ABSTRACT

G5DWLRQDOHIRUWKH'HYHORSPHQW &(77(3$VVD\ VIRUWKH+&(77(3$VVD\ RI$FWLRQRI)OXRUHVFHLQ7(3LQWKH7HVW6\VWHP GWRWKH+XPDQ(\H 5HVXOWV H+&(77(3$VVD\ H

INTRODUCTION

WHVWPHWKRGV WR WKH'UDL]H UDEELW H\H LUULWDWLRQ WHVWPXVW EH IRUPDQFHH[SHFWDWLRQVRID IRUPDOYDOLGDWLRQVWXG\ ,QWHUQD KDYH SURYLGHG VSHFLÀF FULWHULD WKDW DUH WR EH PHW IRU WHVW

PHWKRGYDOLGDWLRQLQ 1,(+6

7KUHH FRQGLWLRQV FRQVLGHUHGYDOLGWKH RIWKHQHZWHVWPHWK RSPHQW RI D QHZ WHV GHYHORSPHQWSUHYDOL IXOÀOOHGE\WKHFRPS RGVLQWKHSURFHVVRI SURFHVVHVGHÀQHGE\ (&9$0WKH2(&' RI$OWHUQDWLYH0HWKR EHHQ SURSRVHG IRU WK IDYRUVWKHFRQWLQXHG XVHIXOUHSODFHPHQWV

7KLVFKDSWHUGHV WKHVFLHQWLÀFUHOHYDQ 7KHVHFRPSRQHQWVR DKXPDQFRUQHDOHSL QDWLYHPHWKRGWRWKH HWDO.UXV]HZ

Background and R

7KH+&(77(3 /DERUDWRULHV*0(/ RFXODU LUULWDWLRQSRWH :DONHU.DKQ DVVD\ PHDVXUHV WKH ÁXRUHVFHLQ WUDQVHSLW FRUQHDOHSLWKHOLDOFH

7KH+&(77(3 FHOOV WKH+& FHOOOLQHLVSUHIHUDEO LQYLWURWR[LFRORJLFD LFDO YDULDELOLW\ SRVV 7KHVHSUREOHPVDUH VXUIDFHHSLWKHOLDOFH ORQJ LQFXOWXUH2Q DYDLODEOH VRXUFH RI YLWURWHVWPHWKRGV

$QXPEHURIKXP L]HGE\*0(/VFLHQ

WKHLUSXEOLVKHGJXLGHOLQHV%DOOVHWDOD2(&'

PXVW EH VDWLVÀHG IRU D QHZ WR[LFRORJLFDO WHVW PHWKRG WR EH VFLHQWLÀFUHOHYDQFHWKHSUHGLFWLYHUHOHYDQFHDQGWKHUHOLDELOLW\ RG1,(+6:RUWKDQG%DOOV7KHSUHIHUUHGGHYHO WPHWKRGZRXOG LQFOXGH VWUDWHJLFDOO\ SODQQLQJ WKH WHVWPHWKRG GDWLRQDQGYDOLGDWLRQVRWKDWDOOFRQGLWLRQVIRULWVYDOLGDWLRQDUH OHWLRQRIWKHLQWHUODERUDWRU\YDOLGDWLRQVWXG\+RZHYHUWHVWPHWK YDOLGDWLRQDWWKLVWLPHZHUHGHYHORSHGEHIRUHWKHIRUPDOYDOLGDWLRQ WKH(XURSHDQ&HQWHUIRUWKH9DOLGDWLRQRI$OWHUQDWLYH0HWKRGV DQGWKH,QWHUDJHQF\&RRUGLQDWLQJ&RPPLWWHHRQWKH9DOLGDWLRQ GV,&&9$0ZHUHÀQDOL]HG7KHUHIRUHWKHÁH[LELOLW\WKDWKDV H YDOLGDWLRQ SURFHVVHV 1,(+6 :RUWK DQG%DOOV HYDOXDWLRQRIVFLHQWLÀFDOO\UHOHYDQWPHWKRGVWKDWDUHOLNHO\WREH RUDGMXQFWVWRFXUUHQWWR[LFLW\WHVWPHWKRGV FULEHVWZRHVVHQWLDOVWHSVLQWKHYDOLGDWLRQSURFHVVHVWDEOLVKLQJ FHRIDQHZWHVWPHWKRGDQGWKHSUHYDOLGDWLRQRIWKHWHVWPHWKRG IWKHYDOLGDWLRQSURFHVVDUHGHVFULEHGIRUWKH+&(77(3DVVD\ WKHOLDOFHOOEDVHGDVVD\WKDWZDVGHYHORSHGDVDQLQYLWURDOWHU 'UDL]HUDEELWH\HLUULWDWLRQWHVW.DKQDQG:DONHU.DKQ VNLHWDO

SCIENTIFIC RELEVANCE

ationale for the Development of the HCE-T TEP Assay

WHVWPHWKRGZDVGHYHORSHGDWWKH*LOOHWWH0HGLFDO(YDOXDWLRQ LQ²DVDQLQYLWURWHVWPHWKRGXVHGWRHYDOXDWHWKH QWLDO RIZDWHUVROXEOH FKHPLFDOV DQG IRUPXODWLRQV .DKQ DQG HWDO.UXV]HZVNLHWDO7KH+&(77(3 GRVHGHSHQGHQW HIIHFW RI D WHVW PDWHULDO RQ WKH VXEVHTXHQW KHOLDO SHUPHDELOLW\ 7(3 DFURVV VWUDWLÀHG FXOWXUHV RI KXPDQ OOVWKH+&(7FHOOOLQH DVVD\LVSHUIRUPHGXVLQJFXOWXUHVRIKXPDQFRUQHDOHSLWKHOLDO (7FHOO OLQH7KHUHDUHQXPHURXV UHDVRQVZK\ WKHXVHRID HWRWKHXVHRISULPDU\FHOOVRUWLVVXHV IRUWKHGHYHORSPHQWRI ODVVD\V3UREOHPVZLWKWKHXVHRISULPDU\FHOOVLQFOXGHELRORJ LEOH LQIHFWLRXV DJHQW FRQWDPLQDWLRQ DQG ODFN RI DYDLODELOLW\ SDUWLFXODUO\HYLGHQWZKHQLWFRPHVWRWKHXVHRIKXPDQRFXODU OOVZKLFKDUHIUHTXHQWO\GLIÀFXOW WRREWDLQDQGGRQRWVXUYLYH

WKHRWKHUKDQGZHOOFKDUDFWHUL]HGFHOO OLQHVSURYLGHD UHDGLO\ FHOOV WKDW FDQ EH XVHG IRU WKH GHYHORSPHQW RI UHSURGXFLEOH LQ

DQFRUQHDOHSLWKHOLDOFHOOOLQHVZHUHGHYHORSHGDQGFKDUDFWHU WLVWVEHWZHHQDQG7KH+&(7FHOOOLQHZDV

GHYHORSHGE\WUDQVIH FRUQHDOHSLWKHOLDOFHO HWDO.DKQDQ UHVXOWLQJFHOOOLQHVZ FRPSDUDELOLW\WRSULP PRGXODWLRQRIPRUSK PHWDOORSURWHLQDVHH[ DPHDVXUDEOHHSLWKHO .DKQDQG:D ED&ORWK DOVRFRQÀUPHGWKHU FHOOOLQH%UDXQVWHLQ DQG+D]OHWW 6 KDYHDQH[WHQGHGOLI FHOOVYHUVXVDSSUR[LP DUHDGLO\DYDLODEOHVR VWXGLHV7KHSURGXFW WKDW KDV SURSHUWLHV V REVHUYDWLRQVLQGLFD FRQWLQXHG XVH LQ WKH +&(7FHOO O $7&&LQ0DQDVVDV

)RU WKH+&(77 PHPEUDQHV ZKHUH WK VWUXFWXUHRIWKHFRUQ WKHKXPDQFRUQHDWK 7FHOOVDUHFDOOHGWKH LQFHOOFXOWXUHPHGLX DWWKHDLU²OLTXLGLQWH GHYHORSPHQWRIPXO EHWZHHQWKHFHOOVZ DVWKH\SUROLIHUDWHDQ

2QHRI WKHPDMR GLIIHUHQWELRORJLFDOH WLYHSHUPHDELOLW\ED HSLWKHOLXP IRUPV D LQWDFW FRUQHDO HSLWKH PROHFXODUZHLJKWÁX 7KXV WKH WHFKQLTXH RSKWKDOPRORJLVWVWR

