centrifugacion humana

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E L S E V I E R Acta Tropica 62 (1996) 183-187

ACTATROPICAS h o r t c o m m u n i c a t i o n

Im proved perform ance of the anion-exchangecentr ifugation techn ique for studies with hu m an infectiveAfr ican t rypanosom esU . Z i l l m a n n a . , , S . M . K o n s t a n t i n o v b, M . R . B e r g e r b, R . B r a u n c

a Z e n t r a l e s T i e rl a bo r , D e u t s c h e s K r e b s f o r s c h u n g s ze n t r u m , I m N e u e n h e i m e r F e l d 2 8 0 ,69120 He ide lberg , Germ anyb . 4 G T o x i k o l o g i e u n d C h e m o t h e ra p i e, D e u t s c h e s K r e b s f o r sc h u n g s z e n t r u m ,I m N e u e n h e i m e r F e l d 2 8 0 , 6 9 1 2 0 H e i de l b e rg , G e r m a n y T e c h n i sc h e A b t ., D e u t s c h e s K r e b s f o r s c h u n g s z e n tr u m , I m N e u e n h e i m e r F e l d 2 8 0 ,69120 He ide lberg , Germ anyReceived 24 November 1995; revision 28 May 1996; accepted 25 June 1996

K e y w o r d s : Human African trypanosomes; Anion-exchange centrifugation technique;StandardisationI n a d d i t i o n t o t h e n a t iv e s mea r a n d imp r o v ed h a ema to c r i t c en t r i fu g a t io n t ec h -n iq u es ( W o o , 1 9 6 9 ; W a lk e r, 1 97 2) , t h e min i a tu r e a n io n -ex c h a n g e c en t ri fu g a t io n

t ec h n iq u e ( min i -A E CT ) ( L a n h a m a n d G o d f r ey , 1 97 0) w a s fo u n d s u it a b le t o a sse sst h e e l i m i n a ti o n o f t r y p a n o s o m e s f r o m m o u s e b l o o d b y n e w c a n d i d a t e t r y p a n o c id a lc o mp o u n d s . Fo r mo r e t h a n 2 5 y ea r s t h e min i -A E CT h a s b een t h e mo s t e f f ec t iv ed i a g n o s t i c s ep a r a t i o n me th o d fo r p a th o g en ic Trypanosoma species ( from subgeneraDuttunella, Nannomonas a n d Trypanozoon) t h a t i n f ec t t h e b lo o d o f v a r io u s h o s t s(L um sde n et a l. , 1977, 1979, 1981; D uk es et a l ., 1984; Sachs , 1984; Stangh el l in i an dR ou x, 1984; M ieza n et a l ., 1994). I n fac t , i ts sensi t iv i ty is con siderab ly grea ter ford e t ec t i n g s u b mic r o s c o p ic p a r a s it a emia s t h a n u s in g t h e h a em a to c r i t m e th o d . By o u ro w n ex p e ri en c e t h e m e th o d i s a b l e t o d e t ec t f ew er t h a n 1 0 t r y p a n o s o m es (T . bruceisub-spp . ) in 1 m l b lood . Th e m eth od i s a lso usefu l fo r the p rep a ra t io n of an t igen(as mid i - o r m axi -A EC T) a nd , to a lesser ex ten t , fo r the in i t ia t ion of b loo ds t reamfo r m c u l tu re s o r t h e p e r fo r m a n c e o f t h e d r u g i n c u b a t io n i n fec t iv i ty t e s t ( D I I T ;Zw eyg ar th e t a l., 1989; K am insk y e t a l ., 1990).

* Corresponding author. T el. + 49 6221 424260," Fa x + 49 6221 424258,E-mail." m. schulz@ dkfz-heide lberg, de0001-706X/96/$15.00 Copyright 1996 Elsevier Science B.V. All rights reservedP I I S0001-706X(96) 00018-6


