Identifying obstetrics patients in whom RHD genotyping can be used to assess risk of D alloimmunization.

CONCLUSIONS: The D antigen is highly immunogenic and may cause alloimmunization to occur after blood transfusion or pregnancy. Some RHD variant alleles express a D antigen that is missing one or more epitopes, thus putting a presumed D+ patient at risk for alloanti-D and hemolytic disease of the fetus and newborn. It is generally accepted that individuals who have a serologic weak D phenotype due to one of three alleles common in Caucasians, RHD*weak D types 1, 2, or 3, are not at risk for alloimmunization. In this study, blood samples from 46 obstetrics patients from a local health system were identified based on discrepant results between automated gel and manual tube testing (n = 20) or based on presentation with a serologic weak D phenotype (n = 26). RHD genotyping was performed using commercial and laboratory-developed tests. Of the 26 serologic weak D samples, 18 (69.2%) were found to carry alleles RHD*weak D type 1, 2, or 3. The remaining eight samples (30.8%) were found to carry partial D alleles. Of the 20 samples submitted because of D typing discrepancy, 7 (35%) carried alleles RHD*weak D type 1, 2, or 3, while 13 (65%) carried partial RHD alleles. This report summarizes the findings of one hospital system and its approach to integrating RHD genotyping into its assessment of risk of alloimmunization in obstetrics patients. It demonstrates that individuals with partial RHD alleles can present with serologic weak D phenotype, such that, without RHD genotyping, these individuals may not be identified as candidates for Rh immune globulin. The study also demonstrates that use of two methods (automated gel and tube testing) allows for identification of partial D cases that would otherwise be missed. I.

Comparison of ABO genotyping methods: a study of two low-resolution polymerase chain reaction assays in a clinical testing laboratory.

CONCLUSIONS: The ABO blood group system is the most clinically significant system in transfusion medicine. Although serologic typing for ABO antigens is routine and reliable, molecular methods can be used to predict an ABO type in the absence of a blood specimen as well as to investigate ABO typing discrepancies often caused by ABO subgroups that cause weakened antigen expression, weak or missing serum reactivity, and/or extra red blood cell reactivity. By detecting single nucleotide variants that are hallmarks of the major ABO alleles, low-resolution genotyping methods can be used to make allele assignments and predict phenotypes. This approach has become a dependable tool, initially to resolve typing discrepancies identified in blood banks and donor centers and, more recently, to predict the ABO group in bone marrow transplant donors and in deceased donors of solid organs. The aim of this report is to compare two different low-resolution polymerase chain reaction (PCR)-based methods: a PCRrestriction fragment length polymorphism (RFLP) implemented based on a publication and a commercially available TaqManbased sequence-specific primer-PCR for resolution of ABO typing discrepancies. Fifty-six peripheral blood samples from 31 patients and 25 blood donors were used to isolate genomic DNA and perform genotyping. Results of 49 of the 56 samples (87.5%) were concordant between methods, three samples yielded an unexpected banding pattern on the PCR-RFLP method, and four sample results were discordant between assays. The discordances all involved group A versus A2 discrepancies. Sanger sequencing was used as a high-resolution genotyping method to resolve discrepancies between the two low-resolution methods. This study demonstrates that, in the majority of cases, a low-resolution genotyping method can resolve an ABO discrepancy. Although there is no U.S. Food and Drug Administration-approved genotyping method for ABO determination, molecular testing for investigation of discrepancies is a useful tool for blood banks and transplant programs.

Novel mutations in KLF1 encoding the In(Lu) phenotype reflect a diversity of clinical presentations.

BACKGROUND: Mutation in the KLF1 gene is the cause of the In(Lu) (Inhibitor of Lutheran) Lu(a-b-) phenotype and more than 60 alleles have been associated with this phenotype. Here we describe findings from investigation of seven cases: six presenting with a Lu(a-b-) phenotype including the historical index case and one referred from a patient with chronic anemia. STUDY DESIGN AND METHODS: Serologic testing was by standard methods. DNA testing included amplification and sequencing of KLF1 and LU coding regions. A StuI polymerase chain reaction-restriction fragment length polymorphism was designed to target c.304T>C in KLF1. RESULTS: Five different KLF1 alleles were identified. Three are new: KLF1*90A (p.Trp30Ter), KLF*911A (p.Thr304Lys), and KLF1*304C,318G (p. Ser102Pro, Tyr106Ter) present in two unrelated individuals. Two, including the index case, had c.954dupG (p.Arg319Glufs*34), that is, KLF1*BGM06. The child with unexplained anemia had c.973G>A (p.Glu325Lys), associated with congenital dyserythropoietic anemia. The common c.304T>C was found in two of the seven samples investigated and in 60 of 100 blood donors. CONCLUSION: Mutations in KLF1 are pleiotropic and although most are benign, others are associated with hematologic abnormalities. We report three new KLF1 alleles associated with benign In(Lu) and document both the molecular basis of the original In(Lu) phenotype using a frozen sample stored for more than 50 years and the cause of unexplained anemia in a child. We also confirm previous observations that c.304C (p.102Pro) is not, by itself, associated with an In(Lu) phenotype in donors self-identified as U.S. minorities.

