원저 Lab Med Online Vol. 2, No. 1: 10-14, January 임상화학 자동화학분석기에서의라텍스면역비탁법의 Autolab HbA 1c 평가 Evaluation of
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1 원저 Lab Med Online Vol. 2, No. 1: 10-14, January 2012 임상화학 자동화학분석기에서의라텍스면역비탁법의 Autolab HbA 1c 평가 Evaluation of HbA1c Levels Via the Latex Immunoturbidimetric Method by Using Chemistry Autoanalyzer 조용준 이소영 박해일 김영식 이제훈 김용구 한경자 Yongjun Jo, M.D., So-young Lee, M.D., Hae-il Park, M.D., YeongSic Kim, M.D., Jehoon Lee, M.D., Yonggoo Kim, M.D., Kyungja Han, M.D. 가톨릭대학교의과대학진단검사의학교실 Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea Background: Measurement of HbA1c levels is widely used to diagnose diabetes mellitus and to evaluate and monitor plasma-glucose concentrations over 6-8 weeks. In this study, we evaluated the diagnostic performance of the newly developed latex immunoturbidimetric method by using Autolab HbA1c. Methods: We analyzed and compared the diagnostic performance of Autolab HbA1c with that of Toshiba 200FR between April 2009 and July According to guidelines (EP5-A2, EP6-P, EP9-A2) of the clinical and laboratory standards institute (CLSI), we compared linearity, precision and correlation of Autolab HbA1c with those of G7 (Tosoh Corp., Kyoto, Japan) by using high-performance liquid chromatography (HPLC) method. Results: Data obtained using Autolab HbA1c showed good linearity in mixtures of samples with low (3.1%) and high (15.1%) levels of HbA1c (r 2 = ). In the analysis of within-run precision of the samples with HbA1c levels of 5.1% and 12.1%, the SDs were 0.04 and 0.06 and covariances of these samples were 0.8% and 0.5%, respectively. In the Deming regression model, the regression equation was as follows: Autolab HbA1c= Tosoh HPLC Conclusions: In this study, Autolab HbA1c method showed better performance characteristics than Tosoh G7 did. In reference review, there was no interference of variant hemoglobin. The data acquisition time of Autolab HbA1c was lower than that of Tosoh G7. The advantages of Autolab HbA1c are that it can be used as an autoanlyzer in routine chemical analysis, it does not require pre-analytical treatment, and the samples are automatically treated with distilled water for hemolysis. Key Words: HbA1c, Immunoturbidimetric method, HPLC 서론 당뇨병은대표적인만성질병으로 2010 년에전세계적으로 3 억여 Corresponding author: Jehoon Lee, M.D. Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea Yeouido St. Mary s Hospital, 62 Yeouido-dong Yeongdeungpo-gu, Seoul , Korea Tel: , Fax: , lyejh@catholic.ac.kr Received: December 10, 2010 Revision received: August 8, 2011 Accepted: August 9, 2011 This article is available from , 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. 명의환자가추산되며 2030년에는 4억 4천여명의환자가존재할것으로예상되는질환으로 [1], 한국에서도유병률이 2009년인구 10만명당 19.6 명으로조사되었고 [2] 2009년한국인의사망원인중 5위를차지하고있다 [3]. 당뇨병으로인한합병증과사망을줄이기위해서는엄격한혈당관리가필요하며 [4, 5] 관리의지표로 HbA 1c 나 fructosamine, 당화알부민등이사용되고있다. HbA 1c 는당화혈색소의일종으로혈색소의 β 사슬의 N-말단에발린 (valine) 에혈당이비가역적으로결합하여형성된것으로최근 6-8주간의혈당조절을반영하며최근미국당뇨학회 (American Diabetes Association) 에서당뇨의진단및고위험군선정기준에포함됐다 [6]. 이진단기준에의하면 National Glycohemoglobin Standardization Program (NGSP) 의인증을받고 Diabetes Control and Complications Trial (DCCT) 참고법으로표준화된검사법의결과가 6.5% 이상이면당뇨병으로진단되며 % 인경우당뇨병의 10 eissn
2 조용준외 : Evaluation of HbA 1c Latex Immunoturbidimetric Method 고위험군으로분류할수있다. HbA 1c 를측정하는방법으로는전하차이를통한방법 (ion-exchange chromatography, high-performance liquid chromatography-hplc, electrophoresis, isoelectric focusing), 구조의차이를통한방법 (affinity chromatography, immunoassay), 화학적분석방법 (photometry, spectrophotometry) 으로나눌수있으며 [7] 1995년에는친화크로마토그래피방법을많이사용하였으나 2007년조사에서는면역분석법과이온교환방식을 99% 이상사용하는것으로나타났다 [8]. 2009년에국내에서시행된대사질환검사신빙도조사에의하면 196기관중 84% 이상의기관에서면역분석법과 HPLC법을사용하고있는것으로조사되었다 [9]. 이러한분석방법의변화는임상검사실에서분석이편리한방법을선호하는것을나타낸다 [8]. 최근 IVD lab회사에서개발된 Autolab HbA 1c (IVD lab, Suwon, Korea) 는라텍스면역비탁법을사용하여 HbA 1c 를측정하는방법으로전용분석장비가필요하지않고자동화학분석기에적용이가능하며전처리과정이필요하지않으며용혈액으로증류수사용이가능하여검사단가가낮아지는장점을가지고있다. HPLC 방법인 Tosoh G7 (Tosoh Corp., Kyoto, Japan) 간의결과를비교하여 Autolab HbA 1c 의분석능력과임상검사실에서의유용성을평가하고자하였다. 대상및방법 1. 대상 2009년 4월부터 7월까지본원검사실에 HbA 1c 검사가의뢰된환자의 EDTA 항응고처리된전혈검체를이용하여직선성, 상관성, 검체침강에따른영향을평가하였다. 채혈한지일주일이내의신선한검체만을사용하였으며평가이외에는 4 의냉장고에보관하였다. 2. 검사법 1) Autolab HbA 1c Autolab HbA 1c 는전혈에서 HbA 1c 를직접적으로측정하는방법으로라텍스면역비탁법을이용한다. 전용장비가필요하지않으며자동화학분석기에서사용이가능하다. 자동화학분석기에항응고제로처리된전혈검체를장착하면검체흡입후증류수로용혈한다. 제1반응을통하여헤모글로빈과 HbA 1c 가라텍스입자와균일한비율로반응하게되며제2반응을통하여 mouse anti human HbA 1c 단클론항체와 anti-mouse IgG 다클론항체복합체가라텍스복합체 HbA 1c 와반응한다. 응집의정도는라텍스에흡착된 HbA 1c 의농도에비례하고이때의흡광도를 660 nm의파장으로측정하여 % 결과값을얻는다. Autolab HbA 1c 는 2010년 10월 1일에 NGSP 로부터 DCCT에대한표준화된방법으로인증을받았으며제조사 가밝힌직선성은 % 이다. 2) Tosoh G7 Tosoh G7은이온교환 HPLC를기본원리로하여 HbA 1c 를포함한혈색소분획을분석하는장비로전혈검체가기기에장착되면장비에서검체를흡입하여용혈희석후분석하게된다. 칼럼을통과하면서여러혈색소성분들이분리되어 415 nm에서의흡광도를측정하여 HbA 1c 의 % 값을계산하게된다. 3. 평가방법 Autolab HbA 1c 의직선성, 정밀도, 상관성은본원에서사용하는 TBA-200FR (Toshiba Medical Systems, Tokyo, Japan) 으로측정하여평가하였고혈액침강에따른변화는 Hitachi 7600 (Hitachi, Tokyo, Japan) 에서 2009년 4월부터 7월까지진행하였다. Clinical and Laboratory Standard Institute (CLSI) 가이드라인 (EP5-A2, EP6-A, EP9-A2) 을참조하여정밀도, 직선성, 상관성을분석하였고, 정도관리물질은시약에동봉된저농도와고농도의정도관리물질을사용하였다. 