ACTA TROPICA

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Acta Tropica 62 (1996) 183-187

Short communication

Improved performance of the anion-exchange centrifugation technique for studies with human infective African trypanosomes
U. Zillmann a.,, S.M. Konstantinov b, M.R. Berger b, R. Braun c
a Zentrales Tierlabor, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b .4G Toxikologie und Chemotherapie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Technische Abt., Deutsches Krebsforschungszentrum, Im Neuenheimer Feld280, 69120 Heidelberg, Germany
Received 24 November 1995; revision 28 May 1996; accepted 25 June 1996

Keywords: Human

African

trypanosomes;

Anion-exchange centrifugation technique;

Standardisation In addition to the native smear and improved haematocrit centrifugation techniques (Woo, 1969; Walker, 1972), the miniature anion-exchange centrifugation technique (mini-AECT) (Lanham and Godfrey, 1970) was found suitable to assess the elimination of trypanosomes from mouse blood by new candidate trypanocidal compounds. For more than 25 years the mini-AECT has been the most effective diagnostic separation method for pathogenic Trypanosoma species (from subgenera Duttunella, Nannomonas and Trypanozoon) that infect the blood of various hosts (Lumsden et al., 1977, 1979, 1981; Dukes et al., 1984; Sachs, 1984; Stanghellini and Roux, 1984; Miezan et al., 1994). In fact, its sensitivity is considerably greater for detecting submicroscopic parasitaemias than using the haematocrit method. By our own experience the method is able to detect fewer than 10 trypanosomes (T. brucei sub-spp.) in 1 ml blood. The method is also useful for the preparation of antigen (as midi- or maxi-AECT) and, to a lesser extent, for the initiation of bloodstream form cultures or the performance of the drug incubation infectivity test (DIIT; Zweygarth et al., 1989; Kaminsky et al., 1990).
* Corresponding author. Tel. +49 6221 424260," Fax +49 6221 424258, E-mail." m. schulz@dkfz-heidelberg,de
0001-706X/96/$15.00 Copyright 1996 Elsevier ScienceB.V. All rights reserved PII S0001-706X(96) 00018-6

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U. Zillmann et al./Acta Tropica 62 (1996) 183-187

The chemical conditions of the mini-AECT are easily adjusted, but the technical performance is left to the experimenter's skill. When performing the method, especially during the last 3 or 4 working steps (see below), the risk of irregularly spreading trypanosomes is considerable. A field kit for the anion-exchange centrifugation technique (Lumsden et al., 1980) has solved some of the problems with respect to the supply of Pasteur pipettes and most of the chemicals needed. However, placing of Pasteur pipettes in unstable racks remained a problem and their stable insertion into closed and fitting centrifuge adapters was difficult, because the leakage rate during centrifugation was reported to be as high as 7% (Lumsden et al., 1981). That value was in the range of our own practical experience. Depending on the origin of Pasteur pipettes (supplier), quality (sealing) and the routine of the technician involved, frustrating losses were between 5 and 10%. We therefore designed a more standardised procedure involving the safer equipment necessary for, e.g., manipulating drug-resistant stocks and clones during further in vivo therapy studies. We first built a rack (Fig. 1, upper panel) consisting of a lower and upper platform of Plexiglass (capacity 4-6 columns). The platforms were vertically connected by two stainless steel rods. Two tightly fitting Teflon rings were integrated on both sides of the upper platform. Movable metal rings offered the possibility to adjust the height of the upper platform (tolerance of syringe shaft and centrifuge tube). In later studies we set up a new working protocol for performing the mini-AECT. An additional aim was to use common articles routinely available in laboratories. Many of them, except the Pasteur pipettes, were intended to be reusable after careful disinfection and rinsing in distilled water. Fig. 1 (lower panel) and Fig. 2 describe the whole equipment and new practical performance of the mini-AECT in detail. During packing of the cellulose column (working phase I) the 50 ml centrifuge tubes (Falcon: 2070) are used without lids. Their central hole of 6 mm diameter is as essential as the shortened blue tips of Eppendorf~ pipettes for the adaptation and centralization of Pasteur pipettes during working phases II-V. The central hole was prepared with a self-made mechanical perforator. Alternatively, this also can be done with sharp scissors. After use and decontamination the perforated lids may be reused several times without reduced performance. To ensure further safety in the modified routine procedure, one has still to concentrate on working phases V and VI (centrifugation, microscopic analysis). Our method pays particular attention to the fact that the heat-sealed Pasteur pipettes have a tendency to break at any time during use; therefore, if a pipette is not properly sealed or ruptures (phases III-V), the suspension of trypanosomes will now be collected in the surrounding centrifuge tube. This can easily be seen and the infective trypanosomes can be inactivated and disinfected in situ. Consequently, the centrifuged intact Pasteur pipettes must be manipulated with great care during the subsequent microscopic inspection (phase VI). This can be accomplished by selecting a support safer than Lumsden's viewing chamber (Lumsden et al., 1981). As seen in Fig. 2, right panel), a slide plus two glass capillaries fixed with glue can be used. Additionally, the opposite end of the Pasteur pipette should be closed with a plastic tip (Eppendorf~ system: 0.2-0.6 ml), to prevent dripping of buffer or

