【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 elucidate the prediction ability of monocyte monolayer assay(MMA)used in hemolytic dis-ease of fetus and newborn(HDFN)caused by IgG anti-M.Methods Plasma from eight pregnant women containing IgG an-ti-M were collected,and were divided into two groups(4 cases with HDFN,with severe clinical symptoms such as fetal hy-drops,and 4 cases without HDFN)according to the clinical outcomes.M antigen positive cells were sensitized with dithioth-reitol(DTT)treated plasma from eight pregnant women respectively.MMA was performed by coincubation with monocytes and sensitized M cells,along with negative and positive control set up.T-test was conducted to compare the difference in phagocytic efficiency between two groups.Results The phagocytic efficiency in group with HDFN were 15.37%,13.05%,9.17%and 24.50%respectively,with the mean value of 15.52%,while the group without HDFN were 8.74%,11.07%,5.12%and 6.23%respectively,with the mean value of 7.79%.There was no significant difference in phagocytic efficiency between two groups(P>0.05).The mean values of both groups were not significantly different from the negative control(P>0.05),but both were significantly lower than positive control(P<0.05).Conclusion The low phagocytic efficiency couldn't convince that the MMA is an effective predictor for the HDFN caused by IgG anti-M,indicating that another mech-anism might be responsible for it rather than monocyte phagocytosis.The assessment of the peak systolic velocity in middle cerebral artery of the fetal should be considered in the management for pregnant women who produce IgG anti-M to estimate the situation of fetal anemia.

Objective To investigate the distribution,phenotype and genotype of D-elute type(Del)in blood donors with RhD negative blood in Guangzhou,so as to understand the molecular biological background of DEL blood group in this area.Methods During the period from November 1,2021 to June 30,2022,the RhD-negative blood initially screened by saline method was confirmed by indirect anti-human globulin test(IAT)serology,and RhCE phenotype was determined by RhCE typing card.A total of 1 146 RhD-negative samples,including all RhD-negative samples with RhCE C+(n=459)and a randomly selected subset of RhCE C-(n=175),were subjected to adsorption-elution(Del)screening(a total of 634 sam-ples).DNA from Del-positive samples was extracted for real-time fluorescent PCR detection of the RHD gene c.1227 locus using high-resolution melting curve analysis(HRM).For samples without mutations detected at the RHD?1227 locus by HRM,restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP)was performed to amplify the prod-uct which was subsequently digested with Pst I enzyme and analyzed by electrophoresis to determine RHD gene haplotypes.Sanger sequencing was performed for exon sequencing(exon 1-10)of the RHD gene,and gene mutations were analyzed u-sing SeqMan software.Suspected Del-positive samples were subjected to RHD whole gene analysis using third-generation sin-gle-molecule sequencing technology.Results Among the 634 confirmed RhD-negative samples,229(36.1%)displayed Del phenotype,accounting for20%(229/1 146)of the total confirmed RhD-negative samples.The RhCE phenotypes of the 229 DEL cases were as follows:Ccee in 181 cases,CCee in 40 cases,CcEe in 7 cases,and ccEe in 1 case.HRM combined with RHD haplotype analysis showed that there were 170 cases with RHD gene as RHD?1227A/01N.01,32 cases with RHD gene as RHD?1227A/1227G,26 cases with RHD gene as RHD?1227A/1227A,and 6 cases with RHD gene as RHD?1227G/1227G(sequencing results included 1 case of weak D type 12,4 cases of D-,and 1 case of RHD?01EL.02).Con-clusion The individual genotype of DEL blood donors in Guangzhou area is mainly characterized by RHD?1227A/01N.01,and their RhCE phenotypes are all C+.HRM can be used as a molecular biology method for routine screening of Asian-type DEL blood type genes.

Objective To develop a solid-phase red cell adherence(SPRC A)reagent kit for unexpected alloantibody i-dentification.Methods A solid-phase carrier for bounding red blood cell(RBC)was prepared by coating microstrip with succinimide,then panel cell suspensions were dispensed to microwells to produce RBC monolayers,after that,1×PBS(pH7.40)was used to lysis the RBC monolayer to form stroma monolayer.Prior to the drying procedure,drying medium suspension was added to microwell to protect stroma monolayer.The solid-phase microplate was successfully produced after the stroma had been dried in refrigerator at 4℃.IgG anti-E and anti-Fya with low titer were tested every two months with the reagent kit to observe its stability.Seventy-two plasma samples containing unexpected antibodies and 152 normal plasma samples were determined with SPRCA reagent kit and column agglutination technique(CAT).Performances including sensi-tivity and specificity of the two reagents were compared by paired chi-square test.Results RBC stroma monolayer could be attached well on microwell bottom coated with succinimide.Drying medium suspension could provide effective protection for stroma monolayer.Both IgG anti-E and anti-Fya with low titer were determined well by the reagent kit during the storage peri-od.Of the 72 positive samples,70 were identified correctly by SPRCA kit and 71 by CAT[Sensitivity:97.22％(70/72)vs 98.61％(71/72)].Of the 152 negative plasma samples,149 were correctly detected by SPRCA kit and 152 by CAT[Spe-cificity:98.02％(149/152)vs 100％(152/152)].Paired chi-square test showed no significant differences between the two reagents(P＞0.05).Conclusion A SPRCA reagent kit for IgG unexpected antibody identification had been developed suc-cessfully.