1) 직선성직선성평가는 CLSI EP06-A의지침에따라실시하였다 [10]. 저농도 (3.1%), 고농도 (15.1%) 검체를희석하여 5가지단계의농도물질 (3.1%, 6.1%, 9.1%, 12.1%, 15.1%) 을제조한후 TBA-200FR에각각 2 회측정하였다. StatisPro v (Analyse-it Software Ltd., Leeds, UK) 으로검증을하였고회귀선이일차선형이고유의한적합성결여가없는경우직선성이있다고판정하였다. 2) 정밀도제조사에서제공되는저농도와고농도정도관리물질을 CLSI EP05-A2를일부변형하여 2가지정도관리물질을하루 2회, 1회에 2번씩총 20일간측정하여검사일내정밀도와검사일간정밀도를시행하였다 [11]. 오전 / 오후에나눠서측정하였으며 1회의검사후에는최소 30분이상의간격을두고시행하였다. 3) 상관성 CLSI EP09-A2 지침에따라 Tosoh G7으로측정된 HbA 1c 의값이 % 인검체 40개를선정하여 HPLC인 Tosoh G7방식과 TBA- 200FR에 Autolab HbA 1c 을사용하여 2회씩측정한후데밍회귀분석을시행하였다 [12]. 4) 혈액침강과 HbA 1c 값의변화측정검사대기시간에따른영향을파악하기위해서 Tosoh G7에서 11
3 조용준외 : Evaluation of HbA 1c Levels Via the Latex Immunoturbidimetric Method by Using Chemistry Autoanalyzer % 의결과값을나타내는 48개검체를대상으로하여 Hitachi 7600에서측정하였다. 측정방식은검체를잘섞은후측정한값을기준으로한후안정화시간을 25분씩증가시키며측정하였다. 측정하기전에검체를흔들어섞어주었다. 5.1% 검체의경우각각표준편차는 0.04, 0.08이었고변이계수는 0.8%, 1.6% 이었다. 제조사에서제시한검사일내정밀도의표준편차와변이계수는각각 4.76% 의농도에서 0.06와 1.26%, 7.29% 농도에서 0.08과 1.1%, 10.9% 농도에서 0.16 과 1.47% 이었다. 4. 분석도구 StatisPro v (Analyse-it Software Ltd., Leeds, UK) 와 Med- Calc v (MedCalc software, Broekstraat, Belgium), Excel 2007 (Microsoft Corporation, WA, USA) 을이용하였다. 1. 직선성 결과 Autolab HbA 1c 는저농도 (3.1%) 와고농도 (15.1%) 의검체를이용 하여 5 단계의농도를분석한결과 r 2 = 의높은직선성을나 타내었고유의한적합성결여가관찰되지않았다 (Fig. 1). 2. 정밀도 농도 12.1% 검체의경우검사일내정밀도와검사일간정밀도의 표준편차는각각 0.06, 0.12 였고변이계수는 0.5%, 1.0% 였다. 농도 HbA1c (%) y=0.995x R 2 = Expected value (%) Fig. 1. Linearity of HbA1c values as measured by Autolab HbA1c. 3. 상관성 비교식은 Autolab HbA 1c = Tosoh G 로나타났 다 (Fig. 2). 기울기와절편의 95% 구간은각각 , 이었다. Tosoh G7 값이 6.4% 미만의경우 0.26 의음의바이 어스가, 6.4% 이상 7.6% 미만에서는 0.05 의음의바이어스, 7.6% 이 상의농도에서 0.07 의양의바이어스가관찰되었다 (Fig. 3). 4. 혈액침강과 HbA 1c 와의관계 총 48 검체를검사하였으며한검체는재검한결과를반영하였다. 처음결과와비교하여최대 ±0.2% 의차이를보였으며평균적으로 Difference (%) Mean TBA-200FR-Mean Tosoh G Mean Tosoh G7 (%) Fig. 3. Difference plot between Autolab HbA1c and Tosoh G7. 11 Average of Autolab and Autolab Average of Tosoh G7 and Tosoh_G min 25 min 50 min 75 min Fig. 2. Deming regression plot of Tosoh G7 vs. Autolab HbA1c. Fig. 4. Changes in HbA1c levels with the length of time after the samples were mixed. 12
