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    Platelet aggregation (reported as AUC measured by impedance aggregometry) versus Hct (A), platelet count (B), plateletcrit (C), and WBC count (D) for blood samples obtained from 24 privately owned healthy adult dogs of various breeds. A single blood sample was obtained from each dog. That blood sample was then allocated into 4 aliquots. One aliquot was analyzed without further manipulation (unmanipulated sample). A portion of the buffy coat fraction was removed from 2 aliquots; the buffy coat fraction was removed more aggressively from 1 sample than the other so that 2 platelet-depleted blood samples with different platelet counts were acquired. The remaining aliquot was used to obtain plasma, which was used to adjust the Hct of the 2 platelet-depleted samples to within 2% of the Hct for the unmanipulated sample. All 3 blood samples (the unmanipulated sample and 2 platelet-depleted samples) underwent impedance aggregometry in duplicate, and the mean was calculated for each sample and used for analysis. One platelet-depleted blood sample from a male dog was unable to be processed within 60 minutes after collection; therefore, the data for that single sample were excluded from all analyses. For each of 2 other dogs, 1 platelet-depleted sample had platelet clumping detected during evaluation of the blood smear, and those samples were also excluded from the analyses. Both platelet-depleted samples from a male dog were excluded, one because of evidence of platelet activation with the addition of saline (0.9% NaCl) solution (control) and the other because the AUC results were outside the instrument's quality control limits for inbuilt imprecision. For a female dog, only a single platelet-depleted sample was prepared. Thus, a total of 66 samples underwent aggregometry, and each dot represents the mean result for 1 blood sample. For the unmanipulated blood samples evaluated, the Hct ranged from 38.1% to 53.5%, platelet count ranged from 48 × 103 platelets/μL to 392 × 103 platelets/μL, plateletcrit ranged from 0.04% to 0.5%, and WBC count ranged from 1.47 × 103 WBCs/μL to 12.23 × 103 WBCs/μL. Because the cell lines were not independent of each other, an individual effect cannot be estimated without linear mixed modeling. AU = Aggregation units.

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In vitro effect of blood cell counts on multiple-electrode impedance aggregometry in dogs

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 3 Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 4 Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.

Abstract

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs.

ANIMALS 24 healthy dogs.

PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models.

RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

Abstract

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs.

ANIMALS 24 healthy dogs.

PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models.

RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

Platelets have a pivotal role in both primary and secondary hemostasis. They are important in the formation of the initial platelet plug, provide a surface for propagation of thrombin and a generation site for fibrin attachment, and facilitate clot retraction. Changes in platelet function can lead to substantial illness and death owing to thrombosis or hemorrhage. Tests of platelet function have been used in human medicine since the 1960s.1 Applications for their use are diverse and include screening certain patient populations, diagnosing congenital or acquired diseases of primary hemostasis, monitoring of patients receiving antiplatelet therapy, and determining risk stratification in patients receiving drugs that affect platelet function who are to undergo invasive procedures.1 Many clinically useful tests for direct and indirect measurement of platelet function have been developed including buccal mucosal bleeding time, clot retraction, light transmission aggregometry, whole blood impedance aggregometry, impact cone and plate analysis, flow cytometry, platelet adhesion assays, thromboelastography, and measurements of the mean platelet component.1–3

Whole blood impedance aggregometers are used extensively in human medicine. Those instruments measure platelet aggregation in a continuous manner in blood samples after the addition of classic platelet agonists such as ADP, arachidonic acid, collagen, ristocetin, and thrombin receptor activator for peptide 6. As platelets attach to the sensor, a change in impedance is detected and transformed into an aggregation-over-time curve. Arbitrary aggregation units are generated. In clinical practice, the most important parameter used for monitoring the overall expression of platelet activity is the total AUC. In addition to AUC, whole blood impedance aggregometers also calculate the height of the aggregation-over-time curve and maximum slope attained, which are used to generate the overall aggregation and velocity, respectively.

