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1 Review Article J Korean Soc Transplant 2015;29: Luminex 를이용한이식면역검사 가톨릭대학교의과대학서울성모병원진단검사의학과 이혜영ㆍ오은지 Luminex-based Immunoassay for Organ Transplantation Hyeyoung Lee, M.D. and Eun-Jee Oh, M.D. Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Development of luminex-based solid phase assays enables advanced measurement of HLA antibody with sensitivity, specificity, and increasing knowledge of unacceptable antigens. In this review, we described the principle of the luminex-based assay and its current applications for organ transplantation including C1q assay, calculated panel reactive antibody, and virtual crossmatching. We also discussed the technical aspects and limitations for clinical utilization. The variables related to measurement of HLA antibody specificities and their clinical relevance remain unclear, therefore the interpretation of results requires comprehensive knowledge and clinical information in critical cases. Key Words: Transplantation, Luminex, HLA antibody, Multiplex bead immunoassay 중심단어 : 이식, 루미넥스, HLA 항체, 다중비드면역측정법 Received June 19, 2015 Revised June 20, 2015 Accepted June 20, 2015 서론 장기이식환자에서 HLA (human leukocyte antigen) 형별시험과 HLA 항체검사는이식의예후판정과거부반응발생예측및면역억제제조절을위해중요하다. 신장이식실패의주요기전인항체매개성거부반응의진단과치료를위해서는공여자특이 HLA 항체 (donor-specific HLA antibodies, DSA) 의적절한검출과해석이요구된다 (1-3). Luminex를이용한 solid-phase assay (SPA) 는 DNA probes 나추출또는재조합된 HLA 항원을다중형광비드에부착 Corresponding author: Eun-Jee Oh Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul , Korea Tel: , Fax: ejoh@catholic.ac.kr 하여검체와반응시킨후 luminex 장비를이용하여 HLA 형별과 HLA 항체를검출하는방법이다. 이러한다중비드면역측정법 (multiplex bead immunoassay) 은검출민감도와특이도가높고상품화된시약을이용할수있는장점이있어, 최근다양한적용보고와함께사용이증가하고있다. 본논문에서는 luminex 를이용한이식면역검사의원리와종류, 임상적적용및결과해석시고려해야할점에대해다루고자한다. 1. 검사원리 Luminex 장비를이용한다중비드면역측정법의원리는 luminex 장비내 2개의레이저를사용하여비드와검체반응을각각검출하는것으로서유세포분석기원리를이용한다. Red laser (633 nm) 를이용하여형광강도수준 (fluorescence intensity level) 이다른 100여가지의 polystyrene microsphere beads를구분하고, 각각의비드에는 DNA 염기서열이나항원등의다른표적물질들이부착가 Copyright c the Korean Society for Transplantation, 2015

2 Hyeyoung Lee and Eun-Jee Oh: Luminex-based Immunoassay for Organ Transplantation 능하다. 검체내항원, 항체, DNA, 효소등의측정을위해서상보적인결합이가능한항체, 항원, DNA probe 등이부착된 luminex beads와검체를반응시키고 PE (phycoerythrin) 형광물질이결합된 2차항체를이용하여검출후 PE 형광시그널을 green laser (532 nm) 를이용하여측정한다. Luminex 비드를사용한 SPA는적은양의검체 ( 혈청 50 L 이하 ) 로최대 100여가지의검사를동시에시행할수있으며, 민감도가높고, median fluorescence intensity (MFI) 값으로반정량분석이가능하며, 각회사에서제공하는개별의고유프로그램을통해검사결과를분석한다. 