NG DNG K THUT LAI HUNH QUANG TI CH

TM TT t vn : Phn ln cc d bi nhim sc th c chn on trc sinh lin quan n cc nhim sc th 13, 18, 21, X v Y. K thut lai hunh quang ti ch (FISH) gip xc nh nhanh cc bt thng v s lng ca nhim sc th trn nhn t bo gian k. Mc tiu: Xc nh tnh hiu qu ca k thut lai hunh quang ti ch (FISH) trong chn on trc sinh cc trng hp lch bi nhim sc th thng gp Phng php: i tng: 1302 sn ph c ch nh chc i kho st tnh trng lch bi nhim sc th (13, 18, 21, X, Y) bng k thut FISH. Thi gian: 01/2004 09/2006. Thc hin k thut FISH theo qui trnh chun ca hng VYSIS. Kt qu: Trong thi gian t 01 / 2004 n 09 /2006 chng ti thc hin k thut lai hunh quang ti ch trn 1302 trng hp thai k c nguy c d bi nhim sc th. 136 (10,45%) trng hp d bi nhim sc th c pht hin. 66 trng hp tam nhim sc th 18, 60 trng hp tam nhim sc th 21, 3 trng hp tam nhim sc th 13, 3 trng hp XO, 2 trng hp XXY, 1 trng hp

XXX v 1 trng hp tam bi c pht hin thng qua k thut FISH. Khng c trng hp m tnh gi hay dng tnh gi no c ghi nhn. Kt lun: K thut lai hunh quang ti ch trn nhn t bo i gian k l mt phng php chn on nhanh v chnh xc cho nhng thai k c nguy c cao d bi nhim sc th. ABSTRACT Introduction: Most of aneuploidies diagnosed prenatally involve the autosomes 13, 18, 21 and sex chromosomes X and Y. Fluorescence in situ hybridization (FISH) technique allows rapid analysis of chromosome copy number in interphase cells. Objective: Identify the effectiveness of applying the FISH tecnique in prenatally diagnosing the common aneuploidies. Method: Subjects: 1302 pregnant women were indicated to have an amniocentesis to detect aneuploidies of chromosome 13, 18, 21, X, Y by using FISH technique. Time of research: from January 2004 to September 2006. Applying FISH technique following the Vysiss standard protocol. Results: From January 2004 to September 2006, we applied FISH technique on 1302 pregnancies having a risk of chromosome aneuploidy. Aneuploidies were detected in 136 (10,45%) samples. 66 cases of trisomy 18, 60 cases of trisomy 21, 03 cases of trisomy 13, 03 cases of monosomy X, 02 cases of XXY, a case of XXX and

a case of triploidy were diagnosed by FISH. There were no false negative or false positive results. Conclusion: We conclude that FISH analysis of interphase, uncultured amniotic cells is an fast and accurate method for routine prenatal diagnosis of the most common aneuploidies in high-risk pregnancies. T VN Bt thng nhim sc th c chn on trc sinh phn ln l nhng dng lch bi ca cc nhim sc th thng (13, 18, 21) v ca nhim sc th gii (X, Y). K thut lai hunh quang ti ch (FISH: Fluorescence in situ hybridization) vi vic s dng nhng on d c hiu cho tng nhim sc th c chng minh l c hiu qu cao cho vic xc nh nhanh s lng ca cc nhim sc th trong nhn t bo giai on gian k[2,3,5,9,10]. Vi c tnh nhanh, nhy v chnh xc, vic s dng k thut FISH trong chn on trc sinh cc lch bi nhim sc th thng gp tr nn ph bin[4,8,12]. Ngoi ra k thut FISH cn c xem nh l mt la chn gip gim s lo u cho cc sn ph c thai k nguy c cao[7]. Ti thnh ph H Ch Minh, k thut FISH bc u c ng dng trn 33 mu dch i vo nm 2003[1]. Tuy nhin vi mt c mu nh nn cha th khng nh c gi tr ca k thut FISH trong chn on trc sinh cc lch bi nhim sc th thng gp.

