Abstract
Acute stress induced by physical restraint can interfere with the validity of laboratory findings. Sedation could minimize such stress. However, it is not known whether sedation can affect hematologic and hemostatic parameters in cats. The purpose of this study was to evaluate hematologic and hemostatic parameters in domestic cats subjected to physical restraint in addition to one of two sedation protocols. In total, 50 cats were subjected to physical restraint and were then randomly divided into two groups of 25 animals, receiving dexmedetomidine (5 µg/kg) and butorphanol (0.3 mg/kg; DB group) or dexmedetomidine (5 µg/kg), butorphanol (0.3 mg/kg) and ketamine (3 mg/kg; DBK group). The cats were assessed for acute stress, sedation level, onset of sedation and duration of sedation. Blood samples were collected after handling and after sedation. The complete blood count (CBC), platelet count, buccal mucosal bleeding time (BMBT), whole-blood clotting time, prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) were determined for each sample, before and after chemical restraint. No statistically significant differences were found in the hematologic parameters. Certain hemostatic parameters (PT, aPTT and TT) were higher in the DB group (P <0.05). The onset of sedation was similar in the two groups, and the duration of sedation was longer in the DBK group. Both sedation protocols were effective for short-duration chemical restraint for blood collection from the studied cats, and no clinically relevant effects on hematologic or hemostatic parameters were detected.
Introduction
Cats are becoming the most popular pets in the world. 1 These animals show characteristic behavior patterns, and they require special handling procedures to maintain their welfare and ensure the safety of the professional during blood sample collection. It has been established that catecholamines, such as epinephrine and norepinephrine, induce splenic contraction in many species, including cats. 2 A stress leukogram may also be observed in this situation, showing evidence of mature neutrophilia resulting from the effects of catecholamines; lymphocytosis; 3 and, eventually, slight thrombocytosis. 4 In contrast, certain drugs, such as alpha-2 agonists, can decrease the red blood cell (RBC) count and hemoglobin concentration by sequestration of erythrocytes within the spleen, 5 which is caused by reduced sympathetic activity of the central nervous system. 6
The use of sedation protocols can be of assistance in the collection of blood samples, especially in cats. The choice of sedation protocol must involve drugs that produce no changes or minimal changes in laboratory test results and that ensure adequate sedation. The purpose of this study was to assess the effects of stress resulting from physical restraint in addition to one of two sedation protocols on the hematologic and primary and secondary hemostatic parameters in cats. We hypothesized that certain anesthetic/sedative drugs (dexmedetomidine, butorphanol and ketamine) could alter the hematological and hemostatic parameters.
Materials and methods
Animals
This study was approved by the ethics committee on animal experimentation (Agroveterinary Sciences Center, Santa Catarina State University protocol number 1.69.12).
We used 50 healthy mixed-breed cats (26 females, 24 males) aged 1–7 years (median 24 months; range 8–84 months), weighing 3.3 ± 0.9 kg, and belonging to owners who agreed to the cats’ participation in this study by providing written consent. All animals were received the day before the study and had been subjected to previous examinations. We included only the cats showing no clinical or laboratory changes (American Society of Anesthesiologists grade I). Highly aggressive animals were not used in this study. Prior to the experiment, the cats were deprived of food for 8 h and deprived of water for 2 h. On the day before the start of the experiment, the animals were shaved in the neck region, in the area of both jugular veins, and an anesthetic cream was applied 2 h before the first collection (EMLA; AstraZeneca). After the experiment, the cats were neutered and returned to their owners.
Treatments
Randomization of treatments
After inclusion in the study, the animals were randomly allocated to one of the experimental groups. We attempted to standardize the number of males (n = 24) and females (n = 26) included in each group; therefore, if a group already had the maximum number of animals of one sex, the new animal was automatically allocated to the other group. The groups were then completed. Because of the random assignment, two experimental groups, each with 13 females and 12 males, were formed. The two groups were subjected to different sedation protocols.
