Agreement of stall‐side and laboratory major crossmatch tests with the reference standard method in horses

Abstract Background Crossmatching is used to prevent life‐threatening transfusion reactions in horses. Laboratory methods are laborious and technically challenging, which is impractical during emergencies. Hypothesis/Objectives Evaluate agreement between a stall‐side crossmatch kit (KIT) and a laboratory method (LAB) in horses with known and unknown blood types. Animals Twenty‐four blood‐typed and alloantibody‐screened healthy adult horses (Aim 1) and 156 adult horses of unknown blood type (Aim 2). Methods Prospective, blinded study. Expected positive (n = 35) and negative (n = 36) crossmatches among 24 antibody and blood‐typed horses were used to determine sensitivity and specificity of KIT and LAB against the reference method. Agreement in 156 untyped horses was evaluated by reciprocal crossmatch (n = 156). Results Sensitivity (95% confidence interval [CI]) for LAB and KIT compared with expected reactions was 77.1% (59.9%‐90.0%) and 91.4% (77.0%‐98.2%), and specificity 77.8% (60.9%‐89.9%) and 73.5% (55.6%‐87.1%), respectively. The KIT was 100% sensitive for Aa reactions; LAB was 100% sensitive for Qab; and both were 100% sensitive for Ca. Cohen's κ agreement for LAB and KIT with expected positive and negative reactions (n = 71) was moderate (0.55 [0.36‐0.74]) and substantial (0.65 [0.47‐0.82]), respectively. Agreement was fair comparing LAB with KIT in Aim 1 (0.30 [0.08‐0.52]) and in untyped horses in Aim 2 (0.26 [0.11‐0.41]). Conclusions and Clinical Importance Agreement between KIT and LAB with expected reactions was blood type dependent. Performance of both methods depends on blood type prevalence.


| INTRODUCTION
Whole blood transfusion is a vital tool in equine critical care, the purpose of which is restoring oxygen delivery to tissues in patients with severe anemia. Transfusions are not necessarily benign, and reactions vary in severity from mild hives to anaphylaxis and death. 1 Knowledge about the donor's blood type and presence of antibodies in the recipient can help prevent adverse reactions. Blood groups are defined by inherited antigens on the red blood cell (RBC) surface. They contribute to recognition of self and can elicit antibody production when introduced into the circulation of an animal with RBCs lacking that antigen. This becomes clinically relevant during blood transfusions, where allogeneic incompatibilities affect patient safety. 2 There are 8 RBC groups in horses (A, C, D, K, P, Q, U, and T). Each group corresponds to a particular gene for which ≥2 alleles exist.
These genes produce surface molecules known as factors, with >30 different factors identified. Consequently, there are over 400,000 possible equine blood types. 3 Approximately 90% of horses have no naturally-occurring alloantibodies. Of the 10% that do, anti-Aa and anti-Ca antibodies occur most often. 4 Anti-Ca antibodies have minimal clinical effects, instead, anti-Aa and Qa are highly immunogenic and associated with severe reactions. 4,5 Pre-transfusion testing is indicated to minimize the risk of incompatible transfusions. 5,6 Blood typing and crossmatching should be performed before transfusion to identify an appropriate donor, but this is challenging in emergency situations. Only a few laboratories perform blood typing in horses. Crossmatching is more readily available as a bench-top laboratory assay, but it is time consuming and requires technical expertise. Therefore, most emergency transfusions are performed without compatibility testing.
A new stall-side crossmatch kit has been developed for horses and has yet to be made available commercially. It uses a gel column agglutination (GEL) method that has been used for crossmatching in dogs. 7 A previous study compared different crossmatch methods in horses, 6 but did not evaluate the particular stall-side kit used in our study. The kit is offered as an efficient way to establish transfusion compatibility. It can be run at all hours by lay individuals, providing results in <20 minutes. Therefore, if deemed a sensitive and specific method, it would allow for safe blood transfusions in emergency situations.
Our objective was to evaluate agreement between this commercial gel-based stall-side crossmatch kit (KIT) and the standard laboratory method (LAB) in horses of known and unknown blood types. Aim 1 compared sensitivity, specificity, and agreement for the KIT and LAB in crossmatch reactions with expected outcomes based on blood type and antibody screening. Aim 1 also determined if method agreement depended on blood type. Aim 2 compared agreement between KIT and LAB in a large population of horses of unknown blood types.
This approach mimicked field situations where unscreened horses may be transfused with untyped blood. We hypothesized that the KIT would be a sensitive and specific crossmatch method and would have good agreement with the LAB method in horses of known and unknown blood types.

| MATERIAL AND METHODS
Blood (20 mL) was collected from the jugular vein of horses into noadditive vacutainer tubes and vacutainer tubes containing K-ethylenediaminetetraacetic acid (EDTA) for blood typing and crossmatch testing by the KIT and LAB methods. As a blinded study, the personnel sampling the horses and identifying crossmatch combinations were different than those performing the crossmatches. In addition, the person performing the KIT method was blinded to the results of the LAB crossmatch, and vice versa, until the end of the study.
All protocols were approved by Cornell University's Institutional Animal Care and Use Committee, and horses were housed in accordance with federal guidelines for the humane care and use of laboratory animals.

| Aim 1
Twenty-one adult clinically healthy horses housed at Cornell University's Equine Park were used in this prospective methods comparison study between July 2017 and June 2018. Additional anti-sera was kindly donated from the University of California, Davis Hematology Laboratory (anti-Qab, n = 1) or identified in-house from incompatible crossmatches performed at Cornell University's Clinical Pathology Laboratory on samples submitted to the Animal Health Diagnostic Center for routine neonatal isoerythrolysis testing (anti-Aa, n = 1; anti-Ca, n = 1; May 2017). Prior transfusion history was unknown and it also was not known if any horse had been diagnosed with neonatal isoerythrolysis as a foal or had produced foals that developed neonatal isoerythrolysis.

| Reference standard method (expected reactions)
Serum and anticoagulated whole blood from Aim 1 horses were submitted for blood typing (for blood groups A, C, D, K, P, Q, and U) and screening for anti-RBC hemolytic and agglutinating antibodies (against Aa, Ab, Ac, Ad, Af, Ca, Da, Dg, Dk, Ka, Pa, Pb, Pc, Qa, Qb, Qc, Ua, and donkey factor) to the University of California-Davis Veterinary Medical Teaching Hospital Hematology Laboratory. Eleven of the 21 horses were blood typed in February 2017 and 10 in January 2018. This laboratory uses a herd of typed horses, previously described standard antisera and macroscopic tube crossmatch methods for determining antibody profiles and blood types. 8 Briefly, screening for antibodies was performed by incubating serial dilutions of a serum sample with a series of equine RBC of known blood types. This procedure was repeated with the addition of complement for the hemolysin assay.
The presence of agglutination and hemolysis was assessed visually, and antibodies were reported as present or absent. If antibodies were detected but could not be further identified (ie, if it could not be determined which RBC antigens they were directed against), they were classified as "unidentified anti-RBC antibodies." 9 Based on the blood types and antibody screens, expected positive and negative crossmatch reactions were set up by 1 investigator (T. Stokol) using different donor-recipient pairs to achieve approximately even numbers of expected positive (n = 35) and negative (n = 36) crossmatches. An example of how expected positive and negative crossmatches were determined is as follows: A recipient with anti-Qabc alloantibodies matched against a donor with Qa antigen on its RBC would be an expected positive (ie, incompatible reaction), but the same recipient against a donor with Aa antigen on its RBC would be an expected negative (ie, compatible reaction). Recipients with unidentified anti-RBC antibodies were placed in an additional "unknown" crossmatch group (n = 19) because we could not know if the donor had the corresponding antigen. Crossmatches using horses with only antidonkey antibodies as recipients were considered negative reactions.

