ORIGINAL ARTICLE Performance Evaluation of Gentian Cystatin C on the Hitachi 7600 Automatic Analyzer Yun-A Jo 1, Mi Kyung Shin 2, Wonkeun Song 1, Han-

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1 ORIGINAL ARTICLE Performance Evaluation of Gentian Cystatin C on the Hitachi 7600 Automatic Analyzer Yun-A Jo 1, Mi Kyung Shin 2, Wonkeun Song 1, Han-Sung Kim 1, and Min-Jeong Park 1 1 Department of Laboratory Medicine, Hallym University College of Medicine, Chuncheon; 2 Department of Laboratory Medicine, Hallym University Kangnam Sacred Heart Hospital, Seoul, Korea Corresponding author: Min-Jeong Park Department of Laboratory Medicine, Hallym University Kangnam Sacred Heart Hospital, 1 Singil-ro, Yeongdeungpo-gu, Seoul , Korea Tel: Fax: mjpark@hallym.or.kr Background: Cystatin C, a 13-kDa protein synthesized in all nucleated cells, has been proposed as a replacement for serum creatinine in assessments of renal function. The Gentian Cystatin C immunoassay (Gentian, Norway) was recently developed using particle enhanced turbidimetric immunoassay. In this study, we evaluated the analytical performance of the Gentian Cystatin C immunoassay on the Hitachi 7600 Automatic Analyzer (Hitachi Ltd., Japan). Methods: We performed precision and linearity studies using Hitachi Clinical Analyzer 7600 with Gentian reagent and compared the results to those obtained with the N Latex Cystatin C (Siemens, Germany) using a particle enhanced nephelometric immunoassay method performed on the Behring Nephelometer II (Siemens, Germany). We also analyzed the traceability of Gentian reagent and Siemens reagent to Cystatin C standard reference material, ERM-DA471/IFCC. Results: The coefficient of variations (CVs) for within-run imprecision at low and high levels were 1.58% and 1.06% and the CVs for total imprecision at low and high levels were 2.53% and 2.09%, respectively. In the linearity test, the coefficient of determination (R 2 ) was (range, 0.23 to 7.50 mg/l), and comparison with the results obtained by Siemens reagent showed an excellent correlation coefficient of In the traceability test, Gentian reagent is more accurate than Siemens reagent and the total accuracy was 96.0%. Conclusions: Gentian reagent provides good analytic performance on the Hitachi 7600 Automatic Analyzer and can be used for the diagnosis, treatment, monitoring, and risk assessment of renal function. ( J Lab Med Qual Assur 2013;35:81-6) Key Words: Cystatin C, Creatinine, Traceability pissn: X eissn: Received September 26, 2013, Revision received November 1, 2013, Accepted November 4, 2013 서론 사구체여과율 (glomerular filtration rate, GFR) 은신장이일정시간특정물질을제거할수있는혈장량으로정의되며기능을하고있는사구체수를볼수있는가장좋은방법으로신기능의단계를구분할수있는가장좋은지표로이용되었다 [1]. GFR 측정의참고방법은 inulin 또는 51 Cr-EDTA, 99m TC-labeled diethylenetriamine pentaacetic acid, 125 I-labeled iothalamate 와같은방사성표지자등외부표지자를주입후제거율을측정하는방법이사용되지만환자에게표지약제를주입해야하고일정시간매우표준화된상태에서소변을모아측정해야하는단점이있어일반검사실에서시행하기는어렵다. 