Effect of dilution of canine blood samples on the specificity of saline agglutination tests for immune-mediated hemolysis.

Abstract

BackgroundSaline agglutination tests (SATs) are widely recommended for diagnosis of immune‐mediated hemolytic anemia in dogs, but there are frequent false‐positive results.ObjectivesSpecificity of SATs will improve at higher saline‐to‐blood ratios.AnimalsOne hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%.MethodsProspective diagnostic accuracy study. Immune‐mediated hemolysis (IMH) was considered present if a gel direct antiglobulin test (DAT) was positive and there was clinical evidence of hemolysis (n = 9), absent if another mechanism for anemia was identified and the DAT was negative or there was no hemolysis (n = 138), and if IMH status was unclear, dogs were excluded (n = 3). Saline agglutination tests were prepared at 1 : 1, 4 : 1, 9 : 1, and 49 : 1 saline‐to‐blood ratios, and microscopic agglutination was considered a positive result.ResultsSpecificity for IMH increased from 29% (95% confidence interval 20‐38) at a 1 : 1 dilution to 97% (93‐99) at a 49 : 1 dilution. Sensitivity was 88% (47‐100) at 1 : 1 and 4 : 1 dilutions and 67% (30‐93%) at 9 : 1 and 49 : 1 dilutions. Diagnostic accuracy increased from 33% (24–42) at 1 : 1 dilution to 95% (90‐98) at 49 : 1 dilution.Conclusions and Clinical ImportanceIf performed using a 49 : 1 saline‐to‐blood ratio, SATs achieve high specificity for IMH. Based on a gold standard of positive DAT and evidence of hemolysis, lower saline‐to‐blood ratio results should not be used because false‐positive results are common.