Y HOC V | 6 T N A M T H A N G 4 - SO PAc BIET/2012
VAI TRO DICHLOROPHENOUNDOPHENOL TEST TRONG SANG LOG BENH HEMOGLOBIN E TREN DAN TOC KHMER
6 MOT SO TINH DONG B A N G SONG CU(U LONG
Le Thi Hoang My*, Pham Quang Vinh**, Huynh Nghla*
T O M TAT:
Myc tieu: Scinh gia vai tro eua DCIP test trong sdng loc benh HbE tren dan toe Khmer a mot so tinh dong bSng song Cu'u Long (DBSCL).
Doi two-ng va phwcng phap MSu mdu cua 426 doi tu'png duac thue hien cac x6t nghiem TPTTBMNV, khao sat sire ben tham thau hong cau vd dichloropheno! indophenol (DCIP) test de sdng lpc benh HbE Bien di Hb dupe sic dung de xdc dinh c h i n dodn benh HbE. Ket qua ti le mang gen HbE IrSn ddn toe Khmer DBSCL la 25,4%. DCIP test eb dp nhay 99.07%, dp dac hi$u 96,86%, ti le duong tinh gia 3 , 1 % va ti 1^ am tinh gia 0,93%. Ket luan: dan tpc Khmer eb ti le mang gen HbE cao, DCIP test la phu'ong phdp tot nhat de Ipa ehpn trong sdng Ige HbE trong epng dong
Tiekhoa: hemoglobin E, DCiP test, thalassemia
S U M M A R Y :
THE ROLE OF DICHLOROPHENOLINDOPHENOL ON SCREENING HEMOGLOBIN E IN
THE KHMER ETHNIC IN SOME PROVINCES OF THE MEKONG DELTA
Objective: Evaluate the role of dichlorophenolindophenol on screening hemoglobin E in the Khmer ethnic in some provinces of the Mekong Delta. Patients and methods: Blood samples of 426 subjects were tested complete blood count, osmotic fragility and
* Truing D^i hoc Y Dwgc Cdn Tha. ** Dai hQC Y Ha '** Dai hoc Y Duac Thdnh pho Hd Chi Minh Phan bien khoa h o c : PGS.TS N g u y i n Ha Thanh 18
diehlorophenolindophenol test for screening of hemoglobin E. Hemoglobin electrophoresis was used to confirm HbE diagnosis Results:
prevalence of HbE gene carriers m the Khmer of Mekong Delta is 25,4% DCIP test had a sensitivity of 99,07%, specificity 96,86%, false positive value 3 , 1 % and false negative value 0,93%. Conclusion: Khmer ethnic has a high prevalence of HbE, DCIP test is the best method for screening of HbE in the community
I. OAT VAN OE'
Hemoglobin E (ai^j") do dot biln chu6i P'^ cua globin, la mot hemoglobin bat thuong pho bien nhat a khu vire dong nam Chau A.
The di hop tu hoac dong hop tu thuong khong bieu hien trieu chung dang kL Tuy nhien, HbE co the di truyen cho th^ he sau, va tuong tac vai p - thalassemia din den mot trang thai di hop tir kep HbE/p - thai. Day la the benh da dang tir gan nhu khong bieu hien trieu chiing den phu thuoc vao truyen mau.
Van de quan trong nhat la han che cae the benh nang xay ra vai cac chucmg trinh sang loc nguai mang gen, tham van di truyen va chan doan truac sinh [3], [6].
Viet Nam nam trong khu virc dong nam chau A, tin su4t luu hanh benh p-thalassemia thay d6i tii 1,5 - 25% va ti le mang gen HbE kha cao dac biet a cac dan toe it nguol. Dan toe Khmer song 6 khap nai tren ca nuae, tuy
Ngi.
Y HOC VIET NAM THANG 4 - SO D A C BIET/2012
nhien chu yeu tap trung a mien Nam va song xen ke vai cae dan toe Kinh, Hoa, Cham trong cong dong. Ty Ie mang HbE trong dan toe Khmer khoang 20 - 30% dan den nguy ca xuat hien nhung the benh nang la rat cao [4].
Hien nay, van de sang Ipc HbE trong cong dong van chua dugc quan tam. Chan doan benh chu yeu dua vao dien di hemoglobin, mot xet nghiem chi co the thuc hien dugc a cac thanh pho Ion. Dichlorophenol indophenol test (DCIP test) la mot phuong phap don gian, dugc ung dung rat hieu qua trong sang Igc HbE gop phan Iam giam eac the benh nang trong cong dong trong nhieu n5m nay tai Thai Lan. Tai Viet Nam, DCIP test chua dugc ung dung, nen chiing toi tien hanh khao sat ''Vai tro dichlorophenolindophenol test trong sdng lpc benh hemoglobin E tren ddn toe Khmer
& mot so tinh ddng bdng song Cuu Long (BBSCLf voi muc tieu:
1. Xac dinh ty Ie mang gen HbE tren dan toe Khmer a mot so tinh DBSCL
2. Xae dinh vai tro DCIP test trong sang lpc benh HbE tren dan tpc Khmer a mot so tinh DBSCL.
II. odi TliONG VA PHaONG PHAP NGHIEN COu
- Do! tu-pTig: 426 nguai dan tpc Khmer sinh s6ng tai 3 tinh Tra Vinh, Soc TrSng, Bac Lieu. Do la nhiJng nguai co ong ba, cha me la nguai dan tpc Khmer.
- C& mSu: ap dung cong thiic tinh ca mauN = Z^.a/2P(l-p)/d^
• Voi p = 0,3; d = 0,2 X p = 0,2 X 0,3 = 0,06 -> N = 224,06 # 225
- Phuong phap:
• ThiSt kk nghien ciiu: mo ta cat ngang.
• Cach chpn mau: chpn mau nhieu giai doan.
Buac 1: chgn mau co chu dich, chpn cac xa tap trung dan toe Khmer a 3 tinh Tra Vinh, Soc Trang va Bac Lieu.
Budc 2: hoc tham ngau nhien 3 xa, tai moi xa, boc tham ngfiu nhien 2 ap,
Budc 3: tai moi ap, chpn mau theo phuong phap ngau nhien he thong den khi du so lugng mau can thiet.
• De khac phue nbuge diem ciia ehpn mau nhieu giai doan, chiing toi chgn he so thiet ke la 2. Nhu vay, ca mau la: 225 x 2 = 450 mau. Tuy nhien, thuc te ehiing toi ehi ehpn dugc 426 mau.
• Cac ky thuat thuc hien:
• Lay mdu mdu: 4mL mau tinh mach ciia moi doi tugng cho vao 2 tube chong dong EDTA.
• Tong phdn tich le bdo mdu ngoai vi (TPTTBMNV): thuc hien tai cho voi may huy^t hpe ban tu dpng 18 thong so MEK- 6420k ciia hang Nihon Kohden - Nhat Ban.
• Khdo sdt sifc ben thdm thau hong cdu (OF test): tien hanh tai cho vai ky thuat khao sat sue ben tham thau hong cau cai tien voi dung dich NaCl 0,35%. [5], [7]. Kit qua dugc dpc bang mat thuong, am tinh khi dung djch trong suot, ducmg tinh khi dung dich ma, khong dpc ro chir tren ta giay dat phia sau lube mau.
• DCIP (Dichlorophenol Indophenol) test: tiln hanh tai ch6 vai KKU-DCIP test kit ciia Thai lan [5], [7]. 20uL mau chong dong ciia doi tugng dugc cho vao tube 2mL dung dich DCIP dirge dieu che san. U tube mau a 37°C trong diing 15 phiit; sau 15 phiit, cho vao 20uL dung dich clear, lac deu va dpc ket qua tuong tir OF test.
• Sdng loc duang tinh khi: 1 trong ba ket qua tong phan tich te bao mau ngoai vi, OF test va DCIP test ducmg tinh.
Y HOC VIET NAM THANG 4 - SO Dv^C BIET/2012
• Dien di hemoglobin: mau mau dugc trii ngay a nhiet dp tir 2 - SOC va van chuyen ve trucmg DHYD C ^ Tha. Cac mhi co kk qua sang lpc duong tinh dugc giri dien di hemoglobin tai Trung tam ehan doan Y khoa TPHCM vai ky thuat dien di mao quan tren may Capillarys Sebia, Phap. Benh HbE dupe
ch4n doan khi co su hien dien ciia HbE tren kat qua dien di. Di hop tir HbE (HbAE) khi HbE < 30% tren dien di. D6ng hop tir HbE (HbEE) khi HbE va HbAi chiSm 85 - 99%
t6ng lugng hemoglobin [4].
- Xfr Iy so lieu: voi phan mem thong ke SPSS 20.0.
