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대한임상검사학회지 : 38 권제 2 호, 117-124, 2006 분석측정범위의실증적평가 녹십자의료재단, 고객지원및 QA 부 1, 화학혈액검사실 2, 면역검사실 3, 김천보건대학, 임상병리과 4 장상우 1 이상곤 2 김영환 1 송은영 3 박용원 2 박병옥 2 류재기 4 An Empirical Study of the Analytical Measurement Range in Clinical Chemistry Sang-Wu Chang 1, Sang-Gon Lee 2, Young-Hwan Kim 1, Eun-Young Song 3, Yong-Won Park 2, Byong-Ok Park 2, and Jae-Gi Lyu 4 Technical Informaton & QA Division 1, Department of Clinical Chemistry 2, and Immunology 3, Green Cross Reference Laboratories, Yongin 449-913, Korea Department of Biomedical Laboratory Science, Gimcheon College, Gimcheon 740-704, Korea 4 The analytical measurement range (AMR) is the range of analyte values that a method can directly measure on a specimen without any dilution, concentration, or other pretreatment not part of the usual assay process. The linearity of the AMR is its ability to obtain test results which are directly proportional to the concentration of analyte in the sample from the upper and lower limit of the AMR. The AMR validation is the process of confirming that the assay system will correctly recover the concentration or activity of the analyte over the AMR. The test specimen must have analyte values which, at a minimum, are near the low, midpoint, and high values of the AMR. The AMR must be revalidated at least every six months, at changes in major system components, and when a complete change in reagents for a procesure is introduced; unless the laboratory can demonstrate that changing the reagent lot number does not affect the range used to report patient test results. The AMR linearity was total protein (0-16.6), albumin (0-8.1), total bilirubin (0-18.1), alkaline phosphatase (0-1244.3), aspartate aminotransferase (0-1527.9), alanine aminotransferase (0-1107.9), gamma glutamyl transpeptidase (0-1527.7), creatine kinase (0-1666.6), lactate dehydrogenase (0-1342), high density lipoprotein cholesterol (0.3-154.3), sodium (35.4-309), creatinine (0-19.2), blood urea nitrogen (0.5-206.2), uric acid (0-23.9), total cholesterol (-0.3-510), triglycerides (0.7-539.6), glucose (0-672.7), amylase (0-1595.3), calcium (0-23.9), inorganic phosphorus (0.03-17.0), potassium (0.1-116.5), chloride (3.3-278.7). We are sure that materials for the AMR affect the evaluation of the upper limit of the AMR in the process system. Key Words : Analytical measurement range, Linearity, Validation, Process 1) 교신저자 : 장상우, ( 우 )449-913 경기도용인시구성읍보정리 314 녹십자의료재단고객지원및 QA 부 Tel : 031-260-9206. HP : 011-9909-7224 E-mail : johnmadams@hanmail.net 117

I. 서론 임상검사실에서사용하는자동분석장비의검사시약은일반적으로키트화된상품으로판매되고있다. 동일한검사원리라도시약의순도, 안정성, 그리고자동분석기의사용년수등에따라서참고치의범위, 분석측정범위 (analytical measurement range: AMR), 직선성, 정확성, 정밀성, 특이도, 그리고분석민감도등에서다르게나타난다 ( 문과장, 2004). AST의경우를보면다양한검사키트가있지만측정방법론적특성들인민감도와특이도, 측정반응, 측정온도, 완충액의유형들, 완충액의분자량, ph, alpha-ketoglutarate 농도, aspartate의농도, NADH 농도, MDH의활성치와순도, MDH의준비과정, LDH의첨가, pyridoxal phosphate의첨가, 맹검, 측정모드, 측정시간, 표준화등에따라서검사결과치및참고치범위, 보고가능범위, 직선성, 정확성, 정밀성, 특이도, 민감도등이다르다. 2001년도 CAP LAP의인증심사점검표 (AGC.23120 Phase 11 N/A YES NO) 에의하면 자동분석기시스템에대한모든자동분석기를위해서분석측정범위 (AMR) 의상한범위와하한범위가정의되고, 검사결과치를보고하기전에필요하면그결과치가관리범위를벗어나면적절하게조사하고재측정하는냐? 라는점검항목이있다 ( 문과장, 2004). 분석측정범위 (AMR) 란검사벙법이어떤희석이나, 농축, 일상적인측정공정에다른전처리가없이직접측정될수있는분석수치의범위이다. 즉각각의진단검사의학과검사실은임상적이용을위해서수용할수있는결과를제공하는분석측정범위 (AMR) 를설정해야하는것이다. 국내, 외검사실인증심사에서는분석측정범위는시약 lot No. 의변화와분석기의중요부품의교체또는최소한매 6개월마다분석측정범위의상한치와하한치를검증하도록요구하고있다. 실제로설정된분석측정범위를초과하는모든검사들은임상보고가능범위를설정해야하고, 희석방법과보고방법이정의되고문서화되어있어야함을인식하고는있으나, 임상화학검사실에서상품화된직선성시료를구매하여사용하면한번실험하는데약 200만원정도가소요되므로경제적실행방법을찾는것이중요한변수이다. 이에좀더경제적이고실행가능한자체직선성의범위산출방법을설립하기위해서본연구를실행하였다. 본연구는임상화학검사실에서일상적으로검사되는 total protein(tp), albumin(alb), total bilirubin(tb), alkaline phosphatase(alp), aspartate aminotranferase (AST), alanine aminotransferase(alt), chloride(cl), creatnine kinase(ck), lactic dehydrogenase(ld), potassium (K), sodium(na), creatinine(cre), blood urea nitrogen (BUN), uric acid(ua), total cholesterol(tc), glucose (GLU), amylase(aml), gamma glutamyltranspeptidase (GGT), triglyceride(tg), calcium(ca), inorganic phosphorus (IP), 그리고 HDL Cholesterol(HDL-C) 등 22검사종목에대하여자체직선성의범위산출을통한측정가능범위의산출을시도하였다. II. 재료및방법 1. 연구방법분석측정범위에대한연구는외부정도관리에참여하는참고장비 ADVIA 1650 System으로 Bayer Diagnostics Corporation에서제조한검사키트와 Boehringer Mannheim GmbH에서제조한검사키트를이용하여분석측정범위의상한치과하한치에근접한 9점시료를 3회측정하고그평균치를사용하였다. 검사항목들은임상화학검사실에서일상적으로사용되는검사로 TP, ALB, TB, ALP, AST, ALT, CL, CK, LD, K, Na, CRE, BUN, UA, TC, IP, GLU, AML, GGT, TG, CA, 그리고 HDL-C 등 22종목을대상으로실시하였다. 1) Calibrator Calibrator는 Bayer Diagnostics Corporation에서제조한 Chemistry CAL(SETpoint) 를사용하여 calibration을하고일내검량보정은매시약의 vial to vial에대한검사변이를감소하기위하여교체할때마다 calibration을하였다. 2) 자동분석장비자동분석장비는 ADVIA 1650 System(Bayer Corp., Health Care Diag..Div., New York, USA) 을사용하여검사를실행하였다. 3) 재료상품화된직선성물질은 enzyme, non-enzyme, lipid 118

