HLA genotyping by next‐generation sequencing is now widely performed. We aimed at evaluating the performance of the One Lambda AllType kit using Thermo Fisher Scientific reagents on the Ion S5 XL platform. Reads were analyzed using the TypeStream Visual software. We performed 15 runs between April and September 2018 to type DNA at the HLA‐A/B/C/DRB1/3/4/5/DQA1/DQB1/DPA1/DPB1 loci from 340 samples and 15 positive controls. We observed only seven (0.1%) critical mistakes among the 6009 alleles typed, corresponding to two allele dropouts, one false heterozygous typing assignment, and four phasing abnormalities. Among the 1793 presumably new alleles detected by the analysis software, 11 displayed exon mismatches, of which nine were confirmed as new alleles and two had been described previously. Intron mismatches were observed among the remaining presumably new alleles, of which 371 were considered as probably new, and 1411 were rejected for at least one sequence feature such as homopolymers (n = 1206), nucleotide doublet repeats (n = 26), low read depth (<200 reads, n = 93), high background (>20%, n = 79), or phasing abnormalities (n = 7). A comparison of the AllType results with those obtained using other methods at the second‐field resolution level showed 99.5% (1497/1504) concordance for the HLA‐A/B/C/DRB1/DQB1/DPB1 loci. Similar agreement was observed between the HLA‐C or HLA‐DRB3/4/5 results and common linkage disequilibrium, with 96.6% (657/680) and 97.2% (530/545) concordance, respectively. Therefore, the AllType kit used with the Ion S5 XL platform displayed satisfactory performance for HLA typing in current clinical practice.
PCR‐sequence‐specific oligonucleotide (PCR‐SSO) is commonly used for HLA‐typing. We recently replaced LabType SSO HD kit for HLA‐C typing with the XR kit (OneLambda, Inc.). We used 137 patients' samples representing unique most likely HLA‐C allele combinations to compare these kits' performance. The XR kit decreased the number of allele ambiguities with a median of 26.1% (first to third quartiles 20% to 36.2%, range 0% to 96.1%). The XR kit did not resolve all the common, intermediate and well‐documented HLA allele ambiguities, which may be important in the clinical setting. The XR kit eliminated 23.6% of null allele ambiguities. In conclusion, the HLA‐C XR kit provides a moderate yet valuable improvement of HLA‐typing resolution in comparison with the HLA‐C HD kit.
The c.1544+1G>A mutation was identified in Gypsy Glanzmann thrombasthenia (GT) patients.
Gypsy GT patients express normal αvβ3 carrying HPA‐1b epitopes.
To demonstrate HPA‐1a alloimmunization by modified antigen capture assays.
Gypsy GT patients could develop anti‐HPA‐1a alloantibodies against β3 and αvβ3.
Abstract
BackgroundGlanzmann thrombasthenia (GT) is a rare bleeding disorder caused by the absence or the dysfunction of the platelet αIIbβ3 integrin. A founder mutation in the ITGA2B gene was previously identified in French Gypsy patients. Interestingly, this mutation was strongly linked to the human platelet antigen‐1b (HPA‐1b). The HPA‐1bb Gypsy patients are at risk of isoimmunization against αIIbβ3, as this complex is not expressed at their platelet surface. Tentatively, they would, however, not have an increased risk of developing anti‐HPA‐1a alloantibodies by exposure of αIIbβ3 on platelets from random platelet transfusions. However, the β3 chain can also associate with the αv subunit expressed at the platelet surface. Because Gypsy GT patients express normal αvβ3 carrying HPA‐1b epitopes, these patients might develop anti‐HPA‐1a alloantibodies reacting with αvβ3 and/or β3.
Objectives/Patients/MethodsTo demonstrate this hypothesis, sera from HPA‐1bb (n = 5) and HPA‐1ab (n = 1) Gypsy GT patients were investigated by modified antigen capture assay using platelets or stable transfected cells. Furthermore, stable transfected cells expressing either αIIbβ3 or αvβ3 together with soluble monomeric chimeric β3 (as absorbent) were used to differentiate anti‐β3 and anti‐αvβ3 reactivity.
ResultsOnly HPA‐1bb patients developed alloantibodies reacting with HPA‐1a cells. Further analysis showed that HPA‐1bb patients developed anti‐HPA‐1a alloantibodies reacting with β3 and/or αvβ3.
ConclusionIn this study, we found that HPA‐1bb patients who failed to express αIIbβ3 on the platelet surface can develop alloantibodies against HPA‐1a reacting with β3 as well as αvβ3. This is of particular importance as anti‐HPA‐1a alloantibodies might cause fetal neonatal alloimmune thrombocytopenia and/or platelet transfusion refractoriness.
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