대한수혈학회지 : 제 24 권제 2 호, 2013 The Korean Journal of Blood Transfusion Vol. 24, No. 2, 161-174, August 2013 ISSN 1226-9336 Original Article LG AdvanSure HBV Real-Time QPCR Kit 의성능평가 문정주 1 ㆍ임채승 1 ㆍ이창규 1 ㆍ오흥범 2 고려대학교의과대학진단검사의학교실 1, 울산대학교의과대학서울아산병원진단검사의학교실 2 Performance Evaluation of LG AdvanSure HBV Real-Time QPCR Kit Jung Joo Moon 1, Chae Seung Lim 1, Chang Kyu Lee 1, Heung Bum Oh 2 Department of Laboratory Medicine, College of Medicine, Korea University 1, Asan Medical Center, University of Ulsan College of Medicine 2, Seoul, Korea Background: Accurate quantitative testing of HBV DNA is very important for choosing antiviral treatment targets and evaluating treatment response in chronic HBV patients. We evaluated the performance of LG AdvanSure HBV Real-Time QPCR kit (LG) utilizing real-time quantitative PCR. Methods: The LG kit was conducted for 201 chronic hepatitis patients undergoing treatment at the Korea University Ansan hospital and 48 normal control volunteers. The precision, limit of detection, sensitivity, and specificity of LG Kit were evaluated. Correlation analysis was done with Abbott Real Time HBV kit (Abbott) and the Cobas Amplicor HBV Monitor kit (Cobas) and the concordances rate of the three methods were calculated. Results: The LG assay showed linear range of detection from 10 2 to 10 6 and coefficient of variation (CV) was 1.10 0.52% at > or =1,000 IU/mL and 1.19% at 100 IU/mL. The coefficient of determination for precision analysis was 0.997. The limit of detection for detection of 95% of positive samples was 9.71 IU/mL (54.4 copies/ml). In 201 clinical samples, the log HBV DNA/ml showed good correlation between Roche vs Abott, Roche vs LG and Abott vs LG, respectively (n=105, 108, 133, r 2 =0.91, 0.89, 0.94, P<0.0001). The overall concordance rate of three methods were 79.1% (159/201), 82.1% (165/201), and 85.1% (171/201), respectively, showing no stastically significant difference (P>0.05). Conclusion: LG AdvanSure HBV Real-Time QPCR kit showed outstanding precision, linearity, limit of detection, good correlation with previous methods, and is a valuable tool in treatment monitoring of chronic HBV infections. (Korean J Blood Transfus 2013;24:161-174) Key words: AdvanSure HBV Real-Time QPCR, Hepatitis B, HBV quantitation, Performance evaluation 서론 B 형간염바이러스 (Hepatitis B Virus, HBV) 는 만성간질환의주요한원인이며, 간경변증, 간암등의합병증을유발시키는것으로알려져있다. 세계보건기구의 2000년통계에의하면, 전세계 Received on April 6, 2013. Revised on May 14, 2013. Accepted on May 14, 2013 Correspondence to: Chae Seung Lim Department of Laboratory Medicine, Korea University Guro Hospital, Guro 2-dong, Guro-gu, Seoul 152-703, Korea Tel: 82-2-2626-3245, Fax: 82-2-2626-1465, E-mail: malarim@korea.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright C 2013 The Korean Society of Blood Transfusion - 161 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 적으로 20억정도의인구가 HBV에감염되어있고그중 3억 5천만명이상이만성간염을가지고있는것으로보고되었다. 1) HBV DNA 정량검사는만성 B형간염환자에게감염의현상태를파악하여만성 B형간염환자를진단하고치료여부를결정하며연속적으로모니터링하는데있어필수적인검사이다. 최근여러연구들에서항바이러스제치료시작후 24 48주에혈중 HBV DNA농도를측정하면항바이러스제에대한내성출현을예측할수있고, 2-4) 또한간경변증으로의진행과간세포암종발생위험을예측하는데있어서도중요하다. 또한 HBV의혈중농도는간경화나간세포암종과같은합병증의발생위험과직접관련이있다는연구도있어 HBV DNA 정량검사는만성 B형간염으로진단받은환자모두에게측정되어야한다. 5) HBV DNA정량검사의결과는혈청 HBV DNA의단위는 IU/mL로표준화하는것이세계적인추세이고, 1 IU/mL는약 5.6 copies/ml이다. 6) 혈중 HBV DNA 정량측정의수치별의의로, 과거에는 HBV DNA의혈중농도가 17,867.1 IU/ ml (10 5 copies/ml) 이하인경우비활동성보유자 (inactive carrier) 로간주되어임상적으로관심이낮았으나, 현재에는 Cobas Amplicor HBV Monitor kits (Roche Molecular System, Pleasanton, CA, USA) 로측정했을때 35.7 IU/mL (200 copies/ml) 이하에서는재발률이 37%, 178.6 IU/mL (1,000 copies/ml) 이상인경우는 73% 에서재발한다는보고가있어관심이높아지고있다. 