Major crossmatch compatibility of rabbit blood with rabbit, canine, and feline blood.

OBJECTIVE: To evaluate the major crossmatch compatibility between rabbit recipients, rabbit donors, and the major canine and feline blood types. DESIGN: Prospective in vitro study in December 2021. SETTING: Academic veterinary teaching hospital. ANIMALS: Whole blood samples were collected from 11 healthy New Zealand White rabbits (Oryctolagus cuniculus) with no previous transfusion history. Three pigtail segments were acquired from dog erythrocyte antigen (DEA)-1-positive, DEA-1-negative, and feline type A blood units. Whole blood was collected from a healthy type B blood donor cat. INTERVENTIONS: Blood from each rabbit recipient underwent a major crossmatch using standard tube crossmatch methodology with itself and the following donor blood types: rabbit, DEA-1-positive, DEA-1-negative, feline type A, and feline type B. MEASUREMENTS AND MAIN RESULTS: Self-crossmatches and crossmatches between rabbit recipients and conspecific donors were negative for hemolysis and agglutination. Crossmatches between rabbit recipients and canine and feline donors yielded no hemolysis but produced varying degrees of macroscopic and microscopic agglutination. Rabbit recipients had 1.4 (95% confidence interval: 1.1-1.8) times the risk of macroscopic agglutination when major crossmatched with canine blood compared to feline blood. No significant difference in agglutination was found between DEA-1-positive and DEA-1-negative or feline type A and type B donors. CONCLUSIONS: These findings support allogeneic blood transfusions between rabbits being highly compatible and suggest rabbits have naturally occurring alloantibodies against both canine and feline red blood cells. However, feline red blood cells had a lower rate of in vitro incompatibility on major crossmatch, suggesting potentially higher in vivo compatibility if an emergency xenotransfusion is needed. Further prospective research is needed to determine if xenotransfusion is associated with a higher incidence of acute and delayed transfusion reactions in rabbits than allogeneic transfusions.

Evaluation of post-transfusion RBC alloimmunization in dogs using a gel-column crossmatch with and without anti-canine globulin enhancement.

A blood crossmatch is essential to ensure RBC compatibility for previously transfused dogs. There is no gold standard crossmatch method for dogs, although the standards used most commonly by academic institutions and reference laboratories are the tube and gel-column crossmatches. Addition of anti-canine globulin (ACG) has been suggested to increase detection of RBC incompatibilities. Our objective was to determine if there is a correlation between results of a standard and an ACG-enhanced gel-column crossmatch in detecting post-transfusion RBC alloimmunization. Pre- and post-transfusion serum or plasma samples were obtained from 33 dogs for major crossmatches to 1-6 (median: 3) blood donors. Crossmatches were performed with (n = 202) and without (n = 202) ACG, with results scored by 4 observers, 3 of whom were anonymized. Ten of 33 (30%) dogs had major crossmatch incompatibilities post-transfusion. RBC incompatibilities (2-4+ agglutination) were detected only with ACG in 4 dogs, only without ACG in 3 dogs, and with both methods in 3 dogs. There was fair correlation between crossmatch methods for determination of compatibility (ρ = 0.34; p < 0.001) and incompatibility (ρ = 0.35; p < 0.001) scores. Among 4 observers, there was near-perfect agreement in determining compatibility (κ = 0.97; p < 0.001) and substantial agreement in overall scoring of incompatibility (κ = 0.77; p < 0.001). Our results suggest that detection of RBC incompatibilities in dogs can be maximized by performing a gel-column crossmatch both with and without ACG enhancement.

Discordance between ABC blood phenotype and genotype in a domestic short-haired cat.

