Abstract
AAmaç: Çalışmada Ankilozan spondilit ve diğer spondiloartropatilerin moleküler tanısında kullanılmak üzere güvenilir, ekonomik ve rutin kullanıma uygun laboratuvar yapımı (in-house) HLA-B*27 PCR-SSP tanı kiti geliştirilmesi amaçlanmıştır.
Gereç ve Yöntem: Çalışmaya, ticari kitler kullanılarak HLA-B*27 pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olmak üzere toplam 565 olgu dahil edildi. HLA-B*27 tipleme için sekansa spesifik primerler ve beta globin gen bölgesinden yararlanılarak dizayn edilen internal kontrol primer çifti kullanılarak PCR optimizasyonu gerçekleştirildi.
Bulgular: HLA-B*27’si pozitif olan olgularda, %2’lik agaroz jelinde 273 bç internal kontrol bandının ilerisinde 149 bç’lik spesifik bant olmak üzere çift bant, B*27 negatif hastalarda ise sadece 273 bç’lik internal kontrol bandı saptandı. Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 bireyin sonuçları ile tarafımızdan geliştirilen PCR-SSP kiti kullanılarak elde edilen sonuçların tamamen uyumlu olması nedeniyle sensitivite ve spesifite %100 olarak belirlendi. Geliştirdiğimiz laboratuvar yapımı PCR-SSP kitinin maliyetinin, ticari kitlere göre en az 50 kat daha ucuz olduğu görüldü.
Sonuç: Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olgunun tamamında, geliştirdiğimiz yöntemle de birebir uyumlu sonuçlar elde edildi. Bu bulgular geliştirdiğimiz kitin güvenilir, ekonomik ve rutin kullanıma uygun olduğunu göstermektedir.
References
- 1. Schwartz BD. The human major histocompatibility HLA complex. In Basic and Clinical Immunology (Eds DP Stites, JD Stobo, JV Wells): 50-64. California, Appleton & Lange, 1987.
- 2. Allele Search Tool. IMGT/HLA-IPD-EMBL-EBI. [Internet]. Available at: https://www.ebi.ac.uk/ipd/imgt/hla/stats.html
- 3. Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet.1973;28(7809):904-7. 4. Bowness P. HLA-B27 in health and disease: a double-edged sword? Rheumatology. 2002;41:857-68.
- 5. Khan MA. Update on spondyloarthropathies. Ann Intern Med. 2002;136:896-907.
- 6. Seipp MT, Erali M, Wies RL, Wittwer C. HLA B27 typing: Evaluation of an allele specific PCR melting assay and two flow cytometric antigen assays. Cytometry B Clin Cytom. 2005;63:10-15. 7. Granfors K, Marker-Hermann E, de Keyser F, Khan MA, Veys EM, Yu DT. The cutting edge of spondylarthropathy research in the millennium. Arthritis Rheum. 2002;46:606-13.
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- 12. Lopez de Castro JA, Alvarez I, Marcilla M, Paradela A, Ramos M, Sesma L, et al. HLA-B27: a registry of constitutive peptide ligands. Tissue Antigens. 2004;63:424-45.
- 13. Khan MA. Update on spondyloarthropathies. Ann Intern Med. 2002;136:896-907.
- 14. Taurog JD. The mystery of HLA-B27: if it isn’t one thing, it’s another. Arthritis Rheum 2007;56:2478-81.
- 15. Oguz FS, Ocal L, Diler AS, Ozkul H, Asicioglu F, Kasapoglu E, et al. HLA B-27 subtypes in Turkish patients with spondyloarthropathy and healthy controls. Dis Markers. 2004;20:309-12.
- 16. Acar M, Cora T, Tunc R, Acar H.HLA-B27 subtypes in Turkish patients with ankylosing spondylitis and healthy controls. Rheumatol Int. 2012;32:3103-5.
- 17. Birinci A, Bilgici A, Kuru O, Durupinar B. HLA-B27 polymorphism in Turkish patients with ankylosing spondylitis. Rheumatol Int. 2006;26:285-7.
- 18. Diyarbakir E, Eyerci N, Melikoglu M, Topcu A, Pirim I. HLA B27 subtype distribution among patients with ankylosing spondylitis in eastern Turkey. Genet Test Mol Biomarkers. 2012;16:456-8.
- 19. Varnavidou-Nicolaidou A, Karpasitou K, Georgiou D, Stylianou G, Kokkofitou A, Michalis C, et al. HLA-B27 in the Greek Cypriot population: distribution of subtypes in patients with ankylosing spondylitis and other HLA-B27-related diseases. The possible protective role of B*2707. Hum Immunol. 2004;65:1451-4.
- 20. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
- 21. Bunce M. PCR-Sequence-Specific Primer Typing of HLA Class I and Class II Alleles. In Methods in Molecular Biology: MHC Protocols (Eds SH Powis, RW Vaughan RW): 143-172. MHC Protocols, Totowa, NJ, Humana Press, 2003.
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- 24. Dunky A, Neumüller J, Hübner C, Fischer GF, Bayer PM, Wagner E, et al. HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay. Rheumatol Int. 1996;16:95-100.
- 25. Coates E, Darke C. Routine HLA-B27 typing by flow cytometry: Differentiation of the products of HLA-B*2702, B*2705 and B*2708. Eur J Immunogenet. 1998;25:29-37.
- 26. Skalska U, Kozakiewicz A, Maslinski W and Jurkowska M. HLA-B27 detection - comparison of genetic sequence-based method and flow cytometry assay. Reumatologia. 2015;53:74-8.
- 27. Levering WH, Wind H, Sintnicolaas K, Hooijkaas H, Gratama JW. 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;54:28-38.
- 28. Dominguez O, Coto E, Martinez-Naves E, Choo SY, Lopez-Larrea C. Molecular typing of HLA-B27 alleles. Immunogenetics. 1992;36:277-82.
Abstract
Purpose: In the present study, we aimed to develop a reliable, cost effective and suitable for routine use in-house HLA-B*27 PCR-SSP typing kit for the molecular diagnosis of Ankylosing spondylitis and other spondyloarthropathies.
Materials and Methods: The study included 565 cases with 446 negative and 119 positive HLA-B*27 subjects. PCR optimization was performed for HLA-B*27 alleles using sequence-specific and internal control primers designed based on HLA-B and beta globin gene sequences, respectively.
Results: The presence of 149 bp specific band with 273 bp internal control band showed B*27 positivity and the presence of only 273 bp control band showed B*27 negativity on 2.0% agarose gel. The HLA-B*27 results show 100% sensitivity and specificity to our in-house SSP kit developted by our laboratory and the commercially available SSP kits. The cost of the in-house PCR-SSP kit we developed was at least 50 times cheaper than the commercial kits. In addition to the faultless correlation between the kits, our in-house SSP kit did not cross-react with other HLA-B alleles such as B*07, B*12, B*13, B*16, B*17, B*22, B*37, B*40, B*41, B*42, B*47 and B*48.
Conclusion: The fully evaluated findings show that in-house SSP kit for HLA-B*27 typing we developed is reliable, cost effective and suitable for routine use in tissue typing and genetic laboratories.
References
- 1. Schwartz BD. The human major histocompatibility HLA complex. In Basic and Clinical Immunology (Eds DP Stites, JD Stobo, JV Wells): 50-64. California, Appleton & Lange, 1987.
- 2. Allele Search Tool. IMGT/HLA-IPD-EMBL-EBI. [Internet]. Available at: https://www.ebi.ac.uk/ipd/imgt/hla/stats.html
- 3. Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet.1973;28(7809):904-7. 4. Bowness P. HLA-B27 in health and disease: a double-edged sword? Rheumatology. 2002;41:857-68.
- 5. Khan MA. Update on spondyloarthropathies. Ann Intern Med. 2002;136:896-907.
- 6. Seipp MT, Erali M, Wies RL, Wittwer C. HLA B27 typing: Evaluation of an allele specific PCR melting assay and two flow cytometric antigen assays. Cytometry B Clin Cytom. 2005;63:10-15. 7. Granfors K, Marker-Hermann E, de Keyser F, Khan MA, Veys EM, Yu DT. The cutting edge of spondylarthropathy research in the millennium. Arthritis Rheum. 2002;46:606-13.