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.

ObjectiveTo study the effect of Tanreqing injection （TRQ） on the pulmonary flora in the rat model of chronic obstructive pulmonary disease （COPD）. MethodWistar rats were randomized into control， model， and TRQ groups. The rats in other groups except the control group were treated by smoking combined with intratracheal instillation of lipopolysaccharide for the modeling of COPD. The TRQ group was intraperitoneally injected with TRQ （2 g·kg^-1）. At the end of the experiment， after blood collection from the abdominal aorta of the rats， the lung tissue was collected for hematoxylin-eosin and picric sirius red staining to reveal the pathological changes. The lung lavage fluid was collected， and the diversity and relative abundance of lung flora in different groups were analyzed by 16S rDNA amplicon sequencing. ResultThe lungs of the control group were normal， and those of the model group showed neutrophil infiltration， telangiectasia， lung hemorrhage and emphysema in individual cases， and thickening of collagen fibers in the trachea. Compared with the model group， the TRQ group showed significantly improved lungs and recovered collagen fibers. The MLI analysis showed that compared with the control group， the model group showcased increased alveolar space （P<0.01）， which was reduced in the TRQ group （P<0.05）. Compared with the control group， the model group showed increased wall thickness （P<0.01）， and the increase was attenuated in the TRQ group （P<0.01）. TRQ increased the Simpson index and altered the α diversity of pulmonary flora. The results of principal co-ordinate analysis showed that TRQ changed the β diversity and reduced the β diversity index of pulmonary flora. At the genus level， the model group showed increased relative abundance of g_Bacillus and g_Brevundimonas and decreased relative abundance of g_Pseudomonas， compared with the control group. After treatment with TRQ， the relative abundance of g_Stenotrophomonas increased， and that of g_Bacillus decreased. The LEfSe of differential taxa between groups showed that the modeling increased the relative abundance of g_Lachnospiraceae_NK4A136_group， and TRQ treatment increased the relative abundance of g_Rhodococcus and g_Stenotrophomonas. ConclusionTRQ can regulate the diversity of pulmonary flora and restore the balance of bacterial genera in the rat model of COPD， which may be one of the mechanisms of the prevention and treatment of COPD with TRQ.

The 19 th Chinese Symposium of Burns and Wounds was successfully held in Foshan of Guangdong Province from June 20 th to 22 nd in 2024. There were more than 700 delegates attending the academic event. The theme of the congress was expansion, integration and standardization, which could promote academic exchanges, multi-disciplinary fusion, and standardization of clinical treatment of burns and wounds. A total of nearly 200 famous experts and scholars had their speeches on the two-day keynote forum and special academic seminars including critical care, wound repair, scar prevention and treatment, rehabilitation nursing, and disciplinary integration sessions. The congress ended successfully with abundant fruits and friendship.

[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.

The proband was a 33-year-old pregnant woman (G4P1) who suffered spontaneous abortion in the first 3 months of pregnancy without a history of blood transfusion or transplantation. The fourth pregnancy was clinically diagnosed with threatened abortion, and a cesarean section was performed on June 28, 2023, at the Obstetrics and Gynecology Department of Dongguan Hospital of Traditional Chinese Medicine. During cross-matching tests, unexpected antibodies were detected in the proband′s plasma, which could not be specifically identified, and no suitable donor red blood cells could be found. The blood samples were sent to the Blood Transfusion Laboratory of Dongguan Blood Center. The laboratory used serology to identify the erythrocyte phenotype of the proband and confirmed the proband as having a rare p blood group. The unexpected antibody was identified as anti-PP1P K, and gene sequencing of the proband revealed that the new allele A4GALT* (c.100G>A+c.418_428delins) was homozygous, which is speculated to cause changes in the polypeptide chains p.Veral34ile and p.GERln140TRPFS *73, and inactivation of α1, 4-galactosyltransferase. At the same time, another new allele A4GALT*c.100G>A was found in family members, and it was predicted that the single change of p.Val34Ile caused by this mutation would not affect protein function or enzyme activity.

【Objective】 To study the effect of RHAG variants identified in Chinese population on mRNA splicing by minigene splicing assay(MSA) in vitro. 【Methods】 The pSplicePOLR2G minigene expression plasmids were constructed for 10 RHAG mutations with relatively high distribution frequency in Chinese population near splicing sites or synonymous mutations by analyzing the RHAG gene data in the KMxD database. Then, the wild-type and mutant plasmids were transfected into HEK 293T cells, and RNA was extracted 48 hours after transfection. After reverse transcription, specific primers were used for PCR amplification, and then agarose gel electrophoresis and capillary electrophoresis were performed to determine whether the mutations will affect the normal splicing of exons. 【Results】 MSA in vitro showed that 2 mutations (c.158-5delT, c. 807+ 3A>C) near the splicing site reduced the amount of normal transcripts slightly. The remaining 8 synonymous mutations(c.312G>A, c. 341+ 3G>A, c. 609C>T, c. 681G>A, c. 861G>A, c. 957T>A, c. 984T>C and c. 1139-7G>A) had no impact on the splicing of RHAG mRNA. 【Conclusion】 This study showed that RHAG gene was conservative in terms of splicing, and the mutations near splicing sites and synonymous mutations were less likely to cause abnormal splicing of RHAG gene.