Value of flowcytometry and pcr - ssp techniques in detection of hla - b27

Nội dung text: Value of flowcytometry and pcr - ssp techniques in detection of hla - b27

1. Journal of military pharmaco-medicine 7-2013 VALUE OF FLOWCYTOMETRY AND PCR-SSP TECHNIQUES IN DETECTION OF HLA-B27 Le Xuan Hai*; Nguyen Dang Dung** Summary HLA-B27 has been demonstrated to be strongly associated with ankylosing spondylitis (AS), a chronic inflammatory disease affecting the axial muscoskeletal system. Routine clinical tests detecting HLA-B27 include cytotoxicity, immunofluorescent, and flowcytometry-based assays, among which the later showed flowcytometry assays are the best advantages of specificity and rapidity in performance. In recent years, however, application of PCR-based DNA typing resulted in a more reliable assay for HLA-B27 detection. In the present study, we detected HLA-B27 in blood samples of patients suspected to have AS, by both flowcytometry-based and PCR-SSP-based assays, in order to assess the value of each method in HLA-B27 detection. The results showed that: - Flowcytometry-based assay can detect cell surface HLA-B27 expression when positive and negative events got a clear cut over the cut-off level (the real positive and negative results). - PCR-SSP-based assay is of much help in detecting HLA-B27 encoding gene when flowcytometry-based assay was unable to determine the expression of HLA-B27 (the undetermined results). * Key words: HLA-B27; Flowcytometry; Polymerase chain reaction (PCR); Ankylosing spodylitis. INTRODUCTION HLA-B27 is a protein encoded by an allele of locus HLA-B, one of three loci within the human class I major histocompatibility complex (MHC). It is well demonstrated that the expression of HLA-B27 strongly associated with ankylosing spondylitis (AS), a chronic inflammatory disease affecting the axial muscoskeletal system in human. According to statistical data, about 1% of people with HLA-B27 positive would develop AS; however, 90 - 95% of patients with AS had HLA-B27 positive expression, while in patients without the disease, this incidence was 5 - 8% only. The role of HLA-B27 in pathogenesis of AS has not been completely understood. A recent study revealed that the HLA-B27 heavy chain had a tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum (ER). In experimental HLA-B27 transgenic rats model, HLA-B27 misfolding generates ER stress and leads to activation of the unfold protein response, which enhances the production of interleukin (IL)-23 [2]. These findings were consistent with the discovery of IL-23R gene as an additional susceptibility gene for AS in humans, and have led to new ideas about the role of HLA-B27 in the AS pathogenesis. Thus, expression of HLA-B27 is considered an important risk factor for the development of AS in humans. [1, 6, 7]. There are different techniques being adopted in detecting HLA-B27 expression, including serological testing (based on lymphocytotoxicity test), immunofluorescent technique, or flowcytometry [5]. Lymphocytotoxicity technique is simple to perform, but has some limitations itself (low sensitivity and specificity; requirement of 90% or more of viable cells in blood sample). Immunofluorescent technique improves the sensitivity in HLA-B27 detection, and allows using sample with low cell viability; yet, the technique is manually performed, which costs time and labor. Application of flowcytometry (FCM) using fluorescent-labeled monoclonal antibody specific to HLA-B27 has overcome all disadvantages of techniques mentioned above. Recently, the development of DNA typing techniques based on PCR amplification of target DNA copies allows the detection of HLA-B27 encoding gene with more reliable results. [3, 8]. In this study, we detected the expression of HLA-B27 in blood samples from patients suspected to have AS, by both FCM and PCR methods, in order to assess the value of each method in HLA-B27 detecting assay.
