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

Management of immune thrombocytopenia (ITP) in dogs and cats is evolving, but there are no evidence-based guidelines to assist clinicians with treatment decisions. Likewise, the overall goals for treatment of ITP have not been established. Immunosuppressive doses of glucocorticoids are the first line treatment, but optimal treatment regimens beyond glucocorticoids remain uncertain. Additional options include secondary immunosuppressive drugs such as azathioprine, modified cyclosporine, and mycophenolate mofetil, usually selected based on clinician preference. Vincristine, human IV immunoglobulin (hIVIg), and transfusion of platelet or red blood cell-containing products are often used in more severe cases. Splenectomy and thrombopoietin receptor agonists are usually reserved for refractory cases, but when and in which patient these modalities should be employed is under debate. To develop evidence-based guidelines for individualized treatment of ITP patients, we asked 20 Population Intervention Comparison Outcome (PICO) format questions. These were addressed by 17 evidence evaluators using a literature pool of 288 articles identified by a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations. These were integrated by treatment domain chairs and then refined by iterative Delphi survey review to reach consensus on the final guidelines. In addition, 19 non-PICO questions covering scenarios in which evidence was lacking or of low quality were answered by expert opinion using iterative Delphi surveys with panelist integration and refinement. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The rigorous consensus process identified few comparative treatment studies, highlighting many areas of ITP treatment requiring additional studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Diagnosis of Immune Thrombocytopenia in Dogs and Cats.

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.

Rectally obtained fecal samples can be used for fecal occult blood testing in dogs, and fecal immunochemical tests do not detect canine or feline blood.

OBJECTIVE: The first objective was to determine if the sample collection method (naturally voided vs digital rectal examination collection) affected fecal occult blood test (FOBT) results. The second objective was to assess the ability of human fecal hemoglobin immunochemical tests to detect canine and feline blood. ANIMALS: 308 privately owned dogs, healthy and sick. METHODS: Guaiac FOBTs were performed on paired voided and rectally obtained canine fecal samples. The kappa statistic was used to assess agreement between the 2 collection methods, and a multivariate regression model was used to identify factors associated with a positive FOBT. Two fecal immunochemical tests (FITs; Hemosure One Step and OC-Light S) were tested with serially diluted human, canine, and feline blood. RESULTS: Voided and rectally obtained samples showed strong FOB-positivity agreement (k = 0.80), with 92.5% concordance and only 13/308 dogs negative on void but positive on rectal. Multivariate analysis showed dogs with gastrointestinal disease (P = .0008, rectal; P = .0001, void) were more likely and heavier dogs (P = .0037, rectal; P = .0022 void) were less likely to test FOBT positive. Health status, fasting status, NSAID use, and age were associated with FOBT results on univariate, but not multivariate, analysis. FITs did not detect canine or feline blood at any concentration while human blood performed as expected. CLINICAL RELEVANCE: Rectally obtained fecal samples can be reliably used for FOBTs. Human FITs may not be suitable for companion animals, but evaluation of other available tests is needed.

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Development of a Ratiometric Tension Sensor Exclusively Responding to Integrin Tension Magnitude in Live Cells.

Integrin tensions are critical for cell mechanotransduction. By converting force to fluorescence, molecular tension sensors image integrin tensions in live cells with a high resolution. However, the fluorescence signal intensity results collectively from integrin tension magnitude, tension dwell time, integrin density, sensor accessibility, and so forth, making it highly challenging to specifically monitor the molecular force level of integrin tensions. Here, a ratiometric tension sensor (RTS) was developed to exclusively monitor the integrin tension magnitude. The RTS consists of two tension-sensing units that are coupled in series and always subject to the same integrin tension. These two units are activated by tension to fluoresce in separate spectra and with different activation rates. The ratio of their activation probabilities, reported by fluorescence ratiometric measurement, is solely determined by the local integrin tension magnitude. RTS responded sensitively to the variation of integrin tension magnitude in platelets and focal adhesions due to different cell plating times, actomyosin inhibition, or vinculin knockout. At last, RTS confirmed that integrin tension magnitude in platelets and focal adhesions decreases monotonically with the substrate rigidity, verifying the rigidity dependence of integrin tensions in live cells and suggesting that integrin tension magnitude could be a key biomechanical factor in cell rigidity sensing.

