Automated HLA‐B27 testing using the FACSPrep/FACScan system

Wm, Reynolds; Pr, Evans; Ac, Lane; Wm, Howell; Pj, Wilson; Wong, Roderick; Smith, J. L.

doi:10.1002/cyto.990180210

Cited by 13 publications

(6 citation statements)

References 23 publications

(8 reference statements)

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“…The cross-reactivity of anti-B27 antibody to HLA-B7positive subjects has been reported with some monoclonal reagents ( 7,10,18). Among the studied samples were three HLA-B7-positive subjects, all of which were found to be B27-negative by our assay.…”

Section: No Cross-reactivity Of Anti-b27 Antibody To Hla-b7 Positive supporting

confidence: 51%

“…FD705 reacted to all seven B27 subtypes (B2701-B2707) tested so far (8). The lack of cross reactivity to B7 antigen of FD705 avoided the possible false positive reaction with B7 homozygotes ( 10,26).…”

Section: Discussionmentioning

confidence: 99%

“…Several studies examining FCM B27 techniques with method comparisons ( 6 ) , types of monoclonal antibodies used (7,8,9), and automated sample preparation (10) have recently been published. However, although the FCM B27 assay utilizes immunofluorescent antibody binding, few reports have addressed the potential for quantitative reporting of the FCM B27 results.…”

mentioning

confidence: 99%

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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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Section: No Cross-reactivity Of Anti-b27 Antibody To Hla-b7 Positive supporting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

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

et al. 1996

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“…However, using the CD3 gating method, the result of 7686 MESF was 4000 MESF higher than the upper limit cutoff of 3986, providing a more confident conclusion as to the weak HLA-B27 positivity of the patient, considering that there is a 5-20% of biological variation of B27 expression (see Validation of FCM Procedure). The cross-reactivity of anti-B27 antibody to HLA-B7-positive subjects has been reported with some monoclonal reagents ( 7,10,18). Among the studied samples were three HLA-B7-positive subjects, all of which were found to be B27-negative by our assay.…”

Section: A B27-positive Sample With Low Mesfmentioning

confidence: 72%

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. © 1996 Wiley‐Liss, Inc.

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Optimisation of HLA-B27 Testing by Association of Flow Cytometry and DNA Typing

Bonnaud

Aupetit

Preux

et al. 1999

Clinical Rheumatology

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HLA-B27 typing contributes to the diagnosis of ankylosing spondylitis. The classical technique of microlymphocytotoxicity is costly and can give false-negative results. We have compared 304 samples using two relatively new methods - flow cytometry and PCR-SSP - and evaluated their respective uses in routine analysis. Flow cytometric HLA-B27 testing was performed using three monoclonal anti-B27 antibodies (HLA-ABC-m3, GS145.2 and FD705 clones). Cut-off values were established to differentiate HLA-B27-positive from HLA-B27-negative samples with ROC curves. Although flow cytometric analysis with a reliable monoclonal antibody (mAb) is valuable for HLA screening, none of the HLA-B27 flow cytometry protocols was sufficient on its own to ascertain the HLA phenotype in 100% of samples. Two false negatives were observed with the FD705 mAb and the use of two different monoclonal antibodies did not increase the accuracy of HLA-B27 typing. HLA-B27 typing using molecular biology is a reliable but costly technique. Therefore we suggest that DNA typing could be used as a complementary technique and applied to samples whose HLA-B27 phenotype cannot be determined by flow cytometry. The association of flow cytometry and DNA typing is, in our experience, an economical and reliable approach.

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Automated HLA‐B27 testing using the FACSPrep/FACScan system

Cited by 13 publications

References 23 publications

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

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

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

Optimisation of HLA-B27 Testing by Association of Flow Cytometry and DNA Typing

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