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Comparative Efficacy of Various Formalin Fixatives for Molecular Diagnosis in Pathological Tissues
Biomed Sci Letters 2022;28:298-306
Published online December 31, 2022;  https://doi.org/10.15616/BSL.2022.28.4.298
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Woohyun Jee1,2,*, Moonhwan Bae1,**, Hyejin Yoon3,*, Inyoung Kang2,*, Myoungjoo Koo2,*, Jaewang Lee2,4,*** and Jin Hyun Jun2,3,4,†,***

1Department of Pathology, Samsung Medical Center, Seoul 06351, Korea
2Department of Biomedical Laboratory Science, Graduate School, Eulji University, Seongnam 13135, Korea
3Department of Senior Healthcare, Graduate School, Eulji University, Seongnam 13135, Korea
4Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea
Correspondence to: Jin Hyun Jun. Department of Biomedical Laboratory Science, College of Health Science, Eulji University, B106, Jicheon B/D, 553 Sanseong-daero, Sujeong-gu, Seongnam-si 13135, Korea.
Tel: +82-31-740-7210, Fax: +82-31-740-7351, e-mail: junjh55@eulji.ac.kr
*Graduate student, **Researcher, ***Professor.
Received October 27, 2022; Revised December 22, 2022; Accepted December 23, 2022.
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.
 Abstract
Pathological tissue fixation using formalin has been widely used for histological samples in many hospitals and institutions. In general, formalin fixatives were either manufactured in laboratories or purchased commercially because of the risks and environmental concerns of handling organic compounds. In this study, the efficacy of three kinds of commercially purchased and one laboratory-made formalin fixative was compared in the PCR-based molecular diagnosis using the extracted DNA from formalin-fixed paraffin-embedded (FFPE) tissues. The quality of extracted DNA from FFPE tonsil tissues with four kinds of formalin solutions was evaluated, and PCR for beta-globin gene and microsatellite instabilities (MSI) tests for pentaplex panel markers were performed using the extracted DNA. There was no difference in PCR and MSI tests as molecular diagnoses regardless of the types of formalin used in this study. However, the total amount and average length of double-stranded DNA extracted from FFPE tonsil tissue showed significant differences according to the type of formalin fixative. Optimized formalin fixatives and methods for DNA extraction might be sophisticated to extract good quality DNA from the small size of specific tissue samples. Further studies are needed to select the most effective formalin fixative for histology and molecular pathology using human FFPE tissues.
Keywords : Formalin, Formalin-fixed paraffin embedded (FFPE) tissues, Histology, Molecular diagnosis, DNA extraction, Polymerase chain reaction (PCR), microsatellite instabilities (MSI) tests
꽌 濡

議곗쭅븰쟻 愿李곗 깮臾쇱껜瑜 援ъ꽦븯怨 엳뒗 뿬윭 議곗쭅쓣 긽쑝濡 븯뒗 湲곕낯쟻씤 뿰援 諛⑸쾿쑝濡 젙솗븳 議곗쭅怨 꽭룷쓽 삎깭븰쟻 솗씤쓣 쐞빐꽌 議곗쭅쓽 蹂꽦쓣 留됰뒗 怨쇱젙씤 怨좎젙(fixation) 議곗쭅븰쟻 뿰援ъ쓽 湲곕낯쟻씤 닚꽌씪 븷 닔 엳떎. 怨좎젙븸쓽 듅꽦뿉 뵲씪 怨좎젙맂 議곗쭅뿉꽌 듅씠 遺꾩옄臾쇱쭏쓽 냼떎, 議곗쭅泥섎━怨쇱젙 以 議곗쭅쓽 뙺李쎌씠굹 닔異, 議곗쭅븰쟻 샊 硫댁뿭議곗쭅븰쟻 뿼깋 젙룄 李⑥씠(variation of quality), 깮솕븰쟻 諛섏쓳 怨쇱젙쓽 젙솗꽦, 꽭룷냼湲곌 援ъ“쓽 蹂댁〈 젙룄 벑뿉꽌 李⑥씠媛 굹궇 닔 엳떎(Kiernan, 2000; Qin et al., 2021). 룷瑜대쭚由 怨좎젙 諛 뙆씪 룷留ㅻ뒗 議곗쭅 蹂댁〈뿉 媛옣 꼸由 궗슜릺뒗 씪諛섏쟻씤 議곗쭅븰쟻 諛⑸쾿씠硫(Bass et al., 2014). 룷瑜대쭚由곗쓣 씠슜븳 議곗쭅 怨좎젙 議곗쭅 蹂꽦쓣 뼲젣븯怨 꽭룷 援ъ“瑜 蹂댁〈븯뒗 슚怨쇨 슦닔븯떎(Peter et al., 2014). 꽭룷 援ъ“瑜 쑀吏븯湲 쐞빐 솕븰쟻씤 議곗쭅쓽 怨좎젙 怨쇱젙쓣 넻빐 꽭룷굹 議곗쭅쓣 엳뒗 洹몃濡 蹂댁〈븯怨 紐⑤뱺 슚냼 怨쇱젙씠굹 궗 怨쇱젙쓣 以묒떆耳 怨좎젙 썑 蹂솕(post-fixation change)瑜 理쒖냼솕븯뿬빞 븳떎. 議곗쭅쓽 怨좎젙 怨쇱젙 떊猶곗꽦 엳뒗 議곗쭅븰쟻 떆猷 以鍮 怨쇱젙쓽 媛옣 以묒슂븳 遺遺 以묒쓽 븯굹濡 뿰援ъ 吏꾨떒뿉꽌 젙솗븳 寃곌낵瑜 젣怨듯븯뒗 뜲 以묒슂븳 슂씤씠떎. 遺덉셿쟾븳 怨좎젙(under-fixation) 議곗쭅쓽 삎깭쟻 蹂댁〈씠 씠猷⑥뼱吏吏 븡븘 빐떦 議곗쭅쓽 젙솗븳 솗씤씠 遺덇뒫빐吏 닔 엳怨, 怨쇨퀬젙(over-fixation) 듅젙 議곗쭅 遺꾩옄쓽 냼떎 삉뒗 鍮꾪듅씠쟻 諛곌꼍 떊샇쓽 끂씠利(noise) 벑쓣 쑀諛쒗븷 닔 엳떎(Lee, 2011; Zeng et al., 2013). 씠긽쟻씤 怨좎젙 議곗쭅씠굹 꽭룷쓽 援ъ“, 삎깭굹 쐞移섎 엳뒗 洹몃濡 쑀吏븯怨 꽭룷 궡쇅쓽 듅젙 遺꾩옄뱾쓽 넀떎쓣 諛⑹븷 닔 엳뒗 諛⑸쾿씠뼱빞 븯硫, 議곗쭅쓽 빆썝꽦(antigenicity)쓣 엳뒗 洹몃濡 쑀吏븯嫄곕굹 빆썝쓽 蹂꽦쓣 理쒖냼솕븯硫 議곗쭅 궡 臾쇱쭏쓽 솗궛씠굹 옱諛곗뿴쓣 諛⑹븯뒗 寃껋씠떎(Spector and Goldman, 2005; Qin et al., 2021).