A Caucasian JK*A/JK*B woman with Jk(a+b-) red blood cells, anti-Jkb, and a novel JK*B allele c.1038delG.

The Kidd blood group on the red blood cell (RBC) glycoprotein urea transporter-B has a growing number of weak and null alleles in its gene SLC14A1 that are emerging from more widespread genotyping of blood donors and patients. We investigated a 64-year-old Caucasian woman of Polish-Czech descent who developed anti-Jkb detected in solid-phase RBC adherence testing within 12 days after 7 units of RBCs were transfused. Her RBCs subsequently typed Jk(a+b­) by licensed reagents and human antisera. Nevertheless, in RBC genotyping (BioArray HEA BeadChip, Immucor, Warren, NJ) performed in our transfusion service on all patients with alloantibodies, her Kidd typing was JK*A/JK*B based on the Jka/Jkb single nucleotide polymorphism in exon 9 (c.838G>A, p.Asp280Asn). Genomic analysis and cDNA sequencing of her JK*B allele revealed a novel single-nucleotide deletion of c.1038G in exon 11, predicting a frameshift and premature stop (p.Thr346Thrfs*5) after translation of nearly 90 percent of the expressed exons 4­11. This allele has been provisionally named JK*02N.14, subject to approval by the International Society of Blood Transfusion Working Party. The site of this variant is closer to the C-terminus than that of any allele associated with the Jk(a­b­) phenotype reported to date. Routine genotyping of patients with RBC alloantibodies can reveal variants posing potential risk of alloimmunization. Continuing investigation of Kidd variants may shed light on the structure of Kidd antigens and the function of urea transporter-B.

A model for integrating molecular-based testing in transfusion services.

BACKGROUND: Molecular-based laboratory tests can predict blood group antigens and supplement serological methods, adding a unique technology to assist in resolving discrepant or incomplete blood group typing or antibody identification. Hospital transfusion services have options for integrating molecular-based methods in their routine operations. We describe here the model of a hospital-reference laboratory partnership. MATERIALS AND METHODS: Blood samples for compatibility testing were obtained from patients in a 609-bed hospital serving an urban multiethnic and multiracial population. When results of blood group phenotyping by serological methods were inconclusive, samples were referred for molecular-based testing. The reference laboratory used several methods for genotyping, including polymerase chain reaction followed by restriction enzyme-linked polymorphism analysis, sequence-specific primer polymerase chain reaction and array-based approaches. Human erythrocyte antigen, RHCE and RHD single nucleotide polymorphism arrays were integrated into the laboratory as they became commercially available. RESULTS: The hospital-reference laboratory model made it possible to integrate blood group genotyping promptly by current technology without the expense of new laboratory equipment or adding personnel with technical expertise. We describe ten cases that illustrate the categories of serological problems that were resolved by molecular methods. DISCUSSION: In-hospital molecular testing for transfusion services has logistical advantages, but is financially impractical for most hospitals. Our model demonstrates the advantages of a hospital-reference laboratory partnership. In conclusion, hospital transfusion services can integrate molecular-based testing in their routine services without delay by establishing a partnership with a molecular blood group reference laboratory. The hospital reference-laboratory model promotes genomic medicine without the expense of new equipment and skilled personnel, while supporting the economy of centralised large-scale laboratory operations.

RHCE*ceAG (254C>G, Ala85Gly) is prevalent in blacks, encodes a partial ce-phenotype, and is associated with discordant RHD zygosity.

BACKGROUND: RHCE*ceAG has the nucleotide change c.254C>G, which encodes p.Ala85Gly associated with altered expression of e antigen. We analyzed serologic and DNA-based testing data on samples with RHCE*ceAG to determine its effect on antigen expression, linkage with RHD, and its prevalence in African Americans. STUDY DESIGN AND METHODS: Serologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction-restriction fragment length polymorphism, RH-specific exon sequencing, and RHD zygosity, and Rh-cDNA was sequenced. Samples from 32 individuals referred for serologic problems, 57 patients with sickle cell disease, and 44 donors positive for c.254C>G were investigated. Allele prevalence was determined in random African Americans. RESULTS: Red blood cells from samples homozygous RHCE*ceAG/ceAG or in trans to RHCE*cE reacted variably with anti-e reagents and 17 samples from the 32 referred patients had alloanti-e in their plasma. The majority of samples with RHCE*ceAG, when tested for RHD zygosity gave discordant results between PstI-RFLP and hybrid box assay. Rare samples with 254C>G had additional allelic changes: one with c.697G (p.233Glu), three with c.733G, 941C (p.245Val, 314Ala), and two with c.307T (p.103Ser) encoding robust C antigen expression in the absence of other C-specific nucleotides. A total of 101 samples with RHCE*ceAG were encountered in 1159 randomly selected African Americans. CONCLUSIONS: RHCE*ceAG (c.254G, p.85Gly) encodes a partial phenotype and the absence of the high-prevalence antigen RH59 (CEAG). The allele was present in one in 11 African Americans and is most often in cis to a RHD deletion associated with discordant RHD zygosity. To further determine clinical significance, detection of this allele should be part of routine RHCE genotyping in this population.