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184 U . Z i l l m a n n e t a l . / A c t a Tr op ic a 6 2 ( 1 9 9 6 ) 1 8 3 - 1 8 7T h e c h emic a l c o n d i t i o n s o f t h e m in i -A E C T a r e ea s il y a d ju s ted , b u t t h e t ec h n ic a l

p e r fo r m a n c e is l ef t t o t h e ex p e r imen te r 's sk ill. W h en p e r fo r m in g th e m e th o d , e s pec i-a l ly dur ing the la s t 3 o r 4 w ork in g s teps ( see be low) , the r i sk o f i r r egu la r ly sp read ingt rypa nos om es is cons iderab le . A f ie ld k i t fo r the an ion-exch ange cen t r i fuga t iont ec h n iq u e ( L u m s d en e t a l ., 1 98 0) h a s s o lv ed so m e o f t h e p r o b lems w i th r e sp ec t toth e s u p p ly o f Pa s t eu r p ip et te s a n d m o s t o f th e c h em ic a l s n eed ed . H o w ev er , p la c in go f Pa s t eu r p ip e t te s i n u n s t a b l e r a c k s r em a in ed a p r o b lem a n d th e i r s ta b l e i n s er t io nin to c losed and f i t t ing cen t r i fuge adap ter s was d i f f icu l t , because the leakage ra ted u r in g c en t r i fu g a t io n w a s r ep o r t ed t o b e a s h ig h a s 7 % ( L u m s d en e t a l ., 1 98 1). T h a tv a lu e w a s i n t h e r a n g e o f o u r o w n p r a c t i c a l ex pe r ien c e . D ep en d in g o n t h e o r ig in o fPa s teur p ipe t tes ( supp l ier ) , qua l i ty ( sea l ing) an d the ro u t ine o f the tech n ic ianinvolved , f rus t r a t ing losses were be twe en 5 an d 10%. W e therefore des igned a m ores t a n d a r d i s ed p r o c ed u r e i n v o lv in g t h e s a fe r eq u ip men t n ec es s a ry fo r, e .g ., m a n ip u l a t -ing d rug-res i s tan t s tocks and c lones dur ing fu r ther in v ivo therapy s tud ies .W e f i rs t bu i l t a r ack (Fig . 1 , uppe r pan el ) cons is t ing of a lower and u pper p la t fo rmof P lexiglass ( c a p a c i ty 4 -6 c o lu mn s ) . T h e p l a t fo r ms w er e v e r ti c a l ly c o n n ec t ed b ytwo s ta inless s teel rods . Two t ight ly f i t t ing Teflon r in g s w er e i n t eg r a ted o n b o ths ides o f the upp er p la t fo rm . M ova ble m eta l r ings o f fered the poss ib i l ity to ad ju s tt h e h e ig h t o f t h e u p p e r p l a t fo r m ( to l e r a n c e o f s y rin g e s h a f t a n d c en t r ifu g e t u b e ). I nl a t e r st u d ie s w e s e t u p a n ew w o r k in g p r o to c o l fo r p e r fo r m in g th e min i -A E C T . A na d d i t i o n a l a im w a s t o u s e c o m m o n a r t i c le s ro u t in e ly av a i la b l e in l a b o r a to ri e s . M a n yof them, excep t the Pas teur p ipe t tes , were in tended to be reusab le a f te r ca refu ld is in fec t ion a nd r ins ing in d is t il l ed wa ter .

F ig . 1 ( l o w er p a n e l ) a n d F ig . 2 d es c rib e t h e w h o le eq u ip m en t a n d n ew p r a c t i c a lp e r fo r ma n c e o f t h e min i -A E CT in d e t a i l . D u r in g p a c k in g o f t h e c e l l u lo s e c o lu mn(work in g phase I ) the 50 m l cen t r i fuge tubes (Fa lcon : 2070) a re used wi th ou t l ids.T h e i r c en t r a l h o l e o f 6 m m d ia m e te r is a s e s s en t ia l a s t h e s h o r t en ed b lu e t ip s o fE p p e n d o r f~ p ip et t es fo r t h e a d a p ta t i o n a n d c en t r a l iz a t i o n o f Pa s t eu r p ip e tt e s d u r in gw o r k in g p h a s es I I -V . T h e c en t r a l h o le w a s p r ep a r ed w i th a s e lf -ma d e mec h a n ic a lper fora to r . Al te rna t ive ly , th i s a l so can be done wi th sha rp sc is sor s . Af te r use andd ec o n ta min a t io n t h e p e r fo r a t ed l i d s ma y b e r eu s ed s ev e r a l t imes w i th o u t r ed u c edp e r fo r ma n c e . T o en s u r e fu r th e r s a fe ty i n t h e mo d i f i ed r o u t in e p r o c ed u r e , o n e h a sstill t o c o n c en t r a t e o n w o r k in g p h a s es V a n d V I ( c en t ri fu g a tio n , m ic r o s c o p ic a n a ly -sis). O u r m e th o d p a y s p a r t i c u l a r a t t en t i o n t o t h e f a c t t h a t t h e h ea t - sea l ed Pa s t eu rp ipe t tes have a tendency to b reak a t any t ime dur ing use ; therefore , i f a p ipe t te i sn o t p r o p e r ly s ea led o r r u p tu r e s ( p h a s es I I I -V ) , t h e s u s p en sio n o f t ry p a n o s o m es willn o w b e c o l l ec t ed i n t h e s u r r o u n d in g c en t r i fu g e t u b e . T h i s c a n ea s i ly b e s een a n d th einfec t ive t rypanosomes can be inac t iva ted and d is in fec ted in s i tu . Consequent ly ,t h e c en t r i fu g ed in t a c t Pa s t eu r p ip e t te s m u s t b e m a n ip u la t ed w i th g r ea t c a r e d u r in gth e s u b s eq u en t mic r o s c o p ic i n s p ec t i o n ( p h a s e V I ) . T h i s c a n b e a c c o mp l i s h ed b ys e lec ti n g a s u p p o r t s a fe r t h a n L u m s d en ' s v i ew in g c h a m b er ( L u m s d en e t a l . , 1 9 8 1).As seen in Fig . 2 , r ight panel) , a s l ide plus two glass capi l la r ies f ixed with glue canbe used . Addi t iona l ly , the oppos i te e nd o f the Pa s teur p ipe t te shou ld be c losed wi tha p l a s t i c t i p ( E p p en d o r f~ s y st em: 0 .2 -0 . 6 m l ) , t o p r ev en t d r ip p in g o f b u f f e r o r