洹쇰옒뿉 議곗쭅븰쟻 떆猷뚯씤 룷瑜대쭚由곗쑝濡 怨좎젙맂(formalin-fixed paraffin-embedded, FFPE) 議곗쭅뿉꽌쓽 DNA 異붿텧怨 씠瑜 씠슜븳 떎뼇븳 遺꾩옄蹂묐━ 寃궗뒗 吏꾨떒肉먮쭔 븘땲씪 뜑 굹븘媛 李⑥꽭 뿼湲곗꽌뿴遺꾩꽍(next generation sequencing, NGS) 寃궗 媛숈씠 젙諛 쓽猷 諛 몴쟻 移섎즺瑜 쐞븳 吏꾨떒뿉룄 솢슜릺怨 엳떎(Norouzi et al., 2021; McDonough et al., 2019; Fujii et al., 2022). 뵲씪꽌, 議곗쭅븰쟻 蹂묐━ 떆猷뚮뒗 떒닚븳 吏꾨떒쓽 紐⑹쟻뿉꽌 移섎즺 諛 삁썑, 삁痢, 몴쟻 移섎즺源뚯 洹 쁺뿭 諛 솢슜룄瑜 넂씠怨 엳떎. 遺꾩옄 蹂묐━쓽 슚슜꽦쓣 넂씠湲 쐞빐꽌뒗 FFPE 議곗쭅뿉꽌 怨좏뭹吏덉쓽 DNA 異붿텧씠 꽑뻾릺뼱빞 븯湲 븣臾몄뿉 씠뿉 븳 뿰援 삉븳 떎뼇븯寃 吏꾪뻾릺怨 엳떎(Kocjan et al., 2016; Panchal et al., 2020). 룷瑜대쭚由 怨좎젙븸쓣 씠슜븳 議곗쭅 怨좎젙 怨쇱젙뿉꽌쓽 DNA 넀긽쓽 씪諛섏쟻씤 삎깭뒗, 옣 湲곌컙쓽 옣湲고솕 議곗쭅 怨좎젙뿉 궗슜릺뒗 룷瑜대쭚由곗쓽 궙 pH濡쒖쓽 궛꽦솕 뿰愿맂 寃껋쑝濡 븣젮졇 엳떎(Ludyga et al., 2012; Groelz et al., 2018; Bauer et al., 2021).

씤泥댁뿉 쑀빐븳 臾쇱쭏씤 룷由꾩븣뜲엳뱶뒗 10%쓽 닔슜븸씤 룷瑜대쭚由곗쑝濡 씗꽍릺뼱 蹂묐━ 議곗쭅 怨좎젙슜쑝濡 留롮씠 궗슜릺怨 엳쑝硫, 紐뉖챺 蹂묒썝 諛 湲곌뿉꽌뒗 吏곸젒 젣議고븯뿬 궗슜븯怨 엳떎. 洹몃윭굹 쑀湲고솕빀臾 痍④툒뿉 뵲瑜 쐞뿕꽦怨 솚寃쎈Ц젣濡 젣議곕릺뼱 떆뙋릺뒗 룷瑜대쭚由곗씠 꼸由 궗슜 以묒씠硫 媛곴컖쓽 룷瑜대쭚由곗쓽 슚슜꽦쓣 뙆븙븯뿬 湲곌留덈떎 쟻빀븳 룷瑜대쭚由곗쓣 꽑깮븯뿬 궗슜븯뒗 寃껋씠 以묒슂븯寃 깮媛곷릺怨 엳떎. 뵲씪꽌 援ъ엯븯뿬 궗슜븷 닔 엳뒗 뿬윭 쉶궗쓽 룷瑜대쭚由 怨좎젙븸뱾怨 湲곌 蹂묐━怨쇱뿉꽌 옄泥 젣議고븳 룷瑜대쭚由 怨좎젙븸 벑쓽 議곗쭅븰쟻 寃궗 遺꾩옄吏꾨떒 寃궗뿉꽌쓽 슚슜꽦뿉 븳 鍮꾧탳 룊媛 뿰援ш 븘슂븳 긽솴씠떎.

蹂 뿰援щ뒗 떆뙋릺뒗 젣뭹뱾怨 떎뿕떎뿉꽌 옄泥 젣議고븳 룷瑜대쭚由 怨좎젙븸 벑쓽 깮솕븰쟻 듅꽦쓣 鍮꾧탳븯怨 議곗쭅븰쟻 愿李 諛 遺꾩옄 吏꾨떒뿉꽌쓽 洹 슚슜꽦쓣 룊媛뒗 寃껋쓣 紐⑹쟻쑝濡 吏꾪뻾븯떎. 긽 룷瑜대쭚由곕뱾쓽 pH瑜 痢≪젙븯怨 怨좎젙븳 FFPE 議곗쭅 젅렪뿉꽌 DNA瑜 異붿텧븯뿬 냽룄 吏(quality)쓣 鍮꾧탳븯怨, 異붿텧븳 DNA瑜 씠슜븯뿬 beta-globin 쑀쟾옄뿉 븳 以묓빀슚냼뿰뇙諛섏쓳(PCR)怨 떎뼇븳 븫(cancer)쓽 吏꾨떒뿉 궗슜릺뒗 쁽誘몃닔泥 遺덉븞쟾꽦(microsatellite instability, MSI) 寃궗瑜 떎떆븯뿬 洹 寃곌낵瑜 솗씤븯떎.

