TW CHi V - DVOC HOC OUAN SV sS «-2014
NGHIEN CLfU ANH H I / 6 N G C D A pH DUNG DjCH OfM VA NONG DO SUDAN B DOI Vdl PHAN LTNG
NHUOM LIPID BACH CAU NGUdl
Trin Van Tinh*
TOIM TAT
Nghien ciiu tdi iru hda quS trinh nhudm mau lipid bjoh clu ngydi bang Sudan B. Quy hojoll hda thyc nghiam theo md hinh mjt hai nhan to. pH (X,) va ndng dd Sudan B (X2); ham muc tiSu 1^ diem nhudm lipid (Y) bang phin mIm Modde 6.0. Phu'O'ng trinh hdi quy thu dygc: Y = 362,7 + 5,2 X X, -10.5 X X2 - 26.3 x X,^ (R^ = 0,985 vdi p > 0,95) va dieu l(ien nhudm toi uu: Nong SO Sudan B id 0,20% pha trong dung dich 6$m vdi pH = 7,42. HiSu suit nhudm tdt nh^t trong diSu kijn toi uu tim dup'C 95,10 ±1,0% (p = 0,95).
* TCr l<hda: Nhudm hda hoc te bSo; Nhudm Sudan B; Nhudm lipid.
EFFECT OF pH AND B SUDAN SOLUTION ON LIPID STRAINING OF HUMAN LEUKOCYTE
SUMIVIARY
A research was conducted on the optimal conditions for the lipid of human leukocyte with Sudan B solution. Experiments were designed by response surface modeling with two factors: pH fX,) and Sudan B concentrate (X2) to target lipid score function; Regression equation presents dependence of lipid score on two factors quoted above as: Y= 362.7 •*• 5.2 x X^-10.5 x Xi- 26.3 xXi2(F^ = 0M5 with p > 0.95) and the optimal conditions of the lipid staining as' pH = 7.42, Sudan B 0,20%.
The best efficiency of the lipid staining In the above optimal conditions is found to be 95.10 ±1.0%
(p = 0.95).
* Key words: Cytochemistry; Sudan B staining; Lipid staining
O A T V A N B E phan Lrng mau r^t l^hac nhau, bao gom 1 i„iH .-A t r „ „ „ tA 1,*., W....I. > • • l i khuech tan vat ly San t h u i n hoac Hdn Lipid CO trong te bao bach cau nguci la j,
este cua axit bdo vdi aneol. xac dinh cac '^®' '"'^^ '^'"= ''"''"= ''®P- '^'°"9 "^^^ 1°?' lipid nay bang phan Crng mau vdi nhieu "ludc nhudm, Sudan la mdt nhdm phlim loai pham mau: dau dd, seharlach R, nhudm du'crc u-a chupng de nhupm lipid rodamin B, nile blue, Sudan... Ca che eua tCr vang (sudan Ml) den den (sudan B) [3],
• B$nh vien 19-8
Ngu^phin hoi (Corresponding): TrSn Vin Tinh ([email protected]) Ngiy nhan bii: 17/06/2014; Ngiy phin biin dinh gia bai bio: 28/07/2014
Ngiy bii bao dwgv ding: 11/08/2014
TRP CHf V - DVOC HOC QURN SV SO' 6-2014 Ddi vdi mau ngoai vi, phan irng nhudm
sudan B cho k^t qua bach eSu hat du'ang tinh manh, monoxit duang tinh yeu, lymphoxit va hdng cau am tinh. Trong tuy xuang binh thu'dng, cae te bao ddng tijy duang tinh tCr ti^n tuy bao d^n t l bao trudng thanh, ddng monoxit chf du'ang tinh d te bao tru'ang thanh; m l u tieu c^u va tieu eau du'ang tinh o' cac giai doan.
Trong nhieu trudng hap mac benh bach Cciu cap, cd the phan biet tuang ddi rd ddng tuy va nhCrng ddng td bao Ithac bang ket qua nhudm sudan B. Ket qua duang tinh Sudan B cua m l u benh phlm thudng cho kit qua du'ang tinh vdi ky thuat nhupm peroxydase. Chinh vl vay, k i t qua nhupm Sudan B la mdt trong nh&ng tieu chuan du'pc irng dung de phdn loai t h i bpnh trong bpnh bach cau e l p theo tieu chuln FAB [2].
Chayen (1958) cho ring, muc dd bat mau chpn Ipc cOa cac thude nhupm ddi vdi lipid phu thupc r l t nhieu vao ban chat lipid va dac tinh oCia thude nhudm, thude dieu trj, hormon va dieu ki#n tien hanh nhupm [3]. Phan irng nhupm sudan B ndi ridng va nhudm lipid ndi chung cd t h i cho kit qua tuang d l i khac nhau trong tCrng dieu kipn cu t h i . VI vay, vi#c nghidn ciru tim ra dilu kipn tdi u'u d l t i l n hanh phan irng, nham dam bao tinh I n djnh v l mat ky thuat, nang cao ehlt lupng nhudm va giam chi phi la v l n de cd y nghTa khoa hpc va thi.rc t i l n eIn thilt. IVluc tieu ciia edng trinh nay: Nghien cuv toi uv hoa quy trinh nhupm lipid te bio b^ch cau nguvi bang pham miu sudan B dung trong xet nghiem de phan io$i ddng te biro ung thwmau.
£)6l Tl/ONG VA PHirONG PHAP NGHliN Ctjru
1. D6i tu'O'ng, dung cu va hda c h i t nghien CCPU.
* Doi tuong nghien ciru: bach c l u lly tCr mau cua ngu'di khoe manh cd chira lipid. IViau sau khi l l y tir tinh mach, tiln hanh dan 14 tieu ban khdng qua chdng ddng, d l khd trong dilu kipn tu" nhien va cd djnh ngay.
* Di,ing cu: may ii nhiet CW-20G 0 - 50°C, dd chinh xac + 0,1°C, may do pH inoLab 730 vdi dd chinh xac d i n 10', can phan tieh Sartorius TE214S ed dp chinh xac 10"", kinh hiln vi Nikon vdi dd phdng dai 1.000 iln, pipet ti,r ddng cua Hang Thermo kem dlu cdn 0,2,1,0 va 10 ml cua Hang Gilson.
* Hoa chat: sudan B {Hang Sigma- Aidrich); phenol (Hang Sigma-Aldrich);
axit citric 99.5% (Hang Sigma-Aldrich);
formaldehyd 37% ACS 99,5% (Hang Sigma-Aldrich); ethanol 96% (Hang Sigma-Aldrich); aceton ACS 99,5% (Hang Sigma-Aldrich); Na2HP04 (Hang Sigma- Aldrich); Giemsa (Hang Sigma-Aidrich).
- Pha ch4 thuoc thCir + Dung dich ed djnh tieu ban:
++ Dung djch citrat: 25 ml (lly tir dung djch 18 mmol axft citric + 9 mmoi natri citrat va 12 mmol NaCI trong 1 lit).
++ Acetone 99,5%: 65 ml.
++ Focmaidehyd 37%: 8 ml.
+ Dung, djch dem phenol-phosphat vdi pH dup'C pha theo bang 1: phenol 16% va NaaHPO, 0,3% dieu chinh pH bang HCI 0,1 M.
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TjP CH11 • DVOC HOC QUflN SV i6 6-2014 + Dung dich nhudm sudan pha theo bang 1 trong ethanol 96% va dem phenol- phosphat d tren theo ty le 6:4.
+ Dung dich nhudm nhan: Giemsa.
2. Phu'cng phap nghien cu'u.
* Thiet ke thi/c nghiSm:
Thi nghipm dupe thilt k l theo md hinh khao sat mat muc tieu (Response Surface Modeling - RSM) cu the: t l hap mSt tam (The Central Composite Face - CCF) 2 nhan to trdn phin mIm iVIODS. Moi nhan to gom 3 miro: a tam (d = 0), d dinh (1) va d mat (d). Md hlnh gom 11 thi nghlpm:
4 thi nghiem d dinh, 4 thi nghipm dilm sao nam d mat khdng gian mdi nhan to va 3 thi nghidm lap a tam. Thilt k l quy hoach hda thuc nghi#m gdm 2 biln la pH dung djch dem (X,) va ndng do sudan B theo g/100 ml (Xj). Anh hudng ciia hai nhan to nay len ham muc tieu (Y) la dilm nhudm tuan theo phuang trinh:
Y = b„ + b,X, + bzXz + b,(Xi)' + b2(X2)^ + bizXiXz cac dilu kidn ma hda d l nhap du' lieu vao chuang trinh phin mem {bang 1):
Bang 1: Ky hieu va mire ma hda thyc nghipm.
BISN
pH dung dich dem Sudan B (%)
Hl§u X, X2
MOC MA HdA
•1
7,2 0,2
0 7,4 0,3
1 7,6 0,4
* Ky thuat tiin hanh nhuom:
- Cd djnh tidu ban trong dung djch c6 dinh tieu ban a tren vdi thdi gian 30 giay.
Sau dd, gdi va dua cae tidu ban vao hpp
chdng I m bae quan 6 nhiet dp 4 - 8 C (thdi gian bao quan cd t h i keo dai 2 thang [81).
- Nhiing tieu ban vao dung dich nhudm Sudan d tren vdi ndng dp va pH khao nhan theo quy hoach thu'c nghiem d bang 1 trong 30 phiit. Sau dd, rCra tieu ban dudi vol nude 5 phiit, d l khd.
- Nhudm nen b l n g dung djch Giemsa 10% trong 15 phiit va d l khd.
- Dpc tieu ban dudi vat kinh dau vdi d^
phdng dai 1.000 i l n .
Cac viing cd chira lipid trong nguyen sinh chat bach c l u se xult hien hat bat mau den. Ddi vdi ddng tuy t l bao cang tru'ang thanh, mire dp du'ang tinh cang manh. Ddi vdl ddng lympho binh thudng am tinh.
Danh gia bach c l u tmng tinh duang tinh thanh 5 mire dd:
Op 0: khdng cd hat bat mdu den.
Bp 1: it hat trdn nguyen sinh chat, hat mm nhd, khdng cd hat tren nhan t l bao.
Dp 2: nhieu hat tp tren nguyen sinh ehlt, khdng cd hat trdn nhan t l bae.
Bd 3: nhilu hat tP trSn toan bp t l bao, nIn xau.
Dd 4: nhieu hat to tren toan bp t l bao, n I n dep.
Cdng thirc tinh dilm nhupm: JY (dilm) = YOxO + Y 1 x 1 + Y 2 x 2 + Y 3 x 3 + Y 4 x 4
Nhu vay, phan irng nhupm se dat tli U'U khi tpan bd t l bao bach cau trung tinh duang tinh d dp 4, tong diem tdi da 400 vahi?u s u i t dat 100%.
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TAP CHf V - D v a c Hpc Q U A N SV s d 6-2014
KET QUA NGHIEN CljIU
Bang 2: K i t qua thyc nghiem nhupm sudan thee theo md hlnh khao sat mat mMC tieu.
THl NGHIEM 8 1 2 7 6 11 10 9 3 4 5
MATRANMAHOA X I
-1
0 0 0 0 0
X2 -1 -1 1 1 0 0 -1 1 0 0 0
N 6 N G Bd THUC NGHIEM (%) X,
7.2 76 7 2 7.6 7.2 7.6 7.4 74 7.4 7.4 7.4
X2 0 2 0.2 04 0.4 0.3 03 02 0.4 0.3 0.3 0.3
OI^M NHUdM (Y) 342 355 322 335 335 340 378 355 360 362 364 Nhgp k^t qua vao ph^n mem Modde 5.0 qua xu" ly, du'p'c phu-cng trinh hoi quy:
Y = 362,7 + 5,2 x Xi -10.5 x X2 - 26,3 x X,^ + 2,7 x Xz^ -5.53x10"^ x Xi x X2.
* Do tin c$y thong ke ciJa cac bien s6:
Bang 3: H$ s6 va do tin cay thong ke cua cac h0 s6.
Y Constant
Xi X.2 X,*X, Xi'Xi X,*X2 N = 11 DF = 5
CCEFF. SO 362,737 5,16673 -10,5 -26,3421 2,65793 -3,15E-06 Q= = R' = R'Adj. =
STD. ERR 1,45222 1,15571 1,15571 1,7786 1,7786 1,41545 0,878 0,985 0,97
P 1.95E-11 0,006576 0,00027 2,54E-05 0,195304 1 Cond. no, =
Y-mIss = RSD = Conf lev. =
CONF. INT (±) 3,73306 2,97085 2,97065 4,57205 4,57205 3,63854 3,0822 0 2,8309
0,95 Nhu vay, l09i bd he s6 X2^va X, x Xj phuang trinh hdi quy la: Y = 362,7 + 5,2 x X, - 10,5 X X2 - 26,3 x Xi^. Phan tieh Anova cho thly phuang trinh md ta tuang ddi tdt k i t qua thire nghiem (he sd tuang quan R^ = 0,985 (p > 0,95)).
