methylene blue stain to the sample may allow greater visibility of any microfilariae. However, the hematocrit tube method will not allow for species differentiation. Modified Knott's test The modified Knott's test is more sensitive because it concentrates microfilariae, improving the chance of diagnosis. A blood sample is mixed with 2% formalin and centrifuged in a tube. The supernatant is removed and methylene blue stain is added to the pellet remaining in the tube for microscopic examination. It allows microfilariae species differentiation based on morphology. Microfilariae can be differentiated between D. immitis and Acanthocheilonema reconditum because of small differences in morphology. The Modified Knott's test is the best method of visual examination when determining presence of microfilaria because it preserves their morphology and size. It is easy to perform, quick, and inexpensive. The potential for a microfilaremic infection is 5 – 67%. The number of circulating microfilariae does not correlate with the number of adult heartworms, so is not an indicator of disease severity. Antigen testing In most practices, antigen testing has supplanted or supplemented microfilarial detection. Combining the microfilaria and adult antigen test is most useful in dogs receiving diethylcarbamazine or no preventive (macrolides like ivermectin or moxidectin typically render the dog amicrofilaremic). Up to 1% of infected dogs are microfilaria-positive and antigen-negative. Immunodiagnostics (ELISA, lateral flow immunoassay, rapid immunomigration techniques) to detect heartworm antigen in the host's blood are now regularly used. They can detect occult infections, or infections without the presence of circulating microfilariae. However, these tests are limited in that they only detect the antigens released from the sexually mature female worm's reproductive tract. Therefore, false-negative results may occur during the first five to eight months of infection when the worms are not yet sexually mature. The specificity of these tests is close to 100%, and the sensitivity is more than 80%. A recent study demonstrated a sensitivity of only 64% for infections of only one female worm, but improved with increasing female worm burden (85%, 88%, and 89% for two, three, and four female worms, respectively). Specificity in this study was 97%. False-negative test results can be due to low worm counts, immature infections, and all-male infections. X-rays X-rays are used to evaluate the severity of the heartworm infection and develop a prognosis for the animal. Typically, the changes observed are enlargement of the main pulmonary artery, the right side of the heart, and the pulmonary