원저 Lab Med Online Vol. 3, No. 1: 29-33, January 2013 진단면역학 2010 ACR/EULAR 류마티스관절염의분류체계에대한항핵주변인자검사의적용에관한예비연구 A Preliminary Study for Applying Antiperinuclear Antibody Test to 2010 ACR/EULAR Classification Criteria for Rheumatoid Arthritis 최종문 1 김신규 2 Jong-Moon Choi, M.D. 1, Think-You Kim, M.D. 1,2 한양대학교의료원진단검사의학과 1, 조기관절염과 2 Departments of Laboratory Medicine 1, Early Arthritis 2, Hanyang University Medical Center, Seoul, Korea Background: The American College of Rheumatology/European League against Rheumatism classification criteria for rheumatoid arthritis (ACR/EULAR criteria) include the rheumatoid factor (RF) test and the anti-citrullinated peptide/protein antibody (ACPA) test as serologic makers for rheumatoid arthritis. Antiperinuclear factor (APF) test, an originator of ACPA, is highly specific for rheumatoid arthritis and can be detected in RF or anti-cyclic citrullinated peptide (anti-ccp) negative rheumatoid arthritis, but it is not included in the serologic criterion of ACR/EULAR criteria. In this study, we investigated the way for applying the APF test to ACR/EULAR criteria. Methods: We analyzed clinical symptoms and laboratory findings of 53 patients who were suspected having rheumatoid arthritis. All patients were negative for the RF and anti-ccp and positive for APF. We classified these patients into 4 groups according to the fluorescence intensity of APF test, and gave 1-4 points for APF positivity. The proportion of patients who scored 6 or greater in ACR/EULAR criteria in relation to APF scores was evaluated. Results: Median scores of ACR/EULAR criteria showed a tendency to increase as the level of fluorescence intensity of APF rises, but ACR/EULAR scores of 4 groups were not different significantly from each other (P >0.05). The proportion of patients who scored 6 or greater in ACR/EULAR criteria were 39.6% and 77.4%, when scores of APF positivity were 2 and 3 points, respectively. Conclusions: We think it is reasonable to include APF test in the ACPA of ACR/EULAR criteria and give 3 points for APF positivity, regardless of its fluorescence intensity. Key Words: Antiperinuclear factor, Rheumatoid arthritis, 2010 ACR/EULAR criteria 서론 지난 2010 년도에류마티스관절염의새로운분류체계인 American College of Rheumatology/European League against Rheuma- Corresponding author: Think-You Kim, M.D. Department of Early Arthritis and Laboratory Medicine, Hanyang University Medical Center, 222 Wangsimri-ro, Seongdong-gu, Seoul 133-792, Korea Tel: +82-2-2290-8975, Fax: +82-2-2298-1735, E-mail: tykim@hanyang.ac.kr Received: September 27, 2012 Revision received: October 8, 2012 Accepted: October 8, 2012 This article is available from http://www.labmedonline.org 2013, Laboratory Medicine Online 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. tism classification criteria for rheumatoid arthritis (ACR/EULAR criteria) 가발표되었다 [1]. 이분류체계는류마티스인자 (rheumatoid factor) 와 anti-citrullinated peptide/protein antibody (ACPA) 검사를통해환자의혈청학적특성을평가하도록하였으며그정량적결과에따라각기다른점수를부여하여진단에도움이되도록하고있다. ACPA 의검출을위해여러검사방법이시도되었으나오늘날대부분의검사실에서는 anti-cyclic citrullinated peptide antibody (anti-ccp) 검사를류마티스관절염의진단에활용하고있다. 반면 ACPA 검사의시초라고할수있는항핵주변인자 (antiperinuclear factor) 검사는류마티스관절염에대한높은진단특이도를가지고있음에도불구하고 [2-4] 검사표준화및기질확보의문제로인하여널리사용되지는않았다. 그러나국내에서상품화된항핵주변인자검사키트 (IT-APF)[5] 가개발되면서일반검사실에서도항핵주변인자를검사할수있는길이열렸다. eissn 2093-6338 www.labmedonline.org 29
항핵주변인자검사는류마티스관절염의관절손상을조기에예측할수있는지표중하나로서 [6] 저자들은대규모연구를통해항핵주변인자검사가 anti-ccp 및류마티스인자음성인류마티스관절염의진단에보완적역할을수행할수있음을밝힌바있다 [7, 8]. 하지만항핵주변인자의진단적가치에도불구하고이를 ACR/ EULAR criteria에적용하여류마티스관절염을진단할수있는지침은아직없는실정이다. 이에저자들은류마티스관절염의심환자를대상으로시행한항핵주변인자검사, anti-ccp 검사및류마티스인자검사결과를평가하여항핵주변인자의 ACR/EULAR criteria에대한적용방안에대해연구하고자본연구를시행하였다. 대상및방법 1. 대상 류마티스관절염이의심되어본원에내원한환자중항핵주변인자검사양성, anti-ccp 및류마티스인자음성인환자 53명을대상으로임상증상및검사결과를평가하였다. 환자들은남성 11명, 여성 42명, 평균연령은 53.9 세였으며질환의이환기간은모두 6개월이상이었다. ACR/EULAR criteria상점수가 6점이상인류마티스관절염환자및전신성홍반성루프스, 섬유근육통등다른류마티스질환으로관절부위의통증이발생한환자는본연구에서제외되었다. 2. 검사방법환자선별시사용한 anti-ccp 검사와류마티스인자검사는각각 2세대 ELISA 검사키트 (ImmuLisa CCP ELISA, Immco Diagnostics, Buffalo, USA) 와 nephelometry (BN II nephelometer, Simens Healthcare Diagnostics Inc., Newark, USA) 검사장비를이용해시행하였으며음성기준은제조사의권고에따라각각 25 U/mL 및 15 IU/mL 이하인경우로하였다. 항핵주변인자검사는환자의혈청을 1:20으로희석하여상품화된검사키트 (IT-APF, ImmunoThink Co., Seoul, Republic of Korea) 를이용해간접면역형광법으로시행하였으며형광현미경 (Nikon Eclipse E80i, Nikon, Tokyo, Japan) 으로관찰했을때핵주변부에위치하는원형입자가관찰되는경우그형광강도에따라아래와같이 1+ 에서 4+ 까지구분하여보고하였다. 4+: Maximal fluorescence, brilliant yellow-green 3+: Less brilliant yellow-green fluorescence 2+: Less brilliant, but definite fluorescence 1+: Very subdued fluorescence 3. ACR/EULAR criteria 점수평가및항핵주변인자검사의점수체계설정대상환자의임상증상및검사결과를조사하고이를통해 ACR/ EULAR criteria의점수를계산하였다. 통증및부종이있는관절의수와종류및위치를조사하고 ACR/EULAR criteria에서제시하는기준 [1] 에따라관절의종류를대관절 (large joint) 과소관절 (small joint) 로분류하였으며 0에서 5점까지점수를측정하였다. 적혈구침강속도 (erythrocyte sedimentation rate, ESR), C 반응성단백 (Creactive protein, CRP) 검사를통해급성기반응물질에대한점수를평가하였으며두검사중하나이상양성인경우 1점, 둘다음성인경우 0점을부여하였다. 질병의이환기간에따른점수는본연구에서조사한환자모두질병이환기간이 6주이상이었으므로전부 1점을부여하였다. 