Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

Establishment of Shaanxi rare blood group information supply platform / 中国输血杂志

【Objective】 To establish a rare blood group information supply platform in Shaanxi Province. 【Methods】 The rare blood group information supply platform consists of sample registration, result registration, donor files and inventory blood. The blood donation codes of voluntary blood donors were recorded for blood typing, and the antigen identification results of each blood group system were registered, all stored in the rare blood type information supply platform. When receiving an application for unusual or rare blood type missing multiple conventional antigens or a certain high-frequency antigen, the corresponding antigen negative blood donors and their blood status (in stock or not) were queried from the donor profile module of the platform, and the inventory of blood of rare blood type was monitored dynamically. 【Results】 The results showed that 5.060% (273/5 398) of rare Rh phenotype donors, 1.540‰ (51/33 010) of donors lacking multiple regular antigens, and 13 O-type donors lacking high-frequency antigens were recorded in the rare blood type information supply platform. Among them, 0.019‰ (3/158 484) of Jk(a-b-) phenotype, 0.436‰ (2/4 586) of Di(a+b-) phenotype, and 4.030‰ (8/1 983) of Fy (a-b+) phenotype were stored in the blood bank for rare blood type. 【Conclusion】 The establishment of rare blood group information supply platform can meet the urgent demand for blood of rare blood types in clinical practice and ensure the safety of blood transfusion.

Comparative analysis of antigen coding genes in 15 red cell blood group systems of Yunnan Yi nationality in China: A cross-sectional study.

Introduction: There are few analyses of the 15 red blood group system antigen coding genes found in the Yunnan Yi nationality. This has caused many poteintial dangers relating to clinical blood transfusion. In this report, the coding genes and distribution of 15 blood group antigens system in the Yi nationality were tested and compared with those of Han nationality and other ethnic minorities. Methods: The samples came from the healthy subjects in the first people's Hospital of Qujing, Yunnan Province. Two hundred and three Yunnan Yi and 197 Han nationality individuals were included. Thirty-three blood group antigens with a low frequency from the 15 blood group systems of Yunnan Yi blood donors were genotyped and analyzed by PCR-SSP. Sanger sequencing was used to detect A4GALT from the Yunnan Yi nationality. The χ 2 test was used to analyze observed and expected values of gene distribution to verify conformation to the Hardy-Weinberg equilibrium law. Fisher's exact test was used to analyze gene frequency distribution, and the statistical significance was set at p < 0.05. Results: The ABO blood group examination results for the Yi nationality and the local Han nationality in Qujing City, Yunnan Province, showed the majority were type A and type O, while the least prevalent was type AB. RhD+ accounts for more than 98% of the Yi and Han populations. There was a significant difference in ABO blood group antigen distribution between these two nationalities (p < 0.05), but there was no significant difference in the composition ratio of D antigen in the Rh blood group system (p > 0.05). Compared with Tibetan (Tibet), Zhuang (Nanning), and Dong (Guangxi), the gene distribution frequencies of Rh blood group system phenotype CC were significantly lower in the Yunnan Yi nationality (p < 0.05). There were significant differences in six erythrocyte phenotypic antigens in the Yi nationality in Yunnan compared with Han nationality, such as LW(a-b-), JK(a-b+), MMSs, Di(a-b+), Wr(a-b-), and Kp(a-b+) (p < 0.05). There were gene phenotypes with a low frequency in the four rare blood group systems: LW, MNS, Wright, and Colton. Several different mutation types occurred in the P1PK blood group system's A4GALT gene. Conclusion: Yunnan Yi nationality has a unique genetic background. There are some significantly different distributions of blood group system genes with a low frequency in different regions and groups in China. Multiple mutations in the A4GALT gene of the P1PK blood group system may be related to their environment and ethnic evolution.

