Rh blood system classification and compatible blood transfusion / 中国输血杂志

【Objective】 To investigate the profiles of RhC, c, E, and e antigens and phenotypes in 4 704 inpatients from multiple regions, i. e. Nanning, Guangzhou and Shenzhen, and provide data information for compatibility blood transfusion of Rh blood group. 【Methods】 The Rh blood group antigens were detected by microcolumn gel cards from three manufactures. If the test and the control results are inconsistent, a third-party reagent would be used, and traditional tube method for confirmation if needed. The Chi-square test and Fisher's exact test were used to analyze antigen frequency and Rh phenotypes in each region. 【Results】 Among the 4 704 inpatients, the frequency of C, c, E, and e antigen was e(91.77%)>C(85.64%)>c(49.62%)>E(41.60%), and Rh phenotypes distribution was CCee(49.40%)>CcEe(27.53%)>Ccee(8.16%)>ccEE(7.74%)>ccEe(4.89%)>CCEe(0.96%)>ccee(0.83%)>CcEE(0.47%)>CCEE(0.02%). There were significant differences in Rh blood type distribution among Nanning, Guangzhou and Shenzhen(P<0.05). Differences in Rh phenotype distribution between male and female were noticed in Shenzhen(P< 0.05), but not in Nanning or Guangzhou. 【Conclusion】 The distribution of Rh blood group in Shenzhen, Nanning and Guangzhou were significantly different from each other, therefore regional characteristics should be considered when carrying out Rh-compatible blood transfusion, so as to guarantee the security of transfusion and reduce the incidence of unexpected antibodies.

Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

β-Lactam antibiotics promoting aging and clearance of RBCs would deteriorate the DIIHA / 中国输血杂志

【Objective】 To analyze the influence of β-lactam antibiotics on RBC aging and clearance by detecting various indicators of aging and clearance on RBCs, as well as the differences in phagocytosis for erythrocytes before and after drugs treated in vitro. 【Methods】 RBCs were treated by β-lactam antibiotics, including Penicillin, Cefepime, Cefoperazone and Ceftazidime, and the changing of phosphatidylserine (PS) and clearance related CD markers, including CD35, CD47, CD55 and CD59 on the surface of the RBCs, were detected by flow cytometry at 0h and 24h after drugs treatment. The proportion of acanthocytes by microscope also at 0h and 24h after drugs treatment was calculated. The phagocytosis of drug-treated RBC was detected by monocyte monolayer assay (MMA). Untreated RBCs were incubated in PBS by the same condition as a negative control.The influence of β-lactam antibiotics on RBC aging and clearance by all the results above was studied. 【Results】 Compare to the untreated RBCs, the drug treated RBCs showed a higher PS level on the cell surface. The results showed by percentage as following(0 h vs 24 h): Penicillin 9.42% vs 93.30%, Cefepime 3.88% vs 57.27%, Cefoperazone 4.71% vs 75.75% and Ceftazidime 3.05% vs 43.19%. The acanthocytes ratio was as following(0 h vs 24 h): Penicillin 7.33% vs 86%, Cefepime 2.67% vs 52.67%, Cefoperazone 3.33% vs 67.67% and Ceftazidime 3.33% vs 90.67%. On the opposite, the clearance related CD markers, showed an obviously lower level after drugs treated(0 h vs 24 h): CD35: Penicillin 7.36% vs 11.87%, Cefepime 0.14% vs 28.51%, Cefoperazone 11.85% vs 21.55% and Ceftazidime 7.63% vs 8.73%; CD47: Penicillin 1.22% vs 9.13%, Cefepime 1.80% vs 0.86%, Cefoperazone 0.08% vs 6.85% and Ceftazidime 1.54% vs 5.50%; CD55: Penicillin 14.46% vs 44.31%, Cefepime 17.27% vs 38.41%, Cefoperazone 19.28% vs 33.28% and Ceftazidime 14.62% vs 34.13%; CD59: Penicillin 4.71% vs 20.56%, Cefepime 4.03% vs 7.60%, Cefoperazone 5.91% vs 22.38% and Ceftazidime 5.93% vs 30.89%. Drug-treated RBCs attached more to monocytes than untreated RBCs. 【Conclusion】 The β-lactam antibiotics could induce the changing of PS and the clearance of related CD markers on surface of RBCs. They also could lead acanthocytes and make the RBCs more susceptible to phagocytosis by monocytes. The β-lactam antibiotics could promote the RBCs aging and clearance, which might deteriorate the DIIHA.

