2.3. Crossmatch methods

A trained veterinarian, undergraduate student, or medical technologist in the Clinical Pathology Laboratory performed the LAB crossmatches (S.E. Mix, A.D. Bortsie‐Aryee, E.M. Wood) and a single veterinarian from Cornell University Hospital for Animals performed all the KIT crossmatches (M.S. Fenn). Results for both methods were verified by independent blinded observers, another medical technologist for LAB and another veterinarian for KIT. A third person was consulted if there were discrepant results within method. All crossmatches were performed within 12 hours of blood collection except for the 3 sources of anti‐sera (Aim 1), which were stored frozen at −80°C. To maintain blinding, additional serum from typed and antibody‐screened horses was stored similarly frozen. Frozen sera were thawed in a warm water bath at 37°C before use.

For both methods, LAB and KIT, reactions of 1‐3+ (details below) were considered positive or an incompatible crossmatch, with 0 equivalent to no agglutination and a compatible crossmatch.

This test was performed using the standard macroscopic and microscopic agglutination and hemolysis method8, 10 used for routine crossmatches in the Clinical Pathology Laboratory. In this assay, no‐additive tubes were centrifuged at 3800g for 5 minutes to harvest serum. EDTA‐blood from the “donor” was centrifuged at 1000g for 1 minute and washed 3 times in phosphate‐buffered saline, creating a final 2% suspension of RBCs. “Recipient” serum (fresh or frozen‐thawed) was diluted 1:2 in 0.9% sodium chloride and added with guinea pig complement (Guinea Pig Serum and Saline Diluent, MP Biomedicals, Solon, Ohio) in a 1:1:1 ratio to the 2% RBC suspension. The complement is necessary to detect hemolyzing antibodies. Auto‐controls were performed using “donor” RBCs and serum. All tubes were incubated at 37°C for 30 minutes and then centrifuged for 1 minute at 1000g. The tubes were examined macroscopically for hemolysis and agglutination and microscopically for agglutination, using a predetermined scale of 0‐3+. Both agglutination and hemolytic reactions were used in the comparison to KIT. The semiquantitative agglutination score was 0 when no agglutination was observed; 1+ for weak agglutination with 2‐3 RBCs per agglutinate or transient RBC adherence; 2+ for moderate agglutination with only microscopic small agglutinins of 4‐10 RBCs per agglutinate; and 3+ for severe agglutination with any microscopic large agglutinins of >10 RBCs per agglutinate or any gross agglutination.

Crossmatches were performed using the gel matrix column KIT test according to the manufacturer's guidelines (Gel Test for Major Equine Crossmatch, Alvedia Veterinary Diagnostic Company, Limonest, France). “Donor” RBCs were allowed to settle by gravity for 5 minutes, the supernatant plasma removed and the “packed” RBCs were collected using the kit strip and then resuspended in the kit buffer, without washing the RBCs. Then, a 1:1 mixture of “donor” RBC suspension and “recipient” serum was added to a test tube. The mixture was lightly agitated by tapping using an index finger for approximately 10 seconds and then incubated at room temperature for 10 minutes. After incubation, the mixture was added to the top of the polypropylene gel column and centrifuged at 400 g for 5 minutes. The extent of RBC retention in the gel, corresponding to agglutination, was graded using a 0‐3+ scale (Figure ​(Figure1).1). Hemolysis was not analyzed. To determine agreement among evaluators, results were archived by photography and were scored by 3 blinded independent evaluators.

Scoring of agglutination in a crossmatch with the stall‐side kit (KIT). The degree of RBC retention in the gel is graded according to the following scale: 0, all RBCs at the bottom of the gel (compatible); 1+, few RBC agglutinates in the lower half of the gel but most RBCs at the bottom of the gel; 2+, RBC agglutinates dispersed throughout the gel, 3+, RBC agglutinates throughout gel and RBCs on upper surface. RBC retention of ≥1+ is considered incompatible