2. HLA 형별검사 Luminex 를이용한 HLA 형별검사의원리는 MHC (Major Histocompatibility Complex) class I의 exon 2, 3, MHC class II의 exon 2 부위를 group specific primer로증폭시킨후 luminex 비드에부착된 sequence specific oligonucleotide probe 와의반응을검출하여 HLA 형별을결정하는 PCR-SSO (Polymerase Chain Reaction-Sequence Specific Oligonucleotide) 방법이다 (4). PCR 증폭에사용되는 primer에 biotin이부착되어있고, probe와증폭된 DNA의 hybridization 후 Streptavidin-Phycoerytherin (SAPE) conjugate 와반응시킨후 luminex 에서검출한다. 현재출시되는 luminex 키트는일부 HLA 유전자의경우고해상도결과까지보고가능한저해상도-중등도해상도검사이며, 적은양의 DNA를사용하여대량검체검사가가능한장점이있어검사실에서의사용이증가하고있다. 3. HLA 항체검사 Luminex 를이용한 HLA 항체검사는 HLA 항원이부착 된비드와혈청내 HLA 항체를반응시키고, 전용장비와프로그램을이용하여 HLA 항체를검출하는방법이다 (5). Luminex 검사는효소면역법이나세포독성법을이용한 HLA 항체검사에비해민감도와특이도가우수하며 (6), 소량의검체를사용하고, 96 well microplate를이용하여검사할수있으므로대량검사가가능한장점이있다. 국내에는두제조사의시약이공급되고있다 (Tepnel/Lifecodes, Stamford, CT, USA and One Lambda, Canoga Park, CA, USA). 비드에결합된표적항원에따라다음의 3가지검사로구분할수있다 (Table 1). 1) class I 또는 class II pooled Ag 패널을비드에부착하여환자혈청내 class I 또는 class II 항체유무를검출하는 HLA 항체선별검사 (pooled Ag bead, HLA antibody screen test), 2) 한명의 HLA class I 또는 class II 항원을한개의비드에부착하여이용하는 HLA 항체동정검사 (phenotype bead, HLA antibody identification test), 3) 재조합된단일항원이부착된단일항원비드 (single antigen bead, SAB) 를이용하는단일항원 HLA 항체동정검사 (single antigen bead, HLA antibody specification). 이중단일항원 HLA 항체검사는정확한항체특이성동정이가능하므로, 항체특이성결과를이용하여 calculated panel reactive antibody (cpra), virtual crossmatch 등에적용가능하다. 또한 HLA 유전자좌의항원을추출및재조합하여사용하므로 HLA-DQ, HLA-DQA, HLA-DPA, HLA-DPB, MICA 항체도검출가능하다. 특히 HLA-DQ 항체는신장이식환자의 33 90% 에서검출되고 (7,8), de novo DSA 의주요원인으로서이식손상과관련이있다는보고가있으므로 (9), 최근 HLA-DQ 형별검사와함께임상적예후판정을위한중요한이식면역검사로대두되고있다. Luminex를이용한 HLA 항체검사키트는정성검사로 Table 1. Characteristics of three beads for luminex-based HLA antibody tests Pooled antigen bead Single patient phenotype (identification) bead Single antigen bead Clinical utility Screening Identification of antigen specificity, % PRA Identification of allele specificity Relative antigen density Low Intermediate High Antigen composition per bead Class I or II phenotype of Class I or II phenotype of one Single allelic HLA antigen several individuals individual Resolution Result Positive/negative Identification % PRA Identification Unacceptable antigen % cpra/ C1q binding Abbreviations: PRA, panel reactive antibody; cpra, calculated panel reactive antibody; HLA, human leukocyte antigen. 55

