N G H I E N CUIU DONG OICH Ctf DjNH DA T H A N H P H A N
TRONG KY THUAT N H U O M HOA HOC it BAOT6M TAT
Dung djch cd djnh da thdnh phin gdm: formal 9.01%. etanol 31.04%, axeton 34.52% vi dim xitmt li 25.39%; d thi d djnh cic tiiu bin miu, tuj' dung cho tit ci cic ky thu$t nhudm hda hpc ti bio, nit ngin th&i gian vi giim ddc hpi
Tir kh6a: nhudm hda hpc ti bdo SUMMARY
The fixation solution has components include:
formaldehyde 9.01%, 31.04% ethanol. acetone is 34.52% and 25.39% citmte buffer, that can fix the blood specimen, bone marrow slide for all cytochemistry of staining, shorten time and mduce toxic
Keywords: cytochemistry of staining DAT V A N D £
Cfi (Knh la mflt budc quan tnpng trong quy trinh nhuflm hba hpe l i bdo. Muc dich ciia cfi djnh tidu bfin la gifit chdt t i bao nhung v i n bdo qudn chOng trong tinh trgng g i n nhit nhu khi t i bdo sfing. Cdc chit co djnh trong ky IhudI nhufim hba hpe tfi bdo thudng su dyng forniol, ethanol, methanol,,. Hifin nay, mfii ky thufit nhudm hda hpe t i bdo phdi su dyng dung dich cfi ^nh ndng, thdi gian cfi dmh khdc nhau nen viec pha chd cung nhu tifin hanh ed i^nh r i i phue tgp, dflc hai eao [2, 3, 4, 6], Vi vdy, nghifln cuu mflt dung dich cfi djnh da thanh p h i n diing cho todn bfl cdc ky thudt nhuflm hba hpe t i bdo trong cung mflt thdi gian Id mflt nghien cuu ung dyng eb y nghTa thue tidn eao,
DOI TU'O'NG VA PHU-ONG P H A P NGHIEN CLTU 1. Obi tup'ng nghidn c u u : Ld lidu ban mdu dan liy tu ngudi khoe manh.
2. Phuong phdp nghidn c i r u :
Tfii uu hba dung d|ch efi djnh b i n g phuong phap quy hogeh hba Ihyc nghlfim don hinh [1 ] sau dd lidn hanh nhubm lidu ban Iheo tam ky thudt nhuflm PAS, Sudan B (SUD), Peroxydaza (PER), Phosphataza kiim (PAL) vd axit (PA), Esteraza dac hidu (EST).
khflng ddc hidu uc ehfi (NEST-NaF) vfi khflng uc e h i (NEST).
Thidt kfi quy hogeh hba thye nghidm gfim 4 bifin la nfing dfi % t h l tich. fomiol (XI), ethanol (X2), axeton (X3) vd dung d|eh dfim xitrat (X4). Chpn thdi gian ed (^nh 1 phut.
Lya ehpn mire gdc t h u e nghidm t h i hifin d being 1.
Bdng 1. Lya ehpn muc gfic thye nghidm (1,5ml)
Nhan 14 Mu'Cqoc
l i
X1,(%) 8 1
X2, (%) 27
5 X3, (%)
40 5
X4 (%) 25 25
TRAN VAN TINH Bdnh vidn 19-8, Bd Cdng an Ap dyng ede cfing thCrc tinh theo phuong phdp quy hogeh hda thye nghidm gdm 4 bifin theo phuong phdp don hinh thu duye ma trdn thye nghidm eda cdc dlnh I h i hidn d bdng 2.
Ky thu$t nhufim Chuin bi
- Di^ng CM: Mdy tfing phdn tieh mdu Cell-dyn 3200, mdy LI nhifit 0-500C, dfl ehlnh xdc ±0,10C, mdy do pH dfi ehlnh xdc d i n 0,001, cdn phdn tieh dfi chinh xdc 0,0001, kinh hifin vi vdi dfl phbng dgi 1.000 l i n .
- Hod c h i t :
+ Co chit: cac loai eo chit dimg eho phdn ung nhuflm enzym cua hang SIgma-Aldrich
+ Cde hba chit co bdn ciia hdng Slgma-AIdrich.
+ Cde hda c h i t nhuflm nhdn eua hdng Sigma- Aldrich
- Bfinh p h i m : L i y mdu, ddn tidu bdn, 6h khfl Irong khflng khi. Mdu dupe phan tieh b i n g may tdng phdn tich Cell-dyn 3200 nhim l i y cbng thue bach c i u dfi tinh didm ly thuyit tfii da cua tidu bdn nhufim hda hpc l i bfio.