7KHVWUDWLÀHGFXO ZKLFKLVUHODWLYHO\LP IXQFWLRQLQWKH+&( SHUPHDELOLW\ WR VRGL &KHPLFDO LUULWDQWV K LQWDFWKXPDQFRUQHD

FWLRQWKHLQVHUWLRQRIYLUDOJHQHVRISULPDU\FXOWXUHVRIKXPDQ OV+&(ZLWKWKH69ODUJH7DQWLJHQSODVPLG5697.DKQ G5KLP-XO\:DONHUDQG.DKQ6HSWHPEHU7KH HUHFKDUDFWHUL]HGXVLQJDQXPEHURIFULWHULDWRGHWHUPLQHWKHLU DU\+&(FHOOVLQFOXGLQJFHOOPRUSKRORJ\FDOFLXPPHGLDWHG RORJ\NHUDWLQH[SUHVVLRQF\WRNLQHUHOHDVHODFWDWHSURGXFWLRQ SUHVVLRQDQGWKHDELOLW\WRIRUPVWUDWLÀHGFXOWXUHVWKDWGHYHORS LDOSHUPHDELOLW\EDUULHUDQGH[SUHVVWLJKWMXQFWLRQV.DKQHWDO ONHU.UXV]HZVNLHWDO:DUGHWDOD LHU HW DO 5HVHDUFKÀ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ÀOP7KHVHLQYLWURFXOWXUHVRIVWUDWLÀHG+&( +&(7PRGHO7KHERWWRPOD\HUVRI+&(7FHOOVDUHLPPHUVHG PDQGWKHWRSOD\HUIDFHVWKHDLUVRWKH\DUHGHVFULEHGDVJURZQ UIDFH)LJXUH$7KLVW\SHRIFXOWXUHIRUPDWSURPRWHVWKH WLSOH FHOO OD\HUV VWUDWLÀFDWLRQ DQG WKH IRUPDWLRQRI MXQFWLRQV KLFKDUHQRUPDOSURFHVVHVWKDWFRUQHDOHSLWKHOLDOFHOOVXQGHUJR GGLIIHUHQWLDWH UIXQFWLRQVRIDQHSLWKHOLDO WLVVXHLV WRIRUPDERUGHUEHWZHHQ QYLURQPHQWVWKXVSURYLGLQJERWKDSURWHFWLYHUROHDQGDVHOHF UULHU 0DFND\HW DO $W WKHRFXODU VXUIDFH WKHFRUQHDO EDUULHU WR SURWHFW WKH H\H IURP WKH H[WHUQDO HQYLURQPHQW7KH OLXP IRUPV D WLJKW EDUULHU WKDW LV LPSHUPHDEOH WR WKH VPDOO RUHVFHQWG\HVRGLXPÁXRUHVFHLQ0DXULFH1RUQ RIYLWDO VWDLQLQJRI WKH FRUQHDE\ÁXRUHVFHLQ FDQEHXVHGE\ LGHQWLI\RFXODUVXUIDFHHSLWKHOLDOGHIHFWV WXUHVRIWKH+&(7PRGHOGHYHORSDQHSLWKHOLDOEDUULHUIXQFWLRQ

SHUPHDEOHWRÁXRUHVFHLQ'LVUXSWLRQRI WKHHSLWKHOLDOEDUULHU

7PRGHOE\DFKHPLFDOLUULWDQWFDQEHPHDVXUHGE\LWVLQFUHDVHG XPÁXRUHVFHLQ :DUGHW DO E.UXV]HZVNL HW DO DYH OLNHZLVH EHHQ IRXQG WR GLVUXSW WKH LPSHUPHDELOLW\ RI WKH WRÁXRUHVFHLQ0DUVKDQG0DXULFH6LQFHGLVUXSWLRQRI

WKHFRUQHDOHSLWKHOLD FKHPLFDOLQGXFHGH\ SDUDPHWHU WKDW VKRX DQGRWKHUH\HLUULWDQ

7KH+&(77(3 DELOLW\ 7(3RI WKH DIWHU WKHLUH[SRVXUH FRQVLVWVRI WKUHHPD )LJXUH$H

OEDUULHU LVRQHRI WKHHDUOLHVWELRORJLFDOHYHQWV WKDWRFFXUV LQ H LUULWDWLRQ 0DXULFH LW KDVEHHQ LGHQWLÀHGDV D OG EH SUHGLFWLYH RI WKH RFXODU LUULWDWLRQ SRWHQWLDO RI FKHPLFDOV WV7FKDR:DUGHWDOE

DVVD\SURYLGHVDPHDQVWRPHDVXUHWKHWUDQVHSLWKHOLDOSHUPH PXOWLOD\HUHGFRUQHDOHSLWKHOLDOFXOWXUHV WRVRGLXPÁXRUHVFHLQ WR LQFUHDVLQJGRVHVRID WHVWPDWHULDO7KH+&(77(3DVVD\ MRU VWHSV GHYHORSPHQWDQGJURZWKRI WKH+&(7FXOWXUHV [SRVXUHRIWKHFXOWXUHVIRUPLQWRGLIIHUHQWFRQFHQWUDWLRQVRI

D WHVW PDWHULDO )LJX SHUPHDELOLW\RIWKHF DPRXQWRIÁXRUHVFHLQ 77(3DVVD\SURYLGH PDWHULDORQWKH+&( FXUYH)LJXUH' IURPWKHGRVHUHVSRQ FLDWHGZLWKÁXR ZDVHPSLULFDOO\GHWH 'UDL]HH\HLUULWDWLRQ

$SUHGLFWLRQPRG VKLSEHWZHHQWKHGDWD PHWKRG %UXQHU HW UHIHUHQFHPHWKRGIR 'UDL]HHWDO E\VWDWLVWLFDOPRGHOLQ YLYRKLVWRULFDO'UDL]H VXUIDFWDQWV DQG VXUID PRGHOFDQEHXVHGWR E\XVLQJWKHWR[LFLW\ )57KHFDOFXODWL HWDO7KHOR VROXEOHFKHPLFDOVDQ WLVVXH VFRUHV VXFK D VFRUH.UXV]HZVNLH

Scientific Basis fo

7KHVWDQGDUGPH LUULWDWLRQ SRWHQWLDO R 'UDL]HHWDO DQG YDULRXV PRGLÀ UHYLHZHGLQ&KDQD S,QJHQHUDO LQ WKH FRQMXQFWLYDO RFXODU UHDFWLRQ WR WK LQWHUYDOVDFFRUGLQJ VWDQGDUGJUDGLQJVFK FRUQHDO UHVSRQVHV F SRLQWVDQGWKH LULV WKH FRQWLQXHG XVH R ODUJHGDWDEDVHRI LQ EHWZHHQWKHUDEELWD WKH'UDL]H WHVW IRU W %HFNOH\HWDO ,/6,$$7

UH % DQG HYDOXDWLRQ RI WHVW PDWHULDO HIIHFWV RQ WKH XOWXUHVWRVRGLXPÁXRUHVFHLQ)LJXUH&%\PHDVXULQJWKH WKDWSDVVHVWKURXJKWKHFXOWXUHVLQWRWKHEDVDOPHGLDWKH+&( VDTXDQWLWDWLYHPHDVXUHIRUWKHGRVHGHSHQGHQWHIIHFWRIDWHVW 7FHOOFXOWXUHV7KHUHVXOWVFDQEHSORWWHGDVDGRVHUHVSRQVH 7KHHQGSRLQWIRUWKHLQYLWURDVVD\WKHORJ)5LVREWDLQHG VHFXUYH7KH)5LVWKHFRQFHQWUDWLRQRIWHVWPDWHULDODVVR UHVFHLQUHWHQWLRQ)5UHODWLYHWRWKHFRQWUROFXOWXUHV7KH)5 UPLQHGWREHWKHHQGSRLQWWKDWSURYLGHGRSWLPDOFRUUHODWLRQWR GDWD.UXV]HZVNLHWDO HOFRQWDLQVDQDOJRULWKPWKDWGHÀ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

r the HCE-T TEP Assay

WKRGDFFHSWHGE\UHJXODWRU\DJHQFLHVIRUGHWHUPLQLQJWKHH\H I D VXEVWDQFH KDV EHHQ WKH 'UDL]H UDEELW H\H LUULWDWLRQ WHVW 7KHRULJLQDOWHVWKDVEHHQPRGLÀHGWRXVHWKUHHWRVL[DQLPDOV HG YHUVLRQV DUH VSHFLÀHG E\ GLIIHUHQW UHJXODWRU\ DJHQFLHV QG+D\HVSS²'L3DVTXDOHDQG+D\HV DVPDOODPRXQWRIWHVWPDWHULDOPORUPJLVSODFHG VDF RI RQH H\H RI D VSHFLÀHG QXPEHU RI DOELQR UDEELWV7KH H WHVWPDWHULDO LV GHWHUPLQHG DQG JUDGHG DW YDULRXV VFRULQJ WRUHVSRQVHVLQWKHFRUQHD WKHFRQMXQFWLYDDQGWKHLULV,QD HPHWKHWRWDO LUULWDWLRQLVUDQNHGRQDVFDOHRIWR WKH RPSULVH SRLQWV WKH FRQMXQFWLYDO UHVSRQVHV UHVSRQVHVSRLQWV7R[LFRORJLVWVIHHOFRPIRUWDEOHZLWK I WKLVPHWKRG EHFDXVH RI LWV IDPLOLDULW\ DQG EHFDXVH RI WKH

IRUPDWLRQDYDLODEOH+RZHYHU FRQVLGHUDEOH GLIIHUHQFHV H[LVW QGWKHKXPDQH\HZKLFKKDVOHGPDQ\WRTXHVWLRQWKHXVHRI KHSUHGLFWLRQRI WKHKXPDQRFXODU UHVSRQVH %HFNOH\ *HUVKEHLQDQG0F'RQDOG*UHHQHWDO:DONHU 3UREOHPVZLWK WKH UHSURGXFLELOLW\RI'UDL]H WHVW UHVXOWV

KDYHDOVREHHQDFRQ DQGLQWHUODERUDWRU\ RI:HLODQG6FDOD

7KHPDMRUVLPLO WHVWPHWKRGDQGWKH

7KHFRUQHDOUHVS WREHDVVHVVHGE\D' DQGFRQVXPHUSURGX RIWKHJOREH7KHH\ WKHH\H OLG DSK\VLF WKH HSLWKHOLXP FRYH EDUULHU7KHFRUQHD WR EH H[SRVHG WR D SURWHFWLYHEDUULHUIX SDUHQWSRUWIRUWKHH -DNRELHF&R LQMXU\WRWKHFRUQHD WRWDO'UDL]HVFRUH

7KHFRUQHDOHSLW WLRQDOEDUULHUWRWR[LF 0F'RQDOG7 VL[ FHOO OD\HUV7KH GHVPRVRPDOLQWHUFHO 7KHVHPXOWLSO EDUULHURI WKHRFXODU WKHSULPDU\MXQFWLRQ DQG WLJKW MXQFWLRQV W\SHDQGGHJUHHRIE +DPDODLQHQHWDO

$QXQGHUVWDQGLQ WKHGHYHORSPHQWRI FRPSRVHG RI WKUHH ZKLFK LV LQWHUVSHUVH FHOOOD\HURIWKHFRUQ KXPRU RI WKH DQWHUL GLIIHUHQWLDOSHUPHDE LVDW OHDVWSDUWLDOO\E 'H6DQWLVDQG3DWLO RIK\GURSKLOLFVXEVWD PRVWRIWKHEDUULHUW ORJLFSURSHUWLHVRIW PRGHORIWKHFRUQHD

%RWKWKHUDEELW WRVXUIDFWDQWRFXODU LQ LVRODWHG UDEELW D VRGLXPGRGHF\O VX

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ÀFDQFHRIRIWKH PD[LPXPFRUQHDOVFRUHRXWRIPD[LPXPWRWDOVFRUH KHOLXPDVWKHRXWHUPRVWOD\HURIWKHFRUQHDVHUYHVDVDIXQF DQWHQWU\LQWRWKHH\H'DVWRQDQG)UHHEHUJ+DFNHWWDQG KHFRUQHDOHSLWKHOLXPLVDVWUDWLÀHGWLVVXHFRPSRVHGRIÀYHWR VXUIDFH FHOOV DUH FRQQHFWHG E\ WLJKW MXQFWLRQV DQG DGGLWLRQDO OXODUMXQFWLRQVH[LVWWKURXJKRXWWKHUHPDLQLQJFHOOOD\HUV)LJXUH HFHOOOD\HUVDQGLQWHUFHOOXODUDGKHVLRQVSURYLGHWKHSURWHFWLYH VXUIDFH7KHRXWHU WLJKW MXQFWLRQRI WKH FRUQHDOHSLWKHOLXP LV WRUHJXODWHHSLWKHOLDOSHUPHDELOLW\7KHLQWHUFHOOXODUDGKHVLRQV DUH RUJDQL]HG LQ D WLVVXHVSHFLÀFPDQQHU SURYLGLQJ D XQLTXH DUULHUIXQFWLRQE\WKHFRUQHDOHSLWKHOLXP0DFND\HWDO JRI WKHVWUXFWXUHDQGSK\VLRORJ\RI WKHFRUQHD LVHVVHQWLDO WR DSSURSULDWHO\VWUXFWXUHG LQYLWURFRUQHDOPRGHOV7KHFRUQHDLV FHOOXODU OD\HUV WKH RXWHUPRVW FRUQHDO HSLWKHOLXP WKH VWURPD GZLWK ÀEUREODVWW\SH FHOOV FDOOHG NHUDWRF\WHV DQG WKH VLQJOH HDOHQGRWKHOLXPZKLFKSURYLGHVWKHLQWHUIDFHZLWKWKHDTXHRXV RU FKDPEHU7KH WKUHH FHOOXODU OD\HUV RI WKH FRUQHD SURYLGH D LOLW\EDUULHUWRWKHSHQHWUDWLRQRIFRPSRXQGVLQWRWKHH\HZKLFK DVHGXSRQWKHSDUWLWLRQLQJEHKDYLRURIDSDUWLFXODUFRPSRXQG 7KHHSLWKHOLXPSURYLGHV WKHEDUULHU WR WKHSHQHWUDWLRQ QFHVLQWRWKHFRUQHDZKLOHWKHVWURPDDQGHQGRWKHOLXPSURYLGH RWKHSHQHWUDWLRQRIPRUHOLSRSKLOLFVXEVWDQFHV7KHVHSK\VLR KHFRUQHDSURYLGHWKHELRORJLFDOUDWLRQDOHIRUXVLQJDQLQYLWUR

OHSLWKHOLXPWRHYDOXDWHVXUIDFWDQWLQGXFHGFRUQHDOHIIHFWV DQGWKHKXPDQFRUQHDKDYHEHHQVKRZQWRUHVSRQGVLPLODUO\ H[SRVXUHV%HUU\DQG(DVW\VKRZHGWKDWFRUQHDOHIIHFWV QG KXPDQ H\HV ZHUH VLPLODU DIWHU H[SRVXUHV WR DQ DQLRQLF OIDWH DQG D QRQLRQLF 7ZHHQ VXUIDFWDQW+RZHYHU RWKHU

VXEVWDQFHVVXFKDV 7KHVHUHVXOWVVXSSR DV WKH UHIHUHQFHPH WKH HYDOXDWLRQ RI VX FRXOGDOVRH[SODLQZ EHWZHHQWKH'UDL]H 2WKHUFRQVLGHUDWLRQ EHWZHHQODERUDWRULH WKH 'UDL]H H\H LUULW GHYHORSPHQWRIQHZ

5HFHQW VWXGLHV L UHVXOWVLQFHOOGHDWK RI RFXODU LUULWDWLRQ WKDWWKHRFXODULUULWDW LQFUHDVHGDUHDDQGG E7KH\IRXQG VXUIDFH HSLWKHOLXP LQFUHDVLQJO\ JUHDWHU DOVR FDXVHG FRUQHDO DSSURSULDWHIRUWKHH ZKHQGLOXWLRQVRIWK UHVXOWVREWDLQHGLQHY 7KH+&(77(3 DV LQVXIÀFLHQWQXPEHU WKHUHVXOWV

5HSURGXFWLRQRI LQWRFRQVLGHUDWLRQG FRQVLVWVRIDPXOWLOD RQDSHUPHDEOHFROOD 7FHOOVRQSHUPHDEO DQHOHYDWHGFRQFHQW GLIIHUHQWLDWH IRUPLQ

HWKDQRODQGDFHWRQHVKRZHGVSHFLHVVSHFLÀFFRUQHDOHIIHFWV UWWKHELRORJLFDOUHOHYDQFHRIXVLQJ'UDL]HUDEELWH\HWHVWGDWD WKRG IRUDKXPDQFRUQHDOFHOOEDVHG LQYLWUR WHVWPHWKRG IRU UIDFWDQWV DQG VXUIDFWDQWEDVHG WHVWPDWHULDOV7KHVH ÀQGLQJV K\VRPHRWKHUFKHPLFDOFODVVHVVKRZHGD ORZHUFRUUHODWLRQ

GDWDDQGWKH+&(77(3DVVD\GDWD.UXV]HZVNLHWDO VVXFKDVWKHODUJHYDULDELOLWLHVREVHUYHGEHWZHHQDQLPDOVDQG VZLWK'UDL]HH\HWHVWGDWD:HLODQG6FDODVWLOOPDNH DWLRQ WHVW OHVV WKDQ RSWLPDO DV WKH UHIHUHQFHPHWKRG IRU WKH WHVWPHWKRGV Q RQH ODERUDWRU\ LQGLFDWH WKDW RFXODU LQMXU\ IURP VXUIDFWDQWV DQGWKDWWKHH[WHQWRIFHOOGHDWKPD\EHSUHGLFWLYHRIWKHGHJUHH -HVWHU HW DO 3UHYLRXV VWXGLHV LQ WKLV ODE GHPRQVWUDWHG LRQSURGXFHGE\VXUIDFWDQWVRILQFUHDVLQJLUULWDQF\LVUHODWHGWR HSWKRIFRUQHDOLQMXU\-HVWHUHWDO0DXUHUHWDO WKDWVOLJKWWRPLOGLUULWDQWVSURGXFHGLQMXU\PRVWO\WRWKHRFXODU ZKLOH WKH GDPDJH IURP PRGHUDWH WR VHYHUH LUULWDQWV VKRZHG GHSWKV RI LQMXU\ WKURXJK WKH FRUQHDO VWURPD 6HYHUH LUULWDQWV HQGRWKHOLDO FHOO GDPDJH 7KXV D FRUQHDO HSLWKHOLDO PRGHO LV YDOXDWLRQRIVOLJKWDQGPLOGLUULWDQWVDQGIRUPRGHUDWHLUULWDQWV HPDWHULDOVDUHHYDOXDWHG7KLVFRQFOXVLRQ LVVXSSRUWHGE\WKH DOXDWLQJVXUIDFWDQWH[SRVXUHVXVLQJWKHVWUDWLÀHG+&(7PRGHO VD\ KDV DOVR EHHQ DEOH WR LGHQWLI\ VHYHUH H\H LUULWDQWV EXW DQ KDYHEHHQHYDOXDWHGWRGHWHUPLQHWKHDFFXUDF\DQGSUHFLVLRQRI

WKHELRORJLFDOFKDUDFWHULVWLFVRIWKHFRUQHDOVXUIDFHZDVWDNHQ XULQJWKHGHYHORSPHQWRIWKH+&(7PRGHO7KH+&(7PRGHO

\HUHGFXOWXUHRI+&(7KXPDQFRUQHDOHSLWKHOLDOFHOOVJURZQ JHQPHPEUDQHDWWKHDLU²OLTXLGLQWHUIDFH&XOWXUHRIWKH+&( HPHPEUDQHVDWWKHDLU²OLTXLGLQWHUIDFHLQPHGLXPFRQWDLQLQJ UDWLRQRIFDOFLXPP0 LQGXFHVWKHFHOOV WRVWUDWLI\DQG J D WLVVXH ZLWK D ZHOOGHYHORSHG EDUULHU IXQFWLRQ %DUULHU

IXQFWLRQ LQ WKH+&( UHVWULFWWKHSHUPHDWL DELOLW\WRÁXRUHVFHLQ WKHOLDO HOHFWULFDO UHV :DUGH

8OWUDVWUXFWXUDO DQ KXPDQFRUQHDOVXUIDF VXUIDFHFHOOOD\HUVLQ FRPSRVHGRIÁDWWHQHG VLEOHIRUUHJXODWLRQR FHOOOD\HUVRIWKH+&(

7KH FURVVVHFWLRQ RI WKHFKHPLFDO WR[L FXOWXUH)LJXUH$ %$& ZDV F VKRZQE\WKHODFNRI )LJXUH%7KH VRPHHGHPDLQGLFDW VSDFHV 3K\VLRORJLFD

A

7PRGHO FDQEHPHDVXUHGE\ WKH DELOLW\ RI WKH FXOWXUHV WR RQRIVRGLXPÁ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

B

\WRWR[LF WR WKH VXSHUÀFLDO FHOOV LQ DQRWKHU+&(7 FXOWXUH DV LQWUDFHOOXODUFRQWHQWLQWKHVXUIDFHFHOORIWKHSKRWRPLFURJUDSK HSLWKHOLDOFHOOVLQWKH%$&WUHDWHGFXOWXUHDOVRDSSHDUWRKDYH HGE\WKHLUSOXPSDSSHDUDQFHDQGE\WKHLQFUHDVHGLQWHUFHOOXODU O DVVHVVPHQW RI WKH FXOWXUHV VKRZHG WKDW %$&FDXVHG

OHVVWKDQRYHUD FHLQ SHUPHDELOLW\ RI WLJKWMXQFWLRQVGHFU

$QRWKHUVLPLODUL LVWKHSURGXFWLRQRI VXUIDFHRIWKHLQWDFW D )LJXUH FRUQHDODQGFRQMXQFW ,QDWRPLHWDO VSUHDGLQJRIWKHDTX IRUWKHVXUIDFHRIWK RI WKH VXUIDFH HSLWK VXEVWDQFHV'H6DQWL 7PRGHOZDVDFFRP PXFLQZDV WKHQ IRX 7KHVHUHVXOWVD HWDO(GHOKDX

7KHPXOWLSOHFH WKRVHRIWKHFRUQHDO E\ WKH PRQROD\HU F :DUG HW DO E KDYHEHHQVKRZQWR WRFKHPLFDO:DUGH 7KHVH ÀQGLQJV VXSS GLPHQVLRQDOFRUQHD QDWLYHWHVWPHWKRG

OOF\WRWR[LFLW\077DVVD\RQO\VOLJKWO\LQFUHDVHGWKHÁXRUHV WKH FXOWXUHV GHFUHDVHG7(3 EXW VLJQLÀFDQWO\ GDPDJHG WKH HDVHG7(5:DUGHWDOE W\EHWZHHQWKH+&(7FXOWXUHVDQGWKHLQYLYRFRUQHDOVXUIDFH PXFLQE\WKHFRUQHDOHSLWKHOLDOFHOOVDQGLWVORFDOL]DWLRQDWWKH FRUQHDDQGRQWKHVXUIDFHRIWKH+&(7FXOWXUHV:DUGHWDO 0XFLQ LV D FRPSRQHQW RI WKH WHDU ÀOP WKDW LV SURGXFHG E\ LYDOHSLWKHOLDOFHOOVDVZHOODVWKHJREOHWFHOOVRIWKHFRQMXQFWLYD 7KHIXQFWLRQVRIPXFLQDWWKHRFXODUVXUIDFHDUHWRIDFLOLWDWH HRXVSRUWLRQRIWKHWHDUÀ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

EHPRUHUHVLVWDQW WKDQPRQROD\HUFXOWXUHVRI WKHVDPHFHOOV WDOEDQGSURWHLQWR[LFDQW:DUGHWDOGDPDJH RUW WKH ELRORJLFDO UHOHYDQFH RI XVLQJ D GLIIHUHQWLDWHG WKUHH OHSLWKHOLDOFHOOPRGHO IRU WKHGHYHORSPHQWRID'UDL]HDOWHU

)OXRUHVFHLQSHUP WXUHVZDVRULJLQDOO\ WRWKHH\HE\7FKDR FXOWXUHV RI 0DQGLQ 0'&.FHOOÁXRUHVF DO $O PRQROD\HU 0'&. 'UDL]H WHVW WR ZDUU *HWWLQJV HW DO 7LJKW MXQFWLR QHRXVO\ ZLWK LQFUHD &ORWKLHU7LJK RIEDUULHUIXQFWLRQG KXPDQ FRUQHDO HSLW LQWHUFHOOXODUMXQFWLRQ $UDLHDQG0DXULFH UHWHQWLRQE\+&(7 &KHPLFDOH[SRVXUHV PHDELOLW\7(3ZDV ,Q WKH VDPH H ÁXRUHVFHLQZDVIRXQ 0'&.ÁXRUHVFHLQ WKH WLVVXHVSHFLÀF I RFXODUVXUIDFH

7KH H[SHULPHQWD FRQFOXVLRQ WKDW WKH PHFKDQLVWLFDOO\EDVH UHVSRQVHWRH\HLUULWD

HDELOLW\DVDPHDVXUHRIEDUULHUGLVUXSWLRQLQHSLWKHOLDOFXO SURSRVHGDVDPRGHOWRHYDOXDWHSRWHQWLDOFKHPLFDOWR[LFLW\ 7KHRULJLQDOÁXRUHVFHLQOHDNDJHDVVD\XVHGPRQROD\HU 'DUE\ FDQLQH NLGQH\ HSLWKHOLDO FHOOV 0'&. FHOOV 7KH HLQOHDNDJHDVVD\LVDXVHIXOVFUHHQIRUH\HLUULWDQWV6KDZHW WKRXJK VWXGLHV DUH RQJRLQJ ÁXRUHVFHLQ SHUPHDELOLW\ DFURVV FXOWXUHV KDV QRW GHPRQVWUDWHG VXIÀFLHQW FRUUHODWLRQ WR WKH DQW LWV UHFRPPHQGDWLRQ IRU YDOLGDWLRQ %DOOV HW DO E %UDGODZ HW DO %UDQWRP HW DO =DQYLW HW DO Q GLVUXSWLRQ LQ 0'&. FXOWXUHV DSSHDUV WR RFFXU VLPXOWD VHG ÁXRUHVFHLQ SHUPHDELOLW\ SHUVRQDO FRPPXQLFDWLRQ 5 WMXQFWLRQGLVUXSWLRQKRZHYHULVRQO\SDUWRIWKHPHFKDQLVP LVUXSWLRQZKHQLWRFFXUVDWWKHRFXODUVXUIDFH,QWKHVWUDWLÀHG KHOLXP WKH PXOWLSOH FHOO OD\HUV WLJKW MXQFWLRQV DQG RWKHU VDFWWRUHWDUGÁXRUHVFHLQSHQHWUDWLRQLQWRWKHFRUQHDOVWURPD 7KHVDPHPHFKDQLVPVDUHUHVSRQVLEOHIRUÁXRUHVFHLQ FXOWXUHV7KHGDWDVKRZQLQ)LJXUHLOOXVWUDWHWKLVSRLQW GLVUXSWHGWKHWLJKWMXQFWLRQV7(5EHIRUHÁXRUHVFHLQLPSHU SHUWXUEHGLQWKH+&(7PRGHO:DUGHWDOE)LJXUH [SHULPHQWV UHFRYHU\ LQ WKH DELOLW\ RI WKH FXOWXUHV WR UHWDLQ GWREHLQGHSHQGHQWRIUHFRYHU\LQ7(57KXVDOWKRXJKWKH OHDNDJHDVVD\LVXVHIXODVDVFUHHQ LWGRHVQRWFRQWDLQDOORI HDWXUHV QHHGHG WR UHSURGXFH WKH LUULWDQW UHVSRQVH DW KXPDQ

