A Gel Column Based Antiglobulin Test to Detect Erythrocytic Auto- and Alloantibodies in Cats
The antiglobulin (Coombs') test detects auto- and alloantibodies directed against erythrocytes and has been used to diagnose immune-mediated hemolytic anemia (IMHA) and to identify blood type incompatibilities. Many techniques including tube and microplate agglutination, as well as flow cytometry and gel-column tests, using varied species-specific reagents, have been developed.
We report here on the use of a novel, commercially available, simple, feline antiglobulin containing gel column assay (DiaMed, Switzerland). Briefly, for the direct antiglobulin test (DAT) patient's ~2% red cell suspension was placed on top of one gel column with pre-loaded antiglobulin reagent and one without (saline control), and then the columns were centrifuged. In the indirect antiglobulin test (IAT) patient's serum or plasma was incubated with a red cell suspension on top of gel columns at 37°C prior to centrifugation. With increasing red cell retention in the gel column the results were graded from 0 to 4+.
Of 64 anemic Coombs' tested cats, 13 were DAT positive (>1+; 12 >2+). Eight of these 13 DAT positive cats were clinically diagnosed with either primary or secondary IMHA. The other 5 DAT positive cats did not show clinical signs of hemolysis and were diagnosed with various underlying illnesses. Among the 51 DAT negative cats 8 were suspected to have a hemolytic anemia: 4 Abyssinians and 2 domestic shorthair cats had increased osmotic fragility of erythrocytes, 1 Somali had a pyruvate kinase deficiency and in 1 case the cause of hemolysis remained unknown. When screening samples from 62 healthy blood donors, all cats were found to be DAT negative.
Antiglobulin enhanced gel column crossmatches were performed on select samples from 42 anemic cats to 264 donor blood units. In addition, samples from 8 healthy non-anemic donor cats with suspected compatibility issues were screened against 65 additional units. The major crossmatch (patient plasma and donor red cells) test revealed 90 incompatibilities, including 2 A-B mismatches and 10 reactions involving the Mik-antigen. The minor crossmatch (donor plasma and patient/other donor red cells) test revealed 53 incompatibilities, of which 6 were related to the presence of Mik-alloantibodies. Among the 143 incompatibilities observed overall, 81 were appreciated only in antiglobulin-containing gel columns and not saline gel columns.
In addition, we used the gel column technique with anti-Mik serum for the Mik typing of 132 cats, of which 4 samples (3%) were found to be Mik negative. All Mik reactions were stronger in the antiglobulin-containing columns than in the saline columns.
In conclusion, the feline antiglobulin gel column test seems simple and standardized to use and helpful in identifying IMHA. Moreover, the use of the antiglobulin column in the major and minor crossmatch test facilitates the identification of transfusion incompatibilities (e.g., A, B, Mik and other antigens and their alloantibodies), although the clinical relevance of some of these incompatibilities is not yet determined.