Vetnuus | August 2024 9 If hemolysis or agglutination occurs in either crossmatch, a potential in vivo serological incompatibility may exist between the recipient and blood donor.5–8 The minor crossmatch is considered less clinically important for component therapy but is recommended when transfusing whole blood or plasma.7 Stored canine and feline blood products are frequently available at referral and veterinary specialty centers, making their potential availability for an emergency xenotransfusion to a rabbit recipient likely. Dog erythrocyte antigen (DEA)-1-positive, DEA-1-negative, feline type A, and feline type B (albeit rare) blood products may be potential options for transfusion of an anemic rabbit. While a recent experimental study evaluated the in vitro serological compatibility of rabbit recipients and canine blood products,9 no study has explored the in vitro serological compatibility of rabbit recipients and rabbit or feline blood donors. Similarly, no current evidence suggests that canine or feline blood products would be more compatible. The objective of this study was to evaluate the in vitro major crossmatch compatibility between rabbit recipients, rabbit donors, and canine and feline blood donors of different major blood types. The authors hypothesized that rabbit blood donors would have better in vitro serological compatibility than canine and feline blood donors. Given a previously successful case report in a ferret,4 the authors also hypothesized that feline blood donors would have better in vitro serological compatibility than canine blood donors. 2. METHODS 2.1 Rabbit recipients Blood samples were collected from 11 (9 females, 2 males) juvenile New Zealand White rabbits (Oryctolagus cuniculus) from 2 institutions previously used for clinical teaching or research. Full ethical approval was granted by the 2 institutions’ Institutional Animal Care and Use Committees prior to phlebotomy. All rabbits were 4–5 months old, weighed 4.1–5.4 kg (median: 4.62 kg), were apparently healthy on physical exam, and had no previous transfusion history. For 5 rabbits, 1–2 mL of blood was collected from the left or right lateral saphenous vein under gentle manual restraint via 1-in 25-Ga needles and 3-mL syringes. For the other 6 rabbits, 2 mL of blood was collected via direct intracardiac sampling under injectable anesthesia immediately before euthanasia via 1.5-in 21-Ga needles and 3-mL syringes. Injectable anesthesia was achieved with ketamine (35 mg/kg, IM)a and xylazine (5 mg/ kg, IM).b All blood samples were placed into EDTA microtainers or vacutainers and refrigerated (2–80C) until crossmatching within 2–4 hours of collection. 2.2 Blood product donors Rabbit recipients also served as conspecific rabbit blood donors using the previously detailed blood samples. Feline blood donors were from the authors’ academic veterinary teaching hospital blood donor cat colony and were previously blood typed using a commercial point-of-care immunochromatographic assay.c Canine blood donors were client-owned animals previously blood typed via a commercial laboratory.d All donors met the criteria outlined in the current American College of Veterinary Internal Medicine consensus statement on canine and feline blood donor screening for bloodborne pathogens.10 Three pigtail segments (aliquots of blood attached to the outside of a blood product unit used for crossmatching) were acquired from pRBC units from DEA-1-positive canine donors, DEA-1-negative canine donors, and feline type A donors collected within 1month prior to this study. Canine pRBC units contained the anticoagulant citrate– phosphate–dextrose, as well as a preservative solution containing adenine, dextrose, sorbitol, sodium chloride, and mannitol (ADSOL), and were stored at 2–50C. Feline pRBC units contained the anticoagulant– preservative solution citrate–phosphate–dextrose–adenine and were stored at 2–50C. Whole blood (~3 mL) was collected from a type B feline blood donor via the external jugular vein and placed into an EDTA vacutainer. 2.3 Major crossmatch procedure The major crossmatch procedure was adapted from a published veterinary protocol6 and the mammalian crossmatch standard operating procedure used by the authors’ veterinary teaching hospital clinical pathology laboratory. All crossmatches were performed and interpreted by an experienced nonblinded individual (NGD) within 2–4 hours of rabbit blood collection. Microtainers or vacutainers containing rabbit blood were acclimated to room temperature and centrifuged at 1000 × g for 10 minutes. Rabbit plasma was pipetted off and placed in a plastic transfer tube. Rabbit plasma hemolysis, icterus, and lipemia were assessed as absent, slight, mild, moderate, and gross compared with a visual key. Recipient 3% RBC suspensions were made for each rabbit. Using a calibrated pipette, 100 μL of blood was removed from the spun down microtainers or vacutainers and dispensed into a 12- × 75-mm glass tube labeled with the rabbit’s identification number. Recipient RBCs were resuspended by adding a small amount of phosphate-buffered saline (PBS) and mixing gently. The tube was filled three-fourths full of PBS, centrifuged for 60 seconds at 1000 × g, and the resulting supernatant was decanted, leaving a washed RBC pellet. This was repeated for a total of 3 washes. After the final wash, approximately 3 mL of PBS was added to the washed RBC pellet to produce a 3% RBC suspension. Donor 3% RBC suspensions were made for each blood type. Pigtail segments were pierced via a segment splitter, and approximately 2–3 drops of blood were squeezed into a 12- × 75-mm glass tube labelled with the respective blood type. For the feline type B 3% RBC suspension, 100 μL of blood was removed from the spun down vacutainer and dispensed into a labeled 12- × 75-mm glass tube. Donor RBCs were resuspended, washed 3 times, and used to produce a 3% RBC suspension as previously described. Blood from rabbit recipients underwent a major crossmatch with blood from each canine and feline blood donor type and 1 rabbit donor. Blood from rabbit recipients and rabbit donors was randomly paired via an online random pair generator.e For internal quality control, each rabbit sample underwent a major crossmatch with itself twice. Using disposable pipettes, 2 drops of rabbit plasma followed by 1 drop of the respective donor’s 3% RBC suspension were placed in a 12- × 75-mm glass tube labeled with the rabbit’s identification number and donor’s blood type. Tubes were gently mixed and centrifuged for 20 seconds at 1000 × g. The presence of hemolysis was interpreted compared with the rabbit recipient’s original plasma. The RBC pellet was gently resuspended, and the presence of macroscopic agglutination was interpreted with the aid of a macroscopic agglutination lamp reader. If no macroscopic agglutination was found, the tube was examined for microscopic agglutination using a tube holder and direct microscopy. Tubes were incubated at 370C for 30 minutes using a heat block and centrifuged for 20 seconds at 1000 × g. The presence of hemolysis and agglutination was interpreted as previously described. Hemolysis was graded as present or absent. Macroscopic agglutination was graded on a previously described scale of absent to 4+.6 Microscopic agglutination was graded as present or absent. >>> 10 Leading Article
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