Clinical Approach to Immune-Mediated Hemolytic Anemia and Thrombocytopenia.

Primary immune mediated hemolytic anemia (IMHA) and thrombocytopenia (IMTP) are rare in horses with the conditions more commonly occurring secondary to underlying disease. Several case reports have suggested a link between neoplasia and immune-mediated destruction of platelets and red blood cells. Diagnostic investigations should therefore focus on identifying possible underlying causes such as infections and neoplasia. Immunosuppressive therapy with corticosteroids and azathioprine is the mainstay of treatment but should be used cautiously in cases where underlying infection has not been excluded. Given the frequent association of secondary IMHA and IMTP cases with neoplasia, primary cases generally have a better prognosis.

Single-dose pharmacokinetics of mycophenolic acid following administration of immediate-release mycophenolate mofetil in healthy Beagle dogs.

Mycophenolic acid (MPA) is an immunomodulating agent commonly used in human medicine for the treatment of immune-mediated diseases. There is growing evidence that the immunomodulating properties of mycophenolate mofetil (MMF), a prodrug of MPA, are therapeutically beneficial for the treatment of immune-mediated diseases in dogs. A narrow therapeutic index and high inter-and intra-patient pharmacokinetic (PK) variability complicate the use of MMF. A better characterization of MPA pharmacokinetics is needed to help establish dosing regimens and standardized treatment protocols for canine patients. The purpose of this study was to evaluate the pharmacokinetics of MPA in dogs. MMF oral suspension (10 mg/kg) was administered to five healthy beagle dogs. Serial blood samples were collected from 0 to 18 hours after administration. The simultaneous quantification of MPA, and its metabolites MPA-7-O-glucuronide (MPAG), and acyl glucuronide (AcMPAG) was determined by liquid chromatography (LC)-mass spectrometry (MS)/MS. MPA peak concentrations were achieved rapidly (median Tmax of 0.5 h). Concentrations fell through 3 hours post-dose and then plateaued around 20% of Cmax. The mean elimination half-life was rapid (5.8 hours) and notable variability was observed in all PK parameters. The PK profiles for the MPAG and AcMPAG metabolites followed a similar pattern as MPA concentration. Future repeat-dose studies will be needed to evaluate steady-state PK parameters and to define therapeutic MPA dose levels.

Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review.

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box-1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

Immune Cytopenias after Ex Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation.

Immune-mediated cytopenias (ICs), such as immune thrombocytopenia and immune hemolytic anemia, are among the adverse events after allogeneic hematopoietic cell transplantation (allo-HCT). Previous reports suggest that in vivo T cell depletion may increase the incidence of IC after allo-HCT. We evaluated whether a strategy that reduces functional donor T cells via ex vivo CD34+-selection associates with the development of IC in a cohort of 408 patients who underwent allo-HCT for hematologic malignancy. The cumulative incidence of IC at 6, 12, and 36 months after the 30-day landmark post-HCT was 3.4%, 4.9%, and 5.8%, respectively. Among 23 patients who developed IC, 7 died of relapse-related mortality and 4 of nonrelapse mortality. A median 2 types of treatment (range, 1 to 5) was required to resolve IC, and there was considerable heterogeneity in the therapies used. In univariable analyses, a hematologic malignancy Disease Risk Index (DRI) score of 3 was significantly associated with an increased risk of IC compared with a DRI of 1 or 2 (hazard ratio [HR], 4.12; P = .003), and IC (HR, 2.4; P = .03) was associated with increased risk of relapse. In a multivariable analysis that included DRI, IC remained significantly associated with increased risk of relapse (HR, 2.4; P = .03). Our findings show that IC events occur with relatively similar frequency in patients after ex vivo CD34+-selected allo-HCT compared with unmodified allo-HCT, suggesting that reduced donor T cell immunity is not causative of IC. Moreover, we noted a possible link between its development and/or treatment and increased risk of relapse.

Effects of aspirin dose escalation on platelet function and urinary thromboxane and prostacyclin levels in normal dogs.

