P R E A N A L Y T I C A L C O N S I D E R A T I O N S
I. PRINCIPLE Membrane filtration methods for recover-ing microfilariae from patients with light infections have been developed. These methods have an advantage over simple centrifugation methods in that large sam-ples of blood (20 ml or more) can be used if necessary. The technique described here is one of the most efficient for the clinical laboratory when other procedures used to
recover microfilariae are unsatisfactory (1–3).
Membrane filtration recovers most spe-cies of microfilariae; however, because of their smallness, Mansonella perstans and Mansonella ozzardi may not be recovered.
Membranes of smaller pore size (3 lm) have been suggested for recovering these two species.
II. SPECIMEN Whole blood collected by using EDTA, heparin, or sodium citrate anticoagulant
III. MATERIALS A. Reagents
䊓 Indicate the expiration date on the label and in the work record or on the manufac-turer’s label.
1. Distilled water
2. Methyl alcohol, absolute
3. Giemsa or hematoxylin stain (see procedures 9.8.5 and 9.8.8) 4. Toluene
B. Supplies
1. Glass syringe, 15 ml (clear plastic is acceptable)
2. Nuclepore membrane filter, 25 mm, 5-lm porosity
3. Swinney filter adapter (attaches to syringe, holds filter)
4. Filter paper pad, 25 mm (used to support the membrane filter) 5. Glass microscope slides, 1 by 3 in.
6. Coverslips, 22 by 22 mm or larger, no. 1 thickness
7. Mounting medium C. Equipment
Microscope, binocular with mechani-cal stage; low-power (10⳯), high dry power (40⳯), and oil immersion (100⳯) objectives; 10⳯ oculars; cal-ibrated ocular micrometer; light source equivalent to 20-W halogen or 100-W tungsten bulb; blue and white ground-glass diffuser filters
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. If possible, check the procedure by using human or canine blood containing microfilariae.
B. If positive blood is not available, follow the procedure carefully in testing the specimen submitted for diagnosis. Examine sediment thoroughly with low- and high-power magnification.
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 A. Wear gloves when performing this procedure.
B. Draw 1 ml of fresh whole blood or anticoagulated blood into a 15-ml syringe containing 10 ml of distilled water.
C. Gently shake the mixture for 2 to 3 min to ensure that all blood cells are lysed.
D. Place a 25-mm Nuclepore filter (5-lm porosity) over a moist 25-mm filter paper pad, and place in a Swinney filter adapter (see illustration in Appendix 9.6.9–
2 of procedure 9.6.9).
E. Attach the Swinney filter adapter to the syringe containing the lysed blood.
F. With gentle but steady pressure on the piston, push the lysed blood through the filter.
G. Without disturbing the filter, remove the Swinney adapter from the syringe, and draw approximately 10 ml of distilled water into the syringe. Replace the adap-ter, and gently push the water through the filter to wash the debris from the filter.
H. Remove the adapter again, draw the piston of the syringe to about half the length of the barrel, replace the adapter, and push the air in the barrel through the filter to expel excess water.
I. To prepare the filter for staining, remove the adapter, draw the piston about half the length of the barrel, and then draw 3 ml of absolute methanol into the syringe. Holding the syringe vertically, replace the adapter, and push the meth-anol followed by the air through the filter to fix the microfilariae and expel the excess methanol, respectively.
J. To stain, remove the filter from the adapter, place it on a slide, and allow it to air dry thoroughly. Stain with Giemsa stain as for a thick film (using 0.1%
Triton X-100) (see procedure 9.8.5) or with Delafield’s hematoxylin (see pro-cedure 9.8.8).
K. To cover the stained filter, dip the slide in toluene before mounting the filter with neutral mounting medium and a coverslip. This will lessen the formation of bubbles in or under the filter.
VI. RESULTS A. If present in the sample, microfilariae are concentrated and will appear on the wet membrane.
B. After being stained with Giemsa or Delafield’s hematoxylin, the microfilariae will stain characteristically. The sheath, if present, may or may not stain with Giemsa.
VII. REPORTING RESULTS A. Report the presence of organisms from the wet Nuclepore membrane.
Example: Microfilariae present.
B. Report the genus and species of organisms from the Giemsa- or hematoxylin-stained membrane.
Example: Wuchereria bancrofti microfilariae present.
IV. QUALITY CONTROL (continued)
Membrane Filtration Concentration 9.8.10.3
VIII. PROCEDURE NOTES A. Gently shake the water-blood mixture to ensure total lysis of blood cells. Some parasitologists prefer to use an aqueous solution of 10% Teepol (Shell Oil Co.) to lyse the blood cells.
B. Motile microfilariae may be seen on the membrane filter after washing with water (step V.G); however, low light will be necessary and the filter must be reassembled before fixing with methanol.
C. The membrane filter must be supported by the moistened filter pad to prevent rupture when the water is expelled through the membrane.
D. If you need to add anticoagulant to blood, mix 9 ml of blood and 1 ml of 5%
sodium citrate in a clear plastic centrifuge tube. Then proceed with centrifu-gation.
IX. LIMITATIONS OF THE
PROCEDURE A. Giemsa or hematoxylin staining may be necessary to identify the organisms to the species level.
B. Identification of microfilariae on filters to the species level may be difficult.
REFERENCES 1. Ash, L. R., and T. C. Orihei. 1987. Parasites:
a Guide to Laboratory Procedures and Iden-tification. American Society of Clinical Pa-thologists, Chicago, Ill.
2. Dennis, D. T., and B. H. Kean. 1971. Isola-tion of microfilariae: report of a new method.
J. Parasitol. 57:1146–1147.
3. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed. ASM Press, Washington, D.C.
SUPPLEMENTAL READING Beaver, P. C., R. C. Jung, and E. W. Cupp.
1984. Clinical Parasitology, 9th ed. Lea and Fe-biger, Philadelphia, Pa.
Smith, J. W., and M. S. Bartlett. 1991. Diag-nostic parasitology: introduction and methods, p.
701–716. In A. Balows, W. J. Hausler, Jr., K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed.
American Society for Microbiology, Washington, D.C.
9.8.11.1