External quality assessment of flow cytometric HLA‐B27 typing

Abstract

A biannual external quality assurance (EQA) scheme for flow cytometric typing of the HLA-B27 antigen is operational in The Netherlands and Belgium since 1995. We report here on the results of the first seven send-outs to which 36 to 47 laboratories participated. With the send-out, four specimens from blood bank donors, who had been typed for HLA Class I antigens by complement-dependent cytotoxicity, were distributed. Subtyping of the HLA-B27 allele was performed by PCR-SSP. Ten samples were HLA-B27 pos (all HLA-B*2705) and 18 were HLA-B27 neg. For flow cytometry, the most widely monoclonal antibody (MoAb) used was FD705, followed by GS145.2 and ABC-m3. The majority of laboratories used more than 1 anti-HLA-B27 MoAb for typing. The HLA-B27 pos samples were correctly classified as positive by the large majority of participants (median 95%; range 85% to 100% per send out); some participants considered further typing necessary and misclassification as negative was only sporadically seen. The classification of HLA-B27 neg samples as negative was less straightforward. Ten samples were correctly classified as such by 97% (82% to 100%) of the participants, whereas 64% (range 53% to 70%) of the participants classified the remaining eight samples as HLA-B27 neg. There was no significant prevalence of a particular HLA-B allele among these eight "poor concordancy" samples as compared to the ten "good concordancy" samples. Inspection of the reactivity patterns of the individual MoAb with HLA-B27 neg samples revealed that ABC-m3 showed very little cross-reactivity apart from its well-known cross-reactivity with HLA-B7, whereas the cross-reactivity patterns of GS145.2 and FD705 were more extensive. The small sample size (n 18) and the distribution of HLA-B alleles other than HLA-B27 did not allow assignment of specificities to these cross-reactions. Finally, we showed that standardized interpretation of the combined results of two anti-HLA-B27 MoAb reduced the frequency of false-positive conclusions on HLA-B27 neg samples. In this series, the lowest frequency of false-positive assignments was observed with the combination of the FD705 and ABC-m3 MoAb. Cytometry (Comm. Clin. Cytometry) 42:95-105, 2000.

Evaluation of a method for monoclonal antibody HLA‐B27 analysis with the CELL‐DYN Sapphire haematology analyser

“…As the result of the high frequencies of HLA-B27 in many population groups, the main clinical use of HLA-B27 as a diagnostic marker for ankylosing spondylitis is as a 'ruleout' test (Hoffmann & Janssen, 1997). Analysis of HLA-B27 was traditionally performed by the classical microlymphocytotoxic test (Terasaki & McClelland, 1964), but this has been superseded by flow cytometry analysis using fluorochrome-labelled monoclonal HLA-B27 antibodies (Levering et al, 2000). In practical terms, flow cytometry analysis of HLA-B27 is relatively straightforward and is variously based on one or two colour fluorescence.…”

HLA‐B27 testing: A journey from flow cytometry to molecular subtyping

“…One interesting feature in flow cytometry is that the cross‐reactivity produced by HLA‐B7 is so strong that it can mask not only other weaker cross‐reactive HLA‐B molecules but also even the presence of HLA‐B27 . Besides B7, the antibody GS145.2 is known to cross‐react to with HLA‐B37, HLA‐B44, and HLA‐B55 . All the 20 euroarray negative cases (18 flow equivocal cases and 02 flow positive cases) turned out to be either HLA‐B7 or HLA‐B37 upon sequencing.…”

“…Besides B7, the antibody GS145.2 is known to cross-react to with HLA-B37, HLA-B44, and HLA-B55 21. All the 20 euroarray negative cases (18 flow equivocal cases and 02 flow positive cases) turned out to be either HLA-B7 or HLA-B37 upon sequencing.…”

HLA-B27 genotyping by Fluorescent Resonance Emission Transfer (FRET) probes in real-time PCR

“…Genotyping is much more accurate than flow-cytometry with monoclonal antibodies to HLA-B27 [24], which gives about a 5% ambiguous result. In addition, positive samples should be confirmed by genotyping.…”