V vy, chng ti thc hin nghin cu ny vi mc tiu Xc nh tnh hiu qu ca k thut lai hunh quang ti ch (FISH) trong chn on trc sinh cc trng hp lch bi nhim sc th thng gp. I TNG V PHNG PHP NGHIN CU i tng nghin cu 1302 sn ph c ch nh chc i kho st tnh trng lch bi nhim sc th (13, 18, 21, X, Y) bng k thut FISH ti phng Di truyn t bo bnh vin Ph sn T d v phng khm thai bnh vin Hng vng trong thi gian t 01 / 2004 n 09/ 2006. Phng php nghin cu Tt c cc sn ph u c tham vn v vic chn la k thut nhanh FISH hay k thut nui cy t bo i, v c gii thch cn k v nhng gii hn ca k thut FISH. Thc hin nui cy t bo i i km vi k thut FISH trong cc trng hp: kim nh kt qu FISH, tin cn gia nh c ngi mang chuyn on nhim sc th lin quan n cc nhim sc th 13, 18, 21, theo yu cu ca sn ph. Thu thp vo bng cu hi cc thng tin v h tn, tui ca sn ph, kt qu siu m sn khoa (ghi nhn loi bt thng siu m: nh, ln hay c hai)[11], kt qu xt nghim sng lc sinh ha (MAFP, bhCG, uE3), kt qu phn tch ca k thut FISH, kt qu phn tch nhim sc th (nu c).

Thu thp 6 8 ml dch i cho cc trng hp thc hin k thut FISH, ti thiu l 10 ml cho trng hp thc hin c hai k thut FISH v nui cy t bo i. Lm phng t bo i bng dung dch KCl 0.075M, nh hnh t bo bng hn hp dung dch methanol : acid acetic (3:1). Nh cn t bo ln phin knh sch. Kh nc bng ethanol 70% - 90% - 100%. S dng cc on d DNA c hiu cho cc nhim sc th 13, 18, 21, X, Y ca hng VYSIS (M). Thc hin x l trc khi lai, tin hnh lai v ra sau lai theo qui trnh hng dn ca VYSIS. Thc hin m ti thiu trn 50 nhn t bo i vi tng loi on d DNA. nh gi kt qu lai l bnh thng khi c # 80% nhn t bo c 2 tn hiu lai i vi tng loi on d DNA. nh gi kt qu l bt thng khi c # 70% nhn t bo c s tn hiu lai bt thng i vi tng loi on d DNA. Kt qu FISH c bao gm cc thng tin: m t loi on d c s dng, s tn hiu ca tng loi nhim sc th c kim tra, tnh trng lch bi ca cc nhim sc th 13, 18, 21, X, Y (nu c). KT QU NGHIN CU 136 / 1302 (10,45%) trng hp d bi nhim sc th c pht hin thng qua k thut FISH. Trong , 66 trng hp tam nhim sc th 18, 60 trng hp tam nhim sc th 21, 3 trng hp tam nhim sc th 13, 3 trng hp XO, 2 trng hp XXY, 1 trng hp XXX v 1 trng hp tam bi.

Bng 1. Kt qu FISH Kt qu S trng hp Bnh thng Bt thng Tam nhim sc th 03 18 03 Tam 02 nhim sc th 21 Tam nhim sc th 13 XO XXY 01 01 136 66 60 10,45 1166 (%) 89,55 T l

XXX Tam bi

Tng

1302

100

C 1 trng hp cn thc hin k thut FISH ln th hai v tn hiu lai hunh quang yu khng th phn tch c. C 2 trng hp nghi ng tam nhim sc th 21 khi ghi nhn c 8 - 10 nhn t bo trong tng s 50 nhn t bo c phn tch c 3 tn hiu hunh quang. C 2 trng hp ny c thc hin k thut lai hunh quang ln th hai v ghi nhn ch c 2 3 nhn t bo c 3 tn hiu hunh quang. Ghi nhn sau sinh, c hai chu b u bnh thng. C 3 trng hp nghi ng tam nhim sc th 18 thuc loi bt thng cu trc nhim sc th (isochromosome: nhim sc th u) khi ghi nhn 45 48 nhn t bo c 1 tn hiu ln v 1 tn hiu nh ca nhim sc th 18, 2 5 nhn t bo c 3 tn hiu ca nhim sc th 18. C 3 trng hp c yu cu lm thm k thut nui cy t bo i phn tch b nhim sc th thai nhi ng thi thc hin k thut FISH ln hai trn mu dch i thu thp ln u. Kt qu c 3 b nhim sc th u bnh thng v s lng v cu trc ca nhim sc th 18, khng ghi nhn nhn t bo c 3 tn hiu nhim sc th 18.

