An optimized method for routine HLA‐B27 screening using flow cytometry

Hulstaert, Frank; Albrecht, J.; Hannet, Irene; Lancaster, P.; Buchner, Leonard; Kunz, J; Falkenrodt, A.; Tongio, M.M.; Keyser, Filip De; Veys, Eric; Noens, Lucien; N, Mir; Costello, Christine; Becker, Robert S.; Strauss, Kenneth

doi:10.1002/cyto.990180106

Cited by 27 publications

(11 citation statements)

References 16 publications

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“…HLA-B27 positivity was analyzed at different laboratories using commonly employed methods. Since results of the different methods for analysis of HLA-B27 are insignificant, we have not stressed this further [19]. …”

Section: Methodsmentioning

confidence: 99%

Ultrasonography and color Doppler of proximal gluteal enthesitis in juvenile idiopathic arthritis: a descriptive study

et al. 2011

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BackgroundThe presence of enthesitis (insertional inflammation) in patients with juvenile idiopathic arthritis (JIA) is difficult to establish clinically and may influence classification and treatment of the disease. We used ultrasonography (US) and color Doppler (CD) imaging to detect enthesitis at the small and deep-seated proximal insertion of the gluteus medius fascia on the posterior iliac crest where clinical diagnosis is difficult. The findings in JIA patients were compared with those obtained in healthy controls and with the patients' MRI results.MethodsSeventy-six proximal gluteus medius insertions were studied clinically (tenderness to palpation of the posterior iliac crest) and by US and CD (echogenicity, thickness, hyperemia) in 38 patients with JIA and in 38 healthy controls, respectively (median age 13 years, range 7-18 years). In addition, an additional MRI examination of the sacroiliac joints and iliac crests was performed in all patients.ResultsIn patients with focal, palpable tenderness, US detected decreased echogenicity of the entheses in 53% of the iliac crests (bilateral in 37% and unilateral in 32%). US also revealed significantly thicker entheses in JIA patients compared to healthy controls (p < 0.003 left side, p < 0.001 right side). There was no significant difference in thickness between the left and right sides in individual subjects. Hyperemia was detected by CD in 37% (28/76) of the iliac crests and by contrast-enhanced MRI in 12% (6/50).ConclusionsAccording to US, the gluteus medius insertion was thicker in JIA patients than in controls, and it was hypoechoic (enthesitis) in about half of the patients. These findings may represent chronic, inactive disease in some of the patients, because there was only limited Doppler flow and MRI contrast enhancement. The present study indicates that US can be useful as an adjunct to clinical examination for improved assessment of enthesitis in JIA. This may influence disease classification, ambition to treat, and choice of treatment regimen.

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Section: Methodsmentioning

confidence: 99%

Ultrasonography and color Doppler of proximal gluteal enthesitis in juvenile idiopathic arthritis: a descriptive study

et al. 2011

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“…Time and cost-saving alternatives include the use of an anti-HLA-B27 MAB that does not cross-react with HLA-B7 [ 13] or a technique using an accurate calibration of the flow cytometer before the measurement [14]. The latter 99 method was carried out whenever discrepant typing results occurred between the MLCT with the small panel of antisera and the EIA.…”

Section: Discussionmentioning

confidence: 99%

“…The FC test was established on flow cytometers from Becton & Dickinson (B&D, San Diego, USA) via a particular HLA-B27 software suitable for automated calibration and analysis. The software is provided for combination with an HLA-B27 kit (also by Becton & Dickinson) containing calibration beads and MAB against HLA-B27 (FITC-conjugated; clone GS 145.2, IgG 1, FITC-conjugated) [14] and CD3 (PE-conjugated). In the FC analysis, CD3 + lymphocytes are gated using a dot blot from the signals of the forward light scatter (FSC) at linear amplification and the red fluorescence at logarithmic amplification.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…In order to overcome the difficulties with the MLCT, other serological methods have been developed such as flow cytometric (FC) tests [10][11][12][13][14] and enzyme immunoassays (EIA) [15][16][17]. Recently, molecular biological methods have also been introduced for the determination of HLA-B27, comprising determinations of restriction fragment length polymorphisms [18,19], the demonstration of sequence-specific DNA fragments after a polymerase chain reaction (PCR) with sequence-specific primers (SSP) [20,21] or PCR methods with exon-specific primers combined with blotting and hybridization with sequence-specific oligonucleotides (SSO) [22,23].…”

Section: Introductionmentioning

confidence: 99%

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HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay

Dunky

Neumüller²,

Hübner³

et al. 1996

Rheumatol Int

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Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

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“…The majority of laboratories use flow cytometry for typing of single human leucocyte antigens (HLA), e.g. HLA‐B27 (1–3). The microlymphocytotoxicity test (LCT) (4) and polymerase chain reaction (PCR)‐based techniques (5–8) may also be used, but these techniques cannot be implemented in most laboratories.…”

Section: Phenotyping Of Blood Donors (N = 118) For Hla‐a2 ‐B7 and ‐Bmentioning

confidence: 99%

Application of the particle gel agglutination assay in the typing of single human leucocyte antigens

et al. 2007

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We describe a simple and rapid particle gel agglutination assay (PaGIA) for typing of the human leucocyte antigens (HLA) HLA-A2, HLA-B7 and HLA-B27. Superparamagnetic streptavidin particles were coated with biotinylated monoclonal antibodies (MoAbs) to HLA-A2, HLA-B7 and HLA-B27. Anticoagulated whole blood samples from healthy blood donors (n = 118) with known HLA patterns were incubated with MoAb-coated particles, transferred into a standard ID-gel card, and subsequently centrifuged. Samples were evaluated macroscopically, with antigen-positive samples resulting in a visible agglutination reaction. A clear distinction could be made between all positive and negative samples tested. Fifty-seven samples were found to be positive for HLA-A2 (48%), 26 samples for HLA-B7 (22%) and 5 samples for HLA-B27 (4%).

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An optimized method for routine HLA‐B27 screening using flow cytometry

Cited by 27 publications

References 16 publications

Ultrasonography and color Doppler of proximal gluteal enthesitis in juvenile idiopathic arthritis: a descriptive study

Ultrasonography and color Doppler of proximal gluteal enthesitis in juvenile idiopathic arthritis: a descriptive study

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay

Application of the particle gel agglutination assay in the typing of single human leucocyte antigens

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