이를관절이환정도를평가한점수및급성기반응물질검사의점수를합산하여혈청학적검사를제외한 ACR/EULAR criteria의전체점수를평가하였다. 항핵주변인자검사의형광강도 (1+, 2+, 3+, 4+) 에따라환자를 4 개의그룹으로분류하고 ACR/EULAR criteria의점수를평가한후그룹간에점수부여방식에따라총점이 6점이상이되는환자의비율양상이어떻게변하는지조사하였다. 4. 통계분석항핵주변인자검사결과에따른이환된관절의수, 급성반응기물질검사, ACR/EULAR criteria 점수를비교하기위하여 Kruskal- Wallis 검정을시행하였다. 통계분석도구로는 SPSS 12.0 (SPSS Inc., Chicago, IL, USA) 를이용하였으며, 통계적유의수준은 P < 0.05로하였다. 결과 1. 항핵주변인자검사결과및환자의임상적특성비교조사대상이된 53명의환자중 31명이항핵주변인자검사상 1+, 16명이 2+, 5명이 3+, 1명이 4+ 였다. ESR 및전체 ACR/EULAR criteria 점수의중앙값 (median) 을비교했을때항핵주변인자검사의형광염색강도에따라그수치가증가하는양상을보였으나통계적으로유의한차이는없었다 (Table 1). 2. 항핵주변인자검사의점수부여에따른 ACR/EULAR criteria 점수변화비교 53명전체의 APF 결과와이에따른 ACR/EULAR criteria 점수의분포및 APF에점수를부여할경우점수변화양상은다음과같았다 (Tables 2, 3). 형광염색강도와상관없이양성일때 1점을부여한경우전체 30 www.labmedonline.org
13.2%, 2 점부여시 39.6%, 3 점부여시 77.4%, 4 점부여시 98.1% 의환자가 6 점이상의 ACR/EULAR criteria 점수를받을수있었다. 항핵주변인자검사의형광강도가 1+ 인경우와, 2+, 3+, 4+ 인경 우에각각 1 점과 2 점을부여했을때는 28.3%, 2 점과 3 점을부여했 Table 1. Median values of CRP, ESR, number of joint involvement and score of ACR/EULAR criteria according to the fluorescence intensity of APF test Fluorescence intensity of APF 1+ (N=31) 2+ (N=16) 3+ (N=5) 4+ (N=1) P value CRP (mg/dl) <0.3 <0.3 <0.3 <0.3 NS ESR (mm/hr) 8 12 23 28 NS Small joint 1 2 1 6 NS Large joint 1 2 1 2 NS A CR/EULAR criteria score 3.0 3.5 4.0 5.0 NS Abbreviations: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; ACR/ EULAR criteria, American College of Rheumatology/European League against Rheumatism classification criteria for rheumatoid arthritis; APF, antiperinuclear factor; NS, not significant. 을때 49.1%, 3점과 4점을부여했을때에 86.8% 의환자가 ACR/ EULAR criteria에서 6점이상을받았다. 항핵주변인자검사의형광강도가 1+, 2+ 인경우와 3+, 4+ 인경우각각 1점과 2점을부여했을때에 18.9%, 2점과 3점을부여했을때에 43.4%, 3점과 4점을부여했을때 77.4% 의환자가 ACR/EU- LAR criteria상 6점이상이었다 (Table 3). 고찰 본연구결과항핵주변인자검사의형광강도에따른 ACR/EU- LAR criteria 점수의유의한차이는없었으나항핵주변인자검사의형광강도등급의증가에따라전반적으로 ACR/EULAR criteria의점수가증가하는양상이관찰되었다. 저자들이시행한이전연구 [9] 에서도항핵주변인자검사의형광강도에비례하여류마티스관절염으로진단되는환자의비율이증가하는결과를얻은바있다. ACR/EULAR criteria의점수가통계적으로유의한차이가없었 Table 2. ACR/EULAR criteria scores and modified scores of 53 patients according to level of the fluorescence intensity of APF test Case No. APF ACR/EULAR criteria score Plus 1* Plus 2 Plus 3 Plus 4 Case No. APF ACR/EULAR criteria score Plus 1* Plus 2 Plus 3 Plus 4 1 1+ 1 2 3 4 5 28 1+ 4 5 6 7 8 2 1+ 2 3 4 5 6 29 1+ 5 6 7 8 9 3 1+ 2 3 4 5 6 30 1+ 5 6 7 8 9 4 1+ 2 3 4 5 6 31 1+ 5 6 7 8 9 5 1+ 2 3 4 5 6 32 2+ 2 3 4 5 6 6 1+ 2 3 4 5 6 33 2+ 2 3 4 5 6 7 1+ 2 3 