원저 Lab Med Online Vol. 7, No. 4: , October 임상화학 한국인에서 Soluble ST2 의참고치설정 Establishing Reference I

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1 원저 Lab Med Online Vol. 7, No. 4: , October 임상화학 한국인에서 Soluble ST 의참고치설정 Establishing Reference Intervals for Soluble ST Assay in a Korean Population 박미경 1 지미숙 김한아 1 문희원 1 허미나 1 윤여민 1 Mikyoung Park, M.D. 1, Misuk Ji, M.D., Hanah Kim, M.D. 1, Hee-Won Moon, M.D. 1, Mina Hur, M.D. 1, Yeo-Min Yun, M.D. 1 건국대학교의학전문대학원진단검사의학교실 1, 중앙보훈병원진단검사의학과 Department of Laboratory Medicine 1, Konkuk University School of Medicine, Seoul; Department of Laboratory Medicine, VHS Medical Center, Seoul, Korea Background: Soluble ST (sst) has emerged as a biomarker of heart failure. Previous studies indicated 35 ng/ml of sst as the clinically prognostic cut-off value. This study aims to establish reference intervals in a Korean population using an sst assay and to evaluate the applicability of the cut-off value. Methods: From March to May 014, sst levels were assayed in serum samples of 55 cardio-healthy Koreans (18 men and 17 women) using the Presage ST ELISA kit (Critical Diagnostics, USA). The reference interval for sst was defined using the nonparametric percentile method according to the CLSI EP8-A3c guideline. Results: The median sst concentrations were 3.8 ng/ml (interquartile range (IQR), ), 6.6 ng/ml (IQR, ), and 1.9 ng/ml (IQR, ) for the entire cohort, men, and women, respectively. sst levels were significantly higher in men than in women (P <0.0001). The 97.5th percentile upper reference limits for sst were 43.8 ng/ml, 49.6 ng/ml, and 35.4 ng/ml for the cohort, men, and women, respectively. Gender-specific upper reference limits were similar to limits reported by other studies. Conclusions: We suggest that gender-specific reference intervals should be used for the Korean population, as application of a single cut-off value of 35 ng/ml may be overcautious of the possibility of false positivity, especially in men. Key Words: Soluble ST, Reference interval, Korean, Heart failure 서론 Suppression of tumorigenicity (ST) 는 interleukin-1 수용체계 열의하나인단백으로세포막에결합되어있는 STL 과혈청내존 재하는수용성 ST (soluble ST, sst), 위장관에주로존재하는 변이형 STV 등 3 가지형태로구성되어있다 [1]. 혈청내 sst 농도 는심장질환및다양한염증질환에서증가한다 [1, ]. 전통적으로 Corresponding author: Yeo-Min Yun Department of Laboratory Medicine, Konkuk University School of Medicine, Konkuk University Medical Center, 10-1 Neungdong-ro, Gwangjin-gu, Seoul 05030, Korea Tel: , Fax: , ymyun@kuh.ac.kr Received: October 19, 016 Revision received: April 0, 017 Accepted: April 1, 017 This article is available from 017, 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. 