Impedance aggregometers can be used to diagnose a wide range of acquired and inherited plate-let disorders. Benefits of that method include use of whole blood samples with minimal sample processing, the small sample volume required, ease of use, and rapid turnaround time for acquisition of results.3,4 Use of impedance aggregometry in clinical veterinary medicine is in its infancy, but multiple reports5,6,a during the past decade describe its use in veterinary species and provide standardized testing protocols for canine blood samples. Research in veterinary and human medicine provides evidence that platelet function, as determined by whole blood impedance aggregometry, has potential as a biomarker in dogs7 and humans8 with septic peritonitis.

In human medicine, impedance aggregometric parameters decrease with platelet counts and are affected by other blood cell counts.9–13 Although reference intervals and standardized test protocols for whole blood impedance aggregometry have been established for healthy dogs with platelet counts within the reference interval,5,6,a to our knowledge, data regarding the effect of thrombocytopenia on impedance aggregometric parameters for canine blood samples are unavailable, which limits use of this technique in dogs with that condition.

The purpose of the study reported here was to assess the effect of circulating blood cell counts on platelet aggregation in canine blood samples as measured by use of a commercially available multiple-electrode impedance aggregometer. We hypothesized that impedance aggregometric parameters would be affected by the platelet count in canine blood samples.

Materials and Methods

Animals

Results of an a priori power analysis indicated that 20 blood samples would be sufficient to detect a 20% change in platelet aggregation parameters. All study protocols were reviewed and approved by the Auburn University Institutional Care and Use Committee.

Twenty-four clinically normal dogs owned by staff and students of the Auburn University College of Veterinary Medicine were enrolled in the study. Written consent was obtained from the owner of each dog prior to study enrollment. Each dog was considered healthy on the basis of results of a physical examination, CBC,b serum biochemical analysis,c prothrombin time, and activated partial thromboplastin time.d Dogs with plasma fibrinogen concentrationsd within or mildly increased from the reference range were included in the study. Great Pyrenees, Doberman Pinschers, and Cavalier King Charles Spaniels were excluded from the study because those breeds are predisposed to the development of Glanzmann thrombasthenia, type 1 von Willebrand disease, and inherited macrothrombocytopenia, respectively.14–16 Dogs that had received clopidogrel, NSAIDs (including aspirin), calcium channel blockers, phosphodiesterase inhibitors (including pentoxifylline, sildenafil, and pimobendan), antimicrobials, or other drugs that might affect platelet function within 6 weeks prior to sampling were also excluded from the study.

Blood sample collection and preparation of platelet-depleted samples

From each dog, a blood sample (9 mL) was collected by jugular venipuncture into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. The sample was divided into 4 equal aliquots. A small sample from 1 aliquot was used to obtain an automated cell countb and prepare a blood smear to assess for platelet clumping; the remainder of that aliquot was stored at room temperature (approx 20.5°C) until further analysis. The other 3 aliquots were centrifugede at 1,000 × g for 10 minutes. One of the 3 samples was retained for plasma donation as needed. For the other 2 samples, the majority of the plasma component was manually removed and saved; then, the platelet-rich buffy coat was partially removed with a manual pipette and discarded, and the saved plasma component and RBC mass were reconstituted. The total buffy coat volume removed was recorded for each of the 2 samples. The buffy coat was more aggressively removed from 1 sample than from the other to yield 2 platelet-depleted samples with different platelet counts.

Automated cell counts were performed for each blood sample designated to be analyzed with the aggregometer, and autologous plasma was added or removed from each sample as necessary to correct the Hct to within 2% of that for the initial (unmanipulated) sample. For samples that required plasma volume adjustment, automated and manual cell counts were repeated before aggregometric analysis. All blood smears were microscopically evaluated for platelet clumping. Blood samples that yielded smears with platelet clumps within the body or along the feathered edge of the smear were excluded from aggregometric analysis because of concerns that the cell counts might be inaccurate in those samples.

