• View in gallery
    Figure 1—

    Representative scattergrams of stimulated (A) and unstimulated (B) polymorphonuclear neutrophils (PMN) from 1 healthy dog acquired via flow cytometry and used to determine the threshold for CD61 positivity in other cells. Left dot plots—Polymorphonuclear cells were identified on the basis of their FSC and SSC characteristics (circles). The cells were stained with a phycoerythrin (PE)-labeled antibody specific for the platelet surface molecule CD61. Right dot plots—CD61 versus SSC of unstimulated cells (A) and cells incubated with AA (1 mmol/L) for 20 minutes (B). For all gated PMNs, the percentage of CD61-positive PMNs (squared regions) is shown.

  • View in gallery
    Figure 2—

    Mean ± SD percentage of CD61-positive neutrophils (CD61 + and polymorphonuclear cells) without stimulation (Nil) and after stimulation with various platelet and neutrophil agonists (PMA, collagen [COL], ADP, and epinephrine [EPI]; A) or various concentrations of LPS (B) or AA (C). ADP/COL = ADP plus collagen. EPI/ADP = Epinephrine plus ADP. a–dDifferent letters indicate significant (paired Student t test P < 0.05) differences between values within graphs.

  • View in gallery
    Figure 3—

    Mean ± SD FSC (A–C) and SSC (D–F) of unstimulated neutrophils and of neutrophils after stimulation with various agonists. Notice that the y-axis does not start at 0. See Figure 2 for remainder of key.

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Platelet-neutrophil aggregate formation in blood samples from dogs with systemic inflammatory disorders

Brigitte Hedwig DircksSmall Animal Clinic, University of Veterinary Medicine Hanover, 30559 Hanover, Germany.

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Reinhard MischkeSmall Animal Clinic, University of Veterinary Medicine Hanover, 30559 Hanover, Germany.

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Hans-Joachim SchuberthInstitute for Immunology, University of Veterinary Medicine Hanover, 30559 Hanover, Germany.

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Abstract

Objective—To evaluate platelet-neutrophil aggregate (PNA) formation and neutrophil shape as indicators of neutrophil activation in dogs with systemic inflammatory diseases and after blood sample incubation with various platelet and neutrophil agonists.

Animals—20 dogs with systemic inflammatory response syndrome (SIRS) and 10 healthy Beagles.

Procedures—Neutrophils were isolated from blood samples directly after blood sample collection and after incubation of blood samples with phorbol myristate acetate, collagen, adenosine diphosphate, epinephrine, or various concentrations of lipopolysaccharide or arachidonic acid. CD61+ neutrophils as an indicator of PNA formation were evaluated, and neutrophil size and granularity were assessed via flow cytometry.

Results—Dogs with SIRS had more PNA formation, larger neutrophil size, and less granularity relative to control dogs, but no differences were evident when these dogs were grouped by whether they had sepsis (n = 6) or disseminated intravascular coagulation (12). A significant increase in PNA formation occurred after neutrophil incubation with all agonists, and incubation with phorbol myristate acetate elicited the strongest response. Neutrophils increased in size and decreased in granularity after incubation with all agonists except epinephrine. Incubation with lipopolysaccharide or arachidonic acid resulted in a dose-dependent effect on PNA formation and neutrophil shape.

Conclusions and Clinical Relevance—SIRS appeared to increase the degree of PNA formation and neutrophil shape change. Similar changes after neutrophil incubation with platelet agonists suggested that platelet activation has a role in PNA formation. Additional studies are necessary to determine the clinical importance and diagnostic value of PNA formation in dogs with SIRS and sepsis.

Abstract

Objective—To evaluate platelet-neutrophil aggregate (PNA) formation and neutrophil shape as indicators of neutrophil activation in dogs with systemic inflammatory diseases and after blood sample incubation with various platelet and neutrophil agonists.

Animals—20 dogs with systemic inflammatory response syndrome (SIRS) and 10 healthy Beagles.

Procedures—Neutrophils were isolated from blood samples directly after blood sample collection and after incubation of blood samples with phorbol myristate acetate, collagen, adenosine diphosphate, epinephrine, or various concentrations of lipopolysaccharide or arachidonic acid. CD61+ neutrophils as an indicator of PNA formation were evaluated, and neutrophil size and granularity were assessed via flow cytometry.

Results—Dogs with SIRS had more PNA formation, larger neutrophil size, and less granularity relative to control dogs, but no differences were evident when these dogs were grouped by whether they had sepsis (n = 6) or disseminated intravascular coagulation (12). A significant increase in PNA formation occurred after neutrophil incubation with all agonists, and incubation with phorbol myristate acetate elicited the strongest response. Neutrophils increased in size and decreased in granularity after incubation with all agonists except epinephrine. Incubation with lipopolysaccharide or arachidonic acid resulted in a dose-dependent effect on PNA formation and neutrophil shape.

Conclusions and Clinical Relevance—SIRS appeared to increase the degree of PNA formation and neutrophil shape change. Similar changes after neutrophil incubation with platelet agonists suggested that platelet activation has a role in PNA formation. Additional studies are necessary to determine the clinical importance and diagnostic value of PNA formation in dogs with SIRS and sepsis.

A part from their role in hemostasis, platelets are involved in the initiation and propagation of inflammatory and immune processes via secretion of cytokines (eg, interleukin-1β and β-thromboglobulin) and interaction with other cells.1–3 Platelet activation leads to expression of adhesion molecules on their surface, mediating platelet aggregation and interaction with other cells such as endothelial cells and leukocytes.4–6 Once activated, platelets release and express P-selectin (CD62P), which allows binding to P-selectin glycoprotein ligand-1 (CD162) expressed on the surface of all leukocytes, thus promoting the initial adhesion between cells.7,8

The interaction of neutrophils with platelets appears to mediate neutrophil accumulation and emigration into inflammatory tissue.9,10 This interaction may also enhance activation, secretion, and aggregation of platelets, thereby amplifying the function of both cell types reciprocally.10 Therefore, PNAs provide an important link between thrombosis and inflammation, and their inhibition might be a target for pharmacological intervention in prothrombotic conditions.11

Higher than typical numbers of PNAs have been found in humans with myeloproliferative disorders,12 coronary artery disease,13 diabetes mellitus,14 and sepsis.15,16 Indeed, detection of interactions between platelets and neutrophils may be a sensitive means of detecting platelet activation.17 For dogs, a whole blood assay to detect platelets bound to monocytes and neutrophils has been described and platelet response to various platelet agonists has been investigated.18 To our knowledge, no data exist regarding the presence of PNAs in dogs with systemic inflammatory diseases.

The very process of measuring PNAs is highly likely to cause in vitro stimulation of the platelets present, causing artificial formation of PNAs and misleading results. To minimize ex vivo PNA formation, we developed a simple and reproducible method in which platelets are separated from neutrophils immediately after blood sample collection. The objective of the study reported here was to measure PNA formation and neutrophil shape (to assess neutrophil activation) with the newly developed test in blood samples from dogs with SIRS and in blood samples from healthy dogs that were incubated with various platelet and neutrophil agonists (PMA, collagen, ADP, epinephrine, LPS, and AA).

Materials and Methods

Animals—Twenty dogs with SIRS evaluated at the University of Veterinary Medicine Hanover Small Animal Clinic between August and December 2010 were prospectively entered into the study. All dogs underwent a CBC, serum biochemical analysis, and coagulation profile as well as extensive diagnostic imaging, including ultrasonography and radiography. Dogs were excluded from the study when they had received whole blood or blood component transfusions within 6 months before study entry. Systemic inflammatory response syndrome was diagnosed when ≥ 2 of the following criteria were present: heart rate > 120 beats/min, respiratory rate > 20 breaths/min, rectal temperature < 38°C or > 39°C, and WBC count > 18,000 or < 5,000 cells/μL.