Anti-Jra developed in Jra(-) pregnant woman, case report with molecular genetic analysis / 中国输血杂志

【Objective】 To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. 【Methods】 ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. 【Results】 The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. 【Conclusion】 In this case, the development of anti-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

Establishment and clinical application of Kidd, Diego, Duffy blood group system, Shaanxi Province / 中国输血杂志

【Objective】 To screen individuals with rare blood type of Kidd, Diego, Duffy blood group system among the voluntary blood donor in Shaanxi province and to establish on-line and physical database of rare blood type. 【Methods】 Jk(a-b-)phenotype donors were screened by 2 mol/L urea hemolysis test. Blood donors with Di(a+ b-) phenotype were screened by genotyping; Fy(a-) and D-- phenotype donors were screened by modified antiglobulin assay. 【Results】 Three cases of Jk(a-b-) phenotype were detected out of 158 484 voluntary blood donors. The distribution frequency of Jk(a-b-) phenotype was 0.019‰. Di(a+ b-) phenotype was detected in 2(0.436‰) cases out of 4 586 voluntary blood donor. Fy(a-) phenotype was detected in 8(4.034‰) cases out of 1 983 voluntary blood donors. D-- phenotype was not detected in 29 430 voluntary blood donors. 【Conclusion】 The on-line database of Kidd, Diego, Duffy blood group system had been established by large-size screening of blood donor samples, which can conclude the region′s population distribution and genetic characteristics of RBC blood group. And physical database could further be established using the technology of red blood cells cryopreservation when the conditionspermit, so as to provide the most compatible blood for the clinical effectively improve blood transfusion safety, and provide data support for blood early warning.

Molecular characterization of a rare Rh phenotype Dc-from the Indian subcontinent.

Unusual Rh phenotypes such as Rhnull, D-- and Dc- etc. are rarely encountered in routine blood bank testing. The Rhnull phenotype is characterized by the absence of all Rh antigens, D-- phenotype does not express any RhCcEe antigens whereas Dc- phenotype individual lacks expression of antithetical E /e antigens. These individuals may produce multiple Rh antibodies against missing antigens. An old woman (B RhD positive) from Bangladesh with end-stage renal disease developed severe anaemia. Cross-matching with ABO and RhD compatible blood units showed +3 agglutination reaction. Detailed immunohaematological investigations showed a lack of C, E and e antigens, thus identifying the rare Rh variant as Dc-. Antibodies against C and e antigens were also detected in the patient's serum. PCR-SSP confirmed the absence of the molecular region defining the C, E and e antigens. Copy number analysis by QMPSF revealed the homozygous state of (RHCE-D(4-9)-CE) allele at the RHCE gene locus. This is the first report of the rare Dc- variant individual from the Indian subcontinent.

Study on gene polymorphism of multiple RBC blood group systems in Mongolian population in Inner Mongolia / 中国输血杂志

【Objective】 To investigate the gene frequency and polymorphism of 12 RBC blood group systems, including RHCE, Lw, Duffy, Kidd, MNS, Scianna, Colton, Dombrock, Kell, Diego, Yt, and Lutheran blood group systems in Mongolian in Inner Mongolia, so as to provide data for the establishment of rare blood group registry in this region. 【Methods】 Twelve blood groups of 220 Mongolian people in Inner Mongolia were genotyped and analyzed by Fluo-PCR. 【Results】 The genes frequency of the 12 rare blood group was as follows: 1)RhCE, C=0.613 6, c=0.386 4, E=0.265 9, e=0.734 1; MNS, M=0.609 1, N=0.390 9, S=0.063 6, s=0.931 8, Mur=0; Duffy, Fya=0.856 8, Fyb=0.143 2; Kidd, Jka=0.522 7, Jkb=0.477 3; Diego, Dia=0.027 3, Dib=0.972 7, Wra=0, Wrb=1; Dombrock, Doa=0.163 6, Dob=0.836 4. 2) Kell, K=0.002 3, k=0.997 7, Kpa=0.009 1, Kpb=0.990 9; Yt, Yta=0.986 4, Ytb=0.013 6. 3) Lw, Lwa=1, Lwb=0; Sc2=0; Colton, Coa=1, Cob=0; Lutheran, Lua=0, Lub=1. The 220 Mongolian people with Lw, Scianna, Colton and Lutheran were all homozygous, and their genotypes were Lwa/Lwa, Sc1/Sc1, Lub/Lub and Coa/Coa, respectively. 【Conclusion】 The RHCE, MNS, Duffy, Kidd, Diego and Dombrock blood types of Mongolian population in Inner Mongolia are polymorphic with certain distribution characteristics. The MNS blood group system does not conform to the Hardy-Weinberg equilibrium(P<0.05), which may be related to the sample size or genetic changes. Kell, Lw, Scianna, Colton, Yt and Lutheran showed a monomorphic distribution.

