원저 Lab Med Online Vol. 5, No. 2: 63-68, April 임상화학 Elecsys Neuron-Specific Enolase 의수행능평가 Performance E

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원저 Lab Med Online Vol. 5, No. 2: 63-68, April 215 임상화학 Elecsys Neuron-Specific Enolase 의수행능평가 Performance Evaluation of the Elecsys Neuron-Specific Enolase Assay 김수경 정태동 이우창 전사일 민원기 Soo-Kyung Kim, M.

Center, Seoul, Korea Background: Neuron-specific enolase (NSE) is an enzyme specifically found in neurons and neuroendocrine tissue.

This study evaluates the performance of Elecsys NSE (Roche Diagnostics, Switzerland), an electrochemiluminescent immunoassay.

1 원저 Lab Med Online Vol. 5, No. 2: 63-68, April 215 임상화학 Elecsys Neuron-Specific Enolase 의수행능평가 Performance Evaluation of the Elecsys Neuron-Specific Enolase Assay 김수경 정태동 이우창 전사일 민원기 Soo-Kyung Kim, M.D., Tae-Dong Jeong, M.D., Woochang Lee, M.D., Sail Chun, M.D., Won-Ki Min, M.D. 울산대학교의과대학서울아산병원진단검사의학과 Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea Background: Neuron-specific enolase (NSE) is an enzyme specifically found in neurons and neuroendocrine tissue. It is a common marker for small cell lung cancer diagnosis and is also useful as a predictor of brain damage. This study evaluates the performance of Elecsys NSE (Roche Diagnostics, Switzerland), an electrochemiluminescent immunoassay. Methods: The precision, linearity, limit of detection, and reference interval of the Elecsys NSE, as well as the correlation between Elecsys NSE and ELSA-NSE (Cis-Bio International, France) were evaluated in accordance with the Clinical Laboratory Standards Institute (CLSI) guidelines. PreciControl Tumor Marker (Roche Diagnostics), patient sera, and sera from healthy individuals were used for the analysis. Results: The measured coefficient of variation for the assay was below 3%, and it demonstrated linearity from.2 to ng/ml. The detection limit was.32 ng/ml and the reference interval ranged from.5 to 16.3 ng/ml. Compared with the ELSA-NSE assay, the correlation coefficient was Conclusions: The Elecsys assay showed suitable precision, linearity, limit of detection and reference range for clinical laboratory use; however, the correlation coefficient of Elecsys NSE as compared to ELSA-NSE was below.975. This result may be associated with the use of different monoclonal antibodies in the two different NSE assays. Elecsys NSE demonstrated a high sensitivity without the use of radioactive reagents; therefore, Elecsys NSE will be quite useful for NSE analysis in the clinical laboratory setting. Key Words: Analytical performance, Elecsys NSE, Neuron-specific enolase 서론 에놀라제는 α, β, γ 의세가지소단위로구성된이합체로 α 소단 위는다양한조직, β 소단위는심장이나가로무늬근, γ 소단위는 신경에주로존재한다 [1]. 신경원특이에놀라제 (neuron specific enolase, NSE) 는신경세포에특이적으로존재하는에놀라제로 γγ 또는 αγ 아이소형 (isoform) 으로이루어져있다 [2]. NSE 는 197 년대 Corresponding author: Woochang Lee Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul , Korea Tel: , Fax: , wlee1@amc.seoul.kr Received: July 18, 214 Revision received: September 29, 214 Accepted: October 1, 214 This article is available from 215, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 뇌에서처음분리했고 [3], 면역세포화학 (immunocytochemical) 검사를통해신경세포에특이적으로존재함이밝혀졌다 [4]. 이어서신경세포뿐만아니라말초신경내분비세포에도 NSE가존재함이확인되었다 [5]. 신경내분비세포란신경자극을받아호르몬을분비하는세포로서뇌하수체의코르티코트로프 (corticotrophs), 부갑상선의 C 세포, 부신수질의크롬친화세포, 췌장의베타세포, 폐의신경상피세포등이대표적이다 [4, 6]. 198년대에소세포폐암환자의 6-81% 에서 NSE 증가가동반되는것이보고된이후소세포폐암의초치료후재발감시및치료반응모니터링에도 NSE를유용하게사용할수있음이밝혀졌다. 현재, NSE는폐암의진단및치료에사용할수있는유용한종양표지자로각광받고있다 [7-11]. 최근에는 NSE가뇌경색이나외상으로인한뇌손상, 세균성뇌수막염등다양한뇌손상질환에서손상용적에비례하여증가하며, 예후예측인자로사용이가능한것으로알려져신경계질환에도 NSE에대한관심이집중되고있다 [12-14]. NSE는초기에방사면역측정법 (radioimmunoassay, RIA) 과효소면역측정법 (enzyme immunoassay, EIA) 을이용하여측정하였다. eissn

