Acidification of plasma for detection of pHdependent antibodies.

CONCLUSIONS: Most antibodies to blood group antigens react optimally at a pH range of 6.5-7.5, similar to that of normal plasma or serum. Some antibodies, however, including anti-M, react preferentially or exclusively in an acidic environment with a pH below 6.5. Antibodies with anti-M specificity often show dosage. They can be weakly reactive and even look like nonspecific reactivity at the antihuman globulin phase especially when immediate spin and/or room temperature testing is not part of routine screening. Acidification of serum or plasma may help to identify these antibodies as clinically insignificant versus as an unidentified antibody for which clinical significance is unknown.

Performance and reliability of a benchtop automated instrument for transfusion testing: a comparative multicenter clinical study in the US population.

BACKGROUND: Heavy workload in hospital transfusion services and blood centers necessitates the implementation of automated platforms. We evaluated the performance of Erytra Eflexis (Diagnostic Grifols), a recently developed midsize automated instrument for pretransfusion testing, in comparison with a US Food and Drug Administration (FDA)-cleared device (Erytra). Reproducibility and repeatability of the results were also investigated. STUDY DESIGN AND METHODS: Studies were conducted using the same card technology and reagents at three US sites. Tests were performed on 9174 specimens from hospital patients (55.61%) and blood donors (43.39%). Evaluations included 18,413 ABO/D/reverse typing; 9084 Rh phenotypes, 4640 K phenotypes, 2052 antibody screenings, 1232 antibody identifications, 469 direct antiglobulin tests, 612 IgG crossmatches, and 700 ABO-compatibility crossmatches. A reference blood panel was also sent to each center, for a total of 3900 replicate tests. Concordance between results with the two instruments and performance among the different centers were statistically evaluated. RESULTS: Agreement between instruments was 99.84% for 37,202 test results, with 61 discrepancies (0.16%). Percentages of positive and negative agreement were 99.82% and 99.85%, respectively. No discrepancies were observed in 12,276 tests for direct ABO/D grouping. Discrepancies were observed during antibody identification (n = 19), antibody screening (n = 15), and reverse grouping (n = 10). Investigations of the discrepancies were resolved in favor of the study instrument in 55.73% of the cases. Erytra Eflexis obtained the expected results in the reproducibility analysis. CONCLUSION: This multicenter study demonstrates that Erytra Eflexis with its gel card technology and reagents is reliable and substantially equivalent to the FDA-cleared instrument used as the reference.

A comparison of methods for the detection of the r'(s) haplotype.

BACKGROUND: The r'(s) haplotype is found in 5% to 15% of individuals of African descent. Persons with this haplotype have a partial C antigen and weakened e and can produce anti-C or other "e-like" alloantibodies. Thus, for these chronically transfused patients, accurate detection of the r'(s) haplotype is important for selection of matched units. STUDY DESIGN AND METHODS: African-American donors were genotyped using the human erythrocyte antigen (HEA) microarray. Samples (n = 125) identified as "possible r'(s) " were then tested by IDCORE XT and RHD and RHCE microarrays. DNA sequencing was used to resolve discordant samples. The genotyping results were compared to serologic testing using a monoclonal anti-C reagent (Clone MS24). RESULTS: Of the 125 possible r'(s) samples identified by HEA, only 94 (75%) were confirmed by both RHD and RHCE microarrays. The IDCORE XT accurately detected 93 of 94 (99%) of the confirmed r'(s) and had no false positives. DNA sequencing of the one discordant sample revealed the presence of a compound heterozygote with RHD* DIII.4/RHCE*ceVS.02 as one haplotype and r'(s) Type 2 as the other. The 31 unconfirmed r'(s) samples carried RHCE*ceVS.03 not linked to the hybrid RHD-CE-D. This occurred most often with RHD*DIIIa (55%) or RHD*01 (19%) and rarely with DIII.4, DIII.6, DAU3, and weak D Type 14. Serologic testing with anti-C gave 100% concordance with the r'(s) samples. CONCLUSIONS: The predominant type of r'(s) in African-Americans is Type 1, which can be detected either by a reagent anti-C containing Clone MS24 or by IDCORE XT. However, serology cannot differentiate between a normal C allele and the hybrid.

Three novel alleles in the Kell blood group system resulting in the Knull phenotype and the first in a Native American.

BACKGROUND: Antibodies to Kell antigens can be clinically important but only limited data are published regarding anti-Ku. Missense nucleotide changes in KEL account for the numerous Kell antigens, the K(mod) phenotype, and even the K(null) phenotype. STUDY DESIGN AND METHODS: DNA and RNA were extracted from white blood cells and polymerase chain reaction-based assays, cloning, and sequencing were done using standard protocols. RESULTS: The anti-Ku in Proband 1, which caused hemolytic disease and anemia of the fetus and newborn, was a mixture of immunoglobulin (Ig)G1 and IgG2 and gave macrophage indexes ranging from 47.8 to 59.3 (>20 is clinically significant) in a monocyte monolayer assay. The proband, her daughter, and compatible sister had a heterozygous deletion of a G in Exon 18 (Nucleotide c.1972_1975delG) in a KEL*02 allele causing a frameshift. The mechanism for silencing of the other KE*02 allele was undetermined. Proband 2 was heterozygous for a nonsense change (KEL*382C/T; Arg128Stop), a missense change (KEL*244T/C; Cys82Arg), and KEL*578T/C (KEL*01/KEL*02). Direct sequencing of cDNA and cloning showed that the KEL*01 allele had 244C, 382C, 578T and the KEL*02 allele carried 244T, 382T, 578C. CONCLUSIONS: We report a novel single-nucleotide deletion, a novel nonsense allele, and a novel missense allele all resulting in the K(null) phenotype. The anti-Ku from Proband 1 was clinically important.

A novel JK null allele associated with typing discrepancies among African Americans.

The Jknun (Jk-3) phenotype, attributable to null or silenced alleles, has predominantly been found in persons of Polynesian descent. With the increased use of molecular genotyping, many new silencing mutations have been identified in persons of other ethnic backgrounds. To date, only two JK null alleles have been reported in African Americans, JK*01N.04 and JK*OlN.OS.A comparative study was undertaken to determine whether JK mutations were present in the regional African American population. Results of donor genotyping were compared with previously recorded results of serologic tests, and discrepant results were investigated. Although the two previously identified polymorphisms were not detected in the discrepant samples, a novel allele (191G>A) was identified and was assigned the ISBT number JK*02N.09. This study illustrates a limitation of using single-nucleotide polymorphisms for prediction of blood group antigens.

A novel JKA allele, nt561C>A, associated with silencing of Kidd expression.

BACKGROUND: The Jk(a-b-) null phenotype is not common but is more prevalent in Polynesian and Asian persons and appears to be rare in blacks. We determined the molecular basis for Jk(a-b-) in an African American family. DNA testing of samples from random African American, Caucasian, and Brazilian blacks was done to estimate the allele frequency. STUDY DESIGN AND METHODS: Standard methods were used for red blood cell (RBC) typing. DNA was isolated from white blood cells, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification and sequencing of the coding regions of JK were performed by routine molecular methods. A MaeIII PCR-RFLP assay was designed to target the nucleotide (nt) change. RESULTS: RBCs from the proband typed as Jk(a-b-) and DNA testing indicated JK*A/JK*A. JK sequencing found that the sample was homozygous for nt561C>A change, predicted to encode a premature stop in the protein (187Stop). The altered allele was present in the heterozygous state in three of six siblings. Testing of 500 African American and 100 Caucasian donors from the same region and 500 African American donors from the southern United States found no additional examples. Screening of 1174 Brazilian blacks revealed seven examples: one homozygote and six heterozygotes. CONCLUSIONS: JK*A (561C>A) is associated with a Kidd-null phenotype in this African American family. The allele was present in approximately one in 168 Brazilian blacks, suggesting that detection of this allele is important to avoid false-positive prediction of Jk(a) status in this population.

Knops blood group polymorphism and susceptibility to Mycobacterium tuberculosis infection.

BACKGROUND: Complement receptor 1 (CR1) protein carries the Knops blood group antigens and is the receptor for the major ligand involved in Mycobacterium tuberculosis (Mtb) adhesion to macrophages. Erythrocyte CR1 binds immune complexes (ICs) formed during Mtb invasion, facilitating their clearance by the host immune system. The occurrence of specific Knops blood group genotypes among African populations was investigated to evaluate their impact on resistance or susceptibility to Mtb infection. STUDY DESIGN AND METHODS: The distribution of the Knops blood group genotypes (McC and Sl) was compared between tuberculosis (TB) patients with confirmed diagnosis of Mtb in isolates and negative controls. Conditional logistic regression was used to access the association between genotypes distribution and susceptibility to Mtb infection. RESULTS: At the McC locus, individuals heterozygous (McC(a) /McC(b) ) were more resistant to Mtb infection (odds ratio [OR], 0.42; 95% confidence interval [CI], 0.22-0.81; p = 0.007). Although less significant, a similar effect was conferred by Sl1/Sl2 genotype (OR, 0.05; 95% CI, 0.28-0.9; p = 0.02). This protective effect was maintained among individuals presenting the McC(b) /Sl2 haplotype (OR, 0.25; 95% CI, 0.08-0.74; p = 0.008). CONCLUSION: Acquisition of McC(b) and Sl2 alleles among African population is correlated with resistance to Mtb infection, adding this bacterium to the list of mechanisms underlying the selection of the Knops blood group polymorphism among these populations.