| Aim 2
Horses of unknown blood types and antibody profiles were used in this prospective study between September 2017 and August 2018.
Horses were enrolled at convenience from Cornell's Equine Park (excluding the 21 already blood typed), Cornell University's equestrian and polo teams, and privately owned horses. Written client consent for privately owned horses was obtained before blood sampling. Eligibility criteria for enrollment included healthy horses >6 months old, with health being determined by physical examination. 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.

| Crossmatch methods
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.

| Laboratory crossmatch procedure
This test was performed using the standard macroscopic and microscopic agglutination and hemolysis method 8,10 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 frozenthawed) 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. Autocontrols were performed using "donor" RBCs and serum. All tubes were incubated at 37 C for 30 minutes and then centrifuged for

| Stall-side gel crossmatch kit
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 1).
Hemolysis was not analyzed. To determine agreement among evaluators, results were archived by photography and were scored by 3 blinded independent evaluators.

| Statistical analyses 2.4.1 | General approach
Results were categorized as yes/no for all tests except for unidentified anti-RBC antibodies, which were considered as unknown (U). Data analysis was performed using JMP statistical software

| Aim 1
Sample size was a convenience sample size and was dependent on availability of 21 antibody and blood-type screened horses and 3 antisera. For this Aim, expected negative and positive reactions were determined based on the reference standard method; sensitivity and specificity with 95% CI were determined independently for LAB and KIT relative to expected reactions. Agreement between LAB and KIT, LAB and expected reaction, and KIT and expected reaction, respectively, were determined using Cohen's κ coefficient for dichotomous (yes/no) outcomes. Additionally, weighted κ for LAB versus KIT by strength of reaction (0 to 3+) was calculated.

| Aim 2
Formal sample size calculation was performed using the following assumptions: an expected prevalence of incompatible crossmatches in a convenience sample population consisting of primarily Thoroughbred (TB) and Warmblood (WB) horses to be 20%. 12 We wanted to determine if the kit was able to detect at least 90% of incompatibilities detected by LAB with 95% CI of 80%-100%. Sample size estimation determined 172 crossmatches as appropriate. Agreement between LAB and KIT was determined using Cohen's κ coefficient for yes/no outcomes and weighted κ for strength of reaction (0 to 3+) as described in Aim 1. Agreement between different evaluators for the KIT was determined accordingly. Agreement for crossmatch reactions in Aim 2 was stratified by recipient and donor gender (male versus female) and separately for TB and WB recipient and donor populations, given their largest representation in the sample set and the breed-dependent prevalence of blood types. Assessment of the influence of recipient and donor age, respectively, on test agreement was explored using logistic regression with KIT as the dependent variable, LAB as independent variable, and LAB multiplied by age interaction as fixed effects. When the P-value for the interaction <.05, data were further stratified and Cohen's κ calculated for strata.

| RESULTS
The KIT took <20 minutes to perform, whereas the LAB method took

| Aim 2
A total of 156 horses of unknown blood types and antibody profiles were recruited for reciprocal crossmatches, resulting in 156 crossmatches.

| DISCUSSION
Our objective was to evaluate the agreement between crossmatch tests done by LAB and KIT in horses with both known and unknown blood types.
The KIT is a rapid, point-of-care test that can be performed in 20 minutes and could be used in a field setting for horses with life-threatening anemia requiring prompt transfusion. We found that, when compared with the reference standard method, the agreement was moderate for LAB and substantial for KIT. Overall, LAB and KIT had similar sensitivity and specificity; however these differed by blood type. For horses of unknown blood types, the agreement between LAB and KIT was similarly fair, indicating that the preselection of reactions in Aim 1 did not bias the result substantially. Aim 1 of our study included serum from a horse with anti-Qa antibodies, which, to our knowledge, has not been reported in recent studies, despite anti-Qa antibodies being considered highly immunogenic and implicated in neonatal isoerythrolysis. 4 similar type crossmatch method as used for LAB. However, there is currently no true gold standard method to better evaluate sensitivity and specificity of an equine crossmatch. Additionally, even if reactions are predicted by blood type, the actual occurrence of transfusion reactions may differ and may be caused by other factors not measured with a crossmatch test, such as leukocytes, platelets, proteins, or poorly documented RBC antigens. 1,4 Therefore, the true gold standard would be to actually transfuse blood between 2 horses after the crossmatch to determine compatibility and clinical relevance of these tests. Doing so was beyond the scope of our study.