옱猷 諛 諛⑸쾿

뿰援 긽 諛 옱猷

蹂 뿰援ъ뿉 궗슜븳 룷瑜대쭚由곗 궪꽦꽌슱蹂묒썝 蹂묐━怨쇱뿉꽌 吏곸젒 젣議고븳 룷瑜대쭚由곌낵 3醫낅쪟쓽 떆뙋릺怨 엳뒗 寃껊뱾쓣 궗슜븯쑝硫, 媛곴컖쓽 議곗쭅쓣 떎삩뿉꽌 12~18떆媛 怨좎젙븯떎. 吏곸젒 젣議고븳 룷瑜대쭚由 怨좎젙븸(laboratory-made formalin, LmF) formaldehyde solution, 35% 썝븸 룷瑜대쭚由[(二)궪쟾솕븰]怨 씪諛 利앸쪟닔瑜 1:9 (10%)濡 씗꽍븳 룷瑜대쭚由곗씠뿀쑝硫, 떆뙋 룷瑜대쭚由 怨좎젙븸 (二)誘몃씫뒪쓽 10% neutral buffered formalin (Mirax neutral buffered formalin, MiN), 뿞뵒룷뒪쓽 룷由꾩븣뜲엳뱶(MD Force formaldehyde, MdF), (二)븳援뵪뿉봽뵪耳誘몄뭡쓽 룷瑜대쭚由(Korea CFC chemical formalin, KoF) 벑쓣 궗슜븯떎.

蹂 뿰援щ뒗 궪꽦꽌슱蹂묒썝 뿰援ъ떖쓽쐞썝쉶쓽 듅씤(듅씤踰덊샇: SMC 2019-07-135-002)쓣 諛쏆븘 떆뻾븯쑝硫, 遺꾩옄吏꾨떒 寃궗瑜 쐞빐 由쇳봽 뿬룷 利앹떇쓣 룞諛섑븳 留뚯꽦 렪룄꽑뿼(chronic tonsillitis with lymphoid follicular hyperplasia) 솚옄뿉꽌 梨꾩랬븳 3媛쒖쓽 렪룄(tonsil) 議곗쭅쓣 궗슜븯떎.

떎뼇븳 룷瑜대쭚由 怨좎젙븸뱾쓽 pH 痢≪젙

媛곴컖쓽 룷瑜대쭚由(LmF, MiN, MdF, KoF)뿉 븳 pH 痢≪젙 Mettler Toledo (Greifensee, Switzerland) 궗쓽 Seven compact S210 옣鍮꾨 씠슜븯뿬 10쉶 諛섎났븯뿬 痢≪젙븯떎. 洹 諛⑸쾿 븘옒 媛숇떎.

쟾洹뱀쓣 calibration 踰꾪띁/몴以슜븸뿉 꽔 뮘 CAL쓣 늻瑜대㈃ Cal 1씠 굹굹硫 떊샇媛 븞젙맂 뮘 삉뒗 READ瑜 늻瑜 뮘 誘몃━ 꽑깮맂 END Point 紐⑤뱶뿉 뵲씪 誘명꽣쓽 痢≪젙쓣 셿猷뚰븳떎. 醫낅즺 썑 calibration쓣 솗씤븳 썑 꽱꽌瑜 痢≪젙 깦뵆뿉 꽔 뮘 READ瑜 닃윭 痢≪젙쓣 떆옉븯怨 END Point 엯씠 源쒕컯嫄곕━硫 痢≪젙씠 吏꾪뻾릺硫 깦뵆쓽 痢≪젙媛믪씠 몴떆맂떎. 씠썑 꽑깮맂 븞젙湲곗뿉 뵲씪 痢≪젙씠 븞젙릺硫 븞젙 븘씠肄섏씠 몴떆맂 썑 理쒖쥌 痢≪젙媛믪쓣 솗씤븯떎.

議곗쭅쓽 怨좎젙 諛 젅렪 젣옉

깮寃맂 렪룄(tonsil) 議곗쭅쓣 怨좎젙븯湲 쟾뿉 1 × 1 cm 겕湲곕줈 옄瑜 썑 媛곴컖쓽 룷瑜대쭚由 怨좎젙븸(LmF, MiN, MdF, KoF)쑝濡 12~18떆媛 룞븞 異⑸텇엳 怨좎젙븯떎. 釉붾줉쓣 젣옉븯뒗 怨쇱젙뿉꽌 議곗쭅 궗씠쓽 뙆씪 移⑦닾뒗 Leica Biosystems (Wetzlar, Germany)쓽 Peloris Quick Tips 옣鍮꾨 궗슜븯뿬 븯猷삳갇 룞븞 吏꾪뻾븯쑝硫 뙆씪씠 移⑦닾맂 媛 議곗쭅뱾 Thermo Fisher Scientific Inc. (Waltham, MA, USA)쓽 議곗쭅 룷留 옣移섏씤 Histostar 옣鍮꾨 궗슜븯뿬 씪젙븳 紐⑥뼇쓽 뙆씪 釉붾줉쓣 젣옉븯떎. 젣옉맂 뙆씪 釉붾줉 Thermo Fisher Scientific Inc. (Waltham, MA, USA)쓽 Finesse ME+ 諛뺤젅湲곕 씠슜븯뿬 3~4 μm쓽 몢猿섎줈 諛뺤젅 썑 Muto Pure Chemicals (Tokyo, Japan)궗쓽 쑀由 뒳씪씠뱶뿉 뙆씪 젅렪쓣 遺李⑺븯떎.

議곗쭅븰쟻 愿李곗쓣 쐞븳 hematoxylin & eosin 뿼깋

Hematoxylin & eosin (H&E) 뿼깋 議곗쭅븰 떆猷뚯뿉꽌 씪諛섏쟻쑝濡 궗슜븯뒗 뿼깋踰뺤쑝濡 뿤留덊넚떎由(hematoxylin)怨 뿉삤떊(eosin)쓽 몢 뿼猷뚮 씠슜븯뿬 꽭룷쓽 빑怨 꽭룷吏덉쓣 援щ텇븯뿬 愿李고븷 닔 엳떎.

