Concentration procedures increase the number of organisms recovered from blood specimens submitted for diagnosis of trypanosomiasis, filariasis, and leish-maniasis. A concentration procedure should be performed routinely on all blood specimens submitted for examination for
trypanosomes or microfilariae when the suspected organisms are not found in thick blood films or when organisms are so few that more are needed to make a positive identification of species (see Appendix 9.8.9–1) (1, 2).
II. SPECIMEN Whole blood collected by using EDTA, heparin, or sodium citrate anticoagulant (regular venipuncture collection tubes are recommended—primarily the lavender/
EDTA or green/heparin stoppers)
III. MATERIALS A. Reagents
䊓 Indicate the expiration date on the label and in the work record or on the manufacturer’s label.
1. Methyl alcohol, absolute
2. Giemsa or hematoxylin stain (see procedures 9.8.5 and 9.8.8) B. Supplies
1. 2 centrifuge tubes, glass, 12 ml (plastic tubes can be used, provided they are clear plastic)
2. Capillary pipette with bulb 3. Glass microscope slides, 1 by 3 in.,
alcohol washed
4. Coverslips, 22 by 22 mm or larger, no. 1 thickness
5. Microhematocrit tube(s) (for alter-nate procedure)
6. Blood collection supplies, if appli-cable
C. Equipment
1. Centrifuge with sealable carrier cups, speed calibrated
2. Microscope, binocular with me-chanical stage; low-power (10⳯), high dry power (40⳯), and oil im-mersion (100⳯) objectives; 10⳯
oculars; calibrated ocular microm-eter; light source equivalent to 20-W halogen or 100-20-W tungsten bulb;
blue and white ground-glass dif-fuser filters
I. PRINCIPLE Leishmania donovani amastigotes are dif-ficult to detect in blood specimens but may occasionally be found within monocytes by fractional centrifugation of compara-tively large amounts of blood. The pro-cedure may also be used to recover try-panosomes and microfilariae, both of which are found in the plasma.
Buffy Coat Concentration 9.8.9.2
A N A L Y T I C A L C O N S I D E R A T I O N S
A. Check calibration of centrifuge.
B. Perform the procedure on “normal” blood. The film should be composed almost exclusively of WBCs, which stain characteristically with Giemsa (see procedure 9.8.5). If parasites are present, they also should stain characteristically (see pro-cedure 9.8.5).
C. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the micro-scope. Post the calibration factors for all objectives on the microscope for easy access (multiplication factors can be pasted right on the body of the microscope) (see procedures 9.1 and 9.3.2). Although there is not universal agreement, the microscope should probably be recalibrated once each year. This recommen-dation should be considered with heavy use or if the microscope has been bumped or moved multiple times. If the microscope does not receive heavy use, then recalibration is not required on a yearly basis.
D. Record all QC results.
V. PROCEDURE
(adapted from reference 3) A. Wear gloves when performing this procedure.
B. Centrifuge the anticoagulated blood specimen in a sealed cup at 100⳯ g for 15 min.
C. Remove the thin creamy layer (buffy coat) between the RBCs and plasma with a capillary pipette, or transfer the creamy layer (buffy coat) and plasma to another tube, and centrifuge in a sealed cup at 300⳯ g for 15 min.
D. Examine buffy coat directly for motile trypomastigotes and microfilariae.
1. Place one-half drop of saline on a clean microscope slide.
2. Remove a drop of sediment, and mix it in the saline.
3. Add a coverslip, and examine for organism motility with the low-power (10⳯) and high dry power (40⳯) objectives.
E. Prepare thin films, dry, fix, and stain with Giemsa stain (see procedure 9.8.5 or, if for microfilariae, procedure 9.8.8).
VI. RESULTS A. If present, L. donovani amastigotes will be found within the monocytes on a Giemsa-stained film. Nuclear material stains dark purple-red, the cytoplasm is light blue, and the kinetoplast may or may not be visible as a dark bluish purple structure.