따라서내부표지자를이용하여 GFR을측정하는방법이사용되고있 으며그중혈청크레아티닌이일반적으로가장많이사용되고있다 [2]. 크레아티닌은크레아틴과근육조직의 phosphocreatine 의대사산물로혈중농도가일정하며, 사구체를자유롭게통과하고근위세뇨관에서재흡수되지않아내부표지자로적합하지만몇가지단점을가지고있다 [3]. 크레아티닌의혈중량은체근육량, 나이, 성별, 식이등에영향을받고측정하는방식에따라서혈당, 요산, 케톤, cephalosporin 등간섭인자에의한영향을받는다 [4]. 또한크레아티닌의혈중량과 GFR은비직선형관계를보여초기 GFR 감소를검출하는데있어민감도가떨어진다 [5]. Cystatin C (Cys C) 는 13 kda의저분자량의 protease inhibitor 로사구체기저막을자유롭게통과하여근위세뇨관에서흡수되나세뇨관상피세포에서대사되어체내혈중 Copyright 2013 The Korean Association of Quality Assurance for Clinical Laboratory 81

2 으로돌아가지않아혈중 Cys C 농도는 GFR에만의존하는특징을가지고있어 GFR 측정의새로운내부표지자로제안되었다 [6]. 또한 Cys C의혈중량은체근육량, 나이, 성별에영향을받지않고초기신손상의검출에크레아티닌에비교해민감도가높아초기신손상의선별검사로임상적유용성이더욱부각되고있다 [7]. Cys C를측정하는방법은 particle enhanced nephelometric immunoassay (PENIA) 법과 particle enhanced turbidimetric immunoassay (PETIA) 법이있다 [8]. 최근에출시된 Gentian Cystatin C immunoassay (Gentian, Moss, Norway) 은 PETIA법을이용하여 Cys C 를측정하는 kit로이에대한분석능력및유용성에대한평가는국내에아직보고된바없다. 이연구에서는연구에서는 Clinical and Laboratory Standards Institute (CLSI) 지침에따라 Gentian Cystatin C immunoassay (Gentian) 의분석능력및 PENIA법과상관성을평가하고자하였다. 대상및방법 1. 장비및시약 Cys C를 Gentian Cystatin C immunoassay 와 Hitachi 7600 Automatic Analyzer (Hitachi Ltd., Tokyo, Japan) 를이용하여분석하였다. 비교분석을위하여기존에강남성심병원에서사용하는 N Latex Cystatin C (Siemens, Marburg, Germany) 를 Behring Nephelometer II (Siemens) 장비를이용한 PENIA법으로 Cys C를측정하였다. 검사는각제조회사의지침에따라시행되었다. 2. 방법 1) 정밀도 (precision) CLSI EP5-A [9] 에따라 20일에걸쳐오전과오후매검사마다높은농도와낮은농도의 Gentian Cystatin C Control (Gentian) 을 2회반복측정하였다. 평균 (mean), 검사간변이계수 (between-run coefficient of variation [CV]), 검사일간변이계수 (between-day CV) 및총정밀도를구하였다. Gentian Cystatin C Control의제조사의목표값범위 (insert target range) 는낮은농도물질은 mg/l였고, 높은농도물질은 mg/l 였다. 2) 직선성 (linearity) 직선성은 CLSI EP6-A [10] 에따라시행하였다. Gentian Cystatin C Calibrator (Gentian) 의 level 1 (target value: 0.23 mg/l) 과 level 6 (target value: 7.50 mg/l) 을사용하여 4:0 ( 계산치 : 0.23 mg/l), 3:1 (2.05 mg/l), 2:2 (3.87 mg/l), 1:3 (5.68 mg/l), 0:4 (7.50 mg/l) 의비율로혼합하였다. 5가지단계의농도를제조한후각각 2 회반복측정한뒤계산치와측정치간의회귀방정식과결정계수 (R 2 ) 를구하였다. 3) 상관성 (correlation) 상관성평가는 CLSI EP9-A2 [11] 에따라시행하였다. 강남성심병원외래, 건강검진, 입원환자의검체를기존 Behring Nephelometer II로측정된값을기준으로 Cys C값이 0.51 mg/l에서 5.10 mg/l에걸쳐고르게분포하도록 52검체를선별하였다. Hitachi 7600 Automatic Analyzer 와 Behring Nephelometer II로 2시간안에 2회반복측정하여단순선형회귀분석으로상관성을평가하였다. 4) 소급성 (traceability) 소급성평가는 Cys C의표준물질인 ERM-DA471/IFCC (Cystatin C Standard Reference Meterial) 을사용하였다. ERM-DA471은동결건조시킨사람혈청으로구성되어있으며그안에 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, sodiumazide, benzamidine chloride, aproinin이첨가되어있다. Target값은 5.48± 0.15 mg/l이며 PETIA, PENIA법에그리고방사면역확장법에이용가능하다. 희석용매로원액, 2배, 3배, 5배, 10 배로희석후 2회반복측정하였다 mg/l까지 5개농도의검체에대해기존시약인 Siemens와평가하려는시약 Gentian을이용하여각농도에서결과값을측정하여측정평균치, 표준물질과의 bias, 정확도 (100- difference) 를구하였다. 5) 통계분석통계분석은 Excel 2007 (Microsoft Co., Redmond, WA, USA) 과 MedCalc ver (Medcalc software, Mariakerke, Belgium) 프로그램을사용하였다. 결과 1. 정밀도 (precision) 검사내, 검사간변이계수및검사일간변이계수는저농 82 J Lab Med Qual Assur 2013;35:

3 Table 1. Precision of Cystatin C measurement on the Hitachi 7600 Automatic Analyzer with Gentian Cystatin C immunoassay Level Mean (mg/l) Total SD (mg/l) Within-run CV Between-run CV Between-day CV Total CV Low High Abbreviation: CV, coefficient of variation. 4. 소급성 (traceability) 표준물질을사용해 5개농도의검체에대해기존시약인 Siemens 와평가하려는시약 Gentian 을이용하여각농도에서결과값을측정하여측정평균치, 표준물질과의 bias, 정확도를평가하였으며 Gentian 시약에서 0.56 mg/l의저농도에서정확도 90.2% 였고나머지농도에서정확도는 95% 이상이었다 (Table 2). 고찰 Fig. 1. Linearity of Gentian Cystatin C values on the Hitachi 7600 Automatic Analyzer. 도와고농도에서모두 2% 이내였으며총정밀도의변이계 수는저농도에서 2.53%, 고농도에서 2.09% 였다 (Table 1). 2. 직선성 (linearity) Gentian Cystatin C Calibrator 의 level 1 과 level 6 물 질로제조한 5 가지농도의검체를 2 회반복측정한결과 mg/l 의 Cys C 범위에서 R 2 = 의직선성 을보였다 (Fig. 1). 3. 상관성 (correlation) 기준장비인 Behring Nephelometer II 와비교장비인 Hitachi 7600 Automatic Analyzer 의 Cys C 측정치는각 각 mg/l 와 mg/l 에서분포하였다. 상관계수 R 2 =0.9982, 기울기 , 절편 으로높 은상관관계를보였고, 평균오차는 이었다 (Fig. 2). 혈청크레아티닌은신기능을평가하는데가장널리사용되는지표이지만나이, 성별, 약물, 체근육량, 식이등에의해영향을받아 GFR을직접적으로반영하지는못한다 [1,2,7,12]. 이러한제약을극복하기위해 Cockcroft- Gault 공식이나 Modification of Diet in Renal Disease 공식을권장하고있고이공식에의해도출된 GFR은 chronic kidney disease (CKD) 의분류와진단에이용되고있다 [12]. 하지만혈청크레아티닌을기초로한공식에서도출된 GFR의경우몇가지제한점이있는데 CKD 환자이면서다른질환을가지고있거나노인, 비만, 그리고초기신손상인경우가대표적이다. GFR은 40세이후 10년에 8-10 ml/min씩저하되며 70 세이상에서는혈청크레아티닌의증가없이 GFR 계산값이감소하는경우가 45% 에달한다 [13]. 또한당뇨병환자군에서혈청크레아티닌이정상이지만신장기능의저하가발생한환자를조기에찾아내는것이중요한데크레아티닌으로도출된 GFR을이용할경우 70 ml/min/1.73 m 2 이하로감소될때까지혈중량에변화가없어초기신손상이있는당뇨병환자를잘찾아낼수없다 [14]. Cys C는혈청크레아티닌에비해신기능을측정하는데더믿을만한지표로알려져왔으며특히초기신손상의더좋은지표로보고되고있다 [1,2,7]. Cys C는 1979년처음으로경쟁적방사면역측정법을이용하여측정된이후형광면역법, 효소면역법등으로개발이되어왔다 [1]. 더욱최근에는 Cys C 특이항체로코팅된라텍스나폴리스티렌입 J Lab Med Qual Assur 2013;35:

