Journal of Veterinary Emergency and Critical Care 24(2) 2014, pp 144–153 doi: 10.1111/vec.12139
Original Study
Thromboelastographic evaluation of hemostatic function in dogs treated for crotalid snake envenomation Robert A. Armentano, DVM, DACVIM; Carsten Bandt, DVM, DACVECC; Michael Schaer, DVM, DACVIM, DACVECC; John Pritchett, DVM and Andre Shih, DVM, DACVAA
Abstract
Objective – To characterize the overall hemostatic changes in dogs envenomated by crotaline snakes via kaolinactivated thromboelastography (TEG), and to determine any prognostic/monitoring value from a TEG tracing on presentation, as well as during treatment with antivenom therapy. Design – Prospective observational, cohort study. Setting – University teaching hospital and primary emergency hospital. Animals – Thirty-eight dogs envenomated by crotaline snakes. Interventions – TEG tracings were evaluated on presentation to the hospital (pre) as well as immediately following (post) and 12 hours (12 h post) after antivenom treatment, if administered. Measurements and Main Results – At presentation, data were available for 38 dogs envenomated by crotaline snakes. Twenty dogs were in Group 1 (Antivenin [Crotalidae] Polyvalent antivenom), 12 dogs were in Group 2 (Antivipmyn antivenom), and 6 dogs in Group 3 that were not treated with antivenom. The average number of vials administered to group 1 and 2 were equal at 2.2. On presentation, based on a G value < TEG reference range, 15/38 (39%) of the dogs had hypocoagulable TEG tracings. There was a significant increase in G and MA value from the pre and 12 hour post measurement (P = 0.0001 and 0.0003, respectively), as well as from the post to 12 hour post measurement (P = 0.003 and, 0.014, respectively). During the study, 5 of 38 dogs died (13%) and of the dogs that died, 4/5 (80%) had angle and MA equal to zero on presentation. A decreased G and MA were significantly associated with mortality (P = 0.02 and 0.04, respectively). Conclusions – A hypocoagulable TEG tracing, particularly a decreased G value and MA, is associated with an increased mortality in crotaline snake envenomation. G and MA also demonstrate a significant increase over treatment time. (J Vet Emerg Crit Care 2014; 24(2): 144–153) doi: 10.1111/vec.12139 Keywords: coagulopathy, snake bite, TEG
Abbreviations
ACP ACT aPTT
Antivenin (Crotalidae) Polyvalent activated clotting time activated partial thromboplastin time
From the University of Florida Small Animal Hospital, Small Animal Clinical Sciences, Gainesville, FL 32610 (Armentano, Bandt, Schaer, Shih) and Affiliated Pet Emergency Services, Gainesville, FL 32607 (Pritchett). The authors declare no conflict of interests. Funding for this project was provided by Boehringer-Ingelheim. Presented as an abstract at the International Veterinary Emergency and Critical Care Symposium, Nashville, TN, September 16, 2011. Address correspondence and offprint requests to Dr. Bandt, University of Florida Small Animal Hospital, Small Animal Clinical Sciences, 2015 SW 16th Avenue, Gainesville, FL 32610. Email: [email protected] Submitted March 19, 2012; Accepted November 10, 2013.