議곗쭅 젅렪湲곕 씠슜븯뿬 4 μm 몢猿섎줈 뙆씪 議곗쭅 젅렪쓣 젣옉븯쑝硫, 씠瑜 Roche Diagnostics (USA)쓽 Ventana HE 600 뿼깋湲곕 궗슜븯뿬 H&E 뿼깋쓣 吏꾪뻾븯떎. 嫄댁“遺꽣 遊됱엯, 뿼깋源뚯쓽 쟾泥 怨쇱젙씠 Ventana HE600 옣鍮꾩뿉꽌 옄룞쑝濡 븳 踰덉뿉 씠猷⑥뼱吏湲 븣臾몄뿉 뿼깋 怨쇱젙뿉꽌 뒳씪씠뱶 씠룞 떆媛꾩쓣 媛먯냼떆궎怨 湲곗〈쓽 H&E 뿼깋 'Dip & Dunk' 諛⑹떇쑝濡 뿬윭 議곗쭅 깦뵆쓣 뿼깋븯湲 븣臾몄뿉 援먯감 삤뿼쓣 理쒖냼솕븯떎. 븣肄붿삱怨 옄씪젋(xylene)쓣 궗슜븯吏 븡怨 泥대Ъ吏덉쓣 씠슜븯뒗 理쒖떊 뿼깋 옣鍮꾨 씠슜븯뿬 뿼깋 썑 愿묓븰쁽誘멸꼍쑝濡 愿李고븯떎.

룷瑜대쭚由 怨좎젙 썑 뙆씪 룷留ㅻ맂 렪룄 議곗쭅뿉꽌 DNA 異붿텧 諛 遺꾩꽍

룷瑜대쭚由곗쑝濡 怨좎젙맂 議곗쭅뿉꽌쓽 DNA 異붿텧 궪꽦꽌슱蹂묒썝 蹂묐━怨쇱뿉꽌 궗슜븯怨 엳뒗 Qiagen DNA FFPE Tissue kit (Qiagen, Hilden, Germany)瑜 궗슜븯뿬 媛곴컖쓽 怨좎젙븸뿉 뵲瑜 뙆씪 釉붾줉쓣 5 μm 몢猿섎줈 4~8옣쓽 젅렪쓣 젣옉븯怨 떊쟾湲곗뿉꽌 嫄댁“ 썑 媛곴컖쓽 깦뵆 뒠釉뚯뿉 꽔뿀떎. 깉뙆씪 썑 proteinase K 20 μL, ATL buffer 180 μL瑜 꽔怨, 56℃ 빆삩 닔議곗뿉꽌 12~15떆媛 泥섎━븯怨, 90℃ 媛뿴 釉붾윮뿉꽌 1떆媛 룞븞 怨좎삩 泥섎━븯떎.

긽湲곗 媛숈씠 泥섎━맂 깦뵆 뒠釉뚮 떒떆媛 썝떖遺꾨━ 븳 썑 AL buffer 200 μL 꽔怨, 蹂쇳뀓떛(vortexing)怨 떒떆媛 썝떖遺꾨━瑜 諛섎났븳 썑 100% Et-OH 200 μL瑜 꽔뿀떎. 洹 썑 8,000 rpm쑝濡 1遺 썝떖遺꾨━ 썑 諛묒뿉 媛씪븠 移⑥쟾臾쇱 젣쇅븯怨 긽痢듭븸留 而щ읆(column)뿉 삷湲대떎. 닔꽭瑜 쐞븯뿬 而щ읆(column)뿉 AW1 buffer 500 μL瑜 꽔뼱二쇱뼱 8,000 rpm뿉꽌 1遺꾧컙 썝떖遺꾨━븯怨, 2李⑤줈 AW2 buffer 500 μL瑜 꽔뼱 14,000 rpm뿉꽌 3遺꾧컙 썝떖遺꾨━ 븯떎. 而щ읆(column)뿉 遺李⑸맂 DNA瑜 遺꾨━븯湲 쐞빐 닔꽭맂 而щ읆(column)쓣 源⑤걮븳 1.5 mL 誘몃땲썝떖遺꾨━ 뒠釉뚯뿉 삷湲곌퀬, AE buffer 200 μL瑜 꽔뼱 1遺꾧컙 諛⑹튂븳 썑 8,000 rpm뿉꽌 1遺꾧컙 썝떖遺꾨━ 븯떎.

씠썑 異붿텧맂 FFPE 議곗쭅쓽 DNA뒗 DU-730 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA) Qubit 2.0 fluorometer (Life Technologies Inc., Grand Island, NY, USA) 옣鍮꾨 궗슜븯뿬 DNA쓽 냽룄(concentration), 닚룄(purity) 諛 珥앸웾(total amount)쓣 痢≪젙븯떎.

異붿텧븳 DNA瑜 궗슜븳 以묓빀슚냼뿰뇙諛섏쓳(PCR)怨 利앺룺 궛臾 솗씤

異붿텧븳 genomic DNA쓽 긽깭瑜 솗씤븯湲 쐞빐 븘옒 媛숈 봽씪씠癒 뙇(forward primer 5'-CAACTTCATCCACGTTCACC-3', reverse primer 5'-GGTTGGCCAATCTACTCCCA-3')쓣 씠슜븯뿬 beta-globin 쑀쟾옄뿉 븳 以묓빀슚냼뿰뇙諛섏쓳(PCR)쓣 吏꾪뻾븯쑝硫, 삁긽릺뒗 PCR product쓽 겕湲곕뒗 216 base pairs 떎.

以묓빀슚냼뿰뇙諛섏쓳(PCR) Maxime PCR premix (i-star Taq; iNtRON Biotechnology, Seongnam-si, Korea)瑜 궗슜븯쑝硫, DW 17 μL, forward primer 1 μL, reverse primer 1 μL, genomic DNA 1 μL濡 20 μL쓣 留욎떠꽌 Bio-Rad C1000 PCR 옣鍮(Bio-Rad Laboratories Inc., Hercules, CA, USA)瑜 궗슜븯뿬 40 cycles PCR쓣 吏꾪뻾븯떎. PCR 궛臾쇱쓽 겕湲곕뒗 QIAxcel Advenced System (Qiagen) 옣鍮꾨 궗슜븯뿬 洹 寃곌낵瑜 솗씤븯떎.

