Diagnostik der primären Hämostase beim Hund unter besonderer Berücksichtigung eines neuen Vollblutimpedanzaggregometers

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Tierärztliche Hochschule Hannover

Diagnostik der primären Hämostase beim Hund unter besonderer Berücksichtigung eines neuen

Vollblutimpedanzaggregometers

INAUGURAL – DISSERTATION

zur Erlangung des Grades einer Doktorin der Veterinärmedizin Doctor medicinae veterinariae

(Dr. med. vet.)

vorgelegt von Kerstin Kalbantner

Schwäbisch Gmünd

Hannover 2009

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Wissenschaftliche Betreuung: Univ.-Prof. Dr. R. Mischke Klinik für Kleintiere

1. Gutachter: Univ.-Prof. Dr. R. Mischke 2. Gutachter: Univ.-Prof. Dr. K. Feige

Tag der mündlichen Prüfung: 12.11.09

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Meinen Eltern

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Wer aufhört, besser zu werden, hat aufgehört, gut zu sein.

Philip Rosenthal, 1916

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Teile dieser Arbeit sind bei folgender Zeitschrift zur Veröffentlichung angenommen oder bereits veröffentlicht:

The Veterinary Journal

Ergebnisse dieser Dissertation wurden in Form von Postern auf folgenden Fachtagungen präsentiert:

16. Jahrestagung der Fachgruppe „Innere Medizin und Klinische Labordiagnostik“

der DVG (InnLab 2008):

Messung der Thrombozytenfunktion beim Hund mit Hilfe eines neuen Impedanzaggregometers – Methode und Referenzwerte

17. Jahrestagung der Fachgruppe „Innere Medizin und Klinische Labordiagnostik“

der DVG (InnLab 2009):

Veränderung der Thrombozytenfunktion bei unterschiedlichen Erkrankungen des Hundes

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Inhaltsverzeichnis

I Einleitung...……….….... 1

II Measurement of platelet function in dogs using a new impedance aggrego- meter - Optimisation of agonist concentration and reference values ..…...3

Abstract ……….4

Introduction ………...5

Materials and Methods ……….….6

Results.………..………...11

Discussion ………...15

References.……….…..19

III Plateletfunction in dogs with portosystemic shunt

….

.……….…...30

Abstract ………..…….…31

Introduction ……….……32

Materials and Methods ………33

Results ……….40

Discussion ………...……41

References ………...………45

IV Übergreifende Diskussion ………...…..………..………...……….50

V Zusammenfassung …………...……….…………62

VI Summary .……….……….………....65

VII Anhang …..………...…...68

VIII Publikationsliste………...…….………..…..71

IX Danksagung ………..……….………...72

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Abkürzungsverzeichnis

AA arachidonic acid

Abb. Abbildung

ADP Adenosindiphosphat AU aggregation unit AUC area under the curve CaCl2 Kalziumchlorid COL Kollagen

CPSS congenital portosystemic shunt CT closure time

CV coefficient of variation et al. et alii (lat. „und andere“) Fig. figure

Max Maximum mg Milligramm min Minute Min Minimum ml Milliliter mm Milimeter mmol Millimol n Anzahl

NaCl Natriumchlorid p P-Wert

PFA Plättchenfunktionsanalysator PFP platelet free plasma

PRP platelet rich plasma PSS portosystemischer Shunt SD standard deviation Tab. Tabelle

TI Hirudin

TRAP Thrombinrezeptor-aktivierende Peptide

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TV total volume µg Mikrogramm µl Mikroliter µmol Mikromol

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I Einleitung 1 _____________________________________________________________________

I Einleitung

Die Analyse von Thrombozytenfunktionsstörungen beim Hund hat eine hohe klinische Relevanz in der Hämostasediagnostik. Sie dient sowohl zum Aufdecken der von-Willebrand-Erkrankung und seltener angeborener Plättchenfunktionsdefekte wie z.B. der Glanzmann-Thrombasthenie und der Basset-Hound-Thrombopathie als auch erworbener Funktionsstörungen, die nicht selten in Folge anderer systemischer Erkrankungen wie z.B. Tumoren, Nieren- oder Lebererkrankungen auftreten. Die am häufigsten verwendeten Standardverfahren sind dabei die kapilläre In-vivo- Blutungszeit (Nolte et al., 1997), die automatische Thrombozytenfunktionsanalyse mittels Plättchenfunktionsanalysegerät (Mischke und Keidel, 2003), die Thrombozytenaggregation nach der Born-Methode (Born, 1962) und die Vollblut- Impedanzaggregometrie.

