대한임상검사학회지 : 41권제1호, 37-41, 2009 ISSN 1738-3544 당화혈색소측정을위한 Norudia R HbA1c 키트의평가 주성대학임상병리과 1, 충북대학병원병리과 2 홍승복 1 김은중 2 Evaluation of Norudia R HbA1c Kit for Glycohemoglobin Assay Seung-Bok Hong 1 and Eun-Joong Kim 2 Department of Clinical Laboratory Science, Juseong College, Cheogwon 363-764, Korea 1 Department of Pathology, Chungbuk National University Hospital, Cheongju 361-711, Korea 2 Measurement of hemoglobin A1c is used as an objective indicator of long-term blood glucose control in diabetic patients. We evaluated recently introduced Norudia R HbA1c (Daiichi Pure Chemical Co. Ltd, Tokyo, Japan) test reagent using enzyme method for HbA1c assay. Linearity, precision and correlation with VARIANT TM II Turbo HbA1c analyzer (BIO-RAD, Hercules, CA, USA) were evaluated. The reference range was determined from 201 healthy subjects. The Norudia R HbA1c test reagent was founded to be linear in a range of 5.6% to 14.0% (r 2 =0.9885). The within-run and between-day precision were 0.954% and 1.03% for low level (HbA1c 5.24%), 0.67% and 1.28% for high level (HbA1c 9.01%), respectively. Comparison study between Norudia R HbA1c test reagent and VARIANT TM II Turbo showed good correlation with a slope of 1.0489. an intercept at -0.9717, and coefficient of correlation was 0.9907. The reference range of HbA1c obtained from this reagent was 4.07-5.50%. The Norudia R HbA1c test reagent showed good linearity, precision and correlation with HbA1c analyzer with HPLC method. In addition, the exclusive analyzer is not required for assay and then this kit may be useful for HbA1c assay in clinical laboratory. Key Words : HbA1c, Norudia R HbA1c test reagent, VARIANT TM II Turbo, Evaluation I. 서론 당화혈색소 (glycosylated hemoglobin) 의 75% 가과거 2 개월내의혈당치에의해결정되므로당화혈색소는과거 2~4 개월동안의평균적인혈당조절상태를반영한다 (Koenig 등, 1976a; Koenig 등, 1976b; Tahara 와 Shima, 교신저자 : 홍승복, ( 우 )363-794 충북청원군내수읍덕암리산 4 주성대학임상병리과 TEL : 043-210-8308, 011-9321-7056 E-Mail : sbhong@jsc.ac.kr 1993). 당화혈색소의측정방법중전하차이에근거한검사법에는이온교환크로마토그래피법, 고속액체크로마토그래피법 (HPLC) 및전기영동법등이있고구조적차이에근거한검사법에는친화크로마토그래피법 (affinity chromatography) 과면역분석법 (immunoassay) 이있다 (Sacks 등, 2006). 현재국내에서는당화혈색소측정은 HPLC 방법을사용하는검사실이가장많은데, 검사건수가많을수록 HPLC 방법을보다많이사용하고있는실정이다 ( 송, 2007). 당화혈색소측정에서검체자체에의한오류와검사방 37
대한임상검사학회지 41(1):37-41, 2009 법에의한오류가발생할수있다. 불안정한중간산물인 Schiff 염기및다양한혈색소유도체는전하차에의한분석법으로측정할경우오류가생길수있는데이는검사방법에의한오류라할수있다 (Sacks 등, 2006). 한편대부분의검사실에서당화혈색소분석기가 1 대인경우가많으며이경우장비의고장에의해검사결과보고가늦어질수도있다. 결국검사방법간오류교정이나기기의백업목적으로서로다른측정법을보유하는것은임상검사실에서매우유용할수있다. 최근에국내에소개된 Norudia R HbA1c(Daiichi Pure Chemical Co. LTD, Tokyo, Japan) 방법의측정원리는효소법이며특이하게당화혈색소만의베타쇄의 N 말단에서유래하는당화이중펩타이드 (glycosylated dipeptide) 를측정하는방법이다. 혈색소베타쇄와 3가지효소 (protease, fructosyl-peptide oxidase, peroxidase) 에의해생성된색고정물질의색을흡광도로측정한다. 이원리는화학분석기의대부분의측정원리와유사하므로전용측정기기가필요없고일반화학분석기기로도측정이가능하다. 그러나아직까지이시약에대한기존방법들과비교된국내의보고가없어이시약을현재국내에서가장많이이용하고있는 HPLC 방법과비교하여보았다. 2. 정밀도평가기존기기로측정한 HbA1c의측정치에따라검체를저농도군 (5.24%) 과고농도군 (9.01%) 으로각각나누어각군의혼주전혈을만든후 8개의동일검체로나누어 4 에냉장보관하면서 8일간연속하여 Norudia R HbA1c로측정하였다. 각군의검체는 2 시간간격을두고 2 회검사하였으며각검사시에두번씩반복측정하여검사내정밀도와검사일간정밀도를각각구하였다 (NCCLS, 1999). 기존기기인 VARIANT TM II Turbo HbA1c 측정기의검사내및검사일간정밀도도같은방법으로측정하였다. 3. 상관성평가기존기기에의한 HbA1c의측정치가 5.3% 에서 16.5% 의범위에속하는 85 검체를선별하여 VARIANT TM II Turbo 방법과 Norudia R HbA1c로각각 2 회씩측정하여상관방정식과상관계수를구하였다 (NCCLS, 1995). 4. 참고치설정건강검진을위해내원한사람중혈당및일반혈액검사결과가정상인 20~60대사이의 201 명 ( 남자 102 명, 여자 99 명 ) 을대상으로 Norudia R HbA1c로측정하여참고치를구하였다. II. 재료및방법 2008년 2-3월사이에한대학병원진단검사의학과에의뢰된 HbA1c 검사검체 85 예와건강검진을위해내원한 201 명 ( 남 102 명, 여 99 명 ) 의검체를이용하였다. 1. 직선성평가이온교환 HPLC 방법인기존의 VARIANT TM II Turbo (BIO-RAD, Hercules, CA, USA) 방법으로측정한 HbA1c 치가 5.6% 와 14.0% 인두검체를저농도와고농도의검체로하여 4:0, 3:1, 2:2, 1:3, 0:4의비율로섞어, 각각을 Norudia R HbA1c로 4 회반복측정한측정치와계산치를회귀분석하여회귀방정식과결정계수를구하였다 (NCCLS, 2003). III. 결과 1. 직선성평가저농도와고농도의검체를 4:0, 3:1, 2:2, 1;3, 0:4의비율로섞어만든검체의계산된 HbA1c 농도는각각 5.6%, 7.7%, 9.8%, 11.9%, 14%, 이었으며, 측정치와계산치로구한직선의회귀방정식은 y=1.0512x - 0.49 이었고결정계수 (r 2 ) 는 0.9885 이었다 (Fig. 1). 2. 정밀도평가 Norudia R HbA1c 검사시약과 VARIANT TM II Turbo HbA1c 측정기의정밀도를 Table 1에나타내었다. Norudia R HbA1c 검사시약의검사내정밀도의변이계수는저농도군이 0.954%(5.24% 농도 ) 이었고고농도군이 0.67% (9.01%) 이었다. 검사일간정밀도는각각 1.03% 와 1.28% 38
Korean J. Clin. Lab. Sci. 41(1):37-41, 2009 Measured value (%) 15 13 11 9 7 y = 1.0512x - 0.4902 R 2 = 0.9885 5 3 3 5 7 9 11 13 15 Expected value (%) Fig. 1. Linearity of Norudia R HbA1c test reagent. 의변이계수를보였다. IV. 고 찰 Table 1. Within-run and between-day precision of Norudia R HbA1c test reagent and VARIANT TM II Turbo HbA1c analyzer Parameter Level of HbA1c Norudia R HbA1c VARIANT TM II Turbo Normal High Medium High Mean (% HbA1c) 5.24 9.01 7.79 9.20 Within-run CV (%) 0.95 0.67 1.09 0.39 Between-day CV (%) 1.03 1.28 2.87 1.68 3. 상관성및회귀분석평가 VARIANT TM II Turbo 방법과의선형회귀분석결과는 y=1.0489x-0.9717 이었고상관계수 (r) 는 0.9907 이었다 (p<0.001)(fig. 2). 4. 참고치설정 102 명및 99 명의건강한남, 여를대상으로설정한참고범위는남자에서 4.07~5.49% 그리고여자에서 4.07~ 5.50% 이었다. 2007년대한임상검사정도관리협회의 HbA1c의검사방법에대한조사에서는현재국내에서가장많은검사방법은 HPLC 방법이며중소병원검사실에서는 POCT 장비가주로이용되고있다 ( 송, 2007). 그외여러가지면역분석법이이용되고있다. 현재당화혈색소의검사량이많은경우대부분당화혈색소전용검사기기를이용하며이때주로한가지방법으로측정하게된다. 그러나대형병원에서가장많이사용되고있는 HPLC 방법은전위차를이용한검사법으로대량검체의처리나안정도에서우수한결과를보이나일부임상상황에서는오류가발생할수있다 (Sacks, 2006). 기기의특성상혈색소유도체 (Hb derivatives) 가생기는다양한임상상황에서는부정확한결과를보일수있다. 1) 요독증환자에서 carbamylated 혈색소 (Chachou 등, 2000; Rose 등, 1995; Weykamp 등, 1993a; Weykamp 등, 1999), 2) 아스피린대량장기복용환자에서 acetyl 혈색소 (Weykamp 등, 1993a; Weykamp 등 1993b), 3) 알코올중독환자에서 acetaldehyde 혈색소 (Nguyen와 Peterson, 1984; Weykamp 39
대한임상검사학회지 41(1):37-41, 2009 19.0 17.0 y = 1.0489x - 0.9717 r = 0.9907 15.0 Norudia (%) 13.0 11.0 9.0 7.0 5.0 3.0 3.0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 Variant II Turbo (%) Fig. 2. Correlation of HbA1c between Norudia R HbA1c test reagent and VARIANT TM II Turbo HbA1c analyzer. 등 1993b), 4) 불안정한중간산물인 Schiff 염기 (Sacks, 2006; Weykamp 등, 1993b), 그리고다양한혈색소변이형 (Lee 등, 2007) 등에의해영향을받을수있다. 그러나면역측정법은이들에대한영향이거의없는것으로알려져있다 (Weykamp 등, 1993a; Weykamp 등, 1993b). Norudia R HbA1c는엄밀한의미에서면역측정법보다는 protease, fructosyl-peptide oxidase(fpo) 및 peroxidase (POD) 를이용한효소법으로최근에소개된당화혈색소측정법이다. 즉 protease에의해 glycosylated dipeptide를생성하고 FPO 및 POD에의해생성된색을흡광도의변화를측정하는방법이다. 따라서혈색소유도체를포함한다양한비특이물질에간섭이적고, 기존에이용하고있는화학분석법의원리와도유사하여이들기기를이용할수있는장점이있다. 물론전용기기를이용하면대량검사도가능하지만기존화학측정기기를이용하는경우는 HPLC와같은전용기기의백업용으로유용할수있을것이다. 본연구에서 Norudia R HbA1c의직선성은 5.6~14.0% 농도에서결정계수 (r 2 ) 0.9885로우수하였으며, 검사중정밀도는저농도 (5.24%) 와고농도 (9.01%) 에서각각 0.954% 와 0.67% 로 HPLC (VARIANT TM II Turbo) 방법과유사하거나오히려우수하였다 (Table 1) 검사일간정밀도도각각 1.03% 와 1.28% 로우수하였다. 기존기기 (VARIANT TM II Turbo) 와상관성은상관계수 (r) 0.9907로우수하였으며회사에서제공된안내서결과 (r=0.994) 와도유사하였다. 그리고 Norudia R HbA1c는다른면역측정법과도우수한상관성을보였다 (r=0.990, 제조사제공 ). 건강한성인 201 명을대상으로시행한참고치는 4.07~ 5.50% 로남 녀간차이가거의없었다. 기기및시약을평가하고자할때여러가지방해요소의영향을평가할필요가있다. 방해요소에는통상적인검사항목에서방해할수있는빌리루빈, 고지혈증및비타민 C 등과당화혈색소에서특이적으로방해할수있는 acetaldehyde, sodium cyanate, acetyl aldehyde 및 glucose 등이알려져있다 (Sacks, 2006; Weykamp 등, 1993b). 본연구에서는이들에대한평가를시행하지는못하였지만앞으로이들에대한평가도진행되어야할것이다. 결론적으로 Norudia R HbA1c 측정시약은직선성및정밀도가우수하였으며, VARIANT TM II Turbo 방법과상관관계도우수하였다. 또한간단한검체전처리과정만 40
Korean J. Clin. Lab. Sci. 41(1):37-41, 2009 거치면전용분석기가필요없이일반자동화학분석기로측정할수있으며 HbA1c 유래의당화펩타이드를효소법으로특이하게측정하므로불안정한당화혈색소및유사당화혈색소에의한영향이없다고알려져있다. 따라서 Norudia R HbA1c 측정시약은기존방법을백업할수있는검사로도이용할수있고전용기기를이용하면대량검체도가능하므로임상검사실에서당화혈색소측정에효율적인방법으로사료된다. 참고문헌 1. Chachou A, Randoux C, Millart H, Chanard J, Gillery P. Influence of in vivo hemoglobin carbamylation on HbA1c measurements by various methods. Clin Chem Lab Med 38:321-326, 2000. 