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.     

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.     

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.     

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.     

Effect of reagent batch on activated partial thromboplastin time in canine plasma and use of the ratio system for standardization

In order to test the variability of the results of the activated partial thromboplastin time (APTT) in different reagent batches, 40 samples (20 from healthy dogs, 15 from patients with prolonged APTT as a result of different congenital or acquired haemostasis disorders, 5 from healthy dogs after in vitro addition of heparin) were used to compare 6 different lot Nos. of two commercial APTT-reagents (Pathromtin, PTT Reagent). Although the Friedman test showed a reagent batch dependency (p < 0.0001) for both reagents, only minor quantitative differences were observed with a variation coefficient of 2.7% (Pathromtin) and 2.4% (PTT Reagent), respectively. A second experiment was based on 105 samples measured with two batches of a third reagent (APTT-FS) with remarkable differences of results. Convergence of the results was achieved by converting into ratio values (quotient measurement value/control). However, statistical comparison still showed a significant difference. The study shows the good reproducibility of the APTT measured with different batches of the reagents Pathromtin and PTT Reagent in canine plasma, indicating that standardization is unnecessary. A standardization based on the ratio system can be used for reagents with a low batch consistency, requiring a high-quality control.     

The activated partial thromboplastin time of diluted plasma: variability due to the method of fibrin detection.

The purpose of this study was to determine the effects of the dilution of plasma (1/3 in saline) on the kinetics of fibrin generation in the activated partial thromboplastin time (APTT) assay. The diluted APTT is considered to increase the sensitivity of the APTT test however, studies in our laboratory using an electro-optical fibrin detection system failed to show significant differences in APTT values obtained with diluted and undiluted canine plasma. Seventeen plasmas, including plasmas moderately and markedly deficient in intrinsic factor activity were assayed in the undiluted and diluted APTT assay using two methods for fibrin endpoint detection; a visual "tilt-tube" technique and an electro-optical detection system. In the former technique the endpoint was the formation of a visible fibrin web or clot; in the latter procedure the end point was the first detection of a change in optical density of the plasma. Optical density changes during fibrin formation were also recorded ( thrombokinetograms ). The results indicated that the electro-optical fibrin detection system failed to identify a prolongation of the APTT as a result of 1/3 plasma dilution; a prolongation that was consistently observed with the visual fibrin detection technique. Plasma dilution however, did significantly reduce the rate of fibrin production as indicated by the thrombokinetogram profile. It was concluded that the dilution of plasma with saline, as has been used to increase the sensitivity of the APTT assay procedure, has little effect on the time of onset of fibrin formation in a given plasma. The major effect appears to be on the way in which fibrin forms in that the polymerization/crosslinkage events associated with macroscopic fibrin production are delayed.     

Effects of storage time and temperature on pH,specific gravity,and crystal formation in urine samples from dogs and cats

OBJECTIVE: To determine effects of storage temperature and time on pH and specific gravity of and number and size of crystals in urine samples from dogs and cats. DESIGN: Randomized complete block design. ANIMALS: 31 dogs and 8 cats. PROCEDURE: Aliquots of each urine sample were analyzed within 60 minutes of collection or after storage at room or refrigeration temperatures (20 vs 6 degrees C [68 vs 43 degrees F]) for 6 or 24 hours. RESULTS: Crystals formed in samples from 11 of 39 (28%) animals. Calcium oxalate (CaOx) crystals formed in vitro in samples from 1 cat and 8 dogs. Magnesium ammonium phosphate (MAP) crystals formed in vitro in samples from 2 dogs. Compared with aliquots stored at room temperature, refrigeration increased the number and size of crystals that formed in vitro; however, the increase in number and size of MAP crystals in stored urine samples was not significant. Increased storage time and decreased storage temperature were associated with a significant increase in number of CaOx crystals formed. Greater numbers of crystals formed in urine aliquots stored for 24 hours than in aliquots stored for 6 hours. Storage time and temperature did not have a significant effect on pH or specific gravity. CONCLUSIONS AND CLINICAL RELEVANCE: Urine samples should be analyzed within 60 minutes of collection to minimize temperature- and time-dependent effects on in vitro crystal formation. Presence of crystals observed in stored samples should be validated by reevaluation of fresh urine.     

大熊猫血液标本放置时间对全血细胞计数的影响

为了探讨血液标本放置时间对大熊猫全血细胞计数的影响,试验采用平行试验的方法测定了28只健康成年大熊猫静脉血在120 min内放置不同时间时,红细胞(RBC)、血红蛋白(HGB)、白细胞(WBC)、血小板(PLT)、红细胞比容(HCT)平均值的差异。结果表明:血液标本放置60分钟时RBC、HGB、WBC基本无变化,与即检结果相比差异不显著(P>0.05),放置15,30,45,90,120分钟时与即检结果相比差异显著(P<0.05)。PLT随着放置时间的延长有上升趋势,但差异不显著(P>0.05)。HCT在血液标本放置30 min以内均无显著差异(P>0.05),放置45 min后均呈显著性差异(P<0.05)。综合分析各项细胞计数测定结果认为,经乙二胺四乙酸二钾(EDTA-K2)抗凝的大熊猫全血标本,若采血后不能及时检测,应在放置60 min后再进行测定,可提高结果的准确性。     

An evaluation of activated coagulation time in warfarin-intoxicated dogs

Activated coagulation time, one stage prothrombin time and activated partial thromboplastin time were compared in warfarin-intoxicated dogs. All coagulation tests were significantly prolonged on the third and fourth days following intoxication. Coagulation assays of all dogs except one returned to preintoxication values one day following vitamin K therapy. Significant correlation was found between all coagulation tests in the intoxicated group. Activated coagulation time may provide a useful coagulation screening test to veterinarians with limited laboratory capabilities.     

Co-oximetry in clinically healthy dogs and effects of time of post sampling on measurements

Objectives : Co‐oximetry is a complex and valuable laboratory method that measures haemoglobin species and oxygenation status by multi‐wavelength spectrophotometry. The purpose of this study was to establish reference intervals for clinically healthy dogs and to determine the effect of time of analyses and sex of animals on the accuracy of results. Methods : Blood was collected from 27 healthy adult dogs of various breeds and sex. Co‐oximetry was performed on a CCX co‐oximeter that measures eight haemoglobin and oxygen transport related parameters: carboxyhaemoglobin (COHb), deoxyhaemoglobin (HHb), oxyhaemoglobin (O₂Hb), methaemoglobin (MetHb), total haemoglobin (tHb), oxygen saturation (SO₂%), oxygen content (O₂Ct) and oxygen capacity (O₂Cap). Results : Results obtained after 2 and 4 hours were not significantly different from those obtained immediately after sampling. But after 48 hours, the results for total haemoglobin, oxygen saturation, oxyhaemoglobin, oxygen content and oxygen capacity were significantly lower, and carboxyhaemoglobin and deoxyhaemoglobin values were significantly higher than determination immediately after sampling. Gender had no significant impact on co‐oximetry values. Clinical Significance : Co‐oximetry offers several advantages compared with other methods, including ease of use, increased accuracy and greater differentiation among haemoglobin species.     

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.     

The effect of ketoconazole on whole blood and skin ciclosporin concentrations in dogs

Background –  Ciclosporin (CSA) is approved for the treatment of canine atopic dermatitis. Ciclosporin is metabolized by liver cytochrome P450 enzymes, a process inhibited by ketoconazole (KTZ). Hypothesis/Objectives –  The aims of this study were to determine skin and blood CSA concentrations when CSA was administered alone at 5.0 (Treatment 1) or 2.5 mg/kg (Treatment 2) and when CSA was administered at 2.5 mg/kg concurrently with KTZ at 5 (Treatment 3) or 2.5 mg/kg (Treatment 4). We hypothesized that skin and blood CSA concentrations in Treatment 1 would not differ from those obtained with T3 or T4. Animals –  In a randomized cross‐over study, six healthy research dogs received each of the treatments (Treatment 1, 2, 3 and 4) once daily for 7 days. Methods –  After the first, fourth and seventh dose for each treatment, a peak and trough skin punch biopsy sample and whole blood sample were collected and analysed with high‐performance liquid chromatography–tandem mass spectrometry. Data were analysed using a repeated measures approach with PROC MIXED in SAS. Pairwise comparisons were performed with least squares means and Tukey–Kramer adjustment for multiple comparisons. Results –  Mean blood CSA concentrations in Treatment 1 were not different from those in Treatment 2 or 4, but were less than in Treatment 3. Mean skin CSA concentrations in Treatment 1 were greater than in Treatment 2, not different from those in Treatment 4, and less than those in Treatment 3. Conclusions and clinical importance –  Administration of CSA and KTZ concurrently at 2.5 mg/kg each may be as effective as CSA alone at 5.0 mg/kg for treatment of canine atopic dermatitis.