584994 research-article2015
VDIXXX10.1177/1040638715584994Whole blood platelet function testing in healthy catsHo et al.
Full Scientific Report
Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests
Journal of Veterinary Diagnostic Investigation 2015, Vol. 27(3) 352–360 © 2015 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638715584994 jvdi.sagepub.com
Kimberly K. Ho, Anthony C. G. Abrams-Ogg,1 R. Darren Wood, M. Lynne O’Sullivan, Gordon M. Kirby, Shauna L. Blois
Abstract. The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11–176), 81 (32–129), and 91 (59–129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL–ADP cartridges, was 69 (46–89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18–92) and 49 (9–96), respectively, using impedance hematology analyzer platelet counts, and 94 (25–98) and 68 (14–119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL–ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL–ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable. Key words: Cats; hemostasis; Multiplate; Plateletworks; platelet function; platelet counts.
Introduction Normal hemostasis requires a highly complex series of coordinated interactions between endothelial cells, platelets, and hemostatic proteins. Tests of hemostasis are commonly used in the diagnosis of hemostatic disorders and are also important in the monitoring of anticoagulant therapy. With respect to primary hemostasis, optical aggregometry using platelet-rich plasma (PRP) has historically been used as the standard for assessment of platelet function.11,21 Optical aggregometry evaluates platelet function based on the ability of platelets to aggregate on addition of platelet agonists, such as adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA). When an agonist is added to PRP, light transmission patterns are altered, which reflect platelet activity. Unfortunately, the preparation of PRP is operator intensive and is too cumbersome for routine clinical application.11,14,21 It is also operator dependent, and may activate platelets and possibly alter their function.11,14,21 Although template bleeding time is a readily available test of primary hemostasis, it does not reflect the function of platelets exclusively, is also subject to operator variation, and may be displeasing to owners.1,21
More convenient point-of-care methods for assessing platelet function in whole blood have been developed for humans, specifically analyzer systems that have been coded as MP,a PF,b and PW.c These tests assess platelet aggregation in response to agonists using different methods, and may be used to assess congenital and acquired platelet function defects, to monitor antiplatelet therapy for cardiac conditions, and to assess for risk of hemorrhage and the need for blood products prior to cardiac surgery.11,28 Platelet function has been variably evaluated with these tests in dogs, pigs, sheep, and horses.3,20,29 Studies have also used these tests to evaluate platelet function in cats; however, the literature is limited.17,22,26
From the Departments of Clinical Studies (Ho, Abrams-Ogg, O’Sullivan, Blois), Pathobiology (Wood), and Biomedical Sciences (Kirby), Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada. 1
Corresponding Author: Anthony C. G. Abrams-Ogg, Department of Clinical Studies, Ontario Veterinary College, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada N1G 2W1. [email protected]
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Whole blood platelet function testing in healthy cats Whole blood aggregometry using electrical impedance has gradually become preferred over PRP-based optical aggregometry as the standard test in some laboratories.9,20,33 Similar to optical aggregometry, impedance aggregometry measures platelet function by determining aggregation after addition of an agonist. However, instead of aggregating freely in liquid phase, the platelets aggregate on 2 electrodes within a test cell, subsequently decreasing the electrical conduction between them.11,21 Impedance whole blood aggregometry requires a smaller sample volume and less manipulation of platelets, thereby decreasing the risk of platelet activation prior to analysis.11,20,33 Furthermore, it may be more representative of platelet aggregation in vivo as cellular components are present. The MP analyzer incorporates the principles of impedance whole blood aggregometry in an automated user-friendly device. This device reports the change in electrical impedance as platelets aggregate in proprietary aggregation units over time, and calculates the area underneath the platelet aggregation curve (AUC). Use of this device in animals has been evaluated in dogs, sheep, and in cats.3,12,26 In the feline study, blood from 10 healthy cats was combined with eptifibatide or abciximab in vitro, and reduced platelet function was demonstrated for the former drug but not the latter.26 The PF analyzer is a point-of-care platelet function test that has been evaluated more extensively in animals.21 It is designed to be an in vitro substitute for template bleeding time. Platelet function is measured in terms of closure time (CT). The CT represents the duration of time, in sec, which is required for a platelet plug to form and prevent flow of blood through an aperture under high shear stress conditions in the presence of agonist combinations of either COL and ADP or COL and epinephrine. This analyzer was used in a previous feline study, where platelet function between 42 healthy cats and 30 cats affected with hypertrophic cardiomyopathy was compared.21 There was no difference in platelet function between the groups, but the study did not compare the results of the assay with optical or impedance aggregometry. The PW assay is approved for use in human medicine to monitor the use of the anti-platelet drugs aspirin and clopidogrel.28,37 This assay is potentially more readily available than other platelet function tests as it makes use of standard hematology analyzers. By measuring and comparing platelet counts between an ethylenediamine tetra-acetic acid (EDTA)–anticoagulated blood sample and a blood sample mixed with a platelet agonist, which decreases the number of individual platelets available for counting, the percentage of aggregated platelets can be calculated.28,37 Use of the PW assay has been described in a previous feline study, which demonstrated that platelet function was impaired in healthy cats that had been given 18.75 mg/cat of clopidogrel.18 However, the PW assay has only been validated for use on impedance-based hematology analyzers, and its utility on analyzers that use flow cytometry is unknown. As hematology analyzers
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based on flow cytometry are becoming more commonplace in research, reference, and point-of-care laboratories, compatibility with PW could make platelet function testing even more accessible. Cats with hypertrophic cardiomyopathy are at risk for thromboembolism and are therefore prescribed anti-platelet drugs.16,34 Evaluating anti-platelet drug effect in these animals in the past has been problematic because of the aforementioned problems with platelet function testing. Less cumbersome platelet function testing is now potentially available for this purpose; however, evaluation in cats is limited. The objectives of our study were therefore to establish institutional feline references intervals (RIs) using 3 tests that assess platelet function in whole blood by different principles: MP by electrical impedance, PF by mechanical aperture closure, and PW by platelet counting. A secondary objective was to compare results of PW when platelet counting is performed using an impedance-based analyzerd and flow cytometry.e
Material and methods Cat selection Healthy cats belonging to staff and students at the Ontario Veterinary College, University of Guelph (Canada), were recruited with informed consent. Cats were considered healthy based on history, physical examination, complete blood cell count,e serum biochemical profile,f and tests for Feline leukemia virus and Feline immunodeficiency virus.g Activated clotting timeh,i was performed if sufficient whole blood was available at the time of collection. A coagulation profile, including prothrombin time,j,k activated partial thromboplastin time,j,l fibrinogen concentration,j,m and j,n d-dimer concentration, was performed if residual citrated plasma was available after platelet function testing. Cats were excluded from the study if drugs other than monthly anti-parasitic treatments were used or if they were not amenable to gentle restraint. Animal use and procedures were approved by the University of Guelph Animal Care Committee in accordance with the standards set by the Canadian Council on Animal Care (http://www.ccac.ca/en_/standards/ guidelines) and the Ontario Animals for Research Act (1990; http://www.omafra.gov.on.ca/english/food/inspection/ahw/ ara-index.htm).
Blood collection In a quiet and controlled environment, fasted cats were sedated with ketamineo (2 mg/kg) and butorphanolp (0.2 mg/ kg) intravenously, via a saphenous vein, for atraumatic jugular venipuncture performed using a 21-gauge butterfly catheter.q All venipunctures were performed by a single investigator (KK Ho). Blood was collected directly into vacuum blood collection tubes in the following order: one 4.0-mL
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serum tuber (1 mL collected), one 4.5-mL 3.2% sodium citrate (0.105 M) tubes ensuring a 9:1 blood-to-citrate ratio, and one 2.0-mL K2EDTA tubet; followed by indirect blood collection of 2–3 mL through the butterfly catheter using a 6-mL plastic syringeu for transfer of 1 mL of blood into each of 2 PW tubes (which do not have a vacuum), containing either ADPc or COL,c to ensure a consistent blood-to-agonist ratio. All blood collection tubes and syringes were handled by a single investigator (ACG Abrams-Ogg). Blood collection tubes were gently inverted during and after blood collection to mix the blood and anticoagulant. For logistical reasons and to standardize time of platelet function analysis for the various tests after blood collection, equilibration at room temperature was limited to 10–15 min on a blood tube rockerv prior to analysis of the first replicate. Patient compliance during venipuncture, venipuncture score, and flow of blood during blood collection were documented.4 Blood smears were made from EDTAanticoagulated blood samples and evaluated microscopically for platelet clumps. All blood collections and tests were performed during two 3-week periods, 1 month apart. All samples were collected between 9:00 and 15:00.
The MP analyzer Analysis with the MP analyzer was performed on citrated blood samples using ADP,w COL,x and AAy as agonists. Each agonist was prepared as instructed by the manufacturer by adding 1 mL of distilled water to lyophilized stock. Reconstituted agonists were stored in 200-µL aliquots at –20°C for a maximum of 28 days (ADP and AA) or 4°C for a maximum of 7 days (COL) and allowed to warm to room temperature just prior to analysis. The MP analysis was carried out as instructed by the manufacturer. Using the automated pipette system on the instrument, 300 μL of isotonic NaCl, prewarmed to 37°C, was mixed with 300 μL of citrated whole blood in the test cellz and allowed to incubate for 3 min at 37°C prior to the addition of 20 μL of reconstituted AA agonist. For ADP and COL analyses, 300 μL of isotonic NaCl with 3 mM CaCl2aa was used in place of isotonic NaCl alone for sample re-calcification. The final agonist concentrations used in each analysis were 6.5 µM ADP, 3.2 µg/mL COL, and 0.5 mM AA. Each test cell performed 2 measurements for a total analysis time of 6 min, and mean AUC were reported and used for analysis.
The PF analyzer Analysis with the PF analyzer was performed using 800 µL of each citrated blood sample with cartridges containing COL–ADP-coated membranes for platelet activation (PFCOL/ADP).bb Cartridges were allowed to warm to room temperature prior to use. Each sample was analyzed in duplicate or in triplicate if a sample error (flow obstruction) was encountered in 1 or both of the first 2 replicates. The mean CT values, in sec, for each cat were used for analysis.
The PW assay Baseline platelet counts from the EDTA-anticoagulated samples and platelet counts from the PW assay agonist tubesc containing either ADP or COL (PW-ADP and PW-COL, respectively) were analyzed using 2 automated hematology analyzers that use different methods for platelet enumeration. Platelet counts were obtained in either duplicate or triplicate by an impedance analyzer,d and a single platelet count was obtained by a flow cytometry analyzer.e The percent platelet aggregation was calculated using the following formula: % aggregation =
EDTA platelet count − agonist-induced platelet count × 100 EDTA platelet count
Mean percent aggregation (% Agg) values calculated using impedance-based platelet counts, and single % Agg values calculated using flow cytometric–based platelet counts, were used for analysis.
Reference interval generation and statistical analysis Data were analyzed using commercial softwarecc unless otherwise indicated. The distribution of data for each platelet function test was assessed using Anderson–Darling and Shapiro–Wilk tests of normality. Additional software programsdd,17 were used to calculate the lower and upper reference limits (2.5th and 97.5th percentile of each data set, respectively) and their associated 90% confidence intervals (CIs) if possible. Robust or parametric methods of RI generation were used for data, which were normally distributed or log-transformed, based on a sample size (N), 40 ≤ N ≤ 120. When data were not normally distributed or transformed, nonparametric methods of RI generation were used without 90% CI. Outliers in each data set were identified as values less than the lower quartile (Q1) minus 1.5 × IQR or greater than the upper quartile (Q3) minus 1.5 × IQR, where IQR is the interquartile range. Outliers were removed and RIs recalculated as above. To assess for analytic variation of PF-COL/ADP, PWADP, and PW-COL, intraindividual and interindividual coefficients of variation (CVa and CVg, respectively) and intraclass correlation coefficients (ICC) were calculated. Spearman rank correlation coefficients were calculated to assess for associations between MP (ADP, COL, AA), PFCOL/ADP, and PW (ADP, COL) results. Spearman rank correlation coefficients were also calculated to assess for associations between baseline platelet counts, as performed on the impedance or flow cytometry analyzers, and results of the 3 platelet function tests. Pearson correlation coefficients were calculated to assess for a relationship between PW performed on the 2 hematology analyzers. Linear regression models were used to assess for correlations between results of platelet counts, with or without agonists,
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Whole blood platelet function testing in healthy cats
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Table 1. Means, medians, ranges, 95% confidence interval (CI) and associated 90% CI for each upper and lower reference limit (excluding outliers), for whole blood platelet function testing in 55 cats using 3 analyzer systems.*
Test/Unit MP AUC (U)
PF Sec PW % Agg (impedance) % Agg (flow cytometry)
Agonist
Median/Mean‡
Range
Lower reference value
ADP† COL† AA†
87/93 (53) 81/80 (49) 91/94 (50)
19–174 30–140 49–130
11 32 59
−3 to 26 23–41 52–65
176 129 129
160–191 119–138 122–135
ADP–COL†
69/68 (46)
44–94
46
42–51
89
85–94
ADP COL† ADP COL†
71/64 (54) 49/53 (55) 94/80 (41) 68/67 (43)
17–94 7–95 25–98 7–98
18 9 25 14
NA 1–16 NA 3–26
92 96 98 119
NA 88–104 NA 114–142
90% CI for lower reference value
Upper reference value
90% CI for upper reference value
* AA = arachidonic acid; ADP = adenosine diphosphate; AUC (U) = area under curve screenshot Units [= AU × min/10 and rounded down]; % Agg = percent aggregation; COL = collagen; MP = Multiplate Analyzer (Roche Diagnostics International Ltd., Rotkreuz, Switzerland); PF = Platelet Function Analyzer-100 (Siemens Canada, Mississauga, Ontario, Canada); PW = Plateletworks (Helena Laboratories, Beaumont, TX); NA = not applicable. † Data normally distributed. ‡ Numbers in parentheses are the number of cats. For MP and PW using impedance-based platelet counts, missing values are due to removal of outliers. For PW, using flow cytometry–based platelet counts, measurements were performed in 43 cats, and missing values are due to removal of outliers. For PF, single values were obtained in 21 cats; duplicate values were obtained in 25 cats; for 7 cats that had PF-COL/ADP analyzed in triplicate, in 6 of those cats a reading was not obtained; the total number of flow obstructions was 41 out of 112 determinations (37%).
and % Agg calculated from values acquired by the impedance and flow cytometry analyzers. The level of significance was set at P < 0.05 for all tests.
Results Sixty cats were enrolled; however, only 55 cats met inclusion criteria. Four cats were excluded due to behavior and 1 was excluded due to azotemia. The 55 cats included 28 female (27 spayed, 1 intact) and 27 males (all neutered) with an age range of 1–15 years, mean of 5.51 years, and median of 4 years. Forty-eight of 55 cats had coagulation profiles performed; all were normal except for 1 cat with FXII deficiency (Hageman trait). Cats without coagulation profiles had normal activated clotting times.4 Blood smear evaluations revealed minimal-to-no platelet clumping in all but 4 samples. Subjective scoring of venipuncture technique and flow of blood during blood collection was similar among all cats. The RIs for the 3 platelet function tests, after removal of outlier data, are summarized in Table 1. The 90% CI for each upper and lower reference limit are included if the RI was calculated using parametric or Robust methods. For MP, CVg ranged from 0.30 to 0.46 using the various agonists. Values for CVa and ICC could not be calculated, as the instrument reports only mean values. For PF-COL/ADP, CVa, CVg, and ICC values were 0.16, 0.11, and 0.66, respectively. For PW-ADP and PW-COL, CVa, CVg, and ICC were 0.31, 0.11, and 0.88, and 0.38, 0.11, and 0.93, respectively, when platelet counts were acquired by impedance. Values for CVa and ICC for PW could not be calculated when platelet
counts were acquired by flow cytometry as only 1 value was obtained. There were low but significant correlations between PFCOL/ADP and MP-COL (ρ = 0.11, P = 0.017), and between PF-COL/ADP and PW-COL using impedance platelet counts (ρ = 0.14, P = 0.009). There were no other significant correlations between tests. Platelet counts and % Agg determined using impedance and flow cytometry for PW are compared in Table 2. Baseline platelet counts measured by both methods were significantly highly correlated, as were PW-ADP and PW-COL platelet counts, but values obtained by impedance were consistently higher than those obtained by flow cytometry. Calculated % Agg from each of these platelet counting methods were significantly moderately correlated, and overall impedance-based % Agg was significantly lower than flow cytometry–based % Agg. There were low but significant correlations between baseline platelet counts obtained by impedance and MP-COL (ρ = 0.19, P = 0.002) and MP-AA (ρ = 0.13, P = 0.009). There were also low but significant correlations between baseline platelet counts obtained by flow cytometry and MPCOL (ρ = 0.02, P = 0.002) and MP-AA (ρ = 0.17, P = 0.004). There were no other significant correlations between baseline platelet counts and tests.
Discussion In our study, 3 whole blood platelet function tests were investigated for use in cats. The study confirmed that methods
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Table 2. Comparison of PW results using impedance-based and flow cytometry–based hematology analyzers: mean (range) platelet counts; bias between platelet counts (mean [95% confidence interval]); and correlation of platelet counts and % aggregation.* Platelet count (×109/liter) Baseline (EDTA tube) No. of cats† Impedance Flow cytometry Bias in platelet counts (×109/liter)‡ Correlation of platelet counts (ρ)‡ Bias in % aggregation‡ Correlation of % aggregation (ρ)‡
51 361 (56–726) 269 (54–616) 93 (77–109) 0.77 NA NA
ADP agonist tube 39 132 (18–439) 63 (6–263) 68 (52–84) 0.70 −11 (–17 to –5) 0.64
COL agonist tube 41 163 (16–379) 76 (5–291) 82 (70–94) 0.76 −12 (–17 to 7) 0.64
* ADP = adenosine diphosphate; CI = confidence interval; COL = collagen, EDTA = ethylenediamine tetra-acetic acid; NA = not applicable. † Platelet counts in EDTA tubes were obtained with both impedance and flow cytometry analyzers in 51 cats. Platelet counts in ADP and COL agonist tubes were obtained with both impedance and flow cytometry analyzers in 43 cats. Missing values are due to removal of outliers. ‡ All biases and correlations significantly different, P < 0.001.
other than optical aggregometry using PRP are feasible for assessment of feline platelet function. The first feline study of the MP analyzer was reported in 2014.26 In that study, hirudin was used as the anticoagulant, and ADP and thrombin receptor activator peptide were used as agonists, in concentrations recommended by the manufacturer. In the current study, MP analysis was performed using citrated whole blood with agonists ADP, COL, and AA in concentrations recommended by the manufacturer. The median (87) and range (155) of AUC values with ADP in citrated blood were lower than the previously reported median (306) and range (534) for the same concentration of ADP agonist (6.5 μM) in hirudinized blood.26 The lower aggregation responses using citrated blood, in comparison to hirudinized blood, is consistent with findings in sheep, dogs, and humans.3,13,23,27,32 Citrate was used in our study to facilitate blood collection for, and comparison between, the 3 platelet function tests. There were no differences in the MP analyzer aggregation responses with the different agonists (Table 1). This finding is different from humans, where responses to ADP were lower than responses to COL and AA, and for dogs, where responses to AA appear to have been lower than responses to ADP and COL.27,33 Furthermore, aggregation responses in our study appear to be lower than responses in dogs and sheep, and only marginally greater than responses in humans, using the same or comparable agonist concentrations.3,27,32 Cats are considered to have highly aggregable platelets,35,40 so it was surprising that platelet aggregation responses were not higher compared to other species. Studies in dogs and sheep have demonstrated that aggregation responses can be enhanced by changing agonist concentrations, and similar studies in cats may identify optimal agonist concentrations.3,13,25 Sample collection methods were similar in the previous26 and current study of the MP analyzer in cats, but, in the previous study, samples were allowed to sit for 30 min prior to
analysis,26 while in our study samples were gently rocked for 15 min because of sample handling methods for the PF analyzer and PW assay. Optimum sample handling for cats for the MP analyzer is not known, but it is possible that rocking the samples affected the results. Based on the findings of our study, further examination of preanalytic and analytic variables of the MP analyzer for cats should be pursued. The use of the PF analyzer with COL–ADP was previously reported in cats.22 The mean CT was 64 sec for healthy cats in the previous report,22 while the median CT was 69 sec in our study. The CT range for healthy cats in the previous report was 43–176 sec (and 10th and 90th percentiles ~50 and 120 sec, respectively), which is wider than the range (44–106 sec) and 2.5th and 97.5th percentiles (45 and 104 sec, respectively) reported herein.22 This appears to be largely due to 4 values in the previous study with values >120 sec.22 These differences may be attributed to natural variation not captured in our study, but may also be due to preanalytical variables in sample collection, as this has been reported in humans.25 Sample handling after collection was not reported in the previous study, but in our study, samples were gently rocked for 10–15 min prior to analysis. This was done in part because, during pilot studies, flow obstructions occurred more frequently with samples that were left undisturbed for 30 min then gently remixed prior to analysis than with samples that were continually rocked for 10–15 min (data not shown). Assay failure due to flow obstruction was encountered with the PF analyzer. Although the number of flow obstructions was not reported in the previous study,22 it appears to have been less than in our study. The manufacturer indicates that flow obstruction errors are most likely attributable to blood clots in the sample.b Of the 55 samples included in the study, 6 were analyzed in triplicate without producing a result. On EDTA-anticoagulated blood smear evaluation of these 6 cats, only 1 showed moderate-to-marked platelet clumping. Thus, flow obstruction errors may either not be all
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Whole blood platelet function testing in healthy cats attributable to microscopic blood clots within the sample, or evaluation of the EDTA-anticoagulated blood smears may not have been a good surrogate for evaluation of microscopic clot formation in the citrated blood samples. It is also possible that rocking of samples prior to analysis contributed to platelet activation and flow obstruction. Based on similar CT of the 2 studies, the PF analyzer may be a platelet function test that can be performed with relative consistency in healthy cats. However, biological variation was not assessed in the current study, and the wide RI of the PF analyzer with the COL–ADP cartridges may limit its clinical utility in assessing whether a cat has altered platelet function based on a single assessment. The use of the PW assay using ADP was previously reported for healthy cats.18 The median % Agg in our study was 71%, while the mean % Agg was 70.55% in the previous feline study when impedance (using a different analyzer) was used as the method for platelet counting.18 The maximum allowable time until sample analysis was reported to be 30 min.18 Normal human RIs of 80–100% and 70–100% for ADP and COL, respectively, are provided by the manufacturer. As with the findings for the MP analyzer, the discrepancies between the lower limits of RIs for humans and those developed in our study for cats are surprising given the aggregability of feline platelets.35,40 This may represent an underestimation of the baseline platelet count in some feline samples, which would result in underestimation of percent aggregation. Following the manufacturer’s instructions and the previous study in cats,18 baseline platelet counts in our study were performed using EDTA-anticoagulated blood. Automated platelet counting in feline EDTA-anticoagulated blood is inherently less accurate than platelet counting in human EDTA-anticoagulated blood and is attributed to platelet aggregation.36,40 The “two-syringe collection method” and prompt analysis used in the current study should have minimized platelet aggregation, and platelet clumping was only readily apparent on blood smear evaluation in 4 cases, but this does not preclude that some degree of aggregation occurred.31,38 The PW assay ADP and COL tubes use citrate as anticoagulant, and it is also possible that feline platelet aggregates in citrate-anticoagulated blood subsequently disaggregate more rapidly than human platelet aggregates. Less stable aggregates would increase platelet counts in the PW assay ADP and COL tubes and thereby underestimate % Agg. Any effect of time, however, should have been minimal as the first replicates were assayed within 10–15 min and the duplicates assayed within 15–20 min (sample analysis time was 4 min).18,37 The upper limit of COL-induced aggregation in our report was >100%, which likely reflects inherent error in platelet enumeration. The use of the PW assay has only been validated for impedance-based analyzers. Platelet counts vary with the method, therefore it is not surprising that baseline platelet counts and ADP- and COL-induced % Agg determined by impedance and flow cytometry were different.2,7 Specifically, baseline
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and post-agonist platelet counts using flow cytometry were consistently lower than counts acquired by impedance. The mean (range) baseline platelet counts by flow cytometry in our study were similar to previous studies on a similar analyzer, which reported a mean of 281 × 109/liter, with 2.5th and 97.5th percentile of 42 and 630 × 109/liter, respectively, and a mean (range) of 289 (16–858) × 109/liter, using EDTAanticoagulated blood.5,30 The RI provided by the manufacturer of the impedance analyzer is 300–800 × 109/liter. In previous studies comparing impedance and flow cytometry– based platelet counts in cats, both comparatively higher and lower impedance-based counts have been reported.2,5,24 Correlations for baseline and postagonist platelet counts between the 2 analyzers ranged from 0.70 to 0.77 (Table 2), likely reflecting some imprecision of platelet counts by both analyzers. A limitation of our study is that only 1 value with the flow cytometry–based analyzer was obtained. This occurred because, logistically, replicates could not be analyzed in 120 reference individuals, as using smaller sample sizes may result in less accurate values.15 While the number of animals used in our study was a conventional number used for generating RIs for veterinary studies, with the knowledge that nonparametric methods are likely to be needed in the analysis of platelet function tests, future studies should ideally incorporate larger sample sizes (>120 cats) in order to calculate more accurate RIs. Ideal RIs help identify abnormal values that have clinical implications. Given that RIs developed in our study are not synonymous with the upper and lower thresholds of platelet function required for normal hemostasis, the tendency toward clinical bleeding or thrombosis cannot be inferred from where an individual animal’s values lie relative to the RIs. A study to examine this issue would require a larger sample population with a range of variable platelet function including animals with evidence of clinical bleeding or thrombosis. Cats in our study were also considered to be healthy; however, echocardiograms were not performed, which may have resulted in inadvertent recruitment of cats with subclinical heart disease, potentially altering RIs toward states of increased platelet activity.6,22,34 As such, the platelet function tests for which RIs were developed in our study may be better suited for monitoring the effects of antithrombotic therapy, as they are in humans, and may be more beneficial when using an animal’s own pretreatment values for comparison.10,28 However, for this purpose, biologic variation must also be accounted for in the interpretation of results, and the effects of biological variation on these tests have not yet been investigated. Platelet function in cats can be assessed with the MP, PF, and PW analyzer systems. However, prior to the routine
clinical use of these tests, further evaluation of preanalytical variables (e.g., sample collection and handling) and analytic variables (e.g., anticoagulant and agonist concentrations) are necessary. Acknowledgements We thank Dr. William Sears, Department of Population Medicine, and Gabrielle Monteith, Department of Clinical Studies, for assistance with study design and statistical analysis; the veterinary and laboratory technicians, Health Sciences Centre, Department of Clinical Studies; and the Clinical Pathology Laboratory, Animal Health Laboratory, for assistance with blood collection and platelet function testing.
Sources and manufacturers a. Multiplate analyzer, Roche Diagnostics International Ltd., Rotkreuz, Switzerland. b. Platelet Function Analyzer-100, Siemens Canada, Mississauga, Ontario, Canada. c. Plateletworks Combo-25 kit (catalog no. 5852), Helena Laboratories, Beaumont, TX. d. Vetscan HM5, Abaxis Veterinary Diagnostics, Union City, CA. e. ADVIA 2120, Siemens Canada, Mississauga, Ontario, Canada. f. Cobas C 501, Roche Diagnostics Canada, Laval, Quebec, Canada. g. SNAP FIV/FeLV Combo Test, IDEXX Laboratories Canada Corp., Toronto, Ontario, Canada. h. Actalyke MINI II, Helena Laboratories, Beaumont, TX. i. MAX-ACT (catalog no. 003054), Helena Laboratories, Beaumont, TX. j. Stago STA Compact CT coagulation analyzer, Diamond Diagnostics, Holliston, MA. k. STA Neoplastine CI Plus 5 (catalog no. 00606), Diagnostica Stago SAS, Asnières sur Seine, France. l. STA C.K. Prest 5 (catalog no. 00597), Diagnostica Stago SAS, Asnières sur Seine, France. m. STA Fibrinogen 5 (catalog no. 00674), Diagnostica Stago SAS, Asnières sur Seine, France. n. STA Liatest D-Di (catalog no. 00515), Diagnostica Stago SAS, Asnières sur Seine, France. o. Vetalar, Bioniche Animal Health Canada, Belleville, Ontario, Canada. p. Torbugesic, Zoetis Canada, Kirkland, Quebec, Canada. q. BD Vacutainer Safety-Lok blood collection set, BD Canada, Mississauga, Ontario, Canada. r. BD Vacutainer serum tube (catalog no. 367812), BD Canada, Mississauga, Ontario, Canada. s. BD Vacutainer sodium citrate tube (catalog no. 366415), BD Canada, Mississauga, Ontario, Canada. t. BD Vacutainer K2EDTA tube (catalog no. 367841), BD Canada, Mississauga, Ontario, Canada. u. Monoject 6-mL syringe with regular Luer tip (catalog no. 8881516911), Tyco Healthcare Group LP, Mansfield, MA. v. Coulter blood mixer, Coulter Electronics Inc., Hialeah, FL. w. Multiplate ADP test kit (catalog no. MP0220), Verum Diagnostica GmbH, Munich, Germany. x. Multiplate COL test kit (catalog no. MP0230), Verum Diagnostica GmbH, Munich, Germany.
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Whole blood platelet function testing in healthy cats y. Multiplate ASPI test kit (catalog no. MP0210), Verum Diagnostica GmbH, Munich, Germany. z. Multiplate test cells (catalog no. MP0027), Verum Diagnostica GmbH, Munich, Germany. aa. Multiplate NaCl/CaCl2 (catalog no. MP0530), Verum Diagnostica GmbH, Munich, Germany. bb. Dade PFA Collagen/ADP test cartridge (catalog no. B417021A), Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany. cc. SAS v. 9.1, SAS Institute Inc., Cary, NC. dd. Analyze it for Excel v. 2.24, Analyse-It Software Ltd, Leeds, United Kingdom.
Declaration of Conflicting Interests The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Two Plateletworks Combo-25 kits were generously donated by Ryan Medical, Burlington, Ontario, Canada.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this work was provided by the Ontario Veterinary College Pet Trust.
References 1. Alatzas DG, et al. Reference values and repeatability of buccal mucosal bleeding time in healthy sedated cats. J Feline Med Surg 2014;16:144–148. 2. Bauer NB, et al. Evaluation of the impedance analyzer PocH100iV Diff for analysis of canine and feline blood. Vet Clin Pathol 2012;41:194–206. 3. Baumgarten A, et al. Measurement of platelet aggregation in ovine blood using a new impedance aggregometer. Vet Clin Pathol 2010;39:149–156. 4. Bay JD, et al. Reference values for activated coagulation time in cats. Am J Vet Res 2000;61:750–753. 5. Becker M, et al. Comparative clinical study of canine and feline total blood cell count results with seven in-clinic and two commercial laboratory hematology analyzers. Vet Clin Pathol 2008;37:373–384. 6. Bédard C, et al. Evaluation of coagulation markers in the plasma of healthy cats and cats with asymptomatic hypertrophic cardiomyopathy. Vet Clin Pathol 2007;36:167–172. 7. Bowles KM, et al. Discrepancy between impedance and immunofluorescence platelet counting has implications for clinical decision making in patients with idiopathic thrombocytopenia purpura. Br J Haematol 2006;134:320–322. 8. Callan MB, Giger U. Assessment of a point-of-care instrument for identification of primary hemostatic disorders in dogs. Am J Vet Res 2001;62:652–658. 9. Cathcart CJ, et al. Lack of inhibitory effect of acetylsalicylic acid and meloxicam on whole blood platelet aggregation in cats. J Vet Emerg Crit Care (San Antonio) 2012;22:99–106. 10. Chen F, et al. A randomized clinical trial comparing point-ofcare platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel. Platelets 2012;23:249–258.
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11. Choi JL, et al. Platelet function tests: a review of progresses in clinical application. Biomed Res Int 2014;456569. 12. Dalby MC, et al. Diurnal variation in platelet aggregation with the PFA-100 platelet function analyser. Platelets 2000;11: 320–324. 13. Defontis M, et al. Optimization of Multiplate® whole blood platelet aggregometry in the Beagle dog and Wistar rat for ex vivo drug toxicity testing. Exp Toxicol Pathol 2013;65: 637–644. 14. Dyszkiewicz-Korpanty AM, et al. Approach to the assess ment of platelet function: comparison between optical-based platelet-rich plasma and impedance-based whole blood platelet aggregation methods. Clin Appl Thromb Hemost 2005;11: 25–35. 15. Friedrichs KR, et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol 2012;41:441–453. 16. Fuentes VL. Arterial thromboembolism: risks, realities, and a rational first-line approach. J Feline Med Surg 2012;14:459– 470. 17. Geffré A, et al. Reference Value Advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel. Vet Clin Pathol 2011;40:107–112. 18. Hamel-Jolette A, et al. Plateletworks: a screening assay for clopidogrel therapy monitoring in healthy cats. Can J Vet Res 2009;73:73–76. 19. Hanke AA, et al. Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate). Eur J Med Res 2010;15:214–219. 20. Hogan DF, et al. Antiplatelet effects and pharmacodynamics of clopidogrel in cats. J Am Vet Med Assoc 2004;225:1406– 1411. 21. Jandrey KE. Assessment of platelet function. J Vet Emerg Crit Care (San Antonio) 2012;22:81–98. 22. Jandrey KE, et al. Platelet function in clinically healthy cats and cats with hypertrophic cardiomyopathy: analysis using Platelet Function Analyzer-100. Vet Clin Pathol 2008;37:385–388. 23. Kalbantner K, et al. Measurement of platelet function in dogs using novel impedance aggregometer. Vet J 2010;185: 144–151. 24. Lara-Garcia A, et al. Evaluation of a point-of-care hematology analyzer for use in dogs and cats receiving chemotherapeutic treatment. J Am Vet Med Assoc 2008;232:1488–1495. 25. Lippi G, et al. Sample collection and platelet function testing: influence of vacuum or aspiration principle on PFA-100 test results. Blood Coagul Fibrinolysis 2013;24:666–669. 26. Magee AN, et al. In vitro effects of the glycoprotein IIb/IIIa receptor antagonists abciximab and eptifibatide on platelet aggregation in healthy cats. Am J Vet Res 2014;75:309–312. 27. Marschner CB, et al. Evaluation of platelet aggregometry in dogs using the Multiplate platelet analyzer: impact of anticoagulant choice and assay duration. J Vet Emerg Crit Care (San Antonio) 2012;22:107–115. 28. Michelson AD, et al. Current options in platelet function testing. Am J Cardiol 2006;98:4N–10N. 29. Mischke R, Keidel A. Influence of platelet count, acetylsalicylic acid, von Willebrand’s disease, coagulopathies, and haematocrit on results obtained using a platelet function analyser in dogs. Vet J 2003;165:43–52.
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30. Moritz A, et al. Canine and feline hematology reference values for the ADVIA 120 hematology system. Vet Clin Pathol 2004;33:32–38. 31. Moritz A, Hoffmann C. Thrombozytenzahlung bei der Katze [Platelet count in the cat]. Tierarztl Prax Ausg K Kleintiere Heimtiere 1997;25:695–700. German. 32. Peerschke EI, et al. Reference range determination for wholeblood platelet aggregation using the Multiplate analyzer. Am J Clin Pathol 2014;142:647–656. 33. Seyfert UT, et al. Variables influencing Multiplate™ whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals. Platelets 2007;18:199–206. 34. Smith SA, et al. Arterial thromboembolism in cats: acute crisis in 127 cases (1992–2001) and long-term management with low-dose aspirin in 24 cases. J Vet Intern Med 2003;17:73–83. 35. Tvedten H, Korcal D. Vortex mixing of feline blood to disaggregate platelet clumps. Vet Clin Pathol 2001;30:104–106.
36. Tvedten HW, et al. Enumeration of feline platelets in ethylenediamine tetra-acetic acid anticoagulated blood with ADVIA 2120 system and two manual methods: Leucoplate and Thrombo-TIC. J Vet Diagn Invest 2013;25:493–497. 37. van Werkum JW, et al. A comparison between Plateletworksassay and light transmittance aggregometry for monitoring the inhibitory effects of clopidogrel. Int J Cardiol 2010;140: 123–126. 38. Weiser MG, Kociba GJ. Platelet concentration and platelet volume distribution in healthy cats. Am J Vet Res 1984;45: 518–522. 39. Welles EG, et al. Platelet function and antithrombin, plasminogen, and fibrinolytic activities in cats with heart disease. Am J Vet Res 1994;55:619–627. 40. Zelmanovic D, Hetherington EJ. Automated analysis of feline platelets in whole blood, including platelet count, mean platelet volume, and activation state. Vet Clin Pathol 1998;27:2–9.
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VDIXXX10.1177/1040638715584994Whole blood platelet function testing in healthy catsHo et al.
Full Scientific Report
Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests
Journal of Veterinary Diagnostic Investigation 2015, Vol. 27(3) 352–360 © 2015 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638715584994 jvdi.sagepub.com
Kimberly K. Ho, Anthony C. G. Abrams-Ogg,1 R. Darren Wood, M. Lynne O’Sullivan, Gordon M. Kirby, Shauna L. Blois
Abstract. The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11–176), 81 (32–129), and 91 (59–129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL–ADP cartridges, was 69 (46–89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18–92) and 49 (9–96), respectively, using impedance hematology analyzer platelet counts, and 94 (25–98) and 68 (14–119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL–ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL–ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable. Key words: Cats; hemostasis; Multiplate; Plateletworks; platelet function; platelet counts.
Introduction Normal hemostasis requires a highly complex series of coordinated interactions between endothelial cells, platelets, and hemostatic proteins. Tests of hemostasis are commonly used in the diagnosis of hemostatic disorders and are also important in the monitoring of anticoagulant therapy. With respect to primary hemostasis, optical aggregometry using platelet-rich plasma (PRP) has historically been used as the standard for assessment of platelet function.11,21 Optical aggregometry evaluates platelet function based on the ability of platelets to aggregate on addition of platelet agonists, such as adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA). When an agonist is added to PRP, light transmission patterns are altered, which reflect platelet activity. Unfortunately, the preparation of PRP is operator intensive and is too cumbersome for routine clinical application.11,14,21 It is also operator dependent, and may activate platelets and possibly alter their function.11,14,21 Although template bleeding time is a readily available test of primary hemostasis, it does not reflect the function of platelets exclusively, is also subject to operator variation, and may be displeasing to owners.1,21
More convenient point-of-care methods for assessing platelet function in whole blood have been developed for humans, specifically analyzer systems that have been coded as MP,a PF,b and PW.c These tests assess platelet aggregation in response to agonists using different methods, and may be used to assess congenital and acquired platelet function defects, to monitor antiplatelet therapy for cardiac conditions, and to assess for risk of hemorrhage and the need for blood products prior to cardiac surgery.11,28 Platelet function has been variably evaluated with these tests in dogs, pigs, sheep, and horses.3,20,29 Studies have also used these tests to evaluate platelet function in cats; however, the literature is limited.17,22,26
From the Departments of Clinical Studies (Ho, Abrams-Ogg, O’Sullivan, Blois), Pathobiology (Wood), and Biomedical Sciences (Kirby), Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada. 1
Corresponding Author: Anthony C. G. Abrams-Ogg, Department of Clinical Studies, Ontario Veterinary College, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada N1G 2W1. [email protected]
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Whole blood platelet function testing in healthy cats Whole blood aggregometry using electrical impedance has gradually become preferred over PRP-based optical aggregometry as the standard test in some laboratories.9,20,33 Similar to optical aggregometry, impedance aggregometry measures platelet function by determining aggregation after addition of an agonist. However, instead of aggregating freely in liquid phase, the platelets aggregate on 2 electrodes within a test cell, subsequently decreasing the electrical conduction between them.11,21 Impedance whole blood aggregometry requires a smaller sample volume and less manipulation of platelets, thereby decreasing the risk of platelet activation prior to analysis.11,20,33 Furthermore, it may be more representative of platelet aggregation in vivo as cellular components are present. The MP analyzer incorporates the principles of impedance whole blood aggregometry in an automated user-friendly device. This device reports the change in electrical impedance as platelets aggregate in proprietary aggregation units over time, and calculates the area underneath the platelet aggregation curve (AUC). Use of this device in animals has been evaluated in dogs, sheep, and in cats.3,12,26 In the feline study, blood from 10 healthy cats was combined with eptifibatide or abciximab in vitro, and reduced platelet function was demonstrated for the former drug but not the latter.26 The PF analyzer is a point-of-care platelet function test that has been evaluated more extensively in animals.21 It is designed to be an in vitro substitute for template bleeding time. Platelet function is measured in terms of closure time (CT). The CT represents the duration of time, in sec, which is required for a platelet plug to form and prevent flow of blood through an aperture under high shear stress conditions in the presence of agonist combinations of either COL and ADP or COL and epinephrine. This analyzer was used in a previous feline study, where platelet function between 42 healthy cats and 30 cats affected with hypertrophic cardiomyopathy was compared.21 There was no difference in platelet function between the groups, but the study did not compare the results of the assay with optical or impedance aggregometry. The PW assay is approved for use in human medicine to monitor the use of the anti-platelet drugs aspirin and clopidogrel.28,37 This assay is potentially more readily available than other platelet function tests as it makes use of standard hematology analyzers. By measuring and comparing platelet counts between an ethylenediamine tetra-acetic acid (EDTA)–anticoagulated blood sample and a blood sample mixed with a platelet agonist, which decreases the number of individual platelets available for counting, the percentage of aggregated platelets can be calculated.28,37 Use of the PW assay has been described in a previous feline study, which demonstrated that platelet function was impaired in healthy cats that had been given 18.75 mg/cat of clopidogrel.18 However, the PW assay has only been validated for use on impedance-based hematology analyzers, and its utility on analyzers that use flow cytometry is unknown. As hematology analyzers
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based on flow cytometry are becoming more commonplace in research, reference, and point-of-care laboratories, compatibility with PW could make platelet function testing even more accessible. Cats with hypertrophic cardiomyopathy are at risk for thromboembolism and are therefore prescribed anti-platelet drugs.16,34 Evaluating anti-platelet drug effect in these animals in the past has been problematic because of the aforementioned problems with platelet function testing. Less cumbersome platelet function testing is now potentially available for this purpose; however, evaluation in cats is limited. The objectives of our study were therefore to establish institutional feline references intervals (RIs) using 3 tests that assess platelet function in whole blood by different principles: MP by electrical impedance, PF by mechanical aperture closure, and PW by platelet counting. A secondary objective was to compare results of PW when platelet counting is performed using an impedance-based analyzerd and flow cytometry.e
Material and methods Cat selection Healthy cats belonging to staff and students at the Ontario Veterinary College, University of Guelph (Canada), were recruited with informed consent. Cats were considered healthy based on history, physical examination, complete blood cell count,e serum biochemical profile,f and tests for Feline leukemia virus and Feline immunodeficiency virus.g Activated clotting timeh,i was performed if sufficient whole blood was available at the time of collection. A coagulation profile, including prothrombin time,j,k activated partial thromboplastin time,j,l fibrinogen concentration,j,m and j,n d-dimer concentration, was performed if residual citrated plasma was available after platelet function testing. Cats were excluded from the study if drugs other than monthly anti-parasitic treatments were used or if they were not amenable to gentle restraint. Animal use and procedures were approved by the University of Guelph Animal Care Committee in accordance with the standards set by the Canadian Council on Animal Care (http://www.ccac.ca/en_/standards/ guidelines) and the Ontario Animals for Research Act (1990; http://www.omafra.gov.on.ca/english/food/inspection/ahw/ ara-index.htm).
Blood collection In a quiet and controlled environment, fasted cats were sedated with ketamineo (2 mg/kg) and butorphanolp (0.2 mg/ kg) intravenously, via a saphenous vein, for atraumatic jugular venipuncture performed using a 21-gauge butterfly catheter.q All venipunctures were performed by a single investigator (KK Ho). Blood was collected directly into vacuum blood collection tubes in the following order: one 4.0-mL
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serum tuber (1 mL collected), one 4.5-mL 3.2% sodium citrate (0.105 M) tubes ensuring a 9:1 blood-to-citrate ratio, and one 2.0-mL K2EDTA tubet; followed by indirect blood collection of 2–3 mL through the butterfly catheter using a 6-mL plastic syringeu for transfer of 1 mL of blood into each of 2 PW tubes (which do not have a vacuum), containing either ADPc or COL,c to ensure a consistent blood-to-agonist ratio. All blood collection tubes and syringes were handled by a single investigator (ACG Abrams-Ogg). Blood collection tubes were gently inverted during and after blood collection to mix the blood and anticoagulant. For logistical reasons and to standardize time of platelet function analysis for the various tests after blood collection, equilibration at room temperature was limited to 10–15 min on a blood tube rockerv prior to analysis of the first replicate. Patient compliance during venipuncture, venipuncture score, and flow of blood during blood collection were documented.4 Blood smears were made from EDTAanticoagulated blood samples and evaluated microscopically for platelet clumps. All blood collections and tests were performed during two 3-week periods, 1 month apart. All samples were collected between 9:00 and 15:00.
The MP analyzer Analysis with the MP analyzer was performed on citrated blood samples using ADP,w COL,x and AAy as agonists. Each agonist was prepared as instructed by the manufacturer by adding 1 mL of distilled water to lyophilized stock. Reconstituted agonists were stored in 200-µL aliquots at –20°C for a maximum of 28 days (ADP and AA) or 4°C for a maximum of 7 days (COL) and allowed to warm to room temperature just prior to analysis. The MP analysis was carried out as instructed by the manufacturer. Using the automated pipette system on the instrument, 300 μL of isotonic NaCl, prewarmed to 37°C, was mixed with 300 μL of citrated whole blood in the test cellz and allowed to incubate for 3 min at 37°C prior to the addition of 20 μL of reconstituted AA agonist. For ADP and COL analyses, 300 μL of isotonic NaCl with 3 mM CaCl2aa was used in place of isotonic NaCl alone for sample re-calcification. The final agonist concentrations used in each analysis were 6.5 µM ADP, 3.2 µg/mL COL, and 0.5 mM AA. Each test cell performed 2 measurements for a total analysis time of 6 min, and mean AUC were reported and used for analysis.
The PF analyzer Analysis with the PF analyzer was performed using 800 µL of each citrated blood sample with cartridges containing COL–ADP-coated membranes for platelet activation (PFCOL/ADP).bb Cartridges were allowed to warm to room temperature prior to use. Each sample was analyzed in duplicate or in triplicate if a sample error (flow obstruction) was encountered in 1 or both of the first 2 replicates. The mean CT values, in sec, for each cat were used for analysis.
The PW assay Baseline platelet counts from the EDTA-anticoagulated samples and platelet counts from the PW assay agonist tubesc containing either ADP or COL (PW-ADP and PW-COL, respectively) were analyzed using 2 automated hematology analyzers that use different methods for platelet enumeration. Platelet counts were obtained in either duplicate or triplicate by an impedance analyzer,d and a single platelet count was obtained by a flow cytometry analyzer.e The percent platelet aggregation was calculated using the following formula: % aggregation =
EDTA platelet count − agonist-induced platelet count × 100 EDTA platelet count
Mean percent aggregation (% Agg) values calculated using impedance-based platelet counts, and single % Agg values calculated using flow cytometric–based platelet counts, were used for analysis.
Reference interval generation and statistical analysis Data were analyzed using commercial softwarecc unless otherwise indicated. The distribution of data for each platelet function test was assessed using Anderson–Darling and Shapiro–Wilk tests of normality. Additional software programsdd,17 were used to calculate the lower and upper reference limits (2.5th and 97.5th percentile of each data set, respectively) and their associated 90% confidence intervals (CIs) if possible. Robust or parametric methods of RI generation were used for data, which were normally distributed or log-transformed, based on a sample size (N), 40 ≤ N ≤ 120. When data were not normally distributed or transformed, nonparametric methods of RI generation were used without 90% CI. Outliers in each data set were identified as values less than the lower quartile (Q1) minus 1.5 × IQR or greater than the upper quartile (Q3) minus 1.5 × IQR, where IQR is the interquartile range. Outliers were removed and RIs recalculated as above. To assess for analytic variation of PF-COL/ADP, PWADP, and PW-COL, intraindividual and interindividual coefficients of variation (CVa and CVg, respectively) and intraclass correlation coefficients (ICC) were calculated. Spearman rank correlation coefficients were calculated to assess for associations between MP (ADP, COL, AA), PFCOL/ADP, and PW (ADP, COL) results. Spearman rank correlation coefficients were also calculated to assess for associations between baseline platelet counts, as performed on the impedance or flow cytometry analyzers, and results of the 3 platelet function tests. Pearson correlation coefficients were calculated to assess for a relationship between PW performed on the 2 hematology analyzers. Linear regression models were used to assess for correlations between results of platelet counts, with or without agonists,
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Table 1. Means, medians, ranges, 95% confidence interval (CI) and associated 90% CI for each upper and lower reference limit (excluding outliers), for whole blood platelet function testing in 55 cats using 3 analyzer systems.*
Test/Unit MP AUC (U)
PF Sec PW % Agg (impedance) % Agg (flow cytometry)
Agonist
Median/Mean‡
Range
Lower reference value
ADP† COL† AA†
87/93 (53) 81/80 (49) 91/94 (50)
19–174 30–140 49–130
11 32 59
−3 to 26 23–41 52–65
176 129 129
160–191 119–138 122–135
ADP–COL†
69/68 (46)
44–94
46
42–51
89
85–94
ADP COL† ADP COL†
71/64 (54) 49/53 (55) 94/80 (41) 68/67 (43)
17–94 7–95 25–98 7–98
18 9 25 14
NA 1–16 NA 3–26
92 96 98 119
NA 88–104 NA 114–142
90% CI for lower reference value
Upper reference value
90% CI for upper reference value
* AA = arachidonic acid; ADP = adenosine diphosphate; AUC (U) = area under curve screenshot Units [= AU × min/10 and rounded down]; % Agg = percent aggregation; COL = collagen; MP = Multiplate Analyzer (Roche Diagnostics International Ltd., Rotkreuz, Switzerland); PF = Platelet Function Analyzer-100 (Siemens Canada, Mississauga, Ontario, Canada); PW = Plateletworks (Helena Laboratories, Beaumont, TX); NA = not applicable. † Data normally distributed. ‡ Numbers in parentheses are the number of cats. For MP and PW using impedance-based platelet counts, missing values are due to removal of outliers. For PW, using flow cytometry–based platelet counts, measurements were performed in 43 cats, and missing values are due to removal of outliers. For PF, single values were obtained in 21 cats; duplicate values were obtained in 25 cats; for 7 cats that had PF-COL/ADP analyzed in triplicate, in 6 of those cats a reading was not obtained; the total number of flow obstructions was 41 out of 112 determinations (37%).
and % Agg calculated from values acquired by the impedance and flow cytometry analyzers. The level of significance was set at P < 0.05 for all tests.
Results Sixty cats were enrolled; however, only 55 cats met inclusion criteria. Four cats were excluded due to behavior and 1 was excluded due to azotemia. The 55 cats included 28 female (27 spayed, 1 intact) and 27 males (all neutered) with an age range of 1–15 years, mean of 5.51 years, and median of 4 years. Forty-eight of 55 cats had coagulation profiles performed; all were normal except for 1 cat with FXII deficiency (Hageman trait). Cats without coagulation profiles had normal activated clotting times.4 Blood smear evaluations revealed minimal-to-no platelet clumping in all but 4 samples. Subjective scoring of venipuncture technique and flow of blood during blood collection was similar among all cats. The RIs for the 3 platelet function tests, after removal of outlier data, are summarized in Table 1. The 90% CI for each upper and lower reference limit are included if the RI was calculated using parametric or Robust methods. For MP, CVg ranged from 0.30 to 0.46 using the various agonists. Values for CVa and ICC could not be calculated, as the instrument reports only mean values. For PF-COL/ADP, CVa, CVg, and ICC values were 0.16, 0.11, and 0.66, respectively. For PW-ADP and PW-COL, CVa, CVg, and ICC were 0.31, 0.11, and 0.88, and 0.38, 0.11, and 0.93, respectively, when platelet counts were acquired by impedance. Values for CVa and ICC for PW could not be calculated when platelet
counts were acquired by flow cytometry as only 1 value was obtained. There were low but significant correlations between PFCOL/ADP and MP-COL (ρ = 0.11, P = 0.017), and between PF-COL/ADP and PW-COL using impedance platelet counts (ρ = 0.14, P = 0.009). There were no other significant correlations between tests. Platelet counts and % Agg determined using impedance and flow cytometry for PW are compared in Table 2. Baseline platelet counts measured by both methods were significantly highly correlated, as were PW-ADP and PW-COL platelet counts, but values obtained by impedance were consistently higher than those obtained by flow cytometry. Calculated % Agg from each of these platelet counting methods were significantly moderately correlated, and overall impedance-based % Agg was significantly lower than flow cytometry–based % Agg. There were low but significant correlations between baseline platelet counts obtained by impedance and MP-COL (ρ = 0.19, P = 0.002) and MP-AA (ρ = 0.13, P = 0.009). There were also low but significant correlations between baseline platelet counts obtained by flow cytometry and MPCOL (ρ = 0.02, P = 0.002) and MP-AA (ρ = 0.17, P = 0.004). There were no other significant correlations between baseline platelet counts and tests.
Discussion In our study, 3 whole blood platelet function tests were investigated for use in cats. The study confirmed that methods
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Table 2. Comparison of PW results using impedance-based and flow cytometry–based hematology analyzers: mean (range) platelet counts; bias between platelet counts (mean [95% confidence interval]); and correlation of platelet counts and % aggregation.* Platelet count (×109/liter) Baseline (EDTA tube) No. of cats† Impedance Flow cytometry Bias in platelet counts (×109/liter)‡ Correlation of platelet counts (ρ)‡ Bias in % aggregation‡ Correlation of % aggregation (ρ)‡
51 361 (56–726) 269 (54–616) 93 (77–109) 0.77 NA NA
ADP agonist tube 39 132 (18–439) 63 (6–263) 68 (52–84) 0.70 −11 (–17 to –5) 0.64
COL agonist tube 41 163 (16–379) 76 (5–291) 82 (70–94) 0.76 −12 (–17 to 7) 0.64
* ADP = adenosine diphosphate; CI = confidence interval; COL = collagen, EDTA = ethylenediamine tetra-acetic acid; NA = not applicable. † Platelet counts in EDTA tubes were obtained with both impedance and flow cytometry analyzers in 51 cats. Platelet counts in ADP and COL agonist tubes were obtained with both impedance and flow cytometry analyzers in 43 cats. Missing values are due to removal of outliers. ‡ All biases and correlations significantly different, P < 0.001.
other than optical aggregometry using PRP are feasible for assessment of feline platelet function. The first feline study of the MP analyzer was reported in 2014.26 In that study, hirudin was used as the anticoagulant, and ADP and thrombin receptor activator peptide were used as agonists, in concentrations recommended by the manufacturer. In the current study, MP analysis was performed using citrated whole blood with agonists ADP, COL, and AA in concentrations recommended by the manufacturer. The median (87) and range (155) of AUC values with ADP in citrated blood were lower than the previously reported median (306) and range (534) for the same concentration of ADP agonist (6.5 μM) in hirudinized blood.26 The lower aggregation responses using citrated blood, in comparison to hirudinized blood, is consistent with findings in sheep, dogs, and humans.3,13,23,27,32 Citrate was used in our study to facilitate blood collection for, and comparison between, the 3 platelet function tests. There were no differences in the MP analyzer aggregation responses with the different agonists (Table 1). This finding is different from humans, where responses to ADP were lower than responses to COL and AA, and for dogs, where responses to AA appear to have been lower than responses to ADP and COL.27,33 Furthermore, aggregation responses in our study appear to be lower than responses in dogs and sheep, and only marginally greater than responses in humans, using the same or comparable agonist concentrations.3,27,32 Cats are considered to have highly aggregable platelets,35,40 so it was surprising that platelet aggregation responses were not higher compared to other species. Studies in dogs and sheep have demonstrated that aggregation responses can be enhanced by changing agonist concentrations, and similar studies in cats may identify optimal agonist concentrations.3,13,25 Sample collection methods were similar in the previous26 and current study of the MP analyzer in cats, but, in the previous study, samples were allowed to sit for 30 min prior to
analysis,26 while in our study samples were gently rocked for 15 min because of sample handling methods for the PF analyzer and PW assay. Optimum sample handling for cats for the MP analyzer is not known, but it is possible that rocking the samples affected the results. Based on the findings of our study, further examination of preanalytic and analytic variables of the MP analyzer for cats should be pursued. The use of the PF analyzer with COL–ADP was previously reported in cats.22 The mean CT was 64 sec for healthy cats in the previous report,22 while the median CT was 69 sec in our study. The CT range for healthy cats in the previous report was 43–176 sec (and 10th and 90th percentiles ~50 and 120 sec, respectively), which is wider than the range (44–106 sec) and 2.5th and 97.5th percentiles (45 and 104 sec, respectively) reported herein.22 This appears to be largely due to 4 values in the previous study with values >120 sec.22 These differences may be attributed to natural variation not captured in our study, but may also be due to preanalytical variables in sample collection, as this has been reported in humans.25 Sample handling after collection was not reported in the previous study, but in our study, samples were gently rocked for 10–15 min prior to analysis. This was done in part because, during pilot studies, flow obstructions occurred more frequently with samples that were left undisturbed for 30 min then gently remixed prior to analysis than with samples that were continually rocked for 10–15 min (data not shown). Assay failure due to flow obstruction was encountered with the PF analyzer. Although the number of flow obstructions was not reported in the previous study,22 it appears to have been less than in our study. The manufacturer indicates that flow obstruction errors are most likely attributable to blood clots in the sample.b Of the 55 samples included in the study, 6 were analyzed in triplicate without producing a result. On EDTA-anticoagulated blood smear evaluation of these 6 cats, only 1 showed moderate-to-marked platelet clumping. Thus, flow obstruction errors may either not be all
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Whole blood platelet function testing in healthy cats attributable to microscopic blood clots within the sample, or evaluation of the EDTA-anticoagulated blood smears may not have been a good surrogate for evaluation of microscopic clot formation in the citrated blood samples. It is also possible that rocking of samples prior to analysis contributed to platelet activation and flow obstruction. Based on similar CT of the 2 studies, the PF analyzer may be a platelet function test that can be performed with relative consistency in healthy cats. However, biological variation was not assessed in the current study, and the wide RI of the PF analyzer with the COL–ADP cartridges may limit its clinical utility in assessing whether a cat has altered platelet function based on a single assessment. The use of the PW assay using ADP was previously reported for healthy cats.18 The median % Agg in our study was 71%, while the mean % Agg was 70.55% in the previous feline study when impedance (using a different analyzer) was used as the method for platelet counting.18 The maximum allowable time until sample analysis was reported to be 30 min.18 Normal human RIs of 80–100% and 70–100% for ADP and COL, respectively, are provided by the manufacturer. As with the findings for the MP analyzer, the discrepancies between the lower limits of RIs for humans and those developed in our study for cats are surprising given the aggregability of feline platelets.35,40 This may represent an underestimation of the baseline platelet count in some feline samples, which would result in underestimation of percent aggregation. Following the manufacturer’s instructions and the previous study in cats,18 baseline platelet counts in our study were performed using EDTA-anticoagulated blood. Automated platelet counting in feline EDTA-anticoagulated blood is inherently less accurate than platelet counting in human EDTA-anticoagulated blood and is attributed to platelet aggregation.36,40 The “two-syringe collection method” and prompt analysis used in the current study should have minimized platelet aggregation, and platelet clumping was only readily apparent on blood smear evaluation in 4 cases, but this does not preclude that some degree of aggregation occurred.31,38 The PW assay ADP and COL tubes use citrate as anticoagulant, and it is also possible that feline platelet aggregates in citrate-anticoagulated blood subsequently disaggregate more rapidly than human platelet aggregates. Less stable aggregates would increase platelet counts in the PW assay ADP and COL tubes and thereby underestimate % Agg. Any effect of time, however, should have been minimal as the first replicates were assayed within 10–15 min and the duplicates assayed within 15–20 min (sample analysis time was 4 min).18,37 The upper limit of COL-induced aggregation in our report was >100%, which likely reflects inherent error in platelet enumeration. The use of the PW assay has only been validated for impedance-based analyzers. Platelet counts vary with the method, therefore it is not surprising that baseline platelet counts and ADP- and COL-induced % Agg determined by impedance and flow cytometry were different.2,7 Specifically, baseline
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and post-agonist platelet counts using flow cytometry were consistently lower than counts acquired by impedance. The mean (range) baseline platelet counts by flow cytometry in our study were similar to previous studies on a similar analyzer, which reported a mean of 281 × 109/liter, with 2.5th and 97.5th percentile of 42 and 630 × 109/liter, respectively, and a mean (range) of 289 (16–858) × 109/liter, using EDTAanticoagulated blood.5,30 The RI provided by the manufacturer of the impedance analyzer is 300–800 × 109/liter. In previous studies comparing impedance and flow cytometry– based platelet counts in cats, both comparatively higher and lower impedance-based counts have been reported.2,5,24 Correlations for baseline and postagonist platelet counts between the 2 analyzers ranged from 0.70 to 0.77 (Table 2), likely reflecting some imprecision of platelet counts by both analyzers. A limitation of our study is that only 1 value with the flow cytometry–based analyzer was obtained. This occurred because, logistically, replicates could not be analyzed in 120 reference individuals, as using smaller sample sizes may result in less accurate values.15 While the number of animals used in our study was a conventional number used for generating RIs for veterinary studies, with the knowledge that nonparametric methods are likely to be needed in the analysis of platelet function tests, future studies should ideally incorporate larger sample sizes (>120 cats) in order to calculate more accurate RIs. Ideal RIs help identify abnormal values that have clinical implications. Given that RIs developed in our study are not synonymous with the upper and lower thresholds of platelet function required for normal hemostasis, the tendency toward clinical bleeding or thrombosis cannot be inferred from where an individual animal’s values lie relative to the RIs. A study to examine this issue would require a larger sample population with a range of variable platelet function including animals with evidence of clinical bleeding or thrombosis. Cats in our study were also considered to be healthy; however, echocardiograms were not performed, which may have resulted in inadvertent recruitment of cats with subclinical heart disease, potentially altering RIs toward states of increased platelet activity.6,22,34 As such, the platelet function tests for which RIs were developed in our study may be better suited for monitoring the effects of antithrombotic therapy, as they are in humans, and may be more beneficial when using an animal’s own pretreatment values for comparison.10,28 However, for this purpose, biologic variation must also be accounted for in the interpretation of results, and the effects of biological variation on these tests have not yet been investigated. Platelet function in cats can be assessed with the MP, PF, and PW analyzer systems. However, prior to the routine
clinical use of these tests, further evaluation of preanalytical variables (e.g., sample collection and handling) and analytic variables (e.g., anticoagulant and agonist concentrations) are necessary. Acknowledgements We thank Dr. William Sears, Department of Population Medicine, and Gabrielle Monteith, Department of Clinical Studies, for assistance with study design and statistical analysis; the veterinary and laboratory technicians, Health Sciences Centre, Department of Clinical Studies; and the Clinical Pathology Laboratory, Animal Health Laboratory, for assistance with blood collection and platelet function testing.
Sources and manufacturers a. Multiplate analyzer, Roche Diagnostics International Ltd., Rotkreuz, Switzerland. b. Platelet Function Analyzer-100, Siemens Canada, Mississauga, Ontario, Canada. c. Plateletworks Combo-25 kit (catalog no. 5852), Helena Laboratories, Beaumont, TX. d. Vetscan HM5, Abaxis Veterinary Diagnostics, Union City, CA. e. ADVIA 2120, Siemens Canada, Mississauga, Ontario, Canada. f. Cobas C 501, Roche Diagnostics Canada, Laval, Quebec, Canada. g. SNAP FIV/FeLV Combo Test, IDEXX Laboratories Canada Corp., Toronto, Ontario, Canada. h. Actalyke MINI II, Helena Laboratories, Beaumont, TX. i. MAX-ACT (catalog no. 003054), Helena Laboratories, Beaumont, TX. j. Stago STA Compact CT coagulation analyzer, Diamond Diagnostics, Holliston, MA. k. STA Neoplastine CI Plus 5 (catalog no. 00606), Diagnostica Stago SAS, Asnières sur Seine, France. l. STA C.K. Prest 5 (catalog no. 00597), Diagnostica Stago SAS, Asnières sur Seine, France. m. STA Fibrinogen 5 (catalog no. 00674), Diagnostica Stago SAS, Asnières sur Seine, France. n. STA Liatest D-Di (catalog no. 00515), Diagnostica Stago SAS, Asnières sur Seine, France. o. Vetalar, Bioniche Animal Health Canada, Belleville, Ontario, Canada. p. Torbugesic, Zoetis Canada, Kirkland, Quebec, Canada. q. BD Vacutainer Safety-Lok blood collection set, BD Canada, Mississauga, Ontario, Canada. r. BD Vacutainer serum tube (catalog no. 367812), BD Canada, Mississauga, Ontario, Canada. s. BD Vacutainer sodium citrate tube (catalog no. 366415), BD Canada, Mississauga, Ontario, Canada. t. BD Vacutainer K2EDTA tube (catalog no. 367841), BD Canada, Mississauga, Ontario, Canada. u. Monoject 6-mL syringe with regular Luer tip (catalog no. 8881516911), Tyco Healthcare Group LP, Mansfield, MA. v. Coulter blood mixer, Coulter Electronics Inc., Hialeah, FL. w. Multiplate ADP test kit (catalog no. MP0220), Verum Diagnostica GmbH, Munich, Germany. x. Multiplate COL test kit (catalog no. MP0230), Verum Diagnostica GmbH, Munich, Germany.
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Whole blood platelet function testing in healthy cats y. Multiplate ASPI test kit (catalog no. MP0210), Verum Diagnostica GmbH, Munich, Germany. z. Multiplate test cells (catalog no. MP0027), Verum Diagnostica GmbH, Munich, Germany. aa. Multiplate NaCl/CaCl2 (catalog no. MP0530), Verum Diagnostica GmbH, Munich, Germany. bb. Dade PFA Collagen/ADP test cartridge (catalog no. B417021A), Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany. cc. SAS v. 9.1, SAS Institute Inc., Cary, NC. dd. Analyze it for Excel v. 2.24, Analyse-It Software Ltd, Leeds, United Kingdom.
Declaration of Conflicting Interests The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Two Plateletworks Combo-25 kits were generously donated by Ryan Medical, Burlington, Ontario, Canada.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this work was provided by the Ontario Veterinary College Pet Trust.
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