Original Article

Ann Clin Biochem 1992; 29: 663-667

Improved flow cytometric method for HLA-B27 typing we M Janssen, J A C N Rouwen and J J M L Hoffmann From the Department oj Clinical Laboratories, Catharina Hospital, Eindhoven, The Netherlands

HLA-B27 is a cell marker of clinical interest because of its high association with certain diseases. The HLA-B27 antigen was detected on lymphocytes using a monoclonal antibody in an indirect immuno-fluorescence assay using a fluorescence flow cytometer. The considerable crossreaction of the monoclonal antibody with the HLA-B7 antigen was effectively suppressed by masking it by means of human anti-HLA-B7 antiserum. The flow cytometric method was evaluated by comparing the results with those obtained by the standard Iymphocytotoxicity test and showed complete agreement in 107 selected patient samples.

SUMMARY.

Additional key phrases: immunofluorescence; lymphocytes; human leucocyte antigen

The human leucocyte antigen (HLA) system is an extremely complex system of over 100 antigens which are expressed on all nucleated and soine non-nucleated human cells. Apart from its great importance for tissue and organ transplantation, the HLA system is known to be related to the occurrence of a variety of diseases. For example, the HLA-B27 antigen displays a high degree of association with rheumatic diseases like ankylosing spondylitis, Reiter's syndrome and acute anterior uveitis.' In the case of ankylosing spondylitis this association is so close that the HLA-B27 phenotype can be used to support the clinical diagnosis. The conventional test for typing HLA antigens is the lymphocytotoxicity test (LCT) described by Terasaki et al.2 It is very time consuming and requires much experience. Recently Albrecht and Muller? and Fizet et al:" demonstrated that it is possible to type HLA-B27 using flow cytometry, In their methods monoclonal antibodies are used which are not entirely specific for the HLA-B27 antigen, but crossreact with HLA-B7 and, possibly, HLA-Bw22 antigens. These authors claimed to be able to distinguish crossreacting antigens from HLA-B27 on the basis of fluorescence intensity.v' In our hands, however, this approach proved to be inadequate in a number of patients because of considerable overlap in fluorescence intensity of homozygous Correspondence: Dr J Hoffmann, Department of Clinical Laboratories, Catharina Hospital, PO Box 13S0, S602 ZA Eindhoven, The Netherlands.

HLA-B7 + and HLA-B7 - 27 + lymphocytes. Therefore, we investigated the possibility of eliminating the crossreaction by masking the HLA-B7 antigen using human anti-HLA-B7 antibodies. MATERIALS AND METHODS Materials Anti-HLA-B27, a mouse monoclonal IgG antibody, described by Trapani et al.,5 was purchased from Behringwerke AG (Marburg, Germany; clone HLA-ABC-m3, Lot No. 0227l6Y). Anti-HLA-B7, a human polyclonal IgG antibody, was obtained from Biotest NV (Brussels, Belgium). Goat Ftab '), anti-human IgG, fluorescein-isothiocyanate (FITC) conjugated, was purchased from Kallestad (Chaska, MN, USA). Goat F(ab')2 anti-mouse IgG, phycoerythrin (PE) conjugated, was purchased from Tago Inc. (Burlingame, CA, USA). Lymphoprep (density 1·077 g/mL), was from Nycomed Pharma AS (Oslo, Norway). Calibrite flow cytometer beads were from Becton Dickinson (San Jose, CA, USA). All reagents and materials for HLA-B27 and B7 typing in the LCT were from Biotest (Brussels, Belgium). Method Blood anticoagulated with tripotassium EDTA (l mg/mL final concentration), was obtained from volunteer subjects: 86 healthy subjects which were typed for HLA-B7 and B27 in our 663

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Janssen, Rouwen and Hoffmann

