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.     

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.     

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.     

Vaccine-Associated Immune-Mediated Hemolytic Anemia in the Dog

Vaccination has been incriminated as a trigger of immune-mediated hemolytic anemia (IMHA) in dogs and in people, but evidence to support this association is lacking. In a controlled retrospective study, idiopathic IMHA was identified in 58 dogs over a 27–month period. When compared with a randomly selected control group of 70 dogs (presented for reasons other than IMHA) over the same period, the distribution of cases versus time since vaccination was different (P < .05). Fifteen of the dogs (26%) had been vaccinated within 1 month (mean, 13 days; median, 14 days; range, 1 to 27 days) of developing IMHA (P < .0001), whereas in the control group no marked increase in frequency of presentation was seen in the first month after vaccination. The dogs with IMHA were divided into 2 groups based on time since vaccination: the vaccine IMHA group included dogs vaccinated within 1 month of developing IMHA; the nonvaccine IMHA group included dogs that developed IMHA more than 1 month after vaccination. The recently vaccinated dogs with IMHA (vaccine IMHA group) had significantly lower platelet counts (P < .05) and a trend towards increased prevalence of intravascular hemolysis and autoagglutination when compared with the nonvaccine IMHA group. Similar mortality rates were seen in the vaccine IMHA group (60%) and the nonvaccine IMHA group (44%), with the majority of fatalities (>75%) occurring in the first 3 weeks after presentation. Persistent autoagglutination was a negative prognostic indicator for survival in both groups (P < .05). Presence of icterus and hyperbilirubinemia were negative prognostic indicators for survival in the nonvaccine IMHA group (P < .0001 and P < .01, respectively) but not in the vaccine IMHA group. In the recently vaccinated dogs, combination vaccines from various manufacturers against canine distemper, adenovirus type 2, leptospirosis, parainfluenza, and parvovirus (DHLPP) were involved in each case. Vaccines against rabies virus, Bordetella spp, coronavirus, and Lyme Borrelia were administered concomitantly to some dogs. This study provides the first clinical evidence for a temporal relationship of vaccine-associated IMHA in the dog.     

Evaluation of Serum Ferritin as a Tumor Marker for Canine Histiocytic Sarcoma

Background: Canine histiocytic sarcoma (HS) is an aggressive malignancy. Hyperferritinemia has been documented in dogs with HS and could serve as a tumor marker aiding in diagnosis and treatment. In people, hyperferritinemia is found in inflammatory diseases, liver disease, and hemolysis, and thus may occur in dogs with these conditions. Objective: To determine if serum ferritin concentration is a tumor marker for canine HS. Animals: Dogs with HS (18), inflammatory diseases (20), liver disease (24), immune‐mediated hemolytic anemia (IMHA) (15), and lymphoma (23). Methods: Prospective, observational, cohort study: Serum ferritin concentration was measured at initial diagnosis. Parametric methods were used to compare mean log ferritin concentrations among disease categories. Receiver‐operating characteristic curves and likelihood ratios were used to evaluate serum ferritin concentration as a tumor marker. Results: Varying proportions of dogs with IMHA (94%), HS (89%), liver disease (79%), lymphoma (65%), and inflammatory diseases (40%) had hyperferritinemia. Dogs with IMHA had significantly higher mean ferritin concentration than dogs in all other categories. Dogs with HS had significantly higher mean ferritin concentration than those in the inflammatory disease and lymphoma categories. Mean serum ferritin concentration was not significantly different between dogs with HS and those with liver disease. Decision thresholds were determined to distinguish IMHA and HS from the other diseases associated with hyperferritinemia. Conclusion: Hyperferritinemia is common in dogs with HS and, after IMHA is ruled out, the degree of hyperferritinemia may be useful in differentiating dogs with HS from dogs with inflammatory diseases, liver disease, and lymphoma.     

A Remote Assay for Measuring Canine Platelet Activation and the Inhibitory Effects of Antiplatelet Agents

Background

Antiplatelet medications are increasingly used in dogs. Remote analysis of platelet activity is challenging, limiting assessment of antiplatelet drug efficacy.

Hypothesis/Objectives

To evaluate a method used in humans for stimulation and remote analysis of canine platelet activity.

Animals

Forty‐five dogs of various ages without a coagulopathy or thrombocytopenia. Six were receiving antiplatelet medication.

Methods

Prospective observational study. Platelets were stimulated with combinations of arachidonic acid (AA) and epinephrine (Epi) or adenosine diphosphate (ADP) and the thromboxane A₂‐mimetic U46619 (U4). PAMFix was added to the blood samples to facilitate delayed analysis of platelet activity. Activity was assessed by flow cytometric measurement of surface P‐selectin (CD62P) expression.

Results

Canine platelets could be stimulated with both AA/Epi and ADP/U4. The levels of P‐selectin were significantly greater than paired, unstimulated samples (P < 0.001). Inhibition of P‐selectin expression occurred after this stimulation by adding antiplatelet drugs in vitro. The efficacy of antiplatelet drugs in samples from treated dogs was also measurable ex vivo using this method. Delayed analysis of platelet activity at time points up to 22 days demonstrated excellent correlation between respective mf values at each time point (r² = 0.92, P < 0.0001).

Conclusions and Clinical Importance

This study evaluated a new method to remotely assess canine platelet activity. It shows that PAMFix can be used for this purpose. This provides opportunities to interrogate the inhibitory action of antiplatelet drugs in clinical settings.     

The diagnostic relevance of NT‐proBNP and proANP 31–67 measurements in staging of myxomatous mitral valve disease in dogs

Background

There is no agreement in current publications regarding the reliability of serum concentrations of natriuretic peptides (NPs) to detect dogs with subclinical myxomatous mitral valve disease (MMVD) and to differentiate between asymptomatic stages.

Objectives

We sought to compare N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) and pro‐atrial natriuretic peptide 31‐67 (proANP) concentrations between various stages of canine MMVD and to investigate the influence of age, weight, and sex.

Methods

In this prospective study, dogs were classified in different disease stages using the modified Canine Heart failure International Expert Forum (CHIEF) system. Serum NP concentrations were compared between groups.

Results

A total of 559 samples from 116 healthy dogs and 236 dogs with MMVD were analyzed. Using cut‐off values (1207 pmol/L for NT‐proBNP, 1578 fmol/mL for proANP), dogs with MMVD with and without congestive heart failure (CHF) could be differentiated with a sensitivity of 83% for both and specificities of 85% and 86%, respectively. Dogs staged in CHIEF B1 and B2 could not be distinguished based on NP concentrations due to wide variation within the groups. Intact females (means 598 pmol/L and 1036 fmol/mL, respectively) had significantly higher values of both NPs than intact males (315 pmol/L and 836 fmol/mL).

Conclusions

NPs in canine MMVD are useful to discriminate between asymptomatic dogs and dogs with CHF. Due to a large overlap of NP‐concentrations between the groups, NPs do not seem to be useful to differentiate between dogs in stages B1 and B2. Interpretation of NT‐proBNP and proANP values should include consideration of sex‐specific differences.     

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.     

Reference intervals for the activity of lactate dehydrogenase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines

Background

Lactate dehydrogenase (LD) exists as 5 isoenzymes (LD‐1 through LD‐5) that are expressed throughout the body and can be detected in both serum and cerebrospinal fluid (CSF). LD and its isoenzymes have been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total LD activity and atypical isoenzyme patterns can indicate disease processes, including neurologic abnormalities.

Objectives

The purpose of this study was to establish RIs for LD and its isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive RI for this enzyme in healthy canines, further study of the clinical and diagnostic usefulness of LD can be undertaken.

Methods

Serum and atlantoaxial CSF were collected from clinically healthy dogs. Total LD activity was measured spectrophotometrically immediately after collection. Isoenzyme distributions were also determined within 8 hours of collection using the QuickGel LD Isoenzyme technique and a densitometric scanner.

Results

The median serum total LD in healthy canines was 69.0 U/L (n = 41; range: 21.0‐217.0 U/L), while the median CSF total LD was 10.0 U/L (n = 40; range: 6.0‐19.3 U/L). LD‐5 is the predominant isoenzyme in canine serum (n = 40), contributing over half of the total enzyme activity. Conversely, in canine CSF (n = 42), LD‐1 is the predominant isoenzyme, followed by LD‐2 and LD‐3.

Conclusions

Knowledge of the distribution and concentration of LD in the serum and CSF of healthy dogs will set the foundation for future studies of canine LD as a potentially clinically useful biomarker.     

Effect of Duration of Packed Red Blood Cell Storage on Morbidity and Mortality in Dogs After Transfusion: 3,095 cases (2001–2010)

Background

Accumulating evidence suggests that transfusion of packed red blood cells (PRBCs) stored for >14 days is associated with increased rates of sepsis, multiple organ dysfunction, and mortality in human patients.

Objective

To determine if duration of PRBC storage has an effect on morbidity and mortality in dogs after transfusion.

Animals

Dogs admitted to the Matthew J Ryan Veterinary Hospital of the University of Pennsylvania.

Methods

A retrospective case review of dogs identified through blood bank logbooks that received PRBC transfusions (minimum, 5 mL/kg) between 2001 and 2010. Dogs were categorized according to major cause of anemia (eg, hemorrhage, hemolysis, ineffective erythropoiesis) for analysis.

Results

A total of 3,095 dogs received 5,412 PRBC units. Longer duration of PRBC storage was associated with development of new or progressive coagulation failure (P = .001) and thromboembolic disease (P = .005). There was no association between duration of PRBC storage and survival for all dogs overall. However, a logistic regression model indicated that for dogs with hemolysis, 90% of which had immune‐mediated hemolytic anemia, longer duration of PRBC storage was a negative risk factor for survival. For every 7 day increase in storage, there was a 0.79 lesser odds of 30 day survival (95% CI, 0.64–0.97; P = .024).

Conclusions and Clinical Importance

Duration of PRBC storage does not appear to be a major contributing factor to mortality in the overall canine population. However, longer duration of PRBC storage may negatively impact outcome in dogs with immune‐mediated hemolytic anemia, thus warranting further investigation with prospective studies.     

Splenectomy in the management of primary immune‐mediated hemolytic anemia and primary immune‐mediated thrombocytopenia in dogs

BackgroundCurrent reports about the use of splenectomy for the management of immune‐mediated hemolytic anemia (IMHA) or immune‐mediated thrombocytopenia (ITP) or both in dogs are limited.ObjectivesTo retrospectively describe the use of splenectomy as part of the management for IMHA, ITP, and concurrent IMHA and severe thrombocytopenia (CIST) in dogs. It was hypothesized that splenectomy would be beneficial in allowing for reduction of dose of immunosuppressive drugs or discontinuation in 1 or more of these groups.AnimalsSeventeen client‐owned dogs (7 with IMHA, 7 with ITP, and 3 with CIST) were identified across 7 UK‐based referral hospitals from a study period of 2005 to 2016.MethodsData were collected retrospectively via questionnaires and included information about diagnosis, management and treatment response before and after splenectomy. Based on clinical outcome, treatment with splenectomy as part of the management protocol was classified as either successful or unsuccessful.ResultsSix of 7 dogs with ITP were managed successfully with splenectomy as part of their management protocol (3 complete and 3 partial responses), although 1 subsequently developed suspected IMHA. Of the 7 dogs with IMHA, splenectomy was part of a successful management protocol in 4 dogs (2 complete and 2 partial responses). In the CIST group, 1 case (1/3) responded completely to management with splenectomy as part of the management protocol.Conclusions and Clinical ImportanceSplenectomy was considered successful and well tolerated in most cases of isolated ITP. Whether there is a benefit of splenectomy in cases of IMHA and CIST could not be determined in the current study.     

Comparison of Force Plate Gait Analysis and Owner Assessment of Pain Using the Canine Brief Pain Inventory in Dogs with Osteoarthritis

Background

Lameness assessment using force plate gait analysis (FPGA) and owner assessment of chronic pain using the Canine Brief Pain Inventory (CBPI) are valid and reliable methods of evaluating canine osteoarthritis. There are no studies comparing these 2 outcome measures.

Objective

Evaluate the relationship between CBPI pain severity (PS) and interference (PI) scores with the vertical forces of FPGA as efficacy measures in canine osteoarthritis.

Animals

Sixty‐eight client‐owned dogs with osteoarthritis (50 hind limb and 18 forelimb).

Methods

Double‐blind, randomized. Owners completed the CBPI, and dogs underwent FPGA on days 0 and 14. Dogs received carprofen or placebo on days 1 through 14. The change in PS and PI scores from day 0 to 14 were compared to the change in peak vertical force (PVF) and vertical impulse (VI).

Results

PS and PI scores significantly decreased in carprofen‐ compared with placebo‐treated dogs (P = .002 and P = .03, respectively). PVF and VI significantly increased in carprofen‐ compared with placebo‐treated dogs (P = .006 and P = .02, respectively). There was no correlation or concordance between the PS or PI score changes and change in PVF or VI.

Conclusions and Clinical Importance

In these dogs with hind limb or forelimb osteoarthritis, owner assessment of chronic pain using the CBPI and assessment of lameness using FPGA detected significant improvement in dogs treated with carprofen. The lack of correlation or concordance between the change in owner scores and vertical forces suggests that owners were focused on behaviors other than lameness when making efficacy evaluations in their dogs.     

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.     

A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

Objectives

Glutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.

Methods

Banked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.

Results

Canine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V_max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.

Conclusions

An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.     

Use of serum concentrations of interleukin-18 and monocyte chemoattractant protein-1 as prognostic indicators in primary immune-mediated hemolytic anemia in dogs

Background: The cytokine response in immune‐mediated hemolytic anemia (IMHA) is poorly characterized and correlation with outcome is unknown. Hypothesis/Objectives: To determine if cytokine activity is correlated with outcome in dogs with IMHA. Animals: Twenty dogs with primary IMHA and 6 control dogs. Methods: Prospective study on dogs with IMHA with blood sampling at admission. Serum activity of interleukin‐2 (IL‐2), IL‐4, IL‐6, IL‐7, IL‐8, IL‐10, IL‐15, IL‐18, monocyte chemoattractant protein‐1 (MCP‐1), granulocyte‐macrophage colony stimulating factor (GM‐CSF), interferon‐inducible protein‐10, interferon‐gamma, and keratinocyte chemoattractant (KC) was assessed. Results: Thirty‐day case fatality rate was 25% (5/20 dogs). Increased concentrations (median [range]) of IL‐2 (45.5 ng/L [0;830] versus 0 ng/L [0;46.8]), IL‐10 (8.2 ng/L [0;60.6] versus 0 ng/L [0;88.2]), KC (1.7 μg/L [0.3;4.7] versus 0.5 μg/L [0.2;1.1]), and MCP‐1 (162 ng/L [97.6;438] versus 124 ng/L [90.2;168]) were observed in dogs with IMHA compared with controls. The cytokine profile was indicative of a mixture of pro‐ and anti‐inflammatory cytokines of various cellular origins. Cytokines/chemokines strongly associated with macrophage/monocyte activation and recruitment were significantly increased in nonsurvivors compared with survivors; IL‐15 (179 ng/L [48.0;570] versus 21.3 ng/L [0;193]), IL‐18 (199 ng/L [58.7;915] versus 37.4 ng/L [0;128]), GM‐CSF (134 ng/L [70.0;863] versus 57.6 ng/L [0;164]), and MCP‐1 (219 ng/L [135;438] versus 159 ng/L [97.6;274]), respectively. Logistic regression suggested increased IL‐18 and MCP‐1 concentrations were independently associated with mortality in this population (P<.05, Wald's type 3). Conclusions and Clinical Importance: A mixed cytokine response is present in dogs with IMHA and mediators of macrophage activation and recruitment might serve as prognostic indicators.     

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 hemolytic anemia and severe thrombocytopenia in dogs: 12 cases (2001–2008)

Objective – To identify and characterize the syndrome of immune‐mediated hemolytic anemia (IMHA) with concurrent severe thrombocytopenia (≤15.0 × 10⁹ platelets/L; [15.0 × 10³ platelets/μL]), and to evaluate prognostic factors, clinicopathologic findings, complications, treatment, outcome, and survival of dogs with this hematologic disorder. Design – Retrospective, observational study. Setting – Veterinary teaching hospital. Animals – Twelve client‐owned dogs with IMHA and severe thrombocytopenia (≤15.0 × 10⁹ platelets/L; [15.0 × 10³ platelets/μL]), without evidence of overt disseminated intravascular coagulation. Interventions – The following data were recorded and analyzed from the electronic medical record: signalment, history, concurrent diseases, clinical signs at presentation, clinicopathologic data, diagnostic testing, radiographic findings, treatment modalities, length of hospitalization, complications, and clinical outcome. All dogs were treated with immunosuppressive doses of corticosteroids. Measurements and Main Results – Twelve dogs were identified with the diagnosis of IMHA and severe thrombocytopenia; of these, 9 (75%) survived, 3 (25%) were euthanized, and none died. Dogs that survived were significantly younger than nonsurvivors (P=0.03). There were no specific clinical signs or therapies associated with survival. Conclusions – Dogs in this study had a mortality rate similar to reported rates for dogs with either disease alone. Overall, younger dogs were more likely to survive. No association between different treatment modalities and overall survival was identified.     

ACVIM consensus statement on the diagnosis of immune‐mediated hemolytic anemia in dogs and cats

Immune‐mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune‐mediated erythrocyte destruction, and adverse consequences of long‐term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence‐based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.     

Prevalence of interfering antibodies in dogs and cats evaluated using a species‐independent assay

Background

Interfering antibodies in human serum and plasma are known to react with mammalian antibodies in immunoassays and cause false‐positive test results. Although this phenomenon was recently shown in companion animals, knowledge regarding immunoassay interference in veterinary medicine is very limited.

Objectives

The aims of this study were to set up a species‐independent immunoassay procedure to detect interference in serum samples, to screen for interference in a cross‐section of canine and feline patient samples from an animal hospital, and to determine if the detected interference could be neutralized using an immunoassay based on nonmammalian reagents.

Methods

A 2‐site sandwich‐type interference assay was set up using commercially available mouse reagents. A total of 369 serum samples from 320 dogs and 263 samples from 218 cats were analyzed using the interference assay. Multiple samples were submitted from 36 dogs and 39 cats. Nineteen samples identified as interference‐positive were analyzed in an assay using chicken antibodies.

Results

Interference was detected in samples from 28 dogs (9%) and 10 cats (5%) screened with the interference assay. Except for 1 cat, consistent results were obtained for all 75 dogs and cats that submitted more than 1 sample. The interference was eliminated when analyzed in the chicken‐based assay (P < .001).

Conclusions

Substances with reactivity toward mouse IgG can be detected in serum samples from dog and cat patients using a 2‐site interference assay. The detected substances are most likely interfering antibodies, possibly originating from immunization with other mammalian species.     

ACVIM consensus statement on the treatment of immune‐mediated hemolytic anemia in dogs

Immune‐mediated hemolytic anemia (IMHA) causes severe anemia in dogs and is associated with considerable morbidity and mortality. Treatment with various immunosuppressive and antithrombotic drugs has been described anecdotally and in previous studies, but little consensus exists among veterinarians as to the optimal regimen to employ and maintain after diagnosis of the disease. To address this inconsistency and provide evidence‐based guidelines for treatment of IMHA in dogs, we identified and extracted data from studies published in the veterinary literature. We developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria, explanation of treatment regimens, and validity of statistical methods. In combination with our clinical experience and comparable guidelines for humans afflicted with autoimmune hemolytic anemia, we used the conclusions of this process to make a set of clinical recommendations regarding treatment of IMHA in dogs, which we refined subsequently by conducting several iterations of Delphi review. Additionally, we considered emerging treatments for IMHA in dogs and highlighted areas deserving of future research. Comments were solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted for publication. The resulting document is intended to provide clinical guidelines for management of IMHA in dogs. These guidelines should be implemented pragmatically, with consideration of animal, owner, and veterinary factors that may vary among cases.