[show abstract][hide abstract] ABSTRACT: Individuals with anti-Jr(a) or anti-Lan are ideally transfused with rare Jr(a-) or Lan- red blood cells. We characterized mutations in Dutch Jr(a-) and Lan- individuals and developed a high-throughput genotyping assay to detect Jr(a-) and Lan- donors.
Six Jr(a-) and seven Lan- persons, who all made anti-Jr(a) or anti-Lan, were sequenced for ABCG2 or ABCB6 and the copy number of ABCG2 and ABCB6 was determined. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput screening assay targeting frequently occurring mutations causing the Jr(a-) or Lan- phenotype.
In the six tested Jr(a-) individuals previously described, c.376C > T, c.706C > T, and c.736C > T nonsense mutations in ABCG2 were detected. In the seven Lan- individuals 12 different mutations, of which 10 underlie the Lan- phenotype, were detected. No copy number variation was detected for ABCG2 and ABCB6. The high-throughput screening assay detected five Caucasian donors heterozygous for the c.706C > T or 736C > T mutation in ABCG2 and nine Caucasian donors heterozygous for the 574C > T mutation in ABCB6. No black or Chinese donors were found positive for a mutation.
We describe eight new mutations in ABCB6 of which seven, including three missense mutations, underlie the Lan- phenotype and determine that a complete gene deletion of ABCG2 or ABCB6 is not responsible for the Jr(a-) or Lan- phenotype, respectively. The extended heterogeneity of mutations causing the Jr(a-) or Lan- phenotype in most populations makes genetic screening for the Jr(a-) and Lan- phenotype inefficient in those populations.
[show abstract][hide abstract] ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Since regular platelet function test cannot be performed in patients with low platelet counts, two new assays were developed to determine platelet function. First, the micro aggregation test measuring in platelets isolated from 10 ml whole blood, the platelet potential to form micro-aggregates in response to an agonist. Second, the platelet reactivity assay, measuring platelet reactivity to ADP, convulxin (CVX) and thrombin receptor activator peptide (TRAP) in only 150 µL unprocessed whole blood. Patients with a severe bleeding phenotype, demonstrated a decreased aggregation potential upon phorbol myristate acetate (PMA) stimulation, decreased platelet degranulation following ADP stimulation and a higher concentration of ADP and convulxin needed to activate the glycoprotein IIbIIIa complex compared to patients with a mild bleeding phenotype. In conclusion, here we have established two functional tests that allows for evaluation of platelet function in patients with extremely low platelet counts (<10(9)). These tests show that platelet function is related to bleeding phenotype in chronic ITP.
[show abstract][hide abstract] ABSTRACT: The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX).
Six sera containing anti-human PLT antigen (HPA)-1a (n = 2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n = 63); glycoprotein (GP) IV antibodies (n = 1); PLT autoantibodies (n = 3); HLA antibodies (n = 45); and samples with no PLT-reactive antibodies (n = 82), were tested in both assays.
Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n = 1) or anti-HPA-5b (n = 3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n = 7), anti-GPIIb/IIIa combined with anti-HPA-3a (n = 1), anti-HPA-1a (borderline, n = 1), and anti-GPIV (n = 1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n = 3 and n = 1, respectively.
For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method.
[show abstract][hide abstract] ABSTRACT: Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPA) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). As antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG-Fc, as the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core-fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n=48), but not in total serum IgG1. Antibodies with low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with high amount of Fc-fucose. Consequently, these antibodies with low amount of Fc-fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) PMN or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core-fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (RT, post-platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.
[show abstract][hide abstract] ABSTRACT: In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation-dependent probe amplification (MLPA) technology.
We analyzed 103 random and 150 selected samples to validate the specificity of the blood-MLPA assay that is able to determine the presence, absence, and copy number of 48 blood group and 112 variant alleles of 18 blood group systems. A total of 4038 serologic typing results, including 52 different antigens, were available for these samples.