2 RIA는상대적으로최소검출한계가낮은편이지만 ( 최소검출한계 =3. ng/ml), 방사성동위원소를사용하므로검사자의건강과안전에문제를일으킬수있고, 안전관리및폐기에시간과비용이많이소요된다 [15]. EIA는방사성동위원소를사용하지않지만, RIA보다최소검출한계가높은문제점이있다 ( 최소검출한계 =12.5 ng/ml)[16]. 이후조작이간편하고, 방사성동위원소를사용하지않으면서최소검출한계가낮은 ( 최소검출한계 =.25 ng/ml) 전기화학발광면역측정법 (electrochemiluminescence immunoassay, ECLIA) 이개발되었다 [17]. 저자들은 Elecsys NSE (Roche Diagnostics, Basel, Switzerland) 의검사성능을평가하고자본연구를시행하였다. 대상및방법 1. 검사대상의특성 Elecsys NSE는마이크로파티클을이용하여 ECLIA 원리로 NSE 를측정한다. Elecsys NSE는 γ-enolase에특이한서로다른에피토프를지닌두개의단클론항체 (18E5 와 DN84B1) 를사용하며, 이는 α 소단위와의교차반응은없는것으로알려져있다 [18]. 검사소요시간은약 18분이며, 2 μl의혈청검체를사용한다. Elecsys NSE는 Roche사의 Elecsys 21, ModularAnlytics E 17, cobas e 411, cobas e 61, cobas e 62 등의장비를이용해분석할수있다. 본연구에서는 cobas e 61 장비를이용하여측정하였다. 2. 정밀도 (Precision) 정밀도는 Elecsys NSE의전용대조물질인 PreciControl Tumor Marker 1 (Lot 17198, Roche Diagnostics) 과 PreciControl Tumor Marker 2 (Lot 17199, Roche Diagnostics) 를사용하여 Clinical and Laboratory Standards Institute (CLSI) EP5-A2 지침에따라평가하였다 [19]. 2일간각농도에대해 1일 2회, 1회 2번씩반복측정하였다. 하루 2회검사는 2시간이상의간격을두고오전과오후로나누어실시하였다. 측정된결과를이용하여각농도별평균, 표준편차, 검사차례내변이계수 (within run coefficient of variation, CV), 총변이계수 (total CV) 를계산하였다. 3. 직선성 (Linearity) 직선성은 CLSI EP6-A의지침에따라평가하였다 [2]. 기존에측정한고농도와저농도환자검체를 CLSI EP6-A 지침에따라 4:, 3:1, 2:2, 1:3, :4의비율로섞어 5가지농도를제조하여검사하였다. 제조사가제시한분석측정범위를고려하여저농도에서고농도까지 5가지농도를 4회반복측정하였다. 측정된결과로다항회귀분석을시행하여평가하였다. 4. 검출한계 (limit of detection, LoD) 설정검출한계는 CLSI EP17-A2 지침을참고하여설정하였다 [21]. 먼저공백한계 (limit of blank, LoB) 를설정하기위해검사시사용되는완충액 (ProCell buffer, Roche Diagnostics) 을 2회반복측정하였고, 측정값의평균과표준편차를구하여아래공식에따라 LoB를설정하였다. LoB =Mean blank (SD blank) 이때, Cobas e 61 장비에서는 NSE를제조사의검출한계인.5 ng/ml까지만표시하므로 NSE가.5 ng/ml로측정된경우에는 calibration 함수 (y =2883x+1897) 를이용하여그농도를추정하였다. 환자검체를희석하여제조사의검출한계 (limit of detection, LoD) 와유사한농도의물질을만들어 2회반복측정하였고, 아래공식에따라 LoD를설정하였다. LoD =LoB (SD low concentration sample) 5. 참고구간검증참고구간검증은 CLSI C28-A3 지침에따라시행하였다 [22]. 건강검진센터에서수집된정상인검체 2개를분석하여제조사의참고치와비교하였다. 전체 2개의검체중제조사의참고구간을벗어나는검체가 2개 (1%) 이하이면제조사의참고구간을사용할수있는것으로판단하였다 [22]. 6. 상관성 (Method comparison) 각검사항목에대하여 CLSI EP9-A2 지침을참고하여 44명의환자검체를대상으로평가하였다. Dream Gamma-1 counter (Sin- Jin Medics Co., Seoul, Korea) 장비를이용하여방사면역측정시약인 ELSA-NSE (Cis-Bio International, Gif/Yvette, France) 로측정한값을기준으로하였다. 각장비에서 2회반복측정한값의평균값으로회귀방정식과상관계수 (coefficient of correlation, r) 를산출하였다. 상관계수가.975 이상인경우상관성이있다고판단하였다. 7. 통계분석통계분석은 EP Evaluator Release 9 (David G. Rhoads Assoc., Kennett square, PA, USA) 과 Microsoft Excel 27 (Microsoft corporation, Redmond, WA, USA) 을사용하였다. 결과 1. 정밀도 PreciControl Tumor Marker 1과 PreciControl Tumor Marker 2 두농도에대해 Elecsys NSE의검사차례내변이계수는모두.8% 이었고, 총변이계수는각각 2.7%, 1.7% 였다 (Table 1). 64

3 2. 직선성 5가지농도의검체를 4회반복측정한결과를예상농도와비교하여선형회귀분석을시행하였다. 평가결과 ng/ml 범위에서결정계수 (coefficient of determination, R 2 ) 가.99 이상으로우수한직선성을보였다 (Fig. 1). 평가한결과, 선형회귀모델은 Fig. 2와같았으며, y =1.61x 수식을따랐다. 두장비간의상관계수는.9128로상관성평가기준인.975보다낮은값을보였다 고찰 3. 검출한계설정 공백검체와저농도검체를반복측정하여산출한 LoB 는.25 ng/ml 였으며, LoD 는.32 ng/ml 였다. 4. 참고구간검증 제조사에서제시한참고구간은 ng/ml 였다. 분석한 2 검체중 1 검체가제조사의참고구간을벗어났다. 참고구간을벗어 난검체수가전체검체수의 1% 미만이었으므로제조사의참고구 간을사용할수있는것으로판단하였다. 5. 상관성 44 개의검체로 Elecsys NSE 와 ELSA-NSE 분석법간의상관성을 Table 1. Precision profile of Elecsys NSE Analyte Level Mean (ng/ml) SD (ng/ml) CV (%) Withinrun Betweenrun Betweenday Total NSE Abbreviations: CV, coefficient of variation; NSE, neuron-specific enolase; SD, standard deviation. NSE의정밀도평가에서, The national academy of clinical biochemistry는종양표지자의검사내변이는 5%, 검사간변이는 1% 라는기준을제시하고있다 [23]. 최근발표된 NSE의생물학적변이에관한연구에서는최적의정밀도기준을 3.4% 로제안하였다 [24]. 본평가에서총변이계수는저농도검체에서 2.7%, 고농도검체에서 1.7% 로위기준을모두만족하므로우수한정밀도를보인다고판단할수있다. 직선성평가결과에서 ng/ml 범위에서결정계수가.99 이상으로우수한직선성을보였다. 제조사에서제시한분석측정범위는 ng/ml이다. 질병과 NSE 결과를비교한기존의연구에서, 폐암환자 222명의 NSE의농도가 -17 ng/ml 에이르며 [25], 급성뇌경색환자 3명을대상으로했을때 NSE 농도가 ng/ml [26], 지주막하출혈이있는 62명의환자를대상으로했을때 NSE 농도가 ng/ml에이른다는보고가있다 [27]. 이를통해볼때 NSE가증가하는질병상태라도 NSE가 Elecsys NSE의 AMR 상한이상으로증가하는경우는드문것으로생각된다. 따라서, 본연구에서직선성을평가한상기범위가임상적으로의미있는범위에포함된다고생각하여평가를진행하였다. 검출한계는.32 ng/ml로제조사가제공한분석측정범위의하한치보다 Scatter plot Residual plot :1 Line Fitted overall 15 1 Measured (ng/ml) 15 1 Residual (ng/ml) Assigned (ng/ml) Assigned (ng/ml) Fig. 1. Linear regression and residual plots of Elecsys NSE. The blue line represents the linear regression and the gray line depicts a theoretical line with a slope of 1. and a y-intercept of. 65