異붿텧븳 DNA뿉 븳 쁽誘몃닔泥 遺덉븞젙꽦 寃궗(microsatellite instability test, MSI test)

媛곴컖쓽 룷瑜대쭚由곗쓣 궗슜븯뿬 怨좎젙븳 렪룄(tonsil) 議곗쭅쓽 뙆씪 釉붾줉뿉꽌 異붿텧븳 DNA瑜 궗슜븯뿬 PCR 諛⑸쾿쓣 湲곕컲쓽 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗瑜 떆뻾븯怨 洹 寃곌낵媛믪쓣 鍮꾧탳븯떎. 異붿텧븳 DNA 떆猷 뱾뿉꽌 pentaplex panel씤 NR-27, NR-21, NR-24, BAT-25, BAT-26뿉 븳 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗瑜 빐떦 깘移⑥옄(probe)瑜 씠슜븯뿬 吏꾪뻾븯떎(Suraweera et al., 2002; Goel et al., 2010).

媛곴컖쓽 PCR 뒠釉뚯뿉 遺꾨━맂 DNA瑜 꽔怨, 媛 몴吏옄 蹂꾨줈 삎愿묒씠 몴吏맂 primer瑜 꽔뼱二쇱뿀떎. Hot Starter Taq Master Mix (QIAGEN)쓣 꽔뼱以 썑 옒 꽎뼱二쇱뿀떎. 떆猷뚮뒗 GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA, USA)뿉 옣李⑺븳 썑 95℃뿉꽌 15遺꾧컙 諛섏쓳븳 썑, 94℃뿉꽌 1遺, 55℃뿉꽌 1遺, 72℃뿉꽌 1遺꾧컙 30쉶쟾쓣 諛섏쓳븯怨, 留덉留됱쑝濡 72℃뿉꽌 5遺꾧컙 諛섏쓳븯떎. 諛섏쓳씠 걹궃 떆猷뚯뿉 GeneScan-500 ROX size Standard (Applied Biosystems) 1 μL Hi-Di formamide (Applied Biosystems) 10 μL瑜 꽔뼱 옒 샎빀븳 썑 95℃뿉꽌 5遺꾧컙 諛섏쓳떆耳곕떎. 깘移⑥옄(probe) 샎빀맂 떆猷뚮 96 well plate뿉 삷湲 썑 3100 Genetic Analyzer (Applied Biosystems)뿉 옣李⑺븯뿬 議곌컖 遺꾩꽍(fragment analysis)쓣 떎떆븯떎. 遺꾩꽍씠 걹궃 寃곌낵臾쇱 GeneScan software (Applied Biosystems)쓣 씠슜븯뿬 寃곌낵瑜 솗씤븯떎.

솗씤맂 寃곌낵쓽 룊媛뒗 5媛쒖쓽 몴吏옄(marker) 以 2媛 씠긽쓽 몴吏옄(marker)뿉꽌 蹂씠媛 愿李곕맆 寃쎌슦뿉 MSI-High, 븯굹쓽 몴吏옄(marker)뿉꽌留 蹂씠媛 엳쓣 寃쎌슦뿉뒗 MSI-Low, 紐⑤뱺 몴吏옄(marker)뿉꽌 蹂씠媛 愿李곕릺吏 븡뒗 寃쎌슦뿉 microsatellite stable (MSS)濡 遺꾨쪟븯떎(Sood et al., 2001; Kim and Park, 2006; Bilbao-Sieyro et al., 2014).

넻怨 遺꾩꽍

넻怨꾩쿂由щ뒗 SPSS Version 27 (IBM SPSS Statistics, Chicago, IL, USA) 봽濡쒓렇옩쓣 씠슜븯떎. 紐낅ぉ 蹂닔뿉 빐꽌뒗 Chi-square test, 뿰냽 蹂닔뿉 빐꽌뒗 Student t-test 삉뒗 씪썝諛곗튂 遺꾩궛遺꾩꽍(one-way ANOVA)쓣 떆뻾븯떎. 넻怨꾩쿂由ъ뿉꽌 P 媛믪씠 0.05 誘몃쭔씤 寃쎌슦瑜 넻怨꾪븰쟻쑝濡 쑀쓽븳 李⑥씠媛 엳뒗 寃껋쑝濡 뙋젙븯떎.

寃 怨

떎뼇븳 룷瑜대쭚由 怨좎젙븸뱾쓽 pH 痢≪젙 寃곌낵

媛곴컖쓽 룷瑜대쭚由곗쓽 pH瑜 10쉶 諛섎났 痢≪젙븯쑝硫, 洹 寃곌낵뒗 Table 1 媛숈븯떎. 痢≪젙 寃곌낵 pH 룊洹좉컪 ± 몴以렪李⑤뒗 KoF뿉꽌 6.873 ± 0.055濡 媛옣 넂븯쑝硫, MiN뿉꽌 6.814 ± 0.016쑝濡 媛옣 궙寃 굹궗쑝굹, 씪썝諛곗튂 遺꾩궛遺꾩꽍(one-way ANOVA)뿉꽌 넻怨꾩쟻씤 쑀쓽꽦 뾾뿀떎.

Mean of pH in various formalin fixatives

Name of fixatives pH (mean ± SD)
Laboratory-made formalin (LmF) 6.84 ± 0.06
Mirax Co. 10% neutral buffered formalin (MiN) 6.81 ± 0.01
MD force formaldehyde (MdF) 6.83 ± 0.07
Korea CFC chemical Co. formalin (KoF) 6.87 ± 0.06

The pH of each formalin was measured 10 times

There was no significant difference in one-way ANOVA



룷瑜대쭚由 怨좎젙븸뿉 뵲瑜 H&E 뿼깋꽦 李⑥씠 遺꾩꽍

媛곴컖쓽 怨좎젙븸쓣 궗슜븯뿬 렪룄 議곗쭅쓣 hematoxylin & eosin 뿼깋쓣 떆뻾 썑 愿묓븰쁽誘멸꼍쑝濡 뿼깋꽦쓣 솗씤븳 寃곌낵 빑 뿼湲곗꽦 뿼猷뚯씤 hematoxylin쑝濡 씤븯뿬 吏숈 泥깋쑝濡 뿼깋릺뿀쑝硫, 꽭룷吏덉 궛꽦 뿼猷뚯씤 eosin뿉 쓽빐꽌 쟻깋쑝濡 뿼깋릺뼱 엳뒗 寃껋쓣 愿李고븷 닔 엳뿀떎. 怨좎젙븸뿉 뵲瑜 議곗쭅 媛꾩쓽 뿼깋꽦뿉꽌 쑀쓽븳 李⑥씠젏쓣 諛쒓껄븷 닔 뾾뿀떎(Fig. 1).

