods】 From Dec 2019 to Jun 2020, anticoagulant blood samples were randomly collected in WuMing district (n=700) and GuiGang district (n=500 ) of Nanning city, and the IgG antibody to clonorchis sinensis in plasma was detected, and the DNA of leukocyte was extracted. The full-length exon and partial intron of GYPA gene were sequenced, mutations were characterized by gene cloning, and the risk of infection was calculated by chi-square test. 【Results】 The yield rate of IgG antibody was 62.7% (439/700) in WuMing district and 3.4% (17/500) in GuiGang district(P<0.05). The insertion of base C at the 54th position of intron-2 in GYPA gene caused the reading frame shift. The mutation was presented in 23.9% (105/439) and 17.6% (3/17) of the population with clonorchis sinensis exposure in WuMing and GuiGang area, respectively, while 49.4% (129/261) and 54.7% (264/483) in the negative population, respectively (P<0.05). 【Conclusion】 The infection rate of clonorchis sinensis in WuMing district was higher than that in GuiGang district. The mutation rate of reading frame shift caused by the insertion of base C at the 54th position of GYPA intron-2 was much lower in the positive population of clonorchis sinensis infection than the negative population, suggesting that the mutation is a protective gene in the negative population of clonorchis sinensis infection. It is necessary to study the mechanism of clonorchis sinensis infection and the mutation point of this gene in order to facilitate the early diagnosis of disease, blood transfusion management, treatment and prevention.

Objective    To explore the diagnostic value of circulating tumor cells (CTC) measured by magnetic nanoparticle method in lung cancer. Methods    (1) We measured binding capability of A549 or NCI-H1965 cell lines with recognition peptide and capture efficiency by adding tumor cells into the whole blood of healthy human. (2) We measured CTC of 34 patients suspected with lung cancer, and the counting results of CTC were compared with the following pathological results. Results    (1) The binding capability was 80.0%±6.0% for A549 and 70.1%±4.8% for H1957, while the capture efficiency was 57.3%±7.0% for A549 and 37.3%±6.1% for H1975. (2) CTCs were identified in 71.9% of patients with lung cancer. The specificity was 83.3%, and area under receiver operating characteristic (ROC) curve was 0.792 (P=0.003). Conclusion    CTC measured by magnetic nanoparticle method has promising application in the diagnosis of lung cancer.

Objective    To explore the efficacy of a novel detection technique of circulating tumor cells (CTCs) to identify benign and malignant lung nodules. Methods     Nanomagnetic CTC detection based on polypeptide with epithelial cell adhesion molecule (EpCAM)-specific recognition was performed on enrolled patients with pulmonary nodules. There were 73 patients including 48 patients with malignant lesions as a malignant group and 25 patients with benign lesion as a benign group. There were 13 males and 35 females at age of 57.0±11.9 years in the malignant group and 11 males and 14 females at age of 53.1±13.2 years in the benign group. e calculated the differential diagnostic efficacy of CTC count, and conducted subgroup analysis according to the consolidation-tumor ratio, while compared with PET/CT on the efficacy. Results     CTC count of the malignant group was significantly higher than that of the benign group  (0.50/ml vs. 0.00/ml, P<0.05). Subgroup analysis according to consolidation tumor ratio (CTR) revealed that the difference was statistically significant in pure ground glass (pGGO) nodules 1.00/ml vs. 0.00/ml, P<0.05), but not in part-solid or pure solid nodules. For pGGO nodules, the area under the receiver operating characteristic (ROC) curve of CTC count was 0.833, which was significantly higher than that of maximum of standardized uptake value (SUVmax) (P<0.001). Its sensitivity and specificity was 80.0% and 83.3%, respectively. Conclusion     The peptide-based nanomagnetic CTC detection system can differentiate malignant tumor and benign lesions in pulmonary nodules presented as pGGO. It is of great clinical potential as a noninvasive, nonradiating method to identify malignancies in pulmonary nodules.