7) 최근미국간학회의만성 B형간염치료가이드라인에서도 1,786.7 17,867.1 IU/mL (10 4 10 5 copies/ml) 인경우지속적인추적검사와함께필요한경우치료가필요하다고하여, 8) 17,867.1 IU/mL (10 5 copies/ml) 이상뿐아니라그이하에서도정확한정량이필요하게되었다. 국내에서 HBV DNA 정량을위해주로이용되고있는검사법은 Versant HBV DNA kit 3.0 (Bayer Diagnostics, Emeryville, CA, USA) 과 Digene HBV Hybrid-Capture II (Murex Diagnostics, Dartfold, UK), Roche COBAS Amplicore (Roche Molecular Systems, Pleasanton, CA), Roche Cobas Ampliprep-Cobas TaqMan HBV test, Abbott Real Time HBV kit (Abbott Molecular, Des Plaines, IL), Qiagen Artus HBV QS-RGQ Assay (Qiagen, Hilden, Germany) 등이다. 9) bdna와 hybrid capture 방법은개발시기에따라차이는있으나일반적으로 178.6 1,785,714.3 IU/mL (10 3 10 7 copies/ml) 범위의고농도에서는정확한정량이가능하나저농도의바이러스혈증은검출하지못한다. 10,11) 또한기존의중합요소연쇄반응법은 10 6 IU/mL 이상의바이러스수치에서는신뢰도가떨어지는단점이있었다. 이를보완하여개발된것이최근에많이사용되는방법인실시간중합요소연쇄반응법 (real-time PCR) 이다. HBV DNA의검출한계가 10 2 IU/mL까지로민감도가높아져서이로인해저농도범위에대해서도검사가가능하도록발전하였다. 국내의 ( 주 )LG생명과학에서개발된 B형간염바이러스 (Hepatitis B Virus, HBV) 진단용시약인 LG AdvanSure HBV Real-Time QPCR kit는현재널리사용되는 Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v. 2.0 (Cobas TaqMan HBV Test, Roche Molecular System, Pleasanton, CA, USA) 와 Abbott사의 Abbott Real Time HBV kit (Abbott Molecular Inc., Abbott Park, IL, USA) 와동일한원리인 real-time PCR법을사용하고있다. 이에 LG AdvanSure HBV Real-Time QPCR kit에대해현재국내외적으로가장널리사용되는 Roche 사 Cobas TaqMan HBV Test (Roche Molecular System, Pleasanton, CA, USA) 와 Abbott사의 Abbott - 162 -
문정주외 : LG AdvanSure HBV Real-Time QPCR Real Time HBV kit (Abbott Molecular Inc. Abbott Park, IL, USA) 와비교하여유용성을확인하고자하였다. 대상및방법 1. 대상 1) 대상검체고려대학교안산병원진단검사의학과 HBV DNA 정량검사를의뢰하는환자를대상으로총 201명의 B형간염환자 (2008년 12월부터 2011년 6월 ) 및정상인지원자 48명을대상으로하였다. 참가연구대상자들에게연구의목적과절차를충분히설명하고동의서를받았으며, 연구내용은본원의임상연구윤리위원회의허락을받은후연구를시행하였다. 대상환자의채혈은 EDTA 및 plain tube로약 10 ml를채혈하였고 24시간내에 800 1,600 g로 20분동안원심분리하여분리된혈장과혈청에서검사를실시하였다. 혈장과혈청은 HBV DNA정량검사전까지 70 o C에서냉동보관하였다가검사직전에상온에두어녹인후사용하였다. 2. 방법 1) 대상기기및원리 (1) LG AdvanSure HBV Real-Time QPCR kit: ( 주 )LG생명과학에서개발된 HBV 진단용시약인 LG AdvanSure HBV Real-Time QPCR kit는, 실시간중합효소연쇄반응법을바탕으로한검사로 Hepatitis B Virus의특정부위에특이적인프라이머를이용하여 PCR을통해반응산물을증폭하며, 이와동시에각표적에특이적인 Taqman 프로브를분해함으로써형광을방출하게되고, 형성된형광신호를실시간형광강도측정방법 (real-time PCR detection system) 으로실시간으로검출및정량을실시하는방법이다. 본검사의결과는 International Units (IU)/mL 또는 copies/ml로보고가능하며, 본연구에서는 IU/mL로결과를산출하였다. (2) 핵산추출 : 혈청또는혈장 300 μl를핵산자동화추출장비인 Labturbo 36 compact system, 타이젠 (TAIGEN corp., Taipei City, Taiwan) 을이용하여핵산을추출하였다. (3) 증폭과검출 : 추출된핵산중 10 μl를 LG AdvanSure HBV Real-Time QPCR kit 시약에혼합하여제조사지침에따라 SLAN real time detection system (Hongshi, Shanghai, China) 을이용하여검출을수행했다. 증폭된 HBV와 Internal control 은형광광증폭튜브 (photomultipler tube) 에의해검출되고 SLAN real time detection system (Hongshi, Shanghai, China) 에서자동으로결과가판독되었다. 매반응마다 PCR의오염을확인하기위해음성대조에서음성의결과가나오는지를확인하며, 양성대조물질을동시검사하여지정된범위내에드는지를확인하였다. (4) Roche Cobas Ampliprep-Cobas TaqMan HBV test (Roche Molecular System, Pleasanton, CA, USA): Roche사 Cobas TaqMan HBV Test기기를이용하여혈청에서 HBV DNA를추출하고반응혼합물 (master mix) 을혼합하고증폭을하는일련의과정이 Roche사 COBAS AmpliPrep과 Cobas TaqMan HBV Test기기내에서자동으로이루어졌다. 이때각검사마다각각하나의 COBAS TaqMan Negative control, COBAS TaqMan HBV Low positive control, COBAS TaqMan HBV positive control을첨가하여추출함으로써핵산추출및증폭과정상의오류를확인할수있도록하였다. (5) Abbott Real Time HBV kit (Abbott Mole- - 163 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 cular Inc., Abbott Park, IL, USA): Abbott m2000rt (Abbott Molecular Inc., Abbott Park, IL, USA) 기기를이용하여 Abbott Real Time HBV kit (Abbott Molecular Inc., Abbott Park, IL, USA) 를평가하였다. Abbott m2000sp System (Abbott Molecular Inc., Abbott Park, IL, USA) 을이용하여혈청 200 μl에서 HBV DNA를추출하였다. 