Validation of an immunoturbidimetric D-dimer assay in canine citrated plasma

Abstract: D-dimer is a neoantigen formed when thrombin initiates the transformation of fibrinogen to fibrin; it is derived from plasmin digestion of cross-linked fibrin. In human medicine, the usefulness of this analyte in diagnosing disseminated intravascular coagulation (DIC) has been assessed in patients fulfilling the clinical and laboratory requirements for this disorder. In canine medicine, the use of D-dimer is relatively new. Detailed studies are needed to understand the relationship between D-dimer concentration in plasma and DIC status in dogs. We validated a D-dimer immunoturbidimetric assay (Tina-quant [a] D-Dimer, Boehringer Mannheim) in canine citrated plasma samples. Intra-assay and interassay variability (coefficient of variation) was 5.63% and 8.82%, respectively. The assay was linear, using 2 samples with low and high D-dimer concentrations (r = .996 and .998). Accuracy was 102.2% and 95.7% based on a recovery study in which 2 samples were assessed. Reference values for D-dimer were established using 70 healthy dogs that were assessed clinically and evaluated on the basis of a complete laboratory workup. The reference range was set between 0.02 and 0.28 μg/mL (chi-square test for normal distribution, P > .05).     

Effects of 2 concentrations of sodium citrate on coagulation test results, von Willebrand factor concentration, and platelet function in dogs

BACKGROUND: Blood collection tubes containing 3.2% (0.109 M) sodium citrate, instead of 3.8% (0.129 M) sodium citrate, have recently become available in the United States. These tubes are visually indistinguishable from the traditional 3.8% sodium citrate tubes, except for wording on the label. Consequently, samples for hemostatic evaluation are frequently collected in tubes containing the lower concentration of sodium citrate. HYPOTHESIS: Results of hemostasis assays are different in samples collected in 3.2% versus 3.8% sodium citrate. ANIMALS: Twenty healthy dogs. METHODS: This study aimed at determining whether results of standard coagulation tests, von Willebrand factor concentration (vWF:Ag), and platelet function with the platelet function analyzer PFA-100a were affected by the different concentrations of sodium citrate. Blood samples were collected in tubes containing either 3.2% or 3.8% sodium citrate concentrations and processed routinely for coagulation assays (one-stage prothrombin time [OSPT], activated partial thromboplastin time [aPTT], fibrinogen concentration, and platelet count), vWF:Ag, and platelet function assays with a PFA-100. RESULTS: There was no significant difference between samples collected in 3.2% versus those collected in 3.8% sodium citrate for OSPT, aPTT, fibrinogen concentration, platelet count, or vWF:Ag. The closure times with collagen/adenosine diphosphate were significantly shorter (66 +/- 8.1 versus 74.8 +/- 9.7 seconds; P < .0001) with the 3.2% than with 3.8% sodium citrate concentration, and the hematocrit was significantly higher (47.9 +/- 5.6 versus 46.0 +/- 4.7 seconds; P = .03) in samples collected in 3.2% than in those collected in 3.8% sodium citrate. CONCLUSIONS AND CLINICAL IMPORTANCE: There is no clinically relevant effect of collection of blood into 3.2% or 3.8% sodium citrate.     

Evaluation of Kits for the Detection of Fibrin(ogen) Degradation Products in Dogs

The sensitivities and specificities of 3 commercial serum fibrin(ogen) degradation product (FDP) kits and 1 plasma FDP kit for the detection of FDPs in dogs were determined. Blood was collected for measurement of serum and plasma FDP concentrations from 30 healthy dogs and from 20 dogs that fulfilled clinical and laboratory criteria for disseminated intravascular coagulation. To determine the effect of hemolysis on FDP results, blood was collected simultaneously into Bothrops atrox venom-based and thrombin-based serum collection tubes for measurement of FDPs using a single serum FDP kit. The sensitivity (80-95%) and specificity (90-100%) for a positive or negative FDP result, regardless of concentration, was similar for all kits. Kits yielded discordant results in individual dogs and FDP concentrations obtained from 1 serum FDP kit were consistently higher than those from the other kits. Serum prepared from venom-based collection tubes was significantly more hemolyzed than serum prepared from thrombin-based collection tubes or citrated plasma. Hemolysis did not affect the FDP results. On the basis of these results, we conclude that commercial latex agglutination kits for detection of FDPs in serum and plasma samples from human patients are valid for use in dogs. The plasma FDP assay is a viable alternative to currently used serum FDP assays and has the advantage of using a single (citrated plasma) sample for measuring coagulation parameters and FDP concentration.     

Assessment of three automated assays for C-reactive protein determination in dogs

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.     

Effect of citrate concentration on coagulation test results in dogs

OBJECTIVE: To determine the effect of citrate concentration (3.2 vs 3.8%) on coagulation tests in dogs. DESIGN: Original study. ANIMALS: 30 clinically healthy dogs and 12 dogs with hereditary hemostatic disorders. PROCEDURE: Blood was collected from all dogs directly into collection tubes containing 3.2 or 3.8% buffered citrate. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration were measured by use of 3 clot-detection assay systems (2 mechanical and 1 photo-optic). Factor VIII and factor IX coagulant activities (FVIII:C and FIX:C, respectively) were determined by use of a manual tilt-tube method and a mechanical clot-detection device. RESULTS: Significant differences were not detected in median PT, fibrinogen concentration, FVIII:C, or FIX:C between 3.2 and 3.8% citrate for any assay system. A significant prolongation in aPTT for 3.2% citrate, compared with 3.8% citrate, was found in 1 mechanical system. CONCLUSIONS AND CLINICAL RELEVANCE: Citrate concentration does not significantly affect results of most coagulation assays, regardless of assay system. The aPTT was mildly influenced by the citrate concentration, although this was animal-, instrument-, and reagent-dependent. The choice of 3.2 or 3.8% citrate as an anticoagulant for coagulation tests has minimal influence on assay results in healthy dogs or dogs with hereditary hemostatic disorders.     

Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons

BACKGROUND: Few studies have been done to evaluate anticoagulants for use with blood samples from birds and reptiles. Heparin currently is the most commonly used anticoagulant in practice, but may adversely affect blood cell staining and quantitation. OBJECTIVE: The purpose of this study was to evaluate the effects of lithium heparin, K3-EDTA, and sodium citrate, with and without the addition of albumin, on hematologic variables in macaw (Ara sp) and python (Python molurus bivittatus) blood samples. METHODS: Blood samples from 10 macaws and 10 Burmese pythons were collected in heparin-coated syringes and placed into tubes containing either lithium heparin, K3-EDTA, or sodium citrate with and without the addition of 0.25 mL of a 22% bovine serum albumin solution. Cell lysis was determined by counting the number of lysed cells/200 WBCs in Wright's-Giemsa-stained blood smears and by qualitative evaluation of pink plasma in microhematocrit tubes. A CBC was done after 3, 12, and 24 hours of storage at 4 degrees C in anticoagulant-containing tubes and results were compared with those obtained at 0 hour for the heparin-coated syringe sample. A biochemical panel also was done at each time point in similarly stored lithium-heparin samples. RESULTS: Hemolysis was significantly increased in citrated samples from both macaws and pythons beginning at 12 hours. At 24 hours, 19 of 30 (63%) macaw samples in all anticoagulants had >100 lysed cells/200 WBCs. There were no significant differences in hematologic values in samples from pythons collected in heparin or EDTA at any time point. No significant differences were found in the number of lysed cells or in other hematologic data in samples with albumin. Glucose concentration decreased and potassium concentration increased significantly over time in heparinized blood samples. CONCLUSIONS: Based on the results of this study, whole blood samples anticoagulated with lithium heparin or EDTA should be evaluated within 12 hours (macaws) or 24 hours (pythons) of collection and stored at 4 degrees C for best results. Citrate should be avoided as it may result in increased cell lysis. The addition of albumin does not prevent cell lysis.     

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood

OBJECTIVE: To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. ANIMALS: 12 healthy dogs and 35 dogs with inflammatory processes. PROCEDURE: CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. RESULTS: Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.     

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.     

Effects of preoperative administration of carprofen on renal function and hemostasis in dogs undergoing surgery for fracture repair

OBJECTIVE: To evaluate effects of preoperative administration of carprofen on renal function and hemostasis in dogs undergoing general anesthesia for fracture repair. ANIMALS: 26 client-owned dogs. PROCEDURE: Anesthesia was induced with levomethadone, diazepam, and propofol and maintained by administration of isoflurane in oxygen-nitrous oxide. Carprofen (4 mg/kg, SC) was administered 1 hour before induction to 13 dogs (group 1) and after extubation to the other 13 dogs (group 2). All dogs also received carprofen (4 mg/kg, SC, q 24 h) for the first 4 days after surgery. Renal function (glomerular filtration rate [GFR], urinary protein-to-urinary creatinine ratio [UP:UC], and results of urinalysis and biochemical analysis of plasma), hemostatic variables (bleeding time, platelet aggregation, prothrombin time [PT], activated partial thromboplastin time [APTT], and platelet count), and Hct were assessed before and at various time points after surgery. RESULTS: Analysis of results for renal function tests, most of the hemostatic and plasma biochemical variables, and Hct did not reveal significant differences between treatment groups. Values for GFR, UP:UC, PT, APTT, and platelet aggregation were outside reference ranges in many dogs before surgery and during the first 6 hours after surgery. In most dogs, these trauma-induced pathologic changes returned to within reference ranges during the 4-day period after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Carprofen did not cause clinically relevant adverse effects in dogs anesthetized for fracture repair after 5 days of treatment, even when it was administered before surgery or given to patients with trauma-induced alterations in renal function or hemostasis.     

Effect of storage conditions on hemostatic parameters of canine plasma obtained for transfusion

OBJECTIVE: To evaluate the effects of various storage conditions on one-stage prothrombin time (OSPT), activated partial thromboplastin time (APTT), and fibrinogen concentration of canine plasma collected for transfusion. SAMPLE POPULATION: Plasma from 9 dogs. PROCEDURE: Whole blood was collected from dogs by means of jugular venipuncture and centrifuged at 7,300 X g for 20 minutes at 0 C. A plasma extractor was then used to generate plasma. Aliquots of plasma were collected in segments of plastic tubing and in microcentrifuge tubes, and plasma collection bags, tubing segments, and microcentrifuge tubes were immediately frozen at -30 C. Additional tubing segments and microcentrifuge tubes were stored at 2 C. After 1 week of storage, all samples were thawed, and OSPT, APTT, and fibrinogen concentration were measured. Collection bags and microcentrifuge tubes were refrozen at -30 C, and values were measured again 30 days after blood collection. RESULTS: Values for OSPT, APTT, and fibrinogen concentration did not vary significantly with storage time, temperature, or container. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that storage for up to 30 days and at 2 C versus -30 C did not have any significant effect on hemostatic parameters of canine plasma obtained for transfusion.     

Validation of 2 commercially available enzyme-linked immunosorbent assays for adiponectin determination in canine serum samples

The aim of this study was to validate 2 commercially available enzyme-linked immunosorbent assays (ELISAs) for adiponectin in dogs, 1 canine-specific and 1 originally designed for measurements in humans. Intra-assay and interassay precision was evaluated by multiple measurements in canine serum samples, and assay accuracy was indirectly determined by linearity under dilution. Interference caused by hemolysis and lipemia was also studied. Both assays were subsequently used for measuring adiponectin concentrations in clinically healthy dogs and those with different grades of obesity. The intra-assay and inter-assay precision was less than 7.5% and 13.5% in serum samples with low and high adiponectin concentrations, respectively. Lipemia and hemolysis did not affect the results of any of the assays. Both assays were able to differentiate lean dogs from those that were overweight or obese on the basis of the measured adiponectin concentrations. From these results it can be concluded that canine adiponectin concentrations can be measured reliably by means of the 2 ELISAs evaluated in this study.     

Overestimation of canine albumin concentration with the bromcresol green method in heparinized plasma samples

Albumin concentrations are routinely measured in dogs with bromcresol green (BCG)-binding assays on automated chemistry analyzers. Several variables affect this assay, including the length of reaction time, sample type, and lack of specificity of BCG for albumin. We observed that albumin concentrations measured with BCG appeared higher in heparinized plasma samples in sick dogs. The objective of this study was to determine the effect of anticoagulant and assay procedure on BCG albumin concentrations in clinically ill dogs. We hypothesized that albumin concentrations would be overestimated in heparinized plasma compared with serum because of the combination of heparin and fibrinogen. Furthermore, we hypothesized that the overestimation would be influenced by assay parameters. Blood was collected from 32 clinically ill dogs into tubes containing heparin, citrate, or no anticoagulant. Citrate was chosen to assess the effect of fibrinogen in the absence of heparin. Albumin concentration was measured in all 3 sample types from each dog using 2 different BCG procedures on an automated chemistry analyzer. The BCG procedures (standard and modified) differed in the wavelengths used for absorbance readings (standard, 600/700; modified, 570/505) and the time point at which absorbance was measured (standard, 100 seconds; modified, 40 seconds). In addition, the modified method incorporated a sample blank. Globulin fractions, fibrinogen concentration, and indices of lipemia, hemolysis, and icterus were evaluated for their contribution to the overestimation of albumin concentration in heparinized plasma compared with serum samples. Albumin concentrations were significantly higher (P 相似文献   

Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs

BACKGROUND: Thromboelastography (TEG) is an analytical method that enables global assessment of hemostatic function in whole blood (WB) with evaluation of both plasma and cellular components of hemostasis. TEG has a largely unused potential in the diagnostic workup and monitoring of dogs with hemostatic disorders and it may be a valuable supplement to traditional coagulation parameters. OBJECTIVES: The objective of this study was to establish a clinically applicable reference interval for a TEG assay using recombinant human tissue factor (TF) as the activator on citrated WB from clinically healthy dogs and to evaluate the stability of citrated WB stored for 30 minutes (T30) and 120 minutes (T120) at room temperature (RT). Additionally, we evaluated the analytical variation in reaction time (R), clotting time (K), angle (alpha), and maximum amplitude (MA). METHODS: Blood was collected from 18 clinically healthy dogs. Duplicate TEG analyses with TF as the activator at a concentration of 1:50,000 were performed on canine citrated WB at T30 and T120. R, K, a, and MAwere analyzed. RESULTS: Mean TEG values at T30/T120 were R = 5.61/4.91 minutes, K = 4.20/3.34 minutes, alpha = 45.33/50.90 degrees , and MA = 47.96/50.19 mm. Significant differences in these values were observed after storage for T30 and T120 at RT, with a tendency towards hypercoagulability at T120. The mean coefficients of variation were low. CONCLUSIONS: Canine citrated WB can be used for TEG analysis with human recombinant TF as the activator when stored at RT for T30 or T120. At both time points, the analytical variation was low, suggesting that TEG analysis may be of value in evaluating dogs with hemostatic disorders. A fixed time point should be chosen for serial measurements.     

Hemostatic biomarkers in dogs with chronic congestive heart failure

BACKGROUND: Chronic congestive heart failure (CHF) in humans is associated with abnormal hemostasis, and abnormalities in hemostatic biomarkers carry a poor prognosis. Alterations in hemostatic pathways can be involved in the pathogenesis of CHF in dogs, and microthrombosis in the myocardium could contribute to increased mortality. HYPOTHESIS: That plasma concentration or activity of hemostatic biomarkers is altered in dogs with CHF and that these factors predict mortality. ANIMALS: Thirty-four dogs with CHF caused by either dilated cardiomyopathy (DCM, n=14) or degenerative valvular disease (CDVD, n=20) compared with 23 healthy age-matched control dogs were included in this study. Dogs with CHF were recruited from 2 referral cardiology clinics, and control dogs were owned by friends or colleagues of the investigators. METHODS: Clinical examination and echocardiography were performed in all dogs. Plasma fibrinogen and D-dimer concentrations, antithrombin and protein C activity, and thrombin-antithrombin complex (TAT) were measured in all dogs. RESULTS: Dogs with CHF had significantly higher fibrinogen (P = .04), D-dimer (P = .002), and TAT concentration (P < .0001), lower antithrombin (P < .0001) and protein C activity (P < .001) compared with control dogs. None of the hemostatic biomarkers were associated with risk of death. CONCLUSIONS AND CLINICAL IMPORTANCE: There is evidence of a procoagulant state in dogs with CHF. The lack of predictive value for survival might be due to the small number of dogs examined. Further studies are necessary to investigate the presence and importance of microthrombosis in dogs with CHF.     

Determination of prothrombin in feline plasma

Determination of the major common pathway protein prothrombin, a vitamin K-dependent protein synthesized in the liver, may be useful for identifying coagulopathies in cats with liver disease or vitamin K antagonism. In people with liver disease, prothrombin is more commonly and more severely decreased than other procoagulant proteins. The purpose of this study was to evaluate a commercial chromogenic assay(DiaPharma Group, West Chester, Ohio, USA) for the determination of prothrombin activity in plasma from healthy cats. The method involves the cleavage of prothrombin by Ecarin, a nonphysiologic enzyme activator that cleaves prothrombin to meizothrombin, which then interacts with a chromogenic substrate. Citrated (n = 20) and EDTA (n = 37) plasma samples from clinically healthy cats were tested using 100-fold and on occasion 200-fold dilutions. The assay was run according to manufacturer's specifications and the relative percentage prothrombin activity was calculated using standard curves generated from a feline citrated plasma pool and human reference plasma. Slope and regression values (r =.998) were similar for feline and human samples, suggesting that Ecarin cleaves prothrombin in both feline and human plasma in an analogous manner. The correlation between results obtained using feline vs human reference plasma was high for both citrated (r =.910) and EDTA samples (r =.998). When prothrombin was determined using human reference plasma, results from citrated feline plasma samples were 75.7% +/- 9.0% of normal compared to 91.6% +/- 7.0% of normal when the feline standard curve was used. Similar results were obtained using EDTA plasma. Our results indicate that the prothrombin chromogenic assay may be useful for evaluating one component of the hemostatic pathway in feline plasma. The prothrombin chromogenic assay utilizes routine instrumentation, requires small sample volume (5 microliter/assay), and may be used on EDTA plasma. To optimize sensitivity, the assay should be run using a standard curve generated with a feline plasma pool.     

The effects of different anticoagulants on routine canine plasma biochemistry

The effects of heparin, ethylenediaminetetraacetic acid (EDTA), sodium citrate and sodium fluoride/potassium oxalate on plasma biochemistry results in dogs were studied and compared with serum. Blood specimens from 10 apparently clinical healthy dogs were collected and placed in different tubes containing each anticoagulant tested. Differences in albumin, acetylcholinesterase, ionized calcium and potassium were found between serum and heparinized plasma. Most metabolites and enzymes did not show any variation, but significant decreases in electrolytes, alkaline phosphatase, acetylcholinesterase, bile acids, fructosamine and albumin were found when EDTA was used. Sodium citrate produced a 10-15% decrease in most metabolites and enzymes, possibly due to a sample dilution effect. Sodium fluoride/potassium oxalate produced haemolysis which may have influenced changes in some biochemical parameters.     

D-dimer concentrations in healthy dogs and dogs with disseminated intravascular coagulation

OBJECTIVE: To determine sensitivity and specificity of assays of D-dimer concentrations in dogs with disseminated intravascular coagulation (DIC) and healthy dogs and to compare these results with those of serum and plasma fibrin-fibrinogen degradation product (FDP) assays. ANIMALS: 20 dogs with DIC and 30 healthy dogs. PROCEDURE: Semi-quantitative and quantitative D-dimer concentrations were determined by use of latex-agglutination and immunoturbidometry, respectively. Fibrin-fibrinogen degradation products were measured by use of latex-agglutination. A reference range for the immunoturbidometric D-dimer concentration assay was established; sensitivity and specificity of the assay were determined at 2 cutoff concentrations (0.30 microg/ml and 0.39 microg/ml). RESULTS: Reference range for the immunoturbidometric D-dimer concentration assay was 0.08 to 0.39 microg/ml; median concentrations were significantly higher in dogs with DIC than in healthy dogs. Latex-agglutination D-dimer and serum and plasma FDP assays had similar sensitivity (85 to 100%) and specificity (90 to 100%); the immunoturbidometric assay had lower specificity (77%) at the 0.30 microg/ml cutoff and lower sensitivity (65%) at the 0.39 microg/ml cutoff. Sensitivity or specificity of the latex-agglutination D-dimer assay was not significantly improved when interpreted in series or parallel with FDP assays. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of D-dimer concentrations by latex-agglutination appears to be a sensitive and specific ancillary test for DIC in dogs. Specificity of D-dimer concentrations in dogs with systemic disease other than DIC has not been determined, therefore FDP and D-dimer assays should be performed concurrently as supportive tests for the diagnosis of DIC in dogs.     

Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration

Background: Serum C-reactive protein (CRP) is an acute phase marker in dogs that is useful for the diagnosis and monitoring of inflammatory disease. Rapid, reliable, and automated assays are preferable for routine evaluation of canine serum CRP concentration.
Objective: The aim of this study was to evaluate whether canine serum CRP concentration could be measured reliably using an automated turbidometric immunoassay (TIA) designed for use with human serum.
Methods: A commercially available TIA for human serum CRP (Bayer, Newbury, UK) was used to measure canine serum CRP concentration. Cross-reactivity of antigen was evaluated by the Ouchterlony procedure. Intra-and interassay imprecision was investigated by multiple measurements on canine serum samples and serum pools, respectively. Assay inaccuracy was investigated by linearity under dilution and comparison of methodologies (canine CRP ELISA, Tridelta Development Ltd, Kildare, UK). Then the assay was applied to serum samples from 14 clinically healthy dogs, 11 dogs with neoplasia, 13 with infections, 8 with endocrine or metabolic diseases, and 10 with miscellaneous diseases.
Results: Cross-reactivity between canine serum CRP and the anti-human CRP antibody was found. Intra-and interassay imprecision ranged from 5.2% to 10.8% and 3.0% to 10.2%, respectively. Serum CRP concentration was measured in a linear and proportional manner. There was no significant disagreement and there was linear correlation of the results in the comparison of methodologies, except for a slight proportional discrepancy at low CRP concentrations (<10 μg/mL). Dogs with infections had a significantly higher concentration of serum CRP than did all other dogs, and dogs with neoplasia had a significantly higher concentration of serum CRP than did clinically healthy dogs.
Conclusions: Canine serum CRP concentration can be measured reliably using the commercially available TIA designed for human CRP.     

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.     

Evaluation of a canine D-dimer point-of-care test kit for use in samples obtained from dogs with disseminated intravascular coagulation, thromboembolic disease, and hemorrhage

OBJECTIVE: To evaluate a canine D-dimer point-of-care (cD-d POC) test kit for use in healthy dogs and dogs with disseminated intravascular coagulation (DIC), thromboembolic disease (TED), and hemorrhage. ANIMALS: 12 healthy dogs, 18 dogs with DIC, 23 dogs with TED (19 acute and 4 chronic), and 18 dogs with hemorrhage. PROCEDURE: The cD-d POC, canine D-dimer ELISA (cD-d ELISA), human D-dimer latex agglutination (hD-d LA), and fibrin degradation product (FDP) tests were performed on citrated plasma. RESULTS: All healthy dogs had negative cD-d POC test results and mean cD-d ELISA value of 0.2 U/mL. All dogs with DIC had positive cD-d POC test results and mean cD-d ELISA value of 44 U/mL. Dogs with acuteTED had a mean cD-d ELISA value of 34 U/mL, and 17 of 19 had positive cD-d POC test results. Mean cD-d ELISA value in dogs with hemorrhage was 14 units/mL, and 15 of 18 had positive cD-d POC test results. The cD-d ELISA values in dogs with hemorrhage were significantly higher than those of healthy dogs but lower than those of dogs with DIC and acute TED. The cD-d POC, cD-d ELISA, and hD-d LA tests were comparable in differentiating healthy dogs from dogs with DIC, acute TED, or hemorrhage and appeared to be superior to measurement of FDPs. CONCLUSIONS AND CLINICAL RELEVANCE: The cD-d POC test kit can be quickly and easily used and reliably detects dogs with DIC or acute TED. Positive results may also be seen in dogs with internal hemorrhage.