III. KET QUA
3.L Ty 1^ mang gen hemoglobin E tren dan t$c Khmer
270 mau sang Ipc duong tinh dugc gui dien di hemoglobin. Ket qua thu dugc:
-•^°o 0.0° <
J
Hlnli J: Phan b6 b$nh thalassemia va hemoglobin E tren dan toe Khmer - Ty 1^ mang gen HbE di hop tu: 24,7%
- Ty 1? mang gen HbE dong h<;yp tu: 0,7%
- Ty 1$ mang gen p-thal: 2,8%
3.2. Vai tr6 DCIP test trong s&ng Ipc benh hemoglobin E
Chiing toi tien hanh dong thai cac xet nghiem TPTTBMNV, khao sat sue ben tham thau hong cau vk DCIP test trSn 426 mau nghien ciiu. Cac kk qua sang loc ducmg tinh deu du^c giri di?n di hemoglobin.
XU nghiem TPTTBMNV dugc dimg ii sang Igc vcri c^c chi so: MCV<80fL hojic MCH<28pg (tieu chuto ciia WHO).
Y HOC VIET NAM THANG 4 - SO Ofi.C BIET/2012
Bang 1: Do nhay, dp dac hieu, ti le duong tinh gia va ti le am tinh gia ciia cac xet nghiem sang lpc
C a c X N sanq loc TPTTBMNV
OF test
DCIP test
D o nhay (%) (93/108) 86,11%
(70/108) 64,81%
(107/108) 99,07%
D o dac hieu
(%)
(224/318) 70,44%
(229/318) 72,01%
(308/318) 96,86%
Ti le dU'O'ng tinh qia (%) 29,56%
27,99%
3 , 1 %
Tr le am t i n h qia (%)
13,89%
35,19%
0,93%
IV. BAN LUAN
4.1. Ty le mang gen hemoglobin E tren dan tpc Khmer
S6 mlu CO ket qua dien di bat thuong chi^m ty le 28,2% (120/426). Trong do, HbE chi6m ti le cao nh^t 25,4% (108/426), co 3 trucmg hgp d6ng hop tir HbE (0,7%); p-thal 2,8% (12/426). Khong ghi nhan trucmg hgp a-thal cung nhu the di hgp tir kep HbE/p-thal nao. K8t qua eiia ehiing toi phii hop voi y van ghi nhSn benh Hb pho bien o Viet Nam la HbE, dac biet a cac dan tpc it nguai, ti Ie nay cang cao khi di chuyen ve phia Nam. Theo Bowmann J. E (1971) nghien ciiu tren dan tpc Khmer a miSn Trung nhan thay co 36,8%
nguW mang gen HbE. Su chenh lech nay co th6 do su khac nhau v^ ca mlu, hem nu'a dan tpc Khmer 6 mi6n Trung chi chiim mot ty le rSt nho nen din din ket qua chua tuong ddng. Khong ghi nhan trucmg hgp a-thal, co the do nghien ciiu ciia chiing toi chi dimg lai a xet nghiem dien di Hb, trong khi mot so dot biln a-thal lai khong gay thay d6i thanh phin Hb tren dien di. Nghien cuu ciing khong ghi nhan truang hop di hgp tir kepHbE/p-thal nao. DiSu nay cung phu hgp vi nghien cuu dugc thuc hien trong cong ddng, cac thi di hgp tu kep HbE/p-thal
thuong CO bieu hien trung binh den nang va can phai nhap vien dieu tri.
4.2. Vai tro DCIP test trong sang loc benh hemoglobin E
Khao sat sue ben tham thau hong cau da dugc cong nhan la mot xet nghiem khong phuc tap, re tien diing de sang Ipc thalassemia trong cong dong tren the gioi (S.
Fucharoen, 1989) cung nhu tai Viet Nam [2], [3]. Sue ben tham thau hong cau trong nghien ciru cua chiing toi eo dp nhay va dp dac hieu chap nhan dugc. So voi Biii Van Vien (1999) nghien cuu tren dan tpc Muong thi ket qua cua chung toi thap hon, ket qua nay co the do khac nhau ve ky thuat xet nghiem va nhan dinh ket qua cung nhu do chenh lech ve co'mlu. Tuy nhien, khao sat sue ben tham thau hong cau lai co ti Ie duong tinh gia va am tinh gia kha cao do do khong the diing don dpc trong sang Ipc benh HbE trong cong dong. K. Prayonggranata va cs, Siripakom S. va cs tai Thai Lan cung ghi nhan tucmg tu [5], [7]. Sue bin tham thin h6ng cdu CO vai tro trong sang lpc benh thalassemia, nghien cuu ciia chiing toi thuc hien trong sang lpc benh HbE va chi xac dinh chan doan bang dien di Hb nen co the chua phat hien ra nhung the benh a/p ket hgp ciang nhu cac doi tugng mang gen a, p thalassemia the an do do chua thay dugc vai tro quan trpng cua xet nghiem nay.
