KMJ Original Article Degree of Agreement between Phadia EliA ENA and Euroimmun line Immunoassay; Comparison of Two Methods to Evaluate the Ability to Detect ENA Antibodies Hyun Yong Hwang Department of Laboratory Medicine, College of Medicine, Kosin University, Busan, Korea Phadia EliA ENA 와 Euroimmun line immunoassay 의일치도분석 ; ENA 항체검출능력을평가하기위한두검사법의비교 황현용 고신대학교의과대학진단검사의학교실 Objectives: The purpose of this study is to compare newly developed assay for identification of ENA antibody, Phadia EliA ENA with Euroimmun line immunoassay by analyzing the degree of agreement and the individual antibodies between two methods. Methods: A total of 82 patient samples were used. Indirect immunofluorescence assay using Hep-2 cell was performed to screen the antinuclear antibody (ANA). Euroimmun line immunoassay (LIA) and Phadia EliA ENA assay were tested to identify the antibodies against extractable nuclear antigens (ENAs). Kappa statistics was used to evaluate the degree of agreement. Results: Mean age of patients was 41.0 (8-79), and the M:F ratio was 21:61. ANA was positive in 74 samples, and negative were 8 samples. Kappa analysis of the 82 tested samples showed a moderate strength of agreement (k = 0.521, P = 0.000). There were differences in the order of identified individual antibodies between two methods (Ro > La = RNP > Centromere >Sm>Scl-70 in Phadia EliA ENA, Ro>RNP>Sm>La>Scl-70>Centromere=Jo-1 in Euroimmun LIA). Ro antibody was most frequently identified in Phadia EliA ENA negative-euroimmun LIA positive specimens (Ro > RNP = Jo-1 > La = Sm = Centromere > Scl-70). Conclusions: A moderate strength of agreement was observed between the Phadia EliA ENA and the Euroimmun LIA. There seemed to be a significant difference in the ratio of individual antibodies, especially in the anti-ro and Sm antibodies. Key Words: Antinuclear antibody, Extractable nuclear antigen 일반적으로 extractable nuclear antigen (ENA) 에대한항체검사는 antinuclear antibody (ANA) 양성인경우시행된다. 즉, 간접면역형광염색법을이용한 ANA 선별검사에서 homogenous pattern, speckled pattern 등의 Hep-2 cell의염색형태에따라추가적으로구체적인 ENA 항체검사가선택되어시행된다. ENA 항체의동정은자가면역질환의진단에결정적인역할을하기때문에정확한항체의검출이임상적으로매우중요하다. 1-3 Passive hemagglutination (HA), double immunodiffusion (DID), enzyme-linked immunosorbent assay (ELISA) 및 luminex-based assay와같은다양한방법에의한 ENA 항체검출이가능하다. 4-6 Corresponding Author: Hyun Yong Hwang, Department of laboratory medicine, College of Medicine, Kosin University, 34 Amnamdong, Seo-gu, Busan, 602-702, Korea TEL: 051) 990-6279 FAX: 051) 990-3010 E-mail: terminom@hanmail.net Received: November 16, 2011 Revised: December 8, 2011 Accepted: December 26, 2011 25