In 4018 (99.5%) of the 4038 serologic typing results the predicted phenotypes by the blood-MLPA were in concordance with serologic typing. Twenty discordant results were due to false-positive serologic results (n = 2), false-negative serologic results (n = 1), inability of routine serologic typing to detect variant antigens (n = 14), or false-positive prediction from the blood-MLPA due to the presence of a null allele (n = 3).
The blood-MLPA reliably predicts the presence or absence of blood group antigens, including almost all clinically relevant blood group antigens, except ABO, in patients and donors. Furthermore, it is the first assay that determines copy numbers of blood group alleles in the same test. It even provides more detailed and accurate information than serologic typing, because most variant alleles are immediately recognized. Since only standard laboratory equipment is needed, this assay finally offers the possibility to comprehensively type recipients and makes extensive matching for selected patients groups more feasible.
[show abstract][hide abstract] ABSTRACT: BACKGROUND AND OBJECTIVES: HPA-1a antibodies account for 70-80% of cases of fetal-neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of αIIbβ3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbβ3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories. MATERIALS AND METHODS: Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present. RESULTS: 2/39 (5·1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by ≥20% in 17/24 (70·8%) laboratories and by ≥50% in 9/24 (37·5%) when using HPA-1a1a platelets (mean: 27·7%, range 0-85·1%); when using HPA-1a1b platelets 3/4 (75%), participants reported ≥50% loss of sensitivity (mean 65·6%, range 0-96·6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0·081, P < 0·01). Insufficient PIFT data were returned to draw firm conclusions. CONCLUSION: Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of αIIbβ3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.
[show abstract][hide abstract] ABSTRACT: Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the 'cut-off' for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI.
[show abstract][hide abstract] ABSTRACT: The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.
[show abstract][hide abstract] ABSTRACT: The main function of platelets is to maintain normal hemostasis. Inefficient platelet production and/or defective platelet function results in bleeding disorders owing to a wide range of genetic traits and acquired pathologies. Several platelet function tests have been developed for use in the clinic and in experimental animal models. In particular, platelet aggregation is routinely measured in an aggregometer, which requires normal platelet counts and significant blood sample volumes. For this reason, the analysis of thrombocytopenic patients, infants and animal models is problematic. We have developed a novel flow cytometry test of platelet aggregation, in which 10- to 25-fold lower platelet counts or sample volumes can be used, either of platelet-rich plasma or whole blood from human subjects or mice. This set-up can be applied to test in small assay volumes the influence of a variety of stimuli, drugs and plasma factors, such as antibodies, on platelet aggregation. The presented principle stands as a novel promising tool, which allows analysis of platelet aggregation in thrombocytopenic patients or infants, and facilitates studies in platelets obtained from experimental animal models without the need of special devices but a flow cytometer.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity. STUDY DESIGN AND METHODS: We analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases. RESULTS: In 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized. CONCLUSION: The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.
[show abstract][hide abstract] ABSTRACT: Fetal blood group genotyping using cell-free fetal DNA from maternal plasma is routinely performed in alloimmunized women and has been introduced for targeted antenatal anti-D prophylaxis. The necessity to control for extraction of fetal DNA in these tests is questioned by many. This review describes the various types of controls for preventing false-negative results and discusses their value.
Polymorphic markers like short tandem repeats, insertion/deletion polymorphisms or single nucleotide polymorphisms can be used as a fetal DNA control in pregnancies in which a Y-chromosome marker is not applicable, but workload is considerable and more than 99% coverage is only reached on platforms allowing high level of multiplexing. Recently, the universal fetal marker RASSF1A has been introduced, enabling the demonstration of fetal DNA after methylation-sensitive digestion of maternal DNA. The analysis of recently published noninvasive fetal blood group genotyping studies showed that false-negative results were only encountered in studies lacking a control for the presence of fetal DNA, albeit at a low frequency of 0.1-0.2%.
Because of the potentially severe consequences of false-negative results in alloimmunized women, a blood group antigen-negative result should in these cases only be issued if fetal DNA is demonstrated. However, the low frequency of false-negative results makes it acceptable to perform screening studies without a fetal marker.
Current opinion in hematology 09/2011; 18(6):467-73. · 5.19 Impact Factor