Prophylactic use of a lyophilized platelet product for rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a dog with a P2Y12 platelet receptor mutation.

OBJECTIVE: To describe the periprocedural use of a lyophilized platelet product during rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a Greater Swiss Mountain Dog with a P2Y12 platelet receptor disorder. CASE SUMMARY: After the development of severe epistaxis, a Greater Swiss Mountain Dog was diagnosed with thrombopathia secondary to a P2Y12 receptor gene mutation. Concurrent primary nasal disease was also suspected due to persistent mucopurulent nasal discharge. One month after the initial presentation for epistaxis, the dog was readmitted for workup of nasal disease. Computed tomography of the head showed turbinate lysis and regional lymphadenopathy. Because of concern for a high risk of bleeding in a thrombopathic patient subjected to rhinoscopy and nasal biopsies, a lyophilized platelet product was administered prior to the procedure. Rhinoscopic exam revealed fungal plaques consistent with Aspergillus spp. that were later confirmed on fungal culture to be Aspergillus fumigatus. Rhinoscopic biopsies were performed as well as debridement of the fungal plaques, followed by topical administration of clotrimazole solution. Bleeding was minimal during and after the procedure, and the dog recovered uneventfully. NEW OR UNIQUE INFORMATION PROVIDED: This is the first report of the prophylactic use of lyophilized platelets in a thrombopathic patient undergoing an invasive procedure with potential for significant hemorrhage. Minimal bleeding occurred during the procedure, suggesting that lyophilized platelets could be used for the prevention of bleeding in thrombopathic patients undergoing invasive procedures.

Evaluation of coated platelets, a subset of highly procoagulant platelets, in healthy dogs and dogs with neoplasia.

OBJECTIVE: To determine if dogs with neoplasia produce more coated platelets, a subpopulation of activated platelets generated by dual stimulation with thrombin and convulxin, a glycoprotein VI agonist, than healthy control dogs. ANIMALS: Client-owned dogs diagnosed with lymphoma (n = 19) or solid tumors (14) and healthy control dogs (14). PROCEDURES: Platelets were stimulated ex vivo with thrombin and convulxin. Flow cytometry was used to quantify the percentage of coated platelets based on high levels of surface fibrinogen. To compare the percentage of coated platelets between the three groups, an ANOVA was performed followed by pairwise 95% confidence intervals (CI) adjusted for multiple comparisons using Tukey's method. RESULTS: We observed a greater mean percentage of coated platelets in dogs with solid tumors, compared with healthy control dogs, by 10.9 percentage points (95% CI: -1.0, 22.8), and a mean percentage of coated platelets in dogs with lymphoma that was less than healthy control dogs by 0.3 percentage points (95% CI: -11.4, 10.8). CLINICAL RELEVANCE: This study provides the first data-based evidence that dogs with solid tumors may have a greater mean coated platelet percentage when compared with healthy control dogs, although there is overlap between groups. Further studies are needed investigating coated platelets in specific subsets of neoplasia and investigating additional mechanisms of hypercoagulability in dogs with neoplasia.

Characterization of post-transfusion anti-FEA 1 alloantibodies in transfusion-naive FEA 1-negative cats.

OBJECTIVES: The aim of this study was to characterize anti-feline erythrocyte antigen (FEA) 1 alloantibodies following sensitization of FEA 1-negative cats, including their rate of appearance, agglutination titer over time and immunoglobulin class. A secondary aim was to obtain polyclonal anti-FEA 1 alloantibodies to increase the availability of FEA 1 blood typing. We also describe a case study documenting an acute hemolytic transfusion reaction in a transfusion-naive FEA 1-negative feline patient that received FEA 1-positive blood. METHODS: In this prospective clinical study, 35 cats with blood group type A underwent extensive blood typing for FEA 1-5. Two cats were identified as FEA 1-negative; these cats were transfused uneventfully with 50 ml of FEA 1-positive, but otherwise compatible, packed red blood cells. Post-transfusion blood samples were collected routinely as long as anti-FEA 1 alloantibodies were detected. Appearance of anti-FEA 1 alloantibodies was detected using a gel column crossmatch method. RESULTS: Anti-FEA 1 alloantibodies were detected as early as 5 days post-transfusion and remained detectable for over 400 days in one cat. Agglutination titers in both cats were relatively weak (1:1 to 1:8). The main immunoglobulin class was IgM. CONCLUSIONS AND RELEVANCE: Transfusion of FEA 1-negative, transfusion-naive cats with FEA 1-positive blood results in production of post-transfusion anti-FEA 1 alloantibodies as early as 5 days post-transfusion. Our results confirm the potential immunogenicity of FEA 1 and support crossmatching prior to a blood transfusion, even in transfusion-naive cats. Further studies are needed to better document the clinical importance of these post-transfusion antibodies, as well as to facilitate routine blood typing for the FEA 1 antigen in cats.

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia.

BACKGROUND: Canine immune thrombocytopenia (ITP) ranges from a mild to severe bleeding disorder, and platelet counts do not reliably predict clinical disease course. The detection of platelet autoantibodies may further define the disease phenotype, but variability in assay configurations and a lack of well-characterized controls limit the diagnostic utility of anti-platelet antibody assays. OBJECTIVES: We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays. METHODS: Silica microspheres were coated with canine IgG and IgM to assess the reactivity of goat and rabbit origin anti-canine immunoglobulin reagents. They were also used as positive controls in the PSAIg assay. Preliminary assay evaluation and determination of sample stability used PRP isolated from seven healthy dogs and 26 dogs newly diagnosed with thrombocytopenia. RESULTS: Blood sample stability was established for up to a 48-hour storage time. The conjugated positive control microspheres demonstrated stable fluorescent labeling over a 2-year observation period. Rabbit and goat origin anti-dog IgM fluorescent antibody labels reacted nonspecifically with canine IgG. Rabbit origin anti-dog IgG antibody demonstrated greater class specificity for canine IgG than a goat origin antibody. Thrombocytopenic dogs had a broad range of membrane-bound immunoglobulin. Median PSAIgG for dogs with primary or secondary ITP (18.4%, 34.1%, respectively) were significantly higher than controls (3.8%, P < .05). CONCLUSIONS: The described assay reagents and procedures provide positive controls and allow consistent thresholding to define a positive test result, suitable for any flow cytometer. A rabbit anti-dog IgG fluorescent label demonstrated specificity for canine IgG and was useful for the detection of PSAIgG in thrombocytopenic dogs.

ß-Cyclodextrin-containing polymer treatment of cutaneous lupus and influenza improves outcomes.

Nucleic acid (NA)-containing damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) are implicated in numerous pathological conditions from infectious diseases to autoimmune disorders. Nucleic acid-binding polymers, including polyamidoamine (PAMAM) dendrimers, have demonstrated anti-inflammatory properties when administered to neutralize DAMPs/PAMPs. The PAMAM G3 variant has been shown to have beneficial effects in a cutaneous lupus erythematosus (CLE) murine model and improve survival of mice challenged with influenza. Unfortunately, the narrow therapeutic window of cationic PAMAM dendrimers makes their clinical development challenging. An alternative nucleic acid-binding polymer that has been evaluated in humans is a linear ß-cyclodextrin-containing polymer (CDP). CDP's characteristics prompted us to evaluate its anti-inflammatory potential in CLE autoimmune and influenza infectious disease mouse models. We report that CDP effectively inhibits NA-containing DAMP-mediated activation of Toll-like receptors (TLRs) in cell culture, improves healing in lupus mice, and does not immunocompromise treated animals upon influenza infection but improves survival even when administered 3 days after infection. Finally, as anticipated, we observe limited toxicity in animals treated with CDP compared with PAMAM G3. Thus, CDP is a new anti-inflammatory agent that may be readily translated to the clinic to combat diseases associated with pathological NA-containing DAMPs/PAMPs.

Microfluidic chip grafted with integrin tension sensors for evaluating the effects of flowing shear stress and ROCK inhibitor on platelets.

Integrins are key players in platelet adhesion and aggregation. Integrin molecular tensions, the forces transmitted by integrin molecules, are regulated by both mechanical and biochemical cues, and the outside-in and inside-out signaling has been extensively studied. While the mechanical properties of platelets at static status have been studied by atomic force microscopy, traction force microscopy and tension sensors, the biomechanical properties of flowing platelets remain elusive. Herein, we report microfluidic chips grafted with integrin tension sensors for microfluidic-force mapping in platelets. Specifically, the process of integrin αIIbß3 mediating tension transmission and platelet adhesion under low flow rates has been obtained, and the process of platelet clustering at post-stenotic regions has been demonstrated. We found that flowing shear force can postpone the integrin-mediated tension transmission and platelet adhesion. We further evaluated the effect of Y-27632, a ROCK inhibitor that has been proven to reduce integrin-mediated platelet adhesion, at a series of concentrations and demonstrated that microfluidic chips with integrin tension sensors are sensitive to the concentration-dependent effects of Y-27632. Given their low cost and scalable throughput, these chips are ideal technical platforms for biological studies of platelets at flowing status and for platelet inhibitor or potential antiplatelet drug screening.

Platelet number and function in response to a single intravenous dose of vincristine.

BACKGROUND: Vincristine might increase circulating platelet numbers but the functional capacity of these newly released platelets is unknown. OBJECTIVE: To evaluate and compare the functionality of mature and immature (reticulated) platelets after a single intravenous dose of vincristine in dogs. ANIMALS: Ten healthy purpose-bred dogs. METHODS: Dogs prospectively received a single IV injection of 0.02 mg/kg vincristine or 0.9% saline. Before and after treatment on days 3, 5, and 7, platelets (resting and after thrombin stimulation) were assessed by flow cytometric determination of P-selectin (CD62P) expression. Reticulated platelets were distinguished using thiazole orange (TO) staining. RESULTS: Relative to saline, vincristine administration increased platelet count from day 0 to day 7 (225 ± 58 to 273 ± 65 × 103 /µL, vs 299 ± 76.4 to 214 ± 20 × 103 /µL, P = .01) and increased percentage of reticulated platelets from day 0 to day 5 (3.9 ± 1.5% to 6.1 ± 1.6%, P = .02). On all days, reticulated platelets had greater resting expression of CD62P than did mature platelets (49.6 ± 4% vs 10.2 ± 1%, P ≤ .001). Across all days, CD62P expression by reticulated platelets in the vincristine and saline-treated groups was not different when unstimulated (P = .7) or after thrombin stimulation (P = .33). CONCLUSIONS AND CLINICAL IMPORTANCE: Reticulated platelets released in response to vincristine administration function similarly to mature platelets.

Ecological level analysis of primary lung tumors in dogs and cats and environmental radon activity.

BACKGROUND: Epidemiologic studies suggest residential radon exposure might increase the risk of primary lung cancer in people, but these studies are limited by subject mobility. This limitation might be overcome by evaluating the association in pets. HYPOTHESIS: Primary pulmonary neoplasia (PPN) rate is higher in dogs and cats residing in counties with a high radon exposure risk (Environmental Protection Agency [EPA] zone 1) compared to zones 2 (moderate radon exposure risk) and 3 (low radon exposure risk). ANIMALS: Six hundred ninety client-owned dogs and 205 client-owned cats with PPN. METHODS: Retrospective review of medical records at 10 veterinary colleges identified dogs and cats diagnosed with PPN between 2010 and 2015. Each patient's radon exposure was determined by matching the patient's zip code with published county radon exposure risk. County level PPN rates were calculated using the average annual county cat and dog populations. The PPN counts per 100 000 dog/cat years at risk (PPN rates) were compared across radon zones for each species. RESULTS: The PPN rate ratio in counties in high radon zone (1) was approximately 2-fold higher than in counties in lower radon zones for dogs (rate ratio zone 1 to 2, 2.49; 95% confidence interval [CI], 1.56-4.00; rate ratio zone 1 to 3, 2.29; 95% CI, 1.46-3.59) and cats (rate ratio zone 1 to 2, 2.13; 95% CI, 0.95-4.79; zone 1 to 3, 1.81; 95% CI, 0.9-3.61). CONCLUSIONS AND CLINICAL IMPORTANCE: Exposure to household radon might play a role in development of PPN in dogs and cats.

Lyophilized platelets versus cryopreserved platelets for management of bleeding in thrombocytopenic dogs: A multicenter randomized clinical trial.

BACKGROUND: Thrombocytopenia in dogs is common in critical care medicine, but availability of fresh platelet concentrates in veterinary medicine can be limiting. Lyophilized platelets have long shelf-lives and can be easily transported, stored, and administered in various settings. OBJECTIVE: To evaluate the efficacy and safety of a novel trehalose-stabilized canine lyophilized platelet product in thrombocytopenic dogs with clinically-evident bleeding. ANIMALS: Eighty-eight dogs with platelet counts <50 × 103 /µL and a standardized bleeding assessment tool (DOGiBAT) score ≥2. METHODS: Multicenter, randomized, non-blinded, non-inferiority clinical trial comparing dimethyl sulfoxide (DMSO)-stabilized cryopreserved platelet concentrates (CPP) with trehalose-stabilized lyophilized platelets (LP) for control of bleeding in thrombocytopenic dogs. Dogs were randomized to receive 3 × 109 platelets/kg of LP or CPP. Primary outcome measures were change in DOGiBAT score, platelet count, need for additional red cell transfusion and all-cause mortality. RESULTS: Fifty dogs received LP and 38 received CPP. Baseline demographics and clinical characteristics of both groups were comparable. At 1-hour post-transfusion, LP were superior for change in DOGiBAT score, and non-inferior at 24-hours post-transfusion. The LP were non-inferior to CPP for change in platelet count, need for additional red blood cell units, and survival to discharge. The LP were superior for change in hematocrit at 1-hour post-transfusion, and non-inferior at 24-hours. No adverse effects were noted in either group. CONCLUSIONS AND CLINICAL IMPORTANCE: A novel trehalose-stabilized canine LP product appears to be logistically superior and is clinically non-inferior to DMSO-stabilized canine CPP for management of bleeding in thrombocytopenic dogs.

Cell Migration Driven by Self-Generated Integrin Ligand Gradient on Ligand-Labile Surfaces.

Integrin-ligand interaction mediates the adhesion and migration of many metazoan cells. Here, we report a unique mode of cell migration elicited by the lability of integrin ligands. We found that stationary cells spontaneously turn migratory on substrates where integrin ligands are subject to depletion by cellular force. Using TGT, a rupturable molecular linker, we quantitatively tuned the rate of ligand rupture by cellular force and tested platelets (anucleate cells), CHO-K1 cells (nucleated cells), and other cell types on TGT surfaces. These originally stationary cells readily turn motile on the uniform TGT surface, and their motility is correlated with the ligand depletion rate caused by cells. We named this new migration mode ligand-depleting (LD) migration. Through both experiments and simulations, we revealed the biophysical mechanism of LD migration. We found that the cells create and maintain a gradient of ligand surface density underneath the cell body by constantly rupturing local ligands, and the gradient in turn drives and guides cell migration. This is reminiscent of the phenomenon that some liquid droplets or solid beads can spontaneously move on homogeneous surfaces by chemically forming and maintaining a local gradient of surface energy. Here, we showed that cells, as living systems, can harness a similar mechanism to migrate. LD migration is beneficial for cells to maintain adhesion on ligand-labile surfaces, and might also play a role in the migration of cancer cells, immune cells, and platelets that deplete adhesive ligands of the matrix.

Neutrophil extracellular traps in stored canine red blood cell units.

BACKGROUND: Neutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion-related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients. HYPOTHESES: (1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients. ANIMALS: Six healthy purpose-bred research dogs were utilized for blood donation. METHODS: Prospective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell-free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed. RESULTS: Markers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant. CONCLUSIONS AND CLINICAL IMPORTANCE: NETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

Performance of a Nageotte hemocytometer method and a flow cytometric assay for residual leukocyte quantification in leukoreduced canine packed red blood cells.

OBJECTIVE: To evaluate the performances of a manual Nageotte hemocytometer method and commercial fluorescent bead-based flow cytometric assay for quantifying [rWBC] in leukoreduced canine packed red blood cell (pRBC) units. DESIGN: Prospective study. Five, commercially purchased, double leukoreduced canine pRBC units were spiked with canine leukocytes to create 6 pRBC standards with the following [rWBC]: < 0.1, 0.375, 1.5, 3.0, 6.0, and 24.0 WBC/µL. [rWBC] of each pRBC standard was measured with the Nageotte hemocytometer and flow cytometric techniques. Limit of detection (LoD), linearity, and bias were determined for each method. For each standard, accuracy and precision were calculated; the cumulative accuracy and mean precision for measurements between the LoD and 24.0 WBC/µL were also determined. SETTING: University veterinary blood bank and clinical pathology laboratory. MEASUREMENTS AND MAIN RESULTS: The Nageotte hemocytometer method had an LoD = 1.48 WBC/µL, inadequate linearity (R2  = 0.92), and a significant negative proportional bias (slope best-fit line = 0.52 ± 0.03). Between [rWBC] 1.5-24 WBC/µL, the technique demonstrated poor cumulative accuracy (6.7%) but acceptable mean precision (17.3%). Relative to a 2 rWBC/µL threshold, at 1.5 WBC/µL the method was inaccurate (6.7%) with acceptable precision (16.6%). The flow cytometric assay had an LoD = 1.3 WBC/µL, acceptable linearity (R2  = 0.99), and a mild positive proportional bias (slope best-fit line = 1.11 ± 0.01). The technique had acceptable cumulative accuracy (80%) and mean precision (10.7%) for measuring [rWBC] between 1.5 and 24 WBC/µL. At 1.5 WBC/µL, this method was acceptably accurate (86.7%) and precise (16.0%). CONCLUSIONS: The flow cytometric assay demonstrated acceptable performance for quantification of [rWBC] in leukoreduced canine pRBC units. The Nageotte hemocytometer method should be used cautiously due to poor accuracy and significant negative bias.

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

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

Inaccurate point-of-care blood glucose measurement in a dog with secondary erythrocytosis.

BACKGROUND: Point-of-care (POC) portable blood glucose meters (PBGMs) are convenient and inexpensive tools for assessing patient blood glucose concentrations. They are often used to quickly diagnose hypoglycemia or collect serial glucose readings in diabetic patients. However, POC meters have been previously identified in human and veterinary literature to be inaccurate when utilized in patients with abnormal HCT. This problem may not be reflected in manufacturer guidelines referenced by practitioners in the POC setting. KEY FINDINGS: A 1.5-year-old dog, previously diagnosed with multiple congenital cardiac malformations, right-to-left cardiac shunting and secondary erythrocytosis, presented to a veterinary emergency center minimally responsive and without detectable pulses. PBGM measurement identified hypoglycemia. Following stabilization of the dog, serial glucose assessments showed discordant results between PBGMs and the reference laboratory biochemistry analyzer. A pathological cause for hypoglycemia was not identified and PBGM readings were determined to be erroneously low due to the dog's abnormally high HCT. SIGNIFICANCE: This case demonstrates the limitations of using PBGMs to assess blood glucose in a dog with secondary erythrocytosis. The report emphasizes the need for judicious use of PBGMs in critically ill patients and that these glucometers may not be reliable in patients with abnormal HCT values.