자가면역질환에서이들 ENA 항체의검출에있어검사방법에따른차이가있기때문에질환의진단에대한민감도와특이도에차이가발생한다. 근래에 Phadia EliA ENA (Phadia, Uppsala, Sweden) 가소개되었으나아직널리사용되지않고또한기존의검사법과비교된자료가없다. 따라서본연구에서는 ENA 항체를검출하기위한 Phadia EliA ENA와현재널리사용되고있는 Euroimmun line immunoassay (EUROIMMUN, Lübeck, Germany) 를비교함으로써두검사법간의대체가능성을가늠해보고개별항체검출능력의차이를평가하고자하였다. 연구대상과방법 2010년 6월부터 2011년 6월까지 ANA 선별검사와 ENA 에대한항체검사를시행한환자중무작위로추출된총 82명의환자검체를사용하였다. Fluoro HEPANA test (MBL, Nagoya, Japan) 를사용하였으며제조자의지시대로시험하였다. 실험방법을간략히설명하면, HEp-2 세포를기질로사용하는슬라이드에환자의혈청을 1:20 으로희석하여떨어뜨린후실온에서 20분간반응시키고세척후 FITC 형광물질이부착된 2차항체를다시반응시킨다. 실온에서 20분간반응시키고세척한다음암실에서형광현미경으로 HEp-2 세포에염색된형광의강도와형태를관찰한다. EUROLINE (EUROIMMUN, Germany) 를사용하였으며제조자의지시대로시험하였다. 실험방법을간략히설명하면, 모두 15개의자가항체 (RNP, Sm, SS-A, Ro- 52, SS-B, Scl-70, PM-Scel, Jo-1, Centromere, PCNA, ds DNA, Nucleosome, Histone, Ribosomal protein 및 AMA-M2) 에대한항원이부착된직사각형의기다란막스트립에관찰하고자하는혈청을떨어뜨린다. 5분간반응을시킨후, 혈청을흡인해내고다시혈청으로 30분간반응시키고세척해낸후효소기질반응을위한효소를떨어뜨린다. 효소의항체부착반응을위해 30분간반응시킨후 3회연속세척한다. 색소원이부착된기질을떨어뜨려효소기질반응을유발시킨다. 10분간반응시킨후증류수로세척하고물기를흡인해낸다. 항체가부착된밴드에색소가염색된것이관찰되며스캐너와판독프로그램을이용하여판독한다. 검사를시행한 15개항체중 Sm, RNP, Ro, La, Scl-70, Centromere 및 Jo-1 항체검사결과를사용하였다. 환자의혈청을사용하여 7종류의자가항체 (Sm, U1RNP, Ro, La, Scl-70, Centromere, 및 Jo-1) 검사를자동화된면역검사장비인 Phadia 250 (Phadia, Uppsala, Sweden) 에서시행하였고 Euroimmun LIA 결과와비교하였다. 두검사법간의일치도 (degree of agreement) 를분석하기위하여 kappa 통계를사용하였다. 각통계적분석에서유의확률 P 값이 0.05 미만인경우통계적으로유의한것으로판단하였다. 통계적분석을위해 SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) 을사용하였다. 결과 총 82 명의환자검체를사용하였으며남녀비율은 M:F = 21:61 이었고평균연령은 41.0세 (8-79) 였다. 74명의환자검체가 ANA 양성이었고 8명의검체는음성이었다. Hep-2 세포의염색형태에따른분류에서는 homogenous pattern 이 30검체, speckled pattern 이 17검체, mixed pattern 이 8검체, discrete speckled pattern이 3검체, nucleolar pattern 이 3검체, 그리고 cytoplasmin 26
Degree of Agreement between Phadia EliA ENA and Euroimmun line Immunoassay; Comparison of Two Methods to Evaluate the Ability to Detect ENA Antibodies pattern이 13검체였다. 비교평가할 7개의개별항체에대해 Phadia EliA ENA 와 Euroimmun LIA 두검사법에서각각양성, 음성으로판정된검체를비교하였다. 이때 Euroimmun LIA에서 trace 로판정된경우는양성으로간주하였고 Phadia EliA ENA 에서판독불가 (equivocal) 로판정된경우는양성혹은음성어떤범주에도포함시키지않았다. 이러한기준으로비교하였을때 74.39% 에서두검사법의결과값이일치하였다 (Table 1). Kappa 통계를이용한일치도분석에서중등도의일치도를나타내었다 (k = 0.521, P = 0.000). 