| Differences between LAB and KIT methods
The LAB procedure uses the standard tube agglutination method. This The LAB method evaluates 2 more aspects of incompatibility than does KIT. The LAB detects both macroscopic and microscopic agglutination, compared to KIT, which only examines agglutination macroscopically. Additionally, LAB detects hemolysis and KIT is not designed to do so. These differences might be expected to result in lower sensitivity for KIT, but we did not observe differences. The gel likely captures microscopic agglutination even though it is evaluated macroscopically.
Additionally, it appears that anti-RBC antibodies that cause hemolysis alone without concurrent agglutination are quite rare, and the inability of the kit to detect hemolysis likely will not markedly affect the sensitivity of the test. 6 Yet, this still remains a limitation of KIT.
Another difference between methods is that LAB dilutes the recipient's serum at 1:2 and KIT does not predilute serum. Additionally, with the LAB method, complement is added to the suspension, further adding to the dilutional effect, which might also explain the numerically lower sensitivity of the LAB method for expected positive reactions in Aim 1 (the reference method used less dilute serum).

| Discrepancies between tests
The dilutional effect could contribute to discrepancies between the LAB and KIT results for the Aa blood group expected positive reactions (ie, LAB had a higher rate of false-negative reactions than did KIT). Anti-Aa antibody-positive recipient serum was available from 2 horses for our study and false-negative reactions for LAB were seen with both antisera.
Because numerical values are not reported for titers, it is possible that the anti-Aa antibody titers were low in these 2 horses and, combined with the predilution of serum, resulted in a false negative with the LAB method. In contrast, the incompatibility was still detectable with the The time from blood typing and antibody screening of horses in Aim 1 to crossmatching ranged from a minimum of 5 months to a maximum of 16 months. Therefore, the discrepancy between screenings could be due to a true change in the presence or absence of antibodies, a change in the capacity of the test to detect antibodies in low concentrations or weak immunoreactivity. 9 The latter study found that most of the discrepancies between tests were associated with horses that had changes in their antibody profile. 9 Similarly, the repeatability of blood typing with the reference method is not known and it is possible that some of the donors were incorrectly blood typed (particularly because weak agglutinins may be missed when microscopic methods are not used to assess for agglutination). Future studies could include repeating the antibody screening and blood typing to better understand differences in performance of the methods.

| Clinical implications of breed on test performance
Expected performance of both LAB and KIT will depend on the prevalence of blood types within the tested horse population. In horses,  Table S1). 12 The frequency of blood types in WB has not been published, but 78% of the 9 tested WB in Aim 1 were Aa positive and 11% were Qa positive (Supplemental Table S1).

| Clinical relevance
As previously mentioned, weak antibody titers could have contributed to discrepant test results. However, the actual clinical relevance of these weak titers is unknown.
Based on studies in foals with neonatal isoerythrolysis, anti-Ca antibodies are reported to not be as clinically relevant as anti-Aa and anti-Qa antibodies. 15 A previous study also showed that 3+ incompatible crossmatches with anti-Ca antibodies only predicted mild febrile and tachycardic transfusion reactions that did not prevent completing the transfusion. 4

| Conclusion
Agreement with expected reactions between KIT and LAB was substantial and moderate, respectively, but only fair when comparing both methods with each other in both blood typed and untyped mixed populations of horses. Agreement was blood type dependent and improved when stratifying data by the 2 most represented breeds (TB and WB). Thus, we conclude that the performance of both methods will depend on the prevalence of blood types within