Screening of rare blood group Lu(a-b-) phenotype and study of its molecular basis in ethnic Han Chinese from Shanghai region / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the frequency of rare blood group Lu(a-b-) phenotype in a population from Shanghai region, and to explore the molecular basis of Lu(a-b-) by detecting the Lu and Lu relative mediator gene EKLF/KLF1.</p><p><b>METHODS</b>Donors from Shanghai region were screened for Lutheran blood group by monoclonal anti-Lub using serological methods. Individuals with Lu(b-) were determined Lua, P1 and i antigens. Fifteen exons of the LU gene and 3 exons of the EKLF/KLF1 gene for the identified Lu(a-b-) samples were amplified and sequenced.</p><p><b>RESULTS</b>Ten Lu(a-b-) donors were obtained from 44 331 donors from Shanghai region. No homozygous or heterozygous mutations were found in the LU gene, whilst 7 mutations in EKLF/KLF1 gene were identified in the 10 samples.</p><p><b>CONCLUSION</b>The frequency of rare Lu(a-b-) blood group in Shanghai was approximately 0.02%, and all the individuals had an In(Lu) phenotype. The molecular basis of such samples may be related to mutations in the EKLF/KLF1 gene.</p>

Study on CIX-M type ESBLs-producing Escherichia coli and Klebsiella pneumoiae in Guangzhou / 中华检验医学杂志

Objective To study phylogenies, epidemiology and genetic environment of CTX-M type of ESBLs produced by Escherichia coli and Klebsiella pneumoniae isolated from nine hospitals in Guangzhou. Methods The phylogenies of CTX-M type of ESBLs were analyzed by PCR Genetic environment of CTX-M-15 encoding gene (bla_(CTX-M-15)) were investigated by conjugation test and plasmid analysis. The clonal relationship of strains producing CTX-M-15 was determined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Results A total of 361 ESBLs-producing isolates of Escherichia coli and Klebsiella pneumoniae were collected. 67.3% of ESBLs strains were detected to produce CTX-M-type ESBLs, and the commonest genotypes in Escherichia coli and Klebsiella pneumoniae were CTX-M-14 (35.4% and 28.3%), CTX-M-15(21.5% and 26.1%) EBIC-PCR products of all CTX-M-15-producing strains show 39 strains of Escherichia coli were classified into 27 genotypes while 43 strains of Klebsiella pneumoniae were divided into 30 genotypes. Furthermore, the genotypes of CTX-M-55, CTX-M-19, CTX-M-27, with ceftazidime-hydrelyzing activity, were detected in this study. The great majority of bla_(CTX-M-15) genes were found to locate on a 65 000 bp-conjugative plasmid, and there was no blaTEM-1, bla_(OXA-1), blaDSA-1 or aac (6')-Ib-cr gene coexisted on the plasmid, ISEcp1-like insertion sequences, relative to mobilization of bla_(CTX-M-15) gene, were detected in all bla_(CTX-M-15) positive strains, and the distances between the end of ISEcp1-like insertion sequences and the start cedon of bla_(CTX-M-15) were equal, with 48 base pairs. Conclusion CTX-M-14 is still the most common genotype of ESBLs in Guangzhou, but high prevalence of CTX-M-15 ESBLs hydrolyzing ceftazidime already appears in south China.

Establishment of platelet antigen panel and its application in the identification of platelet specific antibodies / 中华检验医学杂志

Objective To establish the platelet antigen panel for identifying the specificity of platelet antibodies which cause platelet transfusion refractoriness and neonatal alloimmune thrombocytopenia and provide evidence for clinical therapy and platelet genotyping research.Methods Based on the frequency distribution of human platelet alloantigen (HPA)-1 to HPA-16 gene in China, the frequencies of HPA-1 to HPA-6,HPA-15 alleles in blood group O donors were genotyped by the polymerase chain reaction with sequence-specific primers (PCR-SSP) method, and suitable donors were chosen to establish platelet-specific antigen panel.Using the established platelet-specific antigen panel, the specificity of platelet antibodies caused by alloimmune reaction was identified by using simplified sensitized erythrocyte platelet serology assay (SEPSA).Results Eleven ptatelet donors with blood group O were chosen to establish platelet-specific antigen panel which can identify specificity of HPA-1 to HPA-6, HPA-15 antibodies.One case of HPA-4b (Penb) and two cases of HPA-15a (Govb) platelet specific antibodies were detected in 1 120 samples.Conclusion Identifying the specific platelet antibodies using platelet specific antigen panel has profound significance on increasing the safety and effectiveness of clinical platelet transfusion and prevention of neonatal alloimmune thrombocytopenia.

Different KEL gene mRNA transcripts in reticulocyte and non-reticulocyte cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the difference of the transcripts between reticulocyte and non-reticulocyte cells in human blood.</p><p><b>METHODS</b>Genomic DNA, reticulocyte RNA and total RNA of K-, K+ and Kell-null(K0) were extracted, then PCR, reverse transcription-PCR(RT-PCR) and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene mRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry.</p><p><b>RESULTS</b>In reticulocyte, only one normal KEL transcript faithful to the genomic structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry.</p><p><b>CONCLUSION</b>Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that reticulocyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.</p>

Study on B(A) phenotypes and identification of novel B(A)641 allele in chinese population / 中国输血杂志

C mutation. All 8 samples displayed the B(A) phenotype. Their real genotypes were B(A)/O. Conclusion Three B(A) alleles in the Chinese Han population were detected. Two alleles,B(A)700,B(A)640 were reported previously. One novel allele B(A)641, was first identified in this study.

Investigation of H antigen on AB subgroup RBCs / 中国输血杂志

Objective To find the rule of the distribution of H antigens on AB subgrouperythrocytes.Methods ABO subgroups were confirmed by using serological and molecular biology (PCR-RFLP) methods. AB subgroup with strong H was defined as red cell agglutination by anti-H of 2 scores or more higher than that of B cells.Results Strong H was only found in certain AB subgroups ,CisAB(100%),B(A)(100%), AxB(46.2%) and A2B(43.6%), but seldom in others among Chinese population.Conclusion The fact that H type-3, which comes from A type-2, can hardly be transferred by B and weak A glycosyltransferase can help to explain why some ‘strong’ H combines with ‘weak’ A in AB erythrocytes. Why only little H can be found in 53.8% AxB, 56.4% A2B and all A3B, AmB subgroup samples still cannot been explained.

Molecular study on Ax subgroup in Chinese population / 中国输血杂志

Objective To study Ax subgroup’s molecular characteristics in Chinese Han population. Methods Eight samples suspected as Ax subgroup were analyzed and duplex PCR RFLP test was used to determine the primary ABO genotypes. These samples were then analyzed by another PCR RFLP test to identify whether there was an nt646 “T” to “A” mutation within the exon 7 of ABO gene, which was a known mutation related to most Ax phenotypes. Samples with discrepancy between serological and gene typing were chosen for further T A cloning and sequence analysis. Results Four out of all tested samples had the known nt646 “T” to “A” mutation. An A *weak01 allele including nt407 and nt467 “C” to “T” mis sense mutation was detected in this study. Moreover, a novel Ax allele with a new single nucleotide C to T mutation was detected at nt745. Another 2 unrelated samples were suspected as AxB through serological test, both of which contained higher quantities of anti A and showed strong agglutination with anti H. And their initial genotypes were BO, and sequence analysis clarified that both had normal O gene and novel nt640 “A” to “G” mutation in their B alleles. Conclusion The novel Ax alleles, one kind of novel B(A) allele and one A *weak01 allele in Chinese Han individuals,have been detected. B(A) phenotypes should have their molecular biology bases as well as other ABO subgroups.