이때내부대조 (internal control, IC) DNA를첨가하여추출함으로써핵산추출및증폭과정상의오류를확인할수있도록하였다. 기기내에서추출한 HBV DNA에 50 μl의반응혼합물 (master mix) 을혼합하고반응 plate에옮긴후 Abbott m2000rt로증폭과정을시행하였다. 2) LG AdvanSure HBV Real-Time QPCR kit 성능평가방법 (1) 직선성 (Linearity): 고대안산병원에서 LG AdvanSure HBV Real-Time QPCR kit에대해서만시험을실시하였다. 각각 CLSI EP6-A, CLSI EP5- A2 가이드라인에따라실험을진행하였다. 12,13) 제조사에서제공한정상인검체를혼합제조한정도관리용 pooled serum 검체를 NIBSC international standard (97/750, Hepatitis B Virus DNA for nucleic acid amplification techniques) 와혼합하여농도를교정한 10 7 IU/mL의시료를선정하고, National Committee for Clinical Laboratory Standards (NCCLS) EP6-P지침에따라 Lyphochek Drug Free Serum (Bio-Rad Labaratories, Inc., CA, USA) 을이용하여희석하여 10 6, 10 5, 10 4, 10 3, 10 2 IU/mL의검체를제조하였다. 12) 제조된 5가지단계의농도물질을 3일동안 8회에걸쳐반복측정하여직선성을평가하였다. 이론치와실측치에대한상관분석을실시하여무작위적오차에대한결정계수를구하였다. (2) 정밀도 (Precision, Repeatability): 제조된 5 가지단계의농도물질을 4 회에걸쳐반복측정하 여평가하였다. 한편각농도별로반복한결과에대한평균, 표준편차및변이계수 (%CV) 값을구하여평가하였다. (3) 검출한계 (Limit of Detection): 검출한계 (Lower limit of Detection, LLoD) 는신뢰할수있는값으로측정가능한최저농도로, EP17 CLSI 가이드라인에따라 6개의낮은농도검체를이용한 12번의측정에기반하여 5% 미만의위양성 (α) 및위음성 (β) 에따라결정되었다. NIBSC international standard (97/750, Hepatitis B Virus DNA for nucleic acid amplification techniques) 를 Lyphochek Drug Free Serum (Bio-Rad Labaratories, Inc., CA, USA) 을이용하여 10 3, 10 2.5, 10 2, 10 1.5, 10 1, 10 0.5 IU/mL로희석하여시료를제조하였다. 각농도별로 24회의반복시험을진행하여양성률을측정하였으며, 이를통계처리프로그램인 SPSS 를이용하여 probit analysis를실시하여 LLoD를구하였다. 한편각농도별로반복한결과에대한평균, 표준편차및변이계수 (%CV) 값을구하여평가하였다. (4) Abbott Real Time HBV kit, COBAS AmpliPrep/COBAS TaqMan HBV Test와의상관성평가 : 본연구를위한고려대안산병원에서채혈된 201개의 HBV 의뢰검체에대해 (a) LG AdvanSure HBV Real-Time QPCR kit, (b) Roche Cobas Ampliprep-Cobas TaqMan HBV test, (c) Abbott Real Time HBV kit 검사를시행하였고 CLSI EP9-A2 가이드라인에따라실험을진행하였다. 14) 검사기간동안 201개의환자검체와 48 개의정상인검체를사용한세 kit의개별검사가수행되었으며각 kit간검사시간간격은 2시간이내, 하루 8검체이내로 20일동안분석되었다. 환자의임상상태및각종검사지표는고려대학교안산병원의의무기록을참조하여분석하였다 (Table 1). 현재국내에서사용되는 Roche Cobas - 164 -
문정주외 : LG AdvanSure HBV Real-Time QPCR Table 1. Baseline characteristics of the patients according to the HBV DNA, antibiotic resistance testing data expressed as ratio between positive cases/patients in which resistance testing was done Clinical characteristics All HBV patients (n=201) Viremia group* (n=149) No-viremia group (n=152) Age, yr 48.1±12.1 47.2±12.2 50.7±11.4 Male, n (%) 137±93.9 (68.2) 139.4±93.0 (93.3) 130.1±97.0 (85.5) AST (IU/L) 59.1±66.8 59.6±67.2 57.6±66.5 ALT (IU/L) 51.2±55.0 48.4±12.2 59.7±70.9 HCC 16 9 7 Cholangiocarcinoma 1 1 0 Hepatitis treatment 67 48 19 Adefovir 22 18 4 Dipivoxil 9 2 7 Clevudine 23 6 17 Lamivudine 43 16 27 Telbivudine 3 3 0 Entecavir 40 30 1 No treatment 134 101 33 Adefovir resistance 5/5 4/4 1/1 Lamivudine resistance 4/11 4/10 0/1 Abbreviations: HBV, hepatitis B virus; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HCC, hepatocellular cancer. *Patients positive for HBV RNA. Patients negative for HBV DNA. YMDD motif mutation included. Ampliprep-Cobas TaqMan HBV test, Abbott Real Time HBV kit와의결과일치여부등을비교하여국내에서임상에서의응용가능성등을분석하였다. 결과값이 cutoff level 미만시는이전엔음성이었다가다시 cutoff level 미만으로정량이되는샘플이일부있는것으로보아 primer dimer, probe의안정성등에의한 Non-specific signal일가능성이있으므로 HBV DNA가검출안된것과같게보고통계처리하였다. 세가지 HBV DNA 방법의상관성분석시상관성이있는지 (correlate), 허용범위에드는지 (acceptable) 의판정기준은세검사가모두같은결과를내거나허용범위에드는결과를내는경우를일치하는경우로간주하고, 세검사중한가지만나머지두검사와 다른결과를내는경우를불일치하는경우로간주하고통계처리하였으며아래와같은식으로일치율 (overall concordance rate) 을구하였다. 일치율 (overall concordance rate) 3) 통계분석 = 일치하는경우 (concordant case) 일치하는경우 (concordant case)+ 불일치하는경우 (discordant case) 검사결과에대한통계학적분석은 Microsoft Excel 2002 (Microsoft Corporation, New York, USA) 과 SPSS (version 18.0, SPSS Inc., Chicago, IL, USA) 를이용하였고, Pearson의상관계수 r 값을구해회귀분석검정을시행하였다. - 165 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 결과 1. 대상군의특성 2008년 12월 1일부터 2009년 6월 30일고려대학교안산병원진단검사의학과 HBV DNA정량검사가의뢰된 201명의 B형간염환자및정상인지원자 48명을대상으로두종류의 HBV DNA 정량검사와 LG AdvanSure HBV Real-Time QPCR kit와의방법간비교시험을진행하였다. 남녀비율은남자가 137명으로 68.2%, 여자가 64명으로 31.8% 이고연령분포는 81세이상이 1% (2명), 71 80세가 1.5% (3명), 61 70세가 11.5% (23명), 51 60세가 33.5% (67명), 41 50세가 26.5% (53 명 ), 31 40세가 17.5% (35명), 21 30세가 8.5% (17명), 20세이하가 0.5% (1명) 였다. AST, ALT 수치는각각평균 59.1 IU/L, 51.2 IU/L였고, 또한치료받고있는환자가 78.6% (158명), 치료의종류로는 Adefovir 10.9% (22명), Dipivoxil 4.5% (9 명 ), Clevudine 11.4% (23명), Lamivudine 21.4% (43명), Telbivudine 1.5% (3명), Entecavir 19.9% (40명) 였다. 약물내성검사는약물투여후에도 HBV 바이러스가감소하지않는 16명에서만이루어졌는데, 그결과 Adefovir 내성이 5명 (5/5명), Lamivudine (YMDD motif 돌연변이 ) 내성이 4명 (4/11명) 이었다 (Table 1). 2. LG AdvanSure HBV Real-Time QPCR kit 성능평가방법 Fig. 1. Linearity testing of LG AdvanSure HBV Real-Time QPCR kit. 1) 직선성 (Linearity) 각농도별로반복실험한결과에서변이계수는 0.58 1.19% 정도로 2% 미만의양호한값을나타냈다 (Fig. 1). 2) 정밀도 (Precision, Repeatability) LG AdvanSure HBV Real-Time QPCR kit의이론치와실측치에대한회귀분석을실시한결과결정계수는 0.9968이었다 (Table 2). 3) 검출한계 (Limit of Detection) 양성률을측정한결과를통계처리프로그램인 Table 2. Result of precision analysis of AdvanSure HBV Real-Time QPCR, correlation between theoretical value and estimated value of HBV titer according to the CLSI EP5-A2 guideline 13) Theoretical value (IU/mL) 1,000,000 100,000 10,000 1,000 100 Mean 21.25 24.33 27.91 30.42 34.24 SD 0.23 0.17 0.16 0.16 0.41 CV (%) 1.10 0.70 0.58 0.52 1.19 Estimated value (IU/mL) 1,386,887.8 157,595.6 12,424.7 2,112.0 140.7-166 -
문정주외 : LG AdvanSure HBV Real-Time QPCR Table 3. Result of limit of detection analysis of AdvanSure HBV Real-Time QPCR IU/mL (copies/ml) 10 3 IU (5600) 10 2.5 IU (1769) 10 2 IU (560) 10 1.5 IU (177) 10 1 IU (56) 10 0.5 IU (17.7) Mean 30.76 32.60 34.13 36.53 38.91 39.46 Standard deviation 0.20 0.48 0.59 0.59 0.43 0.35 CV (%) 0.66 1.48 1.74 1.62 1.10 0.88 Number detected 10/10 10/10 10/10 24/24 23/24 9/24 Detection (%) 100.00 100.00 100.00 100.00 95.83 37.50 SPSS를이용하여 probit analysis를실시하여 Limit of Detection을구하였다. LG AdvanSure HBV Real-Time QPCR kit의경우양성검체를 95% 검출가능한 Limit of Detection은 9.71 IU/mL였으며해당 Limit of Detection의 95% 신뢰구간은 7.65 15.08 IU/mL였다 (Table 3). 4) 임상검체에서의 LG AdvanSure HBV Real- Time QPCR kit, Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas Taq- Man HBV test의상관성평가 B형간염양성으로진단되어치료및보균자로지내는대상환자 201명중 Roche Cobas Ampliprep-Cobas TaqMan HBV test 에서는 123명에서 HBV DNA가검출한계인 2.14 IU/mL (12 copies/ml) 이상으로검출되었고, 55명에서는 2.14 IU/mL (12 copies/ml) 이하로검출되었고 23 명의환자에서는 HBV DNA가검출되지않았다. Abbott Real Time HBV kit에서는 149명에서 HBV DNA가검출한계인 9.11 IU/mL (51 copies/ml) 이상으로검출되었고, 33명에서는 9.11 IU/mL (51 copies/ml) 이하로검출되었고 19명의환자에서는 HBV DNA가검출되지않았다. LG AdvanSure HBV Real-Time QPCR kit에서는 149명에서검출한계인 0.71 IU/mL (4 copies/ml) 이상으로검출되었고, 52명에서는 HBV DNA가검출되지않았다 (Table 4). LG AdvanSure HBV Real-Time QPCR kit가 Table 4. Result of 3 HBV DNA quantification assays in 201 clinical specimens (Roche, Abbott, LG) Roche Abbott LG Positive* 123/201 149/201 149/201 Not detected 23/201 19/201 52/201 Less than cutoff level 55/201 33/201 Total, n 201 201 201 *Positive=above cutoff level; Roche Cobas Ampliprep Cobas TaqMan HBV test : 2.14 IU/mL (12 copies/ml); Abbott Real Time HBV kit : 9.11 IU/mL (51 copies/ml); LG AdvanSure HBV Real Time QPCR : 0.71 IU/mL (4 copies/ml). Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test 모두와불일치한경우는환자 201명중총 12명이었다. LG Advan- Sure HBV Real-Time QPCR kit 양성, Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test에서검출한계이하로검출된경우가 7명, LG AdvanSure HBV Real-Time QPCR kit 양성, Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test 음성인경우가 1명이었으며 LG AdvanSure HBV Real-Time QPCR kit 음성, Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test 양성인경우는 4명이었다. Abbott Real Time HBV kit만양성인경우가 11명으로 Roche Cobas Ampliprep- Cobas TaqMan HBV test만양성인경우 1명보다 - 167 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 Table 5. Result of 3 HBV DNA quantification assays comparison in HBV patients (a) and normal controls (b) (LTC: less than cutoff level; Roche Cobas TaqMan HBV Test cutoff: 2.14 IU/mL (12 copies/ml), Abbott Real Time HBV kit cutoff: 9.11 IU/mL (51 copies/ml), LG AdvanSure HBV Real Time QPCR cutoff: 0.71 IU/mL (4 copies/ml)) (a) Positive specimen according to method Roche Abbott LG No. Remark Positive* Positive* Positive* 111 Correlate Positive Positive LTC 4 LG discordance Positive LTC Positive 7 Abott discordance Positive LTC LTC 1 Roche discordance LTC Positive Positive 19 Roche discordance LTC Positive LTC 8 Abott discordance LTC LTC Positive 7 LG discordance LTC LTC LTC 10 Correlate LTC Not detected LTC 11 Acceptable Not detected Positive Positive 4 Roche discordance Not detected Positive LTC 3 Abott discordance Not detected Not detected Positive 1 LG discordance Not detected LTC LTC 8 Acceptable Not detected Not detected LTC 7 Acceptable Total No. 201 HBV patient *Positive=above detection limit. (b) Roche Abbott LG No. Remark Not detected Not detected Not detected 42 Correlate Not detected LTC Not detected 5 Abott discordance LTC Not detected Not detected 1 Roche discordance Total No. 48 Normal control 많았다. Abbott Real Time HBV kit만검출한계이하또는음성인경우가 8명으로 Roche Cobas Ampliprep-Cobas TaqMan HBV test만검출한계이하또는음성인경우 23명보다적었다. 세시약간결과차이중의미있는것으로보이는경우는 LG AdvanSure HBV Real-Time QPCR kit에서는 HBV DNA가검출되지않았으나 Abbott Real Time HBV kit나 Roche Cobas Ampliprep-Cobas TaqMan HBV test에서검출한계이상으로양성인경우가 4명이있었다 (P<0.01). 이환자들의경우모두항바이러스제치료중인상태였다 (Table 5). LG AdvanSure HBV Real-Time QPCR kit의경우비특이적신호에대한확인을위해 Ct38 이하에출현하는 signal값에대해재시험을실시하였 - 168 -
문정주외 : LG AdvanSure HBV Real-Time QPCR 으나, Roche Cobas Ampliprep-Cobas TaqMan HBV test나 Abbott Real Time HBV kit은확인하지않았다. Roche Cobas Ampliprep-Cobas TaqMan HBV test의경우 <2.14 IU/mL (12 copies/ml) 에대해재시험을실시할경우재시험율이현저히높아져서, <2.14 IU/mL (12 copies/ml) 인검체는 53개, >18,965,517 IU/mL (110,000,000 copies/ml) 인검체는 9개로이는 200개중 62개로재시험율이 31% 에달했고 Abbott Real Time HBV kit도재시험율이 28% 였다. LG AdvanSure HBV Real-Time QPCR kit 재시험건수는 200개중 36개로재시험율이 18% 에달했으며여기엔장비교체, 프로토콜변경등이포함되었다. Roche Cobas Ampliprep-Cobas TaqMan HBV test 의경우 850 ul의혈액샘플을이용하여 75 ul로용출받은것중 50 ul를 PCR에사용하여 PCR 투입용량은 100 ul이었다. Abbott Real Time HBV kit는 200 ul의혈액샘플을이용하여 70 ul로용출받은것중 50 ul를 PCR에사용하여 PCR 투입용량은 50 ul이었다. 따라서 Roche Cobas Ampliprep-Cobas TaqMan HBV test은불순물의양이상대적으로높으므로이로인해 <2.14 IU/mL (12 copies/ml) 의결과가비특이적일가능성이높으나둘다재검을통한정량및진단하지는않았다. LG AdvanSure HBV Real-Time QPCR kit의경우 200 ul의혈액샘플을이용하여 150 ul로용출받은것중 5 ul를 PCR에사용하여 PCR 투입용량은 20 ul이었다. 