L Hinh 2.2 Hinh anh tiem trong da
c)Gfiy nhiem va hap phu
Kilm tra tinh t r ^ g ciia tf bio tren cic phifn 24 gifng
N6ng dp giy nhifm: 1/256 ; 1/512 ; 1/1024 ; 1/2048 ; 1/4096 ; 1/8192 (hoic 1/16384,...), lya chpn nong dp giy nhiem niy phu thudc vio kft qui kifm tra huyft thanh
giira ky, nfu huyft tiianh giila ky cao (1/1024 vin buy hoai te bio) — cd tile lua chpn ndng dp pha loang cao hon v i ngupc laị
Gay nhifm: 0,2 ml / gjfng x 2 gifng / 1 ndng dp (ti-pn ky mau ttudc khi gay nhilm) 1/256 1/512 1/1024 1/2048 1/4096 1/8196 Mlul
Mlu 2
d) Chuan d$ ngupc
Myc dich: chuin dp lai khing nguyen di sii dung (200 CCIDso / 0,1 ml) dl trung hda nhim kiem chiing nong dp trung hda cd nim trong khoing 30 - 600 CClDjo / 0,1 ml khdng. Dinh gii chinh xic kSt qui chuin dp hifu gii khing huylt thanh thu dupẹ
Khing nguyen 200 CCIDso / 0,1 ml da qua xit ly budc 5, se dupe tilp tuc pha loing bfc 10 thyc hifn thao tic chuin dp khing nguyen Sdi
10" 10-
KN (200 CCIDso/0,1ml) 4 ml -> 0,3 ml GM 5% BS 2,7 ml Gay nhiem 0,2 ml /1 gilng x 10 gieng
10' 0,3 ml I . 0,3 mil J ^ 2.7 ml J '^ 2,7 ml J 2 3 4 5 10" 10-' 10-^ 10-^
CC: Cell Control - doi chiing tl bio (khdng giy nhiem) Hip phu 37°C/60 phut
e) Them mdi trudng
Kit thdc thdi gian hip phy 37''C / 60 phiit, thuc hien thao tac them moi trudng duy uị Them MEM2%BS: 1 ml / gieng; Nudi d 37°C ; 5%C02. Ngiy thir 7 sau gay nhiem, bo sung tiiem mdi tnrdng duy tri: 0,5 ml MEM2%BS /1 gilng.
0 Dpc vi tinh kit qui
Dpc kit qui tai cic thdi dilm: 7 ngiy, 10 ngiy vi 14 ngay sau giy nhilm
Hinh 2.4.ạ CPE (+): dim te bio bi pha
vS-vich bien thinh te bio khong lo (huy hôi te bio)
Hinh 2.4.b. CPE (-): tl bio vin km 1 lop, hinh dang, vien tl bio binh thudng (nhu
gilng doi chuung te bio)
Tai cic thdi dilm dpc kit qui, tiln hinh soi tl bio dudi kinh hifn vi quan sit tim dim buy hoai tl bio CPE (nhu hinh md ti ttfn). Eiiln bien bin (+ ) nlu gilng quan sit thiy cd hiiy hoai te bio CPE, diln (-) nlu khdng cd CPẸ
Ket qui dpc duoc ngiy 14 se sir dyng dl tinh kit qui theo cdng thirc Reed - Muench vdi don vi CCIDso (lilu huy hoai 50% tl bio) / 0,1 ml.
1 I 2 I 3 i 4 1 5 CC ' I , 2 I 3 4 5 CC 10"
10-' 10-' 10-^
Vi dy cich tinh nhu sau (doi vdi chuin dp ngupc):
Ndng đ giy Kit qui Cdng luy kl . . . nhiem ppp/+i rT)EM Luyklsd Luy kesd gilng ^ *•
(0,1 ml/gieng) ^^'"^ ' ^^'^^'•' gilng CPE (+) CPE(-) (+)/tdngsd
Tyle% gieng CPE 10" 10-' 10-^ I0-' 10 10 20 10 2 0 0 2 10 20 20/20 100% 10/12 83,33 % 2/12 16,67%% 0/20 0 % PD = Ty le % gilng CPE(+) > 50% (+)n50% 83,33 - 50 83,33 - 16,67 33,3 3 66,6 7 Ty If % gilng CPE(+) > 50% DTy If % gilng CPE
(+)<50%
PD: chi sd khoing cich giira nong dp cd CPE (+) > 50% vi nong đ cd CPE (+) < 50% ED: chi so nong dp pha loang cd buy hoai 50% te bao
ED = PD X (chi so nong đ pha loang cd huy hoai < 50% - chi sd nong dp pha loing cd buy hosi >50%) + chi so nong dp pha loing cd huy hoai >50% = 0,49x{(-2) - (-1)) + (-1) = -1,49 (adsbygoogle = window.adsbygoogle || []).push({});
Hifu gii = 1 0 ' " CCIDso/0,1 ml = 303 CCIDso/0,1 ml Tinh kit qui mau chuin tuong tu nhu vaỵ
Bfc ket qui chuin do hifu gii khing huyit thanh
Quan sit tl bio tun dim huy hoai
Difn bifu bin diu (+): nlu quan sit fliiy buy hoai tf bio (cd CPE), dilu niy chiing td khing nguyfn Sdi di khdng bi khing huylt tiianh Sdi mmg hda nghia la khdng cd khing huylt thanh Sdị Dien bien ban (-): nlu quan sit khdng thiy huy hoai tl bao (khdng cd CPE), nghia li khdng cd khing nguyen Sdi, do khing nguyen Sdi da bi tmng hda bdi khing huylt tiianh Sdi cd trong huylt thanh thd.
Trudc khi tinh kit qua hifu gii khing huyft thanh phii tiln hinh dinh gii kit qui mau chuin dp chuan df ngupc khing nguyfn sir dung.
Danh gii:
Nfu khing nguyfn chuin do ngupc nim Ucng khoing 30 ~ 600 CCIDso / 0,1 ml flii khang nguyen pha sit dung cho trung hda mau khing huylt thanh dat yeu ciu, kit qui hifu gii khing huylt thanh thu dupe cd gii trj.
Hifu gii khing huyet thanh Sdi dupe xac dinh t^ ndng dp pha loang cao nhit mi tai đ trung hda hit khing nguyfn Sdi (khdng cd CPE xuit hifn). Gii sit d nong dp pha loang 1/8192 li ndng đ pha loing cudi cimg khdng xuit hifn CPE ci d 2 gifng gay nhifm do viy hifu gii khing huylt tiianh li 8192 don vi
Trong hirdng hpp: 1 gilng nong dp 1/8192 cd CPE, 1 gilng khdng cd, luc đ hifu gii bing tri sd trung binh giiia n6ng dp ed I gifng huy hoai vi nong đ die hon khdng cd buy hoai do viy hifu gii khing huyet flianh li (8192 + 4096)/2 = 6144 don vi
M i u l 1/512 - - 1/1024 - - 1/2048 - - 1/4096 - - 1/8192 + 1/16384 + + + + Miu 2
Vi dy: Mau 1 ed hieu gii khang huyet thanh li 8192 don vi Mau 2 cd hifu gii Ii (8192 + 4096)/2 bang 6144 don vi Thyc hifn 1^ thir nghifm trong cic trudng hpp sau:
1/ Kit qui chuin dp ngupc khing nguyfn su dyng fliip < 30 CCIDso/O,! ml hoic cao > 600 CCIDso/0,1 ml lim cho kit qui hifu gii khing huylt thanh khdng chinh xic do vay phai lim lai tit ci cic miụ
2/ Cic giSng gay nhilm ciia 1 miu (ci 6 ndng dp pha loang) diu khdng cd CPE nhu vay hifu gia khing huyet thanh fliue tf cao hon ndng do da pha loang tai thu nghifm niy do vay chi lim lai miu khdng dat
3/ Cic gilng gay nhilm ciia 1 mau (ci 6 ndng dp pha loang) diu cd CPE nhu viy hifu gii khing huyet thanh thuc tf thip hem ndng dp da pha loang tai thii nghiem do viy chi lim lai miu khdng dat
2.3.2. Xic dinh hifu gii khing huyit thanh Bai liet
2J.2.1. Nguyen vat lifu - Huyet thanh thd
- Tl bio Hep2C (Td chirc Y tl Thf gidi)
- Vmit vicxin Sabm don gii typ I, typ II vi typ in cd hifu gii dn dinh do phdng sin xuit bin thinh phim cung cip
- Huylt thanh Bai lift typ l(antipolio type 1 lot 284, hifu gii 16.000 dcm vi); Huylt thanh B?ii lift typ 2 (antipolio type 2 lot 294, hifu gii 16.000 don vj) vi huylt flianh B?i Uft typ 3 (antipolio type 1 lot 292, hifu gia 8.000 don vi)
- Mdi tiudng: MEM 2% FBS (FBS - huylt tiianh bf bio tiiai) (Cat. Nọ 16140-071 GIBCO); Nude mudi sinh ly 0,9 % (SOP pha nude mudi smh ly)
- Pipet vi lupng: 50 jil, 100 jil, 200 \sX (Gilson)
- Diu tip vd triing tuong ung. (Coming, NY 14831 - Mexico) - Pipet thuy tmh: 2 ml, 5 ml, 10 ml (Trung Qudc)
- Pipet aid; Pipetus-akku (Diic)
-Hood CLF460EC -Tuim37°C,36°C (Memmer - Dire)
- Philn nhya 96 gilng diy phing vd tiling (NUNC - Dan Mach) - Effendorf loai 1,5 ml vd tirmg (Costar Cat. Nọ 3621 - My) - Lp thuy tinh (pinicciline) vd trimg (Vift nam)
- Be dilu nhift (Techne TE-8J) 2J.2.2. Cic buAc tiln hinh
Budc 1: Pha loing l/4va bat boat huyet thanh tho
Nude mudi sinh ly 0,9%, dupe pha them khang smh (Streptomycin vi Pinicillin theo nSng dp 1 ml khing smh /100 ml NaCl 0,9%)
0,1 ml huylt thanh thd + 0,3 ml NaCl 0,9% (ed khing sinh): dung h-ong effendorf vd trimg cd ghi sd thd tuong ling.
Huyet tiianh tiid 1/4 dupe bit boat d 56°C / 30 phiit bing bl dilu nhift. (adsbygoogle = window.adsbygoogle || []).push({});
Bir&c 2. Pha loing huyit Ihanh theo bp 2 tit 1/4 Bute 3: Pha loing khing nguyen chuan typ I, II va / / /
Pha loing hie 10 vuitt Sabm chuin typ I, II vi HI (khing nguyfn typ I, II vi HI) df dupe ndng đ su dung li 1000 CCIDso/O, 1ml
Budc 4: Trung hoa
1 2 3 10 11 12 Mausd B C D E F G H
Chung KN HI Chung te bio
• H3
>H6 H9
Chiing huylt thanh thd
Chimg KN I
Chimg KN U