219 trng hp c thc hin nui cy t bo i i km vi thc hin k thut FISH. Cc bt thng lch bi nhim sc th 13, 18, 21, X, Y u tng ng gia hai k thut. C 2 trng hp bt thng cu trc nhim sc th khng c pht hin bi k thut FISH l chuyn on tng h nhim sc th t(13;14) v t(14;21). Bng 2. Kt qu nui cy t bo i B NST S trng hp 46,XX 46,XY 47,XX/XY,+18 47,XX/XY,+21 47,XX,+13 45,X 47,XXX 46,XY/47,XXY 78 87 30 17 01 01 01 01

69,XXX 45,XX,t(13;14) 45,XY,t(14;21) Tng

01 01 01 219

Ch nh chnh chc i lm k thut FISH l c hnh nh bt thng trn siu m (41,32%) v kt qu xt nghim sng lc sinh ha mu m bt thng (35,87%). Phn ln s trng hp lch bi 5 nhim sc th (13, 18, 21, X, Y) cng c pht hin qua hai ch nh ny. C mt trng hp XO c pht hin tnh c thng qua ch nh chc i l tin cn sy thai lin tip. Bng 3. Ch nh chc i v kh nng pht hin lch bi ca k thut FISH S Ch S T l trng nh chc trng (%) i hp bi Bt thng siu m 538 41,32 74 hp lch

S Ch S T l trng nh chc trng (%) i hp bi Bt thng xt nghim sinh ha M ln tui Bt thng siu m + sinh ha Tin cn sinh 09 0,69 0 135 10,37 41 139 10,68 02 467 35,87 16 hp lch

con Down Tin cn sinh 08 0,61 0

S Ch S T l trng nh chc trng (%) i hp bi con d tt bm sinh Tin cn nh chuyn on NST Tin cn gia 02 0,15 0 gia c 03 0,23 02 hp lch

nh c hi chng Down Sy thai tip lin 01 0,08 01

S Ch S T l trng nh chc trng (%) i hp bi Tng 1302 100 136 hp lch

T l pht hin lch bi nhim sc th 13, 18, 21, X, Y khi c c hai loi bt thng siu m l 40,9% (27/66), khi ch c loi bt thng ln l 15,8% (68/431) v khi ch c loi bt thng nh l 11,4% (20/176). C 1 trng hp bt thng cu trc nhim sc th (chuyn on nhim sc th t(13;14)) c pht hin bng k thut nui cy t bo i vi hnh nh siu m l st mi ch vm hu. Bng 4. Kt qu siu m v kt qu FISH kt qu cy i Loi bt thng siu m Ln Nh hai 68 20 27 115 C Tng

Kt qu FISH cy i

Lch bi (13, 18, 21, X, Y) Bt

01

01

thng cu trc NST Bnh thng Tng 431 176 66 673 362 156 39 557

Vi kt qu xt nghim sng lc cho thy c nguy c tam nhim sc th 18 v 21 th khng c trng hp lch bi no c ghi nhn. Bng 5. Kt qu xt nghim sng lc sinh ha v kh nng pht hin lch bi ca k thut FISH S S Kt qu sinh ha bt trng thng hp bi Nguy c T21 480 78,95 26 Nguy c T18 50 8,22 16 Nguy c T21 + T18 12 1,97 0 T18: T21: (%) hp lch T l trng

AFP gim

35

5,76 03/ 01

T18: T21:

AFP tng

23

3,78 01 01

T18: /T21:

AFP+bhCG/bhCG /bhCG+uE3 gim AFP+bhCG/bhCG/AFP+uE3 tng Tng BN LUN

04

0,66 03

T18:

04

0,66 01

T21:

608

100

52

Trong chn on trc sinh, vic a ra kt lun mt thai nhi bnh thng v s lng hay cu trc ca b nhim sc th i hi phi c tnh chnh xc cao v kt lun s a n hnh ng can thip chm dt thai k hoc s khng cn can thip g thm. K thut nui cy t bo i phn tch b nhim sc th thai nhi c xem l mt k thut chn on mang tnh chnh xc cao v c ng dng trong chn on trc sinh sut mt thi gian di. Tuy nhin vic phn tch b nhim sc

th i hi phi thc hin trn cc nhim sc th k gia c thu hoch sau mt thi gian di nui cy. Trong phn ln trng hp, vic ch i kt qu gy nn tm l lo u, cng thng cho sn ph, v cng tng thm khi sn ph c bo tin mu cy ny khng t, cn thc hin chc i ln hai. K thut lai hunh quang ti ch (FISH) thc hin trn nhn t bo gian k nn c th gip chn on nhanh v khng phi tn thi gian nui cy t bo. Nhiu nghin cu trn th gii thc hin vic so snh gia hai k thut nui cy t bo v k thut FISH chng minh kh nng s dng k thut FISH nh mt cng c chn on c lp trong chn on trc sinh cc trng hp lch bi ca nhim sc th 13, 18, 21, X, Y[2,5,9]. Nhng nghin cu ny cng a ra nhng gii hn ca k thut FISH nh tnh trng ln mu m, lng t bo i thu thp khng phn tch, tnh c hiu ca on d DNA. Trong nghin cu ca chng ti, t l pht hin lch bi nhim sc th 13, 18, 21, X, Y l kh cao (10,45%) vi hu ht cc dng lch bi thng gp. Kt qu ny cng ph hp vi kt qu nghin cu ca Baruch Feldman khi thc hin k thut FISH c chn lc trn nhng thai k c nguy c cao i vi cc lch bi nhim sc th 13, 18, 21, X, Y[9]. Nh vy i vi nhng thai k c nguy c cao i vi cc lch bi nhim sc th 13, 18, 21, X, Y th k thut FISH c th xem nh l la chn hng u. t c hiu qu pht hin lch bi cao, chng ti thc hin mt s hiu chnh thch hp.

Chng ti thu thp th tch dch i ti thiu l 6 - 8 ml v vi lng th tch ny lng t bo i thu thp c nhiu hn v c bit c gi tr i vi cc trng hp tui thai # 20 tun hoc cc trng hp a i. Cc nghin cu ca cc tc gi khc v nghin cu ca chng ti nm 2003[1] cng cho thy khi ch thu thp 2 3 ml dch i th chc chn s c mt s trng hp khng thc hin k thut FISH c v khng t bo phn tch. Cc nghin cu xc nh tnh c hiu ca on d DNA c s dng u cho thy phn ln nhng trng hp lai khng c hiu, sng ca tn hiu khng n nh u lin quan n nhng on d DNA t tng hp hoc nhng on d DNA khng c kim nh cht lng. i vi cc on d DNA ca VYSIS dng cho vic pht hin bt thng s lng nhim sc th 13, 18, 21, X, Y ch ghi nhn m tnh gi nhim sc th 13, 21 do mu ln mu m nhiu v tn hiu lai khng r[6,13]; ghi nhn dng tnh gi nhim sc th 21 cha gii thch c nguyn nhn[13]; ghi nhn m tnh gi nhim sc th 18[6,13] do ln mu m nhiu, do s khng ng b v kch thc ca hai tn hiu nhim sc th 18; ghi nhn dng tnh gi nhim sc th 18 do lai cho vi nhim sc th 22[13]. Trong nghin cu ny, chng ti s dng b on d DNA ca hng VYSIS vi mt s iu chnh nh hn ch nhng sai lm m cc nghin cu trn a ra. Chng ti thc hin phng php ly tm nhiu ln i vi mu ln mu m nhiu gim lng mu m trong mu dch i trc khi tin hnh cc bc tip theo, nh vy trong mu nghin cu ny khng c trng hp tht bi k thut FISH do ln mu m. Chng ti thc hin x l pepsin cc nhn t bo i vi thi gian thch

hp cho tng nhm tui thai, m bo th tch on d DNA cn thit v m bo iu kin lai ti u nhm mc ch gim trng hp tn hiu lai yu. Trong mu nghin cu ny c 1 trng hp tn hiu lai yu do ngi gi mu xt nghim khng nh chnh xc tui thai ca sn ph. Sau khi c thng tin chnh xc v tui thai, chng ti thc hin li k thut FISH trn mu dch i thu thp ln trc vi vic iu chnh thi gian x l pepsin. Kt qu t c tn hiu lai r rng. C 2 trng hp nghi ng tam nhim sc th 21 c loi tr sau khi thc hin li k thut FISH ln th hai. C 3 trng hp nghi ng tam nhim sc th 18 do s khng ng b v kch thc ca tn hiu lai v c mt t l nh nhn t bo c ba tn hiu lai u c loi tr sau khi thc hin k thut FISH ln hai v k thut nui cy t bo i. Nh vy i vi cc trng hp nghi ng tam nhim sc th chng ta nn thc hin k thut FISH ln th hai, nu t l nhn t bo c 3 tn hiu gim nhiu so vi ln u th c th kt lun bnh thng. i vi trng hp khng ng b v kch thc tn hiu nhim sc th 18, nu tt c nhn t bo c phn tch u c hai tn hiu khng ng b v kch thc th c th xem nh bnh thng, nu c mt t l nh nhn t bo c ba tn hiu lai th nn kim nh li bng k thut FISH ln hai. Trong 219 trng hp c thc hin k thut nui cy t bo i i km vi k thut FISH, chng ti cha ghi nhn c s bt tng hp gia hai k thut FISH v nui cy t bo i. iu ny chng t vi nhng iu chnh thch hp th k thut FISH tht s hiu qu trong vic pht hin cc lch bi nhim sc th 13, 18, 21, X, Y. C 2 trng hp bt thng cu trc c pht hin qua nui cy t