O GDWD DQG WKH VFLHQWLÀF OLWHUDWXUH UHSRUWHG KHUH VXSSRUW WKH +&(7 7(3 DVVD\ LV D WHVW PHWKRG WKDW LV ELRORJLFDOO\ DQG GDQGWKXVVKRXOGSURYLGHDJRRGPRGHOIRUWKHKXPDQFRUQHDO QWV

The Mechanism o Compared

2QHRIWKHSULPD LUULWDWLQJ PDWHULDO LV GLVUXSWLRQ FDQ EHP FRUQHDOHSLWKHOLXP LQ WKH SUHYLRXV VHFW LQYROYHGLQWKHPDLQ LQWKHVWUDWLÀHG+&(

7UDQVHSLWKHOLDOÁ RIWKHLQWHJULW\RIWK 6LQJK %XUVWH ÁXRUHVFHLQ LVDVWDQ WKHFRUQHDOHSLWKHOLX ÁXRUHVFHQWDQG\HOO LVZDWHUVROXEOHVKR VLGHUHG WR EH QRQWR ÁXRUHVFHLQDXVHIXO UHVFHLQ FDQ EH TXDQ SHDN DEVRUSWLRQ DW SHUPHDELOLW\ RIÁXR UHSRUWHG WREHv WKHFRUQHDOHQGRWKHO RQVWUDWH WKH VLJQLÀF SHQHWUDWLRQRIZDWHU LQWRWKHH\H

7KH LQ YLYR FRU PHDVXULQJWKHORVVR FKDPEHU 0DXULFH VFDQQLQJÁXRURSKRWR DQWHULRUFKDPEHURIW LVEH\RQGWKHVFRSH LVOLPLWHGE\WKHUHVR VRWKDWDODUJHSRSXOD JURXSV-RVKLHWDO ÁXRURSKRWRPHWU\P GDWD WR WKH LQ YLWUR XQGHUO\LQJWKHWZRW

&RUQHDOSHUPHDE DVF\WRWR[LFLW\DQGM ULFH 0F&DUH GLVUXSWLRQDUHHDUO\ 5DPVHODDU HW DO 0F&DUH\DQG5HDYH SURSRVHG FODVVLÀFDW

f Action of Fluorescein TEP in the Test System to the Human Eye

U\UHVSRQVHVWKDWRFFXUXSRQVXSHUÀFLDORFXODUH[SRVXUHWRDQ GLVUXSWLRQ RI WKH FRUQHDO HSLWKHOLDO EDUULHU IXQFWLRQ %DUULHU HDVXUHG E\ WKH ORVV RI ÁXRUHVFHLQ LPSHUPHDELOLW\ DFURVV WKH ERWKLQWKHKXPDQH\HDQGLQWKH+&(7PRGHO$VH[SODLQHG LRQ WKLV LV GXH WR WKH VLPLODULW\ LQ WKH FHOOXODU PHFKDQLVPV WHQDQFHRIEDUULHUIXQFWLRQLQWKHLQYLYRFRUQHDOHSLWKHOLXPDQG 7FXOWXUHV XRUHVFHLQSHUPHDELOLW\KDVEHHQVKRZQWREHDXVHIXOLQGLFDWRU HLQYLYRFRUQHDOHSLWKHOLDOEDUULHU0DXULFH0DXULFHDQG LQ 9LWDO VWDLQLQJ RI WKH KXPDQ FRUQHD XVLQJ VRGLXP GDUGWHFKQLTXHXVHGE\RSKWKDOPRORJLVWV WRDVVHVVGDPDJHWR P6RGLXPÁXRUHVFHLQLVDVPDOOPROHFXODUPDVVEULJKWO\ RZLVKJUHHQFRORUHGPROHFXOH7KHVRGLXPVDOWRIÁXRUHVFHLQ ZVQHJOLJLEOHELQGLQJWRWKHLQWDFWRFXODUVXUIDFHDQGLVFRQ [LF 0DXULFH ZKLFK DUH SURSHUWLHV WKDW PDNH VRGLXP PROHFXOHIRURSKWKDOPRORJLFXVH,QVROXWLRQWKHFRORUHGÁXR WLWDWHG E\ PHDVXULQJ HLWKHU DEVRUEDQFH RU ÁXRUHVFHQFH ZLWK QP DQG SHDN HPLVVLRQ DW QP 0DXULFH 7KH UHVFHLQ LQ WKHKXPDQ FRUQHDO HSLWKHOLXP LQ RQH VXEMHFWZDV ²FPKUZKLFKLVVLJQLÀFDQWO\ OHVV WKDQWKDWPHDVXUHGIRU LXPv²FPKU 0DXULFH7KRVHÀQGLQJVGHP DQW UROH RI WKH FRUQHDO HSLWKHOLXP DV WKHPDMRU EDUULHU WR WKH VROXEOHGUXJV DQG FRPSRXQGV LQFOXGLQJ VRGLXPÁXRUHVFHLQ

QHDO HSLWKHOLDO EDUULHU WR ÁXRUHVFHLQ KDV EHHQ GHWHUPLQHG E\ IÁXRUHVFHLQIURPWKHWHDUÀOPDQGLWVDSSHDUDQFHLQWKHDQWHULRU GH .UXLMI HW DO $Q LQVWUXPHQW FDOOHG D PHWHULVXVHGWRHYDOXDWHWKHSHUPHDWLRQRIÁXRUHVFHLQLQWRWKH KHH\H(GHOKDXVHUDQG*UHHQ$UHYLHZRIWKLVSURFHGXUH RIWKLVFKDSWHU,WLVZRUWK\WRQRWHKRZHYHUWKDWWKHSURFHGXUH OXWLRQRIWKHLQVWUXPHQWDQGE\RWKHUPHWKRGRORJLFDOSUREOHPV WLRQQHHGVWREHHYDOXDWHGWRGHWHUPLQHHIIHFWVDPRQJWUHDWPHQW 0F1DPDUDHWDO7KHVHWHFKQLFDOGLIÀFXOWLHVZLWK DNHLWXQOLNHO\WKDWWKHFRUUHODWLRQRI LQYLYRÁXRURSKRWRPHWU\ +&(77(3 DVVD\ZRXOG EH IHDVLEOH+RZHYHU WKH SULQFLSOH HFKQLTXHVLVWKHVDPH LOLW\WRÁXRUHVFHLQLVDPHDVXUHRIFHOOXODUGDPDJHPDQLIHVWHG XQFWLRQDOGLVUXSWLRQWRWKHFRUQHDOHSLWKHOLXP$UDLHDQG0DX

\ DQG 5HDYHV (SLWKHOLDO FHOO GDPDJH DQG MXQFWLRQDO HYHQWVWKDWRFFXUGXULQJFKHPLFDOLQGXFHGKXPDQH\HLUULWDWLRQ *REEHOV DQG6SLW]QDV *UDQW DQG 6FKXPDQ V -HVWHU HW DO0DXUHUHW DOE$ UHFHQWO\ LRQ VFKHPH IRU KXPDQ FKHPLFDOLQGXFHG H\H LQMXU\ WKDW ZDV

GHYHORSHGE\DJURX DV RQH RI WKH ÀUVW R 0DXUHUHWDOD WKHGHJUHHRIFRUQHDO LQMXU\WRWKHH\HXQ PRGHORFXODUVXUIDFH SHUPHDELOLW\RFFXUV DELOLW\HSLWKHOLDOFHOO LQFUHDVHLQDGRVHGH )LJXUH6LQFHW 7HSLWKHOLDOEDUULHUL WKDWFDQEHPHDVXUHG FKHPLFDOH[SRVXUH

