Cytologic features of canine melanotroph and corticotroph pituitary adenomas.

BACKGROUND: The introduction of intraoperative cytology revolutionized neurosurgical procedures in human medicine, providing real-time diagnostic guidance to surgeons and contributing to improved patient outcomes. In the realm of veterinary medicine, the understanding of pituitary tumors in dogs and cats remains limited due to challenges in obtaining antemortem samples of central nervous system lesions. OBJECTIVES: The aim of this study was to describe the cytologic features of pituitary adenomas in 12 dogs that underwent hypophysectomy. METHODS: The series included nine melanotroph adenomas and three corticotroph adenomas. Definitive diagnosis was based on histopathology and immunohistochemistry. RESULTS: Cytologically, the adenomas had high numbers of bare nuclei and intact cells that were round to polygonal and situated individually or in small clusters. The intact cells had round to oval, eccentric nuclei with finely stippled chromatin and one to three prominent nucleoli and ample to abundant lightly basophilic to amphophilic, grainy cytoplasm with distinct borders, and variable numbers of discrete vacuoles. Mild-to-moderate anisocytosis and anisokaryosis, occasional binucleation, rare and atypical mitotic figures, and nuclear molding were also observed. CONCLUSIONS: The results suggest that intraoperative cytology of canine pituitary adenomas holds promise as a valuable diagnostic tool, aiding swift differentiation from other sellar masses before histologic confirmation. Cytologic characterization of pituitary adenomas in dogs is exceptionally rare in the scientific literature, making this study one of the first to offer a comprehensive description of these cytologic features.

Evaluation of platelet additive solution for prolonging storage of functional canine platelet concentrate.

OBJECTIVE: To assess storage lesion development, platelet function, and bacterial growth in canine platelet concentrates (PCs) stored in a platelet additive solution (PAS) or a plasma control at 4°C for 21 days. DESIGN: Prospective, ex vivo, experimental controlled study. SETTING: University veterinary teaching hospital. ANIMALS: Ten units of canine PCs collected from blood bank donations. INTERVENTIONS: The PCs were separated into 2 bags, 1 containing 100% plasma and the other containing 35% plasma and 65% of a PAS (Plasma-Lyte A), and stored at 4°C for 21 days. At days 0, 7, 14, and 21, PCs were analyzed for the presence of swirling, aggregate formation, platelet counts, platelet indices, glucose, lactate, lactate dehydrogenase, Pvco2 , Pvo2 , aggregation via light aggregometry, activation percentages using flow cytometry, and bacterial growth. MEASUREMENTS AND MAIN RESULTS: Cold-stored PCs in both PAS and plasma control maintained mean pH >6.8 and mean lactate <9.0 mmol/L over 21 days, with no difference in glucose utilization. Swirl was maintained in both solutions for most days (76/80 combined total samples), with no difference in aggregate formation between solutions. The Pvco2 was higher in plasma on all days (P < 0.001), with no difference in Pvo2 . Platelet indices did not reflect significant storage lesion development in either solution. Lactate dehydrogenase did not differ between solutions but did increase from day 7 to day 21. Mean maximal aggregation percentage was reduced overall but with no significant difference between solutions. The only observed difference in mean activation percentage between solutions was in PAS on day 7, which was significantly higher than plasma (P < 0.05). No bacterial growth occurred during storage. CONCLUSIONS: Cold storage in PAS and plasma allowed PCs to be stored for up to 21 days with minimal storage lesion development, maintenance of platelet function, limited platelet activation, and no bacterial growth within stored bags.

The effects of additive solutions on the development of storage lesions in canine platelet concentrates stored at 4°C.

OBJECTIVE: To assess platelet storage lesion development as evaluated by measurement of metabolic markers, platelet activation markers, and aggregometry, and determine the occurrence of bacterial growth in platelets stored in platelet additive solution (PAS) at 4°C for 7 days. DESIGN: Prospective, ex vivo experimental controlled study. SETTING: Research laboratory of a university veterinary teaching hospital. ANIMALS: Ten units of canine platelet concentrate collected from blood bank donations. INTERVENTIONS: Concentrates were aliquoted into 4 separate bags containing 100% plasma (control) or 30% plasma and 70% of a PAS (Plasma-Lyte A, Isoplate, or InterSol). Samples were stored at 4°C without agitation. At days 0, 3, 5, and 7, samples were analyzed for platelet count, mean platelet volume, glucose, lactate, lactate dehydrogenase, Po2 , Pco2 , degree of swirling, aggregate formation, aggregation via light aggregometry, surface P-selectin via flow cytometry, and bacterial contamination via culture. MEASUREMENTS AND MAIN RESULTS: Development of storage lesions was minimal, demonstrated by maintenance of a mean pH > 7.2 and mean lactate values <6 mmol/L at day 7 in all solutions. Glucose utilization did not vary significantly between any of the solutions. No significant difference was found between plasma and PAS for Po2 and Pco2 . P-selectin expression measured via flow cytometry showed a low platelet activation percent in all the solutions. InterSol had the lowest mean maximum percent aggregation (P < 0.001) and Isoplate the highest (P < 0.05). The mean maximum percent aggregation increased between day 0 and day 7 in all solutions. No bacterial growth was found in any of the solutions. CONCLUSIONS: Overall, PASs were comparable to plasma for the cold storage of platelets. Cold-stored platelets showed minimal storage lesion development with no bacterial growth. Plasma-, Plasma-Lyte A-, and Isoplate-stored platelets maintained function for up to 7 days at 4°C.

