Measurement of prothrombin time (PT) and activated partial thromboplastin time (APTT) on canine citrated plasma samples following different storage conditions

Samples from 75 clinically ill dogs were utilised in the study. APTT and PT tests were performed immediately on fresh citrated plasma samples (Fresh). The remaining plasma was stored at -20 degrees C for less than 4 months (n=36 samples) or between 4 and 7 months (n=39 samples). In batches of five, frozen samples were thawed rapidly and APTT and PT tests were performed on the thawed samples immediately (0RT) and after storage at room temperature (23 degrees C, range: 22-25 degrees C) for 24h (24RT) and 48h (48RT). The median APTT value from the (0RT) samples was significantly longer than that obtained from fresh samples (15s vs. 13.2s) but the PT value was not statistically different (7.8s vs. 7.6s). The median APTT (15s) and PT (7.5s) results from the (24RT) samples were not statistically different to those from the (0RT) samples (APTT: 15s, PT: 7.6s) but both tests were significantly longer (APTT: 16.5s, PT: 9.2s) from the (48RT) samples. We concluded that long term batching and freezing of clinical samples at -20 degrees C is acceptable for measurement of PT but not of APTT. We demonstrated that APTT and PT results do not change following storage of samples at room temperature for 24h but storage for 48h may lead to statistically and clinically significant changes (values at least 25% higher than the high value of the laboratory's reference interval) in both clotting times.     

Effect of storage conditions on prothrombin time, activated partial thromboplastin time and fibrinogen concentration on canine plasma samples

The present study was to assess the effect of storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration in blood samples of healthy dogs. Thirty-five dogs of various breeds were included in the study. Citrated blood samples were obtained and plasma was divided into four aliquots to assess selected clotting parameters by means of a coagulometer. The first aliquot was analysed within 1 h after collection, while the remaining 3 were stored at 8℃ for 4, 8 and 24 h, respectively. One-way repeated measures analysis of variance documented a significant decreasing effect on PT at 24 h compared to 8 h and on fibrinogen concentration after 8 and 24 h compared to sampling time and at 4 and 24 h compared to 8 h post sampling. In conclusion, the results of this study indicate that only fibrinogen appears prone to significant decrease. In fact, aPTT is not substantially affected by refrigeration for at least 24 h post sampling and PT showed a statistical difference that does not necessary indicate biological significance as the results obtained were within reference intervals for the dog.     

Effects of haemolysis, lipaemia and bilirubinaemia on prothrombin time, activated partial thromboplastin time and thrombin time in plasma samples from healthy dogs

Interferences caused by haemolysis, lipaemia and bilirubinaemia on prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT in normal canine plasma samples were studied using commercially available reagents and a steel ball coagulometer. Haemolysis significantly interfered with APTT (P = 0.0076) and TT (P = 0.0292). Regression analysis showed that TT was significantly shortened as haemoglobin concentrations increased. Lipaemia increased as demonstrated by regression analysis. Bilirubin significantly interfered with PT (P=0.0003) and APTT (P=0.002). Although statistically significant, none of the differences found were of clinical relevance.     

Comparison of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration in blood samples collected via an intravenous catheter versus direct venipuncture in dogs

OBJECTIVE: To compare prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration in canine blood samples collected via an indwelling IV catheter and direct venipuncture. ANIMALS: 35 dogs admitted to an intensive care unit that required placement of an IV catheter for treatment. PROCEDURES: Blood samples were collected via IV catheter and direct venipuncture at the time of catheter placement and 24 hours after catheter placement. Prothrombin time, APTT, and fibrinogen concentration were measured. RESULTS: 5 dogs were excluded from the study; results were obtained for the remaining 30 dogs. Agreement (bias) for PT was -0.327 seconds (limits of agreement, -1.350 to 0.696 seconds) and 0.003 seconds (limits of agreement, -1.120 to 1.127 seconds) for the 0- and 24-hour time points, respectively. Agreement for APTT was -0.423 seconds (limits of agreement, -3.123 to 2.276 seconds) and 0.677 seconds (limits of agreement, -3.854 to 5.207 seconds) for the 0- and 24-hour time points, respectively. Agreement for fibrinogen concentration was -2.333 mg/dL (limits of agreement, -80.639 to 75.973 mg/dL) and -1.767 mg/dL (limits of agreement, -50.056 to 46.523 mg/dL) for the 0- and 24-hour time points, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Agreement between the 2 techniques for sample collection was clinically acceptable for PT, APTT, and fibrinogen concentration at time 0 and 24 hours. It is often difficult or undesirable to perform multiple direct venipunctures in critically ill patients. Use of samples collected via an IV catheter to monitor PT and APTT can eliminate additional venous trauma and patient discomfort and reduce the volume of blood collected from these compromised patients.     

Submitting canine blood for prothrombin time and partial thromboplastin time determinations

Practitioners commonly submit samples from dogs for partial thromboplastin time and prothrombin time determinations. Controversy exists as to the necessity for rapid separation of plasma and cells, and submission of the plasma on ice (or frozen). The purpose of this study was to address three questions. First, is it better to submit plasma or is whole blood satisfactory? Second, is it necessary to refrigerate the sample or is maintenance at room temperature (20° C) adequate? Third, does the sample have to arrive at the laboratory within a few hours of collection or can reliable partial thromboplastin time/prothrombin time determinations be made on samples up to 48 hours old?It has been shown by this study that reliable partial thromboplastin time and prothrombin time determinations can be carried out on canine plasma for up to 48 hours after collection regardless of whether or not the plasma is separated immediately; however the samples must be kept at 4°C. If the samples are maintained at room temperature, reliable prothrombin time determinations can be obtained for up to six hours after collection regardless of whether or not the plasma is separated immediately. Reliable partial thromboplastin time determinations can be made on plasma stored at 20°C for up to 24 hours after collection and possibly longer (up to 48 hours) if the plasma has been separated immediately.     

Evaluation of a point-of-care coagulation analyzer for measurement of prothrombin time, activated partial thromboplastin time, and activated clotting time in dogs

OBJECTIVE: To evaluate a point-of-care coagulation analyzer (PCCA) in dogs with coagulopathies and healthy dogs. ANIMALS: 27 healthy and 32 diseased dogs with and without evidence of bleeding. PROCEDURE: Prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were determined, using a PCCA and standard methods. RESULTS: Using the PCCA, mean (+/- SD) PT of citrated whole blood (CWB) from healthy dogs was 14.5+/-1.2 seconds, whereas PT of nonanticoagulated whole blood (NAWB) was 10.4+/-0.5 seconds. Activated partial thromboplastin time using CWB was 86.4+/-6.9 seconds, whereas aPTT was 71.2+/-6.7 seconds using NAWB. Reference ranges for PT and aPTT using CWB were 12.2 to 16.8 seconds and 72.5 to 100.3 seconds, respectively. Activated clotting time in NAWB was 71+/-11.8 seconds. Agreement with standard PT and aPTT methods using citrated plasma was good (overall agreement was 93% for PT and 87.5% for aPTT in CWB). Comparing CWB by the PCCA and conventional coagulation methods using citrated plasma, sensitivity and specificity were 85.7 and 95.5% for PT and 100 and 82.9% for aPTT, respectively. Overall agreement between the PCCA using NAWB and the clinical laboratory was 73% for PT and 88% for aPTT. Using NAWB for the PCCA and citrated plasma for conventional methods, sensitivity and specificity was 85.7 and 68.4% for PT and 86.7 and 88.9% for aPTT, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The PCCA detected intrinsic, extrinsic, and common pathway abnormalities in a similar fashion to clinical laboratory tests.     

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.     

Evaluation of a bench-top coagulation analyzer for measurement of prothrombin time, activated partial thromboplastin time, and fibrinogen concentrations in healthy dogs

OBJECTIVE: To evaluate a bench-top coagulation analyzer for determination of prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration in healthy dogs. ANIMALS: 55 healthy adult dogs. PROCEDURES: PT, APTT, and fibrinogen concentration were determined by use of the coagulation analyzer. Values were compared with results obtained independently by a conventional laboratory. RESULTS: Correlations (with 95% confidence intervals) between the coagulation analyzer and conventional laboratory values were 0.760 (0.610 to 0.857), 0.700 (0.448 to 0.721), and 0.896 (0.878 to 0.918) for PT, APTT, and fibrinogen concentration, respectively. Using linear regression, comparison of data from the coagulation analyzer and the conventional laboratory provided equations relating the coagulation analyzer values with values from the conventional laboratory and suggested that APTT and fibrinogen values from the coagulation analyzer and conventional laboratory were approximately the same within expected random variation. Prothrombin time values for the coagulation analyzer were significantly offset from the PT values for the conventional laboratory but still were correlated reasonably well with the conventional laboratory values. CONCLUSIONS AND CLINICAL RELEVANCE: By use of the mechanical method of analysis, fibrinogen concentrations obtained with a bench-top coagulation analyzer correlated well with results for a conventional laboratory, indicating that the coagulation analyzer is a reliable instrument for determination of this coagulation variable. Coagulation analyzer results for PT and APTT correlated less strongly with those for the conventional laboratory, but they would still be considered clinically reliable.     

Thromboelastography results on citrated whole blood from clinically healthy cats depend on modes of activation

Background

During the last decade, thromboelastography (TEG) has gained increasing acceptance as a diagnostic test in veterinary medicine for evaluation of haemostasis in dogs, however the use of TEG in cats has to date only been described in one previous study and a few abstracts. The objective of the present study was to evaluate and compare three different TEG assays in healthy cats, in order to establish which assay may be best suited for TEG analyses in cats.

Methods

90 TEG analyses were performed on citrated whole blood samples from 15 clinically healthy cats using assays without activator (native) or with human recombinant tissue factor (TF) or kaolin as activators. Results for reaction time (R), clotting time (K), angle (α), maximum amplitude (MA) and clot lysis (LY30; LY60) were recorded.

Results

Coefficients of variation (CVs) were highest in the native assay and comparable in TF and kaolin activated assays. Significant differences were observed between native and kaolin assays for all measured parameters, between kaolin and TF for all measured parameters except LY60 and between native and TF assays for R and K.

Conclusion

The results indicate that TEG is a reproducible method for evaluation of haemostasis in clinically healthy cats. However, the three assays cannot be used interchangeably and the kaolin- and TF activated assays have the lowest analytical variation indicating that using an activator may be superior for performing TEG in cats.     

One-stage prothrombin time, activated partial thromboplastin time, thrombin time, fibrin degradation product concentration, and antithrombin activity in healthy adult alpacas

Background: Alpacas are increasingly presented to veterinarians for evaluation and care. Reports of alpaca reference intervals for one‐stage prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), concentration of fibrin degradation products (FDP), and antithrombin (AT) activities are scarce or nonexistent. Objective: The aim of this study was to determine values for blood coagulation times (PT, aPTT, and TT), FDP concentrations, and AT activities in healthy adult alpacas. Methods: Of blood samples collected from 35 clinically healthy adult alpacas via jugular venipuncture and placed into sodium citrate and FDP tubes, 29 samples were assayable for coagulation testing. PT, aPTT, and TT were determined by physical (mechanical) clot detection; AT activity was determined using a thrombin‐specific chromogenic substrate end‐point assay; and FDP concentrations were determined by the slide agglutination method. Results: Median values and ranges (minimum–maximum) were determined for PT (8.7 seconds, 6.6–11.2 seconds), aPTT (17.3 seconds, 11.9–22.5 seconds), TT (10.2 seconds, 5.4–16.0 seconds), and AT activity (123.3%, 104.8–144.2%). The mean concentration of FDP was <8 μg/mL. Conclusion: These values for coagulation times, FDP concentration, and AT activity will provide a useful starting point in the diagnostic evaluation of ill adult alpacas.     

Effect of sodium heparin and antithrombin III concentration on activated partial thromboplastin time in the dog

Regulation of blood coagulation was studied in 12 dogs, using subcutaneous administration of sodium heparin. Dosage of heparin needed to achieve the desired 1.5- to 2.5-fold increase in the activated partial thromboplastin time (APTT) was 250 to 500 IU/kg of body weight. Increased APTT lasted less than 6 hours. Repeated heparin administration, using the lowest dosage (250 IU/kg) every 6 hours, induced an unacceptable prolongation of clotting times during the first 2 days of treatment. Prolonged administration at a dosage of 200 IU/kg every 6 hours adequately maintained the desired hypocoagulative state initially; after 2 days, however, the prolonged APTT steadily decreased. The decreasing effect was proportionate to a decrease in plasma antithrombin III (AT III). To sustain a correctly balanced hypocoagulative state from prolonged subcutaneous administration of heparin, APTT values should be determined regularly to monitor therapy. In addition, transfusion of AT III-rich donor plasma may be necessary when low plasma AT III reduces the effects of heparin.     

Effects of storage time on chemistry results from canine whole blood,heparinized whole blood,serum and heparinized plasma

The stability and storage characteristics of 24 blood constituents from dogs including nine enzymes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, lipase), 15 metabolites and minerals (albumin, bile acids, bilirubin, calcium, cholesterol, creatinine, fructosamine, glucose, magnesium, phosphate, potassium, protein, sodium, triglycerides, urea) were studied. Conditions studied included storing of nonanticoagulated and heparinized whole blood for 3 days (Part A), and storing of serum and heparinized plasma for 3 days (Part B). The storage temperature for both studies was +4 degrees C from day 0 to day 1, and +20 degrees C, from day 1 to day 2 and 3. Eight of 24 analytes showed no significant differences (p > 0.05) for three days in whole blood. However, the stability of all 24 analytes greatly improved by storing serum or heparinized plasma compared to nonanticoagulated or heparinized whole blood. In stored serum or heparinized plasma, 20 of 24 analytes showed no significant differences (p < 0.05) for 3 days. Nine of 24 analytes showed significant differences (p < 0.05) between serum and heparinized plasma, where CK, LDH, GGT, and potassium showed differences of possible clinical importance. This study strongly supports the practice of separating serum/plasma from clot/cells as promptly as possible to achieve improved stability for most analytes under test.     

Heparin in vitro sensitivity of the activated partial thromboplastin time in canine plasma depends on reagent.

The in vitro heparin sensitivity of 6 different commercial activated partial thromboplastin time (APTT) reagents was investigated based on artificial plasma samples prepared by addition of sodium heparin at different activities (0-1.5 IU/ml) to pooled normal canine plasma. Statistical analysis using 2-way analysis of variance was based on APTT ratios (APTT/mean APTT control). Significant differences between the APTT ratios of different APTT reagents (P < 0.00001) were found, which also depended on heparin activity (interaction between the factors; P < 0.00001). For example, mean APTT ratio at 0.7 IU/ml heparin varied between 1.2 and 2.5. The results of this study indicate that recommendations for the control of heparin therapy in dogs by APTT ratio should be reagent specific.     

Incidence of prolonged prothrombin time in dogs following gastrointestinal decontamination for acute anticoagulant rodenticide ingestion

Objective: To determine the effect of gastrointestinal (GI) decontamination on the incidence of prolonged prothrombin time (PT) in dogs after anticoagulant rodenticide ingestion. Design: Retrospective study. Setting: Urban emergency room. Animals: One hundred and fifty‐one client‐owned dogs. Measurements: Dogs presented to the emergency room within 6 hours of ingestion of an anticoagulant rodenticide and had a PT measured within 2–6 days of toxicant ingestion before initiating vitamin K therapy were included. Dogs were categorized as treated or untreated based on the institution of vitamin K therapy following PT testing. The signalment, body weight, type of rodenticide ingested, time elapsed between ingestion and initial presentation, method(s) of GI decontamination, and the times elapsed between both toxicant ingestion and initial hospital presentation until determination of PT were recorded. The PT results were recorded as well as any treatment received following the recheck examination. Any reported incidents of bleeding or untoward effects between exposure and reexamination were recorded. Main results: Of 151 dogs, only 11 dogs (8.3%) developed prolonged PT requiring vitamin K supplementation. None of the 11 dogs with prolonged PTs exhibited signs of bleeding or required transfusion therapy. No differences in age, weight, or time elapsed between treated and untreated patients were found. Conclusions: The incidence of prolonged PT is low in dogs receiving GI decontamination within 6 hours of anticoagulant rodenticide ingestion. Delaying vitamin K therapy until a PT has been assessed 48–72 hours after initial exposure appears to be safe and sensitive in dogs following anticoagulant rodenticide ingestion.