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25 JULY/AUGUST 2017 ■ TVPJOURNAL.COM HEARTWORM HOTLINE Such techniques rely on denaturing proteins within the complex, allowing for precipitation of antibodies and subsequent freeing of the antigen ( Figure 1 ). In laboratory settings, proteolytic enzymes (eg, pepsin), acid treatment (eg, ethylenediaminetetraacetic acid, citric acid), heat (104°C for 10 minutes), or a combination of these methods is frequently used for immune complex dissociation (ICD). Heat pretreatment (HPT) of serum samples was standard practice in veterinary diagnostic laboratories through the mid-1990s and is still available upon request. However, the demand for simple, cost-effective, commercially available, point-of-care test kits led to its decreased use. HOW DOES HEAT PRETREATMENT AFFECT DIAGNOSIS OF HEARTWORM INFECTION? In recent years, the effect of ICD in the form of HPT has been studied in diagnostic samples from cats and dogs. 1–6 Such reports have demonstrated substantial increases in antigen detection in both species, resulting in greater diagnostic sensitivity ( Table 1 ); however, the antigen detected cannot be identified as coming from living or dead heartworms. It follows that in dogs with heartworm disease that have received adulticidal therapy, a positive result on an antigen test with ICD does not indicate that the therapy was unsuccessful. TABLE 1 Effect of Heat Pretreatment on Antigen Detection REFERENCE POPULATION STANDARD HEAT Ciucă et al 2016 1 194 Romanian stray dogs 8.2% 27% DiGangi et al 2016 2 616 shelter dogs in United States 7.3% 12.3% Drake et al 2015 3 15 owned dogs 0%* 53% Gruntmeir et al 2015 4 34 owned dogs 0%* 67.4% Little et al 2014 5 6 experimentally infected cats 17% 83% Little et al 2014 6 220 shelter cats in United States 0.45% 5.9% *Samples tested negative before study inclusion. FIGURE 1. Immune complex dissociation with sample pretreatment. Figure used with permission from IDEXX Laboratories, Inc. Ag, antigen; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay. Serum Heat plus EDTA Precipitated antibodies Supernatant Immune complex Initial ELISA (no heat) Negative result Sample treatment ELISA after heat treatment Positive result Antibody Antibody © 2017 IDEXX Laboratories, Inc. All rights reserved. • 110441-00 Heartworm antigen Ag Ag