Development and implementation of a hemovigilance program at a university veterinary teaching hospital.

OBJECTIVE: To describe the development and implementation of a small animal hemovigilance program at a university veterinary teaching hospital. DESIGN: Retrospective observational descriptive study performed between October 2014 and March 2019. SETTING: University teaching hospital. ANIMALS: Dogs and cats receiving blood product transfusions . INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS:  A hemovigilance working group composed of veterinary specialists in clinical pathology, internal medicine, and emergency and critical care was established. This group developed evidence-based definitions of transfusion reactions, reaction classification systems, and a transfusion reaction reporting form. The reporting form contained sections for patient information, transfusion information, administration details, and reaction details. Reaction events were classified by reaction type, severity grade, and imputability to the transfusion. Following implementation of the hemovigilance program, transfusion reaction data were collected and examined for the period spanning October 2014 and March 2019. During the study period, 718 canine transfusions (4 whole blood, 400 packed RBC [pRBC], 300 fresh frozen plasma [FFP], 7 platelet rich plasma, and 7 cryoprecipitate) and 124 feline transfusions (5 whole blood, 95 pRBC, and 24 FFP) were administered. There were 32 total reactions (27 canine and 5 feline), with the most common reaction being febrile nonhemolytic transfusion reactions (19/32; 59%). The incidence rate of transfusion reactions was found to be 3.8% in dogs and 4.0% in cats. For the confirmed reactions, classification criteria for case definition, reaction severity grade, and imputability were able to be determined and recorded. This allowed targeted interventions to be implemented in order to potentially reduce future reactions. CONCLUSIONS: A hemovigilance program can be instituted successfully in a veterinary hospital setting and once developed, standardized reporting tools could be utilized by multiple hospitals and provide the basis for more widespread reaction reporting in veterinary medicine.

Association of Veterinary Hematology and Transfusion Medicine (AVHTM) Transfusion Reaction Small Animal Consensus Statement (TRACS). Part 1: Definitions and clinical signs.

OBJECTIVE: To use a systematic, evidence-based consensus process to develop definitions for transfusion reactions in dogs and cats. DESIGN: Evidence evaluation of the literature was carried out for identified transfusion reaction types in dogs and cats. Reaction definitions were generated based on synthesis of human and veterinary literature. Consensus on the definitions was achieved through Delphi-style surveys. Draft recommendations were made available through industry specialty listservs and comments were incorporated. RESULTS: Definitions with imputability criteria were developed for 14 types of transfusion reactions. CONCLUSIONS: The evidence review and consensus process resulted in definitions that can be used to facilitate future veterinary transfusion reaction research.

Association of Veterinary Hematology and Transfusion Medicine (AVHTM) Transfusion Reaction Small Animal Consensus Statement (TRACS) Part 2: Prevention and monitoring.

OBJECTIVE: To systematically review available evidence to develop guidelines for the prevention of transfusion reactions and monitoring of transfusion administration in dogs and cats. DESIGN: Evidence evaluation of the literature (identified through Medline searches through Pubmed and Google Scholar searches) was carried out for identified transfusion reaction types in dogs and cats. Evidence was evaluated using PICO (Population, Intervention, Comparison, Outcome) questions generated for each reaction type. Evidence was categorized by level of evidence (LOE) and quality (Good, Fair, or Poor). Guidelines for prevention and monitoring were generated based on the synthesis of the evidence. Consensus on the final recommendations and a proposed transfusion administration monitoring form was achieved through Delphi-style surveys. Draft recommendations and the monitoring form were made available through veterinary specialty listservs and comments were incorporated. RESULTS: Twenty-nine guidelines and a transfusion administration monitoring form were formulated from the evidence review with a high degree of consensus CONCLUSIONS: This systematic evidence evaluation process yielded recommended prevention and monitoring guidelines and a proposed transfusion administration form. However, significant knowledge gaps were identified, demonstrating the need for additional research in veterinary transfusion medicine.

Dal-induced red blood cell incompatibilities in a Doberman Pinscher with von Willebrand factor deficiency and ehrlichiosis.

OBJECTIVE: To describe a complex case involving the management of a dog with von Willebrand disease (vWD), active ehrlichiosis infection, nonregenerative anemia, and blood type incompatibility related to the Dal antigen. CASE SUMMARY: A 13-week-oldintact male Doberman Pinscher weighing 7.2 kg was presented to the emergency service for a previous hemorrhaging event and progressive nonregenerative anemia. The dog had received a fresh whole blood transfusion 8 days prior to presentation due to severe anemia. Upon presentation, the puppy was tachycardic, and his mucous membranes were pale. A CBC revealed a nonregenerative anemia with a PCV of 0.11 L/L (11%). von Willebrand factor deficiency was suspected and later confirmed. The dog's blood type was dog erythrocyte antigen (DEA) 1 positive, but cross-matching to 4 RBC units, both DEA 1 positive and negative, failed to yield any compatible units. Antibody against a possible Dal RBC antigen was suspected, and 11 blood donors (Dalmatians and Dobermans) were cross-matched to find 2 compatible donors. After an uneventful fresh whole blood transfusion, a bone marrow biopsy revealed a hypocellular bone marrow and erythroid hypoplasia. A SNAP4DxPlus test and subsequent polymerase chain reaction (PCR) testing were positive for Ehrlichia ewingii and E. canis. Treatment with doxycycline was started, and the PCV was 0.17 L/L (17%) at discharge. At the 1-week follow-up, the PCV was 0.24 L/L (24%), and the puppy was doing well. NEW OR UNIQUE INFORMATION PROVIDED: This is a unique case of a dog presenting with several challenging disorders, including vWD resulting in hemorrhage, ehrlichiosis potentially contributing to a nonregenerative anemia, and a blood type incompatibility due to the Dal antigen. Doberman Pinschers have a high prevalence of vWD- and Dal-negative phenotype, which emphasizes the value of cross-matching and the recognition of antigen prevalence in specific breeds.

Development and assessment of a formal learning module to educate veterinary students in an intensive care unit about transfusion reactions.

OBJECTIVE: To develop and assess the instructional efficacy of an online learning module on transfusion reactions in small animals and to evaluate participants' satisfaction of the module. DESIGN: Randomized controlled trial. SETTING: University teaching hospital. SUBJECTS: A total of 55, fourth-year veterinary students, 27 in a treatment group that received the learning module plus standard rotation training and 28 in a control group (no module) who received only standard training INTERVENTIONS: Students received a pretest on transfusion reactions followed by administration of a transfusion reaction learning module covering recognition, treatment, prevention, case examples, and self-assessment questions for 6 common transfusion reactions. Students also received a module satisfaction survey, a post-test at 2 weeks post-module, and a retention test at 6 weeks post-module. MEASUREMENTS AND MAIN RESULTS: Previous transfusion medicine exposure did not affect pretest scores and there was no difference in pretest scores between groups. The module group scored higher on the post-test (P < 0.001) and retention test (P = 0.002) than the control group. Mean post-test scores were 74.4% and 57.7% and mean retention test scores were 80.6% and 56.5% for the module and control groups, respectively. The module group scored higher on posttest and retention questions involving reaction recognition (P < 0.001). Students were overall very satisfied with the module with an average score of 4.8 (1-5). CONCLUSIONS: A transfusion reaction instructional module can be delivered successfully to veterinary students on an ICU-based clinical rotation. Students taking the module scored significantly better on post-assessments up to 6 weeks after module administration as compared to students receiving only conventional clinical rotation training.

Comparison of established and novel methods for the detection and enumeration of microparticles in canine stored erythrocyte concentrates for transfusion.