An adult domestic short-haired feline leukemia virus-infected cat was referred for kidney failure and worsening anemia requiring transfusions. ABC blood typing was performed with an immunochromatographic strip assay at different occasions. Gel column systems were used for the major and minor crossmatching tests, and anti-A and anti-B titers were determined. No discrete A or B bands appeared on the immunochromatographic strips at any time point for the recipient cat. The recipient's plasma agglutinated RBCs from tested type A and B cats. The recipient's RBCs appeared compatible with plasma from 1 type A and 2 B donors, and incompatible with plasma from another type A cat. Genotyping of recipient blood revealed a single homozygous c.179G>T CMAH variant predicting a blood type B. These studies suggest an unusual weak type B or missing all ABC antigens. The latter resembles the exceedingly rare Bombay phenotype in the human ABO blood group system.

Validation of a human paper-based blood typing method for use in pet pigs.

BACKGROUND: Evidence-based medical practices for pet pigs are needed. EldonCard is a human blood-typing card shown to be rapid and reliable in identifying blood phenotypes of pet pigs. The objective of this study was to validate EldonCard by determining its reliability, reproducibility, and robustness for its routine use in the clinical setting. KEY FINDINGS: Twenty-four venous blood samples from pet pigs were collected for a prospective in vitro study. Blood genotypes ("EAAA0 " and "EAA00 ") were identified in 15 samples via polymerase chain reaction (PCR). All samples were phenotyped ("A," "Aweak ," and "0" or "-") via EldonCard. Kappa (κ) statistics measured the level of agreement between 2 raters, and between EldonCard and PCR. McNemar's test determined if an association between the blood types and EldonCard or PCR exists, with significance at P < 0.05. Agreement between raters and methods was perfect (60/60 [100%], κ:1, P < 0.001; 15/15 [100%], κ:1, P < 0.001). There was no difference in the proportions of blood groups based on method. SIGNIFICANCE: In conjunction with previous data, EldonCard is a rapid, accurate, reliable, precise, and robust in-clinic blood-typing method for the A0 system of pet pigs. EldonCard is now a validated blood-typing tool for the A0 system of pet pigs and maybe used for pretransfusion screenings and identification of donors and recipients.

Agglutination and hemolytic crossmatching to determine transfusion reaction differences between large and small breed goats.

BACKGROUND: Blood transfusions are performed frequently in goats, but crossmatches are rarely performed. HYPOTHESIS/OBJECTIVES: Determine differences in the frequency of agglutination and hemolytic crossmatch reactions between large and small breed goats. ANIMALS: Healthy adult goats, 10 large and 10 small breed. METHODS: Two hundred eighty major and minor agglutination and hemolytic crossmatches: 90 large breed donor to large breed recipient (L-L), 90 small breed donor to small breed recipient (S-S), 100 large breed donor to small breed recipient (L-S). A linear mixed model with treatment group (L-L, S-S, L-S) as a fixed effect and individual crossmatch as a random effect was used to identify variations in reaction frequency among groups and individuals. RESULTS: Frequency of major agglutination reactions for L-L, S-S, and L-S were 3/90 (3.3%), 7/90 (7.8%), and 10/100 (10.0%), respectively. Frequency of major hemolytic reactions for L-L, S-S, and L-S were 27/84 (32.1%), 7/72 (9.7%), and 31/71 (43.7%). Individual pairings and groupings had no effect on agglutination reactions. Individual pairings had no effect on the frequency of hemolytic reactions. For major hemolytic crossmatches, pairwise comparisons identified higher frequencies of reactions when comparing L-L to S-S (P = .007) and L-S to S-S (P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Goats experience increased frequencies of hemolytic reactions compared to agglutination. Significant increases in hemolysis were seen between large breed donors and small breed recipients, compared to small breed pairings. Additional studies are required to determine correlations between crossmatches and transfusion reactions.

Validation of a cage-side agglutination card for Dal blood typing in dogs.

