Detection of canine anti-DEA 1 antibodies using flow cytometry in dogs following DEA 1-positive blood transfusion

Abstract

The shortage of blood donor dogs in veterinary emergencies can lead to blood transfusions between animals whose blood type has not been identified. The antibody profile serves as a warning sign for animals that require a second blood transfusion, which is only advisable from compatible donor dogs. This article focuses on the determination of anti-DEA 1 antibodies using the flow cytometry technique in dogs that have undergone a transfusion using DEA 1-positive blood, compared to results obtained from crossmatching. Blood from 18 DEA 1-positive donors ranked according to the chromatographic technique was used to transfuse thirty-three animals with unknown blood types and which demonstrated negative crossmatching to donors. On post-transfusion days 7, 14, 21 and 28, 45% and 27% of the animals tested positive for the anti-DEA 1 antibody, through crossmatching and flow cytometry, respectively. Detecting antibodies using the flow cytometric technique has high specificity and sensitivity, while crossmatching methods are highly sensitive but manifest low specificity. Following the blood transfusion, animals that did not present as positive through crossmatching or flow cytometry were considered different from all other DEA 1-positive blood groups.A escassez de cães doadores de sangue em situações de emergência na Medicina Veterinária pode levar à realização de transfusões de sangue entre animais que não tiveram seu tipo sanguíneo previamente determinado. O padrão de anticorpos serve como um sinal de alerta para animais que serão submetidos a uma segunda transfusão sanguínea, sendo esta recomendável somente se o sangue for proveniente de cães doadores compatíveis. Este artigo aborda a determinação de anticorpos anti-AEC 1 pela técnica de citometria de fluxo em cães que receberam uma transfusão utilizando sangue do grupo AEC 1 positivo, comparando-se os resultados com aqueles obtidos a partir de reação cruzada. Foi utilizado o sangue de 18 animais doadores do tipo AEC 1 positivo classificados por técnica cromatográfica, a fim de transfundir trinta e três animais com tipos sanguíneos desconhecidos e que demonstraram reação cruzada negativa aos doadores. Nos dias 7, 14, 21 e 28 pós-transfusão, 45% e 27% dos animais mostraramse positivos para os anticorpos anti-AEC 1, respectivamente, pela reação cruzada e através de citometria de fluxo. A pesquisa de anticorpos com o emprego da técnica de citometria de fluxo tem alta especificidade e sensibilidade, enquanto a reação cruzada, altamente sensível, tem baixa especificidade. Animais que não apresentaram positividade após a transfusão de sangue na reação cruzada e na citometria de fluxo foram considerados como pertencentes a grupos sanguíneos diferentes do AEC 1 positivo. Palavras-chave: Isoanticorpos. Anticorpos caninos. AEC 1. Transfusão sanguínea em cães.

Blood typing in positive DEA 1 dogs

“…In order to carry out the hemagglutination tests and the flow cytometry, samples of canine antisera from post-transfused dogs with DEA 1 blood type were submitted to the investigation of antibodies by flow cytometry as a follow-up of alloimmunization of these respective dogs (Santos et al, 2018). Each antiserum had its own average fluorescence intensity, ranging from 15 to 538.99 and representing the concentrations of anti-DEA 1 antibodies.…”

“…The sera were then twice washed with saline solution at 2200 x g for 3 minutes. Then, all vials were resuspended in 300 μL of saline and, finally, the readings were taken on the cytometer (Santos et al, 2018).…”

Canine Blood Group Prevalence and Geographical Distribution around the World: An Updated Systematic Review

“…The first method described in 1999 was the Michigan State University test (MSU), in which polyclonal antiserum reagents against DEA 1, DEA 3, DEA 4, DEA 5, and DEA 7 were used [ 21 , 22 ]. Other methods for the determination of blood groups were provided for the use of monoclonal sera [ 22 , 23 , 24 , 25 , 26 ]. According to our bibliographic research, the first reported method featuring the use of monoclonal sera is RapidVet-H, which uses the agglutination reaction between the DEA 1.1 antigen and a monoclonal antibody to classify dogs as either positive or negative for DEA 1.1.…”

“…In 2005, the typing card test and the gel column method were reported—the first as an agglutination-based reaction featuring the use of monoclonal antibodies [ 20 ], and the second based on agglutination in micro-columns using DEA 1.1 monoclonal antibodies within a gel matrix [ 20 , 23 ]. The tube test was used in 2006 with four monoclonal antibodies [ 24 ], and in 2011, flow cytometry was reported as a new method for hematology—not only to identify cell phenotypes but also to detect antibodies via the monoclonal murine anti-DEA 1 antibody [ 12 , 23 , 25 ]. Studies demonstrated that the flow cytometry technique has high specificity and sensitivity compared to other methods [ 12 , 19 , 23 , 25 , 26 ].…”

“…The tube test was used in 2006 with four monoclonal antibodies [ 24 ], and in 2011, flow cytometry was reported as a new method for hematology—not only to identify cell phenotypes but also to detect antibodies via the monoclonal murine anti-DEA 1 antibody [ 12 , 23 , 25 ]. Studies demonstrated that the flow cytometry technique has high specificity and sensitivity compared to other methods [ 12 , 19 , 23 , 25 , 26 ]. Other methods subsequently developed for blood-group determination include the automated canine cartridge dog erythrocyte antigen (DEA) 1.1 blood-typing method (QuickVet/RapidVet) [ 27 ] and the immunochromatographic test, which is based on the migration of red blood cells on a membrane and the reaction with monoclonal antibodies specific to each antigen, where a positive result is characterized by the presence of a red line in front of DEA 1 [ 12 , 15 , 19 , 28 ].…”