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Objective To explore the importance of Rh phenotype detection and homotype blood transfusion. Methods The Rh phenotypes (CcDEe) of 3411 patients with repeated blood transfusions and 4735 blood donors were detected by the microcolumn gel test, and the positive rate of irregular antibodies was counted. A statistical analysis was conducted on the transfusion effectiveness between the Rh homotype transfusion group and the random transfusion group. Results The distribution of Rh antigen and its phenotype in our research is consistent with the reported results. Rh CCDee and Rh CcDEe are high-frequency phenotypes, and the antibodies of the Rh blood group system are high-frequency irregular antibodies, accounting for 62.42% of the detected antibodies. The positive rates of irregular antibodies in Rh system and direct antiglobulin test in random transfusion group were 6.67% and 10%, respectively, while those in Rh homotype transfusion group were 0% and 3.3%, respectively. There were three cases of adverse transfusion reactions in the random transfusion group, whereas no adverse transfusion reactions occurred in the Rh homotype transfusion group. The increase in hemoglobin was more significant after red blood cell transfusion in Rh homotype transfusion group. Conclusion In patients with repeated blood transfusions, Rh phenotype detection and the use of Rh homotype transfusion can avoid the difficulties associated with Rh blood group identification and cross-matching caused by random blood transfusions. It can effectively reduce the risk of immune and adverse reactions related to the Rh blood group system, improve the effectiveness of blood transfusion, and ensure the safety of clinical blood transfusions.

Objective: To establish the absorption and elution test for relatively quantitive obtaining anti-A and anti-B blood group IgG antibodies in the plasma of O-type RhD-positive pregnant women., Methods: 95 cases of the O-type RhD-positive pregnant women plasma samples were randomly selected for obtaining the IgG antibodies of anti-A and anti-B blood group, with absorption test under 37 ℃ and elution test under 56 ℃, and the IgG anti-A and anti-B antibody titers of plasma and elution were determined by the microcolumn gel anti-human globulin test. The differences and correlation between the titers of IgG antibodies in the eluent and plasma were compared and analyzed., Results: After a logarithmic transformation (Log2), there was no statistically difference between IgG antibody anti-A difference value and anti-B difference value in the eluent and plasma ( P >0.05). The titer of IgG antibody in the eluent was positively correlated with the titer of IgG antibody in the plasma ( r =0.914). The linear equation for IgG antibody titers fitted by a scatter plot between the eluent and plasma was Y=-3.55+0.96X., Conclusion: The absorption and elution test can be used to obtain the anti-A and anti-B IgG antibodies in the plasma of O-type RhD-positive pregnant women, whose plasma origin IgG titer is greater than 8. Meanwhile, the acquisition of anti-A antibodies was as effective as anti-B antibodies at the same time, and the antibodies obtained are positive proportional to their respective concentrations in the plasma.

Objective To analyze the serological characteristics and clinical significance of IgG anti-D and anti-C combined antibodies and anti-E and anti-c combined antibodies in patients negative for RhDC and RhEc antigens. Methods The clinical data and laboratory results of 12 cases with two types of irregular antibodies were recorded and analyzed, including age, sex, history of blood transfusion/pregnancy, ABO and RhD blood group identification, Rh antigen typing, irregular antibody screening, antibody-specific identification, absorption-elution tests, antibody titer determination and cross-matching tests. Results Among the 12 patients, the mean age was 51.4±16.9 years. Nine patients had a history of blood transfusion; eight patients had a history of pregnancy; five patients had both. Serological tests showed positive antibody screening and incompatible cross-matching. The results of antibody-specific identification and absorption-elution tests showed the presence of both IgG anti-D and anti-C antibodies in three patients, with anti-D titers at 16-32, and anti-C titers at 8-16. Nine patients had both IgG anti-E and anti-c antibodies, with the titers of anti-E and anti-c antibodies at 8-16. From the patients with combined anti-D and anti-C antibodies, suspended red blood cells of ABO identical type, RhD negative and other Rh antigens as ccee were selected. From patients with combined anti-E and anti-c antibodies, suspended red blood cells of ABO identical type, RhD positive and other Rh antigens as CCee were selected. Cross-matching blood test results showed no agglutination or hemolysis in saline, polycoagulant and anti-human globulin media. Conclusion Blood transfusion and/or pregnancy are the primary causes of irregular antibodies in two Rh systems, leading to positive antibody screening and cross-match incompatibility. Routine compatibility transfusion of Rh antigens, based on ABO homotypic blood transfusion, is of great value and significance for the safety of clinical blood transfusion and the prevention of hemolytic disease of the newborn.

Flow cytometry was previously applied for analysis of Rh(D) antigen density, therefore it was suggested that the flow cytometry may be used for routine detection of weak D positive phenotypes. This study was purposed to evaluate its practicability. Six weak D positive and 7 DEL individuals were detected by using saline, IAT and absorption/elution test from 2010 to 2011 years. By RHD genotyping, zygosity analysis and sequencing, 3 cases of weak D type 15, 3 cases of partial D type DVI-III and 7 cases of DEL carrying RHD1227A alleles were identified. Taking 2 normal Rh(D)-positive and 2 D-negative samples as controls, all the samples were tested by using flow cytometry, and the median fluorescence intensities were observed as well. The results indicated that all weak D type 15 and partial D type DVI samples were detected to be positive by flow cytometry, as compared with 2 Rh(D)-negative samples (P < 0.05). Seven 7 DEL samples were tested to be negative (P > 0.05), although one of 7 DEL was tested as "±" in IAT and strong positive in absorption/elution. The RHD zygosity analysis showed this DEL individual as RHD(+)/RHD(+) homozygote. It is concluded that the sensitivity of detecting D antigen by flow cytometry is similar to that of IAT, but lower than absorption/elution test. As for detecting weak D or partial D antigens, IAT is easier than flow cytometry; as for identifying DEL, the flow cytometry is not sensitive enough.

Objective: To explore the occurrence and clinical significance of erythrocyte allo-antibodies to MNS blood group system among hospitalized patients in China., Methods: The specificity and clinical features of erythrocyte allo-antibodies detected from August 2009 to July 2012 were retrospectively analyzed., Results: A total of 187 erythrocyte allo-antibodies were detected from 66 042 hospitalized patients (0.28%) among which 70 (37.4%) were specific to MNS blood group system. Antibody frequencies were as follows: anti-M, 18.2%; anti-Mi(a), 18.2%; anti-S, 1.1%. Anti-Mi(a) was more frequent among transfused patients (16/34 vs 5/34, P = 0.004), and tended to be accompanied by other allo-antibodies., Conclusions: Antibodies to MNS blood group system are second only to Rh system as the most common erythrocyte allo-antibodies in China. And anti-Mi(a) is an important transfusion-related allo-antibody among the patients of southern ancestry.

In order to analyze the genotype of RhD-negative blood donors with immune antibodies in Harbin, the voluntary blood donors from 1 April 2008 to 30 september 2011 were detected serologically to determine the RhD-negative donors. The blood donors confirmed to be RhD negative were detected to screen the immune antibodies, the samples with immune antibodies were analyzed by PCR-SSP and DNA sequencing to detect RhD genotype. The results showed that the 12 cases of the immune antibodies (0.95%) were screened out from 1265 cases of RhD-negative donors, among which 9 cases showed anti-D-antibody, 3 cases showed anti-(D+C) antibody; 10 cases were RhD-negative, 2 cases were RHD 711D(el)C. It is concluded that RhD negative and RHD 711D(el)C are easy to be immunized to produce the immune antibodies; RhD-negative population, especially women should be highly aware of avoiding mis-transfusion of RhD-positive blood, and also avoiding multiple pregnancies resulting in newborn's hemolytic disease.

Objective: To report a rare case of hemolytic disease of the newborn (HDN) with kernicterus caused by anti-Di(a) diagnosed using high-throughput genotyping multiplex ligation-dependent probe amplification (MLPA)., Methods: Conventional serological methods were used to detect the antibodies related with HDN. The genotypes of more than 40 red blood cell antigens for the newborn and her parents were obtained using the high-throughput MLPA assay. The antibody titers were tested using a standard serological method., Results: The unknown antibody against the low-frequency antigens was predicted based on the primary serological tests. The genotyping results for more than 40 red blood cell antigens of the newborn and her parents showed incompatible antigens of MNS and Diego blood group system, indicating the existence of anti-N or anti-Di(a). Further serological tests confirmed anti-Di(a) existence in the plasma of the newborn and her mother. The titer of anti-Di(a) in the mother's plasma was 1:32., Conclusion: Severe HDN including kernicterus can result from anti-Di(a). High-throughput genotyping MLPA assay can help type some rare antigens in complicated cases. The reagent red cell panels including Di(a)-positive cells are necessary in routine antibody screening test in Chinese population.