BACKGROUND: Although 98% of the canine population is Dal-positive, Dal-negative dogs are more common in some breeds such as Doberman Pinschers (42.4%) and Dalmatians (11.7%), and finding compatible blood for these breeds may be challenging, given limited access to Dal blood typing. OBJECTIVES: To validate a cage-side agglutination card for Dal blood typing and determine the lowest packed cell volume (PCV threshold) at which interpretation remains accurate. ANIMALS: One-hundred fifty dogs, including 38 blood donors, 52 Doberman Pinschers, 23 Dalmatians and 37 anemic dogs. Three additional Dal-positive canine blood donors were included to establish the PCV threshold. METHODS: Dal blood typing was performed on blood samples preserved in ethylenediaminetetraacetic acid (EDTA) <48 hours using the cage-side agglutination card and a gel column technique (gold standard). The PCV threshold was determined using plasma-diluted blood samples. All results were read by 2 observers, blinded to each other's interpretation and to the sample's origin. RESULTS: Interobserver agreement was 98% and 100% using the card and gel column assays, respectively. Overall, the sensitivity and specificity of the cards were 86%-87.6% and 96.6%-100%, respectively, depending on the observer. However, 18 samples were mistyped using the agglutination cards (15/18 by both observers): 1 false-positive (Doberman Pinscher), and 17 false-negative samples including 13 anemic dogs (PCV range, 5%-24%; median, 13%). The PCV threshold allowing reliable interpretation was determined to be >20%. CONCLUSIONS AND CLINICAL IMPORTANCE: Dal agglutination cards are reliable as a cage-side test, but results should be interpreted cautiously in severely anemic patients.

Prevalence of dog erythrocyte antigen 1 in a population of dogs tested in California.

BACKGROUND: Multiple studies have evaluated the breed-specific prevalence of dog erythrocyte antigen (DEA) 1 in various geographic regions. However, few large-scale studies exist that describe breed prevalence of DEA 1 in the United States. KEY FINDINGS: From January 2000 to October 2020, 6469 dogs had their RBC antigen type determined and were included in the study. The overall prevalence of DEA 1 in all dogs was 61.2%. Of 50 breeds with sample sizes ≥20, 8 breeds had a high prevalence (≥90%) of DEA 1-positive blood type: Basset Hound, Bernese Mountain Dog, Brittany, Dachshund, Miniature Pinscher, Miniature Schnauzer, Pug, and Rottweiler. Four breeds had a high prevalence (≥90%) of DEA 1-negative blood type: Boxer, English Bulldog, Flat-Coated Retriever, and French Bulldog. Numerous breeds with a sample size <20 and ≥5 were found to have 100% prevalence of a DEA 1 blood type, although these findings need to be confirmed with a larger sample size. No statistical difference in any breed based on sex was found. The results in this study are consistent with previously reported data in other countries. SIGNIFICANCE: Knowledge of regional breed differences in DEA 1 prevalence can help to improve selection and recruitment of appropriate blood donor dogs.

Effect of donor blood storage on gel column crossmatch in dogs.

BACKGROUND: Compared with fresh blood, stored equine donor blood results in spurious tube crossmatch incompatibilities. Interpretation of blood crossmatch results is considered subjective. OBJECTIVES: We aimed to determine if the duration of canine donor blood storage impacts compatibility testing using a standard gel column crossmatch and evaluate interobserver variation in the interpretation of crossmatch results. METHODS: Observational study. Whole blood segments were obtained from 23 canine packed red blood cell (RBC) units for use in crossmatches after storage for 0, 7, 14, 21, 28, and 35 days. Major and minor crossmatches were performed using serum and RBCs, respectively, from two to three healthy "recipient" dogs per unit. All crossmatch results were interpreted by four observers, of whom three were blinded. RESULTS: All major crossmatches (n = 61) were compatible on day 0 and remained compatible through day 35 of storage. All minor crossmatches (n = 69) were compatible at all time points, except for five donor pairs with 1 to 3+ agglutination. Repeat testing of these five donor pairs confirmed crossmatch incompatibilities on days 0 through 35, with no change in the degree of incompatibility over time. There was substantial agreement among four observers in determining compatibility (κ = 0.94) and scoring incompatibility (κ = 0.76). CONCLUSIONS: The current practice of performing canine crossmatches with whole blood segments stored for up to 35 days is acceptable, with no spurious changes in compatibility expected over time. The substantial interobserver agreement suggests that the gel column is suitable for performing canine crossmatches in a laboratory setting with multiple personnel.