Fig. 1. Paraffin sections of human tonsil tissues were stained with a standard hematoxylin and eosin. Microscopic image of a moderate hyperplasia of a lymphatic follicle. The structure of the tonsil stroma and pathological region was well maintained. There was no difference in cytoplasmic and nuclear morphologies among different formalin fixatives. A and B was used laboratory-made formalin (LmF), C and D was used Mirax Co. 10% neutral buffered formalin (MiN), E and F was used MD force formaldehyde (MdF), G and H was used Korea CFC chemical Co. formalin (KoF), respectively. Magnification of A, C, E and G were ×50, and B, D, F and H ×300, respectively. Black and yellow scale bars respectively indicate 1 mm and 100 μm.

룷瑜대쭚由 怨좎젙븸 醫낅쪟뿉 뵲瑜 議곗쭅 寃泥 쑀옒 DNA쓽 냽룄 諛 긽깭

蹂 뿰援ъ뿉꽌 궗슜븳 4媛吏 룷瑜대쭚由곗쑝濡 怨좎젙븳 렪룄(tonsil) 議곗쭅 젅렪뿉꽌 異붿텧븳 DNA쓽 긽깭뿉 븳 寃곌낵瑜 Fig. 2뿉 굹궡뿀떎. 媛곴컖쓽 떆猷뚯뿉꽌 異붿텧맂 빑궛쓽 냽룄뒗 KoF뿉꽌 媛옣 넂븯怨, LmF, MiN, MdF 닚쑝濡 솗씤릺뿀吏留 넻怨꾩쟻씤 쑀쓽꽦 굹굹吏 븡븯떎. 異붿텧븳 DNA쓽 닚룄瑜 굹궡뒗 A260/280 ratio 媛믪 紐⑤몢 1.94~ 1.99濡 쑀궗븳 寃곌낵瑜 蹂댁떎.

Fig. 2. Differences of DNA quality from formalin-fixed paraffin-embedded (FFPE) tonsil tissues. The concentrations (A) and purity (B) of DNA from FFPE tonsil tissues were similar by various formalin fixatives. The total amount of double-stranded DNA (C) was significantly higher in KoF compared to MdF (*P<0.05). The tape station of double-stranded DNA (D) was significantly higher in MiN compared to LmF and MdF, respectively (**P<0.01). LmF, laboratory-made formalin; MiN, Mirax Co. 10% neutral buffered formalin; MdF, Md force formaldehyde; KoF, Korea CFC chemical Co. formalin.

媛곴컖쓽 룷瑜대쭚由 怨좎젙븸쓣 궗슜븳 떆猷뚯뿉꽌 異붿텧맂 double-stranded DNA (dsDNA)쓽 뼇 KoF뿉꽌 媛옣 넂븯怨, MiN, LmF, MdF 닚쑝濡 솗씤릺뿀쑝硫, KoF瑜 궗슜븳 寃쎌슦뿉 MdF뿉 鍮꾪빐 넻怨꾩쟻쑝濡 쑀쓽븯寃 留롮 dsDNA媛 異붿텧릺뿀떎(P<0.05). 삉븳, 異붿텧맂 dsDNA쓽 룊洹 湲몄씠瑜 쓽誘명븯뒗 tape station MiN뿉꽌 媛옣 湲몄뿀쑝硫, LmF瑜 궗슜븳 寃쎌슦뿉 鍮꾪빐 넻怨꾩쟻쑝濡 쑀쓽븳 李⑥씠媛 굹궗떎(P<0.01).

異붿텧븳 DNA뿉꽌 beta-globin 쑀쟾옄뿉 븳 PCR 寃곌낵

떎뼇븳 룷瑜대쭚由곗쑝濡 怨좎젙븳 렪룄(tonsil) 議곗쭅 젅렪뿉꽌 異붿텧븳 DNA瑜 씠슜븯뿬 떎젣쟻씤 蹂묐━遺꾩옄 吏꾨떒뿉꽌 二쇰줈 궗슜븯뒗 PCR 寃궗瑜 吏꾪뻾븯떎. Beta-globin 쑀쟾옄뿉 븳 PCR 寃곌낵臾쇱쓽 gene scan 씠誘몄뿉꽌 삁긽뻽뜕 216 bp 겕湲곗쓽 쐞移섏뿉꽌 PCR product band瑜 솗씤븷 닔 엳뿀쑝硫, 4媛吏 룷瑜대쭚由 議곗쭅뿉 뵲瑜 PCR 寃곌낵臾쇰뱾怨 positive control 떆猷뚯쓽 利앺룺 궛臾 band 쐞移섍 룞씪븯떎(Fig. 3).

Fig. 3. PCR products of beta-globin gene by the QIAxcel Advanced System (Qiagen). Expected size of PCR products were 216 bp, which were found all lanes of samples. Lower bands of 15 bp and higher bands of 3 Kbp were primer dimer and genomic DNAs, respectively. Lane A1, size markers; A8, MiN (Mirax Co. 10% neutral buffered formalin); A9, LmF (laboratory-made formalin); A10, KoF (Korea CFC chemical Co. formalin); A11, MdF (MD force formaldehyde); A12, positive control.