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U. Zi l lmann e t a l . /Acta Tropica 62 (1996 ) 183-18 7 185

re/movable ~ J~ top of rack Ilulose

metal rings I syringe ube ~, ,~ JfJlter,2 md a~hte~ ,'="""g"t W l.o . mtoleranceelt o , s a n d / s p o n g e ~ d ia me te r ~syringes ) lOmm15C50ml c, PasteurI eentri- ~ ~ pipette

Falcon ~- t I m /

OEAE 52] cellulOSesuspensionblOOd p~USB heparinmax. 0.2mEcapacity

] P.S.G,-buffarpH 80,dilul, acco rd, tryps.10) species and b(ood origin

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1o be ee nlnfugedat2000 g toe 20 rain. ~ --( p h a s e V )

blue i pcentra-lisationand pro-teCl~Oe

F i g . 1 . P i ct u r e ( u p p e r p a n e l ) a n d s c h e m e ( l o w e r p a n e l ) o f a s p e c ia l r a c k d e v e l o p e d f o r s a f e a n d s t a n d a r d i -s e d p e r f o r m a n c e o f t h e m i n i - A E C T . T h e r o m a n n u m b e r s d e s i g n a t e t h e r e s p e c ti v e w o r k i n g p h a s e s ( I =p a c k i n g o f ce l lu l o s e ; I I = a p p l i c a t io n o f b l o o d ; I II = e l u t io n o f p l a s m a a n d t r y p a n o s o m e s ; IV = r e m o v a lo f t h e sy r i n g e a f te r c o m p l e t e e lu t i o n ; V = s e d i m e n t a t i o n o f tr y p a n o s o m e s b y c e n t r i fu g a t i o n )


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186 U . Z i l l m a n n e t a L / A c t a Tr op ic a 6 2 ( 1 9 9 6 ) 1 8 3 - 1 8 7

Fig. 2. Accessories of the stand ardised min i-AE CT . L eft panel: device for centrally perforating tub e lids.Right panel : suppor t of Pasteur p ipet tes aiding safer microscopic inspection.f lo t a t ion o f pe l l e ted t rypanos om es . T hese t ips m ay a l so be reused a f t e rd ec o n t a m i n a t i o n .

Our modi f i ca t ion f ixes the sy r inge sha f t s and comple te ly covers and p ro tec t sPa s t eu r p i p e t te s d u r i n g a p p l i c a t io n o f in f ec ted b l o o d t o t h e c o l u m n ( I I ) a n d t h es ep a r a t i o n p r o c es s o f t r y p a n o s o m es f r o m D E A E 5 2 c e ll u lo s e ( I I I ) . Du r i n g t h ecen t r i fuga t ion phase l eakage (o r comple te des t ruc t ion) i s now reduced to l ess than1%. The advan tages o f t he a l t e red p rac t i ca l cond i t ions dur ing phase V I canno t a sye t be f ina lly a ssessed bu t a re es t im a ted to reduce losses o f p ipe t t es a l so .