$ UHWURVSHFWLYH IROORZLQJ H[SRVXUH GXULQJWKH&RVPHWLF *HWWLQJV HW DO ZHOOZLWK WKH'UDL] 7KHVDPHIRUPXODWLR VKLSEHWZHHQWKHLQ

SRIRSKWKDOPLF UHVHDUFKHUVKDV LGHQWLÀHGFRUQHDOHSLWKHOLXP FXODU WLVVXHV WR EH DIIHFWHG E\ VXSHUÀFLDO FKHPLFDO H[SRVXUH %OD]NDHWDO7KHFODVVLÀFDWLRQVFKHPHLQGLFDWHVWKDW HSLWKHOLDOLQMXU\LQFUHDVHVZLWKWKHRYHUDOOVHYHULW\RIFKHPLFDO WLOWKHHSLWKHOLXPLVWRWDOO\GHVWUR\HG/LNHZLVHLQWKH+&(7 HSLWKHOLDOEDUULHUGLVUXSWLRQDVPHDVXUHGE\DOWHUHGÁXRUHVFHLQ TXLFNO\XSRQVXSHUÀFLDOWR[LFDQWH[SRVXUH)OXRUHVFHLQSHUPH F\WRWR[LFLW\DQGGLVUXSWLRQRILQWHUFHOOXODUDGKHVLRQVMXQFWLRQV SHQGHQWPDQQHUZLWKFKHPLFDOH[SRVXUH:DUGHWDOE KH7(3DVVD\SURYLGHVDPHDVXUHRIWKHLQWHJULW\RIWKH+&( WLVDQDORJRXVWRWKHLQYLYRHYHQWRIHSLWKHOLDOEDUULHUGLVUXSWLRQ E\LQFUHDVHGHSLWKHOLDOSHUPHDELOLW\WRÁXRUHVFHLQIROORZLQJ

HYDOXDWLRQ RI WKH ÁXRUHVFHLQ VWDLQLQJ GDWD IRU UDEELW H\HV WR VXUIDFWDQWFRQWDLQLQJ IRUPXODWLRQV ZKLFK ZDV FROOHFWHG 7RLOHWULHVDQG)UDJUDQFH$VVRFLDWLRQ&7)$3KDVH,,,6WXG\ GHPRQVWUDWHV WKDW FRUQHDO ÁXRUHVFHLQ VWDLQLQJ FRUUHODWHV H VFRUH GDWD FROOHFWHG IURP WKH VDPH DQLPDOV )LJXUH QVZHUHHYDOXDWHGXVLQJWKH+&(77(3DVVD\7KHUHODWLRQ YLYRÁXRUHVFHLQVWDLQLQJGDWDDWKUYHUVXVWKH LQYLWURORJ

)5YDOXHVIRUWKH SUHGLFWLRQ PRGHO L UHODWLRQVKLSEHWZHHQ )LJXUHDQG WKH LQ YLWUR +&(7 DYDLODEOHDORQJZLWK IRUPXODWLRQV HYDOXD 8QIRUWXQDWHO\ÁXRU DWHGZLWKWKH'UDL] DYDLODEOHIRUXVHLQ

7KHUHVXOWV UHSR IDFWDQW H[SRVXUHV R SHUPHDELOLW\ DV DQ 0DUVK DQG 0DXULF GHWHUJHQWVVKRZHGS WUDWLRQVMXVWVOLJKWO\ RWKHUKDQGLQWZR DQGUDEELWH\HVWRV UDEELW H\HVZHUH IR VDPH VXUIDFWDQW FRQ 7KHVHREVHUYDWLRQV SHUPHDELOLW\ DQG VX KXPDQVµ+LVUHVXOW

&7)$IRUPXODWLRQVZKLFKDUHSDUWRIWKH+&(77(3DVVD\ V VKRZQ LQ )LJXUH 7KHVH UHVXOWV GHPRQVWUDWH WKH ÁXRUHVFHLQVWDLQLQJRIWKHUDEELWFRUQHDDQGWKH'UDL]HVFRUHV WKHUHODWLRQVKLSEHWZHHQWKHLQYLYRÁXRUHVFHLQVWDLQLQJDQG 7(3 DVVD\ )LJXUH 7KH ÁXRUHVFHLQ VWDLQLQJ GDWD DUH WKHVWDQGDUG'UDL]HVFRUHGDWDIRU WKHVXUIDFWDQWFRQWDLQLQJ WHG LQ WKH &7)$ 3KDVH ,,, 6WXG\ *HWWLQJV HW DO HVFHLQGDWDDUHQRWDYDLODEOHIRUPDQ\RIWKHPDWHULDOVHYDOX HWHVWDQGVXIÀFLHQWPDWHULDOVZLWKÁXRUHVFHLQGDWDZHUHQRW WHVWPHWKRGYDOLGDWLRQ UWHG LQ WKHRSKWKDOPRORJ\DQG WR[LFRORJ\ OLWHUDWXUH IRUVXU I WKH KXPDQ DQG UDEELW H\H VXSSRUW WKH XVH RI ÁXRUHVFHLQ DSSURSULDWH PHDVXUH IRU VXUIDFWDQWLQGXFHG H\H LUULWDWLRQ H REVHUYHG WKDW KXPDQ H\HV WUHDWHG ZLWK QRQLRQLF HUWXUEDWLRQRIHSLWKHOLDOÁXRUHVFHLQSHUPHDELOLW\DWFRQFHQ OHVVWKDQWKRVHWKDWSURGXFHGPDUNHGH\HLUULWDWLRQ2QWKH GLIIHUHQWH[SHULPHQWVWKDWFRPSDUHGWKHUHVSRQVHVRIKXPDQ XSHUÀFLDODSSOLFDWLRQVRIEHQ]DONRQLXPFKORULGH%$& WKH

XQG WR EHPRUH VHQVLWLYH WKDQ KXPDQ H\HV WUHDWHGZLWK WKH FHQWUDWLRQV %XUVWHLQ %XHKOHU DQG1HZPDQQ OHG%XUVWHLQWRFRQFOXGHWKDW´PHDVXUHPHQWVRIRFXODU UIDFH GDPDJH DUH QRW GLUHFWO\ WUDQVIHUDEOH IURP UDEELWV WR VKRZHYHURQO\LQGLFDWHWKDWWKHUDEELWH\HRYHUSUHGLFWHGWKH

KXPDQRFXODUUHVSR DVVD\ GDWD REWDLQHG VKRZHG WKDW WKH\ DO 'UDL]HWHVWXQSXEOL

7KLVUHYLHZRIW DQG WKH +&(7 7( ÁXRUHVFHLQLPSHUPH PHFKDQLVP RI ÁXRU HSLWKHOLXP)OXRUHVF ORJLFDOUHOHYDQFHRI PXOWLSOH FHOO OD\HUV UHVSRQVLEOH IRU WKH SHUWXUEWKHLPSHUPH E\WKHVDPHPHFKDQ FHOOVWKDWLQFUHDVHVL 77(3DVVD\DQGWK DQLVP IRU ÁXRUHVFH PHFKDQLVPIRUWKHL H[SRVXUHWRVXUIDFWD

$VRQHRIWKHVWHS LQDSUHYDOLGDWLRQVWX DVWDQGDUGL]HGWHVWS RXWFRPHRIWKRVHVWX IXUWKHUYDOLGDWLRQVWX

7KHSUHYDOLGDWLRQ EHSXEOLVKHGLQJUHD

Purpose for the H

)RUWKH3UHYDOLGD WRSUHGLFWWKHH\HLUU ODWLRQVZLWKLQ WKH UD 'UDL]HWRWDOFRUQHDO VWXG\ZHUH WRGHYHOR GHPRQVWUDWH WKH PH SUHOLPLQDU\DVVHVVPH IRULWVVWDWHGSXUSRV

Study Structure

7KHVWXG\FRQVL WLRQVE\WKUHHODERUD

QVHWRWKHFDWLRQLFVXUIDFWDQW%$&3UHOLPLQDU\+&(77(3 DW *0(/ IRU VHYHUDO FDWLRQLF VXUIDFWDQWV LQFOXGLQJ %$& VRZHUH XQGHUSUHGLFWHG LQ WKH LQ YLWUR DVVD\ UHODWLYH WR WKH VKHGREVHUYDWLRQV KHVFLHQWLÀFOLWHUDWXUHDQGWKHUHVXOWVZLWKWKH+&(7PRGHO 3 DVVD\ OHG XV WR FRQFOXGH WKDW WKH PHFKDQLVWLF EDVLV RI DELOLW\SHUPHDELOLW\LQWKH+&(7PRGHOLVDQDORJRXVWRWKH HVFHLQ LPSHUPHDELOLW\SHUPHDELOLW\ LQ WKH LQ YLYR FRUQHDO HLQ LPSHUPHDELOLW\ LQ WKH+&(7PRGHO LV GXH WR WKH ELR WKHVWUDWLÀHGFRUQHDOHSLWKHOLDOFXOWXUHVZLWKWKHLUDQDORJRXV WLJKW MXQFWLRQV DQG RWKHU LQWHUFHOOXODU DGKHVLRQV WKDW DUH LPSHUPHDELOLW\RI WKHHSLWKHOLXP WRÁXRUHVFHLQ6XUIDFWDQWV DELOLW\RIWKHLQYLYRFRUQHDOHSLWKHOLXPDQGWKH+&(7PRGHO LVP³F\WRWR[LFLW\DQGMXQFWLRQDOGLVUXSWLRQWRWKHVXSHUÀFLDO QDGRVHGHSHQGHQWPDQQHU7KXVWKHWHVWV\VWHPWKH+&( HKXPDQH\HWKHVSHFLHVRILQWHUHVWVKDUHDVLPLODUPHFK LQ LPSHUPHDELOLW\ LQ WKH XQGDPDJHG WLVVXHV DQG D VLPLODU QFUHDVHLQÁXRUHVFHLQSHUPHDELOLW\REVHUYHGDIWHUVXSHUÀFLDO QWV