두검사법에서개별항체검사결과가일치하는경우는 82검체중에 65검체 ( 양성, 45검체 ; 음성, 16검체 ) 였고두검사법에서모두양성 (Phadia EliA ENA 양성-Euroimmun LIA trace 이상양성 ) 인 45검체에서개별항체가일치하지않는경우는 2검체였다. 여기서한검체에서는 Phadia EliA ENA에서는 U1RNP 항체가 Euroimmun LIA에서는 Ro 항체가검출되었고나머지한검체에서는 U1RNP, centromere, Ro, La, Scl-70이 Phadia EliA ENA에서 Ro 항체가 Euroimmun LIA에서각각검출되었다. 각검사에서개별항체의분포를조사하였을때 Phadia EliA ENA 에서는 Ro 항체가 53.5% 로가장높았고 La와 RNP 가 18.3% 로두번째로많이검출되었으며나머지는 centromere, Sm 및 Scl-70 순서로검출되었다 (Table 2). Euroimmun LIA에서는 Ro가 35.8% 로가장높았으나 Phadia EliA ENA와달리 RNP (14.2%) 다음으로 Sm (13.5%) 이검출되었으며 La, Scl-70 순으로그리고 Centromere와 Jo-1이가장낮게검출되었다 (Table 3). Phadia EliA ENA에서는음성이었으나 Euroimmun LIA 검사에서는 trace 이상인 17검체에서개별항체의분포를조사하였다 (Fig. 1). 7개의개별항체의검출횟수는중복을허용하여총 46회였다. 한검체에서여러항체가한꺼번에검출되는경우가많았으며 Ro 항체가 11회 (23.9%) 로가장빈번히검출되었고 RNP 와 Jo-1 이 7회 (15.2%), La, Sm 및 Centromere 항체가 6회 (13.0%), 그리고 Scl-70이 3회 (6.5%) 검출되었다. Table 1. Concordance between Phadia EliA ENA and Euroimmun LIA Phadia EliA ENA Positive Equivocal Negative Total Euroimmun LIA Positive 43 0 9 52 Trace 2 2 8 12 Negative 0 2 16 18 Total 45 4 33 82 Table 2. Indivisual antibodies in Phadia EliA ENA ENA Phadia EliA ENA Positive Equivocal Total % Ro 36 2 38 53.5 La 11 2 13 18.3 U1RNP 12 1 13 18.3 Sm 1 1 2 2.8 Centromere 3 1 4 5.6 Scl-70 1 0 1 1.4 Total 64 7 71 100 Table 3. Indivisual antibodies in Euroimmun LIA ENA Euroimmun LIA Positive Trace Total % Ro 44 9 53 35.8 La 13 6 19 12.8 RNP 18 3 21 14.2 Sm 7 13 20 13.5 Centromere 10 1 11 7.4 Scl-70 3 10 13 8.8 Jo-1 7 4 11 7.4 Total 102 46 148 100 27
Fig. 1. Individual antibodies in Phadia EliA negative- Euroimmun LIA positive or trace 고찰 검사실에서 ENA 개별항체의동정은자가면역질환의진단에매우중요하다. 임상적으로흔히동정되는항체인 Ro 항체의경우쇼그렌증후군과같은질환의진단과밀접하게연관되고, 7 간혹간접면역형광법을이용한 ANA 는음성이나 ENA 는양성인경우가있으며간접면역형광법을사용하는경우드물게관찰된다. 8-10 본연구에서도 ANA 검사결과음성인 8검체중 7검체에서 ENA 항체가검출되었다. 따라서개별항체의동정능력이환자의예후에매우중요하게작용할수있다. ENA 항체를검출에 Immunodiffusion assay를사용할경우특이도가떨어지는단점이지적되었고 ELISA 는검사시간이길고노동집약적인단점이있다. 4,11,12 그래서최근에는이러한단점을극복하고자 luminex-based assay 와같은자동화된검사법들이소개되고있다. 13 이러한검사법들을검사실에도입할때가장중요한고려점중하나가기존검사법과의일치도다. 즉, 노동력해소와재현성을높이기위해자동화를원하지만검사결과가기존의검사법과의미있는차이가있다면실제사용하기는어렵다. Table 1에서 Phadia EliA ENA와 Euroimmun LIA 두검사법이 74.4% 에서일치하였지만 kappa 통계를이용한일치도분석에서는중등도의일치도를나타내었다 (k = 0.521, P = 0.000). 따라서두검사법을혼용하는데큰무리는없어보이나 17검체에서 Euroimmun LIA에서양성이나 Phadia EliA ENA 에서는음성이었다. 동정된총항체의검출횟수를보면 Phadia EliA ENA의경우 71회였으나 Euroimmun LIA 의경우 148회로모든개별항체가높은횟수로검출되어후자의검사가좀더민감해보였다 (Table 2, 3). Fig. 1에서 Phadia EliA ENA 에서는음성이나 Euroimmun LIA에서는양성인항체로는 Ro 항체가 23.9% 로가장높았다. Ro 자가항체는 Ro60 혹은 Ro52 항원과반응하는항체의결과물이며반응항원의구성은제조사에따라다르다. Phadia EliA ENA는비록 52 kda과 60 kda 두항원으로구성되어있다고하지만 Euroimmun LIA 처럼 Ro60 과 Ro52 를각각독립된밴드에서검출하고있는경우보다검출력이상대적으로떨어져보였다. 