[Analysis of Relationship between Coagulation Function and Blood Transfusion in Patients with Emergency Rescue].

OBJECTIVE: Through researching preoperative coagulation function in the case of ABO-identical blood insufficient for emergency rescue transfusion according to recommended programs of special emergency rescue transfusion was carried out, the relationship between volume of blood products and coagulation function was analyzed. METHODS: The surgical cases of blood transfusion more than 1 600 ml during operation were collected in our hospitals from Aug 2015 to Dec 2016（n=218）, these cases were divided into the normal coagulation group（Group A） and abnormal coagulation group（Group B）, and the patients of emergency rescue transfusion O type blood group（Group C）. The basic information of cases, the infused volume of red blood cell（RBC）, virus-inactivated frozen plasma（VIFP）, fresh frozen plasma（FFP）, cryoprecipitate（C）and platelets（P）, prothrombin time（PT）, activated partial thromboplastin time（APTT）, fibrinogen（FIB）and international normalized ratio（INR）were analyzed, the relationship between volume of blood transfusion and coagulation function were also analysed. At the same time, the efficiency and safety index were compared before and after transfusion. These indexes, such as hemoglobin（Hb）, indirect bilirubin（IBiL）, direct antiglobulin test（DAT）and irregular antibody were determined at the time-paints of 24 h, 3 d and 7 d after blood transfusion. RESULTS: The differences of age and blood type between group A and B was not statistically significant（P>0.05）. Proportion of A and AB type，transfusion volume of RBC, FFP, C and Plt all were significantly higher in group C （P<0.05）. PT, APTT, FIB and INR in group B and C were significantly different（P<0.05）, which related with the transfusion volume of RBC, FFP and C（P<0.05）. DAT and irregular antibody in every group was all negative before transfusion, No any new irregular antibodies had been detected after transfusion. Hb after blood transfusion was not statistically different before and after transfusion in group C, the IBiL level also was not significantly increased after blood transfusion（P > 0.05）. All those showed that emergency rescue transfusion was safe and effective. CONCLUSION: Preoperative coagulation function is one of factors inflnencing blood products transfusion volume during operation, which also is the basis for evaluating bleeding and blood transfusion. Emergency O type blood and ABO-matched blood transfusions show the same efficiency and safety.

[Experimental Study on Neonatal ABO or RhD Compatible Blood Transfusion].

OBJECTIVE: To investigate the safety and effectiveness of neonatal ABO or Rh(D) by using compatible blood transfusion through retrospective analysis of data from cases received compatible blood transfusion and type matched blood transfusion. METHODS: The clinical data of 26 cases of neonatal compatible blood transfusion in Chinese Nanchang area from January 2014 to October 2016 were collected, and 26 cases of neonatal type-matched blood transfusion were selected according to ratio of 1:1 cases. The efficiency and safety index of 26 patients compatible blood transfusion were compared with that of type-matched blood transfusion. The efficiency indexes included: patients' basic characteristics, red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Hct), and the safety indexes contain Hb level and indirect bilirubin (IBiL) value before and after blood transfusion, irregular antibody screening, direct antiglobulin test (DAT) results and the adverse reactions of blood transfusion. RESULTS: The age, sex, days of hospitalization between compatible blood transfusion and type matched blood transfusion were not statistically significantly different (P>0.05). The Hb level before transfusion, blood transfusion volume and the increase of Hb, Hct and RBC were not statistically significantly different between two groups (P>0.05). The values of Hb, Hct and RBC in 2 groups significantly increased at the day 1 after blood transfusion (P<0.05). No blood transfusion adverse reaction occurred in 2 groups. The IBiL value significantly decreased in compatible blood transfusion patients at the day 1 after blood transfusion (P<0.05). No new irregular antibodies had been detected after transfusion in all patients, and the others' DAT and screening for irregular antibodies were negative except 22 patients with neonatal hemolysis. The values of Hb and IBiL statistically significantly differenence were not in 12 patients between 1d, 3d, 7d after blood transfusion (P>0.05). CONCLUSION: The efficiency and safety between compatible blood transfusion and type matched blood transfusion are the same in neonatal blood transfusion. Compatible blood transfusion is a safe and effective in clinical blood transfusion.

[Study on RHCE genotyping of Rh blood group in Uygur nationality of Xinjiang in China].

This study was aimed to investigate the characteristics of RHCE genotyping of Xinjiang Uygur nationality population in China. Primers for detecting RHCE genes were designed according to the references, 89 Uygur nationality RhD-negative samples, 233 Han nationality RhD-negative samples and 109 Han nationality RhD-positive samples were detected by sequence-specific primer-polymerase chain reaction (SSP-PCR) for RHCE genotyping. All above-mentioned samples were unrelated. The results indicated that RHE/e genotyping results were consistent with the serological test results in the samples of Uygur and Han nationality, regardless of the RhD-negative samples or the RhD-positive samples. The RHC/c genotyping results from 89 RhD-negative samples of Uygur nationality were consistent with serological test results. However, total error of RHC/c genotyping from 233 RhD-negative and 109 RhD-positive samples of Han nationality was 5.05%. In conclusion, this method of RHCE genotyping is suitable for the analysis of the RHE/e genotyping of Uygur nationality, no erroneous RHC/c genotyping of Uygur nationality was found in this study, but this method needs to be further studied.

