[Molecular Mechanism of a Rhesus D Variant Individual with RHD*845A/1227A].

OBJECTIVE: To explore the genetic mutation mechanism of a rare Rhesus D variant individual. METHODS: Regular serological assay was used for determination of Rh type for the sample. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and screen the antibodies. D-screen reagent was used to analyze the RhD epitopes of the sample. RHD genotype and RHD zygosity testing of the sample were detected by palymerase chain reaction with sequence-specific primers (PCR-SSP). The full length coding region of RHD gene was sequenced. RHD mRNA was detected using reverse transcription polymerase chain reaction (RT-PCR). The PCR products were cloned and sequenced. RESULTS: The RhD blood group of the sample was determined as weak D, and the Rh phenotype was CcDEe. The antibody screening was negative. The sample tested with all monoclonal anti-Ds in D-screen showed the D epitope profiles as partial D types. The analysis of RHD gene sequence indicated that the individual with RHD c.845G/A and RHD c.1227G/A base heterozygosis. Three kinds of alternative splicing isoforms were obtained by TA cloning and sequencing. CONCLUSION: The object has RHD c.845G/A and RHD c.1227G/A mutation. This heterozygous mutation is responsible for the low expression of RhD antigen on the red blood cells of the sample.

[Genotyping of Duffy Blood Group by Microchip Capillary Electrophoresis].

OBJECTIVE: To explore a method for rapidly screening the Duffy blood group genotypes and to establish an information bank of rare blood type donors. METHODS: The microfluidic capillary electrophoresis system and PCR-SSP method were used to analyze the Duffy genotype of 3 936 unrelated O-type blood donors in our center from December 2014 to September 2018. The serologic identification and typing of other blood type system phenotypes for FYa-negative specimen were performed. The Fy (a-b-) specimen was sequenced for genotyping. RESULTS: The phenotypes of FYa-negative specimens were consistent with the genotyping results of the microfluidic capillary electrophoresis system. The results of Duffy phenotyping in 3 936 specimens were follows: Fy (a+b-) 3 564 (90.54%), Fy(a+b+) 353 (8.97%), Fy(a-b+) 18 (0.46%), Fy (a-b-) 1 (0.03%). The gene frequency was FY*A (95.03%), FY*B (4.94%), and FY*Null (0.03%). The bank of rare blood types of 19 FYa-negative specimens was established. CONCLUSION: Microfluidic capillary electrophoresis system is suitable for Duffy blood group genotyping screening. It can be used to establish a bank of rare blood type, so as to solve the problem of urgent blood transfusion in patients with rare blood type, and to improve blood transfusion safety.

DEL RBC transfusion should be avoided in particular blood recipient in East Asia due to allosensitization and ineffectiveness.

Previously, both primary and secondary anti-D alloimmunizations induced by "Asian type" DEL (RHD1227A allele) were observed in two incidents. We investigated how often these alloimmunization events occur. The transfusions of any D-negative patients were investigated in the First Affiliated Hospital of Xi'an Jiaotong University Medical College, China, during the entire 2009. The antigens of D, C, c, E, and e were routinely serotyped. The "Asian type" DEL variant was genotyped and the RHD heterozygote was determined through two published methods. The changes in anti-D levels were monitored by the indirect antiglobulin test (IAT) and flow cytometry. Thirty D-negative transfused patients were included in the study. We focused on 11 recipients who were transfused with packed red blood cells (RBCs) from DEL donors at least one time. Of those 11 recipients, seven were anti-D negative before transfusion and four were anti-D positive (one patient with an autoantibody). One of the seven pre-transfusion anti-D negative patients produced a primary-response anti-D after being transfused with 400 ml of DEL blood twice. All four pre-transfusion antibody positive patients were not observed hemoglobin (Hb) levels increased, as expected after transfusions. Two patients had an increase in anti-D from 1:8 to 1:64 by IAT, which was also shown by flow cytometry. None of the patients experienced an acute hemolytic episode. Our data indicated that the primary anti-D induced by DEL transfusion or the secondary anti-D elevated by DEL in a truly D-negative patient might not be unusual. We suggest that a truly D-negative childbearing-aged woman should avoid DEL transfusion to protect her from primary anti-D allosensitization. In addition, anti-D positive recipients should also avoid DEL red cell transfusion due to the delayed hemolytic transfusion reaction (DHTR).

[Molecular polymorphism of gypa gene in association with MN human blood group in Chinese Han population].

This study was purposed to investigate the molecular polymorphism of gypa gene in association with MN human blood group in Chinese Han population. The MN phenotypes of 202 random samples from unrelated Chinese Han volunteers were identified by serology techniques. The primer for gypa gene exon 2 were designed and synthesized according to reference sequences of NG-007470 gene from GenBank, the DNA of 202 samples was amplified by PCR, at the same time, the amplified products were analyzed by direct DNA sequencing. The results showed that all samples had 2 base substitutions at 1st and 56th nt of gypa exon 2, among them the MN phenotype heterozygote exited mainly in the form of 1A > C, 22T/C, 34A/G, 35T/G, 56T > C; the MM phenotype homozygote exited mainly in the form of 1A > C, 22C, 34G, 35T, 56T > C; the NN phenotype homozygote exited mainly in the form of 1A > C, 22T, 34A, 35G, 56T > C. It is concluded that the polymorphism of gypa gene in associated with MN blood group in Chinese Han population is decided by 5 nucleotide sites of 1, 22, 34, 35 and 56. The bases of 1 and 56 are non-functional gypa single nucleotide polymorphism.

Antenatal Rh prophylaxis is unnecessary for "Asia type" DEL women.

BACKGROUND: Generally Rh-negative patients need to be transfused with Rh-negative red blood cells. For pregnant women carrying Rh-positive fetus, the antenatal anti-D detection and Rh immunoglobulin prophylaxis are required worldwide. In East Asia, a RhD variant, termed "Asia type" DEL, was found in approximately 30% of apparent Rh-negative individuals. The antigenic and molecular properties of the DEL were previously defined. Few data discuss whether DEL could be immunized by D antigen clinically although DEL was reported arousing alloimmunization to true Rh-negative patients. STUDY DESIGN AND METHODS: To determine whether the DEL variant can be immunized to the D antigen, we retrospectively evaluated 104 Rh-negative pregnancies with allo-anti-D antibodies, and we also tracked 199 consecutive apparent Rh-negative pregnant women, with a history of gestations or parturitions but not subject to anti-D gamma-globulin prophylaxis, for evidence of allo-anti-D. The DEL variant was first excluded by ccee phenotypes and then identified through PCR analysis or sequencing. RESULTS: In the retrospective study, we expected to find 30 DEL variants, yet none of the anti-D alloimmunized women were DEL-positive. And in the second group, none of 44 DEL-positive women versus 38 of 155 (24.5%) true Rh-negative women (those excluding DEL) formed allo-anti-D. CONCLUSION: The data indicate that the "Asia type" DEL variant does not appear at risk of alloimmunization to the D antigen. It strongly suggests that the antenatal Rh immune globulin prophylaxis is unnecessary for DEL women. Furthermore, it implicates that the "Asia type" DEL may be deemed Rh-positive safely for clinical transfusion therapy.