2. Koenig RJ, Peterson CM, Kilo C, Cerami A, Williamson JR. Hemoglobin AIc as an indicator of the degree of glucose intolerance in diabetes. Diabetes 25:230-232, 1976a. 3. Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A. Correlation of glucose regulation and hemoglobin AIc in diabetes mellitus. N Engl J Med 295:417-420, 1976b. 4. Lee ST, Weykamp CW, Lee YW, Kim JW, Ki CS. Effects of 7 hemoglobin variants on the measurement of glycohemoglobin by 14 analytical methods. Clin Chem 53:2202-2205, 2007. 5. National Committe for Clinical Laboratory Standards. Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline. NCCLS Document EP6-A. Wayne, PA, National Committee for Clinical Laboratory Standards, 2003. 6. National Committe for Clinical Laboratory Standards. Evaluation of the precision performance of clinical chemistry devices; approved guideline. NCCLS document EP5-A. Wayne, PA, National Committee for Clinical Laboratory Standards, 1999. 7. National Committee for Clinical Laboratory Standards. Method comparision and bias estimation using patient samples; approved guideline. NCCLS document EP9-A. Wayne, PA, National Committee for Clinical Laboratory Standards, 1995. 8. Nguyen LB, Peterson CM. Modification of hemoglobin by acetaldehyde: a time course study by high pressure liquid chromatography. Alcohol Clin Exp Res 8:516-521, 1984. 9. Rose AM, Tongate C, Valdes R Jr. A hemoglobin A1c immunoassay method not affected by carbamylated hemoglobin. Ann Clin Lab Sci 25:13-19, 1995. 10. Sacks DB. Carbohydrates. In Burtis CA, Ashwood ER, Bruns DE ed. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. p837-901, Elsevier Saunders, St. Louis, 2006. 11. Tahara Y, Shima K. The response of GHb to stepwise plasma glucose change over time in diabetic patients. Diabetes Care 16:1313-1314, 1993. 12. Weykamp CW, Miedema K, de Haan T, Doelman CJ. Carbamylated hemoglobin interference in glycohemoglobin assays. Clin Chem 45:438-440, 1999. 13. Weykamp CW, Penders TJ, Muskiet FA, van der Slik W. Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods. Clin Chem 39:1717-1723, 1993a. 14. Weykamp CW, Penders TJ, Siebelder CWM, Muskiet FA, van der Slik W. Interference of carbamylated and acetylated hemoglobin in assays of glycohemoglobin by HPLC, electrophoresis, affinity chromatography, and Enzyme immunoassay. Clin Chem 39:138-142, 1993b. 15. 송정한. HbA1c 검사의국내현황및표준화방향. 대한진단검사의학회지 27(S2):S426-S432, 2007. 41