급성호흡곤란의감별진단, 심부전의진단및예후판정을위하여 B-type natriuretic peptide (BNP) 또는 N-terminal pro-bnp (NTproBNP) 가사용되어왔으나 [3], sst가급 만성심부전환자의불량한예후를예측할수있는새로운표지자로최근주목받고있다 [1]. 그러나 sst는질병특이도가없어서심부전의진단보다예후에유용한표지자로평가되고있다 [1, ]. 인간 sst 농도를측정할수있는상업화된제품은 0개이상이나, 이중기존문헌에서주로사용된키트는 4가지이다 [1, ]. 이중 Presage ST ELISA kit (Critical Diagnostics, San Diego, CA, USA) 는혈액내 sst 측정을위한 ELISA 방법중유일하게미국식약처 (Food and Drug Administration) 승인및유럽 Conformité Européenne (CE) Mark를모두획득한방법으로측정한계는 1.3 ng/ ml, 측정범위는 ~00 ng/ml 이다 []. sst는단일기준치 (cut-off value) 를사용함으로써임상적판단이보다용이하다. 그러나현재 sst 농도측정을위해사용중인검사들은표준화가되어있지않으므로, 검사간결과값을호환하기곤란하고해당검사의참고치가중요하다 []. Lu 등 [4] 의연구에근거하여, Presage ST 검사의시약제조사는중앙 95% 참고범위 eissn

2 로남성 ng/ml, 여성 ng/ml를제시하고있고, 심부전예후판정을위한임상적기준치로 35 ng/ml를제시하고있다 [5]. 이임상적기준치는심부전예후판정에사용되어, HF-ACTION 코호트연구에서혈장 sst 농도가 35 ng/ml 보다높은경우사망률이유의하게높은것으로보고되었다 (log rank, P <0.001) [6, 7]. 오스트리아및미국등의참고집단에서 Presage ST 검사를이용하여 sst의참고범위를설정한기존연구들이있으나 [4, 8, 9], 한국의건강한성인에서어느정도의 sst 농도분포가나타나는지잘알려져있지않아, 참고범위설정이필요하다. 따라서본연구에서는한국의건강인참고집단에서 Presage ST 검사의참고범위를 CLSI EP8-A3c [10] 에따라설정하고, 기존의임상적기준치로알려져있는 35 ng/ml와비교하였다. 재료및방법 1. 대상 본연구는건국대학교병원기관윤리심의위원회의승인을받은후, 014년 3월부터 5월까지, 건국대학교병원의건강증진센터를방문한정상성인을대상으로하였다. 연구대상자는기존의연구 [11-13] 에근거하여다음과같은기준으로선택하였다. (1) 고혈압, 심장혈관질환등을포함한심장질환의경력이없을것 () 현재복용하는약물이없을것 (3) Modification of Diet in Renal Disease (MDRD) 방법으로측정한사구체여과율 (estimated Glomerular Fraction Rate, egfr) [14] >90 ml/min/1.73 m (40 세미만 ) >60 ml/min/1.73 m (40 세이상 ) (4) 당화혈색소 (HbA1c) <6.5% (5) B-type natriuretic peptide (BNP) <40 pg/ml 상기조건을만족하는남성 130명, 여성 130명을대상으로선정하였으나, 이중남성 1명이이완기고혈압으로진단받아제외되었고, Dixon-Reed 기준에따른이상치로남성 1명, 여성 3명이대상에서제외되었다 [15]. 최종대상자수는 55명 ( 남성 18명, 여성 17 명 ) 으로, 대상의연령범위는 18세에서 70세였다.. 방법혈청 sst의참고범위설정을위하여장비의분석능을평가하였다. 정밀도 (precision) 를평가하기위해 CLSI EP10-A3-AMD에따라, 저농도, 중간농도, 고농도의 3가지농도의환자의혈청을사용하여 5일에걸쳐 중간농도 - 고농도 - 저농도 - 중간농도 - 중간농도 - 저농도 - 저농도 - 고농도 - 고농도 - 중간농도 순으로검사하였다. 그결과로검사차례내, 검사일간및총정밀도 (within-run, between-day 및 total precision) 변이계수 (coefficient of variation, CV%) 를계산하였다 [16]. 직선성 (linearity) 을평가하기위해, CLSI guideline EP6-A에따라저농도와고농도검체를일정비율로혼합하여총 5가지의농도의검체를준비하여각농도별로 4번반복하여검사하였다 [17]. CLSI EP10-A3-AMD에따라 5일간잔효 (carryover) 를계산하였다 [16]. 총대상자의각각남녀군에서 -80 C에보관된혈청잔여검체에서 Presage ST ELISA Assay (Critical Diagnostics, San Diego, CA, USA) 를사용하여 sst를 번측정하여, 평균값을사용하였다. 혈청 sst의참고범위는 CLSI EP8-A3c에따라설정하였다 [10]. sst의측정값에서 Dixon-Reed 기준에따라이상치를제거한후 [15], 비모수적방법을사용하여.5 퍼센타일에서 97.5 퍼센타일 ( 중앙 95%) 을참고범위로설정하였다. 통계처리는 MedCalc Software version 15.. (MedCalc Software bvba, Ostend, Belgium) 와 Analyse-it (Analyse-it Software Ltd., Leeds, UK) 을이용하였다. 남녀군의혈청 sst의중앙값을비교하기위해, Mann-Whitney U test를사용하였고, P <0.05를통계학적으로유의한차이가있는것으로간주하였다. 결과 검사차례내정밀도 (CV%) 는저농도에서 3.3%, 중간농도에서 1.7%, 고농도에서 3.7% 였다. 검사일간정밀도는저농도에서 3.8%, 중간농도에서 3.8%, 고농도에서.9% 였다. 총정밀도는저농도에서 5.0%, 중간농도에서 4.%, 고농도에서 4.7% 였다. 검사차례내, 검사일간및총정밀도는제조사에서제시하는범위 (.4-9.1%) 내에있었다 [5]. 직선성의평가에서, 4.3 ng/ml에서 19.9 ng/ml 까지직선성을확인하였고 (r = 0.996), 5일간시행한잔효는 -0.54% 부터 +0.4% 로허용범위내에있었다 [17]. 총 55명의대상자중남성은 18명 (50.%) 여성은 17명 (49.8%) 이었다. 연령평균 (mean) 은총대상자 45.0±8.세, 남성 46.0±7.8 세, 여성 44.0±8.6세였다. 사구체여과율 (egfr) 이 90 ml/min/ 1.73 m 를초과한대상자는총 80명 ( 남성 9명, 여성 51명 ) 이었고, ml/min/1.73 m 사이의대상자는총 175명 ( 남성 99명, 여성 76명 ) 이었다. 당화혈색소 (HbA1c) 의평균은총대상자 5.3±0.3%, 남성 5.3±0.3%, 여성 5.3±0.3% 였다. BNP 값이측정한계치미만 (<10 pg/ml) 인대상자는총 140명 ( 남성 94명, 여성 46명 ) 이었고, pg/ml 사이의대상자는총 115명 ( 남성 34명, 여성 81명 ) 이었다. 혈청 sst 참고범위의.5 퍼센타일은총대상자 1.6 ng/ml (90% 신뢰구간, ), 남성 13.4 ng/ml (90% 신뢰구간, ), 여성 1.1 ng/ml (90% 신뢰구간, ) 이었다. 혈청 sst 농도의 97.5 퍼센타일은총대상자 43.8 ng/ml (90% 신뢰구간,

3 50.7), 남성 49.6 ng/ml (90% 신뢰구간, ), 여성 35.4 ng/ml (90% 신뢰구간, ) 이었다 (Table 1). 혈청 sst 농도의중앙 값 ( 사분범위 ) 은총대상자 3.8 ( ) ng/ml, 남성 6.6 ( ) ng/ml, 여성 1.9 ( ) ng/ml 이었고 (Table 1), 남성이여 Table 1. Comparison of previously reported sst reference intervals with values from the Presage ST ELISA assay* Reference Sample size Age (yr) Specimen Unit Dieplinger et al. [8] Lu et al. [4] Coglianese et al. [9] ll Manufacturer [5] Present study N (M)=338 N (F)=190 N (M)=45 N (F)=45 N (M)=46 N (F)=674 N (M)=45 N (F)=45 N (M)=18 N (F)= Median sst (sex) Plasma U/mL 13 (M) Plasma ng/ml 4.9 (M) Plasma, serum Plasma, serum Reference intervals Total Male Female 8 (F) Not reported (F) Not reported ng/ml Not available Not reported (.5th, E) (97.5th, E) (.5th, Q) (97.5th, Q) ng/ml 3.6 (M) 16. (F) 18.8 (T) Serum ng/ml 6.6 (M) 1.9 (F) 3.8 (T) (.5th, E) (97.5th, E) (.5th, Q) (97.5th, Q) *All reference intervals were calculated using a non-parametric percentile method (95%, double sided.) except in only one study [9]. Median sst values were presented except in one study [4]. Median sst values were significantly different according to sex (Mann-Whitney U test, P <0.001). Mean sst concentrations in the male group were significantly higher compared to that in the female group (P <0.0001). ll Reference intervals according to sex and age was estimated using both empirical and quantile regression methods from a healthy reference sample in the Framingham Heart Study [9]. Abbreviations: sst, soluble suppression of tumorigenicity ; ELISA, enzyme-linked immunosorbent assay; M, males; F, females; T, total; E, empirical reference limits; Q, quantile regression reference limits. 