Improvement in HLA‐C typing by a new sequence‐specific oligonucleotides kit

Abstract

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.

Clinical relevance of donor‐specific antibodies directed at HLA‐C: A long road to acceptance

“…25,26 Indeed, anti-HLA antibodies recognize amino acid modifications on surface accessible positions of HLA molecules, named by Duquesnoy et al as "eplets." 27,28 The HLA-C locus being polymorphic, 29,30 dozens of eplets were described for HLA-C alleles and antigens, either locus specific or shared with HLA-A or -B antigens. 25,31,32 Anti-HLA-C antibodies are found in approximately 10% to 15% of patients on SOT waiting lists.…”

“…HLA‐C antigens are undoubtedly immunogenic as they can induce the formation of allo‐antibodies secondary to transfusions, pregnancies or transplantations 25,26 . Indeed, anti‐HLA antibodies recognize amino acid modifications on surface accessible positions of HLA molecules, named by Duquesnoy et al as “eplets.” 27,28 The HLA‐C locus being polymorphic, 29,30 dozens of eplets were described for HLA‐C alleles and antigens, either locus specific or shared with HLA‐A or ‐B antigens 25,31,32 …”

Characterization of the novel HLA‐C*06:314 allele by sequencing‐based typing

“…HLA typing by Luminex reverse sequence-specific oligonucleotide (SSO) was performed (One Lambda Labtype XR, Canoga Park, California). 3,4 With this assay (lot 002, catalog RSSOX1C_002_01), the HLA-typing of the HLA-C*06 allele was HLA-C*06:02:01:01 without any bead modification. Indeed the IPD-IMGT/HLA Database 3.43.0 release shows that there is no other HLA-C allele displaying a GGC sequence in codon 327, explaining why the manufacturer did not include probes recognizing this allele.…”

Characterization of the novel HLA‐C*15:241 allele by sequencing‐based typing

“…This nucleotide change was confirmed using other NGS reagents provided by Immucor (Mia Fora NGS Flex, Norcross, GA) run on the Illumina MiSeq system (San Diego, CA) and analyzed with the Mia Fora Flex software (Immucor, version 4.5). We were very confident in the phasing as the sample displayed a mean read length of 325 base pairs over all the loci, the mismatched T base was attributed 161 times to the new HLA‐C*15 allele and can be only attributed to this allele because it was possible to discriminate from the associated HLA‐C * 16:01:01:01 allele by virtue of two variant positions each distant by less than 100 base pairs.. HLA typing by Luminex reverse sequence‐specific oligonucleotide (SSO) was performed (One Lambda Labtype XR, Canoga Park, CA) 3,4 . With this assay (lot 002, catalog RSSOX1C_002_01), the HLA‐typing of the donor was C * 15:02:01:01 , 16:14 without any bead modification.…”

“…We were very confident in the phasing as the sample displayed a mean read length of 325 base pairs over all the loci, the mismatched T base was attributed 161 times to the new HLA-C*15 allele and can be only attributed to this allele because it was possible to discriminate from the associated HLA-C*16:01:01:01 allele by virtue of two variant positions each distant by less than 100 base pairs.. HLA typing by Luminex reverse sequence-specific oligonucleotide (SSO) was performed (One Lambda Labtype XR, Canoga Park, CA). 3,4 With this assay (lot 002, catalog RSSOX1C_002_01), the HLAtyping of the donor was C*15:02:01:01, 16:14 without any bead modification. Indeed the bead #18 displayed an oligonucleotide targeting the sequence surrounding codon 48 and was indicated by the manufacturer as reactive for the HLA-C*12:40 and C*16:14 alleles which display a TGG sequence in codon 48, thus confirming NGS results.…”