Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Prevalence of dog erythrocyte antigens in retired racing Greyhounds

Background: Blood groups in dogs are designated as dog erythrocyte antigen (DEA) 1.1, 1.2, 3, 4, 5, 7, and Dal. There is limited information about the frequency of different antigens in Greyhound dogs, despite their frequent use as blood donors. Objectives: The aims of this study were to determine the frequencies of DEA 1.1, 1.2, 3, 4, 5, and 7 in Greyhounds, to compare the frequencies with those of non‐Greyhound dogs, and to evaluate the presence of naturally occurring anti‐DEA antibodies. Methods: Blood was collected from 206 Greyhound and 66 non‐Greyhound dogs being screened as potential blood donors. Blood‐typing was performed at Animal Blood Resources International by tube agglutination utilizing polyclonal anti‐DEA antibodies. Results: Of the Greyhound dogs, 27/206 (13.1%) were positive for DEA 1.1, and this frequency was significantly lower (P<.0001) than for non‐Greyhound dogs of which 40/66 (60.6%) were DEA 1.1‐positive. The frequency of positivity for both DEA 1.1 and 1.2 was also lower in Greyhounds (P<.0001). There were no significant differences between Greyhounds and non‐Greyhounds for DEA 1.2, 3, 4, 5, or 7. All 137 dogs (113 Greyhounds and 24 non‐Greyhounds) that were evaluated for naturally occurring anti‐DEA antibodies in serum were negative. A higher percentage of Greyhound dogs (57.3%, 118/206) were considered “universal donors” (negative for all DEAs except DEA 4) compared with non‐Greyhound dogs (28%, 13/46). Conclusion: The frequency of positivity for DEA 1.1 in our population of Greyhounds was significantly lower than previously reported for dogs. Furthermore, a large majority of Greyhounds met the criteria for universal donors.     

Evaluation of the utility and accuracy of body fluids containing red blood cells to determine canine and feline blood types

Objective

To examine the accuracy of using body fluids macroscopically suspected to contain erythrocytes to determine the blood type in dogs and cats by use of an immunochromatographic cartridge (ICC), compared to systemic blood as the reference standard.

Design

Prospective study.

Setting

University teaching hospital.

Animals

Thirty client-owned dogs and 8 cats.

Interventions

Dogs and cats with a sanguineous or serosanguineous body fluid (SBF) that also required a blood sample were eligible for inclusion. PCV and blood type were determined in all blood and fluid samples. For body fluids with a low PCV and discordant blood type results compared to systemic blood, sample concentration and repeat blood typing from the fluid was performed when enough sample was available.

Measurement and Main Results

Body fluid samples consisted of 16 pleural (11 dogs; 5 cats), 12 peritoneal (10 dogs; 2 cats), and 4 canine pericardial effusions, 3 urine samples, and 1 each of feces and epistaxis from dogs and a seroma sample from a cat. Median (range) manual PCV of blood and fluid samples was 34% (14%–66%) and 6% (0.5%–70%) for dogs and 28% (14%–48%) and 14% (0.5%–19%) for cats, respectively. Dogs were correctly classified as being DEA 1 negative, DEA 1 positive, and DEA 1 weak positive when using body fluid for blood typing 13 of 14, 4 of 9, and 5 of 7, respectively. All reference blood type to fluid blood type (FBT) discordant results had a body fluid PCV equal to or below 2%. Subsequently concentrated body fluid samples had a PCV above 8% and repeat FBT matched reference blood type (RBT). All cats were classified as type A by all RBTs and FBTs.

Conclusions

Body fluids containing erythrocytes may be utilized to blood type dogs if sufficiently concentrated and type A cats.     

The breed prevalence of dog erythrocyte antigen 1.1 in the Onderstepoort area of South Africa and its significance in selection of canine blood donors

The blood group antigen Dog Erythrocyte Antigen (DEA) 1.1 is clinically the most important canine blood group as DEA 1.1 antibodies are capable of causing acute haemolytic, potentially life-threatening transfusion reactions. Dogs do not have naturally occurring antibodies to DEA 1.1 but are rapidly sensitised by the first incompatible transfusion. The prevalence of DEA 1.1 in the general dog population is estimated at 42-46%. Canine blood donors registered with the Onderstepoort Animal Blood Bank (n = 93) as well as potential donors (n = 140) were typed for DEA 1.1 using a monoclonal antibody card kit. All dogs came from the Onderstepoort area, near Pretoria, Gauteng province, South Africa. Overall prevalence of DEA 1.1 was 47%. Prevalence was 47% in purebred dogs and 48% in mongrels. Distinct breed differences were noted with less than 20% of German shepherd dogs and Boxers and greater than 75% of Rottweilers, Great Danes, St Bernards and Dalmations testing DEA 1.1 positive. Knowledge of local breed differences will increase effectiveness of blood donor recruitment.     

Comparison of gel column agglutination with monoclonal antibodies and card agglutination methods for assessing the feline AB group system and a frequency study of feline blood types in northern Italy

Background: A new commercial gel column agglutination system is reported to have high sensitivity in detecting cats with blood type AB. Objectives: The aims of this study were to compare gel column agglutination and card agglutination methods for feline blood‐typing and to determine the frequency distribution of feline blood types in northern Italy. Methods: Blood‐typing was performed on 120 cats using both a commercial gel column containing monoclonal antibodies (ID Gel‐Test Micro Typing System) and a card agglutination method (RapidVet‐H Feline). Results were confirmed with back‐typing. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the 2 methods. A second group of 140 Domestic Shorthair (DSH) cats was blood‐typed using the gel column technique to determine the frequency distribution of feline blood types in northern Italy. Results: The card agglutination method demonstrated poor sensitivity in identification of type‐AB cats (61%) and was only 95% specific when identifying type‐B cats. The gel column agglutination technique demonstrated 100% sensitivity and specificity for typing all 3 blood types (A, B, and AB). The frequency distribution study of 140 cats demonstrated that 127 (90.7%) cats were type A, 10 (7.1%) were type B, and 3 (2.1%) were type AB. Conclusion: When blood‐typing cats of breeds with a relatively high frequency of blood types B and AB, methods that use monoclonal antibodies for detection of blood types B and AB are recommended. Alternatively, blood type can be confirmed by more sensitive supplemental testing, such as back‐typing. The high frequency of blood type A in DSH cats in northern Italy was comparable to previously reported frequencies in Italy and world‐wide.