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.     

Serum cardiac troponin I in dogs with primary immune-mediated haemolytic anaemia

Objectives : The assessment of serum cardiac troponin I concentrations in dogs with a range of nonprimary cardiac illnesses has revealed that cardiac myocyte damage is commonplace in many canine diseases. Whilst it is well established that dogs with fatal immune‐mediated haemolytic anaemia frequently have cardiac pathology based on post‐mortem examinations, there is limited information on the incidence of cardiac myocyte damage in this population of dogs. Methods : Serum cardiac troponin I concentrations were measured in 11 healthy dogs, 27 dogs with primary haemolytic anaemia and 49 hospitalised dogs without primary cardiac or haematological disorders. Results : Dogs with primary haemolytic anaemia have higher serum concentrations of cardiac troponin I than hospitalised ill dogs (P<0.005) and healthy dogs (P<0.01). Using a cut‐off of less than 0.1 ng/mL, 20 of 27 dogs with primary haemolytic anaemia had increased serum cardiac troponin I concentrations, which was a significantly higher proportion compared to the hospitalised ill dogs (P<0.001, 16 out of 49 dogs) and healthy dogs (P<0.05, 3 out of 11 dogs). Clinical Significance : Dogs with primary haemolytic anaemia have a higher incidence of subclinical myocyte damage than healthy dogs or dogs with non‐haematological or primary cardiac illnesses. The prognostic significance of increased serum cardiac troponin I concentrations in dogs with primary haemolytic anaemia merits further investigation.     

Concurrent immune-mediated haemolytic anaemia and severe thrombocytopenia in 21 dogs

The medical records of 21 dogs with concurrent immune-mediated haemolytic anaemia (imha) and severe thrombocytopenia (defined as an automated platelet count of less than 50x10(9)/l, confirmed by the examination of a blood smear) were reviewed. Their mean (sd) age was 5.8 (2.5) years. When compared with the 24,759 dogs in the hospital population for the same period Airedale terriers and dobermanns appeared to be over-represented with odds ratios of 22.5 (95 per cent confidence interval [ci] 5.2 to 97.9) and 7.6 (95 per cent ci 1.8 to 32.7) respectively. The median duration of the dogs' clinical signs was seven days, with a range from one to 17 days. Eleven of the dogs had a history of a tendency to bleed, and 15 had evidence of bleeding when examined. Twenty of the 21 dogs had been treated with glucocorticoids, nine with vincristine, and seven with azathioprine. Their median stay in hospital was four days, with a range from one to 17 days. The median period for which they survived after admission to hospital was five days, with a range from one to 558 days, and 16 of the 21 dogs had died or been euthanased within 30 days of their admission.     

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.     

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.     

Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

Background

The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT) for the diagnosis of immune-mediated haemolytic anaemia (IMHA).

Methods

Canine (n = 247) and feline (n = 74) blood samples were submitted for DAT testing to two laboratories. A subset of canine samples was categorized as having idiopathic IMHA, secondary IMHA, or no IMHA.

Results

The kappa values for agreement between the tests were in one laboratory 0.86 for canine and 0.58 for feline samples, and in the other 0.48 for canine samples. The lower agreement in the second laboratory was caused by a high number of positive canine DATs for which the gel test was negative. This group included significantly more dogs with secondary IMHA.

Conclusions

The gel test might be used as a screening test for idiopathic IMHA and is less often positive in secondary IMHA than the DAT.     

Autoimmune haemolytic anaemia in dogs

The clinical, haematological and immunological findings in 24 dogs with Coombs' positive haemolytic anaemia are described; 33% were Old English Sheepdogs. Dogs with intravascular haemolysis had a shorter history of illnesses, more severe clinical signs including vomiting, jaundice and fever, and had a poor survival rate compared to dogs with extravascular haemolysis. The anaemia was severe and regenerative in 18 dogs, and was characterised by spherocytosis and microscopic red cell agglutination, with leukocytosis. Serum IgG levels were elevated in 20 dogs, and changes in IgM, IgA, C3 and C4 were found. Antinuclear antibody was also demonstrated in 13 dogs, of which 7 were Old English Sheepdogs. It is suggested that a distinct multisystem autoimmune syndrome exists within the local Old English Sheepdog population.     

Evaluation of platelet activation in canine immune-mediated haemolytic anaemia

Objectives : To establish whether heightened platelet activation is a common feature of canine immune-mediated haemolytic anaemia, and to evaluate the hypothesis that platelet activation plays a role in the pathogenesis of thromboembolism. Methods : Using whole-blood flow-cytometric analysis, the proportion of activated platelets and platelet-leucocyte aggregates in blood samples from 14 dogs with immune-mediated haemolytic anaemia and 14 healthy dogs was calculated. General linear models with binomial errors were used to compare groups. Results from the immune-mediated haemolytic anaemia-affected dogs were then correlated with established risk factors for thromboembolism in canine immune-mediated haemolytic anaemia, D-dimer concentration and antithrombin activity. Results : There was a strong correlation between platelet activation and severe thrombocytopenia, with heightened platelet activation being observed predominantly in severely thrombocytopenic dogs. Clinical Significance : Dogs with immune-mediated haemolytic anaemia, particularly those with concurrent severe thrombocytopenia, are likely to have heightened platelet activation, which may play a role in the pathogenesis of thromboembolism.     

Evaluation of immunosuppressive regimens for immune-mediated haemolytic anaemia: a retrospective study of 42 dogs

Objectives : Immune‐mediated haemolytic anaemia (IMHA) is a severe disease for which evidence is lacking to make informed choices regarding immunosuppressive regimen. The aims of the current study were to determine the effect of different treatment regimens on outcome in affected animals and to identify parameters that may be used as prognostic factors for the disease. Methods : The records of dogs presenting to a veterinary hospital in the period 2002 to 2010 for treatment of IMHA were examined and follow‐up data were obtained. Statistical tests were performed to establish whether treatment regimen affected outcome and to identify prognostic factors for outcome. Results : Treatment regimen had a significant effect on the outcome (measured as survival of hospitalisation) but there were insufficient subjects to determine the cause of the difference. Serum bilirubin and urea concentrations were found to be significant negative prognostic factors for the outcome of IMHA cases and the concentrations of these parameters were significantly different between animals that survived or died while hospitalised. Clinical Significance : This study presents the first report of a significant difference in outcome comparing animals treated with immunosuppressive drugs which are in widespread clinical usage. Although possible confounding factors should be considered, these findings could have major consequences for the treatment of IMHA.     

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.     

Positive Coombs' test results in two dogs treated with amiodarone

Effects of amiodarone, an antiarrhythmic drug that is effective in suppressing severe ventricular arrhythmias that are refractory to other antiarrhythmic drugs, were evaluated in 2 dogs with cardiac disease. One dog was a Doberman Pinscher with cardiomyopathy that developed severe thrombocytopenia after receiving amiodarone for 7 months. The second was a Giant Schnauzer with acquired mitral valve degeneration that developed regenerative anemia after receiving amiodarone for 5 months. Results of direct Coombs' tests were positive in both dogs. Adverse effects of amiodarone are numerous; in dogs, the most common adverse effects are anorexia and hepatotoxicosis. Frequent CBC and serum biochemical analyses should be performed when amiodarone is administered with the intent of continuing the drug indefinitely.     

Canine idiopathic immune-mediated haemolytic anaemia: a review with recommendations for future research

Idiopathic immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated diseases of dogs. The aim of this article is to review current knowledge of canine IMHA, its etiology, clinical presentation, diagnosis, complications, and treatment, in an attempt to establish why its outcome is still so poor. Clinical signs of anaemia develop within 3 days and dogs present with a median haematocrit of 13%, leucocytosis, a left shift, and reticulocytosis. Coagulation test results support the presence of disseminated intravascular coagulation. About 50% of dogs die in the first 2 weeks after presentation, and analysis of risk factors suggests that mortality is associated with hypercoagulability, inflammatory response, and liver and kidney failure. A positive direct agglutination test, spherocytosis, and true autoagglutination are widely accepted tests to demonstrate anti-erythrocyte antibodies, but are not yet standardized. To date, there is no evidence to support the efficacy of immunomodulators in addition to corticosteroids in the treatment of IMHA. Despite numerous investigations, the prognosis of IMHA remains dismal. There is an urgent need to validate and standardize diagnostic tests and criteria, and clinical trials might benefit from stratifying dogs by mortality risk. Analysis of samples from well-defined cases of canine IMHA might provide insight into the aetiology and pathophysiology of IMHA.     

Immune-mediated haemolytic anaemia in 110 dogs in Victoria, Australia

Objective   Evaluate the survival and prognostic indicators (i.e. breed predilection, season, blood transfusion, and the prevalence of autoagglutination) of dogs with immune-mediated haemolytic anaemia (IMHA) in Victoria, Australia.
Design   Retrospective study of 110 diagnosed with primary IMHA at the University of Melbourne Veterinary Clinic and Hospital.
Results   In total, 80 of the dogs (72.7%) were discharged from hospital and 48 of 65 (73.8%) dogs available for follow-up were known to be alive at 1 year, giving an overall 1-year survival of 48 (50.5%) of 95 dogs for which survival data were available. Regarding breed, 80 (18.2%) of the 110 dogs were Maltese-breed dogs compared with 81 (7.4%) of 1100 control dogs (P < 0.001). Springer Spaniels (P = 0.02), Hungarian Vizslas (P = 0.02) and Airedale Terriers (P < 0.001) were also over-represented compared with the control sample. There was no evidence of an association between the occurrence of IMHA in dogs and season in this study. Receiving one or more blood transfusions did not affect survival to the time of discharge from hospital. On initial blood smear examination, 57 (51.8%) of the 110 dogs had spontaneous autoagglutination and its presence was associated with decreased survival to discharge from hospital (P = 0.03). Packed cell volume, white cell count, platelet count and serum total bilirubin on admission had no effect on survival to the time of discharge from hospital or 1 year later.
Conclusion   Dogs with IMHA have a guarded prognosis as only half are still alive 1 year after the acute event.     

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.     

Immune-mediated haemolytic anaemia: possible association with Ancylostoma caninum infection in three dogs

Immune-mediated haemolytic anaemia (IMHA) may be primary or secondary. In primary IMHA, no underlying cause can be found, whereas secondary IMHA is triggered by an underlying cause, such as neoplasia, infectious diseases, or drugs. This paper describes 3 dogs with typical signs of IMHA that was possibly associated with the intestinal parasite Ancylostoma caninum. As intestinal helminths can be difficult to diagnose on faecal examination, it would be pertinent to perform multiple faecal examinations on any animal that has IMHA with no apparent underlying cause, as part of the therapy.     

Staphyloccocus aureus-associated infarctive purpura haemorrhagica,immune-mediated haemolytic anaemia and myocarditis in a Quarter Horse mare

A 7-year-old Quarter Horse mare was presented for severe left hindlimb oedema and nonweightbearing lameness that was progressive over approximately 48 h. The mare subsequently developed marked and worsening oedema, immune-mediated haemolytic anaemia (IMHA), myositis and myocarditis. The marked oedema, degree of pain and severity of myositis were consistent with infarctive purpura haemorrhagica. Fine needle aspiration of affected tissue resulted in positive bacterial culture of Staphylococcus aureus. Purpura haemorrhagica and associated complications are typically associated with Streptococcal sp. infection or exposure, and have not been previously reported in association with Staphylococcus aureus in the horse. The mare responded favourably to long-term, high-dose corticosteroid therapy, antimicrobials and supportive care.     

The diagnostic significance of the direct antiglobulin test (DAT) in anemic dogs

The direct antiglobulin test (DAT) was positivein 134 (36.1%) of 371 anemic dogs with internal diseases. Four principal types of reaction were recognized: IgG alone in 15 (11.2%), IgG + C' in 41 (30.6%), C' alone in 74 (55.2%) and IgM + C' in 2 (1.5%). Rarely, IgM and/or IgA reactions occurred in association with strong IgG + C' reactions. In 2 (1.5%) DAT-positive dogs the type of reaction was not clear. One or more symptoms of hemolysis, such as hemoglobinemia, indirect type hyperbilirubinemia, increased red cell osmotic fragility, and increased fecal urobilinogen excretion, were demonstrated in 84 DAT-positive dogs. These consisted of 10 of 15 dogs with IgG type DAT, 36 of 41 dogs with IgG + C' type DAT, 36 of 74 dogs with C' type DAT and 2 of 2 dogs with IgM + C' type DAT. Most dogs with IgG + C' type reactions had severe hemolysis, whereas "primary" or "associated" diseases were recognized in only 26 of 56 cases. IgG type incomplete warm antibody, reacting with pooled donor cells, was demonstrated in red cell eluates in each of 3 dogs with IgG type DAT and in 6 of 7 dogs with IgG + C' type reactions. This indicates that dogs with IgG or IgG + C' reactions usually have autoimmune hemolytic anemia. In dogs with C' type DAT, indications of hemolysis were frequently minimal or absent. Symptoms almost always indicated some "primary" disorder. Diagnoses mainly included infections, inflammatory and neoplastic (especially myelo- and lymphoproliferative)diseases. In only 7 (9.5%) of 74 dogs with C' type DAT no diagnosis other than (transient peracute) hemolytic anemia was made. The results of tests for antibodies in the serum and red cell eluates were always negative in dogs with C' type DAT. In one dog with hemolytic anemia and C' + IgM type DAT, there was a high titer of IgM cold agglutinins in the serum and in heat eluates. It is concluded that a positive DAT with anti-IgG antiserum is a strong indication of autoimmune hemolytic anemia but that a reaction of the C' alone type is a rather common phenomenon in canine internal diseases which is seldom associated with serious hemolysis.