BACKGROUND: Microparticles (MP) are submicron, phosphatidylserine (PS)-bearing lipid vesicles with physiologic and pathologic roles in coagulation and inflammation. Microparticles accumulate in packed RBC (pRBC) stored for transfusion, potentially increasing recipient morbidity. Historically, canine MP have been detected with the PS label annexin V in supernatant samples. Other detection methods are available but have not been evaluated in dogs. OBJECTIVES: The purpose of the study was to detect and enumerate MP in canine pRBC using annexin V, lactadherin, and bio-maleimide to compare label performance and assess microparticle accumulation under standard storage conditions. METHODS: Microparticles (0.5-1.0 µm) in canine dog erythrocyte antigen 1.1 positive, nonleukoreduced pRBC were labeled with FITC-annexin V, FITC-lactadherin, and the fluorescent dye bio-maleimide, and were counted using flow cytometry at 3 time points (days 7, 21, and 35) of storage. Unprocessed pRBC, rather than supernatant, were used. RESULTS: Annexin V and bio-maleimide labeling produced comparable microparticle counts (P = .16), while lactadherin labeling resulted in higher microparticle counts than annexin V (P = .002) and bio-maleimide (P = .006), particularly on day 7. Bio-maleimide- and annexin V-based microparticle counts increased significantly from day 7 to 35 (P = .04), and increases from day 21 to 35 approached statistical significance (P = .05). CONCLUSIONS: Bio-maleimide- and annexin V-mediated microparticle counts were comparable in unprocessed canine pRBC using flow cytometry. Whether the increased microparticle counts with lactadherin were due to increased sensitivity for small, PS-bearing MP or due to labeling of membrane fragments and debris requires further investigation.

Coombs' testing and its diagnostic significance in dogs and cats.

The Coombs' test can detect both immunoglobulin and complement on the surface of RBCs, and as such can be of value as an aid in the diagnosis of IMHA. Techniques that may improve sensitivity include use of monovalent reagents, increased dilutions of antiglobulin to avoid a prozone effect, and testing at 4°C. These techniques are not without controversy, and positive tests should always be interpreted in the presence of other clinical and hematologic evidence for IMHA. Alternate techniques, such as flow cytometry, can improve detection of RBC-bound immunoglobulin, but require a flow cytometer and further standardization between laboratories.

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells.

UNLABELLED: There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities. RESULTS: Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45-71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages. CONCLUSION: The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.

Pseudothrombocytopenia secondary to the effects of EDTA in a dog.

Pseudothrombocytopenia (PTCP) secondary to the effects of ethylenediaminetetraacetic acid (EDTA) has been noted in horses and pigs and should be considered in dogs with moderate thrombocytopenia and no clinical bleeding tendency. This type of pseudothrombocytopenia is not a pathological process by itself, but it can be clinically significant if diagnostics and medical treatments are initiated based on the reported thrombocytopenia. Platelet clumping occurs with EDTA-dependent PTCP, resulting in inaccurate hematology analyzer platelet concentrations. A nontraumatic venipuncture may be sufficient to obtain an accurate platelet count. However, rare cases in the dog may require blood drawn into a different anticoagulant, such as sodium citrate, to help discriminate a true thrombocytopenia from PTCP.

Assessment of three automated assays for C-reactive protein determination in dogs.

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.

Detection of activated platelets in canine blood by use of flow cytometry.

OBJECTIVE: To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood. SAMPLE POPULATION: Blood samples from 10 dogs. PROCEDURE: Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine. RESULTS: A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.

The Coombs' test in veterinary medicine: past, present, future.

The Coombs' test, also known as the antiglobulin test, is used most frequently in veterinary medicine as an aid in the diagnosis of immune-mediated hemolytic anemia. The test also is used widely in human medicine to screen for red blood cell alloantibodies. Polyspecific reagents for veterinary use typically contain anti-IgG, anti-IgM, and anti-C3. Monospecific reagents also are available. False-positive and false-negative test results can be obtained. Inadequate sensitivity of the standard test in human and veterinary medicine has necessitated development of alternate, more sensitive technologies.

Systemic Mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat.

Dogs may be infected by Mycobacterium (M.) tuberculosis, M. bovis, and M. avium complex, and the clinical signs associated with each of these infections may be indistinguishable. Rapid speciation of the infecting organism is desirable because of the public health concerns associated with M. bovis and M. tuberculosis infections. A mycobacterial infection was suspected in the dog of this report based on acid-fast staining of organisms in macrophages obtained from liver aspirates and buffy-coat preparations. Polymerase chain reaction (PCR) analysis of a buffy-coat preparation identified M. avium.

Canine and feline blood donor screening for infectious disease.

Thousands of blood transfusions are performed each year on dogs and cats, and the demand for blood products continues to grow. Risks associated with transfusions include the risk of disease transmission. Appropriate screening of blood donors for bloodborne infectious disease agents should be performed to lessen this risk. Geographic restrictions of disease, breed predilection, and documentation of actual disease transmission by transfusion all are factors that might need to be considered when making a decision on what screening program to use. In addition, factors involving general health care and management of blood donors should be employed to further ensure blood safety.