Established "low" aspirin dosages inconsistently inhibit platelet function in dogs. Higher aspirin dosages consistently inhibit platelet function, but are associated with adverse effects. The objectives of this study were to use an escalation in dosage to determine the lowest aspirin dosage that consistently inhibited platelet function without inhibiting prostacyclin synthesis. Eight dogs were treated with five aspirin dosages: 0.5 mg/kg q24h, 1 mg/kg q24h, 2 mg/kg q24h, 4 mg/kg q24h and 10 mg/kg q12h for 7 days. Utilizing aggregometry and a whole-blood platelet function analyzer (PFA-100), platelet function was evaluated before and after treatment. Urine 11-dehydro-thromboxane-B2 (11-dTXB2 ) and 6-keto-prostaglandin-F1α (6-keto-PGF1α ), were measured. Compared to pretreatment, there were significant post-treatment decreases in the maximum aggregometry amplitude and increases in the PFA-100 closure times for all dosages expect 0.5 mg/kg q24h. There was no difference in amplitude or closure time among the 2 mg/kg q24h, 4 mg/kg q24h, and 10 mg/kg q12h dosages. Compared to pretreatment values, there was a significant decrease in urinary 11-dTXB2 -to-creatinine and 6-keto-PGF1α -to-creatinine ratios, but there was no dose-dependent decrease for either metabolite. An aspirin dosage of 2 mg/kg q24h consistently inhibits platelet function without decreasing prostacyclin synthesis significantly more than lower aspirin dosages.

Immune-mediated hemolytic anemia in a bottlenose dolphin: Case report and literature review.

A 15-y-old captive-born female bottlenose dolphin (Tursiops truncatus) had slowly progressive lethargy, inappetence, and pallor of the oral mucosa. Serial hemograms and serum biochemistry analysis revealed hemolyzed plasma, gradually severe anemia with reticulocytosis, spherocytosis, pronounced neutrophilia with left shift and toxic change, and indirect hyperbilirubinemia. Urinalysis identified both hemoglobinuria and bilirubinuria. Hyperglobulinemia with polyclonal gammopathy was evident by protein electrophoresis, and the saline agglutination test was positive. Splenomegaly and hyperechogenic liver were also observed on ultrasound. These findings meet the diagnostic criteria established for immune-mediated hemolytic anemia (IMHA) in small animals, which is extremely rare in cetacean species, and, to our knowledge, has not been fully documented. Several potential triggers of a secondary IMHA were ruled out, including the most common cetacean virus, exposure to new drugs, and recent vaccination events. Following recommendations from companion animal medicine, immunosuppressive therapy was implemented and proved effective in this bottlenose dolphin; the CBC returned to normal after 3 mo of treatment.

Synovial extramedullary hematopoiesis in a dog.

OBJECTIVE: Synovial extramedullary hematopoiesis is a rarely reported condition in humans and, to date, has never been reported in canines. This case report describes the clinical presentation, diagnostic work-up, treatment, and outcome of a canine case confirmed to have hematopoietic tissue within multiple joints. ANIMAL: A client-owned canine. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The clinical presentation was most consistent with immune-mediated polyarthritis, and arthrocentesis was performed in multiple joints for cytological evaluation and culture. Cytology revealed evidence of extramedullary hematopoiesis, and shortly thereafter the dog was diagnosed with immune-mediated hemolytic anemia and thrombocytopenia. TREATMENT AND OUTCOME: Pregabalin, prednisolone, clopidogrel, and cyclosporine were started, and after several recheck appointments and dose adjustments, the dog's clinical signs resolved for all conditions. CLINICAL RELEVANCE: Unusual sites of extramedullary hematopoietic tissue may result in a clinical presentation for which more traditional etiologies and differentials are not applicable.

A case of cold agglutinin disease in a bottlenose dolphin (Tursiops truncatus).

