Effect of hemolysis on canine kaolin‐activated thromboelastography values and ADVIA 2120 platelet activation indices

Background: The impact of hemolysis on thromboelastography (TEG) and platelet activation indices has not been evaluated. Objective: The aim of this study was to investigate the influence of hemolysis induced mechanically (HM) and hemolysis induced by freezing (HF) on TEG, platelet counts (PLT), and platelet activation indicators. Methods: Blood from 17 dogs was divided into the following samples: controls, HM, and HF. HM was induced by 20 repetitions of expulsion of blood through a 23 g needle. Freezing was at −80°C, followed by warming to 37° and dilution with equal parts room temperature blood at 22°C. TEG variables that were examined included reaction time (R), coagulation time (K), angle (α), maximum amplitude (MA), and clot rigidity (G). Platelet indices were measured with the ADVIA 2120 hematology analyzer. Results: Hematocrit (HCT) (mean±SD) for controls, HM, and HF were 0.41±0.02, 0.39±0.03, and 0.25±0.02 L/L, respectively, consistent with decreases in HCT of 4.8% (HM) and 39.0% (HF). HM resulted in decreased R (2.5±0.9 minutes compared with 5.2±1.9 minutes for controls; P<0.001), and HF resulted in increased K (15.2±8.6 minutes compared with 5.3±4.0 minutes in controls; P<0.01) and decreased α (20±11° compared with 46±17° in controls; P<0.001). MA was decreased more in HF samples (26±2 mm) than in HM (38±8 mm) or control samples (49±9 mm; P<0.0001). The same applied to G values. PLT decreased after HM but not after HF. Hemolysis of both types resulted in decreased mean platelet component (MPC) concentration: control, 19.3±2.0, HM 15.5±3.4, and HF 14.3±0.7 g/dL (P<0.0001). Conclusion: In hemolyzed samples decreased MPC and R suggested activated primary and secondary hemostasis, respectively, but decreased MA and G indicated reduced clot firmness, possibly due to hyporeactive platelets. TEG and platelet activation indices should be interpreted cautiously after hemolysis.     

Comparison of quantitative immunoturbidimetric and semiquantitative latex‐agglutination assays for D‐dimer measurement in canine plasma

Background: D‐dimer measurement in dogs is considered the most reliable test for detecting disseminated intravascular coagulation or thromboembolism. Objectives: The purposes of this study were to compare 2 D‐dimer assays, a quantitative immunoturbidimetric and a semiquantitative latex agglutination assay, and to assess the effect of hemolysis and storage conditions on D‐dimer concentration using the quantitative assay. Methods: The immunoturbidimetric assay was validated using canine citrated plasma samples containing different concentrations of D‐dimer. The effect of storage at various temperatures and times was assessed. Hemolysis was produced by adding lysed RBCs to the samples for a final hemoglobin concentration of 0.35 g/dL. Results: For clinically relevant values (>250 μg/L), intra‐assay and interassay coefficients of variation were 6.8% and 7.2%. The assay was linear (r²=1.00), and the tests had good agreement (κ=0.685, P<.001). Storage at 4 °C and ?20 °C and hemolysis had no significant effect on D‐dimer concentrations. In hemolyzed samples stored at room temperature for ≥48 hours, fine clots were noted and often resulted in falsely increased D‐dimer concentrations. Conclusions: Our findings suggest that the immunoturbidimetric assay validated in this study is reliable and accurate for the measurement of D‐dimer in canine plasma. Samples can be stored for up to 1 month at ?20 °C and moderate hemolysis does not significantly affect the D‐dimer concentration in frozen or refrigerated samples.     

Effects of in vitro hemodilution of canine blood on platelet function analysis using the PFA‐100

Background: The platelet function analyzer (PFA)‐100 is a point‐of‐care instrument previously evaluated in humans and dogs. In both species, artificially prolonged platelet closure time (CT) occurs with anemia. Reliability of the analyzer in dogs becomes a concern when the HCT is between 0.25 and 0.35 L/L. Objective: The objective of this study was to further define the level of HCT at which CT is prolonged, using in vitro diluted canine blood. Methods: Citrated whole blood samples were collected from 22 healthy dogs. Initial HCT was determined and autologous platelet‐rich plasma was added to samples to achieve HCTs of 0.33, 0.30, and 0.27 L/L. CT was determined in duplicate on the PFA‐100 using collagen/adenosine‐5′‐diphosphate cartridges. Results: Compared with the initial CT in samples with HCT 0.39–0.54 L/L (CT mean±SD=57.8±5.75 seconds), significantly prolonged CTs were found in hemodiluted samples with HCT 0.33 L/L (61.1±4.64 seconds), 0.30 L/L (64.3±6.79 seconds), and 0.27 L/L (70.8±7.90 seconds) (P=0.029; repeated measures ANOVA). Conclusion: Although statistical differences were found, further studies are needed to determine the clinical significance of the mild prolongation in CT associated with mild anemia. Until then, dogs with HCTs slightly <0.35 L/L should be evaluated cautiously for platelet dysfunction using the PFA‐100.     

Analysis of a commercial dimethyl‐sulfoxide‐stabilized frozen canine platelet concentrate by turbidimetric aggregometry

Objectives – To assess platelet function of a commercial dimethyl‐sulfoxide (DMSO)‐stabilized frozen platelet concentrate (PC) using turbidimetric aggregometry. Design – In vitro analysis. Setting – Research laboratory in a school of veterinary medicine. Animals – Five units of frozen PC in 6% DMSO were studied. Fresh platelet‐rich plasma (PRP), with and without 6% DMSO, from 6 healthy dogs were used as controls. Interventions – Turbidimetric platelet aggregation was measured after initiation of platelet aggregation by addition of adenosine diphosphate (ADP), collagen, or thrombin at concentrations of 30 μM, 20 μg/mL, and 0.5 U/mL, respectively. Measures were performed at thaw and repeated 2 hours after thaw for the frozen PC. Measurements and Main Results – Compared with PRP, the frozen PC showed decreased aggregation in response to thrombin (amplitude of 84% versus 25%, P=0.01), and collagen (amplitude of 13% versus 3%, P=0.05) but not ADP (6.5% versus 18%, P=0.2). Compared with frozen PC at thaw, the frozen PC at 2 hours after thaw showed decreased aggregation in response to thrombin, collagen, and ADP (P<0.05). There was no difference in aggregation between PRP in 6% DMSO and frozen PC. Conclusions – These in vitro data suggest there is a decrease in platelet response to agonists associated with the freeze‐thaw process in the commercially available 6% DMSO canine frozen PC.     

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

Comparison of the VIDAS and IMMULITE‐2000 methods for cortisol measurement in canine serum

Background: Serum cortisol concentration is often measured in dogs for the diagnosis and monitoring of adrenal disease. An enzyme‐linked fluorescent assay (VIDAS method) on the MiniVidas analyzer has been validated for the measurement of cortisol concentration in human serum and could have applications for canine samples. Objective: The aim of this study was to compare canine cortisol results obtained using the VIDAS method with those obtained using the IMMULITE‐2000 immunoassay, which has previously been validated for canine serum. Methods: The concentration of cortisol in 40 canine serum samples was determined concurrently with the VIDAS and IMMULITE methods, the latter as the reference method. Pearson's correlation coefficient, linear, and Deming regression analyses and Bland–Altman analysis were used to compare the 2 methods. Acceptability of the new method was judged using a medical decision chart (MEDx chart). Results: Cortisol concentrations obtained with the IMMULITE method ranged from 23.1 to 1380 nmol/L. Correlation (r=.977) and simple linear regression (slope=1.0722, confidence interval [CI] 0.996–1.148; intercept=?4.799, CI ?42.838 to 33.240) revealed no proportional or constant error. Based on Deming regression and a Bland–Altman plot the 2 methods gave comparable results. The MEDx chart indicated that performance of the new method was good at decision limits of 40, 132, and 480 nmol/L. Conclusion: Results of the VIDAS method were comparable to those of the IMMULITE‐2000 reference method such that the VIDAS may be used as an alternative assay to evaluate serum cortisol concentration in dogs for the diagnosis of adrenal disease.     

Stability of stored canine plasma for hemostasis testing

BACKGROUND: A review of the literature revealed limited information about the stability of samples for coagulation testing in dogs. OBJECTIVE: The aim of this study was to evaluate the stability of individual coagulation factors, clotting times, and other parameters of hemostasis in stored canine plasma. METHODS: Citrated plasma samples were obtained from 21 dogs. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and factor I, II, V, VII, VIII, IX, X, XI, and XII activities were measured on an automated coagulation analyzer with commercially available reagents. Antithrombin (AT) activity and D-dimer concentration were measured on an automated chemistry analyzer using validated kits. Samples were analyzed within 1 hour after collection (initial analysis) and once daily for 2 or 4 consecutive days following storage at room temperature (RT) or 4 degrees C, respectively. RESULTS: Storage time at either temperature did not have any effect on PT, factor II, V, VII, X, or XII activities, D-dimer concentration, or AT activity. In contrast, aPTT was significantly prolonged after 72 and 96 hours at 4 degrees C; fibrinogen concentration was decreased after 48 hours at RT; the activities of factors VIII and IX were decreased after 48, 72, and 96 hours at 4 degrees C; and factor XI activity was decreased after 72 hours at 4 degrees C. CONCLUSIONS: Results suggest that storage of canine plasma for 2 days at RT does not have a significant effect on hemostasis test results with the exception of a slight decrease in fibrinogen concentration. In contrast, aPTT and factors VIII, IX, and XI were unstable in refrigerated plasma after 48 or 72 hours of storage.     

Mean platelet volume artifacts: the effect of anticoagulants and temperature on canine platelets

Improper handling of specimens results in artifactually high Mean Platelet Volume (MPV) measurements limiting their usefulness as a clinical tool. MPV measurement and Scanning Electron Microscopy (SEM) were performed on split specimens collected from normal dogs using two anticoagulants and two temperatures over a period of 4 hours. Platelets exposed to EDTA and maintained at 4 degrees C (39.2 degrees F) exhibited the highest artifactual increase in MPV, while those exposed to citrate and maintained at 37 degrees C (98.6 degrees F) exhibited minimal change. The increase in MPV was accompanied by platelet shape change from a smooth disc to an irregular sphere with filopodia. It is recommended that citrated specimens maintained at 37 degrees C be used in all MPV measurements.     

Comparison of hematologic values and transforming growth factor-beta and insulin-like growth factor concentrations in platelet concentrates obtained by use of buffy coat and apheresis methods from equine blood

OBJECTIVE: To evaluate the buffy coat and apheresis methods for preparation of platelet concentrates from equine blood by comparing platelet and growth factor concentrations. ANIMALS: 15 mature mixed-breed geldings. PROCEDURE: Whole blood samples were collected and processed by use of a buffy coat or apheresis method to obtain platelet poor and platelet concentrated fractions. The PCV, WBC count, and platelet count were compared among whole blood samples, platelet poor fractions, concentrates obtained by use of the apheresis method (ie, apheresis platelet concentrates), and concentrates obtained by use of the buffy coat method (ie, buffy coat platelet concentrates). Concentrations of transforming growth factor-beta (ie,TGF-beta1 andTGF-beta2) and insulin-like growth factor were compared between buffy coat and apheresis platelet concentrates. RESULTS: Platelet concentrations were 8.9-fold and 5.2-fold greater in buffy coat and apheresis platelet concentrates, respectively, compared with whole blood. Platelet concentrations were 13.1-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta1 concentrations were 2.8-fold and 3.1-fold greater in buffy coat and apheresis platelet concentrates, respectively, and TGF-beta1 concentrations were 10.5-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta2 concentrations were 3.6-fold greater in apheresis platelet concentrates, compared with whole blood. Platelet concentrations correlated with growth factor concentrations across all blood and platelet fractions. White blood cell counts had a significant positive correlation with TGF-beta1 concentration in buffy coat platelet concentrates. CONCLUSIONS AND CLINICAL RELEVANCE: Platelets and TGF-beta1 can be concentrated reliably from equine blood by use of buffy coat or apheresis methods, without modification of the protocols used for humans.     

Retrospective comparison of first‐line adjuvant anthracycline vs metronomic‐based chemotherapy protocols in the treatment of stage I and II canine splenic haemangiosarcoma

Splenectomy followed by adjuvant chemotherapy is commonly used to treat canine splenic haemangiosarcoma (HSA), although it is unclear if different treatment protocols may have a similar efficacy. The objective of this retrospective study was to assess outcome in dogs with stage I and II splenic HSA treated with either first‐line adjuvant anthracycline (AC) or metronomic (MC)‐based chemotherapy protocols, by comparing median time to progression (TTP) and median survival time (MST). Medical records of nine institutions were searched for dogs diagnosed with stage I and II splenic HSA that underwent adjuvant treatment with AC‐ or MC‐based protocols following splenectomy. Patients treated with MC following AC were included in an additional group (AMC). Ninety‐three dogs were included: 50 in the AC group, 23 in the AMC group and 20 in the MC group. The overall MST was 200 days (range 47‐3352) and the overall median TTP was 185 days (range 37‐1236). The median TTP of stage I dogs was significantly longer compared to stage II dogs (338 vs 151 days, respectively, P = .028). When adjusting for treatment type, the MST was 154 days for the AC group (range 47‐3352 days), 338 days for the AMC group (range 79‐1623 days) and 225 days for the MC group (range 57‐911 days). The difference in MST and median TTP was not found to be statistically significant between treatment groups. This study suggests that adjuvant MC in canine splenic HSA may result in a similar outcome when compared to other treatment protocols. Further studies are warranted to confirm these findings.     

The microclimate of the canine coat: the effects of heating on coat and skin temperature and relative humidity

The coat of seven ‘Landseer’ Newfoundland dogs was irradiated using an infra-red source for 25 min. In each dog, at a site of each colour (black and white), skin and coat temperatures were monitored, and coat air humidity measured with a specially designed instrument. Almost no differences were noted at sites with differing coat colour. Skin temperature rose from 35 °C to a plateau at 39 °C, whilst coat temperature rose from 30 °C to 41 °C. Relative humidity of coat air initially rose, then fell significantly (P < 0.001). The absolute humidity initially almost doubled (P < 0.001), but then fell, although remaining significantly higher than that of ambient air. It was concluded that an initial burst of sweating was followed by lower but continuing secretion. This was not, however, of great importance in cooling. In a separate study the skin temperature of black coated dogs exposed to bright sunshine was explored. The mean temperature was almost identical to that of the plateau skin temperature noted in Newfoundland dogs.     

Comparison of Multiplate,Platelet Function Analyzer‐200, and Plateletworks in Healthy Dogs Treated with Aspirin and Clopidogrel

Background

Platelet function testing may be warranted to assess response to aspirin and clopidogrel.

Hypothesis/Objectives

To evaluate the effects of aspirin, clopidogrel, or combination therapy using 3 platelet function tests: Multiplate Analyzer (MP), Platelet Function Analyzer‐200 (PFA), and Plateletworks (PW).

Animals

Six healthy laboratory Beagles.

Methods

Randomized double‐blind placebo‐controlled study (crossover design). Dogs were given aspirin 1 mg/kg, clopidogrel 2 mg/kg, or combination therapy for 1 week each, with a washout period of 2 weeks. Platelet function was assessed on days 0 and 7 of each phase using MP (adenosine diphosphate [ADP], arachidonic acid [AA], collagen [COL] agonists), PFA (P2Y, COL‐ADP [CADP], COL‐Epinephrine [CEPI] cartridges), and PW (ADP, AA, COL agonists). Platelet counts were obtained with impedance and optical counters.

Results

For MP, mean aggregation was decreased for COL and AA with combination therapy and for ADP with all treatments. For PFA, mean CT was increased for the CEPI cartridge with aspirin; and for the P2Y and CADP cartridges with clopidogrel or combination therapy. More dogs receiving clopidogrel showed an increase in PFA CT using the P2Y than the CADP cartridge. For PW, mean aggregation was decreased for AA with all treatments; for ADP with clopidogrel or combination therapy; and for COL with clopidogrel. The PW results with the 2 hematology counters showed almost perfect agreement.

Conclusion and Clinical Importance

All platelet function tests detected treatment effects in some dogs and may have utility for monitoring therapy.     

Comparison of doxorubicin–cyclophosphamide with doxorubicin–dacarbazine for the adjuvant treatment of canine hemangiosarcoma

Canine hemangiosarcoma (HSA) is a neoplasm of vascular endothelial origin that has an aggressive biological behaviour, with less than 10% of dogs alive at 12‐months postdiagnosis. Treatment of choice consists of surgery followed by adjuvant doxorubicin‐based chemotherapy. We prospectively compared adjuvant doxorubicin and dacarbazine (ADTIC) to a traditional doxorubicin and cyclophosphamide (AC) treatment, aiming at determining safety and assessing whether this regimen prolongs survival and time to metastasis (TTM). Twenty‐seven dogs were enrolled; following staging work‐up, 18 were treated with AC and 9 with ADTIC. Median TTM and survival time were longer for dogs treated with ADTIC compared with those receiving AC (>550 versus 112 days, P = 0.021 and >550 versus 142 days, P = 0.011, respectively). Both protocols were well tolerated, without need for dose reduction or increased interval between treatments. A protocol consisting of combined doxorubicin and dacarbazine is safe in dogs with HSA and prolongs TTM and survival time.     

Evaluation of a 15‐week CHOP protocol for the treatment of canine multicentric lymphoma

Dose intense CHOP protocols have been shown to improve outcome for people with non‐Hodgkin's lymphoma, but evaluation of dose intense CHOP protocols for canine lymphoma is currently limited. The hypothesis of this retrospective study was that a 15‐week dose intense CHOP protocol would have shorter treatment duration with similar efficacy to other doxorubicin‐based multidrug protocols. Thirty‐one client owned dogs with multicentric lymphoma were treated with a 15‐week CHOP chemotherapy protocol with an overall response rate of 100% and a median progression‐free interval (PFI) of 140 days [95% confidence interval (CI) 91–335 days]. Dogs that had two or more treatment delays had significantly prolonged PFI and overall survival in multivariate analysis. Dose intensity did not correlate with patient outcome. Dogs experiencing multiple treatment delays secondary to adverse events may receive their individual maximally tolerated dose while dogs with no adverse events may be underdosed. Future studies should focus on individual patient dose optimization.