3 J Korean Soc Transplant ㆍ June 2015 ㆍ Volume 29 ㆍ Issue 2 허가되었으나, MFI 결과값을이용한 HLA 항체의반정량결과를항체역가의모니터링과치료효과판정에이용할수있다는보고들이있다 (1-8,10). 그러나이식후급성거부반응이나이식실패를예측할수있는 MFI 값기준에대해서는 1,500 10,000 MFI 등으로보고자마다범위가다양하므로 (11-15), MFI의정량적모니터링은임상정보와함께고려되어야하며, MFI 값에영향을줄수있는시약과검사방법차이에대한평가와표준화가선행되어야할것이다. 이식전 DSA 양성또는 DSA 음성 /high PRA (panel reactive antibody) 양성환자에서그렇지않은환자에비해이식예후가좋지않다는보고가있고 (11,16-19), 이식후 de novo DSA가 개월사이에 6 38% 의빈도로검출되며, 이는급성거부반응과관련있는불량예후인자로간주되지만, 적절한검사기기와간격에대한보고는연구자마다다양하다. 신장이식이전과이후에시행하는 HLA 또는 non-hla 항체검사와관련된최근의권장지침에대한요약은다음과같다 (20). < 신장이식전항체검사 > ㆍ동정된항체특이성과교차시험결과에따라환자의위험그룹을분류한다. ㆍ환자의과거병력 ( 출산, 수혈, 과거이식력 ) 을고려하여양성결과를해석한다. ㆍ보체의존성세포독성검사에서 DSA가검출, 보체의존성세포독성교차시험이양성인경우, 항체매개성거부반응이나이식실패와강한연관성이있으므로이식은금지되어야한다. ㆍSAB를이용한항체검사에서모든 HLA class I과 class II 유전자좌에대한항체가음성인경우, 교차시험없이신장이식을시행할수있다. ㆍ비록정확한양성기준치는정해지지않았지만, DSA 가 luminex 항체검사에서만검출된경우는신이식의절대적금기가아니고, 상대적위험요인이다. ㆍ고감작된환자는신장교환이식이나탈감작요법을고려할수있다. ㆍABO 혈액형불일치의경우, 항체역가를감소시키고이식이가능하다. ㆍ이식전 non-hla 항체검사의역할은명확하지않다. < 신장이식후항체검사 > ㆍ신기능 ( 혈청크레아티닌, 요단백 ) 과조직검사를모니터링한다. ㆍ이식후 DSA 검사는지속시행되어야한다. ㆍ이식전 DSA가이식후지속되거나항체역가가증가하는경우는불량한예후와관련이있다. ㆍ이식후 de novo DSA 발생은불량한예후와관련이있다. ㆍ대부분의 de novo DSA는 HLA class II 항체이다. ㆍ조직검사로항체매개성손상과 C4d 유무를확인해야한다. ㆍHLA 항체검사와조직검사의시기는위험도에따라다르게결정한다. 고위험군 ( 이식전탈감작치료를받았거나 DSA 양성 / 교차시험음성인환자군 ): 이식첫 3개월내에 DSA 검사와조직검사를시행한다. 중등도위험군 (DSA 양성과거력이있으나현재는음성인환자군 ): 이식후 1개월이내 DSA 검사를시행하고, DSA 양성이면조직검사를시행한다. 저위험군 ( 감작력이없고첫번째이식을받은환자군 ): 이식후 3 12개월사이에 DSA 검사를시행하고, DSA 양성이면조직검사를시행한다. ㆍ면역억제제의중단과프로토콜조직검사의역할은불명확하다. ㆍ이식후 non-hla 항체검사의장점은불명확하다. 4. Luminex를이용한검사결과에영향을주는기술적요인 Luminex를이용한 HLA 항체검사는여러기술적인요인에의해 MFI 값이영향을받을수있으므로결과해석에유의해야한다. SAB에부착된 HLA 항원의양은제조사에따라다르고, 동일한키트내에서도각비드마다다르기때문에, 측정된 HLA 항체의 MFI 값에영향을줄수있다 (20). 또한정맥주사용면역글로불린 (IV immunoglobulin) 을투여받는환자나염증, 감염을동반한환자에서비특이적물질에의해 HLA 항체가위양성으로검출될수있다 (21). 이러한비특이적결합이있는경우에는음성대조 (negative control) 물질의 MFI 값도함께증가하기때문에 (21), 결과해석시음성대조물질의결과를유의해서관찰해야한다. 반대로고역가의 HLA 항체를가진환자에서 C1 면역복합체침착이유발되어비드에결합하거나 (22,23), 면역글로불린 M (IgM) 을포함한기타혈청물질이항체대신비드에결합하여위음성결과를초래할수도있다 (24-26). 이런전지대반응 (prozone effect) 에의한위음성이의심될경우검체를희석하거나 EDTA/dithiothreitol 또는열처리를통해문제를해결할수있다 (20,22,27). 비드내에있는각항원들은공통의항원결정부위 (epitope) 를 56

4 Hyeyoung Lee and Eun-Jee Oh: Luminex-based Immunoassay for Organ Transplantation 공유하고있기때문에혈청내 HLA 항체가여러비드에분산되어결합하면실제보다낮은 MFI 값을보일수도있다 (28). 환자가가진 HLA 항체가비드의해당 HLA 항원뿐만아니라교차반응군 (Cross reactive group, CREG) 에속한항원과결합하는경우 MFI 값이낮게측정될수있다. 따라서결과해석시 CREG 항원에대한항체의가능성을염두에두고판독해야한다. 감작력이없는신장이식대기자들에서특정 HLA 항체들이검출된다는보고가있는데, 이는비드의항원과 HLA 항체가결합하는과정에서단백질의삼차원적구조적변화로인해 (29-31) 정상적으로관찰되지않는 neoepitopes 항원결정기가노출되어발생하는위양성결과로알려져있으며 (32-35), 이항체들은 natural HLA 항체로분류되었다 (33,34). Natural HLA 항체는흔하지않은 HLA 항원에대한항체들로이루어졌고 (33) 1,000 5,000 사이의낮은 MFI 값을보이는것이특징이나 (21), MFI 5,000 이상인환자에서도약 25% 에서관찰된다는보고가있으므로 (32), 검사결과해석시염두에둘필요가있다. 이러한 natural HLA 항체들의정확한기전은아직밝혀지지않았지만바이러스, 박테리아항원에대한항체로서발생한것으로예상되고있으며 (29), 임상적인의미에대한추가연구가필요하다 (36,37). 마지막으로, luminex 를이용한 SPA는제품번호 (lot to lot) 간비드구성에차이가있을수있으므로환자의임상적인경과에따른 HLA 항체의 MFI 값의변화를관찰할시염두에두어야한다 (38-40). 따라서, Luminex 방법의 HLA 항체검사결과의정확한해석을위해서는환자의감작력정보와함께다른검사결과 ( 교차시험, luminex-screen) 와의비교가필요하다. 신장이식에서 HLA 또는 non-hla 항체검사방법과관련된최근의권장지침에대한요약은다음과같다 (20). ㆍ보체의존성세포독성교차시험은초급성거부반응을일으킬수있는항체를검출한다. ㆍ유세포분석법을이용한교차시험은림프구에결합하는항체를검출한다. ㆍSPA는 microtiter plate (ELISA) 또는 polystyrene bead 에결합된항원을유세포분석기나 luminex 로검사하는방법을포함한다. ㆍ검사예민도를비교하면, Luminex>ELISA/ 유세포분석>보체의존성세포독성순이다. ㆍCw, DQA, DPA, DPB를포함한 HLA 모든유전자좌에대한항체를검사하기위한이식전검사에 SPA를사용한다. ㆍ세포기반검사 ( 보체의존성세포독성, 유세포분석법을이용한교차시험 ) 와 SPA를함께실행하여두검사 법의상관성을비교하거나양성교차시험결과를예측한다. ㆍLuminex 방법에서는검사결과와임상적해석에영향을줄수있는기술적인측면 ( 예, 비드에부착된항원양의차이나항원의변성 ) 을고려해야한다. ㆍ비드를이용한검사는정성또는반정량검사이다. ㆍ검사실간검사방법과양성결정값에차이가있다. 5. Luminex를이용한 HLA 항체검사의확대적용 1) C1q assay C1q 검사는 luminex 의 SAB를환자혈청과반응시킬때정제된 C1q 보체를첨가하여반응시키고, 형광이부착된항 C1q 항체와반응시켜 HLA 항체중 C1q가결합하는 complement-fixing HLA 항체를검출하는검사법이다 (29,41). C1q+DSA+ 환자군에서 C1q DSA+ 인대조군에비해 C4d 양성, 항체매개성거부반응, 신소실등의불량한예후와관련이있고 (42,43), MFI 역가와 C1q 반응은관련이없다는보고가있다 (44). 그러나, C1q+ 항체는 IgG1/3 의아형인고역가의 HLA 항체에서검출된다는보고들도있으므로 (13-15,43,45), C1q 검사의임상적적용에대한추가연구가필요하다. 2) Calculated PRA (cpra) SAB를이용한 HLA 항체검사의도입으로정확한항체특이성동정이가능해짐에따라, 동정된항체특이성결과를이용하여 cpra 결과를얻을수있다. cpra는특정인종, 국가등집단의 HLA 항원빈도를이용하여, 동정된항체와반응이예상되는항원의빈도를산출하는것이다 (46). 미국장기이식관리기구인 United Network for Organ Sharing (UNOS) 에서는 cpra 80% 이상을보이는환자를고위험군으로간주하고있으나, 국내에서는일부검사실에서자체개발하여사용중에있고 (46), 국내장기이식관리센터에서는 cpra의도입과적용을검토중에있다. cpra는이식전환자의감작정도를간접적으로알수있는객관적지표이므로, 향후장기이식분야에유용하게쓰일것으로생각된다. 3) Virtual crossmatch Virtual crossmatch는 SAB를이용한 HLA 항체검사에서동정된항체특이성결과를이용하여 unacceptable HLA antigen을결정하고제공자의 HLA 형별결과와대조하여, 실제교차시험을시행하지않고결과를예측하는가상의교차시험방법이다. 이식대기자에서검출된 HLA 항체가 57

5 J Korean Soc Transplant ㆍ June 2015 ㆍ Volume 29 ㆍ Issue 2 결합가능한 HLA 항원을가진제공자를 virtual crossmatch 를통해미리배제할수있으므로, 이식제공자선택에이용되거나 (21), high-pra (>85%) 환자에서탈감작치료후 DSA 항체가음성전환또는역가가감소됨을확인하고이식을시행하는데이용될수있다 (47,48). 그러나, HLA 항체검사에서검출되지않는항체에의한교차시험양성이있을수있고, 교차시험음성인낮은역가의 HLA 항체검출로인해이식의기회가줄어들수있으므로 (49,50), 올바른임상적적용을위한 HLA 항체특이성검사방법의표준화와 MFI cut-off 설정이필요하다. 6. 표준화및정도관리 Luminex 를이용한 HLA 항체검사는결과해석에중요한 MFI 값의기준치가검사실간다르게설정될수있고, 각검사실마다프로토콜에차이가있을수있다 (28,51,52). 최근국내 5개병원을대상으로, 동일로트제품을사용하고동일한프로토콜로시행한검사실간비교연구에서는 (53), HLA class I과 class II 항체검출에대한일치율이각각 96.0%, 97.2% 였고, MFI 값은 (HLA class I 항체 ), (HLA class II 항체 ) 의높은상관성을보였다. MFI 값의정밀도평가에서는 1,000 미만의 MFI 값에서그이상의 MFI 값들보다높은변이계수값을보였다. 향후여러제조사에서제조된제품을통한검사실간비교연구가필요할것으로예상되며, 임상적으로유용한 cut-off의공유및정확한결과해석을위해검사실간표준화와지속적인외부정도관리가필요할것이다. 