bo i, c hai trng hp ny u l nhng thai nhi hon ton bnh thng, ch mang bt thng cu trc dng chuyn on cn bng nhim sc th ging b / m. C hai trng hp ny u xc nh b / m mang bt thng cu trc ny. Nh vy, trong trng hp bt thng cu trc nhim sc th dng chuyn on cn bng c lin quan n nhim sc th 13, 21 dng bt thng cu trc c th dn n hnh thnh thai nhi hi chng Down / hi chng Patau th k thut FISH vn l la chn hng u loi tr kh nng thai nhi hi chng Down hoc hi chng Patau, c th thc hin ng thi thm k thut nui cy t bo i xc nh s hin din ca bt thng cu trc nhim sc th ca b / m truyn cho con. Vi nhng tin b khoa hc k thut trong lnh vc siu m v cc xt nghim sinh ha, tui m khng cn l ch nh chnh yu trong chn on trc sinh v s trng hp lch bi nhim sc th c pht hin nhiu hn. Trong nghin cu ca chng ti hnh nh siu bt thng v kt qu xt nghim sng lc sinh ha mu m bt thng l hai l do chnh dn n thc hin chc i lm k thut FISH. Kt qu ny cng ph hp vi cc nghin cu ca cc tc gi khc[9,10,12]. Kh nng pht hin lch bi nhim sc th thng qua siu m l rt cao. Nhng hnh nh bt thng ca thai nhi c pht hin qua siu m c xem l yu t nguy c rt c lin quan n cc bt thng nhim sc th [9, 12], v cng nhiu hnh nh bt thng trn siu m hoc c nhng hnh nh bt thng trn siu m thuc loi bt thng ln th kh nng pht hin bt thng nhim sc th cng

cao [9]. Trong nghin cu ny, vi ch nh chc i l nhng hnh nh bt thng ca thai nhi trn siu m th gip pht hin c 54,41% (74 / 136) trong tng s cc trng hp lch bi nhim sc th c pht hin. 66 trng hp c c hai loi bt thng trn siu m lm gip pht hin cc lch bi nhim sc th nhiu hn (40,9%). Trong nghin cu ny, t l pht hin lch bi nhim sc th nhm c loi bt thng ln khng tng ng k so vi nhm c loi bt thng nh. C l do chng ti a hnh nh d i vo nhm c loi bt thng ln, trong khi hnh nh bt thng ny khng c xem l du hiu chnh gip pht hin lch bi nhim sc th. C mt trng hp chuyn on nhim sc th t(13;14) c pht hin vi hnh nh siu m l st mi ch vm hu. Hnh nh bt thng ny thng lin quan n tam nhim sc th 13. Nhng trong trng hp ny do sn ph l ngi mang chuyn on nhim sc th t(13;14) nn bt thng cu trc nhim sc th ny khng lin quan n hnh nh st mi ch vm hu. Xt nghim sng lc sinh ha mu m cc cht AFP, bhCG, uE3 cng ng gp vai tr trong vic pht hin cc lch bi nhim sc th 18 v nhim sc th 21. Vi vic s dng phn mm tnh ton nguy c lch bi nhim sc th da trn kt qu cc thng s sinh ha lm tng kh nng pht hin lch bi nhim sc th. Trong nghin cu ca chng ti, trn 80% (42 / 52) trng hp lch bi nhim sc th 18, 21 c pht hin khi c s dng phn mm tnh nguy c. KT LUN

Hn hai nm thc hin k thut lai hunh quang ti ch (FISH) trn 1302 trng hp, chng ti : - Pht hin c 136 trng hp d bi nhim sc th, trong 66 trng hp tam nhim sc th 18, 60 trng hp tam nhim sc th 21, 3 trng hp tam nhim sc th 13, 3 trng hp XO, 2 trng hp XXY, 1 trng hp XXX v 1 trng hp tam bi. - Khng c trng hp dng gi hay m gi no c ghi nhn. Chng ti cng rt ra mt s nhn xt sau: - K thut FISH l mt k thut tng i n gin, gip chn on nhanh, chnh xc cc lch bi nhim sc th 13, 18, 21, X, Y. - Hiu qu ca k thut FISH ph thuc vo vic tun th cht ch cc qui nh trong cc bc thu thp, x l mu v phn tch kt qu. - C th s dng k thut FISH nh l mt cng c chn on cho cc lch bi nhim sc th 13, 18, 21, X, Y. - i vi nhng trng hp c tin cn gia nh mang chuyn on nhim sc th th nn thc hin thm k thut nui cy t bo i phn tch b nhim sc th ca thai nhi.