4 Scatter plot Bias plot 25 Regular Regr 1 1:1 Line 2 5 e61 (ng/ml) 15 1 Bias (ng/ml) RIA (ng/ml) RIA (ng/ml) Fig. 2. Method comparison between Elecsys NSE and ELSA-NSE. 낮았다. Woo 등 [28] 은한국성인 844명을대상으로 Elecsys NSE를이용하여 ModularAnlytics E17 장비로분석하였을때 NSE의참고범위는 ng/ml였다고보고하였다. 제조사가주장한참고범위는 16.3 ng/ml 미만으로한국인에서의참고범위와유의한차이가없다고판단하여제조사의참고범위를검증하였다. 분석결과제조사가제시한참고구간을벗어난검체는 5% 로제조사의참고구간인 16.3 ng/ml 미만을사용할수있다고판단하였다. 본연구에서 RIA 원리를사용한기준장비와 ECLIA 방법을사용한 Elecsys NSE의상관계수는.9128로장비간상관성평가기준인상관계수.975 미만이었다. NSE의검사실간및검사키트간낮은상관성은이미보고된바있다 [29]. Štern 등 [3] 의보고에따르면 NSE를측정하는데 RIA 방법을사용하는 Cis-Bio International, Immunotech (Beckman Coulter Company, Prague, Czech Republic), Diasorin (Stillwater, Minnesota, U.S.A), PerkinElmer Life and Analytical Sciences (Wallac Oy, Turku, Finland) 와수기효소면역측정법을사용하는 DRG Instruments GmbH (Marburg, Germany), ECLIA를사용하는 Roche, 면역형광법을사용하는 B.R.A.H.M.S. AG (Hennigsdorf/Berlin, Germany) 사의키트를비교한결과, RIA 원리를사용한키트간의상관계수는모두.979 이상인반면에, RIA와그외원리를이용한키트사이의상관계수는이보다낮은값을보였다 ( 범위 : ). Schmitt 등 [31] 은 ECLIA 방법을이용한 Roche사와 RIA 방법을이용한 Pharmacia 사의 NSE 검사사이의상관계수를.95로보고한바있다. 이를통해볼때, 검사원리의차이로인한낮은상관성은불가피한것으 로보인다. 상관계수가낮은또다른원인으로검사키트마다사용한단일클론항체의차이를생각해볼수있다. 즉, 키트별로사용한단일클론항체마다 NSE에대한친화력이다를수있다 [29, 3]. 또한, 단일클론항체에따라 γγ 아이소형에만부착할수도있고, γγ와 αγ 아이소형모두에부착할수도있다 [29]. 결론적으로 Elecsys NSE의수행능평가결과정밀도와직선성이우수하였으며제조사의참고범위와검출한계를만족하였다. Elecsys NSE는방사성동위원소를사용하지않으며, 민감한검사법이다. 본원에서는본연구를통한평가를바탕으로 NSE를원내도입할예정이며, 이로인해빠른시간내에임상의에게검사결과를제공할수있을것으로예상한다. 요약 배경 : 신경원특이에놀라제 (neuron specific enolase, NSE) 는신경세포와말초신경내분비세포에존재하며, 특히소세포폐암의표지자및뇌손상의예측인자로유용한것으로알려져있다. Elecsys NSE (Roche Diagnostics, Switzerland) 는 NSE를전기화학적발광면역법으로측정하는검사법으로, 본연구는이의수행능을평가하고자하였다. 방법 : CLSI 지침에따라 Elecsys NSE 검사법의정밀도, 직선성, 검출한계, 참고구간, 기존장비와의상관성을평가하였다. 정밀도평가를위해 Elecsys NSE의전용대조물질인 PreciControl Tumor Marker 1과 2 (Roche Diagnostics) 를사용하였고, 직선성평가를 66