Laboratory Testing in Transfusion Medicine.

Canine and feline transfusions are life-saving procedures that have become increasingly common in veterinary medicine. Laboratory testing plays a vital role in transfusion medicine, particularly in the prevention and diagnosis of transfusion reactions. Laboratory tests should be used to screen donors for their general health and for the presence of any blood-borne pathogens. Pretransfusion blood typing and compatibility testing make immunologic reactions less likely, and commercial typing and crossmatching kits are now available. Appropriate diagnostic tests in the face of a potential transfusion reaction are important to tailor effective therapy.

Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs.

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.

Alloimmunization in dogs after transfusion: A serial cross-match study.

BACKGROUND: Cross-matching is performed to determine the serological compatibility of donor and recipient blood. Current guidelines recommend that cross-matching should be performed in dogs when an initial transfusion was performed more than 4 days ago or when the transfusion history is unknown. HYPOTHESIS: Determination at what time point alloantibodies are detected in dogs after transfusion. The hypothesis was that dogs would form alloantibodies within 4 days after a transfusion. ANIMALS: Twenty-one anemic dogs were transfused and monitored for at least 4 subsequent days. Exclusion criteria were persistent red blood cell (RBC) agglutination and a previous transfusion. METHODS: Prospective observational study. Cross-matching was performed before the initial DEA 1-compatible transfusion and on days 1, 2, 3, and 4 and if possible, between day 5 and 28, using the tube method without enhancement (major cross-match, recipient controls); recipients were monitored for transfusion reactions. RESULTS: In 12/21 dogs a positive cross-match (microscopic degree of agglutination [AD] 1+ to 2+) was observed within 4 days after the transfusion. In a nonlinear regression model, no significant association was detected between type of anemia (P = .41), RBC storage time (P = .44), immunosuppressive treatment (P = .75) nor transfusion volume (P = .70) and the occurrence of positive cross-matches within 4 days after transfusion. Another 4 dogs developed a positive cross-match (microscopic AD 1+ to 2+) after 6 to 13 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Because production of alloantibodies was detected as early as 1 day after transfusion, cross-matching should be performed before every subsequent transfusion.

Comparison of a commercial immunochromatographic strip crossmatch kit and standard laboratory crossmatch methods for blood transfusion compatibility in dogs.

OBJECTIVE: To evaluate agreement between 2 standard laboratory (SL) methods and an immunochromatographic strip (ICS) method to crossmatch dogs receiving RBC transfusions. A second objective was to evaluate uninterpretable SL crossmatch results as compared to ICS in the presence of autoagglutination. DESIGN: Prospective observational study (September 2018 to October 2019). SETTING: University teaching hospital. ANIMALS: Forty anemic dogs receiving RBC transfusions. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: All dogs received DEA 1-negative packed RBCs. Three crossmatch methods were evaluated against the same unit transfused to each dog: SL method performed at institutional laboratory (SL-I), SL method sent to a commercial laboratory (SL-C), and a commercially available point-of-care ICS method. Major and minor crossmatches were incompatible for 2.5%/7.5% of ICS tests, 82.5%/52.5% of SL-I tests, and 52.5%/27.5% of SL-C tests. Agreement between ICS and SL-C major (κ = 0.05) and minor (κ = 0.02) crossmatches and between ICS and SL-I major (κ = 0.009) and minor (κ = 0.03) crossmatches was slight. Agreement between SL-C and SL-I major (κ = -0.06) and minor (κ = -0.12) crossmatches was poor. Results of major and minor crossmatches were uninterpretable due to autoagglutination in 38%/38% for SL-I and 29%/18% for SL-C crossmatches. ICS method was interpretable for 93% (major) and 98% (minor) crossmatches. After exclusion of uninterpretable SL pairings, agreement still remained poor to slight between all tests. Only 1 of 40 dogs (2.5%; 95% confidence interval: <1.0%-13.2%) had an immediate immunological transfusion reaction. CONCLUSIONS: Lack of agreement between all methodologies was noted. The high level of incompatibility predicted by SL methods despite lack of clinically relevant reactions suggests a high false incompatibility rate as compared to the ICS test. ICS testing was also able to give results more frequently in the face of autoagglutination. Further work is needed to investigate the ICS method's ability to predict clinically significant transfusion reactions.