This study was purposed to investigate the molecular basis for RhD negative phenotype in Yiwu population in Zhejiang Province of China. The RhD negative samples were screened by saline agglutination test in blood donors. Some real RhD negative and RhDel phenotypes were identified using anti-human globulin test and absorbtion elution test. Ten exons of RHD gene in these samples were amplified by PCR-SSP, and positive exons were DNA sequenced. The results indicated that 30 real RhD negative and 8 RhDel phenotypes were identified in 38 initial RhD negative samples. Ten exons were complete negative in 28 real RhD negative samples and only exon 1, 2 and 10 were positive in 2 real RhD negative samples amplified by PCR. All 10 exons in 8 RbDel samples were positive and a DNA variant (1227G > A) was found in 8 RhDel samples. It is concluded that all exons are absence in most real RhD negative phenotypes, and the partial exons absence is also found in some real negative phenotypes among Yiwu population in Zhejiang province of China. The G to A mutation at position 1227 is found in all RhDel phenotypes.

Aim: To Construct Fab antibody against Rh antigen., Methods: The variable regions of light and heavy chains were amplified by RT-PCR from the PBMCs of volunteers with high titer (1:256-512 by inditect agglutation) antibody to Rh antigen. Meanwhile, the genes of constant regions of light and heavy chains were isolated from pComb3xTT and pComb3xlambda phagmid carrying the templates respectively. Vkappa light chain and Fd heavy chain were linked by the first splicing overlapping extension PCR (SOE), and a full-length Fab gene was created by the second SOE. The Fab gene was ligated to phagmid pComb3HxSS and transformed to E.coli XL1-Blue by electroporation. The obtained human Fab phage antibody library was panned using Rh(-)/Rh(+) RBC four times. the phage antibodies against Rh antigen were highly enriched. Indirect agglutation test, western blot analysis and sequencing analysis were performed to detect the specificity of Fab against Rh., Results: The repertoire of human phage display Fab library was 7.4 x 10(6);. After panning, A Fab clone which could bind to Rh antigen specifically was obtained., Conclusion: A Fab antibody that specifically aggulated Rh(+) RBC is obtained, this makes it possible to produce Rh antibody with high quantity and effection in our country.

The study was aimed to analyze Del phenotype of RhD (-) unrelated blood donors. RhD (-) was initially screened by routine serological test and confirmed by indirect antiglobulin test (IAT). Del phenotype was detected by hot-ether absorption-elution test. The results indicated that 106 RhD (-) samples were confirmed out of 38526 donors, and 28 cases were Del detected by hot-ether absorption-elution test. The incidence of Del in RhD (-) samples was 26.41%, The serological phenotypes of Del were Ccee (78.57%), CCee (14.29%) and CcEe (7.14%) respectively. In conclusion, the detection of Del by using hot-ether absorption-elution test is very important for reasonable application of RhD (-) blood. There is difference in Del phenotypes of populations in different regions of China and Japan.

This study was purposed to establish the method of quantifying RhD antigen on red blood cells (RBC) by flow cytometry (FCM) and to explore the expression of D antigen on RBC of different RhD serotype. RhD(+) RBCs and RhD(-) RBCs were mixed in 1:1 ratio. Cells were stained by the indirect method (IgG anti-D as the first antibody, FITC-anti-IgG F(ab')2 as the second antibody), and the ratio of RhD(+) on RBCs was quantified by FCM. The optimal dosage of IgG anti-D was defined. Expression of RhD antigen on RBC of RhD(+), weak D, RhDel and RhD(-) type were detected by FCM. The results showed that optimal dilution of IgG anti-D monoclonal antibody was 1:4, 1x10(6) cells/50 microl. The percentage of D(+) RBC of RhD(+), weak D, RhDel and RhD(-) type were 96.8+/-2.97%, 79.5+/-9.88%, 47.8+/-11.43%, 3.7+/-2.96%, respectively. The mean fluorescence intensity (MFI) of RhD antigen expression of RhD(+), weak D, RhDel and RhD(-) type were 33.3+/-6.21 Dal, 18.6+/-5.39 Dal, 7.10+/-1.17 Dal, 0.79+/-0.55 Dal, respectively. In conclusion, there are significant differences of RhD antigen expressions among RBC of different RhD serotypes. The level of antigen on RhD(+) RBC is the highest and then weak D the next, while the level of antigen on RhDel RBC is the lowest level.

Aim: To study the prevalence and distribution of anti-Rh blood group antibodies in Chinese population and its clinical significance., Methods: Irregular antibodies were screened and identified by Microcolum Gel Coomb's test. For those identified as positive anti-Rh samples, monoclonal antibodies (anti-D, -C, -c, -E and -e) were used to identify the specific antigen and confirm the accuracy of the irregular antibody tests. The titers, Ig-types and 37 Degrees Celsius-reactivity were tested to confirm its clinical significance. For evaluation of the origin of irregular antibodies, histories of pregnancy and transfusion were reviewed. For the newborns who had positive antibodies, their mothers were tested simultaneously to confirm the origin of the antibodies., Results: 47 out of 54 000 (0.087%) patients were identified as positive with Rh blood group antibodies.Of them, 27 cases had history of pregnancy, 13 had transfusion and 1 had the histories of both. 6 newborns had antibodies derived form their mothers. The specificity of the antibody was as follows: 29 with anti-E (61.70%), 8 with anti-D (17.02%), anti-cE 5(10.64%), 4 with anti-c (8.51%) and 1 with anti-C (2.13%). All the 47 Rh blood group antibodies were IgG or IgG+IgM, and were reactive to red blood cells with corresponding antigens at 37 Degrees Celsius, with a highest titer of 1:4 096., Conclusion: The prevalence of Rh antibodies is lower in Chinese population as compared with that in White population.Of all the antibodies, anti-E is most frequently identified and anti-D was declining. Alloimmunization by pregnancy and transfusion is the major cause of Rh antibody production. Rh blood group antibodies derived from mothers are the major cause of Non-ABO-HDN.

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.

To investigate the frequency of RHD 1227A allele in Rh negative population and random population, an AS-PCR (allele specific-polymerase chain reaction) method was employed to detect RHD 1227A allele. RHD gene copy was determined by D zygosity test and RHD exon 9 nucleotide sequence analysis. The results showed that among 143 Rh negative donors, forty-one RHD 1227A allele carriers were detected, and 8 (19.51%) out of which were RhCCdee, 32 (78.05%) were RhCcdee, and 1 (2.44%) was RhCcdEe. Thirty-five Rh negative RHD 1227A carriers had RHD gene deletion, and the remaining carriers were RHD 1227A homozygous. Seven (1.43%) individuals were detected with RHD 1227A allele among 489 random donors. They were all G/A heterozygous at RHD 1227 site. Serological test indicated that they were normal Rh positive phenotype. It is concluded that the frequency of RHD 1227A allele is 16.43% among Rh negative population and 0.72% among the random population.

The objective of study was to investigate the Rh antigen stability of mPEG-modified RBC. RBC membrane protein SDS-PAGE technology was used to analyze the combination of the mPEG modified RBC membrane protein with mPEG molecules; the RBC ghost coagulation test and 4 degrees C CPD-preserved modified RBC mixed with matched blood were used to observe the stability of RBC Rh antigen camouflaged by mPEG. The results showed that the blood groups of stored mPEG-modified RBC were kept consistency before or after simulating transfusion, i.e. mixture of modified RBC with matched bloods, while the plasma hemoglobin after simulating transfusion was not only within the normal range during the storage, but also less than that before simulating transfusion even after incubation at 37 degrees C. The electrophoresis pattern stained with iodine and Coomassie blue displayed the bands of mPEG combined with RBC membrane protein and the slow mobility of membrane protein. The hemagglutination of PEGylation RBC ghosts did not take place and mPEG still covered the antigen. In conclusion, mPEG-SPA can bind the erythrocyte with its extracted membrane protein in both ghosts and living erythrocytes.

In order to establish a genotyping method for DEL phenotype in Zhejiang Han population, an AS-PCR method was developed according to the RHD 1227A allele sequence. Its specificity and sensitivity were assessed in two Rh negative populations whose RHD 1227A or DEL phenotype status was known. The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method. After re-testing these two samples with serological method and sequence analysis, it was found that original serological method gave false negative results and genotyping method still showed 100% specificity. The minimal target DNA concentration of this genotyping method is 8.13 ng/microl. In conclusion, designed genotyping method can be used to identify DEL phenotype efficiently in Zhejiang Han Rh negative population.

This study was aimed to investigate the molecular genetic basis and serological phenotype of Rh weak D type 15 individuals. Samples were identified by serological tests and genotyped by sequence specific primer-PCR (SSP-PCR), and were sequenced to detect the changes of all ten RHD exons. The number of gene RHD was detected through SSP-PCR. The results showed that in tested individuals of weak D type confirmed by the IAT, 18 cases (56% in weak D) were weak D type 15. Rh factors found in 2 weak D type 15 individuals (11%) were C+c+E+e; Rh factors found in 2 weak D type 15 individuals (11%) were C+c+E-e+; others (78%) were c-c+E+e+. The results by serological tests were consistent with the results genotyped by PCR-SSP method. In all 18 samples, the sequencing result revealed a gene mutation 845G > A at the exon 6 of the RHD and the point mutation changed amino acid G282D of the RhD polypeptide. The zygosity test demonstrated that 2 out of 18 weak D type 15 individuals were RHD(+)/RHD(+) homozygous (two DCe/DcE), 16 cases were RHD(+)/RHD(-) heterozygous (two DCe/dce and fourteen DcE/dce). It is concluded that Weak D type 15 is predominant in weak D individuals of Chinese Han Nationality, and most of them are heterozygous with various RH haplotypes.