Chay chyang trinh tdi yu thu dyac dilu kien tdi yu vdi pH = 7,419 va ndng dp sudan la 0,20 g%, khi dd dy dpan diem nhudm dat 376,1. Kit qua tdi yu hda dypc trinh bay d bang 4 va hinh 1, hinh 2.
TAP CHf V - Dir^C HOC QUflN SV i6 6-2014
Bang 4: Toi u'u hoa theo phu^cng trinh hoi quy,
XI 7,4431 7,4203 7,4431 7,4192 7,4445 7,4189 7,4445 7,4445
X2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2
Y 375,782 376.142 375.782 376,142 375,736 376,14 375,736 375,736
ITER 127 24 127 24 114 24 114 114
LOG(D) -0,564 -0,687 -0,564 -0,687 -0,55
•0,686 -0,55 -0,55
D l khlng dinh dilu kien tdi yu thu dtfijc d tren se cho hipu s u i t nhudm cao nhS, da tiln hanh nhudm 3 thi nghiem.
Bang S: K i t qua thi nghiem lap l?i 6 d i l u ki$n t l i yu.
investigation: Sudan B (MLR) Contour Plot Diemnhuom
SdTHl NGHI$M 1 2 3 Trung binh
BISM S6 (Y) 382 380 379 380,3
Hieu SUAT H%
(YMOO) 96.6%
95.0%
94.8%
95,10%
Sau khi XU' ly d y lieu bang chuong trinh thdng ke SPSS 18, thu dypc kholng gidi han hilu s u i t nhupm trung binh khi cap 95,10% ± 1,0 (p = 0,95) J
Hinh 1: Sa d l dudng ding mire mat toi yu.
Hinh 2: Mat t l i yu thye nghipm.
1Q
Hinh 3: Hinh anh nhudm lipid bach clu it d i l u kien t l i yu vdi sudan B.
BAN LUAN - V l pH dung djch dpm:
pH cua dung djch nhudm ed vai tro quan trpng trong phan irng nhudm, quyet djnh t i e dp khulch tan va giy nguySn hlnh thai eua t l bao eOng nhy lipid it trang thai t y nhien. D l phan irng xay ra tdt nhlt, dung djch sudan B dypc pha chl
TAP CHf V - DVOC HOC OUSN S ^ SO' 6-2014 irong mdi trydng cd dp pH trung tinh
hoac kilm nhp, phenol cd trong dung djch ddng vai trd nhy chit e l djnh mau [4]. K i t iqua thu dyac eua nghien ciru (pH = 7,42) tyang dyang vdi cdng b l cua Hayhoe,
;Sheehan va Storey (pH - 7,4) [1, 3, 4].
- Ndng dd ciia sudan B:
Nong dp sudan B quylt djnh kha nang khulch tan ttr dung mdi vao t l chire ma.
Sudan B nhudm mau lipid theo 2 ea c h l : khulch tan vat ly dan thuin va lien k i t hda hpc. 2 nhdm amino eua sudan B phan U'ng vdi eac nhdm axit cua lipid. Ndng dp Sudan B (0,20 g%) t l i yu thu dyac nhd han 18,9 l l n so vdi k i t qua cua Hayhoe [1,3,4].
K i T L U A N
Qua nghien ciru thyc nghlpm theo md hinh khao sdt m j t mgc tieu 2 biln sd, da tim dypc d i l u ki$n tdi yu cua phan irng nhupm sudan B: pH dung dich dpm nhupm
bang 7,419 va nong dp sudan B la 0,2%
Quy trinh tim dupe dat hidu suit nhupm 95,10% ± 1,0 (p = 0,95), tao dilu kien d l cd t h i triln khai rpng ky thuat nay trong phdng xet nghiem huylt hoc d Vi#t Nam vdi gia thanh thlp.
TAI LIEU THAM K H A O
1. Vl$n Huyit hpc-Truyin miu. Ky thuat xdt nghi#m Huyet hoc-Truyen mSu. NXB Y hoc.
Ha Ndi. 1984.
2. Bennett JM, CatovskyD, Daniel MT, etai.
Proposals for the classification of the acute leukaemias, French-Amencan-British (FAB) co-operative group. J. Haematol. 1976, 33 (4), pp.451-458.
3. F.G.J. Hayhoe &D. Quaglino. Haematological cytochemistry, Churchill Livingstone, London.
1994.
4. Sir John V. Dade; S.M Lewis. Practical Haematology. Longman Group Ltd, Hong Kong 1994.
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