laboratory using the LCT and 21 patients with a completely known, LCT typed, HLAphenotype (kidney transplant candidates). Lymphocytes were isolated from whole blood by density gradient centrifugation using Lymphoprep.P Briefly, 4 mL diluted (2 x ) blood was layered on top of 3 mL Lymphoprep and centrifuged for 20 min at 800 g and ambient temperature. Subsequently, the interphase containing the lymphocytes was collected and washed with phosphate buffered saline (PBS) and adjusted to a concentration of 5 X 106 cells/mL. In the final procedure, to 20 iLL of this lymphocyte suspension, 40 iLL human anti-HLA-B7 serum was added, mixed and incubated for 30 min on melting ice. Then, without washing, the suspension was incubated with 10 iLL diluted (5 x) anti-HLA-B27 serum under identical conditions. After the second incubation, the suspension was washed twice with PBS. The cell pellet was incubated with 25 iLL of a mixture of anti-mouse-IgG PE and anti-human-IgG FITC (final dilutions 1:25 and 1:100, respectively) under the conditions described above. The suspension was washed twice with PBS and the cells were resuspended in 0·2 mL isotonic sheath fluid. For each individual a lymphocyte suspension from which the primary antibodies were omitted served as a negative control. The samples were analysed within 1 h in a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) using the FACScan Research software v2·6. Optimal instrument settings, checked daily, were used, based on calibration using Calibrite microbeads. The fluorescence analysis was confined to the lymphocytes by gating on their forward and side scatter; at least 5000 lymphocytes were counted in each sample. Using the fluorescence parameters FLI (FITC) and FL2 (PE), representing HLA-B7 and HLA-B27, respectively, the results were presented either as a fluorescence histogram (number of cells versus FL2-intensity) or as a two dimensional contour plot of FLI versus FL2. The fluorescence intensity was expressed in arbitrary units, namely channel numbers. In experiments to assess reproducibility we used the mean fluorescence intensity, expressed as channel number. To define a negative reaction, we used the negative control lymphocytes of each subject and set a marker in the fluorescence histogram in such a way that at least 950/0 of all lymphocytes were below this intensity. Using the same marker setting in the corresponding sample containing the HLA-B27 antiserum, we defined lymphocytes as

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HLA-B27 positive when at least 90% of the lymphocytes had a higher fluorescence intensity. RESULTS

Reactivity of the monoclonal anti-HLA-B27 antibody The reactivity of the anti-HLA-B27 antibody was investigated using lymphocytes of donors with the phenotypes HLA-B7 - 27 -, B7 - 27 + and homezygous B7 + 27 -, respectively. In this test the anti-HLA-B7 antiserum was omitted, in contrast to the final method described. In Fig. I the histograms obtained with each of these phenotypes are shown. There was a significant overlap in fluorescence intensity between HLAB27 + and the homozygous HLA-B7 + lymphocytes. In order to investigate the reproducibility of these fluorescence patterns, samples of the HLAB7 - B27 - and B7 - B27 + phenotypes were measured in replicate. The coefficients of variation (CV%) of the mean channel number were 2% and < 1%, respectively (n = 15 each). Masking of the HLA-B7 antigen . In order to find out whether blocking of the HLA-B7 antigen had an effect on the reactivity of the monoclonal anti-HLA-B27 antibody, homozygous HLA-B7 + 27 - lymphocytes were pre-incubated with varying amounts of human anti-HLA-B7 antiserum. The results of these experiments are displayed in Fig. la. It shows a

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Improved flow cytometric method for HLA-B27 typing

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the binding of both the polyclonal human antiHLA-B7 antibody stained with FITC (FLl channel) and the monoclonal mouse anti-HLAB27 antibody stained with PE (FL2 channel). Figure 3 shows the fluorescence patterns of these phenotypes expressed in two-dimensional contour plots .

Validation of the flow cytometric procedure Lymphocytes from 107 subjects with known, LCT typed, HLA-B7 and B27 phenotypes were tested using the flow cytometric procedure described above. Among these subjects were two patients known to be positive for the potentially crossreacting antigen HLA-Bw22. 3 The results of the method comparison are shown in Table 1. Crossreaction of the anti-HLA-B27 monoclonal antibody with other HLA antigens was not found in this study, particularly not in the patients with the HLA-Bw22 phenotype, even if their lymphocytes were tested without human antiHLA-B7 antiserum.

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FL2 Fluorescence intensity (arbitrary units) FIGURE 2. Reactivity profiles of the monoclonal antiHLA-B27 antibody with (a) homozygous HLA-B7+ lymphocytes and (b) HLA-B7 - 27 + lymphocytes, both after preincubation with varying concentrations ofantiHLA-B7 antiserum (- = 0 p.L; ... =20 p.L; --- =40 p.L; . . . = negative control).

decreasing fluorescence intensity of anti-HLAB27 crossreacting with HLA-B7 + 27 - lymphocytes with increasing concentrations of antiHLA-B7 antiserum. Similarly, the effect of increasing concentrations of anti-HLA-B7 antiserum on the reaction between HLA-B7 - 27 + lymphocytes and the monoclonal anti-HLA-B27 antibody is shown in Fig. 2b. No significant change in fluorescence intensities was observed.

