Die vergleichende Statistik wurde mit dem Programm SPSS 16.0 durchgeführt. Initial erfolgte die Überprüfung sämtlicher Stichproben mittels Kolmogorov-Smirnov-Test auf Normalverteilung. Zur Ermittlung der Referenzwerte wurden die 2,5%- und 97,5%- bzw. 5%- und 95%-Quantile berechnet. Im methodischen ersten Teil der Arbeit wurde für jeden Agonisten und die Parameter AUC und maximale Aggregation Mehrfeldertafeln angelegt, die die Anzahl der Messergebnisse unterhalb des, im und oberhalb des Referenzbereichs zu den verschiedenen Messzeiten enthielten. Sie wurden mit Hilfe eines Chi-Quadrat-Tests ausgewertet. Des Weiteren dienten ANOVA mit Messwiederholungen und gepaarte t-Tests dem Vergleich der Referenzwerte zu den verschiedenen Messzeiten. Die mit Clopidogrel behandelten Hunde wurden zu den verschiedenen Zeitpunkten mittels ungepaartem t-Test mit der Kontrollgruppe verglichen.
Im zweiten Teil der Studie wurden die Mittelwerte der Kontrollgruppe und der erkrankten Tiere sowie der Tiere mit und ohne SIRS mittels ungepaarten t-Tests verglichen.
Zum Gruppenvergleich der Daten des PFA-100 wurde der Mann-Whitney-Test herangezogen, da aufgrund der zensierten Werte für diese Messgröße keine Normalverteilung gegeben war.
Wurde bei der Messung die maximale Messzeit von 300 s erreicht, so wurde der Wert zur statistischen Auswertung durch den Wert 301s ersetzt. Als signifikant wurden jeweils Differenzen mit einer Irrtumswahrscheinlichkeit von < 5% (p < 0,05) bezeichnet.
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Influence of test time on results of the Multiplate® impedance aggregometer in dogs
Running title: Optimal test time of the Multiplate® analyser
Monia Abid, Kerstin Kalbantner, Reinhard Mischke
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From the Small Animal Clinic, University of Veterinary Medicine Hanover
1Corresponding Author: Reinhard Mischke, Small Animal Clinic, University of Veterinary Medicine Hanover, Bünteweg 9, D-30559 Hannover, Germany
(Tel: +49 511 9536200; fax: +49 511 6204)
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39 Abstract. The Multiplate® analyser is a relatively new impedance aggregometer designed for human medicine, but was recently evaluated for canine blood. The aim of this study was to determine the influence of test time on reference values and the validity of measurement results in canine blood. Based on blood samples of 83 healthy dogs, reference values for different test times (6, 8, 10 and 12 min) were established for maximum aggregation and area under the curve (AUC) values for ADP-, collagen-, and arachidonic acid-induced aggregation.
The results of 134 samples of 117 dogs with various diseases and 8 samples with reduced platelet function owing to treatment with the platelet aggregation inhibitor clopidogrel were calculated at the different test times and classified as decreased, normal or increased.
Maximum aggregation and AUC values increased significantly with increasing test time. Chi-square test did not show significant differences between the various test times with regard to the number of increased and decreased measurement results for any agonists. All samples of dogs treated with clopidogrel and measured with the agonist ADP revealed decreased AUC values at any time point. The results of our study indicate that test time significantly influences absolute maximum aggregation and AUC values, but has limited influence on sensitivity of measurement results of canine blood using the investigated instrument.
Key words: Whole blood aggregometry; ADP; collagen; arachidonic acid; reference values;
sensitivity; clopidogrel
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Introduction
The measurement of platelet function is an important part of haemostasis diagnostics.
Several laboratory methods have been developed through the last decades, one of them is the impedance aggregometry. This method measures the increase in electrical resistance in whole blood resulting from platelet accumulation between test electrodes.5 Advantages of using whole blood aggregometry in comparison to the turbidimetry on platelet-rich plasma (PRP) are the lacking necessity to prepare PRP. Therefore, the method is less time consuming and no artificial changes of platelet volume composition can occur. In addition, the presence of other blood cells and of a surface may better imitate physiologic conditions.5
One of the newer devices which is based on this principle is the Multiplate® analyser.
As primarily designed for human blood samples, an adjustment of several parameters was made to allow measurements in canine plasma.7 A parameter not elaborated in detail so far is the test time. The manufacturer recommends a test time of 6 minutes for human blood in order to reduce time exposure. In contrast, a recording time of 12 minutes was used for canine blood in the previous study, allowing the curves to almost plateau.7
The aim of this study was to determine the influence of the test time on reference values and on the sensitivity of measurement results of the Multiplate® analyser in canine plasma and to prove whether a reduction of the test time can be performed without negative influence on the validity of the assay.
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41 Materials and methods
Study design
Measurements of 83 healthy dogs and 134 samples from 117 dogs with various diseases were performed using 3 different agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA]) and the test parameters “area under the curve” (AUC) and “maximal aggregation” were calculated after a test time of 6, 8, 10 and 12 minutes. Based on the clinically healthy dogs, reference values for each test time and agonist were calculated. The results of the diseased dogs were classified in relation to reference values as decreased, normal or increased and the distribution among categories between the different test times was compared.
In addition, 8 samples with defined aggregation deficiency which were taken from 4 healthy dogs that were treated with the platelet aggregation inhibitor clopidogrel were measured. Dogs received clopidogrela in doses of 2–4 mg/kg/d orally on three consecutive days and blood samples were collected on the third and fourth day. Aggregation was induced with ADP and AA and the data were analysed as described in experiment 1.
The experimental procedure was approved by the appropriate ethics committee (Lower Saxony State Office for Consumer Protection and Food Safety, reference number 07A 514).
Animals
The clinically healthy dogs included animals of different gender and breeds and were provided by private owners. The age of the dogs varied from 1 to 12 years with a median of
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2.5 years. The health status was defined based on clinical examination, a blood cell count and biochemical profile. An exclusion criterion was treatment with any drug influencing platelet function.
All 117 diseased dogs used in this study were patients of the Small Animal Clinic, University of Veterinary Medicine Hannover. There were 67 patients with neoplasia (22 lymphomas, 10 leukaemias, 15 carcinomas (incl. 4 mammary carcinomas), 7 histiocytic sarcomas, 3 haemangiosarcomas, 2 multiple myeloma and 8 dogs with individually different other neoplasias, respectively), 14 dogs with non-neoplastic hepatopathies (7 portosystemic shunts, 5 degenerative hepatopathies and 2 others), 18 with local or systemic infectious diseases (4 leptospirosis, 2 leishmaniosis, 6 abscesses, 2 pyothorax, 2 bronchopneumonias, 1 pyometra, 1 sepsis), 3 with immunohaemolytic anaemia, 2 with haemophilia A, 2 with von Willebrand diseases, 3 renal insufficiencies, 2 with prostatic cysts, and 6 with miscellaneous disorders. Of 9 dogs, more than one blood samples (up to four) were taken.
The 4 dogs that were treated with clopidogrel in experiment 2 were clinically healthy Beagles owned by the Small Animal Clinic.
Sample collection
Blood samples were taken from the saphenous, cephalic or jugular vein using sterile disposable needles (1.2 x 40 mm). Only gentle pressure was used to raise the vein to minimise stasis potentially activating the platelets. Blood for impedance aggregometry was collected into 4.5 mL plastic tubes covered with hirudinb, which has less influence on platelets than citrate. Blood and anticoagulant were immediately thoroughly mixed by careful swaying.
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43 The samples were tested between 30 minutes and 3 hours after collection, as recommended for human blood. During this period of time, the blood was stored at room temperature and swayed carefully just before starting the measurement.
Test procedure and data analysis
The Multiplate® analyserc is an advancement of the whole blood aggregation by Cardinal and Flower4.2 It has five channels for parallel testing which are assembled with single-use test cells.11
The test was performed as recommended by the manufacturer. After pipetting 300 µl of isotonic sodium chloride solution (prewarmed to 37 °C) into the test cell, 300 µl of hirudin- anticoagulated blood was added. At the end of a three minute incubation time at 37 °C 20 µl of agonist solution was added and the measurement started. The increase of electrical resistance caused by platelets attaching on the surface of the sensors was recorded over 12 minutes and converted into arbitrary “aggregation units” (AU). Aggregation was quantified by the area under the curve (AUC in AU*min) which depends on the velocity (AU/min) of aggregation and maximal aggregation.2 Optimal agonist concentrations which were approved in a previous study7 were used: 10 µmol/L ADPd, 5 µg/mL collagene, and 1 mmol/L AAf.
Aggregation curves were exported from the Multiplate® analyser to a computer on which the spreadsheet software Microsoft Excel™ was installed. The resulting Excel file provided aggregation units in intervals of 0.57 seconds. AUC values were calculated for the different test times (6, 8, 10, 12 minutes) as described by the manufacturer by summarizing the single aggregation values over time and dividing the sum by 60, following an instruction
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provided by the manufacturer. In addition, maximum aggregation values were generated from the Excel files, i.e. the maximal values within a defined test time were chosen.
Statistical analysis
Data were tested for normal distribution using Kolmogorov-Smirnov test. All examined parameters showed normal distribution. Results were expressed as arithmetic mean and standard deviation. In addition, the following reference values were calculated based on results of healthy dogs: 2.5%, 10%, 25%, 50% (median), 75%, 90%, and 97.5% quantiles.
Comparison of the results of normal dogs at different test times were performed with one-way ANOVA (repeated measurements) and t-test for paired observations considering Bonferroni`s correction. Comparison of results of normal dogs and clopidogrel-treated dogs at different test times were performed with student`s t-test. Contingency tables for each agonist were compiled including the numbers of increased, normal and decreased results at the 4 test times.
Evaluation of the contingency table in both experiments was performed with Chi square test.
P < 0.05 was considered significant. Statistical analysis was performed using SPSS 17.0 German.g
Results
Reference values that were calculated for the different agonists at the different test times are presented in Tables 1 and 2. ANOVA revealed significant differences between different test times for both AUC values and maximum aggregation values, independent which agonist was used (P < 0.001). Post hoc analyses revealed significant differences
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45 between all test times for both parameters and all agonists, and thus even a significant increase in maximum aggregation between test times 10 and 12 minutes. Chi square test did not show significant differences between the different test times with regard to the number of samples with AUC or maximal aggregation below, within and above the reference range for any of the agonist (Figs. 1,2).
ADP- and AA-induced AUC and maximum aggregation values of clopidogrel treated dogs were lower when compared to untreated healthy dogs at all test times (P < 0.001 (t test);
Figs. 3–6). Irrespectively of the test time, all AUC values of ADP-induced aggregation from dogs treated with clopidogrel were below the reference range (Fig. 3). All maximal aggregation values performed with the agonist ADP were below the reference range using a test time of 6 or 8 minutes, respectively, but 1/8 value was within the lower limit when the test time was prolonged to 10 and 12 minutes (Fig. 4). After induction with AA, reduced values were also found for the majority of the AUC values (7/8 [6 minutes] or 6/8 [8, 10 and 12 minutes]) and part of the maximal aggregation values (5/8 [6 minutes] or 4/8 [8, 10 and 12 minutes]), without significant influence of the test time (P > 0.05, Chi square test) (Figs. 5, 6).
Discussion
The results of the present study indicate that test time significantly influences test results of the Multiplate® analyser such as maximal aggregation.Therefore, reference values must be established for the used test time. If test time-adjusted reference values are used, the test time does not seem to have a significant influence on the sensitivity and specificity of
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measurements using the Multiplate® analyser. Therefore, a short test time of 6 minutes seems appropriate and may even have a slightly superior sensitivity.
A test time of 6 min is recommended by the manufacturer for human blood samples and in most of the human studies using the Multiplate® analyser this time setting has been chosen.2,3,8,12 Deviating from this, Tòth et al.11 chose a test time of 5 minutes in their study. In the available human and veterinary literature no studies could be found, which systematically compared different test times with respect to the diagnostic accuracy. The results of the present study seem therefore also of interest for the human medicine.
The reason for using a test time of 12 minutes for canine blood in the previous study on canine sample material was to allow the aggregation curves to almost plateau.7 This may, for example, allow to report true “maximum aggregation values”. The main reason to use a standard test time of 6 minutes, which usually does not allow to finish aggregation reaction, seems to be the reduced analysis time.
The main limitation of our study is the lack of a reference method in the first experiment. Therefore, it is undefined whether the results below or above the reference range actually represent true or false positive results. Therefore, we performed a second experiment based on sample material from clopidogrel-treated dogs. Clopidogrel is a potent, non-competitive inhibitor of the platelet ADP membrane receptor P2Y128 which irreversibly inhibits the binding of ADP to its platelet membrane receptors. A significantly reduced ADP-induced platelet aggregation was already described in dogs receiving clopidogrel at the dosage which we administrated to the dogs in the present study.1 Therefore, it can be assumed that all of the blood samples taken from treated dogs and measured with the agonist ADP were below the reference values.
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47 The reduction of AA-induced aggregation in clopidogrel-treated dogs may be surprising, but an influence of clopidogrel on arachidonic acid-induced aggregation was also detected in human platelets.6,9 Although the inhibition is specific and does not significantly affect cyclo-oxygenase (COX) or AA metabolism, clopidogrel can indirectly inhibit platelet aggregation induced by agonists other than ADP by blocking the amplification of platelet activation by released ADP. ADP binding is necessary for activation of the GPIIb/IIIa receptor, which is necessary for linking different platelets together by fibrinogen to form the platelet aggregate.10
In conclusion, the results of our study indicate that the influence of test time on the validity of measurement results in canine whole blood aggregometry is minor, if interpretation is performed based on test time-adjusted reference values. A reduction of test time of canine plasma to 6 minutes – which corresponds to the recommendation in humans – is possible and associated with at least the same validity as longer test times.
Sources and manufacturers
a. Clopidogrel AbZ 75 mg, AbZ-Pharma GmbH, Blaubeuren, Germany.
b. Thrombin Inhibitor blood collection tube 4.5 ml suited for Sarstedt® system, Dynabyte® Informationssysteme GmbH; Munich, Germany
c Dynabyte Informationssysteme GmbH, Munich, Germany.
d. ADPtest, Dynabyte Informationssysteme GmbH, Munich, Germany.
e. COLtest, Dynabyte Informationssysteme GmbH, Munich, Germany.
f. ASPItest, Dynabyte Informationssysteme GmbH, Munich, Germany.
g. SPSS Inc., Chicago, USA.
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References
1. Brainard BM, Kleine SA, Papich MG, Budsberg SC: 2010, Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and the carboxylic acid metabolite SR 26334 in healthy dogs. Am J Vet Res 71:822–830.
2. Calatzis A: 2007, [Analysis of primary hemostasis in whole blood.] J Lab Med 31:239–247 (in German).
3.
assessment of platelet function in the absence of antiplatelet medication: comparison of the PFA-100, multiplate electrical impedance aggregometry and verify now assays. Thromb Res 125:e132–e137.
4. Cardinal DC, Flower RJ: 1980, The electronic aggregometer: a novel device of assessing platelet behavior in blood. J Pharmacol Methods 3:1350–1358.
5. Dyszkiewicz-Korpanty AM, Frenkel EP, Sarode R: 2005, Approach to the assessment of platelet function: Comparison between optical-based platelet-rich plasma and impedance-based whole blood platelet aggregation methods. Clin Appl Thromb Hemost 11:25–35.
6. Eder C, Funke U, Schulze M, et al.: 2007, [Modified platelet aggregation test in patients on ASA and/or clopidogrel] Haemostaseologie 27, 163–176.
7. Kalbantner K, Baumgarten A, Mischke R: 2009, Measurement of platelet function in dogs using a novel impedance aggregometer. Vet J 185:144–151.
8. Mueller T, Dieplinger B, Poelz W, et al.: 2007. Utility of whole blood impedance aggregometry for the assessment of clopidogrel action using the novel Multiplate analyzer – comparison with two flow cytometric methods. Thromb Res 121:249–258.
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49 platelet inhibition by aspirin and P2Y12 antagonists by using multiple electrode aggregometry. Thromb J 8:9.
11. Tóth O, Calatzis A, Penz S, et al.: 2006, Mutiplate electrode aggregometry: A new device
to measure platelet aggregation in whole blood. Thromb Haemost 96:781–788.
12. von Pape KW, Dzijan-Horn M, Bohner J, et al.:
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50 Table 1
Reference values of area under the curve (AUC; AU*min) values measured with the Multiplate® analyser in 83 healthy dogs at different test times using different agonists.
agonist test time
(Min) mean standard deviation
Quantiles
2.5 10 25 50 75 90 97.5
ADP 10 µmol/l
6 965 ± 228 498 681 797 972 1135 1309 1384 8 1479 ± 344 831 1035 1259 1485 1718 1988 2112 10 2022 ± 466 1241 1415 1648 2055 2346 2696 2931 12 2352 ± 577 1167 1548 1907 2393 2741 3139 3443
Collagen 5 µg/ml
6 1060 ± 174 662 845 937 1084 1169 1298 1378 8 1706 ± 265 1050 1379 1514 1739 1861 2060 2219 10 2402 ± 367 1490 1945 2113 2425 2640 2878 3134 12 2810 ± 436 1751 2275 2459 2836 3082 3332 3691
Arachidonic acid 1 mmol/l
6 879 ± 282 417 500 633 898 1091 1288 1450 8 1346 ± 422 630 770 978 1355 1681 1937 2192 10 1837 ± 572 857 1032 1348 1819 2257 2602 3024 12 2137 ± 687 997 1200 1557 2089 2580 3054 3630
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51 Table 2
Reference values of maximal aggregation (AU) values measured with the Multiplate®
analyser in 83 healthy dogs at different test times using different agonists.
agonist test time
(Min) mean standard deviation
Quantiles
2.5 10 25 50 75 90 97.5
ADP 10 µmol/l
6 139 ± 32.0 78.6 96.7 115 140 160 183 200 8 151 ± 34.7 93.0 106 125 153 176 197 220 10 157 ± 35.5 96.3 111 128 159 186 203 226 12 160 ± 36.0 97.0 112 130 163 188 208 232
Collagen 5 µg/ml
6 172 ± 26.5 108 139 153 173 190 205 224 8 191 ± 30.2 121 152 169 193 213 226 253 10 201 ± 32.7 128 161 177 203 222 238 266 12 205 ± 33.8 129 165 179 205 226 243 271
Arachidonic acid 1 mmol/l
6 127 ± 38.9 52.7 73.4 96.8 126 155 177 208 8 137 ± 44.5 56.7 74.9 101 137 167 196 228 10 143 ± 44.6 61.1 85.1 108 141 178 203 237 12 146 ± 46.6 61.7 86.1 110 143 178 204 258
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b)
c)
Fig. 1
Distribution of increased, normal and decreased area under the curve (AUC) results measured with the Multiplate® analyser in 134 samples from 117 dogs with various diseases at 4 different test times. Measurements were performed with the agonists ADP (a), collagen (b) and arachidonic acid (AA) (c).
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b)
c)
Fig. 2
Distribution of increased, normal and decreased maximal aggregation results measured with the Multiplate® analyser in 134 samples from 117 dogs with various diseases at 4 different test times. Measurements were performed with the agonists ADP (a), collagen (b) and arachidonic acid (AA) (c).
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0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000
6 8 10 12
Test time (minutes)
A UC ( A Ux m in )
Fig. 3
Influence of test time on area under the curve (AUC) values (individual values, mean ± standard deviation) of ADP-induced impedance aggregometry using the Multiplate® analyser in 83 samples of healthy dogs and 8 samples from clopidogrel-treated dogs (reference values are highlighted in grey)
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6 8 10 12
Test time (minutes)
0 20 40 60 80 100 120 140 160 180 200 220 240 260
M ax im al a gg reg at io n ( A U )
Fig. 4
Influence of test time on maximum aggregation values (individual values, mean ± standard deviation) of ADP-induced impedance aggregometry using the Multiplate® analyser in 83 samples of healthy dogs and 8 samples from clopidogrel-treated dogs (reference values are highlighted in grey)
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0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000
6 8 10 12
Test time (minutes)
A UC ( A Ux m in )
Fig. 5
Influence of test time on AUC values (individual values, mean ± standard deviation) of arachidonic acid-induced impedance aggregometry using the Multiplate® analyser in 83 samples of healthy dogs and 8 samples from clopidogrel-treated dogs (reference values are highlighted in grey)
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20 40 60 80 100 120 140 160 180 200 220 240 260 280
M ax im al a gg reg at io n ( A U )
6 8 10 12
Test time (minutes)
Fig. 6
Influence of test time on maximum aggregation values (individual values, mean ± standard deviation) of arachidonic acid-induced impedance aggregometry using the Multiplate®
analyser in 83 samples of healthy dogs and 8 samples from clopidogrel-treated dogs (reference values are highlighted in grey)
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Platelet function in dogs with inflammatory diseases
M. Abid, K. Kalbantner, R. Mischke*
Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, D-30559 Hannover, Germany
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* Corresponding author. Tel.: +49 511 9536200
E-mail adress:
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59 Abstract. The aim of this study was to examine the influence of inflammatory diseases on primary haemostasis in dogs. Six different parameters were used for testing platelet function in 25 dogs with bacterial or protozoal infections: capillary bleeding time, automatic platelet function analysis (PFA 100, collagen/ adenosine diphosphate [ADP] and collagen/epinephrine cartridges), turbidimetric platelet aggregation, impedance aggregometry using a multiple electrode aggregometer, platelet count and haematocrit. Results of the 25 diseased dogs were compared to the control group and additionally classified into two subgroups based on criteria of systemic inflammatory response syndrome (SIRS) (groups “SIRS” and
“NonSIRS”) and compared among each other. The following findings were observed in dogs with inflammatory diseases when compared to the control group: a significantly prolonged closure time of the automatic platelet function analyser measured with both cartridges (e.g.
Col/ADP: 83 [55–301] s vs. 65 [47–99]; median [minimum–maximum]; P < 0.0001 ); a significant decrease in maximal aggregation of the turbidimetric aggregometry induced with
Col/ADP: 83 [55–301] s vs. 65 [47–99]; median [minimum–maximum]; P < 0.0001 ); a significant decrease in maximal aggregation of the turbidimetric aggregometry induced with