4 5 6 34 2+ 2 3 4 5 6 8 1+ 3 4 5 6 7 35 2+ 2 3 4 5 6 9 1+ 3 4 5 6 7 36 2+ 2 3 4 5 6 10 1+ 3 4 5 6 7 37 2+ 3 4 5 6 7 11 1+ 3 4 5 6 7 38 2+ 3 4 5 6 7 12 1+ 3 4 5 6 7 39 2+ 3 4 5 6 7 13 1+ 3 4 5 6 7 40 2+ 4 5 6 7 8 14 1+ 3 4 5 6 7 41 2+ 4 5 6 7 8 15 1+ 3 4 5 6 7 42 2+ 4 5 6 7 8 16 1+ 3 4 5 6 7 43 2+ 4 5 6 7 8 17 1+ 3 4 5 6 7 44 2+ 4 5 6 7 8 18 1+ 3 4 5 6 7 45 2+ 5 6 7 8 9 19 1+ 3 4 5 6 7 46 2+ 5 6 7 8 9 20 1+ 3 4 5 6 7 47 2+ 5 6 7 8 9 21 1+ 3 4 5 6 7 48 3+ 3 4 5 6 7 22 1+ 3 4 5 6 7 49 3+ 3 4 5 6 7 23 1+ 4 5 6 7 8 50 3+ 4 5 6 7 8 24 1+ 4 5 6 7 8 51 3+ 4 5 6 7 8 25 1+ 4 5 6 7 8 52 3+ 4 5 6 7 8 26 1+ 4 5 6 7 8 53 4+ 5 6 7 8 9 27 1+ 4 5 6 7 8 *Give 1 point for the positivity of APF test in ACR/EULAR criteria. Give 2 points for the positivity of APF test in ACR/EULAR criteria. Give 3 points for the positivity of APF test in ACR/EULAR criteria. Give 4 points for the positivity of APF test in ACR/EULAR criteria. Abbreviations: ACR/EULAR criteria, American College of Rheumatology/European League against Rheumatism classification criteria for rheumatoid arthritis; APF, antiperinuclear factor. www.labmedonline.org 31
Table 3. Prevalence of patients who were scored 6 or greater when APF tests were applied in ACR/EULAR criteria system APF scoring methods Prevalence of patients who were scored 6 or greater according to APF FI level (%) 1+ 2+ 3+ 4+ Total 1 point for positive APF 9.7 18.8 0.0 100.0 13.2 2 point for positive APF 29.0 50.0 60.0 100.0 39.6 3 point for positive APF 77.4 68.8 100.0 100.0 77.4 4 point for positive APF 96.8 100.0 100.0 100.0 98.1 1 point for 1+ FI/2 point for 2+,3+,4+ FI 9.7 50.0 60.0 100.0 28.3 2 point for 1+ FI/3 point for 2+,3+,4+ FI 29.0 68.8 100.0 100.0 49.1 3 point for 1+ FI/4 point for 2+,3+,4+ FI 77.4 100.0 100.0 100.0 86.8 1 point for 1+, 2+ FI/2 point for 3+,4+ FI 9.7 18.8 60.0 100.0 18.9 2 point for 1+, 2+ FI/3 point for 3+,4+ FI 29.0 50.0 100.0 100.0 43.4 3 point for 1+, 2+ FI/4 point for 3+, 4+ FI 77.4 68.8 100.0 100.0 77.4 Abbreviations: APF, antiperinuclear factor; ACR/EULAR criteria, American College of Rheumatology/European League against Rheumatism classification criteria for rheumatoid arthritis; FI, fluorescence intensity. 던것은이분류방식의점수체계가사용편의성을위해본래점수를반올림하여정수로만들어적용한점과관련이있을것이라생각한다. 정수화된점수체계는검사의진단적가치차이가크지않은경우이를충분히반영하지못하는경향이있다. 실제 ACR/EU- LAR criteria에서도급성기반응물질에대한진단적가치를평가하는초기설정에서는검사의정량적수치의차이에따라각각 1.2와 1.7의차등화된점수가부여되었으나점수의재조정및정수화과정에서정량적수치와관계없이 1점으로통합된바있다 [1]. 