First example of an FY*01 allele associated with weakened expression of Fya on red blood cells.

Duffy antigens are important in immunohematology. the reference allele for the Duffy gene (FY) is FY*02, which encodes Fy(b). An A>G single nucleotide polymorphism (SNP) at coding nucleotide (c.) 125 in exon 2 defines the FY*01 allele, which encodes the antithetical Fy(a). A C>T SNP at c.265 in the FY*02 allele is associated with weakening of Fy(b) expression on red blood cells (R BCs) (called Fy(x)). until recently, this latter change had not been described on a FY*01 background allele. Phenotype-matched units were desired for a multi-transfused Vietnamese fetus with α-thalassemia. Genotyping of the fetus using a microarray assay that interrogates three SNPs (c.1-67, c.125, and c.265) in FY yielded indeterminate results for the predicted Duffy phenotype. Genomic sequencing of FY exon 2 showed that the fetal sample had one wild-type FY*01 allele and one new FY*01 allele with the c.265C>T SNP, which until recently had only been found on the FY*02 allele. Genotyping performed on samples from the proband's parents indicated that the father had the same FY genotype as the fetus. Flow cytometry, which has been previously demonstrated as a useful method to study antigen strength on cells, was used to determine if this new FY*01 allele was associated with reduced Fy(a) expression on the father's RBCs. Median fluorescence intensity of the father's RBCs (after incubation with anti-FY(a) and fluorescein-labeled anti-IgG) was similar to known FY*01 heterozygotes. and significantly weaker than known FY*01 homozygotes. In conclusion, the fetus and father both had one normal FY*01 allele and one new FY*01W.01, is associated with weakened expression of Fy(a) on RBCs.

RHCE variant allele: RHCE*ce254G,733G.

A novel RHCE allele was identified in a 53-year-old African American female blood donor with an Rh phenotype of D+ C­ E­ c+ e+ and a negative antibody screen. The donor's cells typed e+ with all antisera tested. By gel-based genotyping and cDNA analysis, the two RHCE alleles in this donor were characterized. One allele was found to be the known allele RHCE*01.20.01 (RHCE*ce733G) and the second was novel: RHCE*01.06.02 (RHCE*ce254G,733G).

RHCE*ceMO is frequently in cis to RHD*DAU0 and encodes a hr(S) -, hr(B) -, RH:-61 phenotype in black persons: clinical significance.

BACKGROUND: RHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has Nucleotide 1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the red blood cell (RBC) antigen expression, antibody specificity, RHD association, and the prevalence in African-American persons. STUDY DESIGN AND METHODS: Serologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction-restriction fragment length polymorphism and RH-exon sequencing, and for some, Rh-cDNA was sequenced. Seventy-seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African-American persons were screened for allele prevalence. RESULTS: RBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weak or nonreactive with some anti-e and were nonreactive with polyclonal anti-hr(S) and anti-hr(B) . Twenty-three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had alloanti-e, anti-f, anti-hr(S) /hr(B) , or an antibody to a high-prevalence Rh antigen. Three patients with alloanti-c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African-American persons tested and in 69 of 77 (90%) of samples with RHCE*ceMO. CONCLUSIONS: RHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hr(S) - and hr(B) - phenotype, and the absence of a high-prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in one in 50 African-American persons with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion.

RHCE*ceTI encodes partial c and partial e and is often in cis to RHD*DIVa.

BACKGROUND: In the Rh blood group system, variant RhD and RhCE express several partial antigens. We investigated RH in samples with partial DIVa that demonstrated weak and variable reactivity with anti-C. STUDY DESIGN AND METHODS: Standard hemagglutination techniques, polymerase chain reaction-based assays, and RH sequencing were used. RESULTS: DNA analysis showed that six red blood cell (RBC) samples with weak and inconsistent reactivity with anti-C lacked RHCE*C, but all had RHD*DIVa, which encodes partial D and Go(a) . We then tested RBCs from 19 Go(a+) cryopreserved samples (confirmed to have RHD*DIVa) with four anti-C and observed weak variable reactions. RHCE genotyping found all but one of the samples with RHD*DIVa also had RHCE nt 48G>C and 1025C>T, named RHCE*ceTI. Lookback of samples referred for workup and found to have either allele revealed 47 of 55 had both RHD*DIVa and RHCE*ceTI, four had RHD*DIVa without RHCE*ceTI, and four had RHCE*ceTI without RHD*DIVa. Alloanti-c was found in a patient with c+ RBCs and RHCE*ceTI in trans to RHCE*Ce, and alloanti-e was found in a patient with e+ RBC and RHCE*ceTI in trans to RHCE*cE. RHD*DIVa in trans to RHD erroneously tested as RHD hemizygous. CONCLUSIONS: RHD*DIVa and RHCE*ceTI almost always, but not invariably, travel together. This haplotype is found in people of African ancestry and the RBCs can demonstrate aberrant reactivity with anti-C. RHCE*ceTI encodes partial c and e antigens. We confirm that RHD zygosity assays are unreliable in samples with RHD*DIVa.

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.