Article
Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model Dohyeon Yu, Dongho Noh, Jinho Park
Abstract Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and , 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.
Résumé Chez les chiens la septicémie est associée à une morbidité et une mortalité élevée. Les modifications de l’hémostase par une inflammation systémique jouent un rôle important dans la pathophysiologie de la septicémie. Afin d’évaluer les changements hémostatiques fonctionnels lors de septicémie, une évaluation fut faite des profils de coagulation et des mesures par cytométrie en flux de l’expression de P-sélectine (CD62) sur les plaquettes, ainsi que de l’agrégation plaquettes-leucocytes dans un modèle d’endotoxémie induite par le lipopolysaccharide (LPS) chez des chiens (n = 7). Une dose sub-léthale de LPS [1 mg/kg de poids corporel] induisit une thrombocytopénie et augmenta le temps de thromboplastine partielle activée (aPTT), le temps de prothrombine (PT), et les concentrations de dimère-D. L’analyse par cytométrie en flux a montré une augmentation significative de l’expression de P-sélectine sur les plaquettes entre 1 et 24 h du total de 48 h que dura l’expérience. De plus, l’agrégation plaquettes-leucocytes était augmentée de manière significative dans les stages initiaux de l’endotoxémie (à 1 et , 6 h pour l’agrégation plaquettes-monocytes et 3 h pour l’agrégation plaquettes-neutrophiles). Nos résultats suggèrent que l’expression de CD62P sur les plaquettes et l’agrégation plaquettes-leucocytes, telle que mesurée par cytométrie en flux, peuvent être des biomarqueurs utiles de la coagulation intravasculaire disséminée (DIC) lors de septicémie canine. Ces changements fonctionnels contribuent à notre compréhension de la pathophysiologie de l’hémostase lors d’endotoxémie. (Traduit par Docteur Serge Messier)
Introduction Sepsis is defined as a systemic inflammatory response to infection and is associated with a high morbidity and mortality rate in both humans and dogs (1–5). In a state of severe sepsis, inflammatory cytokines and tissue factors lead to acute inflammation and the coagulation cascade becomes activated, with an active consumption of both coagulation factors and platelets. If this systemic inflammation progresses with a continual activation of the blood coagulation system, the systemic hypercoagulable state of the blood may progress toward the hypocoagulable state, with the fulminant clinical signs of hemorrhage, which is a condition known as disseminated intravascular coagulation (DIC). Although DIC is most often caused
by over-activated inflammatory responses such as sepsis, other diseases, such as neoplasia, infections, and immune-mediated diseases, can also trigger DIC in small animals (6). Disseminated intravascular coagulation (DIC) can cause thrombotic occlusion of small blood vessels and is believed to contribute to the development of multiple organ dysfunction syndrome (7). Diagnosis of DIC can be complicated in veterinary clinics, however, especially in dogs with nonovert DIC (6). Disseminated intravascular coagulation (DIC) is a syndrome defined by alterations in primary hemostasis and a secondary hemostasis. Traditionally, DIC has been diagnosed in dogs with an underlying clinical condition capable of inciting DIC, as well as 2 or more of the following laboratory abnormalities: thrombocytopenia; prolonged activated partial
College of Veterinary Medicine, Chonnam National University, Gwangju, 500-757 (Yu), and Chonbuk National University, Jeonju, 561-756 (Noh, Park), Korea. Address all correspondence to Dr. Jinho Park; telephone: 182-63-270-2557; fax: 182-63-270-3780; e-mail: [email protected] The authors declare that there are no conflicts of interest involved in this study. Received August 5, 2013. Accepted January 28, 2014. 52
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Figure 1. Measurement of platelet-leukocyte aggregation from PMA stimulated blood. Canine blood was incubated with phorbol myristate acetate (PMA, 10 mM) at 37°C for 20 min in vitro and platelet-leukocyte aggregation was measured using flow cytometry. CD14 antibody was used as a neutrophil/ monocyte surface marker and CD61 was used as a platelet surface marker. Neutrophils/monocytes were logically gated based on forward/side scatter in flow cytometry (a) and discriminated by CD14 (b). Among the gated cell population shown in Figure 1(a) and (b), CD14 bright1/CD611 double positive cells (upper right quadrant population) represented monocyte-platelet aggregation and CD14 dim1/CD611-double-positive cells (upper left quadrant population) represented granulocyte-platelet aggregation (c).
thromboplastin time (aPTT), prothrombin time (PT), or thrombin clot time; hypofibrinogenemia; decreased antithrombin; elevated markers of fibrinolysis [fibrin(ogen) degradation products or D-dimers]; or evidence of erythrocyte fragmentation on a peripheral blood smear (including schistocytes, keratocytes, and acanthocytes) (8). This approach is aimed at markers of consumption, however, and does not reliably identify nonovert cases of DIC, which highlights the importance of new tools for diagnosing DIC. In order to develop novel diagnostic methods, platelet activation is assessed by a flow cytometry analysis of P-selectin (CD62P) or by a detection of increased numbers of platelet-leukocyte aggregations in human medicine (9,10). In canine sepsis, however, there is a lack of study on the flow cytometric evaluation of hemostatic changes. The purpose of this study was, therefore, to measure CD62P expression and platelet-leukocyte aggregation as indicators of DIC in an endotoxemia model emulating canine sepsis.
Materials and methods Animