Knott's Method

The modified Knotts method is used for the concentration and identification of microfilaria. Procedure: 1. Add 1-ml blood to 10 ml of 2% formalin and mix. 2. Centrifuge for 5 minutes at 1000 to 1500 rpm. 3. Pour off supernatant fluid. Note: The tube may be inverted on a paper towel to allow all the liquid to drain. 4. Mix sediment with equal volume of 1:1000 aqueous methylene blue. 5. Examine as wet mount.

Modified Knott's Heartworm Technique Information

Test Code: KNOTT Test Name: Knotts Heartworm Test

Test Method: Microscopic identification Sample Required: 1 ml of whole blood in anticoagulant, such as EDTA or heparin Collection container: blood tube Transport: Ship on cold packs Test Day: M-F Lag Time: Results same day Species: All species that may have microfilariae in their blood Results Format: Identification of microfilariae, or No microfilariae detected Interpretation This technique is used for the detection and identification of blood-borne microfilariae. It was developed for the detection of Dirofilaria immitis microfilariae in canine blood. In this country the microfilariae of Dirofilaria and those of Dipetalonema reconditum must be differentiated. Dirofilaria immitis microfilariae can persist in a positive animal that has been treated for up to six months after treatment.

The Knotts Technique is not recommended as a stand alone diagnostic test for Dirofilaria immitis because infections may consist of male worms that do not produce microfilariae, or immature female worms that are not yet producing microfilariae. For a more complete evaluation of the Dirofilaria status in a dog or cat, it is recommended that an occult serological test and a microfilarial test be done at the same time. If both techniques are done the results may be interpreted as follows: Antigen Positive and Microfilaria Negative single sex infection (female) immature adult worms (5-6 months postinfection) immune mediated clearance of microfilariae animal on monthly preventives, or after microfilaricide treatment Antigen Negative and Microfilaria Positive microfilariae are not Dirofilaria immitis heartworm antigen not present, or present in levels too low to detect adult worms dead and antigen cleared, but microfilariae still present microfilarial contamination of lysing solution, dye or filter chamber animal transfused with microfilaremic blood prenatal transfer of microfilariae immune mediated clearance of antigen-antibody complexes antigen destroyed due to improper storage or treatment of sample If both a microfilarial test and an occult serological test produce inconclusive results, then other diagnostic tests may be conducted, such as thoracic radiographs, electrocardiogram, blood chemistry, and urinalysis. If the animal is clinically normal, follow up testing in six months with an occult serological test is recommended if the initial testing is inconclusive. In a positive animal that has been treated, heartworm antigen can persist for up to four months. Repeat testing must be done after that time to confirm that treatment has been effective.