항핵주변인자검사의형광강도에따른차등화된점수를적용하는방안이진단정확도의개선하는측면에서더의미가있을것으로생각되나현재의 ACR/EULAR criteria의진단틀을크게변화시키지않는한도내에서항핵주변인자검사의점수를부여하기위해서항핵주변인자검사양성인경우모두동일한점수를부여하는방안이더현실적이라고생각한다. ACPA 검사에부여하는점수인 2점과 3점을항핵주변인자검사에도동일하게적용한경우각각 39.6% 와 77.4% 의환자가총점 6 점이상인 definite rheumatoid arthritis 그룹으로변경되었다. 항핵주변인자검사가 anti-ccp 검사보다류마티스관절염에대해특이도가높은점을고려할때 3점이상의점수를부여하는것이보다합당하다고사료된다. 다만항핵주변인자검사역시 anti-ccp 검사와마찬가지로시트룰린 (citrulline) 을항원결정기 (antigenic determinant) 로서작용한다는공통점을고려할때 [10] ACPA 검사항목에부여할수있는최대점수 (3점) 를초과하는점수를적용하는방안에대해서는추가연구를통해이에대한타당성여부를확인할필요가있겠다. 최근여러연구에서류마티스관절염의진행과정에서발생할수있는관절의영구적손상을방지하기위해조기진단의중요성이강조되고있다 [11-13]. ACR/EULAR criteria를제창한미국및유럽의류마티스연구그룹은 anti-ccp 검사를진단항목에포함시켜 개정한해당분류방식이류마티스관절염의조기진단을위한지표로주목받고있는 anti-ccp 검사 [14, 15] 를진단항목에포함시켜이전에사용하던류마티스관절염분류체계 (1987 revised criteria [16]) 보다류마티스관절염의조기진단면에서유리하다고주장한바있다 [1]. 그러나 Yuko 등 [17] 의연구에따르면류마티스인자검사및 anti- CCP 검사음성인환자에대한 ACR/EULAR criteria의진단민감도는 15.8% 에불과하였다. ACR/EULAR criteria가기존의분류체계보다혈청학적검사에대한의존도가높은점을고려할때 anti- CCP 검사및류마티스인자음성인류마티스관절염의조기진단에현재의 ACR/EULAR criteria는적합하지않으며이를보완할수있는검사지표가필요할것으로사료된다. 저자들은항핵주변인자검사가이를어느정도보완가능할것이라고생각한다. 또한보다정확한진단을위해새로운류마티스관절염의지표로주목받고있는 autoimmune target (AIT) 검사를이용한항 MTOC-MT (microtubule organizing center-microtubule) 항체 [7] 의적용방안에대해서도연구해볼필요가있다고생각한다. 결론적으로저자들은현재의 ACR/EULAR criteria를크게변화시키지않는범위내에서 ACR/EULAR criteria의상기문제점을개선하기위하여항핵주변인자검사를 ACPA 에포함시키고그형광강도결과와상관이없이양성인경우전부 3점을부여하는방안을제안하는바이다. 요약 배경 : 류마티스인자검사와 ACPA검사는 ACR/EULAR criteria에서류마티스관절염의진단을위한혈청학적검사항목으로포함되어있다. 항핵주변인자 (antiperinuclear antibody) 검사는 ACPA 검사의원조에해당하는검사로류마티스관절염에대한높은특이 32 www.labmedonline.org
도를보이면서류마티스인자음성 /anti-cyclic citrullinated peptide (anti-ccp) 음성인류마티스관절염에서자가항체를검출할수있음에도불구하고 ACR/EULAR criteria의혈청학적검사항목에포함되지않았다. 저자들은항핵주변인자검사를 ACR/EULAR criteria에적용하기위한방안을연구하기위해본연구를시행하였다. 방법 : 류마티스관절염이의심되는 53명의환자를대상으로임상증상및검사결과를조사하였다. 대상환자들은전원류마티스인자음성, anti-ccp 검사음성이면서항핵주변인자검사양성이었다. 항핵주변인자검사의형광강도 (1+, 2+, 3+, 4+) 에따라환자를 4개의그룹으로구분하였고항핵주변인자양성소견에대해 1에서 4점까지점수를부여했을때 ACR/EULAR criteria의점수가 6점이상인환자의비율을조사하였다. 결과 : 항핵주변인자검사의형광강도가증가함에따라평균 ACR/EULAR criteria 점수가증가하는양상을보였으나해당 4개의그룹의 ACR/EULAR criteria 점수는유의한차이가없었다 (P > 0.05). 항핵주변인자양성에대해 2점및 3점을 ACR/EULAR criteria에적용하도록하였을때 6점이상이된환자의비율은각각 39.6% 와 77.4% 였다. 결론 : ACR/EULAR criteria의 ACPA 검사항목에항핵주변인자항목도포함시키고항핵주변인자검사양성인경우형광강도와관계없이 3점을부여하는방안이바람직할것으로사료된다. 참고문헌 1. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO 3rd, et al. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010;62:2569-81. 2. Neinhuis RL and Mandema E. A new serum factor in patients with rheumatoid arthritis: the antiperinuclear factor. Ann Rheum Dis 1964; 23:302-5. 