Effect of Dilution of Canine Blood Samples on the Specificity of Saline Agglutination Tests for Immune-mediated Hemolysis

Background: Saline agglutination tests (SATs) are widely recommended for diagnosis of immune-mediated hemolytic anemia in dogs, but there are frequent false-positive results.

Objectives: Specificity of SATs will improve at higher saline-to-blood ratios.

Animals: One hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%.

Methods: Prospective 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.

Results: Specificity 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 Importance: If 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.