결론 HLA형별과 HLA항체검사에이용되는 Luminex-SPA는민감도와특이도가높은우수한검사법이다. Luminex 항체검사의 MFI 결과값을이용하여 HLA항체역가의모니터링이가능하고, 항체특이성결과를이용하여 C1q assay, cpra, virtual crossmatch 등에적용가능하므로장기이식검사로서의임상적유용성은더욱증가할것이다. 그러나, 결과에영향을줄수있는여러기술적요인을고려하고환자의임상정보와다른검사결과와의비교를통한적절한결과해석이필요하며, 검사방법과결과보고의표준화및지속적인외부정도관리가요구된다. 감사의글 이논문은가톨릭대학교서울성모병원의연구비지원 을받아수행되었다. REFERENCES 1) Einecke G, Sis B, Reeve J, Mengel M, Campbell PM, Hidalgo LG, et al. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant 2009;9: ) Sellarés J, de Freitas DG, Mengel M, Reeve J, Einecke G, Sis B, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant 2012;12: ) Süsal C, Döhler B, Sadeghi M, Ovens J, Opelz G. HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report. Transplantation 2009;87: ) Trajanoski D, Fidler SJ. HLA typing using bead-based methods. Methods Mol Biol 2012;882: ) El-Awar N, Lee J, Terasaki PI. HLA antibody identification with single antigen beads compared to conventional methods. Hum Immunol 2005;66: ) Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Innocente A, et al. Luminex technology for anti-hla antibody screening: evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom 2007;72: ) Ozawa M, Rebellato LM, Terasaki PI, Tong A, Briley KP, Catrou P, et al. Longitudinal testing of 266 renal allograft patients for HLA and MICA antibodies: Greenville experience. Clin Transpl 2006; ) DeVos JM, Gaber AO, Knight RJ, Land GA, Suki WN, Gaber LW, et al. Donor-specific HLA-DQ antibodies may contribute to poor graft outcome after renal transplantation. Kidney Int 2012;82: ) Tagliamacco A, Cioni M, Comoli P, Ramondetta M, Brambilla C, Trivelli A, et al. DQ molecules are the principal stimulators of de novo donor-specific antibodies in nonsensitized pediatric recipients receiving a first kidney transplant. Transpl Int 2014;27: ) Murphey CL, Bingaman AW. Histocompatibility considerations for kidney paired donor exchange programs. Curr Opin Organ Transplant 2012;17: ) Caro-Oleas JL, González-Escribano MF, González-Roncero FM, Acevedo-Calado MJ, Cabello-Chaves V, Gentil-Govantes MÁ, et al. Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation. Nephrol Dial Transplant 2012;27: ) Lefaucheur C, Loupy A, Hill GS, Andrade J, Nochy D, Antoine C, et al. Preexisting donor-specific HLA antibodies predict 58

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untitled J Korean Soc Transplant 2012;26:92-100 http://dx.doi.org/10.4285/jkstn.2012.26.2.92 Original Article 장기이식환자에서유세포교차시험결과와 Luminex-PRA 검사로검출되는공여자 - 특이항체의비교평가 서울대학교의과대학검사의학교실 1, 한림대학교성심병원진단검사의학과 2, 인제대학교의과대학서울백병원진단검사의학과