This study was purposed to investigate the molecular basis of Rh DEL phenotype. Rh DEL phenotypes were identified by a serologic adsorption-elution method, the nucleotide sequences of ten RHD exons and exon-intron boundary regions were evaluated by a RHD gene-specific PCR-SSP (PCR-SSP, polymerase chain reaction-sequence specific primer) and sequencing. The results showed that out of 122 random Rh negative donors 35 Rh DEL phenotypes were identified through serologic method, including 6 RhCCdee (17.14%), 28 RhCcdee (80.00%), and 1RhCcdEe (2.86%). Sequence analysis indicated that all DEL phenotypes harbored a RHD 1227 G > A mutation in exon 9. D zygosity test revealed that 29 DEL phenotypes (28 RhCcdee and 1 RhCcdEe) had one RHD gene deleted, and 6 DEL phenotypes (6 RhCCdee) had homogenous RHD gene. It is concluded that RHD 1227A is an important genetic marker for Rh DEL phenotype in Zhejiang Han population.

To investigate the molecular basis of partial D phenotypes in Chinese, D variants with weak D expression was screened by using indirect anti-human globulin test (IAT) method, the polymerase chain reaction-sequence specific primer (PCR-SSP) method was employed to amplify RHD specific exons and their flanking regions. The amplification products were sequenced directly to determine the molecular basis of D variants. The results showed that ten cases of partial D phenotypes, including one case of D Va (Kou.), one case of D Va (Hus.), one case of D Va-like (YH.), and seven cases of D VI type III, were detected from 22 cases of weak D phenotype respectively. All ten cases of partial D phenotypes had one RHD allele deleted. In conclusion, the molecular basis of ten cases of partial D phenotype was confirmed, including D Va (Kou.) and D Va-like (YH.) phenotypes reported firstly in Chinese population.

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.

When we did compatibility testing, we found a case of leukemia with irregular antibody. The antibody specificity was identified as anti-Ce using panel-cell, and was IgG antibody. The anti-Ce antibody had both anti-C and anti-e activity. It is very difficult to find a Ce antigen negative blood for transfusion. The compatible donors rate is very low, only 2%-3%.

Rh is a very important blood group like ABO blood system in transfusion medicine. It causes severe transfusion reaction and hemolytic disease of the newborn (HDN) if RhD blood group does not match between the donor and the recipient. The population of RhD negative is only about 0.2% - 0.5% in Chinese. Conversion of RhD positive RBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located on the surface of A, B, O and AB red blood cells in order to convert RhD positive to RhD negative by the modification of four kinds of methoxypolyethylene glycol (mPEG) derivatives and to observe the effect of mPEG modification on cell morphology, structure and function. The result demonstrated that modification efficiency of mPEG-BTC (mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigen efficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmful effects of mPEG modification on cell morphology, osmotic fragility, hemolysis, AchE, cholesterol, ATP, 2,3-DPG and deformability. It is suggested that success in converting RhD positive RBCs to RhD negative was preliminarily achieved.

Haemolytic transfusion reactions occurred in 4 patients under plastic surgery due to incompatibility of Rh blood groups. The reaction in one of the patient occurred not only abrupt but severe and finally died of renal failure. Other three patients with delayed haemolytic reactions survived after treatment. Since more than 99.5% Chinese population is Rh positive, cross-matching on Rh blood groups is not a routine. The reactions develop usually different from typical ABO blood group haemolytic reactions and are not easy to make an early diagnose. If the surgical patients show profound anemia and haemorrhage following transfusion which could not be explained by bleeding and coagulation abnormalities, haemolytic transfusion reactions due to incompatibility of Rh blood groups might be considered.

The ABO+ Rh blood groups and serum anti- A and anti -B anti bodies were examined in 1944 pregnant women. The results showed that 475 cases (24.4%) were positive for serum immune antibodies resulting in ABO incompatibility, and in 68 (3.4%) newborns haemolytic disease (HDN) occurred. The incidence of ABO blood group incompatibility increased with the increase of maternal serum immune antibody titer, the difference between different titers was significant (chi 2 = 4.13, P less than 0.05). Pregnant women with a high titer of serum immune antibody greater than 1:64 should be followed up regularly and those greater than 1:128 should be treated immediately. The relationship between positive serum immune antibody and blood group was discussed. We consider it is important to do maternal serum immune antibodies examination for antenatal care.