2. Journal of military pharmaco-medicine 7-2013 MATERIALS AND METHODS 1. Subjects. 123 peripheral blood samples (with EDTA as anticoagulant) sent to Immunology Laboratory, National Institute of Hematology and Blood Transfusion, for HLA-B27 detection, during the period of Feb 2013 - April 2013. 2. Materials, reagents. - Anti-HLA-B27 monoclonal antibody labeled with fluorescein isothiocyanate (anti HLA- B27-FITC), and anti-CD3 monoclonal antibody labeled with phycoerythin (anti CD3-PE); red blood cell lysis buffer (Becton Dickinson, USA) - EZHighTM DNA Extraction Kit (Texas BioGene Inc, Taiwan). - MorganTM HLA SSP B27 Typing Kit (Texas BioGene Inc., Taiwan). 3. Methods. * Flowcytometry technique: In this assay, we simultaneously utilized anti -HLA-B27- FITC and anti CD3- PE antibodies for double staining, with steps as followed, - Mixing 100 µl blood sample with 10 µl anti CD3-PE and 10 µl anti HLA-B27-FITC, then incubating reaction tube at room temperature (RT) for 20 min, away from direct light. - Lysing red blood cell (RBC) by adding 2 ml of RBC lysis buffer to the tube, keeping at RT for 10 min, away from direct light. - Analyzing the cell suspension on flowcytometer Cytomics FC500 (Beckman Coulter, USA). Parameters designed for the analysis included: 1. SSC (side scatter) vs. FSC (forward scatter) windows, for isolation of subpopulations of lymphocyte, neutrophil and monocyte in the suspension, thus allowing selection of target lymphocyte subpopulation; 2. CD3-PE vs. HLA-B27-FITC window, allowing analysis of cells double-stained with PE (CD3+ cells) and FITC (HLA-B27+ cell). Combining parameters from 2 windows will bring in the events with cell surface co-expression of CD3+ and HLA-B27+ on lymphocytes. There are 3 possibilities for results of FCM analysis, which depends on density of cell surface HLA-B27 as well as the fluorescent-labeled anti HLA-B27 antibody itself used in the assay (see Figure 1) Positive Negative Undetermined Figure 1: Flowcytometry analysis of cell surface HLA-B27 on TCD3+ lymphocytes. Polymerase chain reaction (PCR): utilizing PCR-SSP (sequence specific primer) technique to detect HLA-B27 encoding gene, with steps as followed: - Extracting DNA by conventional method, using EZHighTM DNA Extraction Kit (BioGene Inc., Taiwan); total time of DNA extraction (for one sample) is about 30 min.
3. Journal of military pharmaco-medicine 7-2013 - Preparing for PCR-SSP: performed as guided by the provider of the reagents, MorganTM HLA SSP B27 Typing Kit (total time for preparation is about 10 min). - Running PCR cycles (including DNA denaturation, annealing and extension steps) with temperature and time parameters as showed in Table 1. Table 1: Time and temperature in PCR cycles. STEPS N0 OF TEMPERATU TIME CYCLES RE 1 1 960C 2,5 min 960C 15 sec 2 10 650C 60 sec 950C 15 sec 3 22 620C 50 sec 720C 30 sec 4 1 40C Until finished - PCR product electrophoresis: to detect copies of HLA-B27 encoding gene (previously amplified by PCR), the product after PCR is electrophoresed on 2% agarose gel in 0.5X TBE buffer (89 mM Tris Base; 89 nM boric acid; 2 mM EDTA, pH 8). + Melting agarose/TBE buffer by microwave (about 1 min), then leaving the gel to cool down to 60 - 700 C (about 5 min). Adding ethidium bromide (0,5 µg/ml) to agarose gel, and then casting the gel with 4 mm thickness, making the wells in gel with 3 mm width. Leaving gel to harden for 40 min. Put the gel in electrophoresis tank, then pouring in the tank with 0.5X TBE buffer to 5mm depth (for the buffer to fully cover the gel surface). + Transferring PCR product into appropriate wells in the gel. Running electrophoresis at 10 V/cm for about 12 min. Visualizing the electrophoresis bands with a UV lamp, and photographing the gel with visualized bands. + The results of PCR-SSP analysis can be expressed as either of the two forms, as followed: 1. positive: a band showed in position specific for HLA-B27 allele; and, 2. negative: no specific band showed. RESULTS