AcknowledgementsDr . S .M. K o n s t a n t i n o v i s a g u es t sc ien ti st fr o m t h e M ed i c a l Un i v e r s it y o f Sof ia ,

De p a r t m en t o f Ph a r m a c o l o g y a n d T o x i c o lo g y , So fi a, B u l ga r i a a n d w a s k i n d ly s u p -p o r t ed b y t h e D .A .A .D . ( Deu t s c h e r A k a d em i s c h e r A u s t a u s c h d i en s t ) . W e a r e a l s ogra tefu l t o Mrs . M. Schu lz fo r typ ing the d ra f t s a s wel l a s t o Mrs . C . Kippenhahnand to M r . J . H ol l a t z fo r m aking the f igures . Fo r the fina l c r i ti ca l r ead ing o r rev i siono f t h e m a n u s c r i p t w e a r e i n d eb t ed t o Dr . R . O w en , P r o f . M. L i tt le ( Deu t s c h esKr eb s f o r s c h u n g s z en t r u m , He i d e l b e r g ) a n d e s p eci al ly t o Dr . R . B r u n(Schweizer i sches Tropen ins t iu t , Base l ) .

ReferencesDukes, P . , Rickman, L .R. , Kil l ick-Kendr ick, R. , Kak om a, J . , Wu rapa, F .K. , de Raadt , P . and M orrow,


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U. Zillmann et al./Acta Tropica 62 (1996) 183-187 187R. (1984) A field comparison of seven diagnostic techniques for human trypanosomiasis in the LuangwaValley, Zambia. Tropenmed. Parasitol. 35, 141-147.Kaminsky, R., Gumm, I.D., Zweygarth, E. and Chnma, F. (1990) A drug incubation infectivity test(DIIT) for assessing resistance in trypanosomes. Vet. Parasitol. 34, 335-343.Lanham, S.M. and Godfrey, D.G. (1970) Isolation of Salivarian trypanosomes from man and otheranimals using DEAE-cellulose. Exp. Parasitol. 28, 521-534.Lumsden, W.H.R., Kimber, C.D. and Strange, M. (1977) Trypanosoma brucei:detection of low parasitae-mias in mice by a miniature anion-exchanger / centrifugation technique. Trans. R. Soc. Trop. Med.Hyg. 71,421-424.Lumsden, W.H.R., Kimber, C.D., Evans, D.A. and Doig, S.J. (1979) Trypanosoma brucei: miniatureanion-exchange centrifugation technique for detection of low parasitaemias: adaptation to field use.Trans. R. Soc. Trop. Med. Hyg. 73, 312-317.Lumsden, W.H.R., Kimber, C.D., Dukes, P. and Doig, S.J. (1980) A field kit for the diagnosis of sleepingsickness by the anion-exchange centrifugation (AEC) technique. Trans. R. Soc. Trop. Med. Hyg.74, 116.Lumsden, W.H.R., Kimber, C.D., Dukes, P., Hailer, L., Stanghellini, A. and Duvallet, G. (1981) Fielddiagnosis of sleeping sickness in the Ivory Coast. I. Comparison of the miniature anion-exchange /centrifugation technique with other protozoological methods. Trans. R. Soc. Trop. Med. Hyg. 75,242-250.Miezan, T.W., Meda, A.H., Doua, F. and Cattand, P. (1994) Evaluation des techniques parasitologiquesutilis6es dans le diagnostic de la trypanosomose humaine ~ Trypanosoma gambienseen C6te d'Ivoire.Bull. Soc. Pathol. Exot. 87/2, 101-104.Sachs, R, (1984) Improvements in the miniature anion-exchange centrifugation technique for detectingtrypanosomes in domestic pigs (Letter). Trans. R. Soc. Trop. Med. Hyg. 78, 561.Stanghellini, A. and Roux, J.F. (1984) Techniques de d6pistage et de diagnostic de la trypanosomehumaine africaine. M6d. Trop. 44, 361-367.Walker, P.J. (1972) Capillary concentration technique applicable to infections of T. congolensein cattle.Trans. R. Soc. Trop. Med. Hyg. 66, 348.

Woo, P.T.K. (1969) The haematocrit centrifuge for the detection of trypanosomes in blood. Can. J. Zool.47, 921-923.Zweygarth, E., Kaminsky, R. and Cheruiyot, J.K. (1989) A simple and rapid method to initiateTrypanosorna brucei bruceiand T. brucei evansi bloodstream form cultures. Acta Trop. 46, 205-206.

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