group의 3가지로나누어시판되므로 experimental materials은경제적부담을경감하기위해서 Bio Rad Laboratory에서제조한 assayed abnormal control과매일아침검사결과치를검색하면서모은환자시료를모아서제조사용하였고, 매일검사공정을확인하기위하여공정설계의설계변수에따른자체통계량의관리허용범위내에들어옴을확인하고시료를검사하였다. 4) 경제적실험시료의농축제조방법 GGT, AST, ALT, CK, LD, AMY, ALP 검사는환자시료를분석측정범위의상한치에근접한시료를모아서사용하고외부정도관리 (external quality control) 에참여하는참고분석기로 3회측정한평균치를 100% 의목표치로하였으며 9점시료에대한희석은 87.5%, 75%, 62.5%, 50%, 37.5%, 25%, 12.5%, 0% 로희석하였다. TP, ALB, TB, CL, K, Na, CRE, BUN, UA, TC, IP, GLU, TG, CA, HDL-C 검사는상품화된 Bio Rad Laboratories에서판매하는 unassayed control serum을농축하여사용하였다. 농축방법은분석측정범위상한치를 assayed control serum 의 target value로나누어주고희석액의첨가량을그배수로나누어주면첨가할희석액의첨가량을구하여농축하였다. III. 결과 1. 기울기, 절편, 상관계수의분석결과분석측정범위의하한치와상한치에고르게걸치는직선성의범위는 9점실험시료를사용하였으며. 각시료를 대상으로 21종목의검사를 3회반복측정한시료집단의결과평균치를 Table 1-1부터 1-4까지표시하였다. 목표치와관측치간의상관그래프 (Fig. 1, Fig. 2, Fig 3, Fig. 4) 를구하였고이로부터 9점시료에대한목표치와관측치의분석측정범위를검증하기위하여기울기, 상관계수, 절편값, 직선성의범위를 Table 2-1과 2-2에나타내었다. 기울기에서가장낮은검사항목은 ALP가 0.8792이었고가장높은검사는 1.0468이였으며평균기울기는 0.9945 로매우양호하였으며, 절편값이가장작은검사는 AST 가 -2.0892이었고가장높은검사는 sodium이 30.007이었고 amylase가 27.63으로나타났으며평균 intercept는 4.674이였으며, 상관계수는 0.983에서 1.0으로매우양호하게나타났다. 분석측정범위의하한치는 NA(35.4), BUN(0.5), TC(-0.3), TG(0.7), IP(0.03), K(0.1), L(3.3) 으로나타나배경잡음 (background noise) 이다소있었으나 15 가지검사에서는 0으로매우양호하게나타났다. 2. 분석측정범위의직선성범위임상화학검사실에서 22개검사항목에대한분석측정범위의직선성범위는 P(0.0-16.5), ALB(0.0-8.1), TB(0.0-18.1), ALP(0.0-1244.3), AST(0.0-1527.9), ALT (0.0-1107.9), GGT(0.0-1527.7), CK(0.0-1666.6), LD (0.0-1342.0), HDL-C(0.0-154.3), NA(35.4-309.0), CREA (0.0-19.2), BUN(0.5-206.2), UA(0.0-23.9), TC (-0.3-510.3), TG(0.7-539.6), GLU(0.0-672.7), AMY (0.0-1595.3), CA(0.0-23.9), IP(0.03-17.00), K (0.1-116.5), CL(3.3-278.7) 로나타났다. 직선성의실험시료의농도가낮은시료를사용할때보다높은시료를사용함으로써분석측정범위의상한을더명확하게확인할수있었다. Table 1-1. Summary mean of population from three times on TP, ALB, TB, ALP, AST, ALT Item TP ALB TB ALP AST ALT Level(%) X Y X Y X Y X Y X Y X Y 100.0 16.50 16.4 8.0 8.1 18.20 18.2 1245.0 1244.3 1533.0 1120.3 1120.3 1107.0 87.5 14.40 14.7 7.0 7.3 15.90 15.9 1089.4 1095.7 1341.3 1341.3 980.3 973.0 75.0 12.40 12.7 6.0 6.4 13.60 13.6 933.8 944.0 1150.0 1149.3 840.0 835.0 62.5 10.30 10.5 5.0 5.5 11.30 11.3 788.1 786.0 959.0 957.3 700.0 698.0 50.0 8.25 8.6 4.0 4.5 9.00 9.0 622.5 621.0 767.0 768.3 560.2 562.7 37.5 6.19 6.6 3.0 3.4 6.80 6.7 466.9 469.3 575.0 573.0 420.0 421.3 25.0 4.30 5.0 2.3 2.4 4.60 4.6 299.0 300.0 299.0 298.0 275.0 276.0 12.5 2.06 2.2 1.0 1.1 2.28 2.2 155.6 154.7 192.0 184.3 140.0 136.0 0.0 0.30 0.3 0.0 0.0 0.00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Legend: X axis is target value, Y axis is observed value 119