Blood donation must go on.

Reacting to the repeated messages on his phone, the blood donor goes to a blood center and donates his AB Rh D negative blood. There, he sees the blood center technician whose mother requires this rare blood unit as she is undergoing treatment at the hospital. The very act of blood donation is soul-satisfying for him as he pens down his experience in the configuration of a poem.

Human induced pluripotent stem cell line banking for the production of rare blood type erythrocytes.

BACKGROUND: The in vitro production of mature human red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has been the focus of research to meet the high demand for blood transfusions. However, limitations like high costs and technological requirements restrict the use of RBCs produced by iPSC differentiation to specific circumstances, such as for patients with rare blood types or alloimmunized patients. In this study, we developed a detailed protocol for the generation of iPSC lines derived from peripheral blood of donors with O D-positive blood and rare blood types (D-and Jr(a-)) and subsequent erythroid differentiation. METHODS: Mononuclear cells separated from the peripheral blood of O D-positive and rare blood type donors were cultured to produce and expand erythroid progenitors and reprogrammed into iPSCs. A 31-day serum-free, xeno-free erythroid differentiation protocol was used to generate reticulocytes. The stability of iPSC lines was confirmed with chromosomal analysis and RT-PCR. Morphology and cell counts were determined by microscopy observations and flow cytometry. RESULTS: Cells from all donors were successfully used to generate iPSC lines, which were differentiated into erythroid precursors without any apparent chromosomal mutations. This differentiation protocol resulted in moderate erythrocyte yield per iPSC. CONCLUSIONS: It has previously only been hypothesized that erythroid differentiation from iPSCs could be used to produce RBCs for transfusion to patients with rare blood types or who have been alloimmunized. Our results demonstrate the feasibility of producing autologous iPSC-differentiated RBCs for clinical transfusions in patients without alternative options.

Immuno-hematological findings in Delayed Hemolytic Transfusion Reaction (DHTR).

Sickle cell disease (SCD) is the most prevalent genetic disorder in France. Many other countries are also affected. Transfusion is still a key treatment for patients suffering from this condition. As a result, SCD patients are much more exposed to transfusions and their risks than the general population. The most feared situation is delayed hemolytic transfusion reaction (DHTR). In certain situations, defined as hyperhemolysis, autologous red blood cells (RBCs) are also targeted and destroyed. This can put the patient in a life-threating situation. Further transfusions worsen the hemolysis. As DHTR will mimic a new or resistant vaso-occlusive crisis, it can be easily underdiagnosed. SCD patients are more likely to be alloimmunized than the general population, due to discrepancies between the recipient's and donor's RBCs phenotypes. Furthermore, they are often transfused in an inflammatory state, and they also frequently harbor partial antigens in the RH system. SCD patients are more prone to develop a new alloantibody than the general population. As a result, patients with DHTR often have complex mixtures of allo and autoantibodies; RH antibodies and those considered as irregular natural antibodies are frequent. Nevertheless, about a third of DHTRs are reported in patients with no previous history of immunization. In addition, a third of SCD patients will not develop an antibody after a DHTR. The evanescence of the antibodies is important. In several studies, DHTRs were reported only in patients who were occasionally transfused. Identifying patients at risk of developing a DHTR is key to managing them properly.

Challenges of Establishing a National Rare Donor Program in Iran.

Background: Over the past decades, interest in establishing a National Rare Donor Program has increased significantly worldwide. The experience of developing countries, however, is still limited. Rare blood is defined as a blood group found in a 1000- 5000 population and donor has an absence of a high-prevalence antigen, or the absence of multiple common antigens. Iranian national rare donor program was established in 2009. This paper reports the experiences and challenges of establishing a national rare donor program in Iran. Materials and Methods: This program provides services to all medical centers that need rare units. The main role of rare donor program is to maintain information of rare donors that are identified at the immunohematology reference laboratory located in Tehran. Good manufacturing practices and standard operating procedures are utilized to all activity. The IRL secures frozen blood to make them available when rare blood is required. Results: As many as 1000 different types of rare donors have been identified in Iran, including several individuals whose blood group had developed clinically significant allo-antibodies. In addition to routine donors' personally identifiable information such as addresses and telephone numbers, we also access to the contact information of their close relatives or friends for emergency situation. Contact data are kept up to date at least twice annually. IRL staff are ready to provide services to patients with rare blood types, 24 hours per day, 7 days per week. To date, more than 80 donors with very rare blood group are listed on the IRL rare donor database in 31 centers. Current practice at IRL is to screen the first and second-degree relatives of any patient found to have a rare blood type for a matching blood donor. Iranian blood services need to establish special departments to provide rare blood RBCs and technical assistance for a quicker and more efficient responses to patients and request of their medical staff for blood transfusion. To achieve this aim, there were several challenges, including situation analysis and justification of the program, allocation of financial support by top managers, engineering and technical maintenance, facility and environmental services, employee awareness and communication between blood centers, technologist training in advanced immunohematology. Conclusion: The results of this survey are encouraging and indicate that the information and database for rare donors will provide services to patients with very difficult and complex serology test results requiring rare blood transfusion. The experience of IRL may be helpful for other transfusion centers in developing countries.