Ky thudt tiin hinh nhudm:
- Mfli tidu bdn cd dinh trong Ihdi gian phut. Cde lidu bdn cbn lai dupc bao quan trong hflp bao quan d -400C cfl thfi kflo ddi 1 t u i n [8]
Sau khi cd dinh tien hdnh nhuflm tidu bdn theo 8 ky thudt nhuflm hba hoc t i bdo.
Ddnh gid k i t qud trdn 100 tfi bdo cfl nhan theo phuong phdp phdn dfl nhu sau'
- Dfl 0: Tfi bdo m i t hinh thdi, k i t qua nhuflm hba hpc tfi bdo dm Hnh.
- Dfl 1: T i bdo truong lo hodc co nhd, kdl qud nhuflm hba hpe ifi bdo dgt db 2 trd xudng.
- Dfl 2: Tfi bdo giu nguydn hinh thdi, kdt qua nhuflm hba hpe l i bdo dgt dfl 2 trd xufing
- Ofi 3: Td bdo bifin dgng It, kdt qua nhuflm hba hpe td bdo dgt dfl 3 trd ldn
- Dfi 4' T i bdo giir nguydn hinh thdi, k i t qua nhuflm hba hpe dgt dfl 3 trd ldn.
K i t qud nhufim hba hpc td bdo dupc phan dfl theo thang dfl cua Sigma.
Tfing d i i m nhuflm cu t h i nhu sau.
SY = N1X1 + N2X2 + N3X3 + N4X4 Trong dd: N Id ty I? % sd id bdo bach c i u duong tinh tai dfl X va Y Id diem danh gid
K£T QUA
Diem Iy thuyet tdi da dupe tinh dya vao cbng thirc bgch c i u eua m i u mau nghien ciru b^ng may long phan tich Cell-dyn 3200 thd hien tren bang 2 [7, 9],
Y HQC THV'C HANH (837) - SO 8/2012
Bdng 2, O i i m ly thuyit tdi da cCia cdc ky thufit nhufim hfia hpc t i bdo
TT 1 2, 3.
4.
5.
Bvch ciu Trunatlnh Monoxit
Axil Baza tymphoxit
%
SO 7 4 1 28
PAS 1 1 1 1 0
PER 4 2
:
2 0
SUD 4 2 2 2 0
PAL 3 0 0 0 0
PA 0 0 0 0 1
EST 4 2 2 2 0
NEST 0 3 0 0 0
ES-NaF 0 0 0 0 0 ' dna diim
Diim ntiuQm 960
ro
2g 7 28 1093 Kfit q u i cdc thl nghlfim theo quy hogeh hfia thyenghlfim don hInh dupc thfi hifin trfin b^ng 2, Bing 3. O i i m thye nghlfim
Don hinh 1
SO
SI S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 821 S22
Dlnh 2 1 2 3 4 5 1- 4' 5' 2' 5- 5"
4"
4'"
1"
2- 4 - 5- y 3- 3"' 5- 5- 5 - 5 7"
1'"
3-' X1
(%)
3 7.76 8,71 8.34 8.02 7,87 8^82 8,75 9,41 8,83 7,97 9.41 6,83 8,75 9,06 8,94 9,14 8,26 9,49 6,34 9.49 10.07 8,26 10.07 8,26 9,01 8,84 8,21X2 4 26,44 26,17 29,31 28,18 27,66 29,08 28,00 28,75 31,13 29,97 26,75 31,12 28,00 29,61 26,22 29,11 28,44 27.19 29,31 27.19 27.70 28.44 27,70 28.44 31,09 28,25 31,04
(%)
X3 5 40,34 39,92 38,23 40,80 39,86 39,07 37,69 37,57 36,66 38,31 37.57 37,90 37,89 35,86 3B.29 37.03 37.12 35.60 38.23 3560 36,28 37.12 36,28 37.12 34,52 36,35 36,84X4
(%)
6 25,48 25,20 24,13 23,20 24.61 23.22 25.36 24.27 23.36 23,75 24,27 22,15 25,36 25,47 26,54 24,73 26.17 27,71 24,13 27,71 25.95 26,17 25,95 26,17 25,39 26,55 23,92Diim nhuQm
10 263 279 419 271 275 301 296 284 305 263 326 296 309 848 831 1005 784 330 364 866 496 678 687 1,026 1086 1,060 1 047
ill
11
1507
1 545 L_1 570 1 579 1 605 1 584 1 647 1647 1660 2 207 2 733 3 429 3 887 3 798 3 832 4 354 4 066 4 248 4 257 4 596 4 851 5 063 5.224 Oon hinh SO cd dinh tfii n h i t Id tfinh sd 1 dgt 264 diim; bd di dlnh ndy, lim ^ n h mdi vd ldm Ihyc nghlfim Ihl thu dupe 6inh 1' ed sfi d i i m Id 303 diim.