따라서투입용량이적으므로대표성이떨어져확률적으로오류를범할가능성이높아서재검을통해확인과정을거쳤다. Roche Cobas Ampliprep-Cobas TaqMan HBV test 음성, LG AdvanSure HBV Real-Time QPCR kit에선정량한경우가 5건으로이중 3건은이전 Roche Cobas Ampliprep-Cobas TaqMan HBV test 결과양성이며, 2건은이전결과가없었다. Roche Cobas Ampliprep-Cobas TaqMan HBV test에서검출한계이하양성, LG AdvanSure HBV Real-Time QPCR kit 음성인경우는 6건으로모두이전 Roche Cobas Ampliprep-Cobas TaqMan HBV test 결과음성이었다. Roche Cobas Ampliprep-Cobas TaqMan HBV test 의검출상한치는 18,965,517 IU/mL (110,000,000 copies/ml) 이상까지 Abbott Real Time HBV kit는 587,931,034 IU/mL (3,410,000,000 copies/ml) 이상까지, LG AdvanSure HBV Real-Time QPCR kit는설정되어있지않았었으며, Roche Cobas Ampliprep- Cobas TaqMan HBV test 검출상한치 18,965,517 IU/mL (110,000,000 copies/ml) 이상인높은 HBV 역가를가진 10명에서는 LG AdvanSure HBV Real-Time QPCR kit가 Abbott Real Time HBV kit 보다낮은 HBV 역가를보였다. 3가지검사결과를양성 ( 검출한계이상 ) 과음성 ( 검출한계이하는음성으로처리 ) 으로구분하여분석한세가지 HBV DNA 방법의일치율 (overall concordance rate) 은 Roche Cobas Ampliprep-Cobas TaqMan HBV test 대 Abbott Real Time HBV kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test 대 LG AdvanSure HBV Real-Time QPCR kit, LG AdvanSure HBV Real-Time QPCR kit 대 Abbott Real Time HBV kit 각각 79.1% (159/201), 82.1% (165/201) 와 85.1% (171/201) 로세검사의전체일치율에는유의한차이가없었다 (P>0.05). HBV감염이없는것으로확인된정상인지원자 48명에서는 LG AdvanSure HBV Real-Time QPCR kit 양성인예는없었고 Abbott Real Time HBV kit 검출한계인 9.11 IU/mL (51 copies/ml) 이하로양성, Roche Cobas Ampliprep-Cobas Taq- Man HBV test 검출한계인 2.14 IU/mL (12 copies/ml) 이하로양성인경우가각기 5예, 1예였다. HBV DNA가정량된 105, 108, 133명의환자에 - 169 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 Fig. 2. Correlation analysis of HBV DNA titer measured by 3 HBV DNA quantification assays (A: Roche Cobas Ampliprep-Cobas TaqMan HBV test (Roche) vs Abbott Real Time HBV kit (Abbott), B: Roche Cobas Ampliprep-Cobas TaqMan HBV test (Roche) vs LG AdvanSure HBV Real-Time QPCR (LG), C: LG AdvanSure HBV Real-Time QPCR (LG) vs Abbott Real Time HBV kit (Abbott)). cf. IU/mL=5.6 copies/ml, log 10 value. 96, 93, 68 samples were negative or less than the cutoff values in one or both of the comparator assays, thus could not be quantitated, and were excluded from the correlation analysis, respectively at A, B, C. 서각각시행한회귀분석결과, Pearson의상관계수는 Roche Cobas Ampliprep-Cobas TaqMan HBV test 대 Abbott Real Time HBV kit은 R 2 =0.9102, Roche Cobas Ampliprep-Cobas TaqMan HBV test 대 LG AdvanSure HBV Real-Time QPCR kit R 2 = 0.8938, LG AdvanSure HBV Real-Time QPCR kit 대 Abbott Real Time HBV kit는 R 2 =0.9367로나타났다 (P<0.0001) (Fig. 2). 고찰정확한 HBV DNA 정량검사는진단과예후평 - 170 -
문정주외 : LG AdvanSure HBV Real-Time QPCR 가, 만성 B형간염환자에서항바이러스치료에대한반응이나바이러스혈증평가와 HBV의복제활성정도와질병경과추적에가장중요한표지자로서사용되고있다. 5) 대한간학회만성 B형간염진료가이드라인에따르면만성 B형간염의항바이러스제치료권고기준은비대상성간경변증이면서 HBV DNA 양성이거나 HBeAg 양성만성 B형간염또는 HBV DNA 양성간경변증으로 20,000 IU/mL, 혹은 HBeAg 음성의 2,000 IU/mL 이상의혈청 DNA 양성값이면서 ALT가정상상한치의 2배이상인경우또는대상성간경변증일때이다. 15) 국내에서사용되는 HBV DNA 정량법은크게증폭법과프로브법으로나뉘며, 증폭법은표적을증폭하는중합효소연쇄반응법, transcription-mediated 핵산증폭법 (TMA) 과신호 (signal) 를증폭하는 branched-chain DNA (bdna) 신호증폭법등이있다. 16) 신호증폭법의알려진민감도 ( 검출한계 ; lower limit of detection) 는방법에따라다양하다. 17-20) 문헌에따르면, 검출한계는 bdna 신호증폭법인 Versant HBV DNA kit 3.0 (Bayer Diagnostics, Emeryville, CA, USA) 는 839.3 IU/mL를, Hybrid capture 신호증폭법인 Digene HBV Hybrid- Capture II (Murex Diagnostics, Dartfold, UK) 는 357.1 IU/mL를보였다. 표적증폭법인 Quatitative PCR 중 Roche COBAS Amplicore (Roche Molecular Systems, Pleasanton, CA, USA) 는 35.7 IU/mL 를, Roche Cobas Ampliprep-Cobas TaqMan HBV test는 6 10 IU/mL를, Abbott Real Time HBV kit (Abbott Molecular, Des Plaines, IL, USA) 는 10 15 IU/mL를, Qiagen Artus HBV QS-RGQ Assay (Qiagen, Hilden, Germany) 는 10.2 IU/mL를보였다고하며, 같은 LG AdvanSure HBV Real-Time QPCR kit에서는약 9.7 IU/mL의검출한계를보였다. 17-20) 여기에필요한검체량은각각 650 μl, 650 μl, 200 500 μl, 1,000 μl, 300 μl에달했다. 20) 본연구에서시약회사가제공한검출한계는 COBAS AmpliPrep/Cobas TaqMan HBV Test 2.1 IU/mL (12 copies/ml), Abbott Real Time HBV kit 9.1 IU/mL (51.0 copies/ml) 이다. 보고가능범위 (Upper reportable range, ULR) 는각기 110,000,000 IU/mL, 3,410,000,000 IU/mL이었고, LG Advan- Sure HBV Real-Time QPCR kit는 ULR가이연구상에서는없었다. 양성이나 LLoD 미만의값이나온경우 COBAS AmpliPrep/COBAS TaqMan HBV Test나 Abbott Real Time HBV kit의경우양성, 음성과따로구분하여분석하였으나 LG AdvanSure HBV Real-Time QPCR kit는 4 미만과음성을하나로처리하였다. 직선성을유지하는분석가능범위는 Roche COBAS Amplicore에서 1.5 8.0 log 10IU/mL를, Roche Cobas Taqman HBV test는 1.3 8.2 log 10IU/ ml를, Abbott Real Time HBV kit는 1.0 9.0 log 10IU/mL를, Qiagen Artus HBV QS-RGQ Assay 는 1.5 7.3 log 10IU/mL를보였다고하며, 본연구에서 LG AdvanSure HBV Real-Time QPCR에서는 2.0 8.0 log 10IU/mL의분석가능범위를보였다. 20) 여기서검증한 LG AdvanSure HBV Real-Time QPCR kit의경우는, 표적을증폭하는중합효소연쇄반응법 (PCR) 으로오염으로인한위양성이나온도가낮거나, DNA의불완전변성이나검체채취나처리등에서의문제등에의한위음성을나타낸다. 21) 그러나본기기평가결과정상인에서의 LG AdvanSure HBV Real-Time QPCR kit의특이도는 100% 로나타났다. 국내에서사용되는 HBV DNA 정량법중가장널리보급된이세검사 kit 간의 HBV DNA copies/ml치는상관계수는 r 2 =0.95 0.97 (P<0.0001) 으로상관성과일치율이매우우수했고, 임상검 - 171 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 체에서시행한비교에서는 LG AdvanSure HBV Real-Time QPCR kit와 Abbott Real Time HBV kit 및 Roche Cobas TaqMan HBV Test와의성능을비교한것이므로비교한 kit들중어느쪽이우월하다는결론을내릴수는없다. 그러나결과를모두종합해본결과세종류의 kit는 HBV DNA 농도측정에있어서서로높은상관관계가있었다. 또한측정값은높은순으로 Roche Cobas TaqMan HBV Test, Abbott Real Time HBV kit, LG AdvanSure HBV Real-Time QPCR kit 순서로나타났다 (Fig. 2). 결론적으로본연구를통하여평가된 LG AdvanSure HBV Real-Time QPCR kit는정밀도, 직선성, 검출한계등의성능에있어우수하며, 기존검사법과의높은일치율을보이고, 다른실시간정량적중합효소연쇄반응 (RQ-PCR) 제품에비해손색이없다고판단되며, HBV DNA 정량을이용한만성 B형간염치료결정및추적관찰에유용하게사용될수있을것으로생각된다. 요약배경 : 정확한 HBV DNA의정량검사는만성 B 형간염환자에대한항바이러스제치료대상선정, 치료반응평가등에매우중요하다. 저자들은최근국내에서개발된실시간정량적중합효소연쇄반응을이용한 LG AdvanSure HBV Real- Time QPCR kit와 Abbott RealTime HBV Quantification kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test를비교평가하였다. 방법 : LG AdvanSure HBV Real-Time QPCR kit 의정밀도, 직선성, 검출민감도를평가하였다. 고려대안산병원에서치료를받고있는만성간염환자간염감염자 201명및정상인지원자 48예를대상으로세종류의실시간정량적중합효소 연쇄반응 HBV검사의상관분석을시행하고일치율을구하였다. 결과 : 직선성평가를위해 10 2 10 6 copies/ml 까지각농도별로 20회반복실험한결과에서변이계수는 0.58 1.19% 정도로 2% 미만의양호한 CV(%) 값을나타냈다. 정밀도판정을위해이론치와실측치에대한상관분석을실시한결과결정계수는 0.997이었다. 양성검체를 95% 검출가능한검출한계는 9.7 IU/mL였다. B형간염양성대상환자 201명에서검출한계이상의값을보인환자는 LG AdvanSure HBV Real-Time QPCR kit, Abbott RealTime HBV Quantification kit, Roche Cobas Ampliprep-Cobas TaqMan HBV test에서각기 74.1%, 61.1%, 74.1% 였다. HBV감염이없는것으로확인된정상인지원자 48명에서검출한계이하의값을보인환자는 LG AdvanSure HBV Real-Time QPCR kit, Abbott RealTime HBV Quantification kit, Roche Cobas Ampliprep-Cobas Taq- Man HBV test에서각기 0%, 2.1%, 10.4% 였으며검출한계이상의값을보인환자는한명도없었다. 세종류의 kit의상관관계는 Roche 대 Abott는 r 2 =0.91, Roche 대 LG r 2 =0.89, Abott 대 LG는 r 2 =0.94로나타났다 (n=105, 108, 133, r 2 =0.91, 0.89, 0.94, P<0.0001). 세가지 HBV DNA 방법의일치율 (overall concordance rate) 은각각 79.1% (159/ 201), 82.1% (165/201) 와 85.1% (171/201) 로세검사의전체일치율에는유의한차이가없었다 (P> 0.05). 결론 : LG AdvanSure HBV Real-Time QPCR kit 는정밀도, 직선성, 검출한계등의성능에있어우수하며, 기존다른실시간정량적중합효소연쇄반응법과의높은상관관계와일치율을보여임상검체를대상으로 HBV DNA 정량에타 RQ- PCR 제품에비해손색이없다고판단되며, 만성 B형간염치료결정및추적관찰에유용하게사 - 172 -
