Evaluation of flow cytometric and automated methods for detection of activated platelets in dogs with inflammatory disease

OBJECTIVE: To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease. ANIMALS: 20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease. Procedures-Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets. Results-12 dogs with inflammatory disease had P-selectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet surface-associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease.     

High intravascular tissue factor expression in dogs with idiopathic immune-mediated haemolytic anaemia

A high mortality occurs in dogs with idiopathic immune-mediated haemolytic anaemia (IMHA) during the first 2 weeks after the diagnosis. The aim of this study was to investigate the inflammatory response and coagulation abnormalities in dogs with IMHA in relation to the prognosis and to establish the contribution of whole blood tissue factor (TF) and IL-8 gene expressions. Gene expressions in dogs with IMHA were compared to healthy dogs, dogs with DIC, dogs with sepsis, and in two groups of dogs that underwent intensive care treatment but had no evidence for either DIC or sepsis. The whole blood TF and IL-8 expressions were up regulated in all non-IMHA groups. Similarly, the TF expression in IMHA dogs was high, but the intravascular IL-8 expression was not increased. The dogs with IMHA had a pronounced inflammatory response that included a high WBC, left shift and monocytosis in comparison to the other disease groups. Coagulation factor activities in IMHA dogs were decreased fitting consumptive coagulopathy and the acute phase proteins FVIII and fibrinogen were increased. The platelet parameters suggested platelet activation and high platelet turnover in IMHA dogs. The model that best explained mortality contained monocytosis, increased activated partial thromboplastin time and elevated creatinine. Whole blood TF gene expression is up regulated and may contribute to consumptive coagulopathy in dogs with IMHA. Increased TF expression by activated platelets is an alternative explanation and should be investigated.     

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.     

Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immune-mediated hemolytic anemia

OBJECTIVE: To determine plasma malondialdehyde (MDA) and serum vitamin E concentrations in dogs with immune-mediated hemolytic anemia (IMHA) and healthy control dogs. SAMPLE POPULATION: Serum and plasma samples from 36 dogs with IMHA and 40 healthy control dogs. PROCEDURE: Blood samples were collected from all study dogs. Plasma MDA concentrations were measured by use of a commercial colorimetric assay, and serum vitamin E concentrations (alpha-, gamma, and delta-tocopherol concentrations) were measured via high-performance liquid chromatography. RESULTS: Plasma MDA concentrations were significantly higher in the dogs with IMHA than in the control dogs. Compared with control dogs, serum alpha-, gamma-, and &tocopherol concentrations were significantly lower in the IMHA-affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated a state of oxidative stress and reduced antioxidant reserve in dogs with IMHA; this finding provides support for further investigation of the potential benefits of antioxidant treatment in dogs with this disease.     

Anti-Endothelial Cell Antibodies in Dogs with Immune-Mediated Hemolytic Anemia and Other Diseases Associated with High Risk of Thromboembolism

Background: Dogs with immune-mediated hemolytic anemia (IMHA) and certain inflammatory diseases are at high risk of developing thromboembolic disease. The presence of anti-endothelial cell autoantibodies (AECA) has been associated with an increased risk of thromboembolism in humans.
Hypothesis: AECA will be detected more often in dogs at risk of thromboembolism than in healthy control animals or dogs with diseases not associated with a higher risk of thromboembolism.
Animals: Ninety-one sick dogs and 22 healthy control dogs.
Methods: Retrospective case-controlled study. Serum was screened for the presence of AECA. Dogs were identified for the study based on the risk of thromboembolism as determined by clinical impression and the underlying disease process. Flow cytometry and normal canine endothelial cells were used to screen serum samples from sick and healthy control dogs for the presence of AECA. In addition, serum from dogs with confirmed thromboemboli was also screened for the presence of AECA by immunohistochemistry.
Results: AECA were detected in 2/91 sick dogs, both with infectious diseases, but were not found in healthy dogs. Anti-endothelial antibodies were not detected in 21 dogs with IMHA and 20 dogs with systemic inflammatory response syndrome, sepsis, or both.
Conclusions: We conclude that AECA are rarely detectable in dogs considered at high risk of thromboembolism. These findings suggest that AECA may not play an important role in the pathogenesis of thromboembolism in dogs with IMHA and other inflammatory diseases.     

Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography

This study investigated the coagulation status of dogs with immune-mediated haemolytic anaemia (IMHA) over time. Thirty animals with primary IMHA were blood sampled on three occasions over a 5 day period and assays performed included prothrombin time, activated partial thromboplastin time, D-dimer and fibrinogen concentration, antithrombin activity and recalcified unactivated thromboelastography (TEG). Based on TEG, dogs with IMHA were significantly hypercoagulable vs. controls (P<0.001) and over the 5 day period, 3/4 of the TEG parameters reflected increased clotting kinetics (P ≤ 0.02). The 30 day survival of these patients was 80% and, at hospital admission, the TEG maximum amplitude (MA) was significantly higher in survivors than non-survivors (P=0.015). Each unit increase in MA was associated with an increased odds of 30 day survival of 1.13 (95%; CI 1.02-1.25). Based on TEG, most dogs with IMHA were hypercoagulable on admission and their clotting kinetics increased with time. Relative hypocoagulability identified by TEG at initial assessment was found to be a negative prognostic indicator.     

C‐reactive protein concentration in dogs with primary immune‐mediated hemolytic anemia

Background: Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C-reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective: The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods: Inclusion criteria for primary IMHA were anemia (PCV<0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results: CRP concentration in the 25 clinically healthy dogs ranged from 0–8.9 μg/mL (median 2.2 μg/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 μg/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 μg/mL on day 0, 69 μg/mL on day 3, 35 μg/mL on day 8, and 2 μg/mL on day 14. In group 2 dogs, median CRP concentration was 194 μg/mL on day 0, 119 μg/mL on day 3, and 41 μg/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=−.598, P=.003). Conclusions: Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients.     

Perinuclear antineutrophil cytoplasmic autoantibodies in dogs infected with various vector-borne pathogens and in dogs with immune-mediated hemolytic anemia

Objective-To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum. Animals-55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens. Procedures-Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status. Results-26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA. Conclusions and Clinical Relevance-pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Characterization of platelet and soluble-porcine P-selectin (CD62P)

P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine P-selectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release.     

Neutrophil Extracellular Traps in Plasma from Dogs with Immune‐mediated Hemolytic Anemia

Background

Neutrophil extracellular traps (NETs) are part of the innate immune response and are essential in local pathogen control, but are associated with pathological inflammation, organ damage, autoimmunity, and thrombosis. Immune‐mediated hemolytic anemia (IMHA) is a pro‐inflammatory, prothrombotic disease associated with high mortality.

Hypothesis/Objectives

Neutrophil extracellular traps (NETs) are a feature of the inflammatory process in dogs with IMHA. The objective of the study was to evaluate plasma from dogs with IMHA for the presence of 2 indirect markers and 1 direct marker of NETs.

Animals

Healthy client‐owned dogs (56) and hospitalized dogs with IMHA (n = 35).

Methods

Prospective study. Plasma samples for all dogs were evaluated for cell‐free DNA using a fluorescence assay, histone‐DNA (hisDNA) complex using an ELISA, and citrullinated histone H3 (specific for NETosis) using Western blot. Reference intervals were generated using plasma from healthy dogs.

Results

In dogs with IMHA, cell‐free DNA concentration was above the reference interval in 17% of samples with a median (range) of 1.0 μg/mL (0.1–17.3), and hisDNA concentration was above the reference interval in 94% of samples with a median (range) of 30.7 × pooled normal plasma (PNP; 0.6–372.1). Western blot for citrullinated histone H3 identified detectable bands in 84% samples from dogs with IMHA.

Conclusions and Clinical Importance

The assay for cell‐free DNA detected evidence of NETs in fewer dogs than did the other approaches. Excessive NETs appears to be a feature of IMHA in dogs and contributions to the prothrombotic state deserve further study.     

Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution

The purpose of this prospective study was to investigate platelet function using in vitro tests based on both high and low shear rates and von Willebrand factor (vWf) multimeric composition in dogs with cardiac disease and turbulent high-velocity blood flow. Client-owned asymptomatic, untreated dogs were divided into 4 groups: 14 Cavalier King Charles Spaniels (Cavaliers) with mitral valve prolapse (MVP) and no or minimal mitral regurgitation (MR), 17 Cavaliers with MVP and moderate to severe MR, 14 control dogs, and 10 dogs with subaortic stenosis (SAS). Clinical examinations and echocardiography were performed in all dogs. PFA100 closure times (the ability of platelets to occlude a hole in a membrane at high shear rates), platelet activation markers (plasma thromboxane B2 concentration, platelet surface P-selectin expression), platelet aggregation (in whole blood and platelet-rich plasma with 3 different agonists), and vWf multimers were analyzed. Cavaliers with moderate to severe MR and dogs with SAS had longer closure times and a lower percentage of the largest vWf multimers than did controls. Maximal aggregation responses were unchanged in dogs with SAS but enhanced in Cavaliers with MVP (regardless of MR status) compared with control dogs. No significant difference in platelet activation markers was found among groups. The data suggest that a form of platelet dysfunction detected at high shear rates was present in dogs with MR and SAS, possibly associated with a qualitative vWf defect. Aggregation results suggest increased platelet reactivity in Cavaliers, but the platelets did not appear to circulate in a preactivated state in either disease.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.     

Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.     

Use of thromboelastography in dogs with immune-mediated hemolytic anemia: 39 cases (2000–2008)

Objective – To analyze thromboelastograms (TEGs) of naturally occurring cases of immune-mediated hemolytic anemia (IMHA) in order to identify whether a hypercoagulable state was present and whether its presence was associated with differences in survival.
Design – Retrospective study spanning January 2000 to June 2008. Medical records of dogs were evaluated. Endpoints were considered death or discharge from the hospital.
Setting – Academic teaching hospital.
Animals – Thirty-nine dogs with a diagnosis of IMHA and at least one TEG performed during hospitalization were included.
Interventions – None.
Measurements and Main Results – Four values were evaluated from the TEG: the R time (R), K time (K), alpha angle (α), and maximum amplitude. From these values, a coagulation index (CI) was calculated to classify patients as normocoagulable, hypercoagulable, or hypocoagulable. Thirty-three of 39 patients were hypercoagulable based on the CI. The 6 remaining dogs were normocoagulable. The patients with a normocoagulable CI had an increased mortality rate (100%) when compared with the hypercoagulable patients using Fisher's exact test ( P =0.02). Additionally, prolongation of partial thromboplastin time did not preclude hypercoagulable TEG values.
Conclusions – The majority of dogs with IMHA were hypercoagulable as measured by TEG. A normal CI was associated with a worse outcome in this patient population. TEG may provide additional and complementary information to prothrombin time and partial thromboplastin time relating to coagulation status in dogs with IMHA and may help predict prognosis and potentially guide clinical decisions to utilize anticoagulant drugs.     

Use of human intravenous immunoglobulin in dogs with primary immune mediated hemolytic anemia

Immune mediated hemolytic anemia (IMHA) in dogs is a severe disease with a high mortality rate. As human immunoglobulin (HIG) was reported to be beneficial for the treatment of IMHA in dogs we examined the influence of HIG on the course of the disease in our dogs with IMHA. Of 22 dogs with primary IMHA 9 dogs received in addition to routine immunosuppressive therapy HIG at a dose of 0.19 to 0.68 g/kg (median 0.35 g/kg), 13 dogs did not receive HIG (-HIG group). Both groups were similar in terms of age, weight, the presence of autoagglutination, spherocytosis, positive Coombs' test, icterus and pigmenturia. The lowest hematocrit measured during the disease was significantly lower in the +HIG group compared to the -HIG group and dogs in the +HIG group received significantly more transfusions than those of the -HIG group. This is an indication for more severe disease signs of the +HIG group dogs. Although mortality during hospitalization and the time from hospital admission to release or death was not significantly different between the two groups, we interpret this similar course of the IMHA despite more severe signs of the +HIG group dogs as a potential positive effect of the HIG therapy.     

Haemoplasma infection is not a common cause of canine immune-mediated haemolytic anaemia in the UK

Objective : The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. Methods : Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results : Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance : Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.     

The utility of screening diagnostic tests in identifying associative immune-mediated haemolytic anaemia in dogs

The study aimed to (1) define the proportion of dogs with immune-mediated haemolytic anaemia (IMHA) that have associative and non-associative disease and (2) evaluate the utility of screening diagnostic tests in identifying potential triggers of associative IMHA. Medical records of 78 dogs diagnosed with IMHA at a specialist hospital in Sydney from July 2008 to August 2017 were reviewed. The original diagnosis was revised according to published guidelines (Garden et al., 2019) as either diagnostic, supportive or suspicious for IMHA. Associative IMHA was confirmed if immunosuppressive therapy was discontinued within six weeks of effective treatment of a potential trigger. Associative IMHA was considered possible when a potential trigger was identified but its significance could not be confirmed. Associative IMHA was confirmed (3) or suspected (7) in 10 dogs (13%, confidence interval [CI] 7.1%–22%), with 68 cases presumed to be non-associative. Associative IMHA was present in 3/29 (10.3%) of dogs with criteria diagnostic for IMHA, 4/42 (9.5%) of dogs with criteria supportive for IMHA and 3/7 (42.9%) of dogs with criteria suspicious for IMHA. Abdominal ultrasound was performed in 68 dogs and identified possible triggers in five (7.3%, CI 3.2% to 16%). Thoracic radiographs were performed in 70 dogs but did not identify any potential triggers (0%, CI 0% to 5.2%). Urine culture was performed in 22 dogs and was positive in three (14%, CI 4.7% to 33.3%). Routine screening tests, particularly thoracic radiographs, have a low yield in identifying potential triggers of associative IMHA, but are more likely to be useful in dogs fulfilling less stringent diagnostic criteria of IMHA.     

Treatment of Immune‐Mediated Hemolytic Anemia with Individually Adjusted Heparin Dosing in Dogs

Background: A major cause of death in dogs with immune‐mediated hemolytic anemia (IMHA) is thromboembolism. Previous studies suggest unfractionated heparin (UH) is not effective in preventing thromboembolism in IMHA; however, subtherapeutic dosing could explain the seeming lack of efficacy. Hypothesis: Providing therapeutic plasma concentration of UH by individually adjusting doses based on antifactor Xa activity would improve survival in IMHA. Animals: Fifteen dogs with primary IMHA. Methods: Randomized, prospective, controlled clinical trial. Dogs received standardized therapy for IMHA and either constant dose (CD) (150 U/kg SC) (n = 7) or individually adjusted dose (IAD) (n = 8) UH, monitored via an anti‐Xa chromogenic assay, adjusted according to a nomogram. UH was administered every 6 hours until day 7, and every 8 hours thereafter. UH dose was adjusted daily in IAD dogs until day 7, weekly until day 28, then tapered over 1 week. Dogs were monitored for 180 days. Results: At day 180, 7 dogs in the IAD group and 1 in the CD group were alive (P= .01). Median survival time for the IAD group was >180 days, and 68 days for the CD group. Thromboembolic events occurred in 5 dogs in the CD group and 2 dogs in the IAD group. Doses of UH between 150 and 566 U/kg achieved therapeutic anti‐Xa activity (0.35–0.7 U/mL). Conclusions and Clinical Importance: This study suggests that IAD UH therapy using anti‐Xa monitoring reduced case fatality rate in dogs with IMHA when compared with dogs receiving fixed low dose UH therapy.     

Case-control study of blood type, breed, sex, and bacteremia in dogs with immune-mediated hemolytic anemia

OBJECTIVE: To determine whether blood type, breed, or sex were risk factors for immune-mediated hemolytic anemia (IMHA) in dogs and whether bacteremia was common in dogs with IMHA. DESIGN: Case-control study. ANIMALS: 33 dogs with IMHA, 1,014 dogs without IMHA for which blood type (dog erythrocyte antigens 1.1, 1.2, 3, 4, 5, and 7) was known, 15,668 dogs without IMHA for which breed was known, and 15,589 dogs without IMHA for which sex was known. PROCEDURE: Blood type, breed, and sex distribution of dogs with IMHA were compared with data for control dogs with Fisher exact tests and by calculating odds ratios (ORs). Results of bacterial culture of blood samples were documented for dogs with IMHA, when available. RESULTS: Dog erythrocyte antigen 7 was associated with a significant protective effect (OR, 0.1) in Cocker Spaniels with IMHA (n = 10), compared with control dogs. Cocker Spaniels, Bichon Frise, Miniature Pinschers, Rough-coated Collies, and Finnish Spitz had a significantly increased risk of IMHA, as did female dogs (OR, 2.1). Blood samples from 12 dogs with IMHA were submitted for bacterial culture, and none had bacteremia. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that blood type, breed, and sex may play a role in IMHA in dogs.