80 Distribution of sst in total subjects (n=55) Median: 3.8 ng/ml.5th percentile (90% CI): 1.6 ng/ml ( ) 97.5th percentile (90% CI): 43.8 ng/ml ( ) Distribution of sst in male subjects (n=18) Median: 6.6 ng/ml.5th percentile (90% CI): 13.4 ng/ml ( ) 97.5th percentile (90% CI): 49.6 ng/ml ( ) Frequency Frequency sst (ng/ml) A sst (ng/ml) B Distribution of sst in female subjects (n=17) Median: 1.9 ng/ml.5th percentile (90% CI): 1.1 ng/ml ( ) 97.5th percentile (90% CI): 35.4 ng/ml ( ) Frequency sst (ng/ml) C Fig. 1. Distributions of sst concentrations and reference intervals measured by the Presage ST ELISA kit in the entire cohort (A), males (B), and females (C). Dotted lines represent the.5th percentile and 97.5th percentile limits. Abbreviations: sst, soluble suppression of tumorigenicity ; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay

4 성보다유의하게높았다 (Mann-Whitney U test, P <0.0001). sst 의심부전예후판정의임상적기준치로알려져있는 35 ng/ml [5-7] 를초과한대상자는총 55명대상자중 7명 (10.6%) 이었고, 이중남성이 4명, 여성이 3명이었다. 한국의건강인참고집단에서 Presage ST 검사를사용하여측정한혈청 sst 참고범위의분포가 Fig. 1에나와있다. 혈청 ST 농도의 97.5 퍼센타일기준치 (43.8 ng/ml) 를초과한대상자총 6명의 hs-tni, 혈액요소질소 (BUN), 크레아티닌 (creatinine), 중성지방 (triglyceride), 총콜레스테롤 (total cholesterol), 저밀도지단백콜레스테롤 (LDL-cholesterol), 고밀도지단백콜레스테롤 (HDL-cholesterol) 의측정값은모두각검사의참고치이내였고, 심질환과관련된특이적인임상병력이없었다. 고찰 성능평가에서재현성이 5% 이내였고, 잔효가없어서혈청 sst 의참고범위설정에문제없음을확인하였다. 본연구는한국의건강인참고집단에서 Presage ST 검사를사용하여혈청 sst 참고범위를설정한첫번째연구이다. Table 1에서보여주는것처럼, 본연구에서성별에따른 sst의참고범위의상한치는한연구를제외하고대체로기존연구들과제조사에서제시한값과비슷하였다 [4, 5, 8, 9]. Lu 등 [4] 은미국의참고집단에서비모수적방법으로설정한혈장및혈청 sst 농도의중앙 95% 참고범위로남성 ng/ml, 여성 ng/ml로제시하였다. Coglianese 등 [9] 은 Framingham Heart 코호트의참고집단 1,136명을대상으로경험적방법으로계산한혈장 sst농도의 97.5% 참고범위를남성에서 ng/ml, 여성에서 ng/ml로제시하였다. Dieplinger 등 [8] 은오스트리아의참고집단에서중앙 95% 의참고범위로남성 4-31 U/mL, 여성 -1 U/mL로보고하였다. 한편, 총대상자의혈청 sst 농도의상한참고치는제조사의상한참고치보다높았다 [5]. 본연구에서는남성의혈청 sst 농도의평균값과상한참고치가여성보다유의하게높았다 (Table 1). 이는기존의연구와크게다르지않았다 [4, 8]. 그리고심부전예후의임상적기준치인 35 ng/ml을초과한총 7명중에서 4명이남성이차지하였다. Lu 등 [4] 의연구에근거한제조사의참고집단에서도 sst가 35 ng/ml를초과한대상자수는남성이여성보다많았다 [5]. 남성이여성보다 sst 농도가높은원인중의하나로안드로겐에의한 sst의합성혹은분비조절의가능성이제기되었다 [1, ] 그러나 Dieplinger 등 [18] 은 sst와성호르몬사이의유의한연관성을발견하지못했다. 성별에따른 sst 농도차이의원인이명확하게밝혀지지않아, 아직더많은연구가필요하다. 남녀의혈청 sst의농도분포에차이 가있으므로, 성별에따라혈청 sst의참고범위를구별하여사용할필요가있다. sst 농도는급성및만성신부전, 관상동맥질환, 만성폐쇄성폐질환, 폐렴, 패혈증, 급성호흡기증후군등의다양한질환에서증가하고, 기존연구에서보고된건강인의혈중 sst의참고범위는심부전환자를포함한다양한질환의혈중 sst 농도범위와중복되어있다 []. 따라서 35 ng/ml의기준치로심부전의예후를판정하는것은특히남성에게서위양성의결과를보일수있으므로유의해야한다. Wu 등 [19] 은 sst의결과해석을위해서는연속적인검사가필요하고, 기준치 (baseline) 에서변할경우심부전진행을나타낼수있으며, 만성심부전환자에서기준치보다 배초과하여증가할경우그결과가성별특이적인참고범위내에있다고하더라도, 의미있는증가로고려되어야할것을제시하였다. 