BACKGROUND: This study compared the estimated costs and times required for ABO/Rh(D) typing and unexpected antibody screening using an automated system and manual methods. METHODS: The total cost included direct and labor costs. Labor costs were calculated on the basis of the average operator salaries and unit values (minutes), which was the hands-on time required to test one sample. To estimate unit values, workflows were recorded on video, and the time required for each process was analyzed separately. RESULTS: The unit values of ABO/Rh(D) typing using the manual method were 5.65 and 8.1 min during regular and unsocial working hours, respectively. The unit value was less than 3.5 min when several samples were tested simultaneously. The unit value for unexpected antibody screening was 2.6 min. The unit values using the automated method for ABO/Rh(D) typing, unexpected antibody screening, and both simultaneously were all 1.5 min. The total cost of ABO/Rh(D) typing of only one sample using the automated analyzer was lower than that of testing only one sample using the manual technique but higher than that of testing several samples simultaneously. The total cost of unexpected antibody screening using an automated analyzer was less than that using the manual method. CONCLUSIONS: ABO/Rh(D) typing using an automated analyzer incurs a lower unit value and cost than that using the manual technique when only one sample is tested at a time. Unexpected antibody screening using an automated analyzer always incurs a lower unit value and cost than that using the manual technique.
ABO Blood-Group System/blood ; Antibodies/analysis ; Automation ; Blood Banks/*economics/*standards ; Blood Grouping and Crossmatching/*economics/instrumentation ; Costs and Cost Analysis ; Humans ; Rh-Hr Blood-Group System/blood ; *Workflow ; Workload

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.
Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

To investigate the RHD gene profiles of RhD-negative individuals in population of Fujian Province, it was to design fourteen pairs of specific primers to amplify RHD exon 1, 3 approximately 7, 9, 10, hybrid Rh box, RHD 1227A allele, RHC allele, RHc allele, RHE allele and RHe allele. Rh genotypes were detected by PCR-SSP in 104 RhD-negative donors, some samples with or without RHD genes were analysed by the absorption-elution test, and two RhD-negative samples with eight RHD exons detected were analysed by DNA sequencing. The results showed that 61.54% RhD-negative individuals lacked all the eight RHD exons detected (RHD-/RHD-), 25.97% carried the RHD 1227A allele (62.96% of which were the heterozygote of RHD+/RHD-, and 37.04% were the homozygote of RHD+/RHD+), 8.65% carried the RHD-CE (2 approximately 9)-D allele (RHD+/RHD-), and 1.92% carried the RHD 710delC allele (RHD+/RHD-). Though the most cases of RHD gene deletion were found in dce haplotype, six cases of RHD gene deletion were found in dCe (their RH genotypes were dce/dCe) and two in dcE haplotype (their RH genotypes were dce/dcE). And it was not accurate to predict the Rh phenotype by detecting a single RHD exon, however, and more accurate when eight RHD exons and RHD 1227A allele were detected (chi2=24.43, p<0.005). It is concluded that RHD genes in population of Fujian Province are polymorphic and the RHD genotyping is not reliable enough to replace the RhD serotyping in China.
Asian Continental Ancestry Group ; genetics ; Blood Donors ; China ; Erythrocytes ; immunology ; Exons ; genetics ; Gene Deletion ; Genotype ; Humans ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

BACKGROUND: Despite the advances in total laboratory automation, a considerable amount of work in blood banks is still done using outdated manual methods. Some automated pre-transfusion testing instruments have recently been developed. Of these, we evaluated and compared the AutoVue Innova (Ortho, USA) and the Techno TwinStation (DiaMed AG, Switzerland). METHODS: Forward and reverse ABO/Rh typing and unexpected antibody screening and identification tests were performed on 4,628 samples using the manual method and the two automated instruments. Two different anticoagulants (EDTA and citrate) were compared in ABO/Rh typing and unexpected antibody screening tests. Titrating studies were conducted on the following 7 dilutions using 5 samples of irregular antibodies with anti-E, anti-E & -c, anti-D, and anti-Le(a) with anti-Fy(a): 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128. The test throughput per hour, the time required to perform 1 and 100 tests, and a simulation test for total events occurring in 1 day were also measured. RESULTS: No erroneous results were reported between the two instruments and the manual method. Discrepancies observed in 10 cases (0.4%) of ABO/Rh typing were of higher intensity with AutoVue Innova than with the manual method. AutoVue Innova exhibited the highest sensitivity in the titrating study and throughput performance compared with the manual method and the Techno TwinStation. Especially in the throughput and time required to complete 100 antibody screening tests, AutoVue Innova had a 3.3- and 3.5-fold higher performance, respectively, than Techno TwinStation. CONCLUSIONS: Because both of the two fully automated instruments (AutoVue Innova and Techno TwinStation) had high levels of accuracy and performance, it is expected that use of fully automated instruments will reduce human labor, turnaround time, and operator error in the blood bank.
ABO Blood-Group System/blood ; Antibodies/blood ; Automation ; Blood Grouping and Crossmatching/*instrumentation ; Blood Transfusion ; Cost-Benefit Analysis ; False Positive Reactions ; Humans ; Rh-Hr Blood-Group System/blood