Cold agglutinin disease, one of the serological classifications of immune-mediated hemolytic anemia, is caused by the production of autoantibodies that react with erythrocytes at low temperatures. A captive bottlenose dolphin presented with regenerative and hemolytic anemia. Anticoagulated whole blood was agglutinated at room temperature (approximately 18°C), with reversal of agglutination on warming to 37°C, indicating the presence of cold agglutinin. Based on these findings, this animal was diagnosed with cold agglutinin disease. Clindamycin, doxycycline, and prednisolone were administered orally to treat the infection and immune-mediated hemolytic anemia. Anemia gradually improved after initiation of pharmacotherapy, and erythrogenesis slowed as erythroblasts disappeared and reticulocyte count decreased in peripheral blood. This represents the first report of cold agglutinin disease in a cetacean.

Determination of optimal storage time and temperature for the detection of red blood cell and platelet surface-associated immunoglobulin by flow cytometry in healthy horses.

Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at 4 °C and PLT-sIg samples were stored at 4 °C and room temperature. RBC-sIg percentages were stable up to 72 h storage. Platelet surface-associated IgG percent positive platelets increased above baseline at all timepoints and percent positive platelets were inconsistent across timepoints for IgM and IgA. PLT-sIg testing should ideally be performed within 4 h of collection. In instances where this is not feasible, samples should be stored at 4 °C and analyzed no later than 24 h after collection. Whereas cutoff values for RBC-sIg remained similar across timepoints, results for PLT-sIg should be compared to time-specific cutoff or reference intervals established by the laboratory running the test.

Preliminary Study on Treatment Outcomes and Prednisolone Tapering after Marine Lipid Extract EAB-277 Supplementation in Dogs with Immune-Mediated Hemolytic Anemia.

Immune-mediated hemolytic anemia (IMHA) is a common autoimmune disorder in dogs with a high fatality rate and it remains a therapeutic challenge. The marine lipid extract, EAB-277, is a natural anti-inflammatory nutraceutical product. However, the effects of EAB-277 in IMHA dogs has rarely been investigated. The objective of this study is to assess the clinical effects of EAB-277 and prednisolone dose-tapering for supplemental therapy in IMHA dogs. Prednisolone was given to 18 anemic IMHA dogs according to a standard regimen. Six dogs were supplementally treated with EAB-277 for 28 days and the remaining twelve dogs were a control group of untreated supplementations. The results demonstrate that the supplement group showed slightly better survival rates (66.7 ± 19.2%) than the control group (16.7 ± 0.7%), but the difference was not statistically significant (p = 0.408). When compared to pre-therapy, the supplement group's blood profiles improved (p < 0.05). The EAB-277 treated group showed a moderate decrease in the incidence rate (4.20 times) of prednisolone tapering compared to the control group. The dosage reduction of prednisolone in supplement group was more than that in the control group (p < 0.0001). Our results suggest that EAB-277 supplementation may enhance clinical outcomes and lessen prednisolone dose-tapering in canine IMHA therapy.

Utility and prognostic significance of leukocyte ratios in dogs with Primary Immune-Mediated Hemolytic Anemia.

Canine immune-mediated hemolytic anemia (IMHA) is a life-threatening condition that is commonly associated with neutrophilia and monocytosis. Leukocyte ratios have been found to have prognostic value in humans and animals affected by a range of inflammatory, infectious, and neoplastic disorders. We hypothesized that in primary IMHA, neutrophil to lymphocyte (NLR), neutrophil to monocyte (NMR), band neutrophil to segmented neutrophil (BNR) and monocyte to lymphocyte (MLR) ratios would be higher in dogs that did not survive to discharge. Medical records of dogs diagnosed with IMHA at two veterinary teaching hospitals were retrospectively reviewed. Twenty-three of the 72 included dogs did not survive to discharge. NLR, NMR, BNR and MLR ratios were compared between dogs that survived to discharge and dogs that died or were euthanized. None of the ratios were significantly different between survivors and non-survivors (P = 0.14-0.99). Area under the Receiver Operating Characteristic (ROC) curve for prediction of non-survival ranged from 0.5 (95% confidence interval 0.38-0.62) for MLR to 0.61 (0.49-0.72) for NMR and was not significantly different from 0.5 for any ratio (P = 0.29-0.99). After exclusion of 31 dogs that received one or both immunosuppressive medications and blood transfusion before presentation, the area under the ROC curve for prediction of survival was significantly different from 0.5 for MLR (0.78, P = 0.01) and NMR (0.78, P = 0.0002). This study suggests that lower MLR and higher NMR may predict poorer prognosis in untreated dogs with IMHA.

