원저 Lab Med Online Vol. 4, No. 1: 28-35, January 임상화학 단클론감마병증에서면역글로불린중쇄및경쇄복합체직접측정의유용성 : 모세관전기영동과의수행능비교 D

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1 원저 Lab Med Online Vol. 4, No. 1: 28-35, January 임상화학 단클론감마병증에서면역글로불린중쇄및경쇄복합체직접측정의유용성 : 모세관전기영동과의수행능비교 Direct Measurement of Serum Immunoglobulin Heavy and Light Chain Pairs for Identification of Monoclonal Gammopathy and a Performance Comparison with Capillary Electrophoresis 강민구 1 신명근 1 김진각 2 장민중 1 이오진 1 김혜란 3 조덕 1 김수현 1 기승정 4 신종희 4 서순팔 4 양동욱 4 Min Gu Kang, M.D. 1, Myung-Geun Shin, M.D. 1, Jin-Gak Kim 2, Min-Joong Jang, M.D. 1, O-Jin Lee, M.D. 1, Hye-Ran Kim 3, Duck Cho, M.D. 1, Soo-Hyun Kim, M.D. 1, Seung-Jung Kee, M.D. 4, Jong-Hee Shin, M.D. 4, Soon-Pal Suh, M.D. 4, Dong-Wook Ryang, M.D. 4 화순전남대학교병원 1, 광양보건대학교임상병리과 2, 전남대학교의생명인력사업단두뇌한국 2 1 3, 전남대학교의과대학진단검사의학교실 4 Department of Laboratory Medicine 1, Chonnam National University Hwasun Hospital, Hwasun; Department of Clinical Pathology 2, Gwangyang Health College, Gwangyang; Brain Korea 21 Project 3, Center for Biomedical Human Resources, Chonnam National University, Gwangju; Department of Laboratory Medicine 4, Chonnam National University Medical School, Gwangju, Korea Background: Determination of monoclonal gammopathy through conventional protein electrophoresis is sometimes difficult because of the presence of large proteins such as haptoglobin and transferrin, which may obscure the results. Ambiguity in an electrophoresis band can give rise to confusion or difficulty in interpretation. The heavy chain/light chain assay (HLC assay) using Hevylite antibody (The Binding Site, UK) has recently been developed for the accurate measurement of monoclonal proteins. We compared the immunotyping (IT) profiles to the immunoglobulin (Ig) heavy/light chain measurements obtained using the HLC assay and observed the ratios between intact Ig kappa and lambda. Methods: We collected 35 and 28 sera from patients with suspicious and definitive monoclonal protein, respectively. Then we performed serum protein electrophoresis (SPEP) and IT by Capillarys2 (Sebia, USA). Monoclonal protein production was investigated using Freelite antibody (The Binding Site) and specific Ig(G, A)κ and Ig(G, A)λ Hevylite antibodies. The results were analyzed using PASW 18.0 for Windows (IBM, USA). Results: Direct measurement of Ig heavy/light chains showed discordant IT results for 12 (34.2%) of 35 patients sera with suspicious SPEP pattern and identical IT results for 28 patients sera with definitive monoclonal peak in the SPEP results. Overall, the results of the HLC assay and IT showed good agreement (κ=0.718, P =0.000 by cross-tabulation Gamma, Kappa analysis). Conclusions:The results of direct measurement of serum Ig heavy chain/light chain pairs were comparable to those of IT and were helpful for determination of monoclonality in the case of ambiguous electrophoresis results. Measurement of the heavy chain/light chain pair ratio also allowed precise quantification of the monoclonal Igs with ambiguous electrophoresis patterns and identification or discrimination of clonality. Key Words: Immunoglobulin, Monoclonal gammopathy, Multiple myeloma, Protein electrophoresis Corresponding author: Myung-Geun Shin Department of Laboratory Medicine, Chonnam National University Hwasun Hospital, 322 Seoyang-ro, Hwasun , Korea Tel: , Fax: , mgshin@chonnam.ac.kr Received: October 26, 2012 Revision received: January 14, 2013 Accepted: January 14, 2013 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. 서론 한가지클론의형질세포가대량으로증식하는경우에환자의혈청에있는특정단백의농도가증가하게되어전기영동에서뚜렷한밴드로보이게되고, 이를 M단백 (M-spike 또는 M-protein) 이라고한다. 이러한파라단백질 (paraprotein) 은단량체, 다량체, 또는유리경쇄나중쇄, 면역글로불린의조각과같은면역글로불린사슬일수있다 [1]. 이러한단클론성면역글로불린은다발골수종, 만성골수백혈병, 버킷림프종, B세포급성림프모구백혈병, 아밀로이드증, 악성종양, 28 eissn

2 자가면역질환등과같은다양한질환과연관되어있다. 또한다발골수종, 발덴스트롬마크로글로불린혈증과같은 B 세포종양에서는그농도가증가하므로, 관련질환의검출이나추적검사에있어중요한임상적의의를가진다. 특히다발골수종의치료반응평가에있어서중요한의미를가지는데, 면역고정전기영동 (IFE) 에서음성소견을보이는것은관해의주요한평가기준으로되어있다. 일반적으로혈청이나소변의단백전기영동 (SPEP) 이나면역고정전기영동 (IFE) 에의해이러한단세포군감마글로불린병증을선별하고있음이현실이다 [1, 2]. 하지만, 낮은농도의 M단백이거나 M단백이다른혈청단백과함께이동하는경우에는검출하기어려운측면이있다. 면역고정전기영동을함으로써이와같은문제점을어느정도해결할수는있으나, 정성검사이므로환자의치료에대한반응을정확히모니터링하는데사용할수없다 [3]. 전기영동의방법으로서최근많이사용되고있는모세관전기영동은자동화기법으로서겔을다루거나염색하는과정없이빠른분석이이루어진다. 이러한모세관전기영동에서는자외선을이용한직접검출방법으로겔전기영동보다나은특이도와재현성을보일수있으며면역고정이아니라특이한항혈청을이용한 immunosubtraction방법으로파라단백질을검출할수있다. 또한모세관전기영동은겔전기영동과유사한민감도와특이도를보이며일치율이높은방법으로서겔전기영동을대체할수있다고보고된바있다 [4, 5]. 한편 SPEP와함께혈청유리형경쇄검사를하게되면, 혈청과소변에서면역고정전기영동 (IFE) 을할때와유사한정도로관련질환의진단을위한분석능력을보일수있는데, 유리형경쇄의카파 / 람다 (κ/λ) 비율을측정함으로써면역고정전기영동보다더낮은농도에서도민감하게잔여질환이나재발을조기에검출할수있는장점이있다 [1]. 최근, 면역글로불린분자의중쇄와경쇄사이의결합부위를항원결정인자로인식하는다클론성항혈청 (Hevylite antibody) 이개발되었는데 (heavy chain/light chain assay, HLC assay), 이를이용하면각면역글로불린의개별적인경쇄형을구별할수있고 ( 예 ; IgGκ, IgGλ), 이것을쌍으로측정하여그결과를비율로나타냄으로써 ( 예 ; IgGκ/IgGλ) 결과적으로는특정한단클론성면역글로불린생산이증가하였는지그형별을구분하고, 정량까지할수있게되었다 [3]. 유리형경쇄검사와위에서언급된 HLC assay는비정상적으로증가하거나감소한 κ/λ 비율이있는지검사함으로써 M단백의존재를확인할수있는데, 비정상적으로증가한경우에는단클론성카파형면역글로불린임을알수있고, 비정상적으로감소한경우에는람다형면역글로불린임을알수있게된다 [6, 7]. 즉, HLC assay에서는 IgG, IgA, IgM 각자의개별적인카파형경쇄와람다형경쇄를각 각측정함으로써각종양클론이어떤형별을가지고있는지평가할수있고, 각자의비종양성면역글로불린이상대적으로얼마나억제되었는지에대한정량적인정보또한얻을수있다 [6]. 본연구에서는이러한혈청면역글로불린의경쇄형에대하여개별적으로측정하고그비율을알아보는것이단클론감마병증의진단에얼마나유용성을가지고있는지, 특히완전면역글로불린형의다발골수종환자에있어서얼마나유용한지에대하여알아보고자하였다. 대상및방법 1. 대상 모세관전기영동검사에서의심스럽거나애매한피크를보인경우혹은확실한단클론성피크를보인환자 57명으로부터얻은총 63개의잔여혈청을이용하였다. 대상환자는다발골수종 ( 완전면역글로불린 IgG 혹은 IgA형 ), 림프종, Castleman병으로화순전남대학교병원에서진단받았거나혹은추적관찰을위해서내원한환자였다. 성별분포는남자 36명, 여자 21명이었고, 연령분포는 43-81세였다. 위대상환자와는별도로다발골수종 ( 완전면역글로불린 IgGκ형 ) 으로진단받은 46세남자환자 1명에대해서는진단시점이후로총 6회의추적검사를시행하였다. 2. 방법모세관전기영동을위한검사장비로서 Capillarys2 (Sebia, USA) 을이용하여시행된혈청단백전기영동과 immunotyping결과를분석하여의심스럽거나애매한피크를보인경우혹은확실한단클론성피크를보인환자에서, 그와동시에시행된혈청유리형경쇄검사, 골수검사, 총면역글로불린, 나이, 성별을의무기록조회를통하여후향적으로검토하였으며, 본연구의흐름을그림으로제시하였다 (Fig. 1). 측정장비로는면역비탁장비인 SPA PLUS (The Binding Site Ltd., UK) 를사용하였다. Hevylite 항체 (The Binding Site Ltd.) 시약으로제조사의지시에따라서대상환자들의잔여혈청검체를이용하여면역글로불린중쇄및경쇄복합체와그비율을각각측정하여 ( 예 ; IgGκ, IgGλ, IgGκ/IgGλ), 정상참고범위 (IgGκ, g/l; IgGλ, g/l; IgGκ/IgGλ, ; IgAκ, g/l; IgAλ, g/l; IgAκ/IgAλ, ) 를벗어나서증가한경우에는 Ig κ 형의클론형성능, 감소한경우에는 Ig λ형의클론형성능이있는것으로판정하였다. 또한제조사에서제공되는고농도의 IgGκ, IgGλ, IgAκ, IgAλ 정도관리물질을정상혼합혈청으로단계별희석하여측정값과예상값을비교하였다. 통계분석에는 PASW 18.0 for Windows (IBM, USA) 을이용하였고, P 값이 0.05 미만인경우에통계적으로유의한것으로판단하였

3 <Phase I> Screening <Phase II> Validation Samples from patients who showed ambiguous SPEP pattern (N=35; MM 33, Lymphoma 1, Castleman s disease1) Samples from MM patients who showed definitive monoclonal peak in SPEP (N=28) Immunotyping: Negative (N=8) Immunotyping: Positive (N=27) Immunotyping: Positive (N=28) HLC assay Analysis of clinical & laboratory characteristics in patients Fig. 1. Schematic diagram and study plan. This study comprised2 phases: in phase I, the laboratory utility of direct measurement of serum immunoglobulin heavy/light chain pairs(the HLC assay) was determined. In phase II, the HLC assay was validated by testing samples showing a definitive monoclonal peak in SPEP and immunotyping. Abbreviations: SPEP, serum protein electrophoresis; MM, multiple myeloma; HLC assay, heavy chain/light chain assay. 다. 전기영동에서의심스럽거나애매한피크를보인군과확실한단클론성피크를보인군간에혈청유리형경쇄검사, HLC assay, 혈청단백 immunotyping에유의한차이가있는지를보기위해 Pearson s chi-square test와 independent sample t-test를시행하였다. 측정된 HLC assay결과와혈청단백전기영동및 immunotyping 결과간의일치성분석을위해서는 Gamma, Kappa 분석을시행하였고, 정도관리물질과정상혼합혈청의혼합에따른측정값과예상값간의상관성평가에는단순회귀분석을시행하였다. 또한추가로 IgGκ형의완전형면역글로불린다발골수종환자 1 명에대해서는, 진단이후로 6회에걸친골수검사소견, 혈청단백전기영동, immunotyping, 총면역글로불린 G, 혈청유리형경쇄검사결과를후향적으로검토하였고, 시기별로보관된잔여혈청 6검체로 HLC assay (IgGκ, IgGλ, IgGκ/IgGλ ratio) 를시행하여검사결과를비교하였다. 결과 모세관전기영동법으로시행된혈청단백전기영동검사에서의심스럽거나애매한피크혹은확실한단클론성피크를보인환자 57명의나이, 성별, 질환, 혈청 immunotyping 결과의분포는 Table 1과같다. 또한 57명의환자로부터얻어진 63개혈청으로시행되었던단백전기영동에서의심스럽거나애매한피크를보인군 (N = 35) 과확실한단클론성피크를보인군 (N =28) 의혈청유리형경쇄검사, HLC assay, 혈청 immunotyping결과의분포는 Table 2와같았다. 두군간에 HLC assay-iggκ, HLC assay-iggκ/iggλ ratio, serum immunotyping 결과에서는유의한차이를보였으나 (P < Table 1. Demographics and clinical characteristics of the study population 0.05), 혈청유리형경쇄검사, HLC assay-iggλ, IgAκ, IgAλ, IgAκ/ IgAλ ratio 의경우에는유의한차이가없었다. 63 개혈청검체의혈청단백전기영동, immunotyping, HLC assay, 혈청유리형경쇄검사결과는다음과같았다. 혈청단백전기영동에 서의심스럽거나애매한피크를보인 35 검체의 immunotyping 과 HLC assay 에의한클론형성능판정을비교해보면 23 검체 (23/35, 66%) 는일치하는소견을보였고, 12 검체 (12/35, 34%) 는 immunotyping 과 HLC assay 에의한클론형성능판정에있어서일치하지 않는소견을보였다. Study population (N=57) Age [yr, median (range)] 66 (43-81) Sex [N (%)] Male/Female 36 (63)/21 (37) Disease type [N (%)] Multiple myeloma 55 (96) Lymphoma 1 (2) Castleman s disease 1 (2) SPEP pattern [N (%)] Ambiguous or suspicious 33 (58) Definitive monoclonal peak 24 (42) Serum immunotyping result [N (%)] IgGκ 30 (53) IgGλ 12 (21) IgAκ 3 (5) IgAλ 4 (7) Negative 8 (14) Abbreviation: SPEP, serum protein electrophoresis

4 위에서언급된, immunotyping 과 HLC assay 간에불일치소견 을보인 12 검체의 HLC assay 와혈청유리형경쇄검사결과를살펴 보았을때 (Table 3), 12 검체중에서 8 검체는 immunotyping 에서는 Table 2. Characteristics of the clinical specimen based on serum protein electrophoresis (SPEP) patterns Suspicious peak (N=35) SPEP pattern Monoclonal peak (N=28) P values sflc assay (mean±sd) FLCκ (mg/l) 42.2± ±1, FLCλ (mg/l) 40.4± ± FLCκ:λ ratio 2.0± ± HLC assay (mean±sd) IgGκ (g/l) 6.8± ± * IgGλ (g/l) 3.5± ± IgGκ/IgGλ ratio 2.7± ± * IgAκ (g/l) 1.6± ± IgAλ (g/l) 1.6± ± IgAκ/IgAλ ratio 2.3± ± Serum immunotyping 0.001* IgGκ [N (%)] 10 (29) 23 (82) IgGλ [N (%)] 10 (29) 4 (14) IgAκ [N (%)] 3 (9) 0 (0) IgAλ [N (%)] 4 (11) 1 (4) Normal [N (%)] 8 (22) 0 (0) *Statistical differences in HLC-IgGκ, HLC-IgGκ/IgGλ ratios, and serum immunotyping results between the suspicious electrophoresis group and the definitive monoclonal peak group were evaluated (P <0.05); Reference range for the HLC assay: IgGκ, g/l; IgGλ, g/l; IgGκ/IgGλ, g/l; IgAκ, g/l; IgAλ, g/l; IgAκ/IgAλ, g/l. Abbreviations: Ig, immunoglobulin; sflc assay, serum free light chain assay; FLCκ, free light chain-kappa; FLCλ, free light chain-lambda; HLC assay, heavy/lightchain assay. IgGκ 또는 IgGλ형의 minor 클론형성능을보였으나 HLC assay에의해서는 8검체모두클론형성능이없었고, 동시에시행되었던혈청유리형경쇄검사에서는그 8검체중 3검체가 κ경쇄형이었다. 또한, immunotyping과 HLC assay 간에불일치소견을보인 12검체중 4검체는 immunotyping에서는정상또는다클론성이거나의심스러운소견을보였지만, HLC assay에의해서는 4검체모두 IgGκ 또는 IgAκ의클론형성능을보였고, 혈청유리형경쇄검사에서는그 4검체중 3검체가 κ경쇄형이었다. 한편혈청단백전기영동과 immunotyping에서확실한단클론성피크가확인된 28검체는 HLC assay에의해서도모두동일한클론형성능을보였으며, 각각의클론형성능의분포와그에따른 Ig κ, Ig λ, Ig κ/ig λ ratio 결과의중위수와범위는 Table 4와같았는데, IgGκ형은 23건, IgGλ형은 4건, IgAλ형은 1건이었고각각 Ig κ/ig λ ratio의중위수는 19.9, 0.21, 0.03를나타내어각검체의클론형성능을잘판정할수있었다. 또한, M단백의검출에있어서모세관전기영동법에의한 immunotyping을기준으로하여비교해보았을때, 혈청유리형경쇄검사와 HLC assay의민감도는각각 58.1%, 83.6% 였으며, 특이도는각각 50%, 37.5% 로나타났다. 위에기술된결과를종합해보면, 혈청단백전기영동에서애매하거나의심스러운피크 (N =35) 혹은확실한단클론성피크 (N =28) 를보인 63개의혈청검체를대상으로 HLC assay를시행한결과 51 건 (51/63, 81%) 에서동일한결과를확인할수있었고, HLC assay와혈청단백전기영동및 immunotyping 결과의일치성분석을위해서시행한 Gamma, Kappa analysis에서일치도 (κ) 는 0.718이었다 (P = 0.000). 또한고농도의정도관리물질 (IgGκ, IgGλ, IgAκ, IgAλ) 과정상혼 Table 3. Samples showing ambiguous serum protein electrophoresis (SPEP) patterns (12 of 35), which also showed discordance in the immunotyping results and HLC ratios Sample SPEP Immunotyping HLC ratios* sflc ratios* (κ/λ) Clinical status 1 Suspicious Minor IgGκ 2.54 FLCκ Diagnosis time point 2 Suspicious Minor IgGκ SD 3 Suspicious Minor IgGκ PR 4 Suspicious Minor IgGλ VGPR 5 Suspicious Normal/suspicious IgGκ 3.7 FLCκ 1.73 SD 6 Suspicious Minor IgGκ 2.28 FLCκ 1.89 PR 7 Suspicious Minor IgGλ PR 8 Suspicious Minor IgGκ 2.72 FLCκ 7.03 SD 9 Suspicious Minor IgGκ SD 10 Suspicious Normal/polyclonal IgGκ SD 11 Suspicious Normal/suspicious IgAκ 2.51 FLCκ 3.62 VGPR 12 Suspicious Normal/suspicious IgGκ 3.76 FLCκ 2.27 VGPR *In the case of only numerical data without Ig κ or FLCκ labeling, this indicates that the result of HLC assay or FLC assay was within the reference range (IgGκ/IgGλ, , IgAκ/IgAλ, ; FLCκ/λ, ); Described as per the response criteria given by the International Myeloma Working Group. Abbreviations: SPEP, serum protein electrophoresis; HLC ratios, heavy/lightchain assay κ:λ ratios; sflc assay, serum free light chain assay; FLCκ, free light chain-kappa; MM, multiple myeloma; SD, stable disease; PR, partial response; VGPR, very good partial response

5 Table 4. HLC concentrations and HLC immunoglobulin κ/λ ratios in patients with multiple myeloma (IgG and IgA) showing a definitive monoclonal peak inserum protein electrophoresis Tumor HLC type (g/l) N Median (range) IgGκ ( ) IgGλ ( ) IgAλ * Non-tumor HLC type (g/l) IgGλ ( ) IgGκ ( ) IgAκ * HLC ratios IgGκ/IgGλ ( ) IgGκ/IgGλ ( ) IgAκ/IgAλ * *Raw data were presented, because only IgA sera from 1multiple myeloma patient was assayed. Abbreviations: HLC, heavy/lightchain assay; SPEP, serum protein electrophoresis. 합혈청의혼합에따른단계별희석을하여실제측정되는값과예상되는값사이의선형회귀분석을해보았을때 (Fig. 2), IgGκ, IgGλ, IgAκ, IgAλ 각각에대해서 r2값은각각 0.981, 0.990, 0.994, 0.999였다 (P = 0.000). 위에언급된대상환자와는별도로, 1명의 IgG형다발골수종환자에대해서진단시점이후로부분관해, 자가조혈모세포이식후재발, 항암치료에의한이차관해에이르기까지기존의검사와 HLC assay 검사결과를비교한내용은다음과같았다. 진단시점으로부터 6회에걸쳐기존에시행되었던혈청단백전기영동, immunotyping, 총 IgG, HLC assay 결과를비교검토해보았을때 (Fig. 3), 일차항암치료로인하여부분적관해에이른때와이식후재발을보였던때에혈청단백전기영동, immunotyping에서 IgGκ형의클론형성능이있었으나, HLC assay에서는클론형성능을보이지않았다. 그후재항암치료에의한이차관해에이른이후에는혈청단백전기영동, immunotyping에서음성이었고, HLC assay에의해서도클론형성능이없다고판정되었다 Observed IgGκ (g/l) y=1.072x (r 2 =0.981, P=0.000) Observed IgGλ (g/l) y=0.968x (r 2 =0.990, P=0.000) Theoretical IgGκ (g/l) A Theoretical IgGλ (g/l) B Observed IgAκ (g/l) y=1.015x (r 2 =0.994, P=0.000) Observed IgAλ (g/l) y=1.006x (r 2 =0.999, P=0.000) Theoretical IgAκ (g/l) C Theoretical IgAλ (g/l) D Fig. 2. Linearity results of Hevylite Ig κ and Ig λ assays performed using a mixture of normal pooled sera with high concentration controls (simulation test by serial dilution). (A) IgGκ assay, (B) IgGλ assay, (C) IgAκ assay, (D) IgAλ assay

6 TCD Relapse Total IgG Monoclonal IgG IgGκ/IgGλ ratio 6 5 Total IgG (g/l) Auto-PBSCT Vel-TCD, PR Vel-vorinostat 2nd response Upper IgGκ/IgGλ NR 4 3 IgGκ/IgGλ ratio 30 45th Vel, CR SPEP/IT;negative Upper IgG NR ,000 1,200 1,400 Time (days) Fig. 3. Serial analysis of sera from an IgGκ multiple myeloma patient; a comparison of serum protein electrophoresis (SPEP), total immunoglobulin G (IgG), monoclonal IgG from SPEP densitometry, and HevyliteTM IgG κ:λ ratio. Abbreviations: IgG, immunoglobulin G; κ, kappa; λ, lambda; TCD, thalidomide plus cyclophosphamide plus dexamethasone treatment; auto-pbsct, autologous peripheral blood stem cell transplantation; Vel, velcade (bortezomib); IT, immunotyping; PR, partial response; CR, complete response; NR, normal range. 고찰 단클론감마병증을보이는질환들중에서다발골수종의대략적인빈도는전체혈액종양의 10% 이며전체종양의 1% 정도를차지한다. 또한그발생빈도는인종에따른뚜렷한차이를보이는데, 동양인에서는다른인종에비해일반적으로발생빈도가낮은것으로알려지고있으며, 우리나라에서다발골수종은드문질환이었다. 그러나, 최근우리나라에서도다발골수종의발생률과사망률이급격하게증가하고있다 [8]. 이러한다발골수종환자의진단, 치료및추적검사에최근활발히사용되고있는혈청유리형경쇄검사는단백전기영동뿐만아니라면역고정전기영동보다훨씬예민하며, 자동화장비를이용하여직접적으로정량보고가가능하고, 육안해석등의주관적인과정이배제된객관적인결과를얻을수있다는점에서 M단백을보이는환자들의진단과추적검사및예후판정에있어서도매우유용한표지자라할수있다 [3, 9]. Legg A 등 [3] 에의하면, 이러한혈청유리형경쇄검사와유사하게 HLC assay를통하여구할수있는 Ig κ/ig λ ratio 또한다발골수종환자의예후판정에있어서중요한의의를가질수있다고하였다. 본연구에서저자들은면역비탁법을이용한자동화장비로면역글로불린의중쇄와경쇄의결합부위를항원결정기로하는특이항체를사용하여, 각면역글로불린의경쇄형에대한개별적인정량검사를시행하여그비율을구하였고 ( 예 ; IgGκ/IgGλ) 이를통해서클론형성능의유무를판정하였다. Bradwell 등 [6] 의연구와 Keren [10] 에의하면, HLC assay는혈청단백전기영동보다 IgG형 M단백을검출하는데있어서약간더낮은분석능을보이며, IgA나 IgM형 M단백의검출에대해서는비슷하거나더나은민감도를나타내었고 M단백의형별결정에있어서도면역고정전기영동과거의동등한결과를보였다고하였다. 본연구에서도혈청단백전기영동, immunotyping과 HLC assay 결과를비교해보았을때, 의심스러운혈청단백전기영동소견을보인 35개의혈청검체중 8건에서 IgGκ형의클론형성능을 immu

7 notyping에서는확인할수있었으나, HLC assay에서는클론형성능이없다고판정되었다. 반대로, immunotyping에서클론형성능이없었으나 HLC assay에서는 IgGκ 또는 IgAκ형의클론형성능을보인 4건이있어서, 상기보고와같이 IgG형의 M단백을검출하는데있어서는 HLC assay가혈청단백전기영동보다는민감도가낮음을알수있었다 (Table 3). 따라서, HLC assay 단독검사를할경우에는단백전기영동보다 M단백검출의민감도가낮을수있으나, 기존의단백전기영동및 immunotyping과함께 HLC assay를하게되면 M단백검출의민감도를좀더높일수있을것으로사료된다. Table 3에서전기영동과 HLC assay 결과간불일치를보였던 12 예는모두전기영동에서애매한피크를보인경우로서, 다발골수종으로진단된시기의검체로검사가시행된 1예를제외하고는임상적으로매우좋은부분관해, 부분관해, 불변상태에해당하였다. 따라서, 재발하거나질환이악화된경우에 HLC assay로 M단백을검출하지못한경우는없었다. 또한, 전기영동및 immunotyping에서확실한단클론성피크를보인 28예에서는 immunotyping 과 HLC assay 모두 M단백검출및형별결정에서동일한결과를보여 (Table 4) HLC assay의유용성을확인할수있었다. 모세관전기영동법으로시행한 Immunotyping결과를기준으로하여혈청유리형경쇄검사와 HLC assay를비교해보았을때, M단백검출민감도는 HLC assay (83.6%) 가더높았으나, 특이도는혈청유리형경쇄검사 (50%) 가더높은것으로나타났다. 요약하자면, 이번연구에서혈청단백전기영동에서의심스럽거나애매한피크를보인경우 (N =35) 혹은확실한단클론성피크 (N =28) 를보인 63개의혈청검체를대상으로 HLC assay를시행한결과, 51건 (51/63, 81%) 에서모세관전기영동에의한 immunotyping과동일한결과를확인할수있었다. Immunotyping과 HLC assay에따른 clonality의판정은상호간에좋은일치도를보였으며 (k= 0.718, degree of agreement), 이는통계적으로유의함을알수있었다 (P = 0.000). 기존보고에서도혈청단백전기영동에서단클론성피크를보이거나의심스러운소견을보인 40개의검체로면역고정전기영동과 HLC assay를시행하여비교한결과, 그중 31 개의검체 (31/40, 78%) 에서동일한클론형성능판정을얻은바있다고하였다 [6, 10]. 또한고농도의정도관리물질 (IgGκ, IgGλ, IgAκ, IgAλ) 과정상혼합혈청의혼합에의해서측정값과예상값사이의회귀분석을한결과 (Fig. 2), IgGκ, IgGλ, IgAκ, IgAλ 각각에대해서 r 2 값은거의 1 에가까운유의한상관성을보였기에 (r 2 = 0.981, 0.990, 0.994, 0.999/ P value = 0.000), HLC assay의분석적민감도가우수함을확인할수있었다. 1명의 IgGκ type의다발골수종환자에대해서진단이후로 6회에걸쳐기존에시행된검사와 HLC assay 결과를비교하였을때 (Fig. 3), 일차항암치료로인하여부분적관해에이르는동안혈청단백전기영동, immunotyping에서는진단시보다는약하더라도여전히 IgGκ형의단클론형성능을보였다. 그러나, HLC-assay에서 IgGκ, IgGλ는모두정상범위보다증가하였더라도, IgGκ/IgGλ 비율은정상범위에있음으로인하여결과적으로는클론형성능이없는것으로판단되었다. 이식후재발한시기에도혈청단백전기영동, immunotyping에서 IgGκ형의클론형성능이있었으나, HLC assay에서는 IgGκ, IgGλ가모두정상범위보다증가하였더라도결과적으로는 IgGκ/IgGλ 비율에의한클론형성능판정에서음성으로간주되었다. 이와같은소견은위에서언급했던 IgG형 M단백의검출에있어서는혈청단백전기영동보다낮은민감도를보인다고했던바와일치하는것으로판단된다. 상기환자는그후재항암치료에의한이차관해에이른상태에서현재까지추적검사로 2번시행된혈청단백전기영동, immunotyping에서클론형성능을보이지않았고, HLC assay에의해서도 IgGκ, IgGλ, IgGκ/IgGλ 비율모두정상범위에속하여클론형성능이없다고판정되었다. 하지만, 바로이전의재발했던시기의 HLC assay-iggκ/iggλ 비율보다는약간씩증가하는양상이어서, 이것이다발골수종클론세포에의한 IgGκ 수치증가에따른것인지에대해서는앞으로의추적검사가더이루어진다음에서야판단할수있을것으로사료된다. 한편 Tovar 등 [11] 에따르면, IgG형다발골수종환자에서 IgAκ/ IgAλ 비율이증가하는것처럼종양세포클론과무관한면역글로불린의 Ig κ/ig λ 비율이상대적으로더증가하게되면총생존율과무진행생존율이유의하게증가함을예측할수있었으며, 이는인체면역체계의회복과관련지을수있는지표가될수있다고하여예후판정에있어서중요함을시사한바있다. 또한면역고정전기영동에서는 M단백과함께이동할수있는혈청내물질에의한간섭을받지않으며, 양클론감마병증, IgD와 IgE 형 M단백, 그리고 IgG/IgA/IgM 중쇄질환까지도검출할수있는등의장점이있기에 HLC assay가면역고정전기영동을완전히대체하기는힘들다 [10]. 하지만, 본연구에서보여진결과에서처럼 HLC assay는 M단백의검출및정량을위해추가적으로필요한, 중요한검사로서 M단백형별결정및정량을위한수치화된정보를재현성있게제공할수있으며, 위에서언급한바와같이다발골수종환자의예후판정에도도움을줄수있는유용한검사방법이라고하겠다. 요약 배경 : 전통적인단백전기영동에의한단클론감마병증의확인은합토글로빈, 트랜스페린과같은큰농도를가진단백에의해가려

8 지므로어려울수있다. 전기영동밴드에서의이와같은불확실성으로인해혼란스럽거나해석에어려움이있을수있다. 최근 M단백의정확한측정을위해서 Hevylite 항체 (The Binding Site, UK) 를사용하는 Heavy chain/light chain assay (HLC assay) 가개발되었기에, 면역전기영동에의한 M단백의형별판정과면역글로불린의중쇄및경쇄쌍의직접측정을하여얻은완전면역글로불린의카파및람다비율에의한형별판정의결과를비교하였다. 방법 : Capillarys2(Sebia, Norcross, USA) 에의한혈청단백전기영동과면역전기영동에서의심스러운소견을보인 35검체와확실한단클론성을보인 28검체를각각모았다. M단백의생산은 Freelite 항체 (The Binding Site, UK) 와특이적인 Ig(G, A)κ and Ig(G, A)λ Hevylite 항체 (The Binding Site) 에의해서도측정되었다. 검사결과는 PASW 18.0 에의해서분석되었다. 결과 : 면역글로불린중쇄 / 경쇄쌍의직접측정은의심스러운전기영동소견을보인 35검체중에서는 12검체 (34.2%) 에서면역전기영동에의한 M단백의형별판정과일치하지않았고, 혈청단백전기영동에서확실한단클론성피크를보였던 28검체에서는면역전기영동에의한 M단백의형별판정과모두일치한소견을보였다. 전반적으로, HLC assay와면역전기영동검사결과는 M단백의형별판정에있어서좋은일치율을보였다 (κ = 0.718, P = 0.000, 일치도분석 ). 결론 : 혈청면역글로불린중쇄 / 경쇄쌍의직접측정은면역전기영동과동등한유용성을보이며, 전기영동에서애매한단클론성을보이는경우형별판정을하는데도움이될수있다. 또한애매한전기영동소견을보이는 M단백의경우, 정량을하여형별판정의확인및감별을하는데유용하다고하겠다. REFERECNCES 1. Hortin GL. Amino acids, peptides, and proteins. In: Burtis CA, Ashwood ER, Bruns DE, eds. Tietz textbook of clinical chemistry and molecular diagnostics. 5th ed. Missouri: ELSEVIER Saunders, 2012: McPherson RA and Massey HD. Laboratory evaluation of immunoglobulin function and humoral immunity. In:McPherson RA, Pincus MR, eds. Henry s clinical diagnosis and management by laboratory methods. 22th ed. Philadelphia: ELSEVIER Saunders, 2011: Legg A, Harding S, Hughes RG, Levoguer AM, Bradwell AR. Serum free light chain and Hevylite analyses in the diagnosis, monitoring and prognosis of B cell disorders. Klin biochem metab2010;18: Yang Z, Harrison K, Park YA, Chaffin CH, Thigpen B, Easley PL, et al. Performance of the Sebia CAPILLARYS 2 for detection and immunotyping of serum monoclonal paraproteins. Am J Clin Pathol 2007;128: McCudden CR, Mathews SP, Hainsworth SA, Chapman JF, Hammett- Stabler CA, Willis MS, et al. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins. Am J Clin Pathol 2008;129: Bradwell AR, Harding SJ, Fourrier NJ, Wallis GL, Drayson MT, Carr- Smith HD, et al. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios. Clin Chem 2009;55: Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, et al. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002;48: Lee JH and Bang SM. Epidemiological change of multiple myeloma in Korea. Korean J Hematol 2006;41: Kang SY, Suh JT, Lee HJ, Yoon HJ, Lee WI. Establishment of serum reference range for free light chains and its clinical usefulness in multiple myeloma. Korean J Lab Med 2004;24: Keren DF. Heavy/Light-chain analysis of monoclonal gammopathies. Clin Chem 2009;55: Tovar N, Fernández de Larrea C, Elena M, Cibeira MT, Aróstegui JI, Rosiñol L, et al. Prognostic impact of serum immunoglobulin heavy/ light chain ratio in patients with multiple myeloma in complete remission after autologous stem-cell transplantation. Biol Blood Marrow Transplant 2012;18:

단클론단백을보이는질환의빈도와특성을살펴보고자한다. 그리고진단명을기준으로각검사들의진단적민감도를평가하고, 이를바탕으로단클론단백검출의선별검사로가장유용한검사또는검사의조합을찾아각선별검사들의진단적유용성을평가하고자한다. 대상및방법 1. 연구대상 1998년부터 2010년까지이대목동병원

단클론단백을보이는질환의빈도와특성을살펴보고자한다. 그리고진단명을기준으로각검사들의진단적민감도를평가하고, 이를바탕으로단클론단백검출의선별검사로가장유용한검사또는검사의조합을찾아각선별검사들의진단적유용성을평가하고자한다. 대상및방법 1. 연구대상 1998년부터 2010년까지이대목동병원 원저 Lab Med Online Vol. 2, No. 2: 80-86, April 2012 임상화학 형질세포종양의진단에서선별검사들의유용성검토 Evaluation of the Screening Tests for the Diagnosis of Plasma Cell Neoplasm 황유선 정화순 홍기숙 Yusun Hwang, M.D., Wha Soon Chung,