[Establishment and Application of a Method for Determination of Glycosyltransferase Activity].

OBJECTIVE: To establish a method for determination of glycosyltransferase and to explore the enzyme A, B glycosyltransferase activity in human serum so as to lay the foundation for the determination of enzyme level and enzyme activity. METHODS: The glycosyltransferase activity kit was used to draw phosphate standard curves in our laboratory. The A and B glycosyltransferase activity were determined by the standard curves. RESULTS: The standard curves (y=2671.3x-0.596 R2=0.9998) for determing glycosyltransferase activity suitable for use in our laboratory were drawn. At the same time the method was set up for determination of A, B glycosyltransferase in human serum. CONCLUSION: The establised method of the determination of glycosyltransferase is suitable for common type of enzyme activity and suitable for the A, B glycosyltransferase in human serum.

[Polymorphism of M, N Allele in MN Blood Group of Chinese Population].

OBJECTIVE: To detect the base sequences of all exons and part of introns in the GYPA gene of the glycophorin GPA and to investigate the polymorphism of M, N alleles in Chinese population. METHODS: A total of 225 blood sample were randomly colleeted from unrelated Chinese volunteers and were detected by serology techniques. The primers were designed by self, the seguencing of GYPA gene related with sample exon 1-7 full length sequences of bases and intron-1-7 partial sequence was performed, the polymorphism of M, N gene mutation in mucleotide sequence was analysed. RESULTS: The results of M and N genotyping were in agreement with the results of serological detection. The 23rd base of intron-2, the 55th base of intron-3, the 63rd base of intron-4, the 55th, 189th and 190th base of intron-6, the 712th base variation of exon-7 in the gene M and N were used to subdivide the gene M and N into the mutant M103, M201, M202, N101, N102, N103, N104, and N201. At the same time, it was found that 42th and 54th base were mutated, the base T was inserted between 59th and 60th base in the intron-2, the new mutations occurred in the alleles 28, 29, 65 and 102 in intron-3 in this study. CONCLUSION: The polymorphism of the the Chinese population's GYPA gene occurs in all the exons and partly in the introns. The gene polymorphism of M and N blood group in Chinese population might provide the theoretical basis for the studies of clinical blood transfusion, human population genetics and molecular biology.

[Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

[Analysis of HLA haplotype frequency and linkage disequilibrium in patients with acute lymphoblastic leukemia from Northern Chinese Han].

OBJECTIVE: To analyze the difference between the frequencies of HLA-A-B, B-DRB1 and A-B-DRB1 haplotype, as well as their linkage disequilibrium pattern in patients with acute lymphoblastic leukemia(ALL) and healthy controls from Northern Chinese Han. METHODS: The frequencies of HLA-A-B, B-DRB1, A-B-DR haplotypes and linkage disequilibrium were estimated by Expectation Maximization method based on the genotypes of 643 patients with ALL and 2 0359 unrelated healthy donors, and the statistical significance between the two groups were estimated by chi-square test. Linkage disequilibrium was analyzed with population genetic methods. RESULTS: The most common HLA-A-B, B-DRB1, and A-B-DR haplotypes were A30-B13, A2-B46, A33-B58, B13-DR7, B46-DR9, B52-DR15, B58-DR17, A30-B13-DR7, A33-B58-DR17 and A1-B37-DR10 in both groups. The frequencies of A30-B13, A2-B46, A33-B44, B13-DR7, A30-B13-DR7 and A2-B46-DR9 haplotypes and linkage disequilibrium value were significantly decreased (P<0.05) in the patient group than that in the control group. On the other hand, the frequencies of A2-B52, A31-B61, A24- B8, B60-DR9, B27-DR4, B52-DR14, B44-DR17, B27-DR12 and A11-B27-DR12 haplotypes and linkage disequilibrium value were significantly increased (P<0.05) in the patient group than that in the control group. CONCLUSION: There are some common and positive linkage disequilibrium haplotypes in both the ALL patients and the healthy donors in Northern Chinese Han. Interestingly, some haplotypes and their linkage disequilibrium patterns had significantly different distributions between the two groups. The study provided basic data for the relationship of ALL and HLA haplotype and for finding the HLA-A, B, DR matching donors.

[Establishment of the method to induce and measure human IL-2 in vitro].

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.

[Comparison between genotyping and serological phenotyping in RhCE blood group].

OBJECTIVE: To genotype the RHCE gene of Hans, Xinjiang's Uigurs and Kazakstans in China, and to compare the results of RHCE genotyping with that of RhCc/Ee phenotyping. METHODS: RHCE genes of 98 Hans with RhD positive and 230 Hans, 72 Uigurs and 18 Kazakstans with RhD/RHD negative were genotyped with PCR-sequence specific primer (SSP) technique. RESULTS: The results of RHE/RHe genotyping from samples with RhD positive and negative were in accord with that of phenotyping. It would result in 4.44% error using C-->G polymorphism at nt48 of RHCE gene to genotype RHCE, and 4.05% failure of detection using the 109 bp insertion to detectRHCE gene in Chinese Hans. The results of RHE/RHe genotyping in unrelated 72 Uigurs and 18 Kazakstans with RhD phenotype were consistent with that of phenotyping, and false positive and false negative were not found in genotyping in Uigurs and Kazakstans tested. CONCLUSION: The results of RHE/RHe and RHc genotyping were correct with PCR-SSP and accordant with that of phenotyping. Using the C48G polymorphism in exon 1 of RHCE to genotype RHC gene would result in false positive resulting from RHc mutation at this locus, and using the 109 bp insertion to genotype RHC gene would result in false negative because of the absence of the 109 bp. Therefore it is necessary to genotype RHC gene using more than two polymorphic loci.

[Effect of platelet CD42a modification by mPEG-SPA with different molecular masses].

OBJECTIVE: To observe the effect platelet antigen modification by mPEG-SPA with different molecular masses. METHODS: Platelet CD42a was modified by 5 kD and 20 kD mPEG-SPA, respectively, and the fluorescence intensity of CD42a was detect by flow cytometry and the three-dimensional structure of CD42a simulated to analyze the distribution of lysine in CD42a molecule. RESULTS: After platelet CD42a modification by 5 kD and 20 kD mPEG-SPA, the fluorescence intensity of CD42a decreased sharply by 85.54% and 88.65%, respectively, and multiple lysine regions were identified on the surface of CD42a molecule. CONCLUSION: Both 5 kD and 20 kD mPEG-SPA allow useful modification of platelet CD42a, but 20 kD mPEG-SPA is more advantageous than 5 kD mPEG-SPA.

[A comparative research on RHD gene structures of Chinese Han and Uigur population].

OBJECTIVE: To research comparatively on the RHD gene structures in unrelated RhD negative individuals of Chinese Uigur and Han population. METHODS: The upstream, downstream, hybrid box and 10 exons of RHD gene were detected with sequence specific primer-PCR technique. RESULTS: The results showed the genotypes of RhD negative individuals to have the significant difference between Chinese Uigur and Han population, that 94.44% Uigur individuals were with RHD(-)/RHD(-) genotype but just 61.40% Han population were with this genotype(94.44% versus 61.40%, P<0.01); 2.78% Uigur individuals were with RHD(+)/RHD(-) genotype but 34.21% Han population were with this genotype(2.78% versus 34.21%, P<0.01). However, there was significantly no RHD(+)/RHD(+) genotype difference between Chinese Uigur and Han population(2.78% versus 4.39%, P>0.05). In 78 cases of RhD negative Chinese Hans with single RHD gene, of which the RHD gene structure showed that 53(67.95%) cases were RHD(1-10) allele(of 53 RHD(1-10) alleles, 14 alleles were unexpressed); 15(19.23%) were RHD-CE(2-9)-D(2) allele; 5(6.41%) cases were RHD-CE(2-7)-D(2) allele; 2(2.56%) were similar to RHD-CE(3-6)D allele; 1(1.28%) case was RHD-CE(5-6)-D allele; and 2(2.56%) were RHD-CE(6)-D or point mutation respectively. Of 2 RhD negative Chinese Uigurs with RhD(-)/RHD(+) genotype, one carried RHD(1-10) allele, another carried RHD-CE(2-9)-D(2) allele. CONCLUSION: The most frequently unexpressed RHD alleles were RHD-CE(2-9)-D(2), RHD(1-10) and RHD-CE(2-7)-D(2) respectively in Chinese Han population who carried single RHD allele with RHD(-) phenotype and RHD(+) genotype. It showed the confluent character of RH gene in Chinese Han and Uigur population that there existed unexpressed RHD-CE(2-9)-D(2) allele in Chinese Uigur nationality, which was infrequent in Chinese Uigur population but frequent in Chinese Han population.

[Comparison of Rhesus boxes in Hans and Uighurs].

OBJECTIVE: To study the difference and similarity between Hans and Uighurs in regard to Rhesus box and its significance. METHODS: The sequence specific primers of upstream, downstream and hybrid Rhesus boxes were designed on the basis of RHD gene sequence. The upstream, downstream and hybrid Rhesus boxes were determined by polymerase chain reaction-sequence specific primer(PCP-SSP) and mismatched PCR. RESULTS: The percentage of RHD-/RHD-, RHD+/RHD- and RHD+/RHD+ genotypes ascertained in the unrelated Hans with RhD(-) were 61.40%, 34.21% and 4.39% respectively, while those in the unrelated Chinese Uighurs with RhD(-) were 94.44%, 2.78% and 2.78% respectively. Furthermore, all 6 cases of some other minorities were RHD-/RHD- types. The percentage of RHD-/RHD- and RHD+/RHD- genotypes ascertained in the unrelated Chinese Uighurs were significantly higher than those in Chinese Hans (P < 0.01), whereas no statistically significant difference in the percentage of RHD+/RDH+ genotype between the two groups was observed (P > 0.05). CONCLUSION: The Rh blood group of Uighurs in Xingjiang possesses both Oriental and Caucasian characteristics, which embodies a special ethnical aspect of the Chinese nation and is in accord with the anthropologic research results.

[Determination of human RHD gene rhesus box and its significance].

The aim was to determine RHD zygosity, further to investigate genetic structure of RHD gene, and to predict hemolytic disease of newborn (HDN). The upstream box, downstream box, and hybrid box of RHD gene were determined by PCR-SSP with 4 primers under the same conditions. The results showed that only hybrid box could be determined in RHD(-)/RHD(-) homozygosity. All the upstream box, downstream box, and hybrid box could be determined in RHD(+)/RHD(-) heterozygosity, while upstream box and downstream box except hybrid box could be determined in RHD(+)/RHD(+) homozygosity. Out of 50 cases of RhD(+), 5 cases (10%) were RHD(+)/RHD(-) heterozygosity, and the others (90%) were RHD(+)/RHD(+) homozygosity. 54 cases (55.1%), 36 cases (36.7%) and 8 cases (8.2%) were RHD(-)/RHD(-) homozygosity, RHD(+)/RHD(-) heterozygosity, and RHD(+)/RHD(+) homozygosity respectively in 98 unrelated cases of RhD(-) Chinese Hans. 2 cases of weak D were proved to be RHD(+)/RHD(-) heterozygosity. Out of 16 D(el) types, the upstream box, downstream box, and hybrid box could be determined in 10 cases (37.5%) and the upstream box and downstream box except hybrid box could be determined in 6 cases. Results detecting of RHD 10 exons in above samples proved the correctness of the method. It is concluded that the method is suitable for clinical application with its simplicity and veracity. There are many noneffective RHD genes (44.9%) in Chinese Hans with RhD(-) phenotype.

[Method of detection of soluble HLA-I and soluble HLA-I level alteration in storage blood].

Aim of this study was to develop the detection method of soluble human leukocyte antigens I (sHLA-I) and to explore sHLA-I level alteration in storage blood and its significance. sHLA-I level in sera of 60 Guangdong normal individuals and sHLA-I concentration in blood components from 20 donors quantitatively were detected by sandwich ELISA. The results showed that sensitivity of this assay was 2.84 ng/ml. Coefficients of variation were 5.80% within assays and 9.00% between assays respectively. The recovery rate was >/= 98.57%. The sHLA-I level of normal individuals in Guangdong was (699.54 +/- 360.10) ng/ml. sHLA-I in red blood cells stored for 28 days and in random-donor platelets were significantly higher than that in other blood components and their amount was proportionate to the number of residual donor leukocytes and to the length of storage. In conclusion, sandwich ELISA assay for detection of sHLA-I is a sensitive, specific and stable technique. Blood components with different concentration of sHLA-I may be chosen for clinical transfusion.

[ABO blood group typing for infants and its application for clinical transfusion].

To study the correct method for determining ABO blood types in infants and its influencing factors, blood types of 33 infants under 6 months old were determined by routine serological method, micro-column gel typing system and PCR-SSP genotyping method. Of the 33 cases with discrepant results of ABO blood type by different methods, the blood types of 32 cases were discrepant between red cell and serological typings in the routine serological method, and a false coincidence in 1 case was caused by bacterial infection resulting in B-like antigen. Correct blood typing was obtained in 27 cases with a correct rate of 84.4% (27/32) by using micro-column gel typing system. PCR-SSP method gave correct results in all of 33 cases. There was a significant difference between the results of micro-column gel typing system and PCR-SSP. It is concluded that to determine ABO blood type for infants < 6 months old, it is recommended to adopt micro-column gel typing system method, and what must be taken into account is the possible false coincidence caused by bacterial infection resulting in B-like antigen. In micro-column gel typing system, if the results of red cell and serological typing are identical, the principle is that blood transfusion must be performed with same ABO blood type between recipient and donor. If not, washed O red blood cells should be used for infants, and then change to transfusion with identical blood group according to PCR-SSP typing results.