Table 1-2. Summary mean of population from three times on GGT, CK, LD, HDL C, NA, CRE Item GGT CK LD HDL-C NA CRE Level(%) X Y X Y X Y X Y X Y X Y 100.0 1529.0 1527.7 1675.7 1666.7 1358.3 1342.0 154.0 154.3 307.7 309.0 19.20 19.2 87.5 1337.9 1353.3 1466.2 1475.3 1188.5 1192.0 134.8 137.7 269.7 270.0 16.80 16.6 75.0 1147.0 1171.3 1257.0 1269.3 1019.0 1049.7 115.5 119.7 230.8 235.7 14.25 13.8 62.5 956.0 987.0 1047.0 1067.7 849.0 877.0 96.3 100.0 192.3 200.3 12.00 11.7 50.0 765.0 800.7 838.0 856.0 679.0 712.3 77.0 79.7 153.9 167.0 9.60 9.6 37.5 573.0 610.3 628.0 645.0 509.0 542.7 57.8 59.0 115.4 2130.7 7.20 7.3 25.0 375.0 376.0 400.0 399.0 717.0 711.0 38.5 38.0 76.9 97.0 4.30 4.9 12.5 191.0 201.7 210.0 216.0 170.0 179.7 19.3 17.0 38.5 62.7 2.40 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 35.4 0.00 0.0 Table 1-3. Summary mean of population from three times on BUN, UA, TC, TG, GLU Item BUN UA TC TG GLU Level(%) X Y X Y X Y X Y X Y 208.6 206.3 24.10 14.0 509.3 510.3 540.7 539.7 670.7 672.7 182.5 184.3 21.10 21.1 445.6 448.0 473.1 478.3 586.9 589.3 156.5 158.8 18.10 19.2 382.0 386.0 406.0 416.0 503.0 506.0 130.4 132.2 15.20 15.2 318.3 321.3 338.0 350.0 419.0 422.0 104.3 107.5 12.10 12.2 254.7 256.7 270.0 282.7 335.0 338.0 78.3 74.8 9.04 9.2 191.0 194.3 203.0 215.0 252.0 253.7 52.2 53.3 6.03 6.1 127.3 127.3 135.0 144.0 168.0 168.3 26.1 25.7 3.01 3.0 63.7 62.3 67.6 71.3 83.8 83.7 0.0 0.5 0.00 0.0 0.0-0.3 0.0 0.7 0.0 0.0 Table 1-4. Summary mean of population from three times on AMY, CA, IP, K, CL Item AMY CA IP K CL Level(%) X Y X Y X Y X Y X Y 100.0 1597.0 1595.3 23.80 24.0 16.7 17.00 16 16.5 279.3 278.7 87.5 1397.4 1423.0 20.80 22.1 14.6 14.80 14 13.7 244.4 239.7 75.0 1197.8 1262.0 17.90 19.6 12.5 12.60 12 11.5 209.5 205.3 62.5 998.1 1092.0 14.90 16.8 10.4 10.40 10 9.6 174.4 169.0 50.0 799.0 923.0 11.90 13.4 8.35 8.30 8 7.5 139.7 133.0 37.5 599.0 710.7 8.90 10.0 6.25 6.50 6 5.5 104.7 100.0 25.0 399.3 488.0 5.92 6.3 4.18 4.00 4 3.6 69.8 65.7 12.5 199.6 208.3 2.90 2.7 2.1 2.00 2 1.7 34.9 34.3 0.0 0.0 0.0 0.00 0.1 0.0 0.03 0 0.1 0.0 3.3 120

Fig. 1. Linearity range of the analytical measurement range on TP, ALB, TB, ALP, AST, and ALT. Fig. 2. Linearity range of the analytical measurement range on GGT, CK, LD, HDL-C, Na, and CRE. 121

Fig. 3. Linearity range of the analytical measurement range on BUN, UA, TC, TG, GLU and AMY. Fig. 4. Linearity range of the analytical measurement range on Ca, IP, K, and CL. 122