[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.

[Investigation of the characteristics of Rh blood group of Uygur nationality in Xinjiang].

The study was to investigate the characteristics of Rh blood group of Uygur nationality in Xinjiang. 1 230 blood samples of Uygur nationality were studied by Rh serological typing, modified antiglobulin test, chloroform/trichloroethylene absorption elution test, upstream, downstream and hybrid Rhesus boxes, 10 exons of D gene, RHD(psi) pseudogene. The results showed that the frequency of RHD negative was 5.8%, and no Del type was found. The further investigation of 72 samples of RhD (-) found that ccee (57.02%) and Ccee (29.08%) phenotype as well as RHD(-)/RHD(-) genotype (94.44%) and complete deletion type of D gene exon (91.12%) were all in high frequency, no RHD(psi) pseudugene was detected. In conclusion, the Rh blood group of Uygurs nationality in Xinjiang possesses both oriental and caucasian Rh characteristics, which enriches the diversity of blood types in chinesenation.

[Screening analysis of irregular antibodies from random donor population in Shaoguan area].

The study was purposed to analyze the frequency and distribution of irregular antibodies in Shaoguan area. Screening 15 033 random donor antibodies in Shaoguan area by screening cells, polybrene and antiglobulin tests. The results indicated that the irregular antibodies were found in 42 samples. The frequency of irregular antibodies in female was higher than that in male (P < 0.001), and Rh blood group antibodies such as anti-D, anti-E, and anti-Ec C were common (47.6%). 2 samples of Le antibodies were failed to be found by polybrene test. 2 samples of irregular antibodies with titer 2 were undiscovered by screening test of 10 pooled samples. In conclusion, because of irregular antibodies resulting in hemolytic transfusion reaction, the investigation of frequency and distribution of irregular antibodies is very important for safe transfusion. Antibody screening must be done for female donors, and especially for massive plasma transfusion of patients with severe and dangerous illness and infants so as to ensure safety.

[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.

[Correlation of newborn hemolytic disease with ABO antibodies in sera of pregnant women].

To investigate the relations between morbidity of hemolytic diseases of newborn and ABO antibodies (HDN) in sera of Han and Yao nationality, pregnant women were examined before and after delivery. Antibodies screen, direct antiglobulin test, free antibodies and elution test were all performed. The results indicated that immunologic ABO antibodies of Han people were found in 673 cases out of 1,471 Han pregnant women, and was also found in 28 cases out of 65 Yao pregnant women, and there was no significant difference of incidences between Han and Yao nationality. 31 cases of HDN were found out of 288 newborn in Han and 3 cases of HDN were found out of 25 newborn in Yao, and there was no significant difference between Han and Yao nationalities. The characteristics of HDN in Han nationality were as same as in Yao nationality. In conclusion, the morbidity of HDN in Han and Yao nationalities of Shaoguan area did not showed essential difference, the immunologic ABO antibodies and its titration test, especially, elution test are important for prognosis of HDN.

[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.

[Dynamic monitoring of graft-versus-host disease after allogeneic peripheral blood stem cell transplantation by soluble HLA-I antigen detection].

OBJECTIVE: To assess the value of dynamic monitoring of soluble human lymphocytic antigen-I (sHLA-I) in the prediction of graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (PBSCT). METHOD: Sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect serum sHLA-I. The serum samples for testing were added into W6 32 monoclonal antibody-coated microtiter plate and incubated with anti-beta2m HRP followed by color development with the addition of the substrate. Serum sHLA-I level was measured in 63 healthy blood donors of Shanghai and in 24 PBSCT recipients before and and at different time points after the operation. RESULT: No changes in sHLA-I levels occurred in allogeneic PBSCT recipients without GVHD or with only grade I GVHD, but sHLA-I reached high levels in patients suffering GVHD II-IV(P<0.05), which was effectively lowered by the application of immunosuppressants. CONCLUSION: Measurements of sHLA-I levels can be valuable for monitoring GVHD after PBSCT.

[Study on detection of samples of Rh-weak D and Del].