The treatment used in the first group (DB group) consisted of dexmedetomidine (Dexdomitor 5 µg/kg; Orion Pharma) and butorphanol (Torbugesic 0.3 mg/kg; Fort Dodge), which were administered by intramuscular injection in combination in the same syringe. The treatment used in the second group (DBK group) consisted of dexmedetomidine (5 µg/kg), butorphanol (0.3 mg/kg) and ketamine (Dopalen 3 mg/kg; Vetbrands), which were administered by intramuscular injection in combination in the same syringe.
Physical restraint and acute stress evaluation
The cats were physically restrained by experienced staff who manually restrained the animals using a facemask, maintaining them in ventral recumbency with slight neck elevation to minimize acute stress. The same person performed the same procedure on all of the animals.
The degree of acute stress was assessed during physical restraint (before sedation) using a stress score. This stress score was developed in an earlier pilot study (Table 1).
Table 1.
Scale used for assessment of acute stress in cats during physical restraint
| Score | Degree of stress | Changes found |
|---|---|---|
| 0 | None | The cat shows no resistance to physical restraint |
| 1 | Mild | The cat shows slight resistance to physical restraint, performs certain movements or becomes anxious, but there is no interference with blood collection |
| 2 | Moderate | The cat shows moderate resistance to physical restraint, performs constant but not vigorous movements, becomes anxious and may vocalize when handled. There is possible interference with blood collection |
| 3 | Severe | The cat shows severe resistance to physical restraint, performs constant and vigorous movements, becomes anxious and vocalizes when handled. Additionally, the cat might become aggressive (may bite and scratch) and might urinate or defecate during handling. There is possible interference with blood collection |
Sedation
Sedation was evaluated and scored based on a numeric descriptive scale ranging from 0–3 (Table 2). This evaluation was performed by a single experienced person who was blinded to the treatment administered. The cats were observed for more than 25 mins after administration of the experimental treatment, and sedation was then scored based on the maximum sedation level observed for each cat within this period of time.
Table 2.
Scale used for assessment of the sedation of the cats in the experimental groups
| Score | Degree of sedation | Changes found |
|---|---|---|
| 0 | None | The score does not indicate sedation, and the cat shows normal movements |
| 1 | Mild | The cat shows mild ataxy but can remain standing by itself |
| 2 | Moderate | The cat has moderate ataxy, presents recumbency and shows mild reactions to stimuli (auditory, visual and tactile) |
| 3 | Intense | The cat presents lateral recumbency, with no responses to stimuli (auditory, visual and tactile) |
The onset of sedation was the time between the administration of the drugs and the observation of the first clinical signs of sedation (a loss of postural tone and a reduced response to external stimuli) (Table 2). The duration of sedation consisted of the interval from the onset of sedation until the cat was able to walk. The sedation score was based on the maximum sedation level observed for each cat.
Blood sampling and storage
Two blood samples were collected from each cat. The first blood collection was performed while the animals were physically restrained, and the blood samples were collected by right jugular vein puncture. At this time, the number of punctures used was the minimum necessary to obtain a suitable volume of blood for all laboratory tests.
The second set of blood samples was collected via left jugular vein puncture. At this time, the collection was performed with the animals under chemical restraint (clinical sedation), after evidence of clinical sedation and within 25 mins of administration of the treatment. Again, the number of punctures was minimized.
Approximately 6 ml of blood was collected at each of the studied time points in both groups. For the blood collections, we used 24 G × ¾" needles (Becton Dickinson), and 3 and 5 ml disposable syringes (Becton Dickinson). Samples were aliquoted into tubes in the following order: a tube with ethylenediaminetetraacetic acid (EDTA) (blood count and platelet count), a tube with sodium citrate (activated partial thromboplastin time [aPTT], prothrombin time [PT] and thrombin time [TT]) and tubes without anticoagulant (whole blood clotting time). The blood samples were stored in polypropylene microtubes containing 10% EDTA, at a proportion of 20 µl to 1 ml of blood, for the complete blood count (CBC) and the platelet count. To determine the aPTT, the PT and the TT, blood was stored in plastic Vacuette tubes (Greiner Bio-One) containing 3.8% sodium citrate, at a proportion of one part anticoagulant to nine parts blood; placed on ice; and immediately centrifuged at 165 g for 10 mins. The plasma was collected and stored in polypropylene microtubes at −20°C until processing. Whole blood clotting time determination was performed using fresh samples of whole blood collected in three numbered glass tubes with no additives.