144
PLT PT SSS TEG
platelet count prothrombin time snakebite severity score thromboelastography
Introduction Pit viper snake (family Crotalidae) envenomation in dogs is a common emergency within certain regions of the United States, particularly in the southeast, southern, and western regions.1 Most crotaline snake envenomations typically cause severe swelling, pain, hematologic abnormalities, hypotension, and occasionally neurologic impairment depending on the particular species of snake. A numerically graded snakebite severity score (SSS) has been validated in people to evaluate C Veterinary Emergency and Critical Care Society 2014
TEG in dogs treated for pit viper envenomation
envenomated patients, and has been recently used in veterinary medicine as a helpful monitoring tool.2, 3 The hematologic assessment of a patient can be difficult since there are multiple venom components that affect primary and secondary hemostasis as well as fibrinolysis. The hematologic tests used for snakebite evaluation include prothrombin time (PT), activated partial thromboplastin time (aPTT), platelet (PLT) count, fibrinogen concentration, D-dimer concentration and assessment of morphologic erythrocyte changes such as echinospherocytosis.4, 5 The activated clotting time (ACT) can be substituted for aPTT to evaluate the intrinsic pathway. Snake venom properties are broadly classified as either cytotoxic, neurotoxic, or coagulopathic.5 The coagulopathic effects of envenomation have potentially lethal consequences for the patient and are therefore one of the most important treatment concerns. These lifethreatening effects depend on the particular species of snake. The most concerning toxins of pit viper snakes that inhabit north central Florida (Eastern diamondback rattlesnake (Crotalus adamanteus), water moccasin (Agkistrodon piscivorus)) are coagulopathic. The coagulopathic properties contribute to a venom-induced consumptive coagulopathy characterized by thrombocytopenia, prolonged clotting times, depletion of fibrinogen and clotting factors, and increased fibrin degradation product concentrations.5 Traditional hemostatic tests require multiple aspects of coagulation: initiation, amplification, and propagation to be tested individually. Thromboelastography (TEG) offers the promise of evaluating all aspects of coagulation in a single, real-time test. TEG has been used in veterinary medicine to evaluate coagulation in several diseases including parvoviral enteritis, neoplasia, disseminated intravascular coagulation, and immune-mediated hemolytic anemia.6–11 TEG is used to assess global hemostasis by measuring the viscoelastic properties of blood from clot formation through fibrinolysis.11, 12 TEG has not yet been evaluated in veterinary patients envenomated by crotaline snakes. However, TEG has been used in the management of children envenomated by snakes in southern Africa.13 In a retrospective study of 51 envenomated children, TEG had a 94% sensitivity of predicting severe bleeding diathesis, and the authors concluded that an abnormal TEG on presentation predicts severe bleeding diathesis in 50% of patients serving as an early indicator for aggressive treatment.13 This study was designed to assess the TEG tracings of dogs presenting for crotaline snake envenomation and to evaluate the ability of TEG parameters to predict outcome in these dogs. A secondary objective of the study was to evaluate the changes in TEG tracings throughout treatment. The C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
mainstay of therapy for crotaline snake envenomation is supportive care including fluid therapy, analgesics, and antivenom, the latter for more toxic venoms.1, 4, 14 Currently ACPa,b Antivenin (Crotalidae) Polyvalent, is the only veterinary approved antivenom. It consists of a whole IgG antibody that is an effective treatment for venom neutralization in dogs.15,b A F(ab’)2 antivenom, Antivipmyn,c has also been evaluated in envenomated dogs and has been found to be safe and effective.16,d We hypothesized that most dogs envenomated by crotaline snakes would have hypocoagulable TEG tracings at the time of their initial evaluation. We further hypothesized that the TEG tracings of dogs treated with antivenom and supportive care would normalize during the first 12 hours of therapy.
Materials and Methods Inclusion criteria consisted of any historically healthy dog diagnosed with a crotaline snake envenomation determined by clinical signs or a history of a witnessed snake bite (Figure 1). In those cases where the pet owner witnessed the snake bite, their familiarity with the indigenous snake population provided an accurate description of the snake. In some situations, the pet owner had killed the snake and brought it to the hospital where identification was confirmed by an experienced clinician. The typical physical abnormalities included focal swelling in the form of painful hemorrhagic lymphedema at a suspected bite wound site and varying degrees of mental depression. The dog’s geographic locale and proximity to snakes in its area were also considered in the history. For inclusion, the dog must have weighed greater than 2 kg and had to be admitted within 12 hours of the observed onset of clinical signs or witnessed envenomation. Dogs were excluded if they were previously treated with any other medication with the exception of isotonic crystalloid fluid therapy. Data were collected between 2010–11 at 2 different locations, 2 miles apart. One site was a university teaching hospital and the other a primary veterinary emergency hospital. The primary author (R.A.) oversaw all cases at both facilities, while a board-certified emergency critical care and internal medicine specialist (M.S.) oversaw all cases at the university hospital. Data collected for each case included signalment, body weight, locations of the wounds, and type of snake (if identified). The Institutional Animal Care and Use Committeeapproved study protocol consisted of collecting whole blood from a cephalic or lateral saphenous vein using an 18-Ga butterfly catheter and 2, 3 mL syringes. Two mL of blood collected in the first syringe was used for the CBC and ACT, and the next 1.8 mL collected in the second syringe was transferred to a citrated tubee for TEG 145