[Methoxypolyethylene glycol-modified HLA-A2 antigen on lymphocytes].

OBJECTIVE: To modify the HLA-A2 antigen on the lymphocytes with methoxypolyethylene glycol (mPEG) so as to block the specific binding site for antibody. METHOD: Different types of mPEG (all with final concentration of 12 mmol/L) were used at different temperatures in PBS with varied pH values for the modification of the HLA-A2 antigen. RESULT: The modification of the antigen was not obviously affected when it was carried out at 4 degrees Celsius or room temperature, but higher temperatures of 30 and 37 degrees Celsius significantly hampered the modification. Better antigen modification was observed with high-concentration mPEG in basic PBS, depending also on the type of mPEGs used for this purpose. CONCLUSION: The specific HLA-A2 binding on the lymphocytes is completely blocked by benzotriazole carbonate-mPEG(mPEG-BTC), which is superior to N-hydroxysuccinimidyl ester of mPEG(mPEG-SPA). Maleimide-mPEG(mPEG-MAL) is incapable of blocking the HLA-A2 ligand-binding site with antibody.

[Camouflage of HLA-I antigen in lymphocyte surface].

The objective of this study was to investigate the method and effect of blocking the specific reaction between lymphocyte HLA-I antigen and its antibody. The lymphocytes were disposed with 12 mmol/L methoxypolyethelene glycol benzotriazol carbonate (mPEG-BTC) in concentration gradient in PBS (pH 7.4) at 22 degrees C. The effect of the modified lymphocytes was detected by microlymphocytotoxicity assay. The results showed that lymphocytes modified by mPEG-BTC did not react with related HLA-I antibodies in microcytotoxicity test. It is suggested that the specific reaction between HLA-I antigen of lymphocyte and HLA-I antibodies can be completely camouflaged by mPEG-BTC in PBS (pH 7.4) under 22 degrees C room temperature.

[Observation on gene polymorphism of Rh blood group in Chinese Han nationality].

To observe the gene polymorphism of Rh blood group in unrelated random individuals and families for Chinese Han nationality, polymerase chain reaction-sequence specific primer (PCR-SSP) was used to amplify the Rh C/E gene, RhD gene, exons, intron 2 and 10, insert and Rh Box in 160 blood samples of RhD positive unrelated individuals and 71 samples of RhD negative unrelated individuals and 7 samples of families whose probands were RhD-negative. The results showed that RhD genes of RhD-negative individuals with C antigens were polymorphism, three forms were found for D exon including intact, partial deletion and complete deletion exons. Insert fragments and Rh Box were found in most cases of families whose probands were RhD-negative and its inheritance accorded with the Mendel's Law, and it did not affect the expression of RhD gene. "Normal" RhD exon 4 amplifying product was not found in all of the samples. It was concluded that gene structure of the RhD-negative in Chinese was polymorphism, intact, partial deletion and complete deletion exons were found in the individuals with C antigen and probably existed specific D (nf) Ce haplotype. The function of insert was uncertain. The Rh gene sequences of Chinese Han nationality are different from those of Caucasian and the Rh gene library based on Han nationality should be established.

Graft survival assessment of MN genotype after allogeneic transplantation of hematopoietic stem cells of umbilical cord blood.

OBJECTIVE: To establish a simple and practical method to assess the outcome of allogeneic transplantation of hematopoietic stem cells from umbilical cord blood. METHODS: The DNA was extracted from the samples collected from the donor and the receptor separately before and 15 and 300 d after transplantation. MN genotype was determined by PCR with sequence-specific primers (PCR-SSP) with the 2 pairs of specific primers designed and synthesized on the basis of reported MN gene sequence. RESULTS: The donor was identified as having NN genotype while the recipient shown to be MM genotype. MN genotype was detected in the recipient (mixed chimerism) at 15 d after transplantation. The genotype reversed to NN (donor origin) at 300 d after transplantation. The NN gene, as the donor's marker and evidence for the graft survival analysis, was eventually identified in the recipient. CONCLUSIONS: With MN genotype test to confirm the graft survival, early engraftment monitor of allogeneic umbilical cord blood transplantation is ensured, with the advantages of better sensitivity and requiring small amount of sample (0.2 ml). It may also facilitate subsequent therapeutic decisions.