PREVALIDATION STUDY RESULTS

VLQWKHYDOLGDWLRQSURFHVVWKH+&(77(3DVVD\ZDVHYDOXDWHG G\3UHYDOLGDWLRQKDVEHHQGHÀQHGDV´WKHSURFHVVGXULQJZKLFK URWRFROLVHYDOXDWHGIRUXVHLQYDOLGDWLRQVWXGLHV%DVHGRQWKH GLHVWKHWHVWSURWRFROPD\EHPRGLÀHGRURSWLPL]HGIRUXVHLQ GLHVµ,&&9$0 VWXG\DQGLWVUHVXOWVDUHRQO\EULHÁ\VXPPDUL]HGKHUHDQGZLOO WHUGHWDLOHOVHZKHUH:DUGHWDOE

CE-T TEP Assay

WLRQ6WXG\WKH+&(77(3DVVD\ZDVHYDOXDWHGIRULWVDELOLW\ LWDWLRQSRWHQWLDORIZDWHUVROXEOHVXUIDFWDQWFRQWDLQLQJIRUPX QJH RI LUULWDWLRQ UHIHUHQFHG E\ D'UDL]H0$6RI WR RU VFRUHRIWR7KHJRDOVRIWKHLQWHUODERUDWRU\SUHYDOLGDWLRQ SDQRSWLPL]HGSURWRFRO IRUYDOLGDWLRQRI WKH WHVWPHWKRG WR WKRG·V WUDQVIHUDELOLW\ WR RWKHU ODERUDWRULHV DQG WR SURYLGH D QWRIWKHUHOLDELOLW\DQGWKHUHOHYDQFHRIWKH+&(77(3DVVD\ H

VWHGRI WKHHYDOXDWLRQRIÀYHVXUIDFWDQWFRQWDLQLQJ IRUPXOD WRULHVZLWKHDFKPDWHULDOWHVWHGIRXUWLPHVLQHDFKODERUDWRU\

IRU D WRWDO RI 7( SDUWLFLSDWLQJ LQ WKH *0(/ LQ *DLWKH *DLWKHUVEXUJ0' PDQG (&%& LQ$ GRXEOHEOLQGSURFHG WHVW PDWHULDOV WR HD ODERUDWRU\ SUDFWLFH UHFRUGHGGDWDXVLQJ E\*0(/ VWDII LQ IRUPHGDVHWRI´WUD EHIRUHLQLWLDWLRQRI JRRGTXDOLW\KLVWRUL VROXEOH VXUIDFWDQW UDQJHRIH\H LUULWDWL IRUPXODWLRQVZHUH* IRUPXODWLRQV *HWWL QHJDWLYHQRQLUULWDQ WKH)HGHUDO+D]DUGR

Data Analysis

%HIRUHWKHEHJLQ WKH VWDWLVWLFDOPRGHO KLVWRULFDOLQYLYR'U FRQWDLQLQJIRUPXODWL HTXDWLRQ 'UDL]H0 SDUDPHWHU ORJLVWLF F ²DQGG SORWLVDQGWK

7KHGRVHUHVSRQ WLRQ)5SHUFHQWDJ RILQLWLDOWHVWPDWHUL WUDWLRQRI WHVWPDWH UHODWLYHWRFRQWUROF LVXVHGIRUFRUUHODWL WKHORJ)5ZDVX HYDOXDWHG LQHDFKD DOVRGHYHORSHGXVLQ &26ZKLFKZHUH WHVWPDWHULDO

7HVWPHWKRGSHU 77(3DVVD\GDWDZL PRGHO DQG E\ GHWHUPLQHGE\WKHLQ 7KH LUULWDWLRQ FODVVL

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µDVVD\VWRGHPRQVWUDWHWKHLUSURÀFLHQF\ZLWKWKHDVVD\ WKHVWXG\7HVWPDWHULDOVZHUHVHOHFWHGIRUWKHVWXG\WKDWKDG FDOUHIHUHQFHPHWKRGGDWD7KHÀYHWHVWPDWHULDOVZHUHZDWHU EDVHG FRQVXPHU SURGXFW IRUPXODWLRQV ZKLFK VSDQQHG WKH RQIURPQRQLUULWDWLQJ WRVHYHUHVHH7DEOH7ZRRI WKH LOOHWWHSURGXFWIRUPXODWLRQVDQGWKUHHZHUH&7)$3KDVH,,, QJV HW DO 7ZR RI WKH WHVWPDWHULDOV DUH FODVVLÀHG DV WVWZRDVSRVLWLYHLUULWDQWVDQGRQHDVLQGHWHUPLQDWHXVLQJ XV6XEVWDQFHV$FW)+6$JXLGHOLQHV)+6$

QLQJRIWKHVWXG\DSUHGLFWLRQPRGHO30ZDVGHYHORSHGE\ LQJRI WKH UHODWLRQVKLSEHWZHHQ WKH LQ YLWUR7(3GDWD DQG WKH DL]H0$6GDWDIRUZDWHUVROXEOHVXUIDFWDQWVDQGVXUIDFWDQW RQV)LJXUHV(DQG7KLVPRGHO LVGHVFULEHGE\ WKH $6 >D H[S EORJ )5 ² F@ G ZKLFK LV D IRXU XUYHZLWK SDUDPHWHU HVWLPDWHV RI D E F 7KHJHQHUDOL]HGFRUUHODWLRQFRHIÀFLHQW5IRUWKH0$630 HRXWHUGDVKHGOLQHVUHSUHVHQWWKHFRQÀGHQFHLQWHUYDOV VHGDWDIURPHDFK7(3DVVD\DUHSORWWHGDVÁXRUHVFHLQUHWHQ HRIFRQWUROYHUVXVWKHORJRIWKHFRQFHQWUDWLRQSHUFHQWDJH DOFRQFHQWUDWLRQ)LJXUH'7KHORJ)5LVWKHFRQFHQ ULDOZKHUH WKH+&(7 FXOWXUHV UHWDLQ RI WKHÁXRUHVFHLQ XOWXUHVDQGLVWKHHQGSRLQWIURPWKHGRVHUHVSRQVHFXUYHWKDW RQZLWKWKHUHIHUHQFHPHWKRGGDWD7KH7(3DVVD\HQGSRLQW VHGWRGHÀQHWKHSUHGLFWHGLQYLYR'UDL]H0$6IRUWKHPDWHULDO VVD\E\XVLQJ WKLVSUHGLFWLRQPRGHO3UHGLFWLRQPRGHOVZHUH JWKH'UDL]HFRUQHDOVFRUH&6DQGWKHFRUQHDORSDFLW\VFRUH XVHGWRREWDLQWKHSUHGLFWHG&6DQGSUHGLFWHG&26IRUHDFK

IRUPDQFHZDVHYDOXDWHGE\HYDOXDWLQJWKHÀWRIWKH+&( WKLQWKHFRQÀGHQFHLQWHUYDOVRIWKHHVWDEOLVKHGSUHGLFWLRQ FRPSDULQJ WKH LUULWDWLRQ FODVVLÀFDWLRQ IRU HDFK WHVW PDWHULDO YLYR'UDL]HWHVWWRWKDWGHWHUPLQHGE\WKH+&(77(3DVVD\ ÀFDWLRQV FDQ EH XVHG WR FDOFXODWH WKH SHUIRUPDQFH VWDWLVWLFV

LQFOXGLQJWHVWPHWK FRQWLQJHQF\WDEOHDQ VHWRIRQO\ÀYHWHVW RIDQDO\VLV

6HYHUDOPHWKRGV WRU\ YDULDELOLW\ DQG $OOPHWKRGVUHVXOWHG FLHQWRIYDULDWLRQ&

Results

2QHPHWKRGIRU GHWHUPLQHZKHWKHUWK PRGHO7KHODEDYHU FRQÀGHQFH LQWHUYDOV 7KHODEDYHUD RIWKHFRUQHDOVFRUH RI WKH FRUQHDO RSDF DQDO\VLVXVHGWRGHWH LFDQW UHODWLRQVKLS EH FODVVLÀFDWLRQREWDLQH

$ VHFRQG ZD\ LUULWDWLRQFODVVLÀFDWL 'UDL]H WHVW WR WKD PDWHULDOV ZHUH FRUU UHODWLYH WR WKHLU FO FODVVLÀFDWLRQVIRUH DQG &DODQGUD ZHUHLQDJUHHPHQWI VKDPSRRZDVXQ DOOWKUHHODERUDWRULH