R. A. Armentano et al.
Figure 1: Schematic representation of study design. Abbreviations: ACT, activated clotting time; PLT, platelet count; SSS, snakebite severity score; TEG, thromboelastography.
determination. Whole blood was used for ACT.f An automatedg PLT count was determined on EDTA stored blood with blood smear PLT estimates reviewed by a clinical pathologist. All samples for TEG and CBC were performed on the same machine at one location. If samples were collected after 5 pm, EDTA blood samples for PLT were refrigerated at 2–8◦ C and submitted for analysis the following morning. Kaolin activated, paired TEGsh,i were produced using recalcified blood with 20 L of 0.2 M calcium chloridej after a 30-minute incubation in a glass viale containing one part 3.2% buffered sodium citrate to 9 parts whole blood stored at room temperature.h After incubation, 1 mL of citrated blood was put into a kaolin tube.k The sample was then sealed and inverted 5 times. Three hundred forty microliters of kaolin-activated blood was transferred to the disposable cupl with calcium chloride. The TEG analyzer was initiated at 37◦ C. Paired samples were performed in 2 disposable cups from the same citrated blood tube as well as the same kaolin activated tube and pipette. Each tracing was run for 120 minutes and then each measured parameter was averaged from the paired samples for analysis. Multiple TEG parameters were recorded and defined as previously reported in other TEG veterinary studies with a reference range based on normal dog TEG values 146
with each parameter averaged ± 2 SDs.7, 8, 12 The reference data consisted of TEG values collected on the hospital machine from clinically healthy dogs (n = 20) that presented for wellness examinations and had normal CBCs, serum biochemical profiles, and urinalyses. The first value, R time, is the reaction or precoagulation time, and is defined as the distance in millimeters from the start of the tracing to where the lines diverge by 2 mm.12 The next value, k, is the clot formation time and is the time in minutes between the end of the R and the point at which the tracing branches diverge 20 mm apart.12 Third is the maximum amplitude (MA), and is the maximum distance in millimeters between the 2 diverging branches and reflects the clot strength.12, 17 Fourth, the angle (␣) is the measurement in degrees between the midline and the tangent to the curve drawn from the 1-mm-wide point, and is indicative of the rate of clot formation.17 Fifth, the G value, assesses global clot strength, and is calculated from the MA (G = 5,000 × MA/(100–MA).7, 18 The G is used as a single measurement to define normo-, hyper-, and hypocoagulability.7, 18 The final value, Ly30, is an indicator of fibrinolysis and is the percent change in the amplitude of the TEG tracing 30 minutes after the peak in the MA.19 A commonly used Ly30 percentage defined in human medicine for hyperfibrinolysis is 7.5% and greater.19,h During this study hypocoagulability is C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
TEG in dogs treated for pit viper envenomation
simply defined as a single G value parameter that is less than the reference range (8.6 dyn/cm2 ). If no tracing was produced, indicating severe hypocoagulability (flat line tracing), R was assigned 120 minutes, with zero assigned to MA, G, and angle and no value assigned to K and Ly30. Three blood samples were taken from each patient that received antivenom, 2 samples were taken if the dog did not receive antivenom. The first sample was collected immediately upon diagnosis at either hospital (pre) (Figure 1). If the dog received antivenom therapy, a sample was collected within 5 minutes after the last antivenom vial was administered (post). A third sample was collected 12 hours after the post-treatment collection sample (12 h post). Only a pre and 12 hours post initial examination sample was performed on dogs not treated with antivenom. At each collection point a modified SSS was documented by the primary investigator (RA). The standard SSS was used with a modified parameter added to assess coagulation (Table 1).2 The standard SSS uses PT, aPTT, and PLT for hematologic assessment. During this study, a PLT and ACT were used to assess hemostasis. The hematologic scoring system was modified to accommodate for ACT instead of PT and aPTT, in which with the scale of 1 to 4, one number was added for every 20% increase in the ACT above the machine reference range (Table 1). All blood collections, TEG samples, and modified SSS were performed by the primary author (R.A.). All dogs received crystalloid fluid therapy and opioids with choice and dose selection at the discretion of the primary emergency clinician. Colloid substitutes and plasma were not used in order to avoid potential adverse effects on platelet function or alter hemostatic testing. It was also at the primary clinician’s discretion whether antivenom was to be administered based on the dog’s clinical signs. The clinicians were blinded to both SSS and TEG findings but not to other testing such as ACT and PLT. The standard of care in this study population included IV crystalloid fluids, analgesics, with or without antivenom. Antivenom was included for some dogs because of the typical snake species (water moccasins and Eastern diamondback rattlesnakes) encountered in the geographic area of the study. In situations involving less venomous snakes such as the copperhead and pygmy rattlesnakes, the need for antivenom is not mandatory unless clinical signs supported its use. The study population was divided into three groups. Group 1 was selected as all patients presenting to the primary emergency hospital that received the ACP antivenom. Group 2 was selected as all patients presenting to the university referral hospital that received the F(ab’)2 antivenom, Antivipmyn. If the F(ab’)2 product was unavailable, the dogs would then C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
be included into Group 1 and receive ACP antivenom. Throughout the study period, each facility was always stocked with 1 of the 2 antivenom products. The primary clinician determined the number of antivenom vials administered based on the rate of progressive swelling at the bite site, the rate of onset and the severity of systemic signs (eg, mental depression, weakness, and prostration), and the hematologic abnormalities, such as a prolonged coagulation tests. Group 3 included dogs that showed mild local bite wound signs without signs of systemic involvement such as mental depression and hypotension. These dogs were not treated with antivenom, but they were hospitalized for supportive care and 24hour monitoring. The decision to use antivenom was not randomized because of the potential lethal effects of the pit vipers involved in this study. For analysis, all 3 groups were evaluated in the pretreatment and 12 hours post group, whereas only groups 1 and 2 were evaluated in the post group since only those groups received antivenom. Dogs receiving antivenom were dosed in 1–2 vial aliquots at the discretion of the clinician. There is no clinical literature that provides the dose of antivenom based on milligram or unit doses despite the fact that in vivo laboratory studies uses the precise units in venom neutralization experiments on mice. The decision determining the number of vials administered was subjectively based on input from the most experienced clinician. Similar subjective dosing recommendations are routinely made in people.20 Subsequent vials were administered based on worsening of the clinical signs. Each vial was reconstituted with 10 mL of 0.9% sterile saline, and then fully dissolved by slow rolling of the vial. Once fully dissolved, the antivenom dose of 1–2 vials was transferred to a 100 mL bag of 0.9% sterile saline and administered via an infusion pump over 30 minutes if the patient weighed more than 5.0 kg. For those less than 5 kg body weight, the antivenom was diluted in a 10 mL aliquot for IV administration over 30 minutes. All patients received antivenom if the clinician decided that it was essential. Finances were not a limiting factor for antivenom treatment in this study because funds were available to subsidize the hospital costs in the few cases where it was necessary and the owners could not afford the treatment. The dosages administered ranged from 1–5 vials for either the ACP or the F(ab’)2 products, and the final amount administered was determined by the most experienced clinician and the dog’s therapeutic response.
Statistical methods The data were not normally distributed, so values are reported as the median value and interquartile range. 147
R. A. Armentano et al.
Table 1: Modified SSS-hematological system. ACT – activated clotting time; aPTT – activated partial thromboplastin time; PT – prothrombin time
Score
Definition
0 1
Parameters within normal limits. Minimal: coagulation parameters slightly abnormal, PT < 20 s, aPTT 180. Very severe: coagulation parameters markedly abnormal with bleeding present or the threat of spontaneous bleeding, including PT unmeasurable, aPTT unmeasurable, platelets
Original Study
Thromboelastographic evaluation of hemostatic function in dogs treated for crotalid snake envenomation Robert A. Armentano, DVM, DACVIM; Carsten Bandt, DVM, DACVECC; Michael Schaer, DVM, DACVIM, DACVECC; John Pritchett, DVM and Andre Shih, DVM, DACVAA
Abstract
Objective – To characterize the overall hemostatic changes in dogs envenomated by crotaline snakes via kaolinactivated thromboelastography (TEG), and to determine any prognostic/monitoring value from a TEG tracing on presentation, as well as during treatment with antivenom therapy. Design – Prospective observational, cohort study. Setting – University teaching hospital and primary emergency hospital. Animals – Thirty-eight dogs envenomated by crotaline snakes. Interventions – TEG tracings were evaluated on presentation to the hospital (pre) as well as immediately following (post) and 12 hours (12 h post) after antivenom treatment, if administered. Measurements and Main Results – At presentation, data were available for 38 dogs envenomated by crotaline snakes. Twenty dogs were in Group 1 (Antivenin [Crotalidae] Polyvalent antivenom), 12 dogs were in Group 2 (Antivipmyn antivenom), and 6 dogs in Group 3 that were not treated with antivenom. The average number of vials administered to group 1 and 2 were equal at 2.2. On presentation, based on a G value < TEG reference range, 15/38 (39%) of the dogs had hypocoagulable TEG tracings. There was a significant increase in G and MA value from the pre and 12 hour post measurement (P = 0.0001 and 0.0003, respectively), as well as from the post to 12 hour post measurement (P = 0.003 and, 0.014, respectively). During the study, 5 of 38 dogs died (13%) and of the dogs that died, 4/5 (80%) had angle and MA equal to zero on presentation. A decreased G and MA were significantly associated with mortality (P = 0.02 and 0.04, respectively). Conclusions – A hypocoagulable TEG tracing, particularly a decreased G value and MA, is associated with an increased mortality in crotaline snake envenomation. G and MA also demonstrate a significant increase over treatment time. (J Vet Emerg Crit Care 2014; 24(2): 144–153) doi: 10.1111/vec.12139 Keywords: coagulopathy, snake bite, TEG
Abbreviations
ACP ACT aPTT
Antivenin (Crotalidae) Polyvalent activated clotting time activated partial thromboplastin time
From the University of Florida Small Animal Hospital, Small Animal Clinical Sciences, Gainesville, FL 32610 (Armentano, Bandt, Schaer, Shih) and Affiliated Pet Emergency Services, Gainesville, FL 32607 (Pritchett). The authors declare no conflict of interests. Funding for this project was provided by Boehringer-Ingelheim. Presented as an abstract at the International Veterinary Emergency and Critical Care Symposium, Nashville, TN, September 16, 2011. Address correspondence and offprint requests to Dr. Bandt, University of Florida Small Animal Hospital, Small Animal Clinical Sciences, 2015 SW 16th Avenue, Gainesville, FL 32610. Email: [email protected] Submitted March 19, 2012; Accepted November 10, 2013.
144
PLT PT SSS TEG
platelet count prothrombin time snakebite severity score thromboelastography
Introduction Pit viper snake (family Crotalidae) envenomation in dogs is a common emergency within certain regions of the United States, particularly in the southeast, southern, and western regions.1 Most crotaline snake envenomations typically cause severe swelling, pain, hematologic abnormalities, hypotension, and occasionally neurologic impairment depending on the particular species of snake. A numerically graded snakebite severity score (SSS) has been validated in people to evaluate C Veterinary Emergency and Critical Care Society 2014
TEG in dogs treated for pit viper envenomation
envenomated patients, and has been recently used in veterinary medicine as a helpful monitoring tool.2, 3 The hematologic assessment of a patient can be difficult since there are multiple venom components that affect primary and secondary hemostasis as well as fibrinolysis. The hematologic tests used for snakebite evaluation include prothrombin time (PT), activated partial thromboplastin time (aPTT), platelet (PLT) count, fibrinogen concentration, D-dimer concentration and assessment of morphologic erythrocyte changes such as echinospherocytosis.4, 5 The activated clotting time (ACT) can be substituted for aPTT to evaluate the intrinsic pathway. Snake venom properties are broadly classified as either cytotoxic, neurotoxic, or coagulopathic.5 The coagulopathic effects of envenomation have potentially lethal consequences for the patient and are therefore one of the most important treatment concerns. These lifethreatening effects depend on the particular species of snake. The most concerning toxins of pit viper snakes that inhabit north central Florida (Eastern diamondback rattlesnake (Crotalus adamanteus), water moccasin (Agkistrodon piscivorus)) are coagulopathic. The coagulopathic properties contribute to a venom-induced consumptive coagulopathy characterized by thrombocytopenia, prolonged clotting times, depletion of fibrinogen and clotting factors, and increased fibrin degradation product concentrations.5 Traditional hemostatic tests require multiple aspects of coagulation: initiation, amplification, and propagation to be tested individually. Thromboelastography (TEG) offers the promise of evaluating all aspects of coagulation in a single, real-time test. TEG has been used in veterinary medicine to evaluate coagulation in several diseases including parvoviral enteritis, neoplasia, disseminated intravascular coagulation, and immune-mediated hemolytic anemia.6–11 TEG is used to assess global hemostasis by measuring the viscoelastic properties of blood from clot formation through fibrinolysis.11, 12 TEG has not yet been evaluated in veterinary patients envenomated by crotaline snakes. However, TEG has been used in the management of children envenomated by snakes in southern Africa.13 In a retrospective study of 51 envenomated children, TEG had a 94% sensitivity of predicting severe bleeding diathesis, and the authors concluded that an abnormal TEG on presentation predicts severe bleeding diathesis in 50% of patients serving as an early indicator for aggressive treatment.13 This study was designed to assess the TEG tracings of dogs presenting for crotaline snake envenomation and to evaluate the ability of TEG parameters to predict outcome in these dogs. A secondary objective of the study was to evaluate the changes in TEG tracings throughout treatment. The C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
mainstay of therapy for crotaline snake envenomation is supportive care including fluid therapy, analgesics, and antivenom, the latter for more toxic venoms.1, 4, 14 Currently ACPa,b Antivenin (Crotalidae) Polyvalent, is the only veterinary approved antivenom. It consists of a whole IgG antibody that is an effective treatment for venom neutralization in dogs.15,b A F(ab’)2 antivenom, Antivipmyn,c has also been evaluated in envenomated dogs and has been found to be safe and effective.16,d We hypothesized that most dogs envenomated by crotaline snakes would have hypocoagulable TEG tracings at the time of their initial evaluation. We further hypothesized that the TEG tracings of dogs treated with antivenom and supportive care would normalize during the first 12 hours of therapy.
Materials and Methods Inclusion criteria consisted of any historically healthy dog diagnosed with a crotaline snake envenomation determined by clinical signs or a history of a witnessed snake bite (Figure 1). In those cases where the pet owner witnessed the snake bite, their familiarity with the indigenous snake population provided an accurate description of the snake. In some situations, the pet owner had killed the snake and brought it to the hospital where identification was confirmed by an experienced clinician. The typical physical abnormalities included focal swelling in the form of painful hemorrhagic lymphedema at a suspected bite wound site and varying degrees of mental depression. The dog’s geographic locale and proximity to snakes in its area were also considered in the history. For inclusion, the dog must have weighed greater than 2 kg and had to be admitted within 12 hours of the observed onset of clinical signs or witnessed envenomation. Dogs were excluded if they were previously treated with any other medication with the exception of isotonic crystalloid fluid therapy. Data were collected between 2010–11 at 2 different locations, 2 miles apart. One site was a university teaching hospital and the other a primary veterinary emergency hospital. The primary author (R.A.) oversaw all cases at both facilities, while a board-certified emergency critical care and internal medicine specialist (M.S.) oversaw all cases at the university hospital. Data collected for each case included signalment, body weight, locations of the wounds, and type of snake (if identified). The Institutional Animal Care and Use Committeeapproved study protocol consisted of collecting whole blood from a cephalic or lateral saphenous vein using an 18-Ga butterfly catheter and 2, 3 mL syringes. Two mL of blood collected in the first syringe was used for the CBC and ACT, and the next 1.8 mL collected in the second syringe was transferred to a citrated tubee for TEG 145
R. A. Armentano et al.
Figure 1: Schematic representation of study design. Abbreviations: ACT, activated clotting time; PLT, platelet count; SSS, snakebite severity score; TEG, thromboelastography.
determination. Whole blood was used for ACT.f An automatedg PLT count was determined on EDTA stored blood with blood smear PLT estimates reviewed by a clinical pathologist. All samples for TEG and CBC were performed on the same machine at one location. If samples were collected after 5 pm, EDTA blood samples for PLT were refrigerated at 2–8◦ C and submitted for analysis the following morning. Kaolin activated, paired TEGsh,i were produced using recalcified blood with 20 L of 0.2 M calcium chloridej after a 30-minute incubation in a glass viale containing one part 3.2% buffered sodium citrate to 9 parts whole blood stored at room temperature.h After incubation, 1 mL of citrated blood was put into a kaolin tube.k The sample was then sealed and inverted 5 times. Three hundred forty microliters of kaolin-activated blood was transferred to the disposable cupl with calcium chloride. The TEG analyzer was initiated at 37◦ C. Paired samples were performed in 2 disposable cups from the same citrated blood tube as well as the same kaolin activated tube and pipette. Each tracing was run for 120 minutes and then each measured parameter was averaged from the paired samples for analysis. Multiple TEG parameters were recorded and defined as previously reported in other TEG veterinary studies with a reference range based on normal dog TEG values 146
with each parameter averaged ± 2 SDs.7, 8, 12 The reference data consisted of TEG values collected on the hospital machine from clinically healthy dogs (n = 20) that presented for wellness examinations and had normal CBCs, serum biochemical profiles, and urinalyses. The first value, R time, is the reaction or precoagulation time, and is defined as the distance in millimeters from the start of the tracing to where the lines diverge by 2 mm.12 The next value, k, is the clot formation time and is the time in minutes between the end of the R and the point at which the tracing branches diverge 20 mm apart.12 Third is the maximum amplitude (MA), and is the maximum distance in millimeters between the 2 diverging branches and reflects the clot strength.12, 17 Fourth, the angle (␣) is the measurement in degrees between the midline and the tangent to the curve drawn from the 1-mm-wide point, and is indicative of the rate of clot formation.17 Fifth, the G value, assesses global clot strength, and is calculated from the MA (G = 5,000 × MA/(100–MA).7, 18 The G is used as a single measurement to define normo-, hyper-, and hypocoagulability.7, 18 The final value, Ly30, is an indicator of fibrinolysis and is the percent change in the amplitude of the TEG tracing 30 minutes after the peak in the MA.19 A commonly used Ly30 percentage defined in human medicine for hyperfibrinolysis is 7.5% and greater.19,h During this study hypocoagulability is C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
TEG in dogs treated for pit viper envenomation
simply defined as a single G value parameter that is less than the reference range (8.6 dyn/cm2 ). If no tracing was produced, indicating severe hypocoagulability (flat line tracing), R was assigned 120 minutes, with zero assigned to MA, G, and angle and no value assigned to K and Ly30. Three blood samples were taken from each patient that received antivenom, 2 samples were taken if the dog did not receive antivenom. The first sample was collected immediately upon diagnosis at either hospital (pre) (Figure 1). If the dog received antivenom therapy, a sample was collected within 5 minutes after the last antivenom vial was administered (post). A third sample was collected 12 hours after the post-treatment collection sample (12 h post). Only a pre and 12 hours post initial examination sample was performed on dogs not treated with antivenom. At each collection point a modified SSS was documented by the primary investigator (RA). The standard SSS was used with a modified parameter added to assess coagulation (Table 1).2 The standard SSS uses PT, aPTT, and PLT for hematologic assessment. During this study, a PLT and ACT were used to assess hemostasis. The hematologic scoring system was modified to accommodate for ACT instead of PT and aPTT, in which with the scale of 1 to 4, one number was added for every 20% increase in the ACT above the machine reference range (Table 1). All blood collections, TEG samples, and modified SSS were performed by the primary author (R.A.). All dogs received crystalloid fluid therapy and opioids with choice and dose selection at the discretion of the primary emergency clinician. Colloid substitutes and plasma were not used in order to avoid potential adverse effects on platelet function or alter hemostatic testing. It was also at the primary clinician’s discretion whether antivenom was to be administered based on the dog’s clinical signs. The clinicians were blinded to both SSS and TEG findings but not to other testing such as ACT and PLT. The standard of care in this study population included IV crystalloid fluids, analgesics, with or without antivenom. Antivenom was included for some dogs because of the typical snake species (water moccasins and Eastern diamondback rattlesnakes) encountered in the geographic area of the study. In situations involving less venomous snakes such as the copperhead and pygmy rattlesnakes, the need for antivenom is not mandatory unless clinical signs supported its use. The study population was divided into three groups. Group 1 was selected as all patients presenting to the primary emergency hospital that received the ACP antivenom. Group 2 was selected as all patients presenting to the university referral hospital that received the F(ab’)2 antivenom, Antivipmyn. If the F(ab’)2 product was unavailable, the dogs would then C Veterinary Emergency and Critical Care Society 2014, doi: 10.1111/vec.12139
be included into Group 1 and receive ACP antivenom. Throughout the study period, each facility was always stocked with 1 of the 2 antivenom products. The primary clinician determined the number of antivenom vials administered based on the rate of progressive swelling at the bite site, the rate of onset and the severity of systemic signs (eg, mental depression, weakness, and prostration), and the hematologic abnormalities, such as a prolonged coagulation tests. Group 3 included dogs that showed mild local bite wound signs without signs of systemic involvement such as mental depression and hypotension. These dogs were not treated with antivenom, but they were hospitalized for supportive care and 24hour monitoring. The decision to use antivenom was not randomized because of the potential lethal effects of the pit vipers involved in this study. For analysis, all 3 groups were evaluated in the pretreatment and 12 hours post group, whereas only groups 1 and 2 were evaluated in the post group since only those groups received antivenom. Dogs receiving antivenom were dosed in 1–2 vial aliquots at the discretion of the clinician. There is no clinical literature that provides the dose of antivenom based on milligram or unit doses despite the fact that in vivo laboratory studies uses the precise units in venom neutralization experiments on mice. The decision determining the number of vials administered was subjectively based on input from the most experienced clinician. Similar subjective dosing recommendations are routinely made in people.20 Subsequent vials were administered based on worsening of the clinical signs. Each vial was reconstituted with 10 mL of 0.9% sterile saline, and then fully dissolved by slow rolling of the vial. Once fully dissolved, the antivenom dose of 1–2 vials was transferred to a 100 mL bag of 0.9% sterile saline and administered via an infusion pump over 30 minutes if the patient weighed more than 5.0 kg. For those less than 5 kg body weight, the antivenom was diluted in a 10 mL aliquot for IV administration over 30 minutes. All patients received antivenom if the clinician decided that it was essential. Finances were not a limiting factor for antivenom treatment in this study because funds were available to subsidize the hospital costs in the few cases where it was necessary and the owners could not afford the treatment. The dosages administered ranged from 1–5 vials for either the ACP or the F(ab’)2 products, and the final amount administered was determined by the most experienced clinician and the dog’s therapeutic response.
Statistical methods The data were not normally distributed, so values are reported as the median value and interquartile range. 147
R. A. Armentano et al.
Table 1: Modified SSS-hematological system. ACT – activated clotting time; aPTT – activated partial thromboplastin time; PT – prothrombin time
Score
Definition
0 1
Parameters within normal limits. Minimal: coagulation parameters slightly abnormal, PT < 20 s, aPTT 180. Very severe: coagulation parameters markedly abnormal with bleeding present or the threat of spontaneous bleeding, including PT unmeasurable, aPTT unmeasurable, platelets