Anticoagulant effects of rivaroxaban, prednisone, alone and in combination, in healthy dogs.

BACKGROUND: The potential effects of glucocorticoid administration on rivaroxaban's anticoagulant bioactivity in dogs, and an appropriate rivaroxaban dosage regimen for dogs receiving glucocorticoid therapy are unknown. HYPOTHESIS/OBJECTIVES: The objective was to determine whether glucocorticoid administration influences the anticoagulant effects of rivaroxaban in healthy dogs. We hypothesized that administration of rivaroxaban and prednisone would reduce the anticoagulant intensity compared with rivaroxaban alone. ANIMALS: Nine healthy dogs. METHODS: Randomized, cross-over study. Dogs were administered prednisone (2 mg/kg, PO, q24h), rivaroxaban (1.5 mg/kg, PO, q24h), or prednisone and rivaroxaban, and the coagulation status was evaluated using prothrombin time (PT), and rivaroxaban-calibrated anti-Xa activity (RIVA, results were log10 transformed for analysis), before drug administration and on days 2, 4, and 8. Linear mixed models and correlation were used to evaluate associations in variables (P < .05 was considered significant). RESULTS: There were no differences in RIVA results for the rivaroxaban and prednisone/rivaroxaban groups on day 8 (P = .599, median 87 [range 45-156] to 167 [56-333], respectively, median difference 90 ng/mL [95% CI:87.3-161.8]) There was a strong correlation between RIVA and PT results when days 2, 4, and 8 were combined (r = .846, P < .001), and increased during drug administration, day 2 (r = .810, P < .001), day 4 (r = .863, P < .001), and day 8 (r = .885, P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Clotting times in the PT correlate with rivaroxaban levels and may prove useful for drug monitoring. Prednisone administration had no apparent influence on the anticoagulant effects of rivaroxaban in healthy dogs, suggesting that combined therapy will not require dosage adjustments.

Pulmonary thromboembolism due to immune-mediated hemolytic anemia in a cat: A serial study of hematology and echocardiographic findings.

Pulmonary thromboembolism (PTE) secondary to immune-mediated hemolytic anemia (IMHA) is rarely diagnosed in cats. In this report, a 3-year-old cat was referred to our private hospital with dyspnea, anorexia, and anemia. On the thoracic radiography, cardiac enlargement and pulmonary edema were noted. Echocardiography revealed right ventricular and right atrium enlargement with mild tricuspid regurgitation (tricuspid regurgitation velocity 3.28 m/s). A thrombus was recognized in the main pulmonary artery on the right parasternal short-axis view. Blood examination suggested regenerative anemia and autoagglutination. The findings suggested immune-mediated hemolytic anemia and PTE. Antithrombotic therapy (dalteparin) and immunosuppressive therapy (prednisolone) were administered under oxygen supplementation in the ICU cage. After treatment, regenerative anemia and right-heart failure were improved. During follow-up, the cat remained hemodynamically stable, and the owner reported no cardiac-related clinical signs. Further blood examination confirmed the anemia was improved. Prednisolone was discontinued on Day 56, and the cat continues in good health, administered only mycophenolate mofetil. The clinical outcome of PTE secondary to the IMHA throughout 100 days of periodical observation was reported.

Comparative study of immunohematological tests with canine blood samples submitted for a direct antiglobulin (Coombs') test.

BACKGROUND: A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. MATERIAL AND METHODS: Anticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics; neutral gel column cards, microtiter plates (at 4°, 22°, and 37°C), capillary tubes, and flow cytometry used in laboratories. RESULTS: Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1-24 weeks after initial assessment. CONCLUSIONS: Based upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable.

Evaluation of membrane-based therapeutic plasma exchange as adjunctive treatment for immune-mediated hematologic disorders in dogs.