- 8. Reveille JD, Ball EJ, Khan MA. HLA-B27 and genetic predisposing factors in spondyloarthropathies. Curr Opin Rheumatol. 2001;13:265-72.
- 9. Lopez-Larrea C, Gonzalez-Roces S, Alvarez V. HLA-B27 structure, function, and disease association. Curr Opin Rheumatol. 1996;8:296-308. 10. Khan MA. Update: the twenty subtypes of HLA-B27. Curr Opin Rheumatol. 2000;12:235-8.
- 11. Akkoc N, Khan MA. HLA-B27 and ankylosing spondylitis. APLAR J Rheumatol. 2005;8:146-53.
- 12. Lopez de Castro JA, Alvarez I, Marcilla M, Paradela A, Ramos M, Sesma L, et al. HLA-B27: a registry of constitutive peptide ligands. Tissue Antigens. 2004;63:424-45.
- 13. Khan MA. Update on spondyloarthropathies. Ann Intern Med. 2002;136:896-907.
- 14. Taurog JD. The mystery of HLA-B27: if it isn’t one thing, it’s another. Arthritis Rheum 2007;56:2478-81.
- 15. Oguz FS, Ocal L, Diler AS, Ozkul H, Asicioglu F, Kasapoglu E, et al. HLA B-27 subtypes in Turkish patients with spondyloarthropathy and healthy controls. Dis Markers. 2004;20:309-12.
- 16. Acar M, Cora T, Tunc R, Acar H.HLA-B27 subtypes in Turkish patients with ankylosing spondylitis and healthy controls. Rheumatol Int. 2012;32:3103-5.
- 17. Birinci A, Bilgici A, Kuru O, Durupinar B. HLA-B27 polymorphism in Turkish patients with ankylosing spondylitis. Rheumatol Int. 2006;26:285-7.
- 18. Diyarbakir E, Eyerci N, Melikoglu M, Topcu A, Pirim I. HLA B27 subtype distribution among patients with ankylosing spondylitis in eastern Turkey. Genet Test Mol Biomarkers. 2012;16:456-8.
- 19. Varnavidou-Nicolaidou A, Karpasitou K, Georgiou D, Stylianou G, Kokkofitou A, Michalis C, et al. HLA-B27 in the Greek Cypriot population: distribution of subtypes in patients with ankylosing spondylitis and other HLA-B27-related diseases. The possible protective role of B*2707. Hum Immunol. 2004;65:1451-4.
- 20. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
- 21. Bunce M. PCR-Sequence-Specific Primer Typing of HLA Class I and Class II Alleles. In Methods in Molecular Biology: MHC Protocols (Eds SH Powis, RW Vaughan RW): 143-172. MHC Protocols, Totowa, NJ, Humana Press, 2003.
- 22. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: a laboratory manuel. 1st Ed., New York: Cold Spring Harbor Laboratory, 1982.
- 23. Mittal KK, Mickey MR, Singal DP, Terasaki PI. Serotyping for homotransplantation. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation. 1968;8:913-27.
- 24. Dunky A, Neumüller J, Hübner C, Fischer GF, Bayer PM, Wagner E, et al. HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay. Rheumatol Int. 1996;16:95-100.
- 25. Coates E, Darke C. Routine HLA-B27 typing by flow cytometry: Differentiation of the products of HLA-B*2702, B*2705 and B*2708. Eur J Immunogenet. 1998;25:29-37.
- 26. Skalska U, Kozakiewicz A, Maslinski W and Jurkowska M. HLA-B27 detection - comparison of genetic sequence-based method and flow cytometry assay. Reumatologia. 2015;53:74-8.
- 27. Levering WH, Wind H, Sintnicolaas K, Hooijkaas H, Gratama JW. 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;54:28-38.
- 28. Dominguez O, Coto E, Martinez-Naves E, Choo SY, Lopez-Larrea C. Molecular typing of HLA-B27 alleles. Immunogenetics. 1992;36:277-82.
Details
| Primary Language | Turkish |
|---|---|
| Subjects | Health Care Administration |
| Journal Section | Research |
| Authors | |
| Publication Date | December 29, 2018 |
| Acceptance Date | July 31, 2018 |
| Published in Issue | Year 2018 Volume: 43 Number: Supplement 1 |
