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Modified Knott’s Technique and Direct Blood Smear

The modified Knott’s method is used for the concentration and identification of microfilariae, specifically the heartworm Dirofilaria immitis. It must be differentiated from the non-pathogenic microfilaria of Dipetalonema reconditum (“Dipet” for short).

A direct blood smear can be done at the same time. It is a quick, non-concentration test for microfilaria. Dirofilaria and Dipet are differentiated by their motility pattern.

These tests should always be done whenever a patient tests heartworm antigen positive, or there is any reason to suspect the dog currently has or has had heartworm disease in the past. The presence of Dirofilaria will impact the course and/or choice of treatment.

The Knott’s test can be done without doing a direct smear, but never do a direct smear without doing a Knott’s test. The Knott’s is a more sensitive test because it concentrates the microfilaria so they are less likely to be missed during microscopic examination.

Procedure for direct smear:

Simple. Place a drop of blood, preferably from an EDTA tube or heparinized syringe, in the center of a slide. Drop a cover slip over top and examine under the microscope on 10x power. Dirofilaria will have a stationary, writhing movement; Dipetalonema will exhibit a more rapid, directional movement and will often shoot right off of the field.

Procedure for the modified Knott’s technique:


Figure 1: Using a 15ml centrifuge tube, add approximately 10ml of 2% formalin (this can be purchased commercially) to 1 ml of anticoagulated blood. Place your thumb over the top and invert several times to thoroughly mix. Centrifuge for 5 minutes at 1000 to 1500 rpms.


Figure 2: You should be able to visualize a whitish plug at the bottom of the tube after spinning. Discard the supernatant.


Figure 3: Using a long glass or plastic pipette, add a drop of new methylene blue stain


Figure 4: and use the pipette to mix the stain with the sediment

Add a drop of this mix to the center of a slide. Drop a cover slip on top. Examine on the microscope under 10x power.

Unlike with the direct smear, the microfilaria will present as stationary. Dirofilaria must be distinguished from Dipetalonema based on size and shape. Use the following table to help you differentiate:

Parameter

D. immitus

D. reconditum

Length (microns)

286-340

258-292

Width  (microns)

5-7.5

4.5-5.5

Shape (anterior end)

Tapered

blunt

Shape (body)

Straight

curved

Shape (tail)

Straight

Button hook

Number present

Few to many

few

Movement

Stationary

Moves across slide

(Cornell University)

Another cool test for microfilaria

Fill a microhematocrit tube and spin down as if doing a pcv.

Lay the tube on a slide and put it on the microscope stage. Focus in on the buffy coat on 10x power – if a dog is heartworm positive and has circulating microfilaria, you can see the microfilaria squirming around in the buffy coat!