Impedance aggregometry

Platelet aggregation was determined in duplicate for each blood sample by use of an impedance aggregometerf between 30 and 60 minutes after sample collection. Briefly, 300 μL of warmed saline (0.9% NaCl) solution containing 3mM calcium chloride was added to each of 2 test cells. The tube containing the blood sample was gently inverted 3 times, and then 300 μL of the sample was added to the 2 test cells. The test cells were incubated for 3 minutes in accordance with the manufacturer's instructions to allow for admixture and recalcification of the reaction mixture. Then, 30 μL of saline solution (control) was added to 1 test cell and 30 μL of 0.2mM ADPg was added to the other test cell such that each test cell had a final concentration of 9.5μM. The intervals from blood sample collection to aggregometric analysis and determination of the platelet AUC, aggregation, and velocity were recorded for each sample. Results from blood samples that underwent aggregometric analysis > 60 minutes after collection or that had evidence of platelet activation with the addition of saline solution (control) were excluded from statistical analyses.

Statistical analysis

Statistical analyses were performed by use of linear mixed-effects models as described17 with an open-access statistical software program.h Measures of platelet function were the outcome (dependent) variables of interest and underwent logarithmic transformations when necessary to normalize data distributions prior to analysis. Each model included fixed effects for platelet count, plateletcrit, WBC count, and Hct and a random intercept for dog. Subject sex and age were included in models to control for confounding when necessary. Confounding was defined as a ≥ 10% difference in regression coefficients between the adjusted (model with sex and age) and reduced (model without sex and age) models. Results were reported as the percentage change from reference values, and F tests were reported for fixed effects. Values of P < 0.05 were considered significant for all analyses.

Results

Dogs

The 24 dogs enrolled in the study included 12 spayed females and 12 neutered males that had a mean ± SD body weight of 26.4 ± 8.5 kg and age of 5.2 ± 2.6 years. The study population included 19 mixed-breed dogs, 2 American Pitbull Terriers, 1 Australian Shepherd, 1 Labrador Retriever, and 1 Pembroke Welsh Corgi.

Hematologic and aggregometric results

One platelet-depleted blood sample from a male dog was unable to be processed within 60 minutes after collection; therefore, the data for that single sample were excluded from all analyses. For each of 2 other dogs, 1 platelet-depleted sample had platelet clumping detected during evaluation of the blood smear, and those samples were also excluded from the analyses. Both platelet-depleted samples from a male dog were excluded, one because of evidence of platelet activation with the addition of saline solution (control) and the other because the AUC results were outside the instrument's quality control limits for inbuilt imprecision. For 1 female dog, only a single platelet-depleted sample was prepared.

The mean Hct, WBC count, platelet count, and platelet AUC, aggregation, and velocity for the unmanipulated blood samples did not differ between spayed female and neutered male dogs (Table 1); therefore, data from male and female dogs were pooled for analyses. A total of 66 processed blood samples were included in the final analysis. For the samples evaluated, the platelet count ranged from 48 × 103 platelets/μL to 392 × 103 platelets/μL (median, 150 × 103 platelets/μL), plateletcrit ranged from 0.04% to 0.5% (median, 0.18%), Hct ranged from 38.1% to 53.5% (median, 46.9%), and WBC count ranged from 1.47 × 103 WBCs/μL to 12.23 × 103 WBCs/μL (median 5.8 × 103 WBCs/μL). The relative percentage change in each aggregometric parameter (AUC, aggregation, and velocity) associated with each cell count was summarized (Table 2). Platelet count, plateletcrit, and Hct were not significantly associated with platelet AUC, aggregation, or velocity within the ranges evaluated. However, the WBC count was positively associated with platelet AUC, aggregation, and velocity. The effect of each cell count on the AUC was depicted (Figure 1).

Figure 1—
Figure 1—

Platelet aggregation (reported as AUC measured by impedance aggregometry) versus Hct (A), platelet count (B), plateletcrit (C), and WBC count (D) for blood samples obtained from 24 privately owned healthy adult dogs of various breeds. A single blood sample was obtained from each dog. That blood sample was then allocated into 4 aliquots. One aliquot was analyzed without further manipulation (unmanipulated sample). A portion of the buffy coat fraction was removed from 2 aliquots; the buffy coat fraction was removed more aggressively from 1 sample than the other so that 2 platelet-depleted blood samples with different platelet counts were acquired. The remaining aliquot was used to obtain plasma, which was used to adjust the Hct of the 2 platelet-depleted samples to within 2% of the Hct for the unmanipulated sample. All 3 blood samples (the unmanipulated sample and 2 platelet-depleted samples) underwent impedance aggregometry in duplicate, and the mean was calculated for each sample and used for analysis. One platelet-depleted blood sample from a male dog was unable to be processed within 60 minutes after collection; therefore, the data for that single sample were excluded from all analyses. For each of 2 other dogs, 1 platelet-depleted sample had platelet clumping detected during evaluation of the blood smear, and those samples were also excluded from the analyses. Both platelet-depleted samples from a male dog were excluded, one because of evidence of platelet activation with the addition of saline (0.9% NaCl) solution (control) and the other because the AUC results were outside the instrument's quality control limits for inbuilt imprecision. For a female dog, only a single platelet-depleted sample was prepared. Thus, a total of 66 samples underwent aggregometry, and each dot represents the mean result for 1 blood sample. For the unmanipulated blood samples evaluated, the Hct ranged from 38.1% to 53.5%, platelet count ranged from 48 × 103 platelets/μL to 392 × 103 platelets/μL, plateletcrit ranged from 0.04% to 0.5%, and WBC count ranged from 1.47 × 103 WBCs/μL to 12.23 × 103 WBCs/μL. Because the cell lines were not independent of each other, an individual effect cannot be estimated without linear mixed modeling. AU = Aggregation units.

Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1380

Table 1—

Mean ± SD values for Hct, WBC count, platelet count, and impedance aggregometric results for unmanipulated blood samples from 24 privately owned healthy adult dogs of various breeds.

VariableAll dogsSpayed femalesNeutered malesP value*
Hct (%)46.8 ± 4.246.9 ± 3.746.6 ± 4.90.86
WBC count (× 103 WBCs/μL)8.0 ± 1.68.2 ± 1.87.8 ± 1.40.50
Platelet count (× 103 platelets/μL)228.7 ± 55.8242.7 ± 62.2213.5 ± 45.70.21
AUC26.9 ± 11.225.8 ± 9.128.0 ± 13.50.66
Aggregation57.6 ± 16.256.3 ± 13.559.0 ± 19.30.71
Velocity7.0 ± 2.66.6 ± 1.87.5 ± 3.30.46

The study population consisted of 12 spayed females and 12 neutered males; there were 19 mixed-breed dogs, 2 American Pitbull Terriers, 1 Australian Shepherd, 1 Labrador Retriever, and 1 Pembroke Welsh Corgi.

Comparison of means between spayed females and neutered males; values of P < 0.05 were considered significant.

Table 2—

Relative percentage change (95% CI) in AUC, aggregation, and velocity associated with each 25 × 103-platelets/μL change in platelet count, 0.05% change in plateletcrit, 1 × 103-WBCs/μL change in WBC count, and 5% change in Hct for the unmanipulated and platelet-depleted samples obtained from the dogs of Table 1.

 AUCAggregationVelocity
VariablePercentage change (95% CI)P valuePercentage change (95% CI)P valuePercentage change (95% CI)P value
Platelet count (per each 25 × 103 platelets/μL)3 (−2.9 to 5.8)0.524 (−1.7 to 4.8)0.35−2 (−4.7 to 2.6)0.59
Plateletcrit (per each 0.05%)−2 (−6.7 to 5.2)0.80−3 (−5.7 to 3.3)0.607 (−2.1 to 8.2)0.25
WBC count (per each 1 × 103 WBCs/μL)13.2 (6.8 to 20.0)< 0.00110.1 (5.4 to 14.9)< 0.0015.5 (0.7 to 10.5)0.027
Hct (per each 5%)−3 (−23.5 to 20.6)0.903 (−15.3 to 17.5)0.90−15 (−24.9 to 11.2)0.46

A single blood sample was obtained from each dog. That blood sample was then allocated into 4 aliquots. One aliquot was analyzed without further manipulation (unmanipulated sample). A portion of the buffy coat fraction was removed from 2 aliquots; the buffy coat fraction was removed more aggressively from 1 sample than the other so that 2 platelet-depleted blood samples with different platelet counts were acquired. The remaining aliquot was used to obtain plasma, which was used to adjust the Hct of the 2 platelet-depleted samples to within 2% of the Hct for the unmanipulated sample. All 3 blood samples (the unmanipulated sample and 2 platelet-depleted samples) underwent impedance aggregometry in duplicate, and the mean was calculated for each sample and used for analysis. One platelet-depleted blood sample from a male dog was unable to be processed within 60 minutes after collection; therefore, the data for that single sample were excluded from all analyses. For each of 2 other dogs, 1 platelet-depleted sample had platelet clumping detected during evaluation of the blood smear, and those samples were also excluded from the analyses. Both platelet-depleted samples from a male dog were excluded, one because of evidence of platelet activation with the addition of saline (0.9% NaCl) solution (control) and the other because the AUC results were outside the instrument's quality control limits for inbuilt imprecision. For a female dog, only a single platelet-depleted sample was prepared. Thus, a total of 66 samples underwent aggregometry.

CI = Confidence interval.

See Table 1 for remainder of key.

Discussion

The mean Hct, WBC count, platelet count, and platelet aggregometric variables (AUC, aggregation, and velocity) did not differ between the healthy adult spayed female and neutered male dogs of the present study. Interestingly, in human medicine, platelet aggregation (as determined by both light transmission and impedance aggregometry) in women is significantly greater than that in men regardless of the anticoagulation method used.11,18 The mechanism by which that occurs is unknown, although theories include differences in the Hct between men and women, which lead to different plasma anticoagulant concentrations in anticoagulated samples, as well as a possible effect of estrogen on platelet aggregation.11 In a study19 involving healthy Beagles, the RBC count for male dogs was significantly greater than that for female dogs, whereas the platelet count for female dogs was significantly greater than that for male dogs. The effect of reproductive status and cycle on RBC and platelet counts was not evaluated in that study.19 In mature women prior to menopause, platelet aggregation results vary significantly among the stages of the menstrual cycle.20 Because all dogs of the present study had been gonadectomized, it was not particularly surprising that the blood cell counts and platelet aggregation results did not differ significantly between males and females.

Results of the present study indicated that there was a significant positive association between WBC count and platelet aggregation. Results of in vitro studies11,12,21,22 involving human blood samples indicate that there is a significant positive association between WBC count and platelet aggregation when various agonists are used, whereas results of other studies22,23 indicate that the WBC count has an inhibitory effect on platelet aggregation. Theories for the positive association between WBC count and platelet aggregation include the reduction of ATP to ADP by leukocyte-derived ATPases and production of reactive oxygen species such as hydrogen peroxide and cathepsin G.12,24 Conversely, lymphocyte-produced cytokines such as interleukin-2 inhibit platelet aggregation.23 Neutrophils have variable effects on in vitro platelet aggregation that are dependent on experimental conditions.24,25 In a study7 in which aggregometric variables were compared between healthy dogs and dogs with septic peritonitis, neutrophil count was not significantly associated with platelet aggregation initiated by arachidonic acid, ADP, or collagen; however, the association between the total WBC count and platelet aggregation was not assessed. Despite the conflicting results regarding the association between WBC count and platelet aggregation in the literature, the balance of results appears to support a positive association between WBC count and platelet aggregation, as was observed in the present study. That association may become clinically relevant for dogs with severe leukopenia because results of this study indicated that the AUC changed by 13.2% for each change in the WBC count of 1 × 103 WBCs/μL; the effect of leukocytosis on platelet aggregation was not evaluated in this study.

The fact that neither platelet count nor plateletcrit was significantly associated with platelet aggregation was an unexpected finding. Although the present study was the first in which investigators evaluated the effect of platelet count on impedance aggregometric parameters in dogs, results of multiple in vivo4,10–12,26 and in vitro9,13,27 studies involving human blood samples indicate that platelet count has a significant positive effect on platelet aggregometric parameters. In some studies,12,27 that effect was observed when platelet counts were within reference limits. In another study,9 platelet aggregation was significantly inhibited at platelet counts < 150 × 103 platelets/μL, although substantial variation was observed among individual samples, and platelet aggregation was within reference limits for some samples with platelet counts as low as 50 × 103 platelets/μL. In yet another study,26 platelet aggregation was not significantly decreased until platelet counts were < 50 × 103 platelets/μL. Impedance aggregometers measure changes in electrical resistance between 2 electrodes suspended in whole blood, and resistance increases as platelets accumulate on the electrodes. The extent of platelet aggregation is thought to be partially dependent on platelet mass, because a markedly decreased platelet mass provides limited platelets for accumulation.10 In the present study, plateletcrit was analyzed separately from platelet count because it is believed to be a more accurate indicator of total platelet mass, which may be a more biologically important indicator of primary hemostasis than platelet count alone and can be used as a corrected measure for patients with altered mean platelet volumes.28 A marked decrease in platelet count and plateletcrit can affect platelet activation independent of the physical effect of platelet mass owing to an overall decrease in concentrations of predominantly platelet-derived agonists such as thromboxane and ADP.9 Decreases in the concentrations of those agonists attenuate the initiation and amplification phases of the platelet aggregation response. The reason for the lack of an independent effect of platelet count or plateletcrit on aggregometric parameters in the present study was unclear, but it is possible a significant effect would have been observed had more severely platelet-depleted blood samples than those created in this study been evaluated. Additionally, the present study was designed to detect a 20% difference in aggregometric parameters, and smaller differences may have been present but undetected. Although Hct was not significantly associated with the platelet aggregation response in the present study, it was within the reference range for all samples evaluated. In human medicine, clinically significant attenuation of the platelet aggregation response is observed as Hct increases.12,29 Postulated mechanisms for that phenomenon include erythrocyte metabolism of ADP as well as the effect of RBC mass on the resultant plasma concentration of anticoagulant in anticoagulated whole blood samples.12,18,30 The present study was not designed to evaluate the effect of an increasing or decreasing Hct on platelet aggregation; therefore, the lack of a significant association between Hct and platelet aggregation should be interpreted cautiously.

Other studies5,6,a have been conducted to evaluate the effect of anticoagulant or agonist selection on impedance aggregometric parameters for dogs. In a prospective study by Kalbantner et al,5 hirudin-and citrate-anticoagulated blood samples from 20 healthy dogs were evaluated with various agonists at various concentrations, and the coefficients of variance for within-run precision were calculated. Hirudin-anticoagulated blood samples yielded significantly higher measurement signals than did citrate-anticoagulated blood samples, and ADP was determined to be a reliable platelet agonist at a concentration of 10 μmol/L.5 Additionally, aggregometric results for citrate-anticoagulated blood samples had greater analytic variation than did hirudin- and heparin-anticoagulated blood samples. In another prospective study6 of 20 healthy and 3 sick dogs, the effects of various anticoagulants, platelet agonists, and times before sample processing were assessed. Contrary to the results of the Kalbantner et al study,5 aggregometric results for heparin- and citrate-anticoagulated blood samples were more reliable than those for hirudin-anticoagulated blood samples.6 Adenosine diphosphate yielded the strongest aggregation response, with 10 μmol/L being the optimal concentration evaluated.6 Results of a pilot studya conducted at our institution indicate that ADP is a reliable platelet agonist, and consistent aggregometric results can be obtained from citrate-anticoagulated blood samples despite mixed success with that anticoagulant in other studies.

An ADP concentration of 9.5μM was used to initiate platelet aggregation in the present study, which was greater than the ADP concentration (6.5μM) recommended by the aggregometer manufacturer for use in human blood samples. That concentration was purposely selected for this study in an attempt to amplify platelet response because of concerns about inadequate platelet aggregation in canine blood samples with markedly decreased platelet counts and was similar to the optimal ADP concentration (10μM) for use with canine blood samples identified in a previous study.5 Additional in vitro studies to investigate the effect of various ADP concentrations on aggregometric parameters for blood samples from dogs with naturally occurring thrombocytopenia and more severely platelet-depleted blood samples than those created in this study are warranted before recommendations for use of this technique can be made for clinical patients.

A major limitation of the present study was the inability to decrease the platelet count without also decreasing the WBC count in the platelet-depleted blood samples. To account for that inability, linear regression models were used to independently evaluate the effect of WBC count on each aggregometric parameter.

Another limitation was the inability to correct for an increase in time to processing for blood samples with progressively depleted platelet counts, plateletcrits, and WBC counts. The aggregometer manufacturer recommends that human blood samples be analyzed between 30 and 180 minutes after collection. Results of a pilot study6 indicate that aggregometric parameters for canine blood samples were unaffected when the samples were analyzed between 30 and 120 minutes after collection. In the present study, although only blood samples that were analyzed between 30 and 60 minutes after collection were retained for statistical analyses, unmanipulated samples (ie, samples with unaltered platelet and WBC counts) were analyzed sooner after collection than were the platelet-depleted samples. We believe it unlikely that the difference in the interval from collection to aggregometric analysis between the unmanipulated and platelet-depleted blood samples had a significant effect on our results.

Centrifugation and manipulation of blood samples can cause premature platelet activation and clumping, which can interfere with automated cell counts. It is also possible that centrifugation of the platelet-depleted blood samples might have resulted in the loss of subpopulations of large, hyperactive, or hypoactive platelets.30,31 We believe that premature platelet activation was minimal for the platelet-depleted blood samples of the present study because each sample was tested in duplicate, with 1 test run with the addition of saline solution rather than ADP to evaluate potential handling-induced endogenous platelet activation. Evidence of endogenous platelet activation was observed for only 1 sample, which was excluded from statistical analyses. Additionally, a blood smear for each sample was microscopically evaluated for the presence of platelet clumping, and samples with platelet clumps were excluded from further analysis.

The present study was the first in which investigators evaluated the effect of circulating blood counts on impedance aggregometric parameters in healthy dogs. Results indicated that platelet count and plateletcrit were not significantly associated with platelet aggregation, but those findings are inconsistent with results obtained for human blood samples, and additional investigation of aggregometric parameters for blood samples from dogs with naturally occurring thrombocytopenia and of more severely platelet-depleted blood samples than those created in this study is necessary to confirm our results. The effect of thrombocytosis on aggregometric parameters was not assessed in this study. Results also indicated that WBC count, or at least in vitro leukopenia, was positively associated with platelet aggregation when ADP was used to activate blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies. Currently, it is unclear whether leukocytosis affects platelet aggregation, and further studies are needed to examine this. Regardless, the effect of WBC count should be considered when assessing platelet function of dogs by use of impedance aggregometry.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Nash to the Auburn University Department of Biomedical Sciences as partial fulfillment of the requirements for a Master of Science degree.

Supported by the American Kennel Club Canine Health Foundation (EAS).

Presented in abstract form at the Auburn University College of Veterinary Medicine Annual Phi Zeta Research Emphasis Day, Auburn, Ala, November 2016.

The authors thank Brooklyn Samons and Donna Shiver for technical assistance and Dr. Rachel M. Burke for assistance with statistical analyses.

ABBREVIATIONS

AUC

Area under the aggregation curve

Footnotes

a.

Dettmer WR, Spangler EA. Establishing reference intervals in dogs using the Multiplate platelet analyzer (abstr), in Proceedings. 15th Annu Merial-NIH Vet Scholar Symp 2014;20.

b.

ADVIA 120 hematology system, Siemens Diagnostics, Deerfield, Ill.

c.

Cobas c311 analyzer, Roche Diagnostics GmbH, Mannheim, Germany.

d.

Sysmex CA-500 series, Siemens Healthcare, Tarrytown, NY.

e.

Dynac III, Becton Dickinson, Franklin Lakes, NJ.

f.

Multiplate analyzer, Roche Diagnostic International Ltd, Rotkreuz, Switzerland.

g.

Multiplate ADPtest, Roche Diagnostics Ltd, Basel, Switzerland.

h.

R, version 3.2.2, R Foundation for Statistical Computing, Vienna, Austria. Available at: www.R-project.org/. Accessed Jul 31, 2016.

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Contributor Notes

Dr. Nash's present address is VETMED, 20610 N Cave Creek Rd, Phoenix, AZ 85024.

Address correspondence to Dr. Nash (kjnash1@outlook.com).