ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

Comparison of visual assessments of anisocytosis in canine blood smears and analyzer-calculated red blood cell distribution width.

Red blood cell distribution width (RDW) and visual assessments of anisocytosis assess variability in erythrocyte size. Veterinary studies on the correlation between the two methods and on observer agreement are scarce. The objectives were to assess the correlation of the grading of anisocytosis by means of conventional microscopy of canine blood smears to RDW, and to assess intra- and inter-observer variation in assessing the degree of anisocytosis. The study included 100 canine blood samples on which blood smear examination and RDW measurement were performed. RDW was measured on the Advia 2120i analyzer. The degree of anisocytosis was based on a human grading scheme assessing the ratio between the size of the representative largest red blood cell and that of the representative smallest red blood cell (1+ if <2x, 2+ if 2-3x, 3+ if 3-4x, and 4+ if >4x). Three observers participated and assessed the blood smears by conventional microscopy twice, 3 weeks apart by each observer. The correlation was assessed for each observer on each occasion using Kendahl-tau-b analysis. Intra-observer agreement was assessed using quadratically weighted kappa. Inter-observer agreement was assessed using free-marginal multi-rater kappa. Anisocytosis graded on blood smears correlated significantly with RDW values as assessed by Kendahl-tau-b ranging between 0.37 and 0.51 (p < 0.0001). Intra-observer agreement ranged from weak to moderate with resulting kappa-coefficients being 0.58, 0.68, and 0.75, respectively. Inter-observer agreement was weak (Kappa-values 0.44). The weak to moderate observer agreement in the visual assessment of anisocytosis indicates that the more precise and more repeatable RDW measurement should be used for clinical decision-making.

Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy.

BACKGROUND: Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs (miRNAs) have promise as non-invasive biomarkers in serum and feces for diagnosis of GIC. HYPOTHESIS/OBJECTIVES: Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats. ANIMALS: Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client-owned. METHODS: Cats were recruited for an international multicenter observational prospective case-control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Serum miR-223-3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval [CI], 0.760-1.0), sensitivity of 90% (95% CI, 59.6-99.5%), and specificity of 77.8% (95% CI, 45.3-96.1%). Serum miR-223-3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE. CONCLUSION AND CLINICAL IMPORTANCE: Serum miR-223-3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.

Association of fecal and serum microRNA profiles with gastrointestinal cancer and chronic inflammatory enteropathy in dogs.

BACKGROUND: Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as diagnostic and prognostic markers of GI disease in humans and dogs. HYPOTHESIS/OBJECTIVES: Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. AIMS: (a) identify miRNAs that differentiate GIC from CIE, (b) use high-throughput reverse transcription quantitative real-time PCR (RT-qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs. ANIMALS: Twenty-four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client-owned. METHODS: An international multicenter observational prospective case-control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT-qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE. RESULTS: The best diagnostic performance for distinguishing GIC from CIE was fecal miR-451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR-223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR-27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR-20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR-148a-3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR-652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR-451 and miR-223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%). CONCLUSIONS AND CLINICAL IMPORTANCE: When used as part of a diagnostic RT-qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.

Investigation of two different human d-dimer assays in the horse.

BACKGROUND: D-dimer has value as a marker of thrombosis in critically ill horses and can provide additional information about prognosis. However, there are currently no equine species-specific d-dimer assays available, nor has there been any formal investigation of the applicability of human d-dimer assays in horses, so it is unknown, which assay performs best in this species. The aim of this study was therefore to evaluate and compare two human d-dimer assays for their applicability in horses. The study included four groups of horses: clinically healthy horses, horses with gastrointestinal (GI) disease and mild systemic inflammation based on low serum amyloid A (SAA) (low SAA group), horses with GI disease and strong systemic inflammation based on high SAA (high SAA group) and, horses with thrombotic GI disease caused by Strongylus vulgaris (also called non-strangulating intestinal infarction (NSII)) (NSII group). The assays evaluated were the STAGO STA-Liatest D-di + (Stago) and NycoCard™ D-dimer (NycoCard). Intra- and inter-coefficients of variation (CV) were assessed on two d-dimer concentrations, and linearity under dilution was evaluated. A group comparison was performed for both assays across the four groups of horses. A Spaghetti plot, Spearman Correlation, Passing Bablok regression and Bland-Altman plot were used to compare methods in terms of agreement. RESULTS: Ten horses were included in the clinically healthy group, eight in the low SAA group, eight in the high SAA group, and seven in the NSII group. For the Stago assay, intra- and inter-CVs were below the accepted level except for one inter-CV. The NycoCard assay did not meet the accepted level for any of the CVs. The linearity under dilution was acceptable for both the Stago and NycoCard. In the group comparison, both methods detected a significantly higher d-dimer concentration in the high SAA and NSII groups compared to the clinically healthy group. Method agreement showed slightly higher d-dimer concentrations with NycoCard compared to Stago. The overall agreement was stronger for the lower d-dimer concentrations. CONCLUSION: Both the Stago and the NycoCard were found to be applicable for use in horses but were not directly comparable.

Comparison of automated total nucleated cell counts vs manual cell counts in the amniotic fluid for the classification of disease status in healthy and diseased pigs.

BACKGROUND: An increase in the total nucleated cell count (TNCC) of amniotic fluid can detect inflammation in the amniotic cavity. Traditionally, a manual count is employed, but the technique suffers from inaccuracy and is labor intensive. Automated cell counting could enhance precision and timeliness. OBJECTIVES: We aimed to analyze the cell counts in the amniotic fluid of preterm pigs using an automated method and compared the results with manual cell counts. We also tested if increased TNCC is associated with an inflammatory group in a porcine model of chorioamnionitis. METHODS: Amniotic fluids from 34 preterm pigs were analyzed blindly using two automatic settings of the ADVIA 2120i hematology analyzer (whole blood [WB] and cell poor [CP] settings) and manually by two observers using Neubauer chambers. Inter- and intra-variability were calculated. The correlation analysis of TNCC/µL in amniotic fluid was determined between the methods, including Deming and Bland-Altman analyses. Fischer's exact tests compared the known health status of the pigs to the outcomes of the automatic and manual TNCC. RESULTS: Inter- and intra-observer variability of the manual TNCC were high, although the correlation of TNCC between (r = .95, P < .0001) and within observers (r = .98, P < .0001; r = .89, P < .0001) was good. Correlation between the manual and CP TNCC was moderate and significant (r = .50, P = .014). Manual and WB TNCC were not correlated. The CP (P = .003) and manual (P = .0001) analyses accurately classified the disease state of the pigs. CONCLUSIONS: An acceptable correlation between automatic CP TNCC and manual counting was demonstrated. Both methods could accurately classify the disease state of the pigs.

Hemostatic Dysfunction in Dogs Naturally Infected with Angiostrongylus vasorum-A Narrative Review.

This narrative review aims to describe Angiostrongylus vasorum-induced hemostatic dysfunction of dogs with emphasis on clinical and laboratory findings as well as potential therapeutic strategies for the bleeding patient. Canine angiostrongylosis (CA) is a disease with potentially high morbidity and mortality in endemic areas and with fatal outcome often associated with either severe respiratory compromise, pulmonary hypertension and right-sided heart failure, or hemostatic dysfunction with severe bleeding. The most common signs of hemorrhage are hematomas, petecchiation, ecchymoses, oral mucosal membrane bleeding and scleral bleeding, while intracranial and pulmonary hemorrhage are among the most severe. The pathophysiological mechanisms underlying hemostatic dysfunction in these patients are presently researched. While the larval effect on platelets remains unknown, the parasite appears to induce dysregulation of hemostatic proteins, with studies suggesting a mixture of pro-coagulant protein consumption and hyperfibrinolysis. Importantly, not all dogs display the same hemostatic abnormalities. Consequently, characterizing the hemostatic state of the individual patient is necessary, but has proven difficult with traditional coagulation tests. Global viscoelastic testing shows promise, but has limited availability in general practice. Treatment of A. vasorum-infected dogs with hemostatic dysfunction relies on anthelmintic treatment as well as therapy directed at the individual dog's specific hemostatic alterations.

Diagnosis of primary hyperfibrinolysis and in vitro investigation of the inhibitory effects of tranexamic acid in a group of dogs with sarcomas - A pilot study.

Primary hyperfibrinolysis is not well characterised in canine cancer. This prospective case-control pilot study aimed to evaluate tissue plasminogen activator-modified thromboelastography (tPA-TEG) for diagnosis of primary hyperfibrinolysis in dogs with cancer and establish the in vitro therapeutic concentration of tranexamic acid (TXA). Nine dogs with sarcomas and normocoagulable thromboelastograms and 11 healthy dogs were included. For each a whole blood tPA-TEG, and four tPA-TEGs with added TXA in different concentrations were analysed. Lysis percentage at 30/60 min following maximal amplitude (LY30/60), clot lysis index (CL30/60), maximum rate of lysis (MRL), and total lysis (L) were investigated as diagnostic parameters of primary hyperfibrinolysis. In vitro TXA concentrations needed to inhibit 50% (IC50) and 90% (IC90) of the fibrinolytic potential were compared between groups. Significant primary hyperfibrinolysis (LY30 (P = 0.0001), LY60 (P = 0.003), CL30 (P = 0.01), and L (P = 0.02)) was observed in dogs with sarcomas. IC50 and IC90 of in vitro TXA for normalizing LY30 were 13.34 (SE 1.52) and 31.10 (SE 3.01) mg/L for dogs with sarcomas and 4.41 (SE 5.84) and 20.00 (SE 6.18) mg/L for healthy dogs. IC50 and IC90 for normalizing LY60 were 22.18 (SE 1.27) and 58.94 (SE 5.47) mg/L for dogs with sarcomas and 11.25 (SE 2.82) and 56.20 (SE 11.61) mg/L for healthy dogs. The IC50 for LY60 was significantly increased for dogs with sarcomas (P = 0.0003). Primary hyperfibrinolysis was documented by tPA-TEG in dogs with sarcomas. In vitro IC50 and IC90 for TXA were established. Clinical studies are required to establish therapeutic dosages in vivo.

Stability and profiling of urinary microRNAs in healthy cats and cats with pyelonephritis or other urological conditions.

BACKGROUND: Specific biomarkers of pyelonephritis (PN) in cats are lacking. MicroRNAs (miRNAs) have diagnostic potential in human nephropathies. OBJECTIVES: To investigate the presence/stability of miRNAs in whole urine of cats and the discriminatory potential of selected urinary miRNAs for PN in cats. ANIMALS: Twelve healthy cats, 5 cats with PN, and 13 cats with chronic kidney disease (n = 5), subclinical bacteriuria (n = 3), and ureteral obstructions (n = 5) recruited from 2 companion animal hospitals. METHODS: Prospective case-control study. Expression profiles of 24 miRNAs were performed by quantitative PCR (qPCR). Effect of storage temperature (4°C [24 hours], -20°C, and -80°C) was determined for a subset of miRNAs in healthy cats. RESULTS: Urinary miR-4286, miR-30c, miR-204, miR4454, miR-21, miR-16, miR-191, and miR-30a were detected. For the majority of miRNAs tested, storage at 4°C and -20°C resulted in significantly lower miRNA yield compared to storage at -80°C (mean log2fold changes across miRNAs from -0.5 ± 0.4 SD to -1.20 ± 0.4 SD (4°C versus -80°C) and from -0.7 ± 0.2 SD to -1.20 ± 0.3 SD (-20°C versus -80°C)). Cats with PN had significantly upregulated miR-16 with a mean log2fold change of 1.0 ± 0.4 SD, compared with controls (-0.1 ± 0.2, P = .01) and other urological conditions (0.6 ± 0.3, P = .04). CONCLUSIONS: Upregulation of miR16 might be PN-specific, pathogen-specific (Escherichia coli), or both.

Evaluation of fecal microRNA stability in healthy cats.

BACKGROUND: Gastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats. OBJECTIVES: We aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and -20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats. METHODS: Healthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to -20°C, stored for 24 hours at 4°C and then transferred to -20°C, or were immediately placed at -20°C on day 1 or at -20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real-time PCR. RESULTS: Ten miRNA assays worked well, and one, let-7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR-26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time. CONCLUSIONS: Fecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

Analytical validation of a conventional cardiac troponin I assay for dogs and cats.

BACKGROUND: Cardiac troponins are gold-standard biomarkers of myocardial injury. There is a need for validation of assays with higher availability and lower costs in veterinary medicine. OBJECTIVES: The primary aim of the present study was to perform an analytical validation of the IMMULITE 2000 TnI assay for use in dogs and cats. A secondary aim was to evaluate its agreement with the previously validated and sensitive Siemens ADVIA Centaur TnI-Ultra assay. METHODS: Intra- and inter-assay variation, detection limits, the linearity under dilution, and a sample addition study (modified spike-and-recovery analysis) were investigated to assess analytical performance in 15 canine and 15 feline serum samples. Agreement between the assays was evaluated by correlation and Bland-Altman analyses including an additional 99 canine serum samples. RESULTS: Intra-assay variation of cTnI in canine and feline serum was 3.71% and 4.68%, while inter-assay variation was 5.88% and 6.54%, respectively. The assay performed with acceptable linearity within a clinically relevant range of serum cTnI concentrations. The sample addition study revealed insufficient recovery in the range of 71.9%-81.4% for dogs and 62.6%-75.7% for cats. This was considered to be due to a negative matrix effect. A significant correlation between the assays was found, and the Bland-Altman analysis showed acceptable agreement for a wide range of concentrations, but revealed a proportional error, with the IMMULITE TnI assay consistently measuring a higher concentration than the Centaur TnI-Ultra assay. This was relevant only at high serum cTnI concentrations. CONCLUSIONS: The IMMULITE TnI assay is considered acceptable for clinical use in dogs and cats.

Analytical validation of platelet microparticle quantification in cats.

BACKGROUND: Cardiogenic embolism (CE) in cats is a devastating condition primarily associated with hypertrophic cardiomyopathy (HCM). Hypercoagulability may pose a risk for thrombus formation; however, no single test can predict CE development. Platelet microparticles (PMPs) released from platelet membranes are associated with thrombosis in humans. OBJECTIVES: The aims were to validate flow cytometric PMP quantification in cats analytically and, in a pilot study, evaluate the procoagulant annexin V (AnV) positive PMP concentration in healthy cats and cats with asymptomatic HCM. METHODS: With CD61 as a platelet marker, CD61+ AnV+ PMPs (0.3-1.0 µm) were quantified in citrated whole blood (WB) and platelet-poor plasma (PPP) using flow cytometry. Analyses were performed in 6 healthy cats and 5 cats with asymptomatic HCM. The coefficient of variation (CV) for duplicate (intra-assay) and parallel (inter-assay) analyses were calculated. RESULTS: PMP concentrations were quantified with acceptable intra-assay CV for WB (CD61+ /AnV- ; 2.4%, 0.2%-8.4% (median, range), CD61+ /AnV+ ; 3.8%, 0.1%-12.5%) and PPP (CD61+ /AnV- ; 5.0%, 0.7%-12.8%, CD61+ /AnV+ ; 7.4%, 0.5%-15.3%), and acceptable inter-assay CV for WB in 10/11 cats (CD61+ /AnV- ; 6.2%, 1.4%-13.3%, CD61+ /AnV+ ; 6.4%, 0.7%-17.2%), but unacceptable for PPP (CD61+ /AnV- ; 15.6%, 5.8%-42.7%, CD61+ /AnV+ ; 27.8%, 8.4%-77.1%). For WB PMP concentrations, the pilot data demonstrated no differences between healthy cats and cats with asymptomatic HCM (4/5 with left ventricular outflow obstruction) for either the CD61+ /AnV- or the CD61+ /AnV+ PMPs. CONCLUSIONS: Only WB PMP concentrations could be quantified reliably in cats in a clinical setting. PMP concentrations did not differ between healthy and asymptomatic HCM cats in this pilot study.

Evaluation of microRNA stability in feces from healthy dogs.

BACKGROUND: Gastrointestinal cancer accounts for approximately 8% of all canine malignancies. Early detection of cancer may have a tremendous impact on both treatment options and prognosis. MicroRNAs (miRNAs), a class of noncoding RNAs that can be found stably expressed in body fluids and feces, have been suggested as valuable human cancer biomarkers. OBJECTIVES: The purpose of the study was to investigate the feasibility of detecting miRNAs in canine feces and to determine the miRNA stability in fecal samples stored at different temperatures for different duration. METHODS: The levels of 4 Canine familiaris (cfa) miRNAs (cfa-miR-16, cfa-miR-20a, cfa-miR-21, and cfa-miR-92a) were investigated by quantitative real-time PCR(qPCR) in fecal samples from 10 healthy dogs. Fecal samples were collected at 3 different time points and samples from the first time point were stored at different temperatures and for a different duration. RESULTS: A statistically significant difference was found in miRNA levels from samples stored at room temperature compared with samples stored at -20°C for cfa-miR-16 and cfa-miR-21. No significant difference was found in the level of the investigated miRNAs over time. CONCLUSIONS: Overall, miRNAs are present in dog feces at measurable levels. Some miRNAs seem to be subject to a higher degree of degradation in samples stored at room temperature for 24 hours compared with samples frozen after collection at -20°C. The investigated miRNAs were stably expressed over time. This study provides the basis for further research on miRNA expression profiles as biomarkers for gastrointestinal cancer in dogs.

Evaluation of microRNA Stability in Plasma and Serum from Healthy Dogs.

BACKGROUND: Early and specific detection of cancer is of great importance for successful treatment of the disease. New biomarkers, such as microRNAs, could improve treatment efficiency and survival ratio. In human medicine, deregulation of microRNA profiles in circulation has shown great potential as a new type of biomarker for cancer diagnostics. There are, however, few studies of circulating microRNAs in dogs. Extracellular circulating microRNAs have shown a high level of stability in human blood and other body fluids. Nevertheless, there are still important issues to be solved before microRNAs can be applied routinely as a clinical tool, one of them being their stability over time in media commonly used for blood sampling. OBJECTIVE: Evaluation of the stability of microRNA levels in plasma and serum from healthy dogs after storage at room temperature for different time points before being processed. METHODS: The levels of four microRNAs (cfa-let-7a, cfa-miR-16, cfa-miR-23a and cfa-miR-26a) known to be stably expressed from other canine studies, have been measured by quantitative real-time PCR (qPCR). RESULTS: MicroRNA levels were found sufficiently stable for gene profiling in serum- and plasma stored at room temperature for 1 hour but not for samples stored at room temperature for 24 hours. CONCLUSION: Storage at room temperature of serum and plasma samples intended for microRNA profiling should be kept for a minimum period of time before proceeding with RNA isolation. For the four microRNAs investigated in the present study, we did not find significant differences in microRNA levels between serum and plasma samples from the same time point.