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Y HOC V l | T NAM THANG 4 - SOOAC BIET/2012
Ngoai sue ben tham thau hong cau, co rat nhieu eong thire img dung cac chi so hong eau de sang Ipc benh thalassemia trong cong dong, tuy nhien cac cong thuc nay it dugc ung dung tren Iam sang. Do do, chiing toi sir dung tieu chu^n ciia WHO voi MCV <80fL hoae MCH<28pg la cong thuc dupe sir dung pho 'bien nhat de sang Ipc. Chiing toi nhan thay, TPTTBMNV co dp nhay va dp dac hieu kha cao 86.11% va 70,44% trong viec sang Ipc HbE. Tuy nhien, tuang tu siic ben tham thau hong cau, cae ehi so hong cau co vai tro chii yeu trong sang Igc thalassemia. Cong thire nay lai co ti Ie duong tinh gia va am tinh gia kha cao do do kho co the sir dung don dpc trong viec sang Igc benh HbE trong cong dong.
Voi DCIP test, ket qua tir nghien ciiu cho thdy day la test co dp nhay va dp dac hieu rat cao trong sang Ipc benh HbE. DCIP test lai dugc ghi nhan co ti Ie ducmg tinh gia va am tinh gia rat thap, dac biet la ti le am tinh gia.
Vcri dp nhay 99,07% va ti Ie am tinh gia la 0,93%; cac benh nhan co kit qua DCIP test ducmg tinh gan nhu dugc chan doan chinh xac la CO mang gen HbE. Va vol dp dac hieu 96,86% va ti Ie ducmg tinh gia la 3,1%; chi CO khoang 3 - 4/100 benh nhan bj chan doan nh4m la mang HbE khi co DCIP test duong tinh. Ghi nhan cua chung toi cung tucmg tu cac nghien cuu tai Thai Lan [5], [7]. Xet nghiem lai dugc thuc hien kha don gian, nhanh chong va khong can trang bj cac may moc hien dai.
V. KET LUAN
Dan tpc Khmer a cac tinh Tra Vinh, Soc Trang, Bac Lieu co ti le mang gen HbE di hgp tu la 24,7%, ty Ie mang gen dong hgp tii la 0,7%.
Dichlorophenolindophenol (DCIP) test la mot xet nghiem don gian. nhanh chong, de thuc hien c6 dp nhay la 99,07% va dp dac hieu la 96,86%. Day la xet nghiem tot de lira chpn trong chien luge sang Ipc benh hemoglobin E.
TAI LIEU THAM KHAO
1. Nguyen Cong Khanh (1993), Tan so benh hemoglobin a Viet Nam, Nhi khoa, 174(8): 11-16.
2. Biii Van Vien, Nguyen Cong Khanh (1999), Sang Ipc nguai mang hemoglobin E the di hpp tir (HbAE), Nhi khoa. 8(2):
76 - 84.
3. Pham Quang Vinh (2010), Quan ly benh huyet sac to. Mot so ehuyen de Huyet hpc - Truyen mau. Tap 3: 193-202.
4. Barbara J. Bain (2006), Other significant hemoglobinophathies,
Haemoglobinopathy diagnosis, Blackwell, 201-9.
5. Prayongratana K. et al (2008), Low cost combination of DCIP and MCV was better tan that of DCIP and OF in the screening for hemoglobin E. J Med Assoc Thai. 91(10): 1499-503.
6. Weatherall DJ and J. B. Clegg (2001), Inherited haemoglobin disorders: an increasing global health problem".
Bulletin of the World Health Organization. 79: 704-12.
7. Sangkitporn S. et al (2005), Validation of osmotic fragility test and dichlorophenol indophenols precipitation test for screening of thalasemia and hemoglobin E. Southeast Asian J Trop Med Public Health. 36 (6): 1538-42.