Blood Parasite Examination

Blood is collected for two basic parasitological procedures:

1. Smears - to detect protozoal and rickettsial infections (e.g., Trypanosoma, Babesia,  Anaplasma). Smears must be fixed and stained to reveal organisms.

2. Concentration - to detect microfilaria (i.e., Dirofilaria and Dipetalonema).

NOTE: If blood is not to be processed immediately upon removal from the patient, an anticoagulant must be added to the sample. Among those commonly used include Heparin (effect lasts only for a matter of hours) and EDTA (effect lasts several days).

1. Blood Smear Procedure (thin films):

1. Clean slide by wiping with alcohol. Handle slides by edges only. (Any grease on the slide will cause the dried blood to flake off during staining).
2. Place a very small drop of blood near the end of a slide.
3. Place the end of another slide (the "spreader") on the sample slide so that the edge of the spreader is just ahead of the drop of blood.
4. Holding the spreader at an angle of about 30 degrees (relative to the sample slide), draw it back until its edge just touches the drop of blood. The blood will then run along the entire edge of the spreader slide.
5. Push the spreader briskly in one fluid motion completely across the sample slide. Note that the blood is being dragged behind the spreader, not pushed in front of it.
6. If the correct amount of blood was applied, the smear should end before the end of the slide, and the smear should end in a "feathered edge" a region where the blood cells are well separated.
7. Air dry slide.
8. Fixation and staining - various methods can be used. Normally a commercial staining kit is utilized following the manufacturer's instructions.

Click here for a video demonstration of the Blood Smear Technique

2. Procedures for concentration of blood:

    A. Modified Knott Method

1. Add 1 ml freshly-drawn blood to 9 ml 2% formalin (aqueous) in a centrifuge tube.
2. Mix well to lyse red blood cells.
3. Centrifuge for 5 minutes at 1500 rpm.
4. Pour off supernatant fluid. Note: Invert the tube completely when decanting the supernatant. Remember, the blood sample you are using is dilute so you won't see a large pellet.
5. Add a drop of 0.1% aqueous methylene blue. (Adjust the amount to suit yourself; it stains the microfilariae blue and makes them much easier to see.) Then stir or mix up the sediment in the bottom of the tube.
6. Mix again and place a drop of the stained mixture on a microscope slide and add a cover slip.
7. Examine slide under a microscope.

NOTE: As a further modification, a microfilaria count can be made if a measured amount of the stained mixture is counted. Although it is only a generality, D. immitis microfilaremias are often characterized by having high concentrations of microfilariae, whereas D. reconditum microfilariae are often found in low concentrations.

Click here for a video demonstration of the Modified Knott Method

    B. Filtration Method

1. Collect a 1 ml blood sample into EDTA or heparin and add to 10 ml lysing solution within a syringe. Mix thoroughly. (Lysing solution consists of 5.0 ml Triton X-100, 8.0 grams NaCO3, 1 liter water.)
2. Attach syringe to a filter unit (see diagram). The lysed blood solution is pushed through an 8 m pore filter membrane.
3. Remove the filter from the filter holder, place it on a microscope slide and add one drop of 1:10,000 Methylene Blue Stain. Cover filter with a cover glass and examine under microscope.

Click here for a video demonstration of the
Filtration Method for Detecting Microfilariae

Miscellaneous

It is frequently difficult to distinguish microfilariae of D. immitis from microfilariae of D. reconditum using the morphologic characteristics outlined above. More definitive techniques for differentiation are available, but they are not usually practical for routine use in the practitioner's laboratory.

The first technique employs a histochemical (acid phosphatase) stain of microfilariae. D. immitis stain positive in certain zones only and D. reconditum stain over the entire microfilariae. See J. Am. Vet. Med. Assoc. 158:601-605, 1971 or consult a parasitologist.

The second technique exploits the fact that D. reconditum microfilariae have a cephalic hook and D. immitis microfilariae do not. Again, since this technique requires good microscopic capability, it may not be suited for routine use. See Proceedings Helminthol. Society of Washington 32(1):15-20, 1965, or Georgi's Parasitology for Veterinarians or consult a parasitologist.