또한 ANA 선별검사가음성인경우 Ro60 은음성이지만 Ro52 는양성인경우가있기때문에임상적으로 Ro52 와연관된자가면역질환이의심되는경우에 ANA 선별검사의결과와관계없이 Ro 자가항체검사의결과가상대적으로매우중요하게된다. 14 따라서 Ro 항체의검출능력이쇼그렌증후군과같이 Ro 항체와밀접하게연관된질환의진단에매우중요하다. RNP 의경우 Phadia EliA ENA 에서두번째로많이검출되기는하였으나 Phadia EliA ENA 음성이고 Euroimmun LIA 양성인검체에서도두번째로많아두검사법에서차이가있어보였다. Sm 항체가있는경우거의모든경우에서 RNP 항체가함께존재한다. 15 또한 RNP 항체가높은역가로있으면서 Sm 항체가없는경우가종종 mixed connective tissue disease (MCTD) 에서관찰된다. 16,17 따라서 RNP 항체와 Sm 항체의정확한검출은진단에매우중요하다. 대략 40% 의 RNP 항체는 U1 RNP 에결합하며, 18 RNP 항체는 U1 snrnp-specific polypeptides (68K, A, 및 C 항원 ) 와반응하는데 68K U1 RNP 가 MCTD RNP 항원의주요구성성분이며, 19 U1 RNP A와 C는 Sm 항원과유사하다. 20 Euroimmun LIA 에서 21검체에서 RNP 가양 28
Degree of Agreement between Phadia EliA ENA and Euroimmun line Immunoassay; Comparison of Two Methods to Evaluate the Ability to Detect ENA Antibodies 성이었으며이들검체중 17검체에서 Sm 항체도검출되었다 (81.0%). 반면에 Phadia EliA ENA에서는 U1 RNP 양성인 13검체중 2검체에서만 Sm이함께검출되었다 (15.4%). 따라서두검사법에서 RNP 와 Sm 항체비율의차이는두검사법에사용된 RNP 항원의구성에따른효과로사료된다. 결론적으로 Phadia EliA ENA 를사용하여개별항체를동정하는경우 Euroimmun LIA 검사법과비교하였을때개별항체의검출에있어일부항체의차이가있으나전반적인일치도를감안할때두방법간에의미있는차이는없었다. 다만, Ro 항체및 RNP/Sm 항체간의비율을포함하는일부 ENA 항체의검출에차이가있는부분에대해서는추가적인연구가필요할것으로사료되었다. 참고문헌 1. Orton SM, Peace-Brewer A, Schmitz JL, Freeman K, Miller WC, Folds JD. Practical evaluation of methods for detection and specificity of autoantibodies to extractable nuclear antigens. Clin Diagn Lab Immunol 2004;11:297-301. 2. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 1989;44:93-151. 3. von Muhlen CA, Tan EM. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum 1995;24: 323-58. 4. Kim Y, Park Y, Lee EY, Kim HS. Comparison of automated multiplexed bead-based ANA screening assay with ELISA for detecting five common anti-extractable nuclear antigens and anti-dsdna in systemic rheumatic diseases. Clin Chim Acta 2012;413:308-11. 5. Lora PS, Laurino CC, Becker BS, Monticielo OA, Brenol JC, Xavier RM. Clinical diagnostic performance of different methods for the detection of antibodies to extractable nuclear antigens in connective tissue diseases: a cohort study. Clin Lab 2011;57:625-9. 6. Siracusano A, Agelli M, Ioppolo S, Quintieri F, Bombardieri S. Detection of anti-extractable nuclear antigens in connective tissue diseases: comparison between passive hemagglutination, counterimmunoelectrophoresis and double immunodiffusion. Ric Clin Lab 1985;15:33-8. 