BACKGROUND: Therapeutic plasma exchange (TPE) has been used increasingly to treat immunological diseases in dogs, although data concerning its efficacy are lacking. HYPOTHESIS/OBJECTIVES: To describe the clinic and the outcome of dogs with immune-mediated hematological disorders (IMHD) treated with membrane filtration TPE. ANIMALS: One hundred forty-six dogs diagnosed with IMHD, including 17 dogs treated with and 129 control dogs treated without TPE. METHODS: Retrospective study of cases selected with a search of all medical records for dogs diagnosed with IMHD (2010-2019). Primary outcomes included the assessment of safety and efficacy of adjunctive TPE compared to medical treatment alone. RESULTS: The TPE group included 7 dogs with immune-mediated hemolytic anemia (IMHA), 5 dogs with immune-mediated thrombocytopenia (IMT), and 5 dogs with combined IMHA-IMT; the control group included 63 dogs with IMHA, 47 dogs with IMT, and 19 dogs with IMHA-IMT. Dogs treated with TPE were more often refractory to initial immunosuppression (71%) than controls (43%; P = .04). Complications were observed in 15/43 TPE treatments (35%). The response rate of dogs treated with TPE was 83% in IMHA (controls, 65%; P = .5) and 80% in IMT (controls, 70%; P = .71). Overall, 12/17 dogs (71%) treated with TPE reached complete remission, 2/17 (12%) partial remission, and 3/17 (18%) died or were euthanized. Eighty-two percent of the dogs treated with TPE survived to discharge (controls, 69%; P = .4). CONCLUSIONS AND CLINICAL IMPORTANCE: Despite a bias toward dogs refractory to initial immunosuppression, dogs treated with adjunctive TPE had a similar outcome as dogs treated medically.

Precursor-targeted immune-mediated anemia in a dog with a stage IV mast cell tumor and bone marrow infiltration.

A 12-year-old spayed female Shiba Inu dog was referred to our hospital for a suspected mast cell tumor (MCT) of the bone marrow (BM). Laboratory abnormalities included severe nonregenerative anemia (packed cell volume or PCV: 12.5%; reference interval (RI): 37.3-61.7%; reticulocytes: 35.1 × 103 /µL; RI: 10-110 × 103 /µL), and a few mast cells were visualized in the blood smear examination. The BM was hypercellular with hematopoietic cells, a decreased myeloid:erythroid (M:E) ratio (0.77; RI, 0.9-1.8), and no dysplastic hematopoietic cells. Mast cells accounted for 11.5% of the total nucleated BM cells. Neoplastic mast cells and histiocytes phagocytizing erythroid progenitor cells were occasionally noted. The dog was diagnosed with precursor-targeted immune-mediated anemia (PIMA) concurrent and a stage IV MCT infiltrating the BM. Multimodal treatment included toceranib, imatinib, vinblastine, lomustine (CCNU), prednisolone, cyclosporine, mycophenolate mofetil, and a blood transfusion. The dog died due to MCT progression lasting 139 days after the initial BM examination. To the best of our knowledge, this is the first report of a dog presenting with PIMA and a stage IV MCT infiltrating the BM.

Presumptive hemophagocytic syndrome associated with immune-mediated anemia in two Miniature Dachshunds.

This report describes the cases of two Miniature Dachshunds who were suspected to have immune-mediated hemolytic anemia (IMHA) and were treated with immunosuppressive therapy. However, progression of anemia, increases in C-reactive protein (CRP) and total-bilirubin (T-Bil) levels, splenomegaly, transition to nonregenerative anemia, and thrombocytopenia occurred after the treatment. Splenectomy and bone-marrow aspirations were performed subsequently. Both dogs were diagnosed with hemophagocytic syndrome (HPS) associated with IMHA. Unfortunately, they died 9 and 6 days later. These findings indicate that some cases of refractory IMHA have the pathogenicity of HPS. HPS should be included as a differential diagnosis of refractory IMHA concurrent with thrombocytopenia. Continuously elevated CRP and T-Bil levels may be helpful indicators in the detection of HPS associated with IMHA.

Immunoglobulin G and phosphatidylserine in regenerative and nonregenerative immune-mediated anemias of dogs.

BACKGROUND: Although precursor-targeted immune-mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA. HYPOTHESIS: Dogs with PIMA have increased nRBC IgG and PS, and dogs with immune-mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs. ANIMALS: Blood from 20 healthy dogs and from dogs with IMHA (11) or other (non-IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non-IMHA, non-PIMA) conditions (7). METHODS: Marrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS. RESULTS: Red blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non-IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non-IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid- or late-stage fractions, whereas all 7 non-PIMA dogs were negative. Mid- and late-stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity. CONCLUSIONS: Immunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.

Effects of clopidogrel and prednisone on platelet function in healthy dogs.

BACKGROUND: Glucocorticoids cause hypercoagulability, but it is unknown if they counteract clopidogrel's antiplatelet effects. HYPOTHESIS/OBJECTIVES: Determine the effects of clopidogrel and prednisone on platelet function. ANIMALS: Twenty-four healthy dogs. METHODS: Double-blinded, placebo-controlled randomized trial. Platelet function was evaluated using a platelet function analyzer and impedance aggregometry (days 0, 14, and 28) for dogs treated with placebo, clopidogrel (2-3 mg/kg/d), prednisone (2 mg/kg/d), or prednisone with clopidogrel PO for 28 days. Results were categorized as nonresponder versus responder (platelet function analyzer), and inadequate, ideal, or excessive response (aggregometry). Results were compared using mixed model, split-plot repeated measures analysis of variance and generalized estimating equation proportional odds models. P < .05 was considered significant. RESULTS: Closure times differed by treatment (F [3, 20] = 10.5; P < .001), time (F [2, 40] = 14.3; P < .001), and treatment-by-time (F [6, 40] = 3.4; P = .01). Area under the curve (AUC) differed by treatment (F [3, 20] = 19.6; P < .001), time (F [2, 40] = 35.4; P < .001), and treatment-by-time (F [6, 40] = 13.5; P < .001). Based on closure times, 5/6 dogs each in the clopidogrel and prednisone/clopidogrel groups were responders. All dogs in the prednisone/clopidogrel group were overcontrolled based on AUC (days 14 and 28), whereas 5/6 (day 14) and 2/6 (day 28) dogs treated with clopidogrel were overcontrolled. Compared to clopidogrel, dogs receiving prednisone/clopidogrel were 11 times (P = .03) more likely to have an excessive response. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of clopidogrel/prednisone increases platelet dysfunction in healthy dogs.

Effect of dilution of canine blood samples on the specificity of saline agglutination tests for immune-mediated hemolysis.

BACKGROUND: Saline agglutination tests (SATs) are widely recommended for diagnosis of immune-mediated hemolytic anemia in dogs, but there are frequent false-positive results. OBJECTIVES: Specificity of SATs will improve at higher saline-to-blood ratios. ANIMALS: One hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%. METHODS: Prospective diagnostic accuracy study. Immune-mediated hemolysis (IMH) was considered present if a gel direct antiglobulin test (DAT) was positive and there was clinical evidence of hemolysis (n = 9), absent if another mechanism for anemia was identified and the DAT was negative or there was no hemolysis (n = 138), and if IMH status was unclear, dogs were excluded (n = 3). Saline agglutination tests were prepared at 1 : 1, 4 : 1, 9 : 1, and 49 : 1 saline-to-blood ratios, and microscopic agglutination was considered a positive result. RESULTS: Specificity for IMH increased from 29% (95% confidence interval 20-38) at a 1 : 1 dilution to 97% (93-99) at a 49 : 1 dilution. Sensitivity was 88% (47-100) at 1 : 1 and 4 : 1 dilutions and 67% (30-93%) at 9 : 1 and 49 : 1 dilutions. Diagnostic accuracy increased from 33% (24-42) at 1 : 1 dilution to 95% (90-98) at 49 : 1 dilution. CONCLUSIONS AND CLINICAL IMPORTANCE: If performed using a 49 : 1 saline-to-blood ratio, SATs achieve high specificity for IMH. Based on a gold standard of positive DAT and evidence of hemolysis, lower saline-to-blood ratio results should not be used because false-positive results are common.