HLA-H27 typing by masking the HLA-H7 antigen Lymphocyte suspensions from four donors with selected HLA-B phenotypes were used to visualize

The major crossreaction of the monoclonal antiHLA-B27 antibody with the HLA-B7 antigen is a potential source of false positive results in the flow cytornetric methods of HLA-B27 typing.3,4 The results presented in Fig. 1 confirm that the monoclonal anti-HLA-B27 serum does crossreact to a considerable degree with the HLA-B7 antigen.' Masking the HLA-B7 antigen is effective in reducing this crossreaction. The reactivity profiles of the monoclonal antiHLA-B27 antibody with the HLA-B7 antigen, presented in Fig. 2a, show decreasing fluorescence intensities when increasing quantities of human anti-HLA-B7 serum were added. When 40 iLL of human anti-HLA-B7 serum was used, the reactivity of anti-HLA-B27 serum with HLAB7 + 27 - lymphocytes was no longer different from the negative control (Fig. 2a). This proves that blocking of the HLA-B7 antigen with human anti-HLA-B7 antiserum reduces or even eliminates the crossreaction of the anti-HLA-B27 antibody. This step has no significant influence on the fluorescence intensity of HLA-B27 + lymphocytes as shown in Fig. 2b. We consider 40 iLL anti-HLA-B7 as optimal, at least for the antiserum batch used in this study. The method presented allows recognition of the different HLA-B7, B27 phenotypes when using a double staining technique, in which the human anti-HLA-B7 antiserum is stained with a

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3. Fluorescence patterns of HLA-B27 (FL2, PE) typing in combination with visualization of HLA-B7 (FLI, FITC). Markers wereset on individual negative controls. (a) HLA-B7 - 27 -; (b) homozygous HLA-B7 + 27 -; (c) HLA-B7-27+; (d) HLA-B7+27+.

FIGURE

1. Comparison of the results from the flow cytometric (FCM) method with the standard lymphocytotoxicity test (LCT)

TABLE

HLA phenotype (by LCT) 7-277+27- , 7+27- b 7-27+ 7+27+ 7-27-w22+

HLA-B27 (by FCM) n

62 23 2 17 1 2 'Heterozygous or unknown. "Homozygous.

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FITC-conjugated second antibody and the mouse anti-HLA-B27 antiserum with a specific PEconjugated secondary antiserum. Illustrative examples of the fluorescence patterns obtained are shown in Fig. 3. Although it was not our intention to perform HLA-B7 typing, this figure illustrates that the method might be used for simultaneous HLA-B27 and B7 typing. The results summarized in Table 1 show a complete agreement of our procedure with the lymphocytoxicity test in 107 subjects with relevant phenotypes. In our opinion this adequately validates our method for routine application

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Improved flow cytometric method for HLA-B27 typing in clinical laboratories. From Table 1 it also can be concluded that the monoclonal anti-B27 antibody apparently did not react with other HLA-B antigens. This was explicitly found for other antigens which can be expected to be present in our population on the basis of gene frequencies. This finding agrees with other studies in comparable, Caucasian groups.v' The discrimination between HLA-B27 + and HLA-B27 - phenotypes using the procedure presented here is better than the results described by others suggest. 3,4 This improved discrimination is the consequence of suppressing the interference of HLA-B7 antigens in our assay. The procedure described by Fizet et al:" is performed in whole blood instead of on isolated lymphocytes. However, when we repeated this in conjunction with HLA-B7 blocking, we obtained unsatisfactory results, probably due to interference by cells other than lymphocytes. A potentiallirnitation in our method is that it depends on the quality and specificity of the antiHLA-B7 antiserum used. Although we obtained satisfactory results with all sera used so far, it may be wise to select an anti-HLA-B7 antiserum with

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a high titer and a low degree of cross-reactivity with the HLA-B27 antigen. We conclude that the method presented here is reliable, relatively fast and allows objective and reproducible interpretation. Therefore it is well suited for routine use in clinical laboratories equipped with a flow cytometer. REFERENCES 1 Svejgaard A, Platz P, Ryder LP. HLA and disease 1982-A survey. Immunol Rev 1983; 70: 193-218 2 Terasaki PI, McClelland JD. Microdroplet assay of human serum cytotoxins, Nature 1964; 2.04: 998-1000 3 Albrecht J, Miiller HAG. HLA-B27 typing by use of flow cytofluorometry, Clin Chem 1987; 33: 1619-23 4 Fizet D, Hitte C, Ferrer AM, Vezon G. Identification de l'antigene HLA B27 par cytometrie de flux. Ann Bioi Clin 1989; 47: 408-11 5 Trapani JA, Vaughan HA, Sparrow RL, Tait BD, McKenzie IF. Description of a mouse monoclonal anti-HLA-B27 antibody HLA-ABC-m3. Human Immunol 1983; 7: 205-16 6 Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21: (suppl. 97): 1-109

Accepted for publication 20 March 1992

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