HLA-B27 typing by use of flow cytofluorometry.

Albrecht, J.; Müller, Holger

doi:10.1093/clinchem/33.9.1619

Cited by 31 publications

(11 citation statements)

References 4 publications

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“…Expectedly, our results confirmed the widely observed cross-reactivity of the ABC-m3 MoAb with HLA-B7 (15,(22)(23)(24)(25)(26)(27)(28). Our suggestion that ABC-m3 might cross-react with HLA-B73 is consistent with Coates' results (27).…”

Section: Discussionsupporting

confidence: 93%

External quality assessment of flow cytometric HLA‐B27 typing

Levering,

van den Beemd,

te Marvelde

et al. 2000

Cytometry

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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.

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Section: Discussionsupporting

confidence: 93%

External quality assessment of flow cytometric HLA‐B27 typing

Levering,

van den Beemd,

te Marvelde

et al. 2000

Cytometry

View full text Add to dashboard Cite

show abstract

“…Expectedly, our results confirmed the widely observed cross‐reactivity of the ABC‐m3 MoAb with HLA‐B7 (15, 22–28). Our suggestion that ABC‐m3 might cross‐react with HLA‐B73 is consistent with Coates' results (27).…”

Section: Discussionsupporting

confidence: 87%

External quality assessment of flow cytometric HLA-B27 typing

Levering¹,

Beemd

Marvelde

et al. 2000

Cytometry

View full text Add to dashboard Cite

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‐B27pos (all HLA‐B*2705) and 18 were HLA‐B27neg. 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‐B27pos 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‐B27neg 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‐B27neg. 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‐B27neg 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‐B27neg 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. © 2000 Wiley‐Liss, Inc.

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“…merase chain reaction-sequence specific oligonucleotide probing (PCR-SSOP) (4), or human anti-B27 or mouse monoclonal antibody based cytotoxicity techniques (5). The classic B27 determination is through tissue typing, which involves time consuming steps of mononuclear cell isolation and subjective visual reading of microcytotoxicity plates.…”

mentioning

confidence: 99%

Flow cytometric HLA‐B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

et al. 1996

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The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.

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HLA-B27 typing by use of flow cytofluorometry.

Cited by 31 publications

References 4 publications

External quality assessment of flow cytometric HLA‐B27 typing

External quality assessment of flow cytometric HLA‐B27 typing

External quality assessment of flow cytometric HLA-B27 typing

Flow cytometric HLA‐B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

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