異붿텧븳 DNA뿉 븳 쁽誘몃닔泥 遺덉븞젙꽦(microsatellite instability, MSI) 寃궗 寃곌낵

媛곴컖쓽 룷瑜대쭚由 怨좎젙븸쓣 궗슜븯뿬 怨좎젙븳 렪룄 議곗쭅쓽 젅렪뿉꽌 異붿텧븳 DNA瑜 씠슜븯뿬 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗瑜 吏꾪뻾븯떎. 쁽誘몃닔泥 遺덉븞젙꽦(MSI)쓣 솗씤븯湲 쐞빐 PCR 諛⑸쾿쑝濡 利앺룺븳 5媛쒖쓽 pentaplex panel markers (NR-27, NR-21, NR-24, BAT-25, BAT-26) 寃궗 寃곌낵뒗 Fig. 4뿉꽌 媛숈씠 뼇꽦 떆猷뚯 媛곴컖쓽 異붿텧맂 DNA 떆猷뚯뿉꽌 쑀궗븳 寃곌낵瑜 굹깉떎.

Fig. 4. Gene scan results of microsatellite instability (MSI) tests for pentaplex panel markers. Similar peak height was obtained in isolated DNAs by FFPE with various formalin fixatives. All samples showed similar with positive sample, however different with negative sample in NR 21 marker. MiN, Mirax Co. 10% neutral buffered formalin; MdF, Md force formaldehyde; LmF, laboratory-made formalin; KoF, Korea CFC chemical Co. formalin; positive control; negative control.
怨 李

蹂묐━議곗쭅寃궗 怨쇱젙쓽 泥 踰덉㎏ 떒怨꾩씤 怨좎젙 怨쇱젙뿉 궗슜븷 닔 엳뒗 뿬윭 怨좎젙븸뱾씠 엳吏留, 洹 以 룷瑜대쭚由곗씠 媛옣 꼸由 궗슜릺怨 엳쑝硫 쇅怨 蹂묐━, 硫댁뿭, 遺꾩옄 寃궗 벑뿉 몢猷 궗슜릺뒗 媛옣 蹂댄렪쟻씤 怨좎젙븸씠떎(Han, 2017). 씠윭븳 룷瑜대쭚由 以 씪諛섏쟻쑝濡 궗슜릺뒗 以묒꽦 셿異 룷瑜대쭚由(neutral buffered formalin) 怨좎젙븸 議곗쭅쓽 삎깭 쑀吏 슚쑉쟻씤 援먯옟 諛섏쓳뿉 媛옣 쟻빀븯떎怨 븣젮졇 엳쑝硫, 怨좎젙 썑 젣옉맂 뙆씪 釉붾줉 빑궛怨 떒諛깆쭏쓣 븞젙솕맂 긽깭濡 옣湲곌컙 蹂댁〈븯湲 쟻빀븯떎怨 븣젮졇 엳떎(Bramwell et al., 1988; Turashvili et al., 2012).

룷瑜대쭚由곗쓽 二쇱꽦遺꾩씤 룷由꾩븣뜲븯씠뱶뒗 議곗쭅 떒諛깆쭏怨 硫뷀떥젋 떎由(methylene bridge)瑜 삎꽦븯硫 湲猷⑦瑜댁븣뜲엳뱶媛 議곗쭅怨 삎꽦븯뒗 援먯감寃고빀蹂대떎뒗 떎냼 빟븳 寃고빀쓣 蹂댁씤떎. 씠쑀뒗 룷由꾩븣뜲엳뱶뒗 븘二 옉 遺꾩옄濡 議곗쭅 궡 援먯감寃고빀쓣 삎꽦븯뒗 遺遺꾧낵 援먯감寃고빀쓣 삎꽦븯吏 븡뒗 遺遺꾩씠 議댁옱븯硫 援먯감寃고빀쓣 삎꽦븯뜑씪룄 떎냼 媛뿭꽦(reversible)쓣 쓣뼱 怨좎젙 썑 꽭泥숆낵젙 벑쑝濡 깉怨좎젙(unfixed) 맆 닔 엳떎. 씠 媛숈씠 룷瑜대쭚由곗 떎냼 빟븳 怨좎젙슚怨쇰 媛吏誘濡 議곗쭅븰쟻 洹몃━怨 硫댁뿭꽭룷솕븰쟻 뿰援ъ뿉 꼸由 씠슜릺怨 엳쑝硫, 듅엳, 빑궛(nucleic acid)쓽 怨좎젙뿉 슚怨쇱쟻씠씪怨 븣젮졇 엳떎(Spector and Goldman, 2005; Sengüven et al., 2014).

洹쇰옒뿉 洹 쟻슜 踰붿쐞媛 솗릺怨 엳뒗 蹂묐━븰쟻 遺꾩옄 吏꾨떒(molecular diagnostics) 씤瑜섏쓽 嫄닿컯, 떇뭹, 솚寃 벑쓽 遺꾩빞뿉꽌 留ㅼ슦 以묒슂븳 遺꾩빞씠硫 DNA RNA 媛숈 빑궛뱾(nucleic acids) 떎뼇븳 遺꾩옄 吏꾨떒쓽 二쇱슂 몴쟻씠 릺뼱 솕떎.

룷瑜대쭚由 怨좎젙 뙆씪 룷留 議곗쭅(FFPE) 떆猷뚮뒗 以묎컻 엫긽 뿰援ъ 遺꾩옄 泥댁쇅 吏꾨떒 벑뿉꽌 쑀슜븳 寃泥대줈 궗슜븯怨 엳쑝硫, 遺꾩옄 吏꾨떒 諛 엫긽 뿰援ъ뿉 媛옣 꼸由 궗슜릺뒗 떆猷뚯씠떎. 룷瑜대쭚由 怨좎젙 썑 젣옉맂 뙆씪 釉붾줉쓣 씠슜븳 遺꾩옄蹂묐━寃궗뒗 蹂 뿰援ъ뿉꽌 吏꾪뻾븳 PCR 諛 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗 肉먮쭔 븘땲씪 理쒓렐뿉뒗 吏꾨떒 寃곌낵, 移섎즺, 삁썑源뚯 삁痢≪씠 媛뒫븳 룞諛 吏꾨떒 諛 李⑥꽭 뿼湲곗꽌뿴遺꾩꽍(NGS)源뚯 떎뼇븳 寃궗踰뺤쑝濡 諛쒖쟾릺怨 엳떎(Lee, 2019; Newton et al., 2020; Norouzi et al., 2021; DiGuardo et al., 2021; Fujii et al., 2022).