B. Trypomastigotes will be found extracellularly (motile in the wet smear). Mor-phological detail will be seen in the Giemsa-stained film. The stain reaction is like that of L. donovani; the kinetoplast will be visible.
C. Microfilariae may be found in the wet smear. Morphological detail will be seen in a Giemsa- or hematoxylin-stained film. The stain reaction is typical for each stain (see procedures 9.8.5 and 9.8.8).
P O S T A N A L Y T I C A L C O N S I D E R A T I O N S
A. Report the presence of organisms from the wet smear.
Examples: Trypomastigotes present.
Microfilariae present.
B. Report the genus and species of organisms from the Giemsa-stained film.
Examples: Trypanosoma cruzi trypomastigotes present.
Leishmania donovani amastigotes present.
IV. QUALITY CONTROL
VII. REPORTING RESULTS
VIII. PROCEDURE NOTES A. If you need to add anticoagulant to blood, mix 9 ml of blood and 1 ml of 5%
sodium citrate in a glass centrifuge tube. Then proceed with centrifugation.
B. This procedure can be performed in a microhematocrit tube if the tube is care-fully scored and broken at the buffy coat interface and if the WBCs are prepared and stained as for a thin blood film.
C. Also, the tube can be examined microscopically (high dry magnification) at the buffy coat layer for motile trypomastigotes and microfilariae before the tube is scored and broken.
IX. LIMITATIONS OF THE
PROCEDURE A. When examined as a wet smear, the intracellular leishmaniae are very difficult to see.
B. Although trypomastigote and microfilarial motility may be visible on the wet smear, specific identification may be difficult.
REFERENCES 1. Ash, L. R., and T. C. Orihel. 1987. Parasites:
a Guide to Laboratory Procedures and Iden-tification. American Society of Clinical Pa-thologists, Chicago, Ill.
2. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed. ASM Press, Washington, D.C.
3. Young, C. W., and H. Van Sant. 1923. Leish-mania, donovani in the peripheral blood. J.
Exp. Med. 38:233.
APPENDIX 9.8.9–1 QBC Capillary Blood Tube
Recently, a centrifugation procedure which yields a buffy coat has been used to concentrate and detect Plasmodium spp. (1–3). The technique uses a commercially available capillary tube (QBC Capillary Blood Tube; Becton Dickinson, Franklin Lakes, N.J.) which is coated with acridine orange stain and fitted with a buoyant plastic insert. The stain causes the malaria organisms to fluoresce, and the plastic insert forces the RBCs containing stained parasites, because they differ in buoyancy from uninfected RBCs, to be concentrated just under the buffy coat. After centrifugation, the tube is examined under the microscope at the plastic insert level for the presence of malaria parasites. Some laboratorians have been able to accurately identify species by using this method. At this time, however, this technique re-quires more trials, and appropriate thin and/or thick films are recommended for the positive identification of Plasmodium spp.
References
1. Long, G. W., T. R. Jones, L. S. Rickman, R.
Trimmer, and S. D. Hoffman. 1991. Acri-dine orange detection of Plasmodium falci-parum malaria: relationship between sensitiv-ity and optical configuration. Am. J. Trop.
Med. Hyg. 44:402–405.
2. Rickman, L. S., G. W. Long, R. Oberst, A.
Caranban, R. Sangalang, J. I. Smith, J. D.
Chulay, and S. L. Hoffman. 1989. Rapid di-agnosis of malaria by acridine orange staining of centrifuged parasites. Lancet 1:68–71.
3. Spielman, A., J. P. Perrone, A. Teklehai-manot, F. Balcha, S. C. Wardlaw, and R. A.
Levine. 1988. Malaria diagnosis by direct ob-servation of centrifuged samples of blood. Am.
J. Trop. Med. Hyg. 39:337–342.
9.8.10.1