한편, sst 의참고범위를해석하고, sst의농도로심부전의진행을예측하기위해서는생물학적변이의특성을고려할필요가있다. Wu 등 [19] 의연구에서 17명의건강인을대상으로 Presage ST 검사를사용하여혈장 sst 농도를 주간격으로 8주간측정하였다. 혈장 sst 평균값은 9 ng/ml로 1-75 ng/ml 의분포를보였고, 개인내변이와개인간변이및참고변화값 (Reference Change Value) 은각각 11%, 46%, 30% 였다. sst농도는심부전치료에의해감소할수있고, 심부전예후의기준치로 30% 의참고변화값이사용될수있다 [, 0]. 이연구는몇가지제한점을가지고있다. 첫째로, 대상자선정기준에건강력, 사구체여과율, 당화혈색소, BNP만을포함하였다. sst가증가할수있는상황을배제할수있는다양한검사들을포함하지않았다. 따라서이연구의건강인대상자중에, 특히남성에서, 무증상의심장질환등의환자가일부포함되었을가능성이있다. 그리고연령의증가에따른참고치증가, 특히여성의경우에 65세이상에서많이증가한다는보고 [9] 를고려할때에연령군에따른참고치설정은고려하지못했다는한계점이있다. 둘째로, 기존연구들에비하여대상자수가적다는것이다. Dieplinger 등 [8] 은 58명, Lu 등 [4] 은 490명, Coglianese 등 [9] 은 1,136명의참고집단을대상으로한것에비해, 이연구에서는총 55명을대상으로하였다. 따라서, 이연구의대상자군이전체한국인집단을대표하지못할가능성이있다. 결론적으로, Presage ST 검사를사용한한국의건강인참고집단에서혈청 sst의성별에따른상한참고치는기존연구들의결과와유사하였다. 성별에따라혈청 sst의농도분포에차이가있으므로, 성별특이적인참고치를사용할것이권장된다. 남성의경우혈청 sst의결과값이건강한성인에서도심부전예후기준으로사용되는 35 ng/ml 보다높은경우가있으므로, 임상적적용시이에대한고려가필요하다

5 요약 배경 : Soluble ST (sst) 는새로운심장표지자이다. 기존연구들에서, 심부전예후를판정하는임상적기준치로 35 ng/ml가사용되었다. 이연구의목표는한국인에서 sst의참고범위를설정하고, 임상적기준치의진단적적용을평가하는것이다. 방법 : 014년 3월부터 5월까지, 총 55명의건강인 ( 남성 18명과여성 17명 ) 의혈청잔여검체로 Presage ST ELISA assay (Critical Diagnostics, USA) 를사용하여 sst을측정하였다. CLSI EP8-A3c 가이드라인에따라비모수적퍼센타일방법을사용하여 sst의참고범위를설정하였다. 결과 : 혈청 sst 농도의중앙값 ( 사분범위 ) 은총대상자, 남성, 그리고여성에서각각 3.8 ( ) ng/ml, 6.6 ( ) ng/ml, 1.9 ( ) ng/ml 였다. 혈청 sst 농도는남성이여성보다유의하게높았다 (P <0.0001). 혈청 sst의 97.5 퍼센타일상한참고치는총대상자, 남성, 그리고여성에서각각 43.8 ng/ml, 49.6 ng/ml, 35.4 ng/ml 였다. 성별에따른상한참고치는기존연구들의결과와유사하였다. 결론 : 성별에따라혈청 sst의농도분포가다르므로, 성별특이적인참고범위를사용할필요가있다. 건강인에서심부전의임상적기준치인 35 ng/ml을적용하는것은, 특히남성에서위양성결과를보일수있으므로유의해야한다. 이해관계 저자들은본연구와관련하여해당회사와이해관계가없음. 참고문헌 1. Dieplinger B and Mueller T. Soluble ST in heart failure. Clin Chim Acta 015;443: Mueller T and Jaffe AS. Soluble ST-analytical considerations. Am J Cardiol 015;115:8B-1B. 3. Yoo B-S. Biomarkers in heart failure: focus on B-type natriuretic peptide. Korean J Med 01;8: Lu J, Snider JV, Grenache DG. Establishment of reference intervals for soluble ST from a United State population. Clin Chim Acta 010;411: Critical diagnostics. Use of ST in Conjunction with Traditional clinical evaluations. Presage_ELISA_Indication_For_Use.pdf (Updated on July 017). 6. US Food and Drug Administration. 510(k) Premarket Notification. (Updated on July 017). 7. Felker GM, Fiuzat M, Thompson V, Shaw LK, Neely ML, Adams KF, et al. Soluble ST in ambulatory patients with heart failure: association with functional capacity and long-term outcomes. Circ Heart Fail 013; 6: Dieplinger B, Januzzi JL, Steinmair M, Gabriel C, Poelz W, Haltmayer M, et al. Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble ST in human plasma--the Presage ST assay. Clin Chim Acta 009;409: Coglianese EE, Larson MG, Vasan RS, Ho JE, Ghorbani A, McCabe EL, et al. Distribution and Clinical Correlates of the Interleukin Receptor Family Member Soluble ST in the Framingham Heart Study. Clin Chem 01;58: Clinical and Laboratory Standards Institute. Defining, establishig, and verifying reference intervals in Clinical laboratory; Approved guideline-third edition. CLSI document EP8-A3C. Wayne PA: Clinical and Laboratory Standards Institute, McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Böhm M, Dickstein K, et al. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 01: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 01 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur J Heart Fail 01;14; Gillett MJ. International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes, Diabetes Care 009;3: Clin Biochem Rev 009;30: Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 01 clinical practice guideline for the evaluation and management of chronic kidney disease. uploads/017/0/kdigo_01_ckd_gl.pdf (Updated on July 017). 14. Levey AS, Goresh J, Greene T, Stevens LA, Zhang YL, Hendriksen S, et al. Using standardized serum creatinine values in the modification of diet in renal disease study equation for estimating glomerular filtration rate. Ann Intern Med 006;145: Reed AH, Henry RJ, Mason WB. Influence of statistical method used on the resulting estimate of normal range, Clin Chem 1971;17: Clinical and Laboratory Standards Institute. Preliminary evaluation of quantitative clinical laboratory measurement procedures; Approved guideline-third edition. CLSI document EP10-A3-AMD. Wayne PA: Clinical and Laboratory Standards Institute, Clinical and Laboratory Standards Institute. Evaluation of the linearity

6 of quantitative measurement procedures: a statistical approach; Approved guideline. CLSI document EP06-A. Wayne PA: Clinical and Laboratory Standards Institute, Dieplinger B, Egger M, Poelz W, Gabriel C, Haltmayer M, Mueller T. Soluble ST is not independently associated with androgen and estrogen status in healthy males and females. Clin Chem Lab Med 011;49: Wu AH, Wians F, Jaffe A. Biological variation of galectin-3 and soluble ST for chronic heart failure: implication on interpretation of test results. Am Heart J 013;165: Mueller T and Dieplinger B. Soluble ST and Galectin-3: What We Know and Don t Know Analytically. EJIFCC. 016;7:

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