Characterization of post-transfusion anti-FEA 1 alloantibodies in transfusion-naive FEA 1-negative cats.

OBJECTIVES: The aim of this study was to characterize anti-feline erythrocyte antigen (FEA) 1 alloantibodies following sensitization of FEA 1-negative cats, including their rate of appearance, agglutination titer over time and immunoglobulin class. A secondary aim was to obtain polyclonal anti-FEA 1 alloantibodies to increase the availability of FEA 1 blood typing. We also describe a case study documenting an acute hemolytic transfusion reaction in a transfusion-naive FEA 1-negative feline patient that received FEA 1-positive blood. METHODS: In this prospective clinical study, 35 cats with blood group type A underwent extensive blood typing for FEA 1-5. Two cats were identified as FEA 1-negative; these cats were transfused uneventfully with 50 ml of FEA 1-positive, but otherwise compatible, packed red blood cells. Post-transfusion blood samples were collected routinely as long as anti-FEA 1 alloantibodies were detected. Appearance of anti-FEA 1 alloantibodies was detected using a gel column crossmatch method. RESULTS: Anti-FEA 1 alloantibodies were detected as early as 5 days post-transfusion and remained detectable for over 400 days in one cat. Agglutination titers in both cats were relatively weak (1:1 to 1:8). The main immunoglobulin class was IgM. CONCLUSIONS AND RELEVANCE: Transfusion of FEA 1-negative, transfusion-naive cats with FEA 1-positive blood results in production of post-transfusion anti-FEA 1 alloantibodies as early as 5 days post-transfusion. Our results confirm the potential immunogenicity of FEA 1 and support crossmatching prior to a blood transfusion, even in transfusion-naive cats. Further studies are needed to better document the clinical importance of these post-transfusion antibodies, as well as to facilitate routine blood typing for the FEA 1 antigen in cats.

Collection and administration of blood products in horses: Transfusion indications, materials, methods, complications, donor selection, and blood testing.

BACKGROUND: Blood transfusion is a lifesaving treatment for horses with acute hemorrhage and other causes of anemia. Transfusions improve oxygen delivery to the tissues via increased blood volume and hemoglobin concentration. Certain aspects of equine blood transfusion are challenging, especially in the field situation, and practitioners may be unfamiliar or feel overwhelmed with the process. An understanding of the indications, materials, methods, and techniques as well as donor selection and possible complications will help practitioners successfully implement blood transfusion in clinical practice. PROCEDURES: Blood transfusion involves several steps including appropriate donor selection, cross-matching, blood collection, and administration, as well as monitoring and handling of transfusion reactions. Guidance for each of these steps are detailed in this review. SUMMARY: Blood transfusion is an effective and often lifesaving treatment for managing diseases of blood loss, hemolysis, and decreased RBC production. Equine practitioners require a thorough understanding of the indications for blood transfusion, the immunological principles behind compatibility testing and transfusion reactions, and the technical skills to aseptically collect and administer blood products KEY POINTS: Equine practitioners require a thorough understanding of the indications for blood transfusion, the immunological principles behind compatibility testing and transfusion reactions, and the technical skills to aseptically collect and administer blood products. Because there are over 400,000 possible equine RBC phenotypes, no universal donor exists, and some blood type incompatibilities are likely between any donor and recipient. Therefore, prior to any blood transfusion, donor and recipient blood should be cross-matched Inadequate delivery of oxygen (Do2 ) to the tissues, resulting from low hemoglobin (Hb) concentration, is the most important indication for blood transfusion Neonatal isoerythrolysis most commonly occurs following an anamnestic response in late gestation; it rarely occurs following a primary exposure because the immune response is not strong enough to produce clinically significant alloantibody titers.