Table 2-1. Slope, intercept, correlation coefficient and linearity range for TP, ALB. TB, ALP, AST, ALT, GGT, CK, LD. HDL-C, and Na Item TP ALB TB ALP AST ALT GGT CK LD HDL-C NA Slope 0.9927 1.028 0.9981 1.0035 1.0003 0.9907 1.0043 1.0003 0.9938 1.0248 0.8935 Intercept 0.3155 0.1536-0.0369-0.5171-2.0892 2.2404 13.811 7.8317 17.414-0.5436 30.007 Correlation coefficient 0.9983 0.9969 0.999 0.9999 1.0000 0.9999 0.9991 0.9997 0.9982 0.9999 0.9991 Linearity range 0-16.5 0-8.1 0-18.1 0-1244.3 0-1527.9 0-1107.9 0-1527.7 0-1666.6 0-1342 0-154.3 35.4-309 Table 2-2. Slope, intercept, correlation coefficient and linearity range for TP, ALB. TB, ALP, AST, ALT, GGT, CK, LD. HDL-C, and Na Item CRE BUN UA TC TG GLU AMY CA IP K CL Slope 0.9759 1.002 0.9974 1.0064 0.9999 1.0044 1.0232 1.0468 1.0183 1.0125 0.9863 Intercept 0.2126 0.2938 0.0669-0.0646 7.1639 0.2201 27.63 0.33 0.0916-0.3556-1.1585 Correlation coefficient 0.9985 0.9991 0.9999 0.9999 0.9992 1.0 0.9853 0.9938 0.9995 0.9961 0.9991 Linearity range 0-19.2 0.5-206 0-23.9-0.3-510.3 0.7-539.6 0-672.7 0-1595.3 0-23.9 0.03-17 0.1-16.5 3.3-278.7 IV. 고 찰 1. 분석측정범위와임상보고가능범위의관련성 검사항목의측정치에대하여임상보고가능범위의상한치경계를정의해야한다. 이때분석측정범위의하한치는분석민감도인최소검출농도와관련된다. CAP LAP의검사실인증을위한분석측정범위는검사실인증심사위원회에서는최소한 5점평가를선호하나 (CAP, 2004) 미국임상병리개선법률 (clinical laboratory improvement amendments, CLIA) 에서는 3개로명시되어있다. 최소한매 6개월마다한번은분석측정범위를평가해야하며최고낮은수치와최고높은수치그리고이들을함께결합한중간치인 3점 (three points) 을취하도록하고있다. 또한과거의검사실인증심사의점검표에서는심사자들이매 6개월마다검량보정을충분하게실행하고있다고보고분석측정범위를요구하지않았다. 그러나새로운인증심사점검표에서는분석측정범위를실행할때에는좀더정밀하고상세하게묘사하고있다 ( 문과장. 2004). 이러한검량보정검증과분석측정범위가정량적검사영역으로서임상화학, 독물학, 면역혈청등의분야에서적용하고있다. 분석측정범위를벗어나는검사결과를보고하기위해서는정의된보고방식을가져야하며적용할때에는희석액과규정을명시해야하며 임상보고가능범위 (clinically reportable range, CRR) 의하한치는더작다, 또는 " 임상보고가능범위의상한치는더크다 " 라고보고할때에숫자적으로의미를부여해준다. 즉각기관의검사실은각 2. 분석측정범위의자체설정시약의역가, 분석기나램프의열화, 검량물질의상이, 검사원리, 화학성분의등급등에따라서측정가능범위는다르게마련이다. 질병의경과관찰과진단의정확한평가를위해서는측정하고자하는검사물질의최대측정치까지측정할수있어야한다. 그러나경제적효율성과 turn around time의단축등으로임상진단에가장이상적인분석측정범위를설정하고초과하는검사에대하여희석범위를규정하고반복검사하여보고하도록하는것이좀더정확한결과를제공할수있게된다는것을인식해야한다. 본연구에서분석측정범위의자체설정시에는필히과학적인진단과경과관찰을고려하여임상보고가능범위를규정하고희석방법을각검사항목에따라서설정하는것이중요하다고판단되었다. 주기적으로실행되는분석측정범위는각검사항목의직선성의범위를재설정하여결정하는것이권장된다. 임상시료를이용한제조혈청의사용은일반적으로비전해질검사에서는분석민감도가매우양호한실험결과를보였으나전해질검사항목인 Na( 목표치, 0; 측정치 123

35.4) 와 CL( 목표치, 0; 측정치, 3.3) 으로약간의차이를보였으며이는전해질측정방법의특성으로사료된다. 그러나전체적으로실용가능한결과를보였다고판단되며, 특히고가의 calibrator 물질을최소한으로사용하면서, 임상검체중, 고농도혈청을수집하여사용함으로써저비용으로직선성을검증할수있었다. 검량보정검증과분석측정범위측정을위한정기적실행사이의검증에사용하는것도의미가있을것으로판단된다. 이러한분석측정범위의설정은환자의결과치에직접적으로는영향을주므로임상보고가능범위를동시에규정하여그범위를현장에배치하고분석자들이즉시참고하여조치하고검색하도록하는것이매우중요하다는것을인식하였다. 참고문헌 1. College of American Pathologists(CAP), Chemistry and toxicology checklist, p23-29, Northfield, IL, 2004. 2. 문해란, 장상우. 6-시그마정도경영 : 인증중심의정도관리. p158-234, 펴냄홍, 서울, 2004. 3. 문해란, 장상우. 6-시그마정도관리 : 인증중심의정도관리. p428, 펴냄홍, 서울, 2004. 124