Comparative study of immunohematological tests with canine blood samples submitted for a direct antiglobulin (Coombs’) test

Abstract

Background A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. Material and methods Anticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics; neutral gel column cards, microtiter plates (at 4°, 22°, and 37°C), capillary tubes, and flow cytometry used in laboratories. Results Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1–24 weeks after initial assessment. Conclusions Based upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable.

The Indirect Antiglobulin (Coombs’) Test Is Specific but Less Sensitive Than the Direct Antiglobulin Test for Detecting Anti-Erythrocytic Autoantibodies and Thereby Immune-Mediated Hemolytic Anemia in Dogs

“…None of the EDTA blood samples were autoagglutinating after three saline washes based upon macro- and microscopic examinations, and thus a DAT could be performed and interpreted [ 10 ]. Leftover serum samples were incubated with washed DAT− erythrocytes, and five IAT techniques were performed (in part with different reagents and temperatures): microtiter plate (MICRO), microcapillary (CAPIL), laboratory gel column (GEL LAB), gel minitube kit (GEL KIT), and immunochromatographic strip kit (STRIP KIT) described in detail below.…”

“…To prepare DAT− erythrocytes from control dogs, EDTA-anticoagulated blood samples, collected <1 day prior to analysis and kept refrigerated after collection, were washed three times with physiological saline, as previously described [ 10 , 13 ]. The DAT was performed using previously published techniques [ 10 ]. All control RBC samples tested were found to be DAT−.…”

“…Hyperbilirubinuria (hyperbilirubinemia), reticulocytosis (polychromasia), macrocytosis, and anisocytosis are typically seen with all hemolytic anemias, not just IMHA [ 1 , 9 ]. There are, however, three diagnostic markers for immune-mediated erythrocytic destruction accompanying IMHA: Persistent autoagglutination of erythrocytes after three saline washes [ 2 , 10 ]. Marked spherocytosis (≥5/high power microscopic field at × 1000 magnification) in >60% of dogs with IMHA [ 11 ], which otherwise only occurs in rare hereditary spherocytosis [ 12 ].…”

“… Marked spherocytosis (≥5/high power microscopic field at × 1000 magnification) in >60% of dogs with IMHA [ 11 ], which otherwise only occurs in rare hereditary spherocytosis [ 12 ]. A positive direct antiglobulin (Coombs’) test (DAT) [ 10 , 13 ]. …”

“…The DAT recognizes the presence of auto- (and allo-) antibodies (and complement) on the surface of erythrocytes, while the indirect antiglobulin test (IAT) is used to detect free anti-erythrocytic antibodies in serum or plasma [ 16 , 17 ]. Our recent studies showed the strong clinical value of various in-clinic and laboratory DAT techniques in the diagnosis (and monitoring) of IMHA in dogs [ 10 , 13 ].…”