Laboratory assays
CBC
Blood smears were prepared after blood sample collection and stained using the Diff-Quik Staining System (Laborclin). 7 RBC and white blood cell (WBC) counts and hemoglobin determination were performed in an automatic counter (CC510-Celm; Barueri) that uses the impedance method and has the following performance parameters: a coefficient of variation (CV) of 0.30% for RBCs, a CV of 0.36% for hemoglobin and a CV of 1.20% for WBCs. The packed cell volume (PCV) was measured by the microhematocrit procedure. 8 A differential leukocyte count was performed on blood smears. The mean cell volume (MCV) and the mean cell hemoglobin concentration (MCHC) were calculated.
Total plasma protein (TPP) was measured by refractometry (Digit-Biosystems).
Hemostasis tests
All of the tests for hemostatic evaluation were performed using blood samples collected at the first puncture.
Primary hemostasis
A platelet count was performed in a hemocytometer chamber (Neubauer chamber) using a 1% ammonium oxalate solution as the diluent. The CV for platelet counting in a hemocytometer is 8–10%. 8 Platelet cell morphology was observed via blood smears stained with the Diff-Quick Staining System. 7 The samples with large amounts of platelet aggregates in blood smears were excluded from the statistical analysis of the platelet count, buccal mucosal bleeding time (BMBT), whole blood clotting time, aPTT, PT and TT. The other tests were performed as usual.
The BMBT was determined with a commercial lancet (Triplett; Helena Laboratories). 9 Each animal was positioned in lateral recumbency. After positioning, the upper lip was everted, and the lancet was positioned perpendicularly to the canine tooth. Timing began after shooting the lancet and was continued until the end of bleeding. This test was performed only after chemical restraint because it is not possible to perform the test in unsedated animals.
Secondary hemostasis
The aPTT (Labtest), PT (Labtest) and TT (Helena Laboratories) were determined with commercial kits. The whole blood clotting time was determined by the Lee–White method. 10
The total procedure time was evaluated from the beginning of handling until the end of the second blood collect after chemical restraint.
Statistical analysis
All analyses were performed using specific software (SigmaStat 3.1; Systat Software). The data were compared between groups and between time points. Normality was tested by the Shapiro–Wilk test. Certain data were not normally distributed (age; the counts of total leukocytes, neutrophils, lymphocytes, eosinophils, basophils and monocytes; the aPTT; the PT; the TT; the whole blood clotting time; the BMBT; the onset of sedation; the puncture number; and the stress and sedation scores). For determination of statistically significant differences between time points and groups, the data were analyzed by Student’s t-test. In contrast, data that were not normally distributed were analyzed by the Mann–Whitney test. Differences were considered statistically significant if P <0.05.
Results
There were no significant differences in the weight of the animals between groups, but evaluation of age indicated that the animals in the DB group (median 24 months; range 8–84 months) had greater values compared with the DBK group (median 15 months; range 12–60 months) (P = 0.02).
Acute stress, puncture number, sedation, onset of sedation, duration of sedation and total procedure time
Stress evaluation indicated that the animals in the DB group had greater stress scores, with a greater number of cats (52%, 13/25) with a score of 2 or 3 on the stress scale (moderate-to-intense stress), whereas most cats in the DBK group (76%, 19/25) had a score of 0 or 1 (no or slight stress) during physical restraint. However, no significant difference in the stress score was found between the groups.
In the DB group, a greater number of punctures was necessary at the time of physical restraint (median 3; range 1–4) than when under chemical restraint (median 2; range 2–4). This difference was statistically significant (P <0.01). The DBK group showed no statistically significant differences between the number of punctures necessary under physical restraint (median 2; range 2–6) and the number needed under chemical restraint (median 2; range 2–4). Moreover, the number of punctures did not differ between the sedation groups (DB and DBK) when under chemical restraint.
In the DB group, 64% of the cats showed moderate-to-intense sedation (scores of 2 and 3), and 36% of the cats showed no or slight sedation (scores of 0 and 1). In the DBK group, 72% of the cats showed moderate-to-intense sedation (scores of 2 and 3), whereas 28% of the cats showed no sedation or slight sedation (scores of 0 and 1). No difference in the sedation scores was found between the sedation groups.
The onset of sedation did not differ significantly between the DB group (median 12 mins; range 5–25 mins) and the DBK group (median 11 mins; range 8–19 mins). In contrast, the duration of sedation was significantly longer in the DBK group (median 31 mins; range 20–43 mins) than in the DB group (median 17 mins; range 6–26 mins) (P <0.01). The total procedure time was significantly longer in the DB group (median 20 mins; range 10–31 mins) than in the DBK group (median 17 mins; range 12–31 mins) (P <0.01).
Hematology
There were no statistically significant changes in the RBC count, hemoglobin concentration, PCV, hematimetric indices (MCV and MCHC), total leukocyte count or TPP values between groups or between time points.
The differential leukocyte count showed no significant differences, except in the basophil count, which was significantly greater under physical restraint than under chemical restraint in the DBK group (P = 0.03). Similarly, there was a significant difference between the sedation groups, with a significantly greater basophil count in the DB group than in the DBK group (P = 0.02) (Table 3).
Table 3.
Values (mean ± SD) for the erythrogram, leukogram, platelet count, total plasma protein [TPP], and coagulation tests (whole blood clotting time, activated partial thromboplastin time [aPTT], prothrombin time [PT] and thrombin time [TT]) in the cats (n = 50) in the studied groups at the studied time points
| Group | Variable | Physical restraint | Chemical restraint | Reference interval 8 |
|---|---|---|---|---|
| Dexmedetomidine and butorphanol | RBCs (×106/µl) | 7.7 ± 1.6 | 7.0 ± 1.5 | 5.0–10.0 |
| Hb (g/dl) | 11.9 ± 2.3 | 11.0 ± 2.0 | 8–15 | |
| PCV (%) | 36.0 ± 6.3 | 33.2 ± 5.8 | 24–45 | |
| MCV (fl) | 47.4 ± 4.3 | 47.5 ± 4.3 | 39–55 | |
| MCHC (%) | 33.0 ± 1.4 | 33.25 ± 1.63 | 30–36 | |
| WBCs (/µl) | 11,595 ± 4629 | 9500 ± 3.893 | 5500–19,500 | |
| Band neutrophils (/µl) | 0.00 ± 0.00 | 0.00 ± 0.00 | 0–300 | |
| Segmented neutrophils (/µl) | 7415 ± 3754 | 6185 ± 3554 | 2500–12,500 | |
| Lymphocytes (/µl) | 2859 ± 2093 | 2211 ± 1422 | 1500–7000 | |
| Eosinophils (/µl) | 1008.0 ± 620.6 | 842.5 ± 576.8 | 0–1500 | |
| Basophils (/µl) | 12.8 ± 36.1 | 45.2 ± 64.3* | Rare | |
| Monocytes (/µl) | 299.0 ± 249.4 | 215.3 ± 195.3 | 0–850 | |
| Platelets (x103/µl) | 406.8 ± 146.0 | 399.0 ± 154.4 | 400–800 | |
| TPP (g/dl) | 7.0 ± 0.6 | 6.6 ± 0.5 | 5.5–8.1 | |
| Whole blood clotting time (mins) | 8.5 ± 2.2 | 8.8 ± 1.8 | Approximately 8 10 | |
| aPTT (s) | 14.9 ± 2.8* | 14.7 ± 3.0* | 10–20 11 | |
| PT (s) | 12.4 ± 1.7* | 12.5 ± 2.0* | 10–15 11 | |
| TT (s) | 12.2 ± 2.1* | 12.2 ± 1.5* | 5–8 12 | |
| Dexmedetomidine, butorphanol and ketamine | RBCs (×106/µl) | 7.6 ± 1.3 | 7.3 ± 1.2 | 5.0–10.0 |
| Hb (g/dl) | 12.1 ± 1.5 | 11.6 ± 1.2 | 8–15 | |
| PCV (%) | 35.9 ± 4.8 | 34.4 ± 3.9 | 24–45 | |
| MCV (fl) | 47.5 ± 4.3 | 47.9 ± 5.2 | 39–55 | |
| MCHC (%) | 33.3 ± 1.6 | 33.8 ± 1.8 | 30–36 | |
| WBCs (/µl) | 12,296 ± 6173 | 11,428 ± 4863 | 5500–19,500 | |
| Band neutrophils (/µl) | 0.00 ± 0.00 | 4.9 ± 24.9 | 0–300 | |
| Segmented neutrophils (/µl) | 7998 ± 4874 | 6990 ± 4155 | 2500–12,500 | |
| Lymphocytes (/µl) | 2876 ± 2063 | 2686 ± 1613 | 1500–7000 | |
| Eosinophils (/µl) | 1051.0 ± 919.1 | 957.8 ± 849.4 | 0–1500 | |
| Basophils (/µl) | 78.2 ± 141.7 † | 14.0 ± 44.6* † | Rare | |
| Monocytes (/µl) | 391.5 ± 224.3 | 334.2 ± 309.2 | 0–850 | |
| Platelets (×103/µl) | 429.5 ± 129.2 | 419.8 ± 133.5 | 400–800 | |
| TPP (g/dl) | 7.2 ± 0.5 | 6.9 ± 0.5 | 5.5–8.1 | |
| Whole blood clotting time (mins) | 8.9 ± 2.3 | 9.8 ± 1.5 | Approximately 8 10 | |
| aPTT (s) | 12.8 ± 2.0* | 13.3 ± 2.0* | 10–20 11 | |
| PT (s) | 10.9 ± 0.8* | 11.0 ± 0.9* | 10–15 11 | |
| TT (s) | 10.5 ± 1.4* | 10.9 ± 1.5* | 5–8 12 |
Means with a statistically significant difference between protocols
Means with a statistically significant difference between time points
RBCs = red blood cells; Hb = hemoglobin concentration; PCV = packed cell volume; MCV = mean cell volume; MCHC = mean cell hemoglobin concentration; WBCs = white blood cells
Coagulation tests
During evaluation of the blood smears, platelet aggregates were observed in 14 samples (six in the DB group and eight in the DBK group). These samples were excluded from the statistical analysis of the platelet count, BMBT, whole blood clotting time, aPTT, PT and TT.
Primary hemostasis
The platelet count showed no statistically significant differences between groups or between time points, although it was higher at the time of physical restraint in both groups (Table 3). The BMBT showed no statistically significant differences between the groups (DB group 74.1 ± 32.0 s; DBK group 70.1 ± 22.9 s).
Secondary hemostasis
The whole blood clotting time did not differ significantly between groups or between time points. The aPTT, PT and TT values showed no statistically significant differences between time points but differed significantly between the sedation groups, with greater aPTT, PT and TT values in the DB group than in the DBK group (P <0.01) (Table 3).
Discussion
In general, the protocols used in this study did not result in statistically significant changes in the hematological values of healthy cats. However, we did find differences in certain parameters, but these changes had no clinical relevance.
The onset of sedation was similar in the two groups, and this onset may be considered as acceptable for chemical restraint in a veterinary clinic. Similarly, the duration of sedation was acceptable. The statistically significant increase in the duration of sedation in the DBK group compared with the DB group occurred because ketamine can enhance the action of certain alpha-2 agonists. 13 This effect can prolong the duration of sedation, as shown in the DBK group. The use of drugs that antagonize the effects of alpha-2 agonists (atipamezole and yohimbine) is indicated in cases with prolonged recovery. These drugs decrease the duration of sedation and are clinically used to reverse the sedative and analgesic effects of alpha-2 agonists. 14 The significantly lower number of punctures observed under chemical restraint compared with physical restraint in the DB group was owing to the sedation of the cat, which facilitated puncturing. This result is consistent with the findings of a previous study. 5 Moreover, the animals in the DB group showed higher stress scores (scores of 2 and 3), further hampering the performance of punctures under physical restraint. The DBK group showed no significant differences between time points (physical restraint and chemical restraint), most likely because of lower stress levels (scores of 0 and 1), which facilitated the performance of punctures under physical restraint. Thus, a smaller number of punctures occurred during physical restraint, with no significant difference between physical restraint and chemical restraint. The total procedure time was higher in the DB group than in the DBK group, which could be explained by the higher number of punctures under physical restraint in the DB group; this situation could have hampered the collection of blood samples and may be a contributing factor to the greater total procedure time.
Physical restraint may cause an increase in the RBC count, hemoglobin concentration and PCV owing to the release of catecholamines, resulting in splenic contraction,15,16 as found in a previous study. 5 However, the reason for the absence of statistically significant differences could be that most of the cats in the study (31/50, 62%) had a lower stress score during physical restraint (scores of 0 and 1), as also observed in a previous study, 17 prior to sedation. Accordingly, it is likely that a more stressed population of cats would show statistically significant differences between the studied time points. Additionally, the action of the drugs could have influenced the outcome. Alpha-2 agonists, such as dexmedetomidine, can be used individually or in combination with other drugs to induce sedation, muscle relaxation and analgesia. Dexmedetomidine is an imidazole derivative with high selectivity for the alpha-2 adrenergic receptor and is the dextro-enantiomer of the racemate medetomidine. 18 Dexmedetomidine, similar to other alpha-2 agonists, can cause hemodynamic variations that coincide with decreases in plasma catecholamine,19,20 contributing to the reduction in the PCV and RBC counts caused by spleen vasodilatation and RBC sequestration.6,15 These events should lead to a significant difference between physical and chemical restraint, which was not observed in this work because the increase in RBCs, hemoglobin and the PCV under physical restraint was not substantial. Ketamine is a dissociative anesthetic with rapid effect, 18 and it has a direct adrenergic effect that can cause vasoconstriction, resulting in spleen contraction and, consequently, an increase in erythrogram parameters. 21 However, ketamine likely did not have vasoactive activity capable of changing the erythrogram results in this study. This conclusion is supported by the absence of statistically significant differences between the studied time points.
Catecholamine release due to physical restraint causes an increase in the number of total leukocytes, neutrophils and lymphocytes,16,22–24 but this change was not found in the present study, which can be explained by the lower degree of stress of the evaluated cats under physical restraint compared with chemical restraint. Similar results were found in certain experimental groups in a previous study. 5
The basophil count showed significant differences between groups and between time points (DBK group), but should be characterized as an occasional finding with individual variation owing to the imprecision of differential leukocyte counts, especially for a minority cell population. 10
Acute stress derived from physical restraint can cause thrombocytosis because epinephrine release causes splenic contraction and a transient increase in platelet numbers or physiologic thrombocytosis. 25 These effects were not observed in the present study, in contrast to a previous study in which a significantly higher platelet count was observed prior to sedation compared with the count following chemical restraint. 5 Multiple punctures could also cause vascular damage, in vivo platelet aggregation and subsequent in vitro platelet aggregation, and a decrease in platelet numbers. 26 These effects could have hampered the observation of thrombocytosis in the studied cats under chemical restraint.
The use of the two sedation protocols did not change the values of whole blood clotting time, aPTT, PT or TT in the present study. A previous study using ketamine and diazepam found statistically significant differences in the aPTT before and after sedation. 17 The statistically significant differences found between groups for the aPTT, PT and TT could be explained by individual variation because these variables were significantly greater under physical restraint. In a previous study, 17 no statistically significant changes in the aPTT or PT were found between the studied groups (midazolam and butorphanol; midazolam, butorphanol and ketamine; midazolam, butorphanol and dexmedetomidine; and ketamine and dexmedetomidine).
Conclusions
The drugs used in the sedation protocols in this study did not show statistically significant effects on the studied hematological parameters, and the differences found in the hemostatic parameters were not clinically relevant to healthy cats.
Footnotes
The authors do not have any potential conflicts of interest to declare.
Funding: This research received no specific grant from any funding agency in the public, commercial or not-for-profit sector.
Accepted: 4 August 2014
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