4 Fig. 2. Comparison of Cystatin C levels measured by Gentian Cystatin C Immunoassay (Gentian) on a Hitachi 7600 Automatic Analyzer and N Latex Cystatin C (Siemens) on a Behring Nephelometer II. Table 2. Traceability of Gentian Cystatin C Immunoassay and N Latex Cystatin C with ERM-DA471/IFCC (Cystatin C standard reference material) Cystatin C standard reference material (mg/l) Gentian Siemens Mean Bias Accuracy Mean Bias Accuracy Total 자를이용하여측정하는방식이개발되어혼탁법을사용하는 PETIA와비탁법을사용하는 PENIA법이있이있으며이두방법은초기개발된방법들에비해더정확하다고알려져있다 [1]. Cys C의초기참고범위의연구에서는그범위가다양했었고원인분석결과 calibration 물질, 참여자의나이그리고측정방법등에의해차이를보인것으로생각되었으며그중 PETIA법으로측정된값은 PENIA법으로측정된값에비해 20-30% 높은값을보인다는연구들이있었다 [15,16]. 또한최근까지도 Cys C 측정의표준화가되어있지않았으며 2010년 International Federation of Clinical Chemistry (IFCC) 에서 Cys C의참고물질 (ERM- DA471/IFCC) 을생산하여 Cys C의표준화연구가진행되 고있다 [16]. 본연구에서는 PETIA법으로측정되는 Gentian시약의분석능을평가하고 PENIA법으로측정되는 Siemens사시약과의상관성을평가하였다. 정밀도검사에서 Gentian 시약의검사내정밀도의변이계수는저농도에서 1.58%, 고농도에서 1.06% 였으며, 총정밀도의변이계수는저농도에서 2.53%, 고농도에서 2.09% 로우수하였다. 직선성검사에서도 R 2 =0.99로높은직선성을보였다. 기존시약인 PENIA 법을이용한 Siemens 사시약과의상관성비교를시행한결과, 높은상관관계를보였으나 (R 2 =0.9982), 평균오차는 0.244로기존연구의결과와유사한값을보였다 [15,16]. 소급성검사는표준물질인 ERM-DA471/IFCC을사용하 84 J Lab Med Qual Assur 2013;35:

5 여시행하였다. Gentian Cystatin C시약은모든농도에서 Siemens 사시약보다높은정확도를보였으며총정확도는 96.0% 로 Siemens사시약의 87.2% 보다정확한값을보였다. 혈청 Cys C는신기능을평가하기위한이상적인지표로서조건을가졌지만크레아티닌보다측정하는데비용이더많이들고아직까지정확한표준화가이루어지지않았다는단점이있었다. 본연구에서는최근제시된표준물질로혈청 Cys C의정확도를평가해보았으며, 이러한시도가 Cys C의표준화에도움을줄수있는결과로생각된다. 결론적으로, Hitachi 7600 Automatic Analyzer 를이용한 Gentian Cystatin C immunoassay 는 PETIA법을이용한 Cys C 측정시약으로서정밀도와직선성이우수하였고 PENIA법을이용한 Siemens 사시약과비교하여상관성이높았다. 또한표준물질을통해평가한정확도가높아임상검사실에서더욱유용하게사용될수있을것이다. REFERENCES 1. Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin Chem 2002;48: Hoek FJ, Kemperman FA, Krediet RT. A comparison between cystatin C, plasma creatinine and the Cockcroft and Gault formula for the estimation of glomerular filtration rate. Nephrol Dial Transplant 2003;18: Heymsfield SB, Arteaga C, McManus C, Smith J, Moffitt S. Measurement of muscle mass in humans: validity of the 24-hour urinary creatinine method. Am J Clin Nutr 1983;37: Spencer K. Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin Biochem 1986;23(Pt 1): Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992;38: Coll E, Botey A, Alvarez L, Poch E, Quinto L, Saurina A, et al. Serum cystatin C as a new marker for noninvasive estimation of glomerular filtration rate and as a marker for early renal impairment. Am J Kidney Dis 2000;36: Ha JS, Ryoo NH, Kim JR, Jun DS, Kim HC, Kim YJ. Cystatin C as a marker for early renal impairment. Korean J Lab Med 2004;24: Finney H, Newman DJ, Gruber W, Merle P, Price CP. Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems (BNA, BN II). Clin Chem 1997;43(6 Pt 1): Clinical and Laboratory Standards Institute. Evaluation precision performance of quantitative measurement methods: approved guideline. 