7. Fujimoto M, Shimozuma M, Yazawa N, Kubo M, Ihn H, Sato S, et al. Prevalence and clinical relevance of 52-kDa and 60-kDa Ro/SS-A autoantibodies in Japanese patients with systemic sclerosis. Ann Rheum Dis 1997;56:667-70. 8. Thomson KF, Murphy A, Goodfield MJ, Misbah SA. Is it useful to test for antibodies to extractable nuclear antigens in the presence of a negative antinuclear antibody on Hep-2 cells? J Clin Pathol 2001;54:413. 9. Manoussakis MN, Garalea KL, Tzioufas AG, Moutsopoulos HM. Testing for antibodies to ENA and to dsdna is not indicated in FANA-negative sera. Clin Rheumatol 1988;7:465-9. 10. Malleson PN, Sailer M, Mackinnon MJ. Usefulness of antinuclear antibody testing to screen for rheumatic diseases. Arch Dis Child 1997;77:299-304. 11. Sinico RA, Bollini B, Sabadini E, Di Toma L, Radice A. The use of laboratory tests in diagnosis and monitoring of systemic lupus erythematosus. J Nephrol 2002;15 (Suppl. 6):S20-7. 12. Fenger M, Wiik A, Hoier-Madsen M, Lykkegaard JJ, Rozenfeld T, Hansen MS, et al. Detection of antinuclear antibodies by solid-phase immunoassays and immunofluorescence analysis. Clin Chem 2004;50:2141-7. 13. Desplat-Jego S, Bardin N, Larida B, Sanmarco M. Evaluation of the BioPlex 2200 ANA screen for the detection of antinuclear antibodies and comparison with conventional methods. Ann N Y Acad Sci 2007;1109:245-55. 14. Hwang HY, Kim JK. Is there no need to perform an additional ENA antibody test when the ANA screening test is negative? : Association analysis between the results of ANA screening test by IIFA and those of Ro60 and Ro52 autoantibody tests by LIA. Kosin medical journal 2007;22:120-5. 15. Van Venrooij WJ, Sillekens PT. Small nuclear RNA associated proteins: autoantigens in connective tissue diseases. Clin Exp Rheumatol 1989;7:635-45. 16. Sharp GC, Irvin WS, Tan EM, Gould RG, Holman HR. Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 1972;52:148-59. 17. Sharp GC, Irvin WS, May CM, Holman HR, McDuffie FC, Hess EV, et al. Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease, systematic lupus erythematosus and other rheumatic diseases. N Engl J Med 1976;295:1149-54. 18. Fritzler MJ and Elkon KB. Autoantibodies in SLE. In: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH. Rheumatology. 3rd ed. NY: Mosby; 2003. P1340-1. 29
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