AND DISCUSSION Results of FCM analysis to detect cell surface HLA-B27 on 123 blood samples were shown in table 2. Table 2: Results of FCM analysis to detect cell surface HLA-B27. RESULT N0 OF SAMPLES INCIDENCE (%) Negative 27 22.0 Undetermined 34 27.6 Positive 62 50.4 Total 123 100 In this FCM analysis to detect HLA-B27, we have set a cut-off value for the expression of HLA-B27 on cell surface, as followed: samples with more than 85% of CD3+ cells locating in HLA-B27+ area will be defined as HLA-B27 positive; samples with less than 15% of CD3+ cells in HLA-B27+ area will be defined as HLA-B27 negative; finally, samples with 15 - 85% of CD3+ cells in HLA-B27+ area will be defined as undetermined (see Figure 1). [5] Before the application of FCM technique, the detection of HLA-B27 was based on cytotoxicity technique, using antibody specific to HLA-B27 in combination with rabbit complement: cells with HLA-B27 on their surface will be bound by the specific antibody, and then complement fixation will attack and get the cells die, making them stained with trypan
4. Journal of military pharmaco-medicine 7-2013 blue or eosin red – the dead cell staining reagents). The principle of the technique makes it dependent on percentage of viable cells in the sample before testing: there should be at least 90% of viable cell in the sample for the assay to be validated. Compared to this technique, the immunofluorescent technique in general, and FCM technique in particular (with the use of fluorescent-labeled anti HLA-B27 antibody), showed many advantages. Especially, the result of FCM technique in detecting HLA-B27 is somewhat quantitative, and independent from cell’s viability in the sample. Due to this advantage, FCM technique helps reduce false positive (which is sometimes relatively high in cytotoxicity testing). However, data in Table 2 showed that 34/123 tested sample (27.6%) were undetermined in terms of HLA-B27 expression. The reason for this indetermination might be due to cross reactivity of anti HLA-B27 antibody with other HLA-B antigens, such as HLA-B7 [4]. Other groups have published similar findings about a certain rate of samples giving indetermination in HLA-B27 expression when analyzed by FCM [1, 4]. PCR-SSP allows detecting the expression of HLA-B27 at the level of encoding gene, and thus, may help avoiding cross reactivity seen in cytotoxicity-based assays, or in assays using labeled antibodies. In this study, in order to assess the ability in detecting HLA-B27 expression of FCM and PCR methods, we intentionally selected 30 samples out of 123 samples tested for HLA-B27 expression by FCM, among which 10 samples had positive results, 10 had negative results, and 10 had undetermined results with cell surface HLA-B27 expression. Then we performed PCR-SSP using the primers pair specific for the HLA-B27 allele, to detect the HLA-B27 encoding gene in 30 selected samples. The results were presented in Table 3 and 4: Table 3: Results in detecting expression of HLA-B27 by PCR-SSP. FCM NEGATIVE UNDETERMINED POSITIVE TOTAL ( 85%) PCR-SSP Positive 0 4 10 14 Negative 10 6 0 16 Total 10 10 10 30 Table 4: Comparisons of the 2 techniques in HLA-B27 detection. FCM PCR-SSP RESULTS INCIDENCE % Positive (> 85%) + 10/10 100 - 0/10 0 Negative (< 15%) + 0/10 0 - 10/10 100 Undetermined (15 - 85%) + 4/10 40 - 6/10 60 Data in table 4 showed that 10/10 (100%) samples with HLA-B27 positive detected by FCM also gave positive results for HLA-B27 allele when tested by PCR-SSP technique; similarly, 10/10 (100%) samples with HLA-B27 negative by FCM had HLA-B27 allele negative. However, PCR-SSP technique had detected HLA-B27 allele in 4/10 (40%) samples undetermined of HLA-B27 expression by FCM; in addition, PCR-SSP technique gave negative result for HLA-B27 allele in 6/10 remaining samples undetermined of HLA-