寃궗떎뿉꽌 쟻젅븳 鍮꾩쑉濡 吏곸젒 젣議고븯뒗 룷瑜대쭚由곗 洹 寃쎌젣꽦씠굹 蹂묐━ 寃곌낵쓽 쑀슜꽦 엳쓣 닔 엳쑝굹, 쑀룆븳 臾쇱쭏뿉 븳 怨쇰룄븳 끂異쒕줈 씤빐 寃궗옄쓽 씤泥댁뿉 誘몄튂뒗 쁺뼢씠 겙 留뚰겮 젣議곕릺뼱 떆뙋릺뒗 룷瑜대쭚由 궗슜씠 쟻洹뱀쟻쑝濡 沅뚯옣릺怨 엳떎. 씠윭븳 떆쟻 쓲由꾩뿉 留욎텛뼱 寃궗떎뿉꽌 吏곸젒 젣議고븯뿬 궗슜븯뒗 룷瑜대쭚由(LmF)쓣 鍮꾨’빐꽌 쁽옱 떆뙋릺뒗 3媛 쉶궗쓽 룷瑜대쭚由(MiN, MdF, KoF)쑝濡 蹂묐━븰쟻씤 遺꾩옄 寃궗뿉꽌쓽 슚슜꽦 솗씤쓣 쐞븳 뿰援щ 吏꾪뻾븯떎.

寃곌낵瑜 醫낇빀빐 蹂대㈃, 吏곸젒 젣議고븯뒗 룷瑜대쭚由곗쓣 鍮꾨’빐꽌 3醫낅쪟쓽 떆뙋릺뒗 룷瑜대쭚由곗뿉꽌 蹂묐━議곗쭅븰쟻 뿼깋꽦 겙 李⑥씠媛 뾾뿀쑝硫, 蹂묐━遺꾩옄寃궗뿉꽌 以묒슂븳 룷瑜대쭚由곗쑝濡 怨좎젙맂 議곗쭅뿉꽌 異붿텧븳 DNA쓽 냽룄 닚룄룄 쑀궗븯寃 솗씤릺뿀떎. 洹몃윭굹 dsDNA 珥앸웾怨 dsDNA쓽 룊洹 湲몄씠瑜 굹궡뒗 tape station 寃곌낵쓽 씪遺뿉꽌 넻怨꾩쟻쑝濡 쑀쓽븳 李⑥씠瑜 蹂댁떎. 떎뿕떎뿉꽌 吏곸젒 젣議고븳 룷瑜대쭚由곗뿉 鍮꾪빐 떆뙋릺뒗 룷瑜대쭚由곕뱾쓣 궗슜븯쓣 븣 遺꾩옄 吏꾨떒쓣 쐞빐 異붿텧븯뒗 DNA쓽 긽깭뿉꽌 而ㅻ떎 李⑥씠媛 뾾쓬쓣 솗씤븯떎. 洹몃━怨 떎젣쟻씤 遺꾩옄吏꾨떒 寃궗瑜 蹂븯뒗 듅젙 쑀쟾옄뿉 븳 PCR怨 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗뿉꽌 媛곴컖쓽 룷瑜대쭚由곕뱾濡 怨좎젙븳 떆猷뚯쓽 뙆씪 釉붾줉 젅렪뿉꽌 異붿텧븳 DNA瑜 궗슜븳 寃쎌슦 쑀궗븳 寃곌낵瑜 蹂댁떎.

寃곕줎쟻쑝濡 떎뼇븳 룷瑜대쭚由 怨좎젙븸쓣 궗슜븳 媛곴컖쓽 떆猷뚮뱾뿉 븳 議곗쭅븰쟻씤 H&E 愿李곗뿉꽌 씪諛 뿼깋 젙룄쓽 李⑥씠뒗 뾾뿀쑝硫, 遺꾩옄 吏꾨떒뿉꽌쓽 PCR怨 쁽誘몃닔泥 遺덉븞젙꽦(MSI) 寃궗뿉꽌룄 쑀쓽븳 李⑥씠媛 굹굹吏 븡븯떎. 洹몃윭굹 怨좎젙븸뿉 뵲씪 렪룄(tonsil) 議곗쭅뿉꽌 異붿텧맂 DNA쓽 珥앸웾怨 湲몄씠뿉꽌 넻怨꾩쟻쑝濡 쑀쓽븳 李⑥씠媛 굹궗떎. 씪諛섏쟻씤 씤泥 옣湲 떆猷뚯뿉꽌 룷瑜대쭚由 怨좎젙븸쓽 醫낅쪟뿉 뵲瑜 議곗쭅븰쟻 寃궗 遺꾩옄蹂묐━寃궗뿉꽌쓽 쑀쓽븳 李⑥씠뒗 솗씤븷 닔 뾾뿀吏留, 냼웾쓽 떆猷뚮 씠슜븳 젙援먰븳 遺꾩옄吏꾨떒 寃궗뿉꽌뒗 理쒖긽쓽 DNA瑜 異붿텧븷 닔 엳뒗 理쒖쟻솕맂 룷瑜대쭚由 怨좎젙븸怨 異붿텧 諛⑸쾿뿉 븳 뿰援щ뱾씠 슂援щ맂떎. 씠윭븳 寃곌낵뱾쓣 뮮諛쏆묠븯湲 쐞빐 FFPE뿉꽌 異붿텧븳 DNA瑜 씠슜븯뿬 real-time PCR怨 媛숈 젙웾쟻씤 寃궗룄 븘슂븷 寃껋쑝濡 깮媛곷맂떎. 뼢썑 떎뼇븳 FFPE 議곗쭅 떆猷뚮뱾쓣 씠슜븳 異붽쟻씤 遺꾩옄吏꾨떒 뿰援щ 넻빐 議곗쭅븰쟻 寃궗 遺꾩옄蹂묐━吏꾨떒 寃궗뿉 媛옣 슚怨쇱쟻씤 룷瑜대쭚由 怨좎젙븸쓣 꽑깮븯怨 怨좎젙 諛⑸쾿쓣 媛쒖꽑븷 븘슂꽦씠 엳떎.

ACKNOWLEDGEMENTS

This project was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2020R1F-1A1071918).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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