Alloimmunization of a dog erythrocyte antigen 1- dog transfused with weakly dog erythrocyte antigen 1+ blood

Abstract

Background: Acute hemolytic transfusion reactions because of dog erythrocyte antigen (DEA) 1 sensitization after mismatched transfusions are serious complications. Dog erythrocyte antigen 1 expression varies from negative to weakly to strongly positive.

Objectives: To assess alloimmunization after transfusion of weakly DEA 1+ blood to a DEA 1- dog.

Animals: One DEA 1- recipient and 1 weakly DEA 1+ donor, and 106 control dogs.

Methods: Long-term follow-up study. Matched for DEA 3, 4, 5, and 7, Dal, and Kai 1 and 2, weakly DEA 1+ donor packed red blood cells (RBCs) were transfused 3 times (0.45 mL/kg at Day 0, 16, and 37) to a DEA 1- recipient. Alloantibodies against RBCs from donor and 106 controls were determined in recipient's plasma samples using a commercial antiglobulin-enhanced immunochromatographic strip and gel tube crossmatches. Alloantibody titers were determined.

Results: The DEA 1- recipient was sensitized after 16 days to ≥1657 days after transfusion to weakly DEA 1+ and otherwise matched RBCs. Strong to moderate crossmatch incompatibilities were observed between recipient's plasma and all 61 DEA 1+ crossmatched controls. Moderate to weak incompatibilities were also observed to DEA 1- controls. Anti-DEA 1 and other alloantibodies were detected over the 4.5 year observation period.

Conclusions and clinical importance: Blood from a weakly DEA 1+ donor induces a strong and durable alloimmunization in a DEA 1- recipient dog. Additional alloantibodies developed against yet to be defined RBC antigens. Those results support the recommendation of typing dogs against DEA 1, considering weakly DEA 1+ as immunogenic, and crossmatching all previously transfused dogs.

Keywords: alloantibodies; blood compatibility; canine; dog erythrocyte antigen; hemolytic transfusion reaction.

Figure 1

Timeline of compatibility testing between Recipient and Donor or Controls throughout immunization and follow‐up period. The time line of the study took place from Day 0 (October 2013) to Day 1657 (April 2018). Recipient plasma samples from Days 106 and 891 were frozen to be crossmatched later with Control red blood cells (RBCs)

Figure 2

Expression of dog erythrocyte antigen (DEA) 1 in Recipient, Donor, and 2 Controls assessed by A, immunochromatographic and B, flow cytometric techniques. The DEA 1 immunochromatographic typing strip has 1 line with a control antibody (labeled “C”) present on all canine red blood cells (RBCs) (thus, a red band at “C” indicates that the test is valid) and any band intensity at the “DEA 1” position is considered positive (graded 1+ to 4+). Dog erythrocyte antigen 1 expression was classified as negative (mean fluorescence intensity [MFI] <10), weakly positive (10 ≤ MFI < 100), moderately positive (100 ≤ MFI < 300), and strongly positive (MFI ≥ 300) by flow cytometric analysis

Figure 3

Major crossmatch incompatibilities between Recipient and Donor after transfusion of weakly DEA 1+ blood to a DEA 1− dog using an antiglobulin‐enhanced immunochromatographic strip crossmatch (AIC) and an antiglobulin‐enhanced gel tube crossmatch (AGC) technique. Antiglobulin‐enhanced immunochromatographic crossmatch test has a band with a control antibody on the strip (labeled “C”) specific to all canine red blood cells (RBCs) (thus, a red band at “C” indicates that the test is valid). Any band intensity at “XM” is considered positive (graded 1+ to 4+) indicating alloantibodies are present against IgG, IgM, and/or C3 on the RBCs surface and thus indicating incompatibility. Agglutination reactions in AGC tests are recorded as follows for negative reactions: 0, all RBCs at the bottom of the tube. For positive reactions: 1+, RBCs' agglutinates disperse in the gel but most at the bottom of the tube; 2+, RBCs' agglutinates disperse in the gel; 3+, some RBCs' agglutinates disperse in upper part and most are forming a red line on the top of gel; and 4+, all RBCs agglutinates form a red line on the gel's surface. Recipient plasma samples from Day 48 to Day 1657 crossmatched with Donor RBCs using AIC and AGC tests: strong to moderate reactions with AIC and 4+ incompatible reactions with AGC at Day 48 and 106. Weak reaction with AIC and 3+ incompatible reactions with AGC at Day 891 and 1657

Figure 4

Alloantibody titration between Recipient plasma and Donor red blood cells (RBCs) using the antiglobulin‐enhanced gel crossmatch (AGC) technique. Recipient plasma samples from Day 106 to 1657 in serial 2‐fold dilutions crossmatched with Donor RBCs. Agglutination reactions with AGC technique were recorded similarly as described in Figure 3

Figure 5

Major crossmatch incompatibilities between Recipient's plasma and a panel of Control red blood cells (RBCs) using the antiglobulin‐enhanced gel crossmatch (AGC) technique. Agglutination reactions with AGC technique were recorded similarly as described in Figure 3. Recipient's plasmas from Day 106 to Day 1657 crossmatched with Control RBCs using AGC displays 2+ to 4+ reactions against DEA 1+ Controls and negative to 3+ reactions with DEA 1− Controls

Figure 6

Recipient's alloantibodies specificity study utilizing adsorbed recipient's plasma from Day 106 in antiglobulin‐enhanced immunochromatographic strip crossmatch (AIC) tests. The presence of anti‐dog erythrocyte antigen (DEA) 1 alloantibodies was assessed by crossmatching (XM) differently treated Recipient's plasma from Day 106 against the same DEA 1+ Control red blood cells (RBCs). Strip 1: Recipient autologous crossmatch (negative autocontrol). Strip 2: Crossmatch of DEA 1+ Control RBCs versus Recipient's plasma (positive control). Strip 3: Crossmatch of DEA 1+ Control RBCs versus Recipient's plasma previously adsorbed against DEA 1− Control RBCs. Strip 4: Crossmatch of DEA 1+ Control RBCs versus Recipient's plasma previously adsorbed against DEA 1+ Control RBCs. Antiglobulin‐enhanced immunochromatographic crossmatch tests results were recorded similarly as described in Figure 3