Failure of the serological determination of HLA-B27 due to antigen masking in patients with ankylosing spondylitis

Abstract

In patients suffering from ankylosing spondylitis (AS) HLA-B27 determination by means of the microlymphocytotoxicity test (MLCT) sometimes gives equivocal or false-negative results even though it has been performed with meticulous care. These failures of the test did not arise when the isolated mononuclear cells (MNC) were incubated in lymphocyte culture medium at 37 degrees C under sterile conditions for 24 h. To objectify these observations two methods of HLA class I typing were implemented before and after incubation of the test MNC in culture medium: a bioluminescence method based on the loss of adenosine triphosphate (ATP) in lysed cells in a modification of the usual MLCT and a flow cytometric (FC) test using direct immunofluorescence with an anti-HLA-B27 monoclonal antibody (MAB). In this study 50 patients with AS and 12 healthy volunteers were typed by the usual MLCT according to the NIH standard method and with both of the quantitative methods. In most of the AS patients the discrimination between positive and negative typing results became more distinct after 24 h incubation in culture medium. In the entire group of AS patients tested three false-negative typing results were prevented by this method. Although the MAB against HLA-B27 is cross-reactive with HLA-B7 and HLA-B22, errors in the FC analysis could be avoided by calibration of the flow cytometer with standard calibration beads. Possible explanations for masking of the HLA-B27 in AS patients are discussed.

Yersinia enterocolitica serotype O:3 alters the expression of serologic HLA-B27 epitopes on human monocytes

“…Earlier studies have shown that certain HLA-B27 epitopes are modified in patients with reactive arthritis and ankylosing spondylitis (2,20,29). In the present study, we wanted to characterize the mechanisms behind this modification.…”

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

“…These alternative methods showed that the MLCT can fail in some instances if only a small panel of commercially available antisera against HLA-B27 and CRS is used. Typing errors can be caused by several mechanisms: earlier investigations [28] have shown that due to cross-reacting antibodies or substances present in the blood of the patients that interact with epitopes of the HLA-B27 molecule, serological HLA-B27 determination can be false-negative in patients suffering from ankylosing spondylitis. This problem can be eliminated by carrying out the MLCT only after incubation of isolated MNC at 37 °C in sterile culture medium for 24 h.…”

HLA‐B*27 subtypes in Northern and Northeastern Thais, Karens, and Bamars determined by a high‐resolution PCR‐SSP technique

“…Serological techniques such as microlymphocytotoxicity, flow cytometry, and enzyme immunoassay could also be used. These methods may give false‐negative results as they are based on the detection of cell surface antigens by antibodies (4, 5). If HLA‐B27 antigen is downregulated or conformationally changed, the sensitivity and specificity of these methods may be decreased.…”

“…Fresh viable blood cells are also required (4). In addition, a major disadvantage of serological identification is cross‐reactivity with different HLA antigens, such as B7 (5). On the other hand, molecular methods do not require viable cells and are more accurate, sensitive, and specific.…”