7KHVWDWLVWLFDODQ RUDWRU\DQGZLWKLQO FRHIÀFLHQWRIYDULDWLR WR 7DEOH UHOLDELOLW\ZLOOEHSU

Conclusions

7KHSUHYDOLGDWLR SHUIRUPDQFHDQGLQ

7KH UHVXOWV LQGL ODERUDWRULHVKDV SHUIRUPHG E\ WUDLQH JRRGSHUIRUPDQFHL

RGVHQVLWLYLW\DQGVSHFLÀFLW\IRUDWHVWPHWKRGXVLQJDv DO\VLV&RRSHUHWDO,&&9$0+RZHYHUDGDWD PDWHULDOVGLGQRWSURYLGHDVXIÀFLHQWVDPSOHVL]HIRUWKLVW\SH

ZHUHXVHGWRDVVHVVWKHUHSHDWDELOLW\RUZLWKLQLQWUDODERUD WKH UHSURGXFLELOLW\ RU EHWZHHQ LQWHU ODERUDWRU\ YDULDELOLW\ LQWKHVDPHFRQFOXVLRQVDQGRQO\WKHUHVXOWVIURPWKHFRHIÀ 9DQDO\VLVZLOOEHVKRZQKHUH

HYDOXDWLQJWKHSHUIRUPDQFHRIDQHZWR[LFLW\WHVWPHWKRGLVWR HQHZWHVWUHVXOWVIROORZWKHDOJRULWKPRIWKHH[LVWLQJSUHGLFWLRQ DJHDVVD\UHVXOWVIRUDOOÀYHWHVWPDWHULDOVIHOOZLWKLQWKH &,RI WKH0$630RI WKH LQGLYLGXDO DVVD\V )LJXUH JHUHVXOWVIRUIRXURXWRIÀYHPDWHULDOVIHOOZLWKLQWKH&, &630DQGÀYHRXWRIÀYHPDWHULDOVIHOOZLWKLQWKH&, LW\ VFRUH &26 30 QRW VKRZQ$QRWKHU W\SH RI VWDWLVWLFDO UPLQHGDWDUHODWLRQVKLSVDFKLVTXDUHDQDO\VLVVKRZHGVLJQLI WZHHQ LUULWDWLRQ FODVVLÀFDWLRQ REWDLQHG E\ WKH0$6 30 DQG GE\WKH'UDL]HWHVWS WR HYDOXDWH WHVW PHWKRG SHUIRUPDQFH LV E\ FRPSDULQJ WKH RQIRUHDFKWHVWPDWHULDOGHWHUPLQHGE\WKHUHIHUHQFHPHWKRG W GHWHUPLQHG E\ WKH QHZ WHVW PHWKRG 7(3 DVVD\$OO ÀYH HFWO\ FODVVLÀHG DV LUULWDQW RU QRQLUULWDQW E\ WKH 7(3 DVVD\ DVVLÀFDWLRQ E\ WKH 'UDL]H WHVW 7DEOH 7KH LUULWDWLRQ DFKWHVWPDWHULDOEDVHGRQWKHIRXUFODVVHVDVVLJQHGE\.D\ DUH VKRZQ LQ 7DEOH 7KHVH LUULWDWLRQ FODVVLÀFDWLRQV RUIRXURIWKHÀYHWHVWPDWHULDOV7KHPRVWLUULWDWLQJPDWHULDO GHUSUHGLFWHGE\ WKH7(3DVVD\E\RQHFODVVLÀFDWLRQ OHYHO LQ V DO\VHVVKRZHGQRVLJQLÀFDQWGLIIHUHQFHVLQERWKEHWZHHQODE DERUDWRU\YDULDELOLW\IRUWKHÀYHWHVWPDWHULDOVQRWVKRZQ7KH Q&9IRUWKHEHWZHHQODERUDWRU\SUHGLFWHG0$6UHVXOWVZHUH $ GLVFXVVLRQ RI WKH VWDWLVWLFDO DQDO\VLV IRU WHVW PHWKRG HVHQWHGLQJUHDWHUGHWDLOHOVHZKHUH:DUGHWDOE

QVWXG\UHVXOWVSURYLGHDSUHOLPLQDU\DVVHVVPHQWRIWHVWPHWKRG

WUDODERUDWRU\DQGLQWHUODERUDWRU\DVVD\YDULDELOLW\ FDWH WKDW WKH +&(7 7(3 DVVD\ LV WUDQVIHUDEOH WR RWKHU DFFHSWDEOHLQWUDODERUDWRU\DQGLQWHUODERUDWRU\YDULDELOLW\ZKHQ G VWDII IROORZLQJ WKH GHVLJQDWHG SURWRFRO GHPRQVWUDWHG QSUHGLFWLQJWKH'UDL]HVFRUHVIRUÀYHVXUIDFWDQWFRQWDLQLQJ

IRUPXODWLRQVLQWKUH ULVN DVVHVVPHQW GHF WDLQLQJIRUPXODWLRQ

7KH+&(77(3 DQGDFFHSWDEOHZLWKL DVVHVVPHQW7KHUHIRU IRUHYDOXDWLRQLQDOD EHIXUWKHUDVVHVVHG7 ZHUHUHSRUWHGWRWKH *LOOHWWH

HODERUDWRULHVDQGKDVJRRGSRWHQWLDOIRUXVHLQPDNLQJ LVLRQV IRU WKH RFXODU LUULWDWLRQ SRWHQWLDO RI VXUIDFWDQWFRQ V DVVD\GHPRQVWUDWHGJRRGSUHGLFWLYHFDSDELOLW\SHUIRUPDQFH QDQGEHWZHHQODERUDWRU\DVVD\YDULDELOLW\ LQWKLVSUHOLPLQDU\

B

A

HWKHPDQDJHPHQWWHDPUHFRPPHQGHGWKH+&(77(3DVVD\ UJHULQWHUODERUDWRU\YDOLGDWLRQVWXG\ZKHUHLWVSHUIRUPDQFHZLOO KHUHVXOWVRIWKHSUHYDOLGDWLRQVWXG\IRUWKH+&(77(3DVVD\ ,&&9$02FXODU7R[LFLW\:RUNLQJ*URXS27:*LQ-DQXDU\

7KLV FKDSWHU GH HVVHQWLDOVWHSVLQWKH QHZWHVWPHWKRGDQG

$QHZWR[LFLW\W UHOHYDQWWRWKHELROR RUFKHPLFDOPL[WXUH UHOHYDQFH RI WKH +& QXPEHURIGLIIHUHQW VXSSRUWLQJ LQIRUPDW 7(3 DVVD\ DFKLHYHV FRUQHDOVXUIDFHZLWK H\H LUULWDWLRQ 7KH VXSSRUWWKHFRQFOXV LFDOO\DQGPHFKDQLV KXPDQFRUQHDOUHVS

SUMMARY

VFULEHV IRU WKH +&(7 7(3 DVVD\ WKH UHVXOWV IRU WZR RI WKH YDOLGDWLRQSURFHVVHVWDEOLVKLQJWKHVFLHQWLÀFUHOHYDQFHRIWKH WKHSUHYDOLGDWLRQRIWKHPHWKRG HVWPHWKRGWRHYDOXDWHKXPDQH\HLUULWDWLRQSRWHQWLDOPXVWEH JLFDODQGWR[LFRORJLFDOSURFHVVHVWKDWRFFXUZKHQDFKHPLFDO PDNHVFRQWDFWZLWKWKHKXPDQRFXODUVXUIDFH7KHVFLHQWLÀF (7 7(3 DVVD\ ZDV GHÀQHG XVLQJ UHVXOWV REWDLQHG IURP D H[SHULPHQWVFRQGXFWHGXVLQJWKHWHVWV\VWHPDQGZLWKUHOHYDQW LRQIURPWKHRSKWKDOPRORJLF UHVHDUFK OLWHUDWXUH7KH+&(7 ELRORJLFDO UHOHYDQFH E\ FRPELQLQJ D PRGHO RI WKH KXPDQ

D WHVWPHWKRG WKDWKDVDPHFKDQLVWLF UHODWLRQVKLS WRKXPDQ H[SHULPHQWDO GDWD DQG WKH VFLHQWLÀF OLWHUDWXUH UHSRUWHG KHUH LRQWKDWWKH+&(77(3DVVD\LVDWHVWPHWKRGWKDWLVELRORJ WLFDOO\EDVHGDQG WKXV VKRXOGSURYLGHDJRRGPRGHO IRU WKH RQVHWRH\HLUULWDQWV

$SUHYDOLGDWLRQV 77(3DVVD\IRUWKH IRUPXODWLRQV7KHWH RI WKH LQWHUODERUDWRU DQGUHOHYDQWPHWKRG FRQWDLQLQJIRUPXODWL

WXG\ZDVFRQGXFWHGWRHYDOXDWHWKHSHUIRUPDQFHRIWKH+&( SUHGLFWLRQRIWKHRFXODULUULWDWLRQSRWHQWLDORIVXUIDFWDQWEDVHG VWPHWKRGZDVWUDQVIHUUHGWRRWKHUODERUDWRULHVDQGWKHUHVXOWV \ DVVHVVPHQW VXJJHVW WKDW WKH+&(77(3DVVD\ LV D UHOLDEOH IRUWKHSUHGLFWLRQRIWKHH\HLUULWDWLRQSRWHQWLDORIVXUIDFWDQW RQV

REFERENCES