We evaluated the clinical usefulness of simultaneous LISS/Coombs and NaCl/Enzyme testing using the gel method for screening and identification of unexpected antibodies in 15,014 samples. When unexpected antibodies were detected by either screening test, those antibodies were identified using both the LISS/Coombs and the NaCl/Enzyme gel test. The positive screening rates of the LISS/Coombs, NaCl/Enzyme, and combined tests (excluding 25 autoantibody cases) were 0.48%, 1.29%, and 1.39%, respectively. Among the 57 samples positive by both screening methods, the antibodies in 19.3% could be identified only by the NaCl/Enzyme method. Among the 137 samples positive only by NaCl/Enzyme screening, 74.5% showed positive results in antibody identification only by the NaCl/Enzyme test, although 7.3% were also positive in the LISS/Coombs test. The NaCl/Enzyme method thus showed about threefold higher detection rates than the LISS/Coombs method, especially in screening for Rh antibodies, and higher exact identification rates and discriminatory power for identifying mixed antibodies. Addition of the NaCl/Enzyme method to routine laboratory procedures may detect and identify considerable numbers of significant antibodies that might be missed if only the LISS/Coombs method is used.
Antibodies/*analysis/immunology ; *Coombs' Test ; Erythrocytes/*immunology ; Hemagglutination Tests/*methods ; Humans ; Isoantibodies/analysis ; Reagent Kits, Diagnostic

BACKGROUND: Although single antigen bead assays (SAB) are approved qualitative tests, the median fluorescence intensity (MFI) values obtained from SAB are frequently used in combination with quantitative significances for diagnostic purposes. To gauge the reproducibility of SAB results, we assessed the interlaboratory variability of MFI values using identical kits with reagents from the same lot and the manufacturer's protocol. METHODS: Six serum samples containing HLA-specific antibodies were analyzed at five laboratories by using Lifecodes LSA Class I and Class II SAB kits (Immucor, USA) from the same lot, according to the manufacturer's protocol. We analyzed the concordance of qualitative results according to distinct MFI cutoffs (1,000, 3,000, 5,000, and 10,000), and the correlation of quantitative MFI values obtained by the participating laboratories. The CV for MFI values were analyzed and grouped by mean MFI values from the five laboratories (<1,000; 1,000-2,999; 3,000-4,999; 5,000-9,999; and > or =10,000). RESULTS: The categorical results obtained from the five laboratories exhibited concordance rates of 96.0% and 97.2% for detection of HLA class I and class II antibodies, respectively. The Pearson correlation coefficients for MFI values of class I and class II antibodies were between 0.947-0.991 and 0.992-0.997, respectively. The median CVs for the MFI values among five laboratories in the lower MFI range (<1,000) were significantly higher than those for the other MFI ranges (all P<0.01). CONCLUSIONS: Analysis of SAB performed in five laboratories using identical protocols and reagents from the same lot resulted in high levels of concordance and strong correlation of results.
Analysis of Variance ; HLA Antigens/immunology ; Histocompatibility Testing ; Humans ; Isoantibodies/*blood ; Laboratories ; Reagent Kits, Diagnostic ; Reproducibility of Results

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

BACKGROUNDHumoral immunity is an important factor for long-term survival of renal allograft. Here we performed a four-year follow-up to explore the clinical significance of monitoring anti-human leukocyte antigens (HLA) and anti-major histocompatibility complex class I-related chain A (MICA) antibody expression after kidney transplantation.

METHODSWe obtained serial serum samples from 84 kidney transplant patients over a four-year period. All patients were followed up at least 6 months after transplantation and had at least two follow-up points. Anti-HLA and anti-MICA antibody titres and serum creatinine (SCr) levels were evaluated at each follow-up. Patients were divided into 4 groups: HLA(+) MICA(-), HLA(-)MICA(+), HLA(+)MICA(+) and HLA(-)MICA(-). The impact of post-transplant antibody level on kidney allograft function was evaluated.

RESULTSAntibodies were detected in 38.1% (32/84) of the renal allograft recipients. HLA, MICA and HLA+MICA expression was observed in 18.89%, 14.44% and 5.93% of the recipients respectively. The most frequent anti-HLA and anti-MICA specific antibodies identified were A11, A24, A29, A32, A33, A80; B7, B13, B37; DR17, DR12, DR18, DR52, DR53, DR1, DR4, DR9, DR51; DQ7, DQ4, DQ8, DQ2, DQ9, DQ5, DQ6 and MICA02, MICA18, MICA19, MICA07, MICA27. As the time after transplantation elapsed, more recipients developed de novo antibody expression. Total 11.91% (10/84) of the recipients had de novo antibody expression during the follow up. The average level of SCr and the percentage of recipients with abnormal allograft function were significantly higher in recipients with anti-HLA and/or anti-MICA antibody expression than those without. The appearance of anti-HLA and anti-MICA antibody expression always preceded the increase in SCr value.

CONCLUSIONSAnti-HLA and anti-MICA antibody expression has predictive value for early and late allograft dysfunction. The presence of donor specific antibody is detrimental to graft function and graft survival.

Female ; Follow-Up Studies ; Graft Survival ; HLA Antigens ; immunology ; Histocompatibility Antigens Class I ; immunology ; Humans ; Isoantibodies ; analysis ; Kidney Transplantation ; Male ; Minor Histocompatibility Antigens

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

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.
Animals ; Base Sequence ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Female ; H-Y Antigen ; analysis ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Isoantibodies ; genetics ; immunology ; Male ; Mice ; Molecular Sequence Data ; Peptide Library

BACKGROUND: Duffy (FY) blood group genotyping is important in transfusion medicine because Duffy alloantibodies are associated with delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. In this study, FY allele frequencies in Thai blood donors were determined by in-house PCR with sequence-specific primers (PCR-SSP), and the probability of obtaining compatible blood for alloimmunized patients was assessed. METHODS: Five hundred blood samples from Thai blood donors of the National Blood Centre, Thai Red Cross Society, were included. Only 200 samples were tested with anti-Fy(a) and anti-Fy(b) using the gel technique. All 500 samples and four samples from a Guinea family with the Fy(a-b-) phenotype were genotyped by using PCR-SSP. Additionally, the probability of obtaining antigen-negative red blood cells (RBCs) for alloimmunized patients was calculated according to the estimated FY allele frequencies. RESULTS: The FY phenotyping and genotyping results were in 100% concordance. The allele frequencies of FY*A and FY*B in 500 central Thais were 0.962 (962/1,000) and 0.038 (38/1,000), respectively. Although the Fy(a-b-) phenotype was not observed in this study, FY*B(ES)/FY*B(ES) was identified by PCR-SSP in the Guinea family and was confirmed by DNA sequencing. CONCLUSIONS: Our results confirm the high frequency of the FY*A allele in the Thai population, similar to that of Asian populations. At least 500 Thai blood donors are needed to obtain two units of antigen-negative RBCs for the Fy(a-b+) phenotype.
Adult ; Alleles ; Asian Continental Ancestry Group/genetics ; Base Sequence ; Blood Donors ; DNA/chemistry/genetics/metabolism ; Duffy Blood-Group System/*genetics/immunology ; Female ; Gene Frequency ; Genotype ; Humans ; Isoantibodies/blood/immunology ; Male ; Middle Aged ; Phenotype ; Polymerase Chain Reaction ; Receptors, Cell Surface/genetics/*immunology ; Sequence Analysis, DNA ; Thailand ; Young Adult