3. Youinou P, Le Goff P, Dumay A, Lelong A, Fauquert P, Jouquan J. The antiperinuclear factor. I. Clinical and serologic associations. Clin Exp Rheumatol 1990;8:259-64. 4. Manera C, Franceschini F, Cretti L, Braga S, Cattanec R. Clinical heterogeneity of rheumatoid arthritis and the antiperinuclear factor. J Rheumatol 1994;21:2021-5. 5. Kim SG, Jung KY, Suh HS, Choe JY, Chung SH, Kim CG, et al. Is it feasible to adopt a commercialized APF kit as a routine diagnostic tool for rheumatoid arthritis. Arthritis Rheum 2001;44:S364. 6. van Jaarsveld CH, ter Borg EJ, Jacobs JW, Schellekens GA, Gmelig- Meyling FH, van Booma-Frankfort C, et al. The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis. Clin Exp Rheumatol 1999; 17:689-97. 7. Kim DA and Kim TY. Anti-microtubule organizing center with microtubule by autoimmune target test is also useful serological marker in rheumatoid arthritis evaluation. Rheumatol Int DOI 10.1007/s00296-011-2228-9. 8. Kim DA, Jearn LH, Kim TY. Antiperinuclear factor test is more useful than anti-sa assay when used with anti-cyclic citrullinated peptide test in diagnosis of rheumatoid arthritis. J Rheumatol 2007;34:1944-5. 9. Kim TY, Oh J, Park IK, Yoo DH, Kim SY. Fluorescence intensity scoring of antiperinuclear factor at the 1:20 threshold in rheumatoid arthritis. Arthritis Rheum 1999;42(S9):S348. 10. Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest 1998;101:273-81. 11. Bijlsma JW and Weinblatt ME. Optimal use of methotrexate: the advantages of tight control. Ann Rheum Dis 2007;66:1409-10. 12. Pincus T, Yazici Y, Sokka T, Aletaha D, Smolen JS. Methotrexate as the anchor drug for the treatment of early rheumatoid arthritis. Clin Exp Rheumatol 2003;21(S31):S179-85. 13. Visser K and van der Heijde D. Optimal dosage and route of administration of methotrexate in rheumatoid arthritis: a systematic review of the literature. Ann Rheum Dis 2009;68:1094-9. 14. Nielen MM, Schaardenburg D, Reesink HW, van de Stadt RJ, van der Horst-Bruinsma IE, de Koning MH, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum 2004;50:380-6. 15. Rantapää-Dahlqvist S, de Jong BA, Berglin E, Hallmans G, Wadell G, Stenlund H, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003;48:2741-9. 16. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24. 17. Kaneko Y, Kuwana M, Kameda H, Takeuchi T. Sensitivity and specificity of 2010 rheumatoid arthritis classification criteria. Rheumatology (Oxford) 2011;50:1268-74. www.labmedonline.org 33