4 조용준외 : Evaluation of HbA 1c Latex Immunoturbidimetric Method 25분경과시 0.008%, 50분경과시 0.037%, 75분경과시 0.037% 낮게측정되었다 (Fig. 4). 고찰 HbA 1c 는 6-8주간의혈당을반영하고당뇨병의경과와진단, 고위험군분류에사용되고있다 [6, 13]. HbA 1c 를측정하는여러방법들이존재하지만방식과제조회사에따라서측정된결과의차이를보인다 [14]. 이를해결하기위해 2001년 7월에 International Federation of Clinical Chemistry (IFCC) 에의해표준화된방식이도입되었다. NGSP방식은 % 로, IFCC방식은 mmol/mol 으로결과가다르게표현되는데 IFCC방식의경우 DCCT에서사용된방법보다 1.5-2% 정도낮게측정된다 [15]. 이러한차이때문에 IFCC 결과수치를 NGSP방식으로보고할때는 NGSP = [ IFCC] 의식에의해변환해주어야하며최근의 consensus에의하면두가지방식의결과를동시에보고하도록권장하고있다 [16]. 그러나 IFCC의표준방식은매우특이적이고분석시간이오래걸리며기술적으로복잡하고, 검사비용이비싸므로일반임상분석에서는사용하기어렵고여러검사법에대한기준치로사용하고있다. Autolab HbA 1c 는직선성, 정밀도, 상관성에서좋은수행능력을보였다. Tosoh G7과비교하여농도에따른바이어스가다른것으로나타났지만변환식을통해서교정이가능한것으로생각된다. 검체침강시간에따른평가결과에의하면침강시간에따라 HbA 1c 농도의감소가관찰되지만 75분후에측정하여도 0.05% 이내로임상적해석에서는무시할수있는수준으로생각된다 [13]. HbA 1c 를측정함에있어서중요한변수중의하나는변이형혈색소에따른 HbA 1c 결과의차이이다. 본연구에서변이형혈색소에의한간섭은평가하지못했지만 HbA 1c 측정방식과변이형혈색소에따른간섭을고찰한논문에따르면같은분석방식에서도측정기기에따른간섭이다른것으로나타났다 [17]. 본연구에서는평가한 Autolab HbA 1c 와유사한라텍스면역비탁법을사용하는 [18] Hitachi Tina Quant 방식은 HbAS, HbAC, HbAE, HbAD에의한간섭이없는것으로조사되었는데이는 Autolab HbA 1c 방식이 HbA 1c 의 N-말단의당화아미노산염기에특이적인항체를사용하기때문으로생각되나전세계적으로흔한 HbS, HbE, HbC, HbD뿐만아니라 [17] 한국인에게흔한 HbG Coushatta, Hb Queens에대한평가가필요할것으로생각된다 [19]. Autolab HbA 1c 는기존에널리사용되고있는 Tosoh G7와비교하여좋은수행능력을나타냈고용혈과정에서증류수를사용할수있고검체마개제거과정이필요하지않으며전용분석장비를필요치않고일반자동화학분석장비에이용이가능하다. 따라서관련시약구입비용이감소하게되며검사방법이용이하다. 다만 Tosoh G7에서는 10검체를검사하는데 13분정도소요되는데 Autolab HbA 1c 는 15분정도소요되어결과획득시간이지연되는것으로나타났다. 그리고검체침강으로인해혈장이일부분리되어장비가검체를채취할때오류가발생할수있기때문에분석전에잘섞은후검사를시행해야검사대기시간에따른오류를줄일수있을것으로생각된다. 요약 배경 : HbA 1c 의측정은최근 6-8주간의혈당조절을평가하고당뇨를진단하기위해사용되고있다. 최근라텍스면역비탁법을사용하는 HbA 1c 측정법이개발되어분석능력과임상검사실에서의유용성을평가하였다. 방법 : 2009년 4월부터 7월까지 TBA-200FR에 Autolab HbA 1c 을장착하여 CLSI guideline (EP5-A2, EP6-P, EP9-A2) 에따라직선성, 정밀도, 상관성을평가하였다. 결과 : Autolab HbA 1c 는고농도 (15.1%) 와저농도 (3.1%) 의검체의혼합에서 r 2 = 의높은직선성을보였다. 12.1% 의경우검사일내정밀도와검사일간정밀도의표준편차는각각 0.06, 0.12였고변이계수는 0.5%, 1.0% 였다. 5.1% 의경우각각표준편차는 0.04, 0.09이었고변이계수는 0.8%, 1.6% 이었다. 비교식은 Autolab HbA 1c = HPLC 이었다. 결론 : 본연구에서 Autolab HbA 1c 는 Tosoh G7과비교하여좋은수행능력을보였다. 문헌고찰에서변이형혈색소에따른간섭은없는것으로알려졌다. Tosoh G7에비해결과획득시간은지연되는단점이있지만 Autolab HbA 1c 의장점은일반화학분석기에적용이가능하고검체의전처리가필요없으며증류수를사용하여자동적으로용혈과정이이루어진다는점이다. 감사의글 본연구는지식경제부산업원천기술개발사업의지원으로이루어졌습니다. 참고문헌 1. International diabetes federation (Eds), Global burden: prevalence and projections and (Updated on Nov 2010). 2. Statistics Korea (Eds), Chronic disease statistics of Korea. index.go.kr/egams/stts/jsp/potal/stts/po_stts_idxmain.jsp?idx_cd= 1438 (Updated on May 2011). 13