Feasibility and safety of preoperative autologous blood donation for pregnant patients / 医学研究生学报

Objective Preoperative autologous blood donation ( PABD) may reduce the need for allogeneic blood , but it may also cause a short massive blood loss in pregnant women , and its fetal and maternal safety has to be adequately assessed .This study was to evaluate the feasibility and safety of PABD for pregnant women and their fetuses . Methods A prospective observational study was conducted among the women who met the inclusion criteria and gave birth in Nanjing Drum Tower Hospital between January and December 2013 .According to the clinical validation of risk stratification criteria for peripartum hemorrhage of California 2013 , the ca-ses were classified into a low-, a medium-, and a high-risk group.Data on blood donation procedures , obstetric outcomes, and blood transfusions were collected after delivery for analysis . Results Totally, 92 pregnant women accomplished 115 blood donations .The median volumes of the donated blood were 300, 300, and 400 mL in the low-, medium-, and high-risk groups, respectively ( P>0.001).There were no significant changes in HR , SBP and SpO2 during the blood donation procedures (P>0.05) except for the fall of diastolic blood pressure by an average of 3.4 mmHg (P0.05), which were similar to those in the cases who donated twice , with no significant differences before and after the donation (P>0.05). Homologous blood transfusion was performed for 5 cases (17.9%) in the high-risk group, with the volume of blood loss >2000 mL in all the cases.All the newborns survived without asphyxia and there was no perinatal death . Conclusion PABD can provide timely autologous whole blood donation for pregnant women .Under strict management , PABD is feasible and safe for pregnant patients who are at a high risk for massive blood loss during delivery or have a rare type of blood no readily available .

A Study to Establish Rare Blood Type Donor Registration System / 대한수혈학회지

BACKGROUND: To find compatible rare bloods, the Korean Red Cross (KRC) blood center is currently testing many units randomly selected from the units in storage. Since this procedure is usually very time-consuming, it is necessary to establish a new donor management system for the rare blood types. METHODS: We evaluated 261 units of red blood cells (RBCs), which were supplied as compatible bloods to the requests of hospitals in 2003. RESULTS: A total of 14 hospitals requested 248 units of compatible RBCs for their 39 patients through 64 occasions and 261 units were supplied. The blood types of 35 patients were identified using the medical records of the hospitals. A total of 19 kinds of specific antigens were negative and, among them, 10 kinds of single specific antigens were negative and 9 kinds of multiple antigens were negative. The frequencies of the negative antigens were C+e(17.9%), E+c(10.3%) and Jka(10.3%), respectively. The number of tested blood units was 1,894 and the rate of compatibility was 13.8%. The mean age of 257 blood donors was 22.5 and, among them, 220 donors (85.6%) were multiple donors with 7.5 collections on average. CONCLUSION: We conclude that the rare blood type donor registration system is necessary and suggest the following preconditions. First, the results of compatible blood test and irregular antibody screening test for the blood donor need to be registered automatically by the blood information management system(BIMS). Second, the test system should be improved to enhance the irregular antibody detection rate. Third, establishment of a frozen-thawing system for RBCs is needed. Fourth, the donors need to be informed of the results of their rare blood type, if any, including irregular antibody. Fifth, a new organization should be established to manage the rare blood type donor registration system. Finally, well cooperation with hospitals and KRC blood center is needed to ensure the stability of blood service for rare blood type RBCs.