5 위해고농도환자검체와 PreciControl Tumor Marker 1을사용하였다. 검출한계는완충액및희석한환자검체를사용하였다. 참고구간검증은정상인검체를사용하였다. 기존장비와의상관성평가는환자검체를사용하여 RIA 방법인 ELSA-NSE (Cis-Bio International, France) 와비교하였다. 결과 : NSE의총정밀도는저농도와고농도에서모두 3% 이내였고, ng/ml 범위에서직선성을나타내었다. 검출한계는.32 ng/ml였다. 참고구간은 ng/ml 로제조사가제시한구간을만족하였다. RIA와의상관성평가에서상관계수는.9128 이었다. 결론 : Elecsys NSE는 NSE 측정에있어정밀도, 직선성이우수하였고, 제조사의검출한계와참고구간을만족하였다. 다만, 기존 RIA 장비와의상관성이낮았는데, 이는검사키트마다서로다른단일클론항체를사용한것이원인일수있다. Elecsys NSE는방사성동위원소로인한위험이없으며, 민감도가높아일선검사실에서의 NSE 분석장비로써적합할것으로판단되었다. REFERENCES 1. Pancholi V. Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci 21;58: Tapia FJ, Polak JM, Barbosa AJ, Bloom SR, Marangos PJ, Dermody C, et al. Neuron-specific enolase is produced by neuroendocrine tumours. Lancet 1981;1: Moore BW. Chemistry and biology of two proteins, S-1 and , specific to the nervous system. Int Rev Neurobiol 1972;15: Marangos PJ, Polak JM, Pearse AGE. Neuron-specific enolase: a probe for neurons and neuroendocrine cells. Trends Neurosci 1982;5: Schmechel D, Marangos PJ, Brightman M. Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 1978;276: Kaltsas GA, Besser GM, Grossman AB. The diagnosis and medical management of advanced neuroendocrine tumors. Endocr Rev 24;25: Carney DN, Ihde DC, Cohen MH, Marangos PJ, Bunn PA Jr, Minna JD, et al. Serum neuron-specific enolase: a marker for disease extent and response to therapy of small-cell lung cancer. Lancet 1982;1: Ariyoshi Y, Kato K, Ishiguro Y, Ota K, Sato T, Suchi T. Evaluation of serum neuron-specific enolase as a tumor marker for carcinoma of the lung. Gan 1983;74: Akoun GM, Scarna HM, Milleron BJ, Bénichou MP, Herman DP. Serum neuron-specific enolase. A marker for disease extent and response to therapy for small-cell lung cancer. Chest 1985;87: Molina R, Holdenrieder S, Auge JM, Schalhorn A, Hatz R, Stieber P. Diagnostic relevance of circulating biomarkers in patients with lung cancer. Cancer Biomark 21;6: Ebert W, Hoppe M, Muley T, Drings P. Monitoring of therapy in inoperable lung cancer patients by measurement of CYFRA 21-1, TPA- TP CEA, and NSE. Anticancer Res 1997;17: Inoue S, Takahashi H, Kaneko K. The fluctuations of neuron-specific enolase (NSE) levels of cerebrospinal fluid during bacterial meningitis: The relationship between the fluctuations of NSE levels and neurological complications or outcome. Acta Paediatr Jpn 1994;36: Cunningham RT, Watt M, Winder J, McKinstry S, Lawson JT, Johnston CF, et al. Serum neurone-specific enolase as an indicator of stroke volume. Eur J Clin Invest 1996;26: Ondruschka B, Pohlers D, Sommer G, Schober K, Teupser D, Franke H, et al. S1B and NSE as useful postmortem biochemical markers of traumatic brain injury in autopsy cases. J Neurotrauma 213;3: Cunningham RT, Johnston CF, Irvine GB, McIlrath EM, McNeill A, Buchanan KD. Development of a radioimmunoassay for neurone specific enolase (NSE) and its application in the study of patients receiving intra hepatic arterial streptozotocin and floxuridine. Clin Chim Acta 199;189: Fu X, Meng M, Zhang Y, Yin Y, Zhang X, Xi R. Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum. Anal Chim Acta 212;722: Muley T, Ebert W, Stieber P, Raith H, Holdenrieder S, Nagel D, et al. Technical performance and diagnostic utility of the new Elecsys neuron-specific enolase enzyme immunoassay. Clin Chem Lab Med 23;41: Sterk M, Oenings A, Eymann E, Roos W. Development of a new automated enzyme immunoassay for the determination of neuron-specific enolase. Anticancer Res 1999;19: Clinical and Laboratory Standards Institute, eds. Evaluation of precision performance of quantitative measurement methods; approved guideline. CLSI document EP5-A2. 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standards Institute, ed. Evaluation of the Linearity of Quantitative Measurement Procedures: a Statistical Approach; Approved Guideline. CLSI document EP6-A. Wayne, PA: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standards Institute, ed. Evaluation of precision 67

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untitled ORIGINAL ARTICLE http://dx.doi.org/1.15263/jlmqa.215.37.2.11 Evaluation of Analytical Performance of the Cobas 8 Analyzer Series Module e62 Kiwoong Ko, Min-Jung Kwon, Hee-Yeon Woo, and Hyosoon Park Department