Here we report a severe case of hemolytic anemia of the newborn with kernicterus caused by anti-Di(a) antibody. A full term male infant was transferred due to hyperbilirubinemia on the third day of life. Despite single phototherapy, the baby's total bilirubin had elevated to 30.1 mg/dL. After exchange transfusion, total bilirubin decreased to 11.45 mg/dL. The direct antiglobulin test on the infant's red cells was positive. The maternal and infant's sera showed a negative reaction in routine antibody detection tests, but were positive in Di(a) panel cells. The frequency of the Di(a) antigen among the Korean population is estimated to be 6.4-14.5%. Anti-Di(a) antibody could cause a hemolytic reaction against transfusion or hemolytic disease of the newborn. We suggest the need for reagent red blood cell panels to include Di(a) antigen positive cells in antibody identification test for Korean.
Alleles ; Bilirubin/blood ; Erythroblastosis, Fetal/*diagnosis/*immunology ; Humans ; Infant, Newborn ; Isoantibodies/*analysis ; Male ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System/*analysis/blood

In the present day, pretransfusion tests include ABO and RhD grouping, antibody screening, antibody identification, and cross matching. Although error rates for these tests have decreased compared to those in the past, clerical errors still occur. When exposed to RhD positive RBCs, a RhD negative person can produce anti-D that causes a severe hemolytic disease of the fetus and the newborn in addition to hemolytic transfusion reactions. Therefore, administration of RhD positive RBCs to a RhD negative person should be avoided. We experienced a RhD negative patient who had been misidentified as positive and transfused 35 units of RhD positive RBCs eight years ago, but did not have detectable anti-D in present. The red cells of the patient showed no agglutination with the anti-D reagent and a negative result in the standard weak D test. The multiplex PCR with sequence-specific priming revealed that the patient was RhD negative.
*Blood Group Incompatibility ; Blood Transfusion ; Erythrocytes/*immunology ; Humans ; Isoantibodies/*analysis/immunology ; Male ; Middle Aged ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System/*analysis/immunology

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.
Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Chromosome Deletion ; Cloning, Molecular ; Gene Deletion ; Gene Frequency ; genetics ; Humans ; Molecular Sequence Data ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology ; Sequence Analysis, DNA

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.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Blood Donors ; China ; ethnology ; Erythrocytes ; immunology ; Exons ; genetics ; Humans ; Molecular Sequence Data ; Phenotype ; Point Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology ; Sequence Analysis, DNA

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.
Asian Continental Ancestry Group ; genetics ; Base Sequence ; Blood Donors ; China ; ethnology ; Erythrocytes ; immunology ; Exons ; genetics ; Genotype ; Haplotypes ; Humans ; Molecular Sequence Data ; Phenotype ; Point Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology ; Sequence Analysis, DNA

OBJECTIVETo elucidate the molecular background of Del phenotype in the Chinese population and explore new Del alleles.

METHODSFive hundred and fifteen RhD negative blood samples was tested by Rh typing test, indirect antiglobulin test and adsorption and elution assay to screen the Del phenotype. DNA of all the Del samples was analysed by multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by sequence-specific primer polymerase chain reaction (PCR-SSP) for Del alleles: RHD 1227A and RHD 885T. Samples which showed the negative result by PCR-SSP, were additionally analysed by genomic DNA sequencing and cDNA sequencing.

RESULTSSeventy-nine Del samples were found by adsorption and elution assay. All these samples had RHD exons 3, 4, 5, 6, 7 and 9. Except 4 Del samples, other 75 Del samples carried the RHD 1227A allele. None of the samples had the RHD 885T allele. Four novel RHD alleles were found in these four Del sample. There were RHD 3G-->A (GenBank DQ310735), RHD 28C-->T, RHD 53T-->C (GenBank DQ451877,DQ451878), RHD 251T-->C (GenBank DQ310734).

CONCLUSIONfnRh blood group system is very complex. New D variation phenotypes and new RHD alleles may be discovered ceaselessly.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genetics, Population ; methods ; Genotype ; Humans ; Molecular Sequence Data ; Mutation ; Phenotype ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA