Maureen McMichael

Viscoelastic coagulation testing: Technology, applications, and limitations

Use of viscoelastic point-of-care (POC) coagulation instrumentation is relatively new to veterinary medicine. In human medicine, this technology has recently undergone resurgence owing to its capacity to detect hypercoagulability. The lack of sensitive tests for detecting hypercoagulable states, along with our current understanding of in vivo coagulation, highlights the deficiencies of standard coagulation tests, such as prothrombin and partial thromboplastin times, which are performed on platelet-poor plasma. Viscoelastic coagulation analyzers can provide an assessment of global coagulation, from the beginning of clot formation to fibrinolysis, utilizing whole blood. In people, use of this technology has been reported to improve management of hemostasis during surgery and decrease usage of blood products and is being used as a rapid screen for hypercoagulability. In veterinary medicine, clinical use of viscoelastic technology has been reported in dogs, cats, foals, and adult horses. This article will provide an overview of the technology, reagents and assays, applications in human and veterinary medicine, and limitations of the 3 viscoelastic POC analyzers in clinical use.

RECOVER evidence and knowledge gap analysis on veterinary CPR. Part 7: Clinical guidelines.

OBJECTIVE To present a series of evidence-based, consensus guidelines for veterinary CPR in dogs and cats. DESIGN Standardized, systematic evaluation of the literature, categorization of relevant articles according to level of evidence and quality, and development of consensus on conclusions for application of the concepts to clinical practice. Questions in five domains were examined: Preparedness and Prevention, Basic Life Support, Advanced Life Support, Monitoring, and Post-Cardiac Arrest Care. Standardized worksheet templates were used for each question, and the results reviewed by the domain members, by the RECOVER committee, and opened for comments by veterinary professionals for 4 weeks. Clinical guidelines were devised from these findings and again reviewed and commented on by the different entities within RECOVER as well as by veterinary professionals. SETTING Academia, referral practice and general practice. RESULTS A total of 74 worksheets were prepared to evaluate questions across the five domains. A series of 101 individual clinical guidelines were generated. In addition, a CPR algorithm, resuscitation drug-dosing scheme, and postcardiac arrest care algorithm were developed. CONCLUSIONS Although many knowledge gaps were identified, specific clinical guidelines for small animal veterinary CPR were generated from this evidence-based process. Future work is needed to objectively evaluate the effects of these new clinical guidelines on CPR outcome, and to address the knowledge gaps identified through this process.

Effect of leukoreduction on transfusion-induced inflammation in dogs

BACKGROUND Removal of leukocytes (LR) has been shown to eliminate or attenuate many of the adverse effects of transfusion in experimental animals and humans. HYPOTHESIS/OBJECTIVES Transfusion of stored packed red blood cells (pRBCs) is associated with an inflammatory response in dogs and prestorage LR attenuates the inflammatory response. ANIMALS Thirteen random-source, clinically healthy, medium and large breed dogs. METHODS Experimental study. On day 0, animals were examined and baseline blood samples were collected for analysis. Whole blood was then collected for processing with and without LR, and stored as pRBC. Twenty-one days later, stored pRBCs were transfused back to the donor. Blood samples were collected before and 1 and 3 days after transfusion. RESULTS In the dogs that received non-LR pRBCs (n = 6) there was a significant increase from baseline in white blood cell count from a mean (SD) of 8.20 (2.74) to 13.95 (4.60) × 10(3) cells/μL (P < .001) and in segmented neutrophil count from a mean (SD) of 5.76 (2.70) to 11.91 (4.71) × 10(3) cells/μL (P < .001). There were also significant increases in fibrinogen from a mean (SD) of 129.7 (24.2) to 268.6 (46.7) mg/dL (P < .001) and C-reactive protein from a mean (SD) of 1.9 (2.1) to 78.3 (39.3) μg/mL (P < .001). There was no significant increase from baseline in any of the markers in the dogs that received LR pRBC (n = 5). CONCLUSIONS AND CLINICAL IMPORTANCE There is a profound inflammatory response to transfusion in normal dogs, which is eliminated by LR of the pRBC units.

Clot formation in canine whole blood as measured by rotational thromboelastometry is influenced by sample handling and coagulation activator.

The objective of the present study was to systematically evaluate the impact of methodology on thromboelastometry with canine whole blood. Thromboelastometry was performed on citrated blood using a variety of combinations of clotting activators [ex-tem (tissue factor or TF), in-tem (ellagic acid), diluted TF from Innovin, or Ca (recalcification only)] and storage times. Thromboelastometry was also performed using diluted TF from Innovin on blood collected into a contact inhibitor. Ex-vivo contact activation was compared between canine and human blood. Clotting activator had a marked impact on coagulation time, a minor impact on alpha angle, and no impact on clot formation time or maximum clot firmness. When ex-tem or in-tem was the clotting activator, sample storage up to 30 min did not affect results. With diluted TF from Innovin or Ca, sample storage was associated with the development of increased coagulability (as indicated by shorter coagulation time and clot formation time and higher alpha angle) due to ex-vivo contact activation. Canine blood underwent markedly more ex-vivo contact activation than did human blood. Canine blood undergoes significant ex-vivo contact activation during and after collection, which influences thromboelastometry results when a weak clotting activator (such as low TF or recalcification) is used. Thromboelastometry with a strong activator (such as ex-tem or in-tem) is less influenced by ex-vivo changes, and, therefore, likely to be more reflective of in-vivo hemostatic capabilities and to provide consistently interpretable and comparable results.

Correlation of hematocrit, platelet concentration, and plasma coagulation factors with results of thromboelastometry in canine whole blood samples

OBJECTIVE To evaluate the components of canine whole blood samples that contribute to results of thromboelastometry (TEM). ANIMALS 127 healthy dogs. PROCEDURES For each dog, a blood sample was collected from a jugular vein into tubes containing no anticoagulant, EDTA, or citrate anticoagulant. Citrated whole blood samples underwent TEM with tissue factor and TEM with ellagic acid. Indicators of RBC mass and platelet concentration were evaluated, and plasma coagulation tests were performed; data obtained were compared with results of TEM. For technical reasons, samples were not available from all dogs for all tests. RESULTS Coagulation time was correlated with concentrations of primarily extrinsic pathway coagulation factors for TEM with tissue factor and with most factors via TEM with ellagic acid. Clot formation time, α angle, and maximum clot firmness were highly correlated with fibrinogen and platelet concentrations and some individual factor concentrations. Sample Hct was strongly correlated with most measured variables; low Hct was associated with relative hypercoagulability, and high Hct was associated with relative hypocoagulability. CONCLUSIONS AND CLINICAL RELEVANCE For TEM of canine blood samples, coagulation time was primarily a function of coagulation factor concentrations, whereas other variables were dependent on platelet and fibrinogen concentrations. Sample Hct strongly influenced the results of TEM, likely because RBCs act as a diluent for plasma coagulation factors. Thromboelastometry appeared to be affected by abnormalities of coagulation factors, platelet concentrations, and RBC mass. In samples from anemic patients, results of TEM indicative of hypercoagulability may be artifactual because of low RBC mass.

Infusion of a lipid emulsion to treat lidocaine intoxication in a cat

CASE DESCRIPTION A 5-year-old castrated male domestic shorthair cat was examined because of presumptive lidocaine intoxication. Thirty minutes earlier, the cat had received an SC injection of approximately 140 mg of lidocaine hydrochloride (20 mg/kg [9.1 mg/lb]) to facilitate closure of a wound on the left pelvic limb. CLINICAL FINDINGS Initial physical examination revealed severe lethargy and respiratory distress; erratic, poor-quality pulses with severe hypotension; and pulmonary edema. TREATMENT AND OUTCOME Initial supportive treatment included administration of oxygen and IV administration of lactated Ringers solution. Additional treatment with a 20% lipid emulsion (1.5 mL/kg [0.68 mL/lb], IV) delivered over a 30-minute period resulted in dramatic improvement in cardiovascular and behavioral variables. No adverse effects from lipid emulsion were detected on routine hematologic evaluation, thoracic radiography, or computed tomography. CLINICAL RELEVANCE IV administration of a lipid emulsion was used in the treatment of lidocaine intoxication in a cat. Rapid infusion of a lipid emulsion may be a therapeutic option for veterinary patients with toxicosis attributable to local anesthetics or other lipid-soluble drugs.

Microparticles in Health and Disease

Microparticles (MPs), small membrane-derived vesicles, are derived from many cell types and released into the circulation. Microparticles can express antigens, and contain cell surface proteins, cytoplasmic contents, and nuclear components from their cell of origin that determines their composition, characterization, and transfer of biologic information. Certain prompts for this release include shear stress, complement activation, proapoptotic stimulation, cellular damage, or agonist interaction with cell surface receptors. Release can be physiologic or pathologic and is associated with proinflammatory and procoagulant effects and has been implicated in thrombotic states. Microparticles also contribute to systemic inflammation and cardiovascular, hematologic, and oncologic disease states. The study of MPs in human medicine is rapidly advancing and extends into the physiology of health, the pathophysiology of disease, and the role of MPs in transfusion medicine. In veterinary medicine, published work on MPs has been limited to the area of inherited disorders, blood storage, and leukoreduction (LR). Microparticle research is still in its infancy, and this review should be seen as a snapshot of what is currently known. As research continues important limitations, including variations in preanalytic variables such as collection, storage, or centrifugation, and limitations of quantitation are coming to the forefront. Correlation of quantitation of MPs with assays of activity will hopefully shed light on the true nature of MPs in health and disease. This review will focus on the role of cellular exocytic vesiculation in health, disease, and transfusion medicine.

Partnership on Rotational ViscoElastic Test Standardization (PROVETS): Evidence‐based guidelines on rotational viscoelastic assays in veterinary medicine

Objective To systematically examine the evidence relating to the performance of rotational viscoelastic testing in companion animals, to develop assay guidelines, and to identify knowledge gaps. Design Multiple questions were considered within 5 parent domains, specifically system comparability, sample handling, assay activation and test protocol, definitions and data reporting, and nonstandard assays. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence and assessed for quality. Consensus was developed regarding conclusions for application of concepts to clinical practice. Setting Academic and referral veterinary medical centers. Results Databases searched included Medline, Commonwealth Agricultural Bureaux abstracts, and Google Scholar. Worksheets were prepared evaluating 28 questions across the 5 domains and generating 84 assay guidelines. Conclusions Evidence-based guidelines for the performance of thromboelastography in companion animals were generated through this process. Some of these guidelines are well supported while others will benefit from additional evidence. Many knowledge gaps were identified and future work should be directed to address these gaps and to objectively evaluate the impact of these guidelines on assay comparability within and between centers.OBJECTIVE To systematically examine the evidence relating to the performance of rotational viscoelastic testing in companion animals, to develop assay guidelines, and to identify knowledge gaps. DESIGN Multiple questions were considered within 5 parent domains, specifically system comparability, sample handling, assay activation and test protocol, definitions and data reporting, and nonstandard assays. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence and assessed for quality. Consensus was developed regarding conclusions for application of concepts to clinical practice. SETTING Academic and referral veterinary medical centers. RESULTS Databases searched included Medline, Commonwealth Agricultural Bureaux abstracts, and Google Scholar. Worksheets were prepared evaluating 28 questions across the 5 domains and generating 84 assay guidelines. CONCLUSIONS Evidence-based guidelines for the performance of thromboelastography in companion animals were generated through this process. Some of these guidelines are well supported while others will benefit from additional evidence. Many knowledge gaps were identified and future work should be directed to address these gaps and to objectively evaluate the impact of these guidelines on assay comparability within and between centers.

Microparticles in stored canine RBC concentrates

BACKGROUND Transfusion of RBC concentrates may cause adverse effects in the recipient, particularly when stored > 2 weeks. Prestorage removal of WBCs and platelets (leukoreduction, LR) improves clinical outcome in the human recipient. As blood ages during storage, progressive alterations in the structure and function of the cells occur. Changes in cell membranes may lead to formation of microparticles (MPs) in stored blood. OBJECTIVES The aim of the study was to quantify MP concentration in supernatants from canine RBC concentrates from 11 clinically healthy dogs. METHODS Whole blood units (n = 11) were collected and randomized either to be stored without LR (n = 5), or to be subject to prestorage LR (n = 6). Whole blood was processed for the generation of RBC concentrates, from which aliquots were aseptically collected weekly for 5 weeks. Supernatants from the concentrates were evaluated for phosphatidylserine-expressing MPs by flow cytometry using staining with Annexin-V-phycoerythrin. RESULTS Microparticle counts were similar between non-LR and LR units on storage days 0 and 7, but were significantly higher in non-LR units on days 14, 21, 28, and 35. MPs increased during the 35-day storage by a mean (SD) of 1.8 (1.4)-fold in LR units and 5.5 (3.1)-fold in non-LR units. CONCLUSIONS There was marked formation of phosphatidylserine-expressing MPs during storage beyond 7 days in canine RBC concentrates. Prestorage LR attenuated the generation of MPs.

In-vitro hypocoagulability on whole blood thromboelastometry associated with in-vivo expansion of red cell mass in an equine model.

In several species, there is a strong correlation between indicators of red cell mass (RCM) and thromboelastometry results. The horse has a reliable, temporary, polycythemia in response to phenylephrine infusion. Our objective was to evaluate the effects of an in-vivo increase in circulating RCM on thromboelastometry results in an equine model of transient polycythemia. Six healthy research horses had whole blood thromboelastometry with contact activator and tissue factor initiation after recalcification of citrated samples. Additional samples were frozen for thrombin-antithrombin (TAT). Complete blood count biochemical analysis, fibrinogen, activated partial thromboplastin time (aPTT), and prothrombin time (PT) were performed. Additional samples were taken at 5 min and 2 h after phenylephrine infusion. Thromboelastometry was performed separately on four horses not receiving phenylephrine with the samples divided and spiked with phenylephrine ex vivo. Red cell count (P < 0.001) and hematocrit (P < 0.001) were significantly higher at 5 min after phenylephrine compared with baseline and 2 h. There was no change in platelet count, fibrinogen, PT, aPTT, or TAT at any time point. Both ex-tem and in-tem parameters were hypocoagulable at 5 min after phenylephrine compared to baseline and 2 h. There was no effect of phenylephrine in the ex-vivo spiking studies on any of the thromboelastometry parameters. Whole blood thromboelastometry results were hypocoagulable in this equine model of in-vivo transient polycythemia only during the polycythemic phase. All other coagulation parameters were unchanged. In the absence of other indicators of hypocoagulability, this may point to an artifact of thromboelastometry. Alternatively, the data may reflect true in-vivo hypocoagulability in patients with increased circulating RCM.