U. Zillmann et al./Acta Tropica 62 (1996) 183-187

185

F" III

IV

re/movable top of rack

Ilulose

~J~

- buffer

metal rings ( toleranceel to,sand syringes )

'''="""g"t
/

I syringe tube k~,,~ JfJlter, 2 md a~hte~,

Wl.o.
c, ~

m
Pasteur ~ pipette

nge tube pored

sponge~ 15C50 ml I eentriFalcon~-t

diameter~ lOmm blue tip centralisation and proteCl~Oe

] B ] 10)

OEAE 52 cellulOSe suspension blOOdp~US heparin max. 0.2 mE capacity P.S.G,-buffarpH 80, dilul, accord, tryps. species and b(ood origin

rotator see Piet. 2a)

1o be eenlnfugedat 2000 g toe 20 rain. ~ (phase V)

--

Fig. 1. Picture (upper panel ) and scheme (lower panel) of a special rack developed for safe and standardised performance of the mini-AECT. The roman numbers designate the respective working phases (I = packing of cellulose; II = application of blood; III = elution of plasma and trypanosomes; IV = removal of the syringe after complete elution; V = sedimentation of trypanosomes by centrifugation)

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U. Zillmann et aL/Acta Tropica 62 (1996) 183-187

Fig. 2. Accessories of the standardised mini-AECT. Left panel: device for centrally perforating tube lids. Right panel: support of Pasteur pipettes aiding safer microscopic inspection.

flotation of pelleted trypanosomes. These tips may also be reused after decontamination. Our modification fixes the syringe shafts and completely covers and protects Pasteur pipettes during application of infected blood to the column (II) and the separation process of trypanosomes from DEAE 52 cellulose (III). During the centrifugation phase leakage (or complete destruction) is now reduced to less than 1%. The advantages of the altered practical conditions during phase VI cannot as yet be finally assessed but are estimated to reduce losses of pipettes also.

Acknowledgements
Dr. S.M. Konstantinov is a guest scientist from the Medical University of Sofia, Department of Pharmacology and Toxicology, Sofia, Bulgaria and was kindly supported by the D.A.A.D. (Deutscher Akademischer Austauschdienst). We are also grateful to Mrs. M. Schulz for typing the drafts as well as to Mrs. C. Kippenhahn and to Mr. J. Hollatz for making the figures. For the final critical reading or revision of the manuscript we are indebted to Dr. R. Owen, Prof. M. Little (Deutsches Krebsforschungszentrum, Heidelberg) and especially to Dr. R. Brun (Schweizerisches Tropeninstiut, Basel).

References
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187

R. (1984) A field comparison of seven diagnostic techniques for human trypanosomiasis in the Luangwa Valley, 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 other animals using DEAE-cellulose. Exp. Parasitol. 28, 521-534. Lumsden, W.H.R., Kimber, C.D. and Strange, M. (1977) Trypanosoma brucei: detection of low parasitaemias 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: miniature anion-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 sleeping sickness 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) Field diagnosis 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 parasitologiques utilis6es dans le diagnostic de la trypanosomose humaine ~ Trypanosoma gambiense en C6te d'Ivoire. Bull. Soc. Pathol. Exot. 87/2, 101-104. Sachs, R, (1984) Improvements in the miniature anion-exchange centrifugation technique for detecting trypanosomes 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 trypanosome humaine africaine. M6d. Trop. 44, 361-367. Walker, P.J. (1972) Capillary concentration technique applicable to infections of T. congolense in 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 initiate Trypanosorna brucei brucei and T. brucei evansi bloodstream form cultures. Acta Trop. 46, 205-206.