In vitro compatibility testing of canine and rabbit blood.

OBJECTIVE: To determine the in vitro compatibility of rabbit and canine blood using both a tube and slide agglutination crossmatch technique and to compare the results obtained from these 2 methods. DESIGN: Prospective observational laboratory study from January to March 2020. SETTING: University veterinary teaching hospital. ANIMALS: Six client-owned rabbits ≥3.5 kg undergoing phlebotomy for a clinical reason. "Pigtail" blood samples from 3 dog erythrocyte antigen (DEA) 1-positive and 3 DEA 1-negative canine packed red blood cell units. INTERVENTIONS: Blood from each rabbit was crossmatched with a single unit of canine blood using both a standard laboratory tube agglutination technique and a simple slide agglutination method with each rabbit/canine unit serving as its own intraassay control. Tube crossmatches were evaluated for agglutination both macro- and microscopically and assessed for hemolysis. Slide crossmatches were assessed for the presence of agglutination both macro- and microscopically.  MEASUREMENTS AND MAIN RESULTS: All crossmatches were incompatible. Varying degrees of agglutination were seen for all crossmatches. Hemolysis was observed with all minor tube crossmatches. Results of both crossmatch techniques were in close agreement.  CONCLUSIONS: The crossmatch results in this present study strongly demonstrate in vitro incompatibility between canine and rabbit blood. Agreement between the 2 techniques in this study indicates that the slide agglutination technique may be quicker, require less blood, and provide reliable results in exclusively assessing the compatibility of canine and rabbit blood. Based on the results of this study, emergency xenotransfusion of canine blood to rabbits cannot be recommended.

Comparison of serum versus EDTA plasma in canine major crossmatch reactions.

BACKGROUND: Protocols for crossmatch reactions vary in veterinary medicine, particularly regarding the use of recipient serum vs plasma. Sources suggest that major crossmatch results might differ when recipient plasma is used instead of serum, but there are conflicting reports as to the exact effects on the results. OBJECTIVES: The aim of this study was to determine the frequency and degree of discrepancy, if any, between canine major crossmatch reactions using serum versus EDTA plasma, performed via a standard tube method. METHODS: One hundred duplicate canine major crossmatch reactions were performed with both serum and EDTA plasma from 100 different "recipient" dogs against erythrocytes from a single, healthy "donor." Decreasing concentrations of a rabbit anti-dog erythrocyte antibody were added to generate strong positive, weak positive, and negative results for each crossmatch reaction. Crossmatch results were followed through the following phases: immediate spin, cold, warm, albumin, and Coombs. Semi-quantitative results were compared between reactions using serum vs EDTA plasma. RESULTS: Weak positive, major crossmatch reactions were significantly more likely to demonstrate stronger agglutination in EDTA plasma compared with serum in the immediate spin phase, cold phase, warm phase, and albumin phase (P < 0.001). There was no statistically significant difference between serum and EDTA plasma results in the Coombs' phase (P = 0.313). CONCLUSIONS: In this experimental setting, EDTA plasma and serum were both deemed acceptable for use in canine major crossmatch reactions. EDTA plasma might be preferable to detect weak agglutination with more sensitivity and reduce recipient blood volumes needed to complete the crossmatch reaction.