To study the detection of weak D and Del from samples initially screened RhD(-), RhD phenotype was initially screened by routine serological test, out of which weak D phenotype was detected by indirect antiglobulin test (IAT) and Del phenotype was detected by chloroform-trichloroethylene absorption-elution test. The results showed that 56 samples were RhD(-) confirmed by routine serology test, which were screened out of 26 200 donors, among them 5 samples were typed as weak D by IAT and 9 cases samples were typed as Del by absorption-elution test. In conclusion, the samples which typed as RhD(-) by routine serological test must be identified by IAT and chloroform-trchloroethylene absorption test is order to detect weak D and Del phenotype. It is important for clinical transfusion safely.

[Association of interleukin-6 induction and the novel gene LX3].

OBJECTIVE: To analyze the association between the expression of the novel gene LX3 and interleukin-6 (IL-6) induction, and explore a new target gene for action mechanism of IL-6. METHODS: The total RNA was extracted from U937 cells induced by IL-6 at different concentrations and varied lengths of time. Reverse transcriptional (RT)-PCR and Northern blotting were employed to determine the expression of LX3 and IL-6 induction. RESULTS: The expression of novel gene LX3 was increased while concentration of IL-6 was improved gradually. High expression was induced by IL-6, which was highest at the concentration was 500 ng/ml and no expression at 0 ng/ml. Time- expression pattern proved that expression of novel gene LX3 of higest at 8 hour after induced by IL-6. Northern blotting confirmed that the expression quantity of LX3 increased in U937 cells induced by IL-6. CONCLUSION: The quantity of LX3 expression is associated with the dose and duration of IL-6 induction, suggesting that LX3 gene is a novel gene correlated with IL-6 induction.

[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.

[A method for Rhesus box test].

To study the method for Rhesus box test and its significance, the sequence specific primers of upstream, downstream and hybrid Rhesus boxes were designed according to RhD gene sequence; the upstream, downstream and hybrid Rhesus boxes were determined by PCP-SSP and mismatched PCR. The results showed that this method was confirmed by DNA Standard test. It was shown that in unrelative RhD positive individuals RHD(+)/RHD(-), RHD(+)/RHD(+) genotype accounted for 9.00%, 91.00% respectively, and in RhD negative individuals RHD(+)/RHD(-), RHD(+)/RHD(+), RHD(-)/RHD(-) genotype were 26.14%, 3.92%, 69.94% respectively. It is concluded that the method of Rhesus box test was confirmed to be reliable and can be used for the identification of RhD haplotype gene structure, as well as for study on inheritance, clinical transfusion and neonatal hemolytic diseases.

[Study on blood ABO typing in patients with autoimmune hemolytic anemia].

To explore effect of autoantibody on identification of ABO and RhD blood group, the blood samples of 38 patients with autoimmune hemolytic anemia (AIHA) were identified by routine typing and typing after chloroquine elution test as well as PCR. The results showed that out of 38 patients with AIHA, 11 cases (31.6%) of ABO blood group were difficulty typed, indirect antiglobulin test were positive, and contradiction between cells typing and sera typing were observed. 1 case of RhD(-) was mistyped as RhD(+) and anti-D was found in its serum. The blood group of these cases were typed correctly by chloroquine elution test. It is concluded that blood group identification of patients with AIHA can be interfered by autoantibody, and the correct typing for blood group of these patients may be determined by using combination of several methods to ensure safe transfusion.

[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.

[Exon polymorphism of human RHD gene].

OBJECTIVE: To study exon polymorphism of human RHD gene and investigate the genetic mechanism of RhD-negative individuals. METHODS: PCR using sequence-specific primers (PCR-SSP) was performed on 40 RhD-positive, 120 RhD-negative and 2 weak D blood samples. RESULTS: All 10 exons could be detected in the 40 RhD-positive and 2 weak D samples. Out of the 120 RhD-negative samples, 28 (23.33%) carried 10 exons, 19 (15.83%) lost most of the 10 exons (with mainly intermediate deletion), and 73 (60.83%) had deletion of all the 10 exons; 19 samples of Del phenotype identified from the 120 RhD-negative samples had all the 10 exons. CONCLUSION: Polymorphism of the exon structure of RHD gene is present in RhD-negative individuals, characterized chiefly by gross deletion, partial deletion and non-deletion.

[Observation of antibody screen of patients with autoimmune hemolytic anemia].

To observe of alloantibodies of patients with autoimmune hemolytic anemia (AIHA), the alloantibodies masked by autoiantibody were detected by using chloroquine elution test, and the specificity of autoantibody was identified by ether elution test. The results showed that 19 cases out of 38 patients with AIHA were positive detected by indirect antiglobulin test and in 7 cases of them alloantibodies in sera cases were found (1 case of anti-D, 4 cases of anti-E and 2 cases of anti-CE), in 5 cases of them alloantibody were detected carried blood group specificity (3 cases of anti-E, anti-C and anti-c 1 case respectively). In conclusion, detections of alloantibodies by chloroquine elution test and ether elution test were very important for transfusion safety in therapy of patients with AIHA.

[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.