5 조용준외 : Evaluation of HbA 1c Levels Via the Latex Immunoturbidimetric Method by Using Chemistry Autoanalyzer 3. Statistics Korea. Cause of death statistics of Korea. portal/korea/kor_nw/2/6/1/index.board?bmode=read&bseq=&aseq= &pageNo=1&rowNum=10&navCount=10&currPg=&sTarget=titl e&stxt (Updated on Nov 2010). 4. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329: UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998;352: American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2011;34(S1):S Burtis CA, Ashwood ER, et al. eds. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. Washington, D.C: Saunders, 2006: Molinaro RJ. Targeting HbA 1c: standardization and clinical laboratory measurement. MLO Med Lab Obs 2008;40:10-4, Song J, Kwon KC, Kim JH, Kim JW, Min WK, Lee SY, et al. Annual report on external quality assessment in metabolic disorders in Korea (2009). J Lab Med Qual Assur 2010;32: Clinical and Laboratory Standards Institute. Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline. Document EP6-A. Wayne, PA: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline. 2nd ed. Document EP5-A2. Wayne, PA: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standards Institute. Method comparison and bias estimation using patient samples; approved guideline. 2nd ed. Document EP9-A2. Wayne, PA: Clinical and Laboratory Standards Institute, Little RR and Sacks DB. HbA 1c: how do we measure it and what does it mean? Curr Opin Endocrinol Diabetes Obes 2009;16: Caragher TE, Dohnal JC, Lomont ME. Cautionary note regarding HbA 1c methods predicting the clinical status of diabetic patients. Diabetes Care 2000;23: Kilpatrick ES. HbA 1c measurement. J Clin Pathol 2004;57: Hanas R, John G, International HbA 1c Consensus Committee consensus statement on the worldwide standardization of the hemoglobin A1C measurement. Diabetes Care 2010;33: Little RR and Roberts WL. A review of variant hemoglobins interfering with hemoglobin A1c measurement. J Diabetes Sci Technol 2009;3: Jones TG, Warber KD, Roberts BD. Analysis of hemoglobin A1c from dried blood spot samples with the Tina-quantR II immunoturbidimetric method. J Diabetes Sci Technol 2010;4: Lee ST, Kim MS, Choi DY, Kim SK, Ki CS. Incidence of variant hemoglobin (Hb) and increased fetal Hb concentrations and their effect on HbA 1c measurement in a Korean population. Clin Chem 2006;52:
1. Korea Centers for Disease Control and Prevention. The fifth Korea National Health and Nutrition Examination Survey (KNHANES V-1) 2010. Cheongwon: Korea Centers for Disease Control and Prevention; 2012.
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