In der folgenden Arbeit wird ein neues bislang vorwiegend beim Menschen eingesetztes Vollblut-Impedanzaggregometer (Multiplate-Analyser) auf seine Anwendbarkeit bei Hunden getestet. Dieses Messgerät wäre im Vergleich zu bisher genutzten Impedanzaggregometern aufgrund seiner einfacheren Handhabung sowie probenmaterial- und zeitsparender Messtechnik nicht nur für Einsätze in der Forschung, sondern auch als fester Bestandteil der Hämostasediagnostik in Routinelaboren geeignet (Calatzis et al., 2006). Es sollen die Eignung unterschiedlicher Agonisten (ADP, Kollagen, Arachidonsäure, Ristocetin und Thrombinrezeptor-aktivierende Peptide [TRAP]) in unterschiedlichen Konzentrationen, die Präzision des Gerätes, Referenzwerte und Einflüsse unterschiedlicher Antikoagulantien untersucht werden.

In einem weiteren Teil dieser Dissertation soll der Einfluss von portosystemischen Shunts (PSS) auf die primäre Hämostase bei Hunden untersucht werden. Der portosystemische Shunt ist eine bei jungen Hunden verbreitete Gefäßmissbildung, die unter anderem mit einer eingeschränkten Leberfunktion und postoperativer Blutungsneigung einhergeht (Willis et al., 1989; Kummeling et al., 2006). Während beim Hund in größeren Studien bisher in erster Linie die sekundäre Blutgerinnung im

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I Einleitung 2 _____________________________________________________________________

Zusammenhang mit PSS untersucht wurde (Niles et al., 2001; Kummeling et al., 2006), gibt es zu einer möglichen Thrombozytenfunktionsveränderung wenige Untersuchungen mit teilweise widersprüchlichen Ergebnissen (Willis et al., 1989;

Schulze, 1998; Keidel, 2001).

Das Ziel des zweiten Teils der vorliegenden Studie ist es daher den Einfluss von PSS bei jungen Hunden auf die primäre Hämostase zu untersuchen. Hierbei sollen sowohl das im ersten Teil der Dissertation beschriebene und evaluierte neue Impedanzaggregometer, sowie die kapilläre Blutungszeit, die automatische Thrombozytenfunktionsanalyse und die Thrombozytenaggregation nach der Born- Methode zum Einsatz kommen.

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II Whole blood platelet aggregation in dogs 3 _____________________________________________________________________

Measurement of platelet function in dogs using a novel impedance aggregometer

K. Kalbantner, A. Baumgarten und R. Mischke*

Small Animal Clinic, Hannover School of Veterinary Medicine, Bischofsholer Damm 15, D-30173 Hannover, Germany

• Corresponding author: Tel.: +49 511/856-8165; Fax: +49 511/856-7686; E- mail address: Reinhard.Mischke@tiho-hannover.de (Reinhard Mischke)

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Abstract

The aim of this study was to optimise the technique and establish reference values for whole blood aggregometry in dogs using a novel multiplate analyser.

Measurements were performed on the hirudin-anticoagulated blood of healthy dogs using a wide range of agonists. Optimal agonist concentrations were 10 µmol/L of

adenosine diphosphate, 5 µg/mL of collagen, and 1 mmol/L of arachidonic acid.

Ristocetin (at 0.2 and 1 mg/mL) and thrombin receptor activating peptide (TRAP-6 at 32 and 160 µmol/L) did not consistently induce platelet aggregation. Coefficients of variance for within-run imprecision (n = 10 repetitions) varied from 5% to 18%.

Measurement signals were significantly higher when analyses were performed on standard samples (hirudin-anticoagulated blood) compared to citrated blood or blood samples anticoagulated with citrate buffer, regardless of whether or not re- calcification was performed (P < 0.05). The findings indicate that the analyser is suitable for the investigation of platelet aggregation in dogs and analysis should be performed on hirudin-anticoagulated blood using optimised agonist concentrations.

Keywords: Platelet aggregation; Impedance aggregometry; Dog; Reference values;

Precision; Anticoagulant

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Introduction

The measurement of platelet aggregation is one of the most important in vitro methods of assessing platelet function (Dyszkiewicz-Korpanty et al., 2005). There are two main methods of measuring platelet aggregation in vitro following the addition of agonists (Ries et al., 1986; Mascelli et al., 1997; Dyszkiewicz-Korpanty et al., 2005).

The turbidimetric method measures the increase of light transmission in platelet-rich plasma during the aggregation (Born, 1962; Dyszkiewicz-Korpanty et al., 2005), while impedance aggregometry measures the increase in the electrical resistance in blood resulting from platelet accumulation (Dyszkiewicz-Korpanty et al., 2005; Tóth et al., 2006).

Although the turbidimetric method has the advantage that the platelet count can be adjusted to a defined value (Dyszkiewicz-Korpanty et al., 2005), the preparation of the platelet rich plasma required is time consuming, requires large sample volumes and experienced personnel (Mascelli et al., 1997). This preparation procedure can also alter the quality of the sample through loss of large platelets with increased or decreased reactivity (Dyszkiewicz-Korpanty et al., 2005). In contrast, the fact that impedance aggregometry does not require cell separations means that shorter preparation times and smaller sample volumes can be achieved (Ingerman-Wojenski et al., 1984; Mascelli et al., 1997; Calatzis 2007; Görlinger et al., 2007). Furthermore, lipaemic samples are more effectively evaluated by impedance aggregometry (Toth et al., 2006). Impedance aggregometry on whole blood may also better reflect in vivo platelet functionality, given that platelets can interact with other blood cells.

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While neither assessment method successfully mimics in vivo platelet aggregation, the electrical probes of the impedance method create an artificial surface that may more successfully replicate in vivo platelet aggregation, which typically occurs on injured or inflamed vascular surfaces. This contrasts with platelet in liquid phase in the turbidimetric method (Mascelli et al., 1997; Dyszkiewicz-Korpanty et al., 2005). The aim of this study was to optimise a novel multiplate analyser for platelet aggregometry on whole blood in dogs and in particular to establish reference values, proof the imprecision and test the influence of anticoagulant.

Materials and methods

Study design

In a pilot experiment, five agonists (adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin, and thrombin receptor activating peptide (TRAP;

TRAP-6 (Ser-Phe-Leu-Leu-Arg-Asn)) were tested over a wide range of concentrations in hirudin-anticoagulated blood from 20 (ADP at 1, 1.5, 2, 2.5, 3, 4, 5, 7.5, 10 and 20 µmol/L, collagen at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 10 and 20 µg/mL and AA at 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 1 and 2 mmol/L) or 10 healthy dogs (ristocetin at 0.2 and 1 mg/ml and TRAP at 32 and 160 µmol/L), respectively Based on these results, additional measurements were performed on samples from further 20 dogs with the following agonist concentrations: ADP at 5, 7.5, 10 and 20 µmol/L, collagen at 2, 2.5, 3, 4, 5 and 10 µg/mL and AA at 0.1, 0.2, 0.3, 0.4, 0.5 and1 mmol/L.

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Reference values were calculated for selected agonist concentrations based on the results of analysis of samples from 50 healthy dogs (see below). Within-run imprecision was investigated at selected agonist concentrations. At each agonist concentration, series of 10-fold measurements were performed on the hirudin- anticoagulated blood samples of two healthy dogs and one thrombocytopenic dog (platelet count 33,000/ µL).

In 10 further dogs, the influence of anticoagulant was studied at selected agonist concentrations. Citrate- and buffered citrate-anticoagulated blood samples were collected from these animals in addition to the standard hirudin-anticoagulated sample. Platelet aggregation analyses in samples anticoagulated with citrate or citrate buffer were performed with or without the addition of calcium chloride.

The experimental procedure was approved by the appropriate ethics committee (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, reference number 07A 514).

Animal selection

Blood was collected from 60 clinically normal male and female adult dogs of various breeds, all had normal haematological and biochemical profiles.

The 50 dogs used in calculating the reference values included 14 mongrels (28%), seven Labrador Retriever (14%), three Golden Retriever (6 %), two (4 %) of each of the following breeds: Australian Shepherd, Beagle, Boxer, Flat-coated Retriever,

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Husky, Nova Scotia Duck Tolling Retriever, and one (2 %) each of the following breeds: Bernese Mountain dog, Bolonka Zwetna, Dalmatian, French bulldog, German Short-haired Pointer, German Spaniel, German Wire-haired Pointer, Irish Wolfhound, Jack Russel Terrier, Lagotto Romagnolo, Romanian Mioritic sheepdog, Rottweiler, Shar Pei and Weimaraner. Twenty-seven of the dogs (54%) were male (14 intact, 13 castrated) and 23 (46 %) were female (12 intact, 11 neutered). The dogs were between 1 and approximately 12 years old (median age of 3 years and 4 months).

Blood sample collection

Samples were collected from the saphenous or cephalic vein using sterile disposable needles (1.2 x 40 mm) with minimal pressure used to raise the vein. The blood was collected into either 4.5 mL S-Monovette plastic tubes covered with hirudin (Sarstedt) as recommended by the manufacturer of the multiplate analyser, or, to facilitate the comparison of different anticoagulants, into plastic tubes containing citrate solution (one part 0.11 mol/L sodium citrate to nine parts blood) or citrate buffer (a 3.8 mL S- Monovette tube containing 0.129 mol/L tri-sodium citrate/citric acid buffer solution, pH 5.5, (Sarstedt)). Blood and anticoagulant were immediately thoroughly mixed by careful swaying.

Following the manufacturer`s recommendations for the analysis of human blood, measurements were performed between 30 min and 3 h after sample collection, during which time the samples were held at room temperature.

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Platelet aggregation measurements

The novel impedance aggregometer under assessment (Multiplate analyser, Dynabyte Medical) has five parallel test channels (Calatzis et al., 2006; Toth et al., 2006;

Calatzis, 2007; Görlinger et al., 2007). The single-use test cell has a pipette-intake, a cup portion with the dual sensor unit protruding into the blood which is stirred by a Teflon-coated stirring magnet and a jack portion which connects the test cell to the device and records the electrical resistance between the sensor wires. The dual sensor units consist of silver-coated, highly conductive copper sensor wires of 0.3 mm diameter and 3.2 mm length. The impedance change determined by each sensor is recorded independently and provides an internal control. During the analysis the sample-reagent mixture was stirred at 800 rpm.

The test was performed as recommended by the manufacturer. Isotonic sodium chloride solution (300 µL) was pre-heated to 37 ^oC and pipetted into the test cells and 300 µL of blood sample anticoagulated with hirudin, citrate or citrate buffer was added to achieve a 1:2 dilution. To test the influence of re-calcification, samples of citrate or citrate-buffer-anticoagulated blood were also measured using 300 µL of sodium chloride solution containing 3 mmol/L CaCl2. After 3 min incubation and stirring at 37°C, measurements were initiated by adding 20 µL of the appropriate agonist solution (sourced from Dynabyte Medical). The impedance change caused by the adhesion and aggregation of platelets on the electrodes was continuously detected.

As distinct from the standard 6 min test time for human blood, a recording time of 12 min was used in our pilot study allowing the curves to almost plateau.

The data registered by the sensors created two aggregation curves (Fig. 1),

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serving as an internal control. If the area under the aggregation curve (AUC) differed by >20 % for the two curves and/or their correlation coefficient was < 0.98, the aggregometer gave a warning signal and the measurement was repeated. The mean values of the two determinations were expressed in arbitrary ‘aggregation units’ (AU) reflecting the increased impedance during the measurement. The results automatically provided by the system are the AUC (unit: AU*min) which expresses the aggregation response over the registration interval, the maximum aggregation (in AU), the velocity (in AU/min - the maximum rise of the aggregation curve) and the

‘difference’ (based on AUC values (%)) and Pearson`s correlation coefficient between the two curves.

All parameters except ‘difference’ were based exclusively on measurements obtained without a warning signal. In cases where measurements were repeated,

‘difference’ was based on a mean value with measurements resulting in a warning signal also included.

Statistical analysis

Data were presented in modified box- and whisker-plots. Outside values (>upper quartile + 1.5 x inter-quartile range or <lower quartile - 1.5 x inter-quartile range) and far outside values (> upper quartile + 3 x inter-quartile range or <lower quartile – 3 x inter-quartile range) were marked separately. Reference values were defined on the basis of the 2.5 % and 97.5 % quantiles of the results on analysis of the samples from 50 dogs.

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Data were tested for normal distribution using Kolmogorov-Smirnov test.

Since all the parameters in the different experiments had a normal distribution, comparison of different experimental groups was performed using one way analysis of variance (ANOVA). With the exception of the ‘difference’ parameter, ANOVA was performed with the option of repeated measurements. Where indicated, paired t tests were performed for comparison of individual agonist concentrations or anticoagulants. P values < 0.05 were considered significant. Results of repeated measurements (within-run imprecision) were summarised by their mean, standard deviation, and coefficient of variance (CV).

Results

Optimisation of agonist concentrations

The range of concentrations of ristocetin and TRAP did not induce significant platelet aggregation in healthy dogs (n = 10). Median values for the AUC were ≤ 134 AU*min (0.2 mg/mL risotcetin, 71.0 AU*min; 1mg/mL risotcetin, 58.5 AU*min; 32 µmol/L TRAP, 74.0 AU*min; 160 µmol/L TRAP, 134 AU*min).

One-way ANOVA revealed significant differences between the AUC obtained with the different concentrations of the agonists ADP, collagen and AA in the pilot experiment on 20 healthy dogs (Fig. 2) as well as for the results from the 40 samples treated with various agonist concentrations (P < 0.05) (Tables 1–3). In addition,

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II Whole blood platelet aggregation in dogs 12 _____________________________________________________________________

significant differences were found between selected concentrations of ADP and AA for velocity (P < 0.05) (Tables 1 and 3). In contrast, maximum aggregation values or imprecision based on ‘differences’ (between AUC values measured by the two sensors within test cells) did not indicate significant differences between the selected concentrations of any of the agonists.

The pilot experiment on samples from 20 dogs indicated lower AUC values and/or higher inter-individual variation at ADP concentrations <5 µmol/L (Fig. 2a).

Comparisons of selected ADP concentrations measured in samples from 40 dogs demonstrated that the AUC and velocity values were significantly higher at 10 µmol/L than with 5, 7.5 or 20 µmol/L (P < 0.05). The lowest inter-individual variation was observed at concentrations of 7.5 and 10 µmol/L (Table 1).

The pilot experiment on samples from 20 dogs indicated lower AUC values and/or higher inter-individual variation at collagen concentrations between <2 µg/mL when compared to concentrations of 2–10 µg/mL. The highest collagen concentration (20 µg/mL) also showed significantly lower AUC values when compared to collagen concentration tested (20 µg/mL) also produced significantly lower AUC values when compared to collagen concentrations of 3–10 µg/mL (P < 0.05) (FIG. 2b). The partially significant differences between the AUC values obtained with selected collagen concentrations (2–10 µg/mL) on the 40 samples did not appeared to conform to any particularpattern; the lowest inter-individual variation was observed for measurements with collagen concentrations of 3–5 µg/mL (Table 2).

Samples from 20 dogs indicated significantly lower AUC values at AA

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concentrations <0.1 mmol/L (0.05 and 0.075 mmol/L) or at 2 mmol/L compared to 0.1–1 mmol/L (P > 0.05) (Fig. 2c). For measurements at selected concentrations (0.1–

1 mmol/L) on the samples from the 40 dogs, significant differences between individual AA concentrations mainly reflect higher values of the AUC and velocity for the concentrations 0.4–1 mmol/L compared to lower concentrations (P < 0.05).

The lowest inter-individual variation was observed with 0.5 and 1 mmol/L AA.

The number of repeat measurements required because of an instrument alarm signal was low (≤3/40 measurements) and was not influenced by the type or concentration of agonist (Tables 1–3).

Reference values

Reference values calculated for selected concentrations of agonists ADP, collagen and AA on samples of 50 healthy dogs are detailed in Table 4. In terms of maximum values and minimal inter-individual variation 10 µmol/L ADP, 5 µg/ml collagen and 1 mmol/l AA performed best. The highest inter-individual variation was found using AA where AUC values varied by more than a factor of three, whereas with ADP and collagen these values varied by approximately two-fold.

Within-run imprecision

Mean CVs for measurements (AUC) of two normal samples using different concentrations of the agonists ADP, collagen and AA varied between 5 and 14 %.

Equivalent measurements for the thrombocytopenic animal were between 7% and

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18% (Table 5). The maximum aggregation and velocity parameters exhibited similar degrees of imprecision. In general the highest level of precision was obtained using collagen as an agonist.

Comparison of anticoagulants

Aggregation (AUC) values were significantly higher when analyses were performed on samples of hirudin-anticoagulated blood compared to samples in citrate or citrate buffer (P < 0.05), regardless of whether re-calcification was performed before the measurement or not (Fig. 3). The values for citrate-anticoagulated blood were slightly higher values (P < 0.05) than for citrate-buffer-anticoagulated blood when ADP or collagen were used as agonists, the exception being re-calcified samples were treated with 20 µmol/L ADP. Re-calcification of the citrate-anticoagulated samples had a limited and varying effect on the measurement signal. The AUC values of the ADP- induced platelet aggregation on citrate-anticoagulated blood (with 10 µmol/L ADP only) and citrate-buffer-anticoagulated blood were significantly higher with re- calcification. In contrast, measurements without re-calcification werethan for re- calcified samples when measurements were performed in citrate-anticoagulated blood with AA. There was no statistically detectable effect of re-calcification on AA- induced platelet aggregation in citrate-buffer-anticoagulated blood or on collagen- induced platelet aggregation in citrate- or citrate buffer-anticoagulated blood.

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Discussion

Our results indicate that 10 µmol/L of ADP, 5 µg/mL of collagen and 1 mmol/L of AA are the most appropriate agonist concentrations to use in assessing platelet aggregation with this novel impedance aggregometer. At concentrations assessed in this study ristocetin and TRAP-6, a shortend form of the human TRAP- peptide, did not induce consistent platelet aggregation.

In the pilot study ADP, collagen and AA were used at concentrations in a wide range around those used with human blood samples and only those agonist concentrations that induced significant aggregation in all healthy animals were used in the further experiments. Given that the sensitivity of detecting platelet function disorders is higher near the threshold concentration (the lowest agonist concentration resulting in maximum aggregation) (Mischke and Schulze, 2004), agonist concentrations remarkable higher than the suspected threshold level were also excluded from further measurements. Surprisingly, collagen and AA added in high concentrations (20 µg/mL and 2 mmol/L, respectively), induced lower AUC values than many of the remaining agonists at lower concentration. This may have been the result of the formation of platelet aggregates in the liquid phase of the sample prior to reaching the electrodes.

Since there was no statistical significant difference between different pre- selected concentrations of the agonists with respect to imprecision based on difference values and CVs, the optimal agonist concentration was determined as that which induced maximum aggregation and minimal inter-individual variation. Although such

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criteria would appear ideal in optimising the sensitivity of detecting canine patients with reduced platelet function, this finding requires further clinical verification.

The findings that low agonist concentrations were associated with a wide reference value variation and maximum values were found within the upper reference range achieved with higher agonist concentrations, suggest the method is unsuitable for detecting increased platelet reactivity in individual patients.

A reduction in the reference range may be achieved by the establishment of breed-related ranges, as ADP-induced platelet aggregation varies between dog breeds (Nielsen et al., 2007). Age and gender also influence platelet aggregation in humans and laboratory animals (Gleerup and Winther, 1995; Okazaki et al. 1998). However, it is debatable if breed- and age-related reference values are practicable in veterinary medicine, particularly as such reference ranges should preferable be laboratory- specific (Zhou and Schmaier, 2005).

The optimal agonist concentrations determined by the this study are slightly higher than those cited for human diagnostics (Calatzis et al., 2006). Previous studies in dogs (albeit using a different impedance aggregometer) used significantly lower collagen concentration (1 µg/ml) (Soloviev et al., 1999; Sato and Harasaki, 2002), although no optimisation for individual species was carried out. It must also be considered that although equine tendon collagen-based reagents were used in the above and in the current studies, different sources of reagents may result in variations in quality. In contrast, the optimal ADP and AA concentrations in the current study were very similar to those found previously (Soloviev et al., 1999; Sato and Harasaki, 2002; Nielsen et al., 2007).

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The results of our experiment indicate that ristocetin, an effective agonist of platelet aggregation in humans, sheep, pigs, horses or buffalos (Soloviev et al., 1999;

Pelagalli, et al. 2002; Sato et al., 2002; Scharbert, et al. 2006), does not consistenly induce aggregation in dogs. This was even at concentrations of up to 1 mg/mL, five times the concentration recommended for use with human blood samples. This finding is supported by that of previous studies using both impedance aggregometry (Soloviev et al., 1999) and turbidimetry (Mischke and Schulze, 2004. The fact that TRAP-6 did not induce significant platelet aggregation in dogs probably reflects species specific differences in the thrombin receptor.

Our findings that hirudin-anticoagulated canine whole blood produced higher AUC values than did citrate- or citrate-buffer-anticoagulated samples is in line with studies of human blood (Wallén et al., 1997; Tóth et al., 2006). In these studies, performed on the same device as in the present study (Tóth et al., 2006) or on a Chrono-Log instrument (Wallén et al., 1997), ADP- and collagen-induced aggregation was significantly less in whole blood anticoagulated with citrate than in hirudin- anticoagulated blood. It is unlikely that the slightly lower percentage of blood in the citrated sample contributed significantly to the difference between the two anticoagulants. Furthermore, calcium binding must not be a major mechanism underpinning this discrepancy given that re-calcification had little effect and did not influnce AA-induced aggregation in citrated blood-

Despite the 10 repetitions to investigate within-run imprecision in the present study, the mean CVs of 5–14 % on blood of healthy dogs using different agonists in optimal

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concentrations were similar to those reported for smaller series of 4–5 repetitions on human blood (8–11%) using different agonists and a Chrono-Log instrument (Ivandic et al., 2006, 2007). The internal duplicate measurements within the test cells and the calculation of mean values by the machine probably reduced imprecision. When considering the relatively delicate mechanism of platelet aggregation, the imprecision values defined in the present study can be regarded as acceptable.

Conclusions

Our study has demonstrated that a novel whole blood impedance aggregometer can be used to reproducibly assess platelet aggregation in dogs. For such assessements, hirudin-anticoagulated blood has been identified as the material sample of choice and the study has defined the optimal concentrations at which various agonists should be used.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

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II Whole blood platelet aggregation in dogs 22 _______________________________________________________________________________________________________________________

Table 1

Reference values of parameters of impedance aggregometry in 40 healthy dogs using different concentrations of the agonist ADP. The table includes the difference between the AUC values of the measurement in duplicate within test cells as well as P values of the statistical comparison of the different ADP concentrations using ANOVA and t-test.

Area under the curve [AU*min]

Maximum aggregation [AU]

Velocity [AU/min]

Difference

[%]

ADP

concentration [µmol/L]

Median

Inter- quantile-

range†

5%- −95%-

quantile Median

range†

5%-

−95%- quantile

Median

range†

5%-

−95%- quantile

Median Minimum− maximum

Repeated measurements°

(numbers)

5^a 2070 2348 960−3308 145 153 66.0−219 16.0 21.2 8.0−29.2 4.0 0.4−15.6 2

7.5^b 2188 1776 1397−3173 151 119 93.6−212 16.7 26.1 9.8−35.9 4.6 0.1−17.7 1

10^c 2341 1943 1489−3432 164 128 105.3−233 18.5 26.2 12.0−38.2 2.7 0.1−15.8 0

20^d 2218 2121 1231−3352 151 131 87.1−218 17.4 21.6 10.3−31.9 4.2 0.3−11.6 0

P-value ANOVA

0.044

0.068

0.003

0.131

Significant differences

t-test

a/c**, b/c**, c/d**

---

a/c**, b/c***, c/d*

---

AU = Aggregation unit

Number of required measurement repetitions when the area under the curve (AUC) differed by more than 20& for the two curves and /or their correlation coefficient was <0.98.

° if difference > 20% or/and correlation coefficient of the two aggregation curves created within the test cell < 0.98

†difference between 95% and 5% quantile

* P < 0.05 ** P < 0.01 *** P < 0.001

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Table 2

Reference values of parameters of impedance aggregometry in 40 healthy dogs using different concentrations of the agonist collagen. The table includes the difference between the AUC values of the measurement in duplicate within test cells as well as P values of the statistical comparison of the different ADP concentrations using ANOVA and t-test. .

Difference

[%]

Collagen

concentration [µg/ml]

Median

range†

5%- −95%-

range†

5%-

−95%- quantile

Median

Inter- quanitle-

range†

5%- −95%-

quantile Median Minimum− Maximum

(numbers)

2^a 2666 2921 450−3371 198 306 39.6−346 21.8 29.2 5.3−34.5 5.4 0.6−13.6 1

2,5^b 2595 2127 1436−3563 203 140 111−251 21.3 17.1 11.1−28.2 3.3 0.0−17.4 2

3^c 2685 1733 1759−3492 204 112 143−255 20.6 18.3 11.9−30.2 3.0 0.0−16.0 2

4^d 2629 1449 1955−3404 198 111 141−252 21.2 16.7 15.5−32.2 5.3 0.4−19.8 0

5^e 2769 1407 2023−3430 207 91 155−246 23.2 22.2 15.9−38.1 3.4 0.1−28.7 1

10^f 2609 1709 1792−3501 191 111 138−249 21.9 20.2 14.1−34.3 3.5 0.0−15.6 0

P-value ANOVA

0.019

0.243

0.267

0.773 Significant

differences t-test

a/c*, b/c*, b/e*, a/f*

---

--- ---

* P < 0.05 ** P < 0.01 *** P < 0.001

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Table 3

Reference values of parameters of impedance aggregometry in 40 healthy dogs using different concentrations of the agonist arachidonic acid. The table includes the difference between the AUC values of the measurement in duplicate within test cells as well as P values of the statistical comparison of the different ADP concentrations using ANOVA and t-test.

Difference

[%]

Arachidonic acid [mmol/L]

Median

range†

5%- −95%-

range†

5%- −95%-

range†

5%- −95%-

quantile Median Minimum− maximum

(numbers)

0.1^a 1997 3049 54−3103 159 209 9.0−218 14.8 20.8 3.2−24.0 2.8 0.2−63.3 3

0.2^b 2047 2390 500−2890 156 152 61.2−213 14.9 20.9 5.1−26.0 3.4 0.1−26.4 1

0.3^c 2178 2204 636−2840 162 162 55.7−217 15.7 18.1 5.6−23.7 2.6 0.0−11.8 0

0.4^d 2210 2188 737−2925 161 129 76.7−206 16.4 20.7 6.5−27.2 2.6 0.1−16.9 2

0.5^e 2197 2000 942−2942 155 149 73.9−223 16.5 19.3 6.3−25.6 3.4 0.0−16.7 0

1^f 2290 2112 1116−3228 154 153 74.7−228 17.6 18.6 8.8−27.4 3.5 0.1−14.5 2

P-value ANOVA

< 0.001

0.305

< 0.001

0.095

Significant differences

t-test

a/c**, a/d**, b/d*, a/f**, b/f**, c/f*, a/e***, b/e**, c/e*, d/e*

---

a/c**, a/d**, b/d**, a/e**, b/e**, a/f***, b/f***, c/f*, d/f**, e/f*

---

* P < 0.05 ** P < 0.01 *** P < 0.001

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II Whole blood platelet aggregation in dogs 25 _____________________________________________________________________________________________________________________

Table 4

Reference values of parameters of impedance aggregometry in 50 healthy dogs using different concentrations of the agonists ADP, collagen and arachidonic acid.

Agonist (concentration)

Median

2.5%- –

97.5%-quantile Median

2.5%- –

97.5%-quantile Median

2.5%- – 97.5%-quantile ADP

(5 µmol/l) 2070 700–3327 144 56.3–219 16.7 7.0–29.3

ADP

(7.5 µmol/l) 2164 1185–3186 149 81.4–213 16.7 9.8–36.2

ADP

(10 µmol/l) 2341 1481–3448 157 105–234 18.5 12.0–38.4

ADP

(20 µmol/l) 2218 1229–3361 149 87–219 17.4 10.2–32.1

Collagen

(3 µg/ml) 2614 1479–3500 197 122–255 20.6 6.2–30.3

Collagen

(5 µg/ml) 2736 2017–3460 205 155–246 23.1 15.9–38.4

Collagen

(10 µg/ml) 2590 1780–3501 190 138–249 21 14.0–34.5

Arachidonic acid

(0.5 mmol/l) 2161 631–2954 152 51.6–223 16.2 5.0–26.5

Arachidonic acid

(1 mmol/l) 2279 1011–3457 150 73.9–228 17.6 8.8–27.4

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Table 5

Within-run imprecision (10 repetitions) of parameters of whole blood aggregometry in dogs. The mean, standard deviation (SD), coefficient of variation (CV), and mean values of the CV of samples from two healthy dogs and from a dog with thrombocytopaenia.

Area under the curve

(AU*min) Maximum aggregation

(AU) Velocity

(AU/min)

Normal Abnormal Normal Abnormal Normal Abnormal

Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3

Agonist (concentration)

Mean

± SD CV (%)

Mean

± SD CV (%)

Mean

CV (%)

Mean

± SD CV (%)

Mean

± SD CV (%)

Mean

± SD CV (%)

Mean CV (%)

Mean

± SD CV (%)

Mean

± SD CV (%)

Mean ± SD CV

(%) Mean

CV (%)

Mean

± SD CV (%) ADP

(5 µMol/L)

2227

±144 6.5 1886

±230 12.2 9.3 1377

±241 17.5 160

±10.4 6.5 119

±14.8 12.4 9.5 94.5

±14.5 15.3 16.2

±1.5 9.1 17.9

±2.5 13.7 11.4 10.2

±1,9 18.4 ADP

(7.5 µMol/L)

2160

±152 7.0 2202

±309 14.0 10.5 1505

±271 18.0 158

±12.7 8.0 149

20.9 14.0 11.0 100.6

±17.9 17.8 15.7

±1.3 8.5 19.2

±2.7 14.0 11.2 11.5

±1.7 14.8 ADP

(10 µMol/L)

2212

±132 6.0 2252

±395 17.5 11.8 1419

±215 15.2 160

±9.6 6.0 143

±28.3 19.8 12.9 95.1

±13.4 14.1 16.9

±1.4 8.1 19.8

±3.2 16.2 12.2 11.1

±1.8 16.4 ADP

(20 µMol/L)

2266

±126 5.6 2107

±255 12.1 8.8 1423

±191 13.4 153

±9.9 6.4 137

±16.3 11.9 9.2 94.5

±13.0 13.7 17.3

±1.3 7.6 18.4

±3.4 18.7 13.2 11.6

±0.87 7.5 Collagen

(3 µg/mL)

2372

±238 10.0 2233

±176 7.9 9.0 1735

±129 7.4 176

±15.4 8.7 170

±14.4 8.5 8.6 133

±10.5 7.9 20.3

±2.6 12.8 17.7

±2.5 14.1 13.5 10.1

±0.53 5.3 Collagen

(5 µg/mL)

2600

±123 4.7 2478

±135 5.5 5.1 1623

±147 9.1 191

±10.1 5.3 184

±12.2 6.6 6.0 128

±11.0 8.6 21.5

±1.6 7.3 18.1

±1.7 9.2 8.3 10.1

±0.86 8.5 Collagen

(10 µg/mL)

2650

±154 5.8 2343

±77 3.3 4.6 1596

±198 12.4 193

±13.2 6.9 173

±7.6 4.4 5.7 123

±16.1 13.1 20.3

±1.7 8.5 17.2

±1.6 9.4 9.0 9.7

±1.0 10.5 Arachidonic

acid (0.5 mMol/L)

2772

±305 11.0 2097

±344 16.4 13.7 1612

±211 13.1 205

±19.5 9.5 149

±24.2 16.3 12.9 111

±13.9 12.5 17.1

±3 17.7 13.9

±2.2 16.0 16.9 12.2

±1.6 13.2 Arachidonic

acid (1 mMol/L)

2166

±381 17.6 2500

±150 6.0 11.8 1356

±172 12.7 168

±38.3 22.9 176

±12.4 7.0 15.0 89.9

±9.3 10.3 14.9

±2.8 19.0 19.2

±1.9 10.0 14.5 11.0

±1.9 17.6 AU = Aggregation unit.

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Fig. 1 Schematic diagram of the two aggregation curves of one single-use test cell of the multiplate impedance aggregometer illustrating the area under the curve (AU*min), the maximum aggregation, and velocity (AU/min) relative to time in min.

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0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500

1 1.5 2 2.5 3 4 5 7.5 10 20

ADP (µMol/L)

AUC (AU*min)

Outside values Far outside values

a

0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500

0.5 1 1.5 2 2.5 3 4 5 10 20

Collagen (µg/mL)

AUC (AU*min)

b

0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500

0.05 0.075 0.1 0.2 0.3 0.4 0.5 1 2

Arachidonic acid (mMol/L)

AUC (AU*min)

c

Fig. 2. Area under the curve (AUC) values of platelet aggregation in hirudin-anticoagulated blood of 20 healthy dogs using different concentrations of ADP (A), collagen (B) and

arachidonic acid (C) as agonist. AU = aggregation unit