Twelve of these dogs were suspected of having DIC on the basis of an abnormal coagulation profile (≥ 2 of the following findings: prolonged activated partial thromboplastin time, prolonged prothrombin time, and thrombocytopenia) and detection of an underlying disease known to be associated with DIC. In 6 of the dogs with SIRS, sepsis was diagnosed on the basis of positive results of microbial culture of a blood sample (n = 1 dog) or detection of a septic focus (5). The 5 dogs in which a septic focus was identified had pyoabdomen due to intestinal wound dehiscence, pyometra and subsequently ruptured uterus, or abdominal perforating bite wound (n = 3); phlegmon due to esophageal perforation (1); or septic pyothorax (1).

Ten university-owned healthy Beagles were used as control dogs. The study design was approved by the animal welfare officer of the University of Veterinary Medicine Hanover and by the competent authority (Lower Saxony State Office for Consumer Protection and Food Safety).

Blood sample collection—Blood samples were collected via cephalic venipuncture by use of a 20-gauge needle. The first 2 mL was discarded; the remainder was collected into was collected into tubes containing sodium heparina and processed immediately. This type of tube was chosen because preliminary tests (data not shown) revealed the lowest amount of PNA formation in blood samples from healthy control dogs when sodium heparin was used as an anticoagulant, compared with when EDTA and sodium citrate were used instead.

Sample processing—To minimize ex vivo PNA formation, a flow cytometric test for the measurement of PNA formation after early separation of platelets from neutrophils was developed. Two milliliters of PBSb solution was added to 2 mL of blood and gently mixed. The mixture was subsequently layered over 5 mL of Ficoll-Hypaque solution,c and centrifuged for 30 minutes at 1,100 × g and 4°C with no brake applied. Plasma, interphase, and Ficoll medium were then removed.

Red blood cell lysis was initiated by incubation of the remaining neutrophil-RBC pellet with 4 mL of distilled water and stopped after 20 seconds by adding 4 mL of 2× PBS solution. This lysis procedure was repeated twice, and each lysis step was followed by centrifugation for 10 minutes (4°C) at 300 × g, 250 × g, and 150 × g. Cells were washed with PBS solution and then centrifuged at 100 × g, washed again with PBS solution, and centrifuged at 80 × g.

Isolated and washed neutrophils were incubated for 30 minutes at 4°C with 25 μL of murine anti-human phycoerythrin-conjugated CD61d (2 μg/mL) or 25 μL of murine anti-pig phycoerythrin-conjugated CD163e (IgG1− isotype control). After 2 washing steps with staining buffer (PBS solution, 0.5% bovine serum albuminf, and 0.01 NaN3g), cells were suspended in 300 μL of sterile-filtered PBS solution.

Flow cytometry—Cells were analyzed by means of flow cytometry.h At least 10,000 events (nucleated cells) were acquired for each sample. Separated neutrophil populations contained between 2% and 12% lymphoid cells. In FSC versus SSC plots, neutrophils were identified on the basis of their characteristic FSC and SSC profiles. After gating to segregate neutrophils (individual gates for each sample), the mean FSC (correlating with cell size) and SSC values (correlating with granularity and complexity) were recorded from FSC and SSC plots as variables indicative of neutrophil shape.

After cells were stained with a phycoerythrin-labeled anti-CD61 monoclonal antibody, the percentage of CD61-positive neutrophils was recorded in fluorescence (FL2) detector SSC dot plots. The voltage of the FL2 detector was adjusted so that > 98% of CD61-stained cells of a healthy dog were obtained within the first decade of the FL2 channel. Regular weekly evaluations for quality control performed with fluorescence calibration beadsi were conducted to ensure reproducibility of the voltage settings. The threshold for CD61 positivity was based on stained and gated neutrophils of healthy dogs (Figure 1). Acquired data were analyzed by use of a software package,j and the percentage of CD61-positive cells after quadrant analysis was recorded.

Figure 1—
Figure 1—

Representative scattergrams of stimulated (A) and unstimulated (B) polymorphonuclear neutrophils (PMN) from 1 healthy dog acquired via flow cytometry and used to determine the threshold for CD61 positivity in other cells. Left dot plots—Polymorphonuclear cells were identified on the basis of their FSC and SSC characteristics (circles). The cells were stained with a phycoerythrin (PE)-labeled antibody specific for the platelet surface molecule CD61. Right dot plots—CD61 versus SSC of unstimulated cells (A) and cells incubated with AA (1 mmol/L) for 20 minutes (B). For all gated PMNs, the percentage of CD61-positive PMNs (squared regions) is shown.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.939

Agonist stimulation and PNA measurement—Aliquots of blood (1.8 mL) from healthy Beagles were incubated at 37°C for 20 minutes with 0.2 mL of PBS solution (control samples) or 0.2 mL of the following agonist solutions: PMAk (final concentration, 5 μmol/L), collagenl (20 μg/mL), ADPm (20 μmol/L), epinephrinen (20 μmol/L), collagen (20 μg/mL) with ADP (20 μmol/L), epinephrine (20 μmol/L) with ADP (20 μmol/L), LPSo (1, 2, 5, and 10 μg/mL), or AAp (0.5, 1, and 2 mmol/L). Afterward, 2 mL of PBS solution was added to each vial and the blood was gently mixed. Sample processing and flow cytometric analysis were performed as described for the other dogs.

Statistical analysis—Flow cytometric data were confirmed to be normally distributed with the Kolmogorov-Smirnov test and are reported as mean ± SD. The unpaired Student t test was used to compare findings among dog groups. The paired Student t test was used to assess the effects of agonists on PNA formation and to compare those effects among various concentrations of agonists. Values of hematologic variables between groups were compared via the Mann-Whitney U test. Analysis was performed by use of a spreadsheet application.q Values of P < 0.05 were considered significant.

Results

Animals—Four of the 20 dogs with SIRS were of mixed breed; the remainder represented 16 breeds. These dogs had a median age of 6.4 years (range, 1.5 to 15.6 years). Ten were male (8 castrated and 2 sexually intact), and 10 were female (6 spayed and 4 sexually intact).

Age and sex distributions were similar between septic and nonseptic dogs and between dogs with and without DIC. Dogs with SIRS and DIC had significantly lower platelet counts and prolonged activated partial thromboplastin time and prothrombin time, compared with dogs with SIRS but no DIC (Table 1).

Table 1—

Median (range) hematologic values in dogs with SIRS (n = 20) classified as having DIC (12) versus no DIC (8) or sepsis (6) versus no sepsis (14).

VariableReference limitsDICNo DICSepsisNo sepsis
Neutrophil count (× 103 neutrophils/μL)3–920.9 (1.3–34.5)a25.9 (2.9–45.6)a30.9 (1.3–43.5)a20.1 (1.4–45.6)a
Band neutrophil count (× 103 neutrophils/μL)0–0.51 (0–2.4)a1.4 (0–7.1)a1.6 (0.9–7.1)a0.9 (0–2.5)a
Platelet count (× 103 platelets/μL)150–500129 (96–197)a424 (193–1,040)b239 (96–441)a160 (104–1,040)a
Activated partial thromboplastin time (s)14.5–1923.8 (13.8–38.5)a12 (10–22)b14.4 (12–38.5)a21.2 (10–38.3)a
Prothrombin time (%)75–13044 (29–110)a88 (65–150)b67 (35–95)a74 (29–150)a

Prothrombin time is reported as percentage activity (100% = activity of pooled plasma from healthy dogs); reduced activities correspond to prolonged prothrombin times.

Excluding the reference limit values, values with different superscript letters within a row are significantly (Mann-Whitney U test P < 0.05) different.

PNA formation and neutrophil shape—The percentage of CD61-positive neutrophils was significantly (P = 0.003) higher in the dogs with SIRS than in healthy control dogs. The mean FSC (ie, size) of neutrophils was significantly (P < 0.001) larger and the mean SSC (ie, granularity and complexity) of neutrophils was significantly (P = 0.013) lower in dogs with SIRS than in dogs without SIRS.

When subgroups of dogs with SIRS (with DIC, without DIC, with sepsis, and without sepsis) were compared with healthy control dogs, a significantly higher percentage of CD61-positive neutrophils and mean FSC were evident in all SIRS subgroups. Dogs with SIRS and sepsis, DIC, or no DIC had a significantly higher mean SSC than did healthy dogs, but no significant difference was evident between nonseptic and healthy dogs. Comparison between subgroups (DIC vs no DIC and sepsis vs no sepsis) revealed no significant difference with regard to the percentage of CD61-positive neutrophils, FSC, and SSC.

Blood sample incubation with platelet and neutrophil agonists—A significant increase in the percentage of CD61-positive neutrophils was detected in blood samples from healthy dogs after the samples were incubated with PMA, collagen, ADP, and various concentrations of LPS and AA (Figure 2). The greatest increase in the percentage of CD61-positive neutrophils (89.4 ± 9.8%) occurred after sample incubation with PMA (P < 0.001), compared with the percentage in control blood samples to which no agonist was added (4.7 ± 0.6%).

Figure 2—
Figure 2—

Mean ± SD percentage of CD61-positive neutrophils (CD61 + and polymorphonuclear cells) without stimulation (Nil) and after stimulation with various platelet and neutrophil agonists (PMA, collagen [COL], ADP, and epinephrine [EPI]; A) or various concentrations of LPS (B) or AA (C). ADP/COL = ADP plus collagen. EPI/ADP = Epinephrine plus ADP. a–dDifferent letters indicate significant (paired Student t test P < 0.05) differences between values within graphs.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.939

Addition of ADP to collagen in the incubation mixture resulted in no additional increase in the percentage of CD61-positive neutrophils, compared with the percentage achieved with collagen alone, whereas the addition of ADP to epinephrine did result in an additional increase. Blood sample incubation with LPS and AA at various concentrations resulted in a significant dose-dependent increase in the percentage of CD61-positive cells, compared with the percentage in control samples. Incubation with all tested agonists except epinephrine resulted in a higher mean FSC and a lower mean SSC of the neutrophils, with PMA having the most prominent effect (Figure 3). Addition of ADP to collagen or epinephrine resulted in no additional stimulation. Incubation with LPS and AA resulted in a significant dose-dependent increase in FSC and decrease in SSC, compared with control values.

Figure 3—
Figure 3—

Mean ± SD FSC (A–C) and SSC (D–F) of unstimulated neutrophils and of neutrophils after stimulation with various agonists. Notice that the y-axis does not start at 0. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.939

Discussion

The study reported here was designed to evaluate methods through which neutrophil activation could be detected in blood samples from dogs with SIRS. On the basis of the flow cytometric measurement of CD61 associated with neutrophils, high numbers of PNAs were detected in samples from dogs with septic and nonseptic systemic inflammatory diseases with and without DIC. This finding is in concordance with the increase in PNA formation reported for humans who develop SIRS and uncomplicated sepsis16 or have other inflammatory conditions.13,19

Measurement of platelet adhesion to leukocytes is suggested as a sensitive and reliable method for evaluation of in vivo platelet activation.20 Platelet adhesion could also be of pathophysiologic importance. Platelet-neutrophil aggregates may represent an important link between hemostasis and inflammation and may be crucial for initiation and progression of local and systemic inflammatory responses.

No significant difference in PNA formation was detected between dogs with SIRS that did or did not have sepsis in the present study, whereas in a study16 involving humans with SIRS, those with uncomplicated sepsis had a higher degree of PNA formation and those with septic shock had a considerably lower degree. Indeed, a negative correlation was found between severity of sepsis and formation of PNAs in humans. In contrast, findings of the present study suggest it cannot be concluded that sepsis is present in dogs with SIRS on the basis of the degree of PNA formation. Because of the small number of dogs with sepsis, no attempt was made to differentiate them by severity of sepsis, and it is possible that other studies might identify a correlation between severity and PNA formation similar to that in humans, if one exists in dogs.

A higher degree of platelet activation within PNAs has been identified in septic humans, compared with activation in nonseptic critically ill patients,15 which might result in more PNAs adhering to the endothelium and thereby disappearing from circulation. Such a phenomenon might explain the failure to detect differences between the diseased dog groups in the present study. Indeed, PNAs bound to the injured endothelium may be important in promoting tissue migration of neutrophils and recruitment and activation of additional platelets and neutrophils, thereby amplifying local thrombosis and inflammation.

In the present study, dogs with DIC had higher numbers of PNAs than did control dogs, but no difference was found between dogs with SIRS that had or did not have DIC. Given that DIC is a clinical syndrome characterized by systemic activation of the hemostatic system, leading to fibrin and microthrombus formation throughout the microcirculation,21 one would suspect a higher degree of PNA formation subsequent to systemic activation of platelets. Possible explanations for this lack of difference are the depletion of platelets and an increase in the integration of activated platelets and PNAs into formed microthrombi in dogs with DIC.

In contrast to microscopic evaluation, which was not performed in the present study, flow cytometry appears to be a reliable and straightforward method for the identification and quantification of PNAs.22 Flow cytometry was performed after platelets had been immediately separated from neutrophils following blood sample collection to minimize ex vivo formation of PNAs. Healthy control dogs had a mean percentage of PNAs of 2.3%, which is comparable to findings in other canine and human studies18,23–25 that involved the use of a whole blood assay.

Most of the agonists used in the present study (ie, ADP, collagen, AA, and epinephrine) reportedly cause platelet aggregation in dogs.26,27 Activated platelets release and express P-selectin (CD62P), which promotes adhesion to each other and, via binding to the P-selectin glycoprotein ligand-1 (CD162), to neutrophils.7,8 The observed increase in PNA formation after stimulation with various platelet and neutrophil agonists in the present study was likely attributable to platelet activation, considering that some of the agonists used (ie, ADP and collagen) are not known to have direct effects on neutrophils. Activated platelets have been detected in dogs with systemic inflammatory disorders by means of flow cytometric analysis, which can measure platelet aggregation, P-selectin surface expression, and microparticle formation, as well as through automated methods that determine mean platelet component concentration and mean platelet component distribution width.28,29

In concordance with other findings in dogs,18 the most potent agonist for PNA formation in the present study was the phorbol ester PMA, which is an exogenous synthetic activator of protein kinase C. Although no significant increases in PNA formation were evident after incubation of blood with ADP, epinephrine, or collagen in another study,18 we found mild but significant increases in PNA formation after blood incubation with ADP and epinephrine and a moderate increase after incubation with collagen, when comparable concentrations were used. In the present study, only Beagles were included; however, the other study18 included various breeds. Other than differences in sample preparation and other methods used, interindividual and breed-related variations in platelet reactivity30 might explain the difference in PNA formation. Control blood samples from healthy dogs of the present study had low percentages of PNAs, which makes the formation of PNAs due to sample handling and technique an unlikely explanation for our findings.

During sepsis or infection with gram-negative bacteria, LPS can be present in the bloodstream.31 The LPS concentrations that induced PNA formation in vitro in the present study were comparable with those measured in humans with severe sepsis.31 Lipopolysaccharide is capable of inducing platelet P-selectin expression,32 which plays an important role in platelet-neutrophil interaction.33 Neutrophils in human whole blood samples also express the signaling receptor for LPS: toll-like receptor 4.34 Therefore, LPS appears to induce platelet and neutrophil activation, thereby contributing to coagulation and inflammation during sepsis. Similar to the findings of the present study for dogs, LPS reportedly initiates interactions between platelets and neutrophils in human blood.35

Arachidonic acid causes platelet activation and aggregation in canine blood.36 In neutrophils, AA induces the production of leukotrienes, including leukotriene B4, which is a potent chemotactic and chemokinetic agent that stimulates neutrophil activation and aggregation.37 In addition to causing platelet activation, blood sample incubation with LPS and AA in the present study may have contributed to an increase in PNA formation by inducing direct neutrophil activation. Indeed, after stimulation with various agonists, an increase in neutrophil size and a decrease in neutrophil granularity were detected, both of which likely indicated neutrophil activation. Other than functional changes such as integrin expression density, inflammatory cytokine production, and enhanced oxygen radical production, structural changes occur in activated neutrophils.38–40 An increase in cell size is possibly the result of cell swelling41 or the presence of band neutrophils, which are larger than segmented neutrophils.42 However, the present study did not reveal whether a change in neutrophil shape, which is believed to be an indicator of neutrophil activation, was induced directly or indirectly (eg, via platelet activation) because blood samples were not incubated with agonists selective for neutrophils.

A significant increase in neutrophil size and decrease in neutrophil granularity were detected in dogs with SIRS with and without sepsis. A greater percentage of neutrophils with increased size in dogs with septic and nonseptic inflammation, compared with that in healthy dogs, was found in another study.39 However, that study revealed a higher percentage of neutrophils with decreased granularity only for dogs with sepsis and not for dogs with nonseptic inflammation. Because SIRS is a syndrome and includes a wide variety of underlying disorders, the discrepancy may be explained by differences in the composition of the sample groups in both studies. The possibility of an undetected septic focus in the dogs of the present study cannot be completely ruled out.

A limitation of the present study is that university-owned Beagles were used as control group, whereas client-owned dogs with SIRS consisted of various breeds. Therefore, breed-related variations might be at least in part responsible for the differences reported in the present study. Despite this limitation, the method described provided a simple and reproducible way of assessing platelet interaction with neutrophils. Additional studies are necessary to evaluate the importance and potential diagnostic value of PNA formation in dogs with SIRS and sepsis. The prognostic value of PNA detection should be addressed by analyzing the possible association between PNA formation and survival rate in dogs.

ABBREVIATIONS

AA

Arachidonic acid

DIC

Disseminated intravascular coagulation

FSC

Forward-angle light scatter

LPS

Lipopolysaccharide

PMA

Phorbol myristate acetate

PNA

Platelet-neutrophil aggregate

SIRS

Systemic inflammatory response syndrome

SSC

Side-angle light scatter

a.

Natrium-Heparin-Monovetten, Sarstedt, Nümbrecht, Germany.

b.

Dulbecco phosphate buffered saline solution, Biochrom-Seromed KG, Berlin, Germany.

c.

Lymphocyte Separation Medium LSM 1077, PAA Laboratories GmbH, Pasching, Austria.

d.

Clone Y2/51, product No. MCA2588PE, AbD Serotec, Kidlington, Oxfordshire, England.

e.

Clone 2A10/11, product No. MCA2311PE, AbD Serotec, Kidlington, Oxfordshire, England.

f.

Albumin Fraction V, Roth GmBH, Karlsruhe, Germany.

g.

Sodium azide ReagentPlus, Sigma-Aldrich Corp, Steinheim, Germany.

h.

FACScan, Becton-Dickinson, Franklin Lakes, NJ.

i.

CaliBRITE Beads, Becton-Dickinson, Franklin Lakes, NJ.

j.

FCS Express 3, De Novo Software, Los Angeles, Calif.

k.

Phorbol 12-myristate 13-acetate, Sigma-Aldrich Corp, Steinheim, Germany.

l.

COLtest, Dynabyte GmbH, Munich, Germany.

m.

ADPtest, Dynabyte GmbH, Munich, Germany.

n.

Suprarenin-Ampullen, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany.

o.

Lipopolysaccarides from Escherichia coli O55:B5, Sigma-Aldrich Corp, Steinheim, Germany.

p.

ASPItest, Dynabyte GmbH, Munich, Germany.

q.

Excel 2007, Microsoft Corp, Redmond, Wash.

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Contributor Notes

Presented in part at the 19th Annual Meeting of the FG InnLab Deutsche Veterinärmedizinische Gesellschaft, Leipzig, Germany, February 2011.

Address correspondence to Dr. Mischke (reinhard.mischke@tihohannover.de).