T i i p tyc ldm nhu vdy dfin don hinh S22 Ihl dung thi nghiflm, eoi d i i u kien Ihyc nghiem d dlnh 2'" Id tdi uu bdi cdc tfim nhufim cua ede don hinh lif S19-S22 ed xu hudng quay xung quanh difim tdi uu. Ngodi ra didm nhuflm ciia don hinh S22 thu duoe khd cao (1,086) vd dgt 99.36% so vdi II thuyit Nhu vfiy dung djch cd djnh tfii uu efi ndng dfl % thfi tich nhu sau' Formol 9,01%, etanol 31,04%, axeton 34,52% va dfim xitrat la 25,39%.
Sy s i p xdp difim sfi cua cdc don hinh t h i hifin trdn hinh 1,
Hinh 1 Biiu didn (Tiim cua cdc don hinh (S) tmng qud trinh tii uv hod
o i khing djnh cde d i i u kifin nhufim mdu dd nim trong m i i n tfii u u dd t i i n hdnh ba thl nghlfim l$p d dinh ndy vd thu d u c c k i t qud nhu t h i hifin d bdng 4
Bdng 4 K i t qud thi nghifim Ifip Igi d dinh tdi uu SA thi nghidm
1 2 3 Tnjng binh
Oi6m s6 (Y) 1084 1089 1062 1 065
Hi«usuitH(%) 99.16%
99 63%
96,99%
99.27%
Sau kht xir ly dO* lifiu tifing chuong trinh thfing kd [5], da Ihu dup'c gidi hgn tin cfiy p=0,95 cua hidu suit nhufim Id: 95,27% ± 3,61 vdi p = 0,95. Kit qud nhufim esteraza d$e hifiu sii' dyng chit cfi dinh 4 thdnh p h i n dudc t h i hifin tren hinh 2.
Hinh 2 Hinh inh nhudm esteraza d$c hidu
Y HQC THUC HANH (837) - SO 8^012
BAN LUAN 1. Nfing dfi etanol
Etanol dupc biit d i n nhu mflt c h i t cd djnh tidu ban mdu ngogi vj phfi b i i n nhit vi tinh It dfle hgi, d i bay hoi. S u dyng etanol trong bude cfi djnh lifiu bdn mdu hofie tuy thudng dOng phuong phdp phu Ifin tifiu bdn mflt Idp mdng. Ti6u bdn dudc dyng mfit gfic 450C ehd khfi hodn todn trude khi nhuflm. Etanol s u dung trong ed d|nh Ihudng diing d nfing dfi t u 70- 100% Ld mflt c h i t khit oxy, gdy kfit lua mgnh protein nhung kfit Ida kdm cde nueieoproteln vd khbng ldm dflng Chung Hba tan tfit mflt sfi lipil, kfit lua nhung khflng efi djnh glycogen. Etanol eb yfiu d i i m ldm co tfi bdo mgnh, VI thfi viflc sij" dyng d nfing dfl t h i p sd khic phyc duve nhudc difim eo tfi bdo. Nfing dfl etanol Ihu Oupc trong qud trinh tfii uu hba Id 31,04%.
2. Nflng dfi formol
Trong ky thufit nhufim, formol Id mflt trong nhO'ng chit ed tfnh tfit n h i t vl nfl cd vai trfl gidl c h i t tfi bdo, cfi tfnh protein thfing qua ede iidn kfit nfli bdo vd ngogi bao tir db bao qudn dupc hinh thai tfi bfio cQng nhu cdc chit hfia hpe khde trong bdo tuong, Formol cd t h i diing dudi hai hinh thuc, mfil Id bay hoi bdo hba; hai Id trfln vdi cac hfla ehdl cfi d|nh khac nhu etanol, melanol . Nfing dfl formol cao cb I h i phdn i>ng vdl hematein cb trong hfing c i u vi tgo ndn s i c id mdu ndu ho$c den K i t qud thue nghidm tim Ihiy ty le formol tfii uu Id 9,01% cao hon so vdi ky thudt ciia hdng Sigma (8,16%) nhung t h i p hon cua Gomon (10%) Nhifiu enzym bi dc ehfi bdi mfll luyng nhd Iham chi d dang vdl formol, n6n sau khi cfi djnh lufln dfli hdl phai n>a ky lidu bdn dudi vbi nude [6, 8,
3. Nbng dfi axeton
Gan day s u dyng axeton dfi efi tfnh lifiu ban mdu ngoai vi cung nhu tieu bdn tuy 6uac ung dung rfing rdl. Ddc tinh Idn nhit cua axeton la cd kha ndng bay hoi nhanh, tdc db I h i m vao Ihdnh tfi bdo Idn ldm giam thdi gian cd dmh Nhupc d i l m cua axeton Id cfl kha nang hba lan ldl cdc lipoprotein lam m i t tf cac cau Inlc hba hpe td bao nhu khi cfln sdng Tuy vdy nb cung lam bbe Ifl mdng le bdo ldm eho cac thdnh p h i n khdc eiia dung d|eh ed tfnh nhu formol va etanol cd the tham nhap co dmh tot hon Nong dfl axelon tdi uu thu dupc Id 34,52% t h i p hon so vdi hdng Sigma 66,33% Nguydn nhdn cb thfi la do hang Sigma t i i n hdnh CO tfnh tidu ban trong thdl gian n g i n hon mflt nira (30 giay) so vdi nghien ciru (60 gidy) [8]
4. Dung djch dem
Dung dich dem xitral cb vai trd nhu Id chat fin dmh mang te bao giiip cho hinh thai te bdo dupe bao tfin tot hon Vai trfl ciia dung dich dem ehu ydu la giir cho
mdi trudng d i n g difin ldm eho protein It bj bifin tinh nhit tir 66 mdng tfi bdo It b| tnj'ong phfing, ede thdnh p h i n t i bdo trong bdo tuong dupc gid nguydn vgn.
Nfing dfl tfii uu hda eho I h i y dung dieh ddm xitrat ndn duy tri d nfing dfi 25,39%. Tgi nfing dfi ndy khd ndng gid fin tfnh eda dung djch Id luong dfii tfit. Nfing dfi tfii uu thu dupc eao hon so vdi eda hdng Sigma (25,51%) [8].
K£T LUAN
Qua nghidn cii'u k i hogeh hda thye nghidm bdng phuong phdp don hinh da tIm dupc ede d i i u kidn tdi uu eua dung djch cfi tfnh cho cdc phdn dng nhufim hoa hpe l i bdo nhu sau: Formol 9,01%, etanol 31,04%, axeton 34,52% vd ddm xitrat Id 25,39%. Vdi viec sir dyng dung djch efi tfnh duy nhit bfin thdnh phin eho l i t ed ede ky thudt nhuflm hba hpe l i bdo dd don gidn hba budc efi tfnh, riil n g i n thdi gian pha ehfi, gidm dflc hgi, tgo tfiu kifln dfi tnin khai rflng r3i kl thudt ndy trong cdc phbng xdt nghlfim huyfit hpc Igi Viflt Nam.
TAI LIEU THAM KHAO
1. Nguyin Cdnh. Quy hoaeh thye nghidm, NXB Dpi hpc Quic gia TP. HCM. 2004, tr 99-102
2 Trudng Dgi hoc y khoa, Ky thudt hifin vi hoc thflng Ihudng, Nhi xuit bin y hpc. 1976, tr.9-25.
3. Vidn Huyit hpc-Tnjyfin mdu. Ky thudl x6t nghiflm Huyfit hpc-Truyfin mau, NXBYH, HB Ndi, 1984. tr. 70-71
4. Cari A. Burlls, Edward R Ashvifood, DBmns.
Tietz textbook of Clincal chemistry and molecular diagnostics, Elsevier Saunders, USA, 2006, p. 191- 218.
5. David M. Levine, Marit L. Berenson, David Stephan. Statistics for managers using microsoft®
excel, Prentice-Hall, inc, 1997. p.146-148.
6. Divyen Palel and et al. Subcellular Fractionation studies Indicate an intracellular localzallon for human specific esterase (MSE), Bntish Journal of Haematology, Volume 84 (4), 2008, p 608- 614.
7. Merck Enzyme cytochemislry Leucognosl®/
HematoGnost Fe®; Working inlructions for simpified cytoehemlcal staining for differentiation of leukaemias and myelodysplastic syndromes, £ Merck. D-64271 Darmstadt, 1995, p.9.16-17
8. Sigma-AIdneh Catalougue. Naphthol AS-D Chloracetate Esterase (Pmcedum No.91), Slgma- AIdrich, 2003. p. 1-3
9. Sir John V. Daeie, S.M Lewis, Practical Haematology, Longman Gmup Ltd. Hong Kong, 1994. p. 153-154
V HQC T H U C HAINH (837) - SO 8/2012