5. Journal of military pharmaco-medicine 7-2013 B27 by FCM technique (Figure 2). Figure 2: Results of PCR-SSP product electrophoresis. H2O: distilled water; neg: negative control (a sample without HLA-B27 by PCR-based HLA typing); pos: positive control (a sample without HLA-B27 by PCR-based HLA typing); M: DNA ladder markers. Samples giving HLA-B27 positive include: pos, 810, 870, 871, 902, 923, 934; samples with negative results: neg, 812, 924, 927. It was revealed by these data that in cases of HLA-B27 indetermination by FCM technique, the use of PCR-SSP technique proved to be a good option, which helped giving clear results. In fact, PCR-based HLA typing assays (known as DNA typing) have been replacing conventional HLA typing assays (the serological assays, using either HLA-specific monoclonal or polyclonal antibodies), both nationally and internationally. Within the framework of this study, the results once again highlighted the value of PCR-SSP technique in HLA typing, this time with a specific allele, namely HLA-B27, making the assay reliable, and thus, helpful in diagnosis of AS. Table 5: Comparisons between FCM and PCR-SSP in their performance. TECHNIQUE FCM PCR-SSP Sample preparation 20 - 30 min (including RBC 2 hours (including DNA extraction, PCR lysis) running time) Analysis on ~2 min 30 min (electrophoresis, interpretation of analyzer results) Ability to run single Easy, flexible Inappropriate; batch assay preferable test Cost ~ 300.000 VND/test ~ 350.000 VND/test Requirement of Flowcytometry and popular lab PCR equipment and others equipment equipment (electrophoresis system, UV lamp) Table 5 showed several comparisons between the FCM and PCR-SSP methods in detecting HLA-B27, in terms of assay performance. In general, FCM method is simple to perform. FCM method is more flexible than PCR method, and FCM can be performed on single sample without inconvenience. With less total testing time, FCM method gives faster assay result than PCR method does. Taking all advantages of FCM method together, this method can be used for HLA-B27 screening assay. Meanwhile, PCR-SSP technique, though taking longer time for assay compared to that in FCM, can help avoiding indetermination in HLA-B27 expression. Thus, PCR-based assay for HLA-B27 detection should only be used when FCM-based assay gave undetermined result in HLA-B27 expression.
6. Journal of military pharmaco-medicine 7-2013 CONCLUSIONS In clinical HLA-B27 detection assays, - Flowcytometry-based assay can detect cell surface HLA-B27 expression when positive and negative events got a clear cut over the cut-off level (the real positive and negative results). - Polymerase chain reaction-based assay is of help in detecting HLA-B27 encoding gene when flowcytometry-based assay was unable to determine the expression of HLA-B27 (the undetermined results). References 1 Acar M, Cora T, Tunc R, et al. HLA-B27 subtypes in Turkish patients with ankylosing spondylitis and healthy controls. Rheumatol. Int. 2012, 32 (10), pp.3103-3015. 2 Colbert RA, DeLay ML, Klenk EI. From HLA-B27 to spondyloarthritis: a journey through the ER. Immunol Rev. 2010, 233 (1), pp.181-202 3 Downing J, Coates E, Street J et al. A high-resolution polymerase chain reaction - sequence specific primer HLA-B27 typing set and its application in routine HLA-B27 testing. Genet. Test. 2006, 10 (2), pp.98-103. 4 Levering WH, Wind H, Sintnicolaas K et al. Flow cytometric HLA-B27 screening: cross- reactivity patterns of commercially available anti-HLA-B27 monoclonal antibodies with other HLA-B antigens. Cytometry B: Clin. Cytom. 2003, 54 (1), pp.28-38. 5 Lingenfelter B, Fuller TC, Hartung L et al. HLA-B27 screening by flow cytometry. Cytometry: Comm. Clin. Cytom. 1995, 22, pp.146-149. 6 Malaviya AP, Ostor AJ. Early diagnosis crucial in ankylosing spondylitis. Practitioner. 2011, 255 (1746), pp.21-24. the pathogenesis of ankylosing spondylitis. Exp. Med. Biol. 2009, 649, pp.159-176. Vanvi P, Raja P, Trivedi S. Optimization of in-house PCR-SSP technique for HLA-B27 detection in saurashtra patients. Int. J. Modern Eng. Res. (IJMER). 2012, 2 (3), pp.996-1000.