문정주외 : LG AdvanSure HBV Real-Time QPCR 용될수있을것으로생각된다. References 1. WHO. WHO web sites on infectious diseases. Hepatitis B. Fact Sheet No. 204. http://www. who.int/mediacentre/factsheets/fs204/en/ [Online] (last visited on 28 March 2013). 2. Di Bisceglie A, Lai CL, Gane E, Chen YC, Thongsawat S, Wang YM, et al. Telbivudine globe trial: maximal early HBV suppression is predictive of optimal two-year efficacy in nucleoside-treated hepatitis B patients. Hepatology 2006;44:230-1 3. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al; Adefovir Dipivoxil 438 Study Group. Longterm therapy with adefovir dipivoxil for HBeAgnegative chronic hepatitis B for up to 5 years. Gastroenterology 2006;131:1743-51 4. Yuen MF, Sablon E, Hui CK, Yuan HJ, Decraemer H, Lai CL. Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001;34:785-91 5. Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN, et al; REVEAL-HBV Study Group. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006;295:65-73 6. Pawlotsky JM. Hepatitis B virus (HBV) DNA assays (methods and practical use) and viral kinetics. J Hepatol 2003;39 Suppl 1:S31-5 7. Lee HC, Suh DJ, Ryu SH, Kim H, Shin JW, Lim YS, et al. Quantitative polymerase chain reaction assay for serum hepatitis B virus DNA as a predictive factor for post-treatment relapse after lamivudine induced hepatitis B e antigen loss or seroconversion. Gut 2003;52: 1779-83 8. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45:507-39 9. Kim SH, Ki CS, Park HK, Cho HJ, Kim JW, Park SS, et al. Annual report on external quality assessment in diagnostic genetics in Korea (2006). J Lab Med Qual Assur 2007;29: 153-74 10. Konnick EQ, Erali M, Ashwood ER, Hillyard DR. Evaluation of the COBAS amplicor HBV monitor assay and comparison with the ultrasensitive HBV hybrid capture 2 assay for quantification of hepatitis B virus DNA. J Clin Microbiol 2005;43:596-603 11. Pawlotsky JM, Bastie A, Hézode C, Lonjon I, Darthuy F, Rémiré J, et al. Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays. J Virol Methods 2000;85: 11-21 12. Clinical and Laboratory Standards Institute. Evaluation of the linearity of quantitative measurement procedures: a statistical approach: approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; EP6-A, Vol. 23, No. 16, April 2003 13. Clinical and Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; EP5-A2, Vol. 24, No. 25, August 2004 14. Clinical and Laboratory Standards Institute. Method comparison and bias estimation using patient samples; approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; EP9-A2, Vol. 22, No. 19, September 2002 15. KASL (President: Byung Chul Yoo). KASL clinical practice guidelines: management of chronic hepatitis B. Clin Mol Hepatol 2012;18: - 173 -
Korean J Blood Transfus Vol. 24, No. 2, 161-174, Aug. 2013 109-62 16. Lee WK. Characteristics of Hepatitis B Virus (HBV) and HBV DNA quantitative tests. Korean J Clin Microbiol 2003;6:89-96 17. Lindh M, Hannoun C. Dynamic range and reproducibility of hepatitis B virus (HBV) DNA detection and quantification by Cobas Taqman HBV, a real-time semiautomated assay. J Clin Microbiol 2005;43:4251-4 18. Kimura T, Rokuhara A, Matsumoto A, Yagi S, Tanaka E, Kiyosawa K, et al. New enzyme immunoassay for detection of hepatitis B virus core antigen (HBcAg) and relation between levels of HBcAg and HBV DNA. J Clin Microbiol 2003;41:1901-6 19. Kim MH, Cha CH, An D, Choi SE, Oh HB. Performance evaluation of Abbott RealTime HBV Quantification Kit for HBV viral load by real-time PCR. Korean J Lab Med 2008;28:144-50 20. Chevaliez S, Rodriguez C, Pawlotsky JM. New virologic tools for management of chronic hepatitis B and C. Gastroenterology 2012;142: 1303-13.e1 21. Quint WG, Heijtink RA, Schirm J, Gerlich WH, Niesters HG. Reliability of methods for hepatitis B virus DNA detection. J Clin Microbiol 1995;33:225-8 - 174 -