2nd ed. Wayne: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standard Institute. Evaluation of the linearity of quantitative measurement procedures: a statistical approach: approved guideline. 2nd ed. Wayne: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standard Institute. Method comparison and bias estimation using patient samples: approved guideline. 2nd ed. Wayne: Clinical and Laboratory Standards Institute, Ta g l ieri N, Ko en ig W, Kaski J C. Cystatin C and cardiovascular risk. Clin Chem 2009;55: Delaney MP, Price CP, Newman DJ, Lamb E. Kidney disease. In: Burtis CA, Ashwood ER, Bruns DE, eds. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. St. Louis: Elsevier Saunders, 2006: Newman DJ, Thakkar H, Edwards RG, Wilkie M, White T, Grubb AO, et al. Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine. Kidney Int 1995;47: Uhlmann EJ, Hock KG, Issitt C, Sneeringer MR, Cervelli DR, Gorman RT, et al. Reference intervals for plasma cystatin C in healthy volunteers and renal patients, as measured by the Dade Behring BN II System, and correlation with creatinine. Clin Chem 2001;47: Voskoboev NV, Larson TS, Rule AD, Lieske JC. Importance of cystatin C assay standardization. Clin Chem 2011;57: J Lab Med Qual Assur 2013;35:

6 Hitachi 7600 자동분석기를이용한 Gentian Cystatin C 의분석능평가조윤아 1 신미경 2 송원근 1 김한성 1 박민정 1 1 한림대학교의과대학진단검사의학교실, 2 한림대학교강남성심병원진단검사의학과 배경 : Cystatin C (Cys C) 는 13 kda 의분자량의모든유핵세포에서생성되는단백질로신기능평가에혈청크레아티닌을대체할지표로제시되고있다. 최근에 particle enhanced turbidimetric immunoassay 법을이용해 Cys C 를측정하는 Gentian Cystatin C immunoassay (Gentian, Norway) 가개발되었다. 본연구에서는 Hitachi 7600 Automatic Analyzer (Hitachi Ltd, Japan) 에서 Gentian 시약을사용하여 Cys C 의측정수행능을평가하였다. 방법 : Hitachi 7600 Automatic Analyzer 에서 Gentian 시약의정밀도, 직선성을평가하였고 particle enhanced nephelometric immunoassay 법으로 Behring Nephelometer II (Siemens, Germany) 에서측정되는 N Latex Cystatin C (Siemens) 와의상관성을평가하였다. 또한 Cys C 표준물질인 ERM- DA471/IFCC 을사용하여 Gentian Cystatin C 와 Siemens 사시약의소급성을분석하였다. 결과 : 검사내정밀도의변이계수는저농도와고농도에서 1.58% 와 1.06% 였고, 총정밀도의변이계수는저농도와고농도에서 2.53% 와 2.09% 였다. 직선성은 mg/l 범위에서 R 2 이 이었고, 상관성검사에서 Gentian 시약과 Siemens 사시약간의상관계수는 로좋은상관관계를보였다. 소급성검사에서는총정확도가 96.0% 로 Gentian 시약이 Siemens 사시약보다더정확하였다. 결론 : Gentian Cystatin C immunoassay 는 Cys C 측정에서우수한분석능을보였고기존의 Siemens 시약과도우수한상관성을보였으므로, Cys C 를이용한신기능평가에유용하게사용될수있을것으로생각된다. (J Lab Med Qual Assur 2013;35:81-6) 교신저자 : 박민정우 ) 서울시영등포구신길로 1, 한림대학교강남성심병원진단검사의학과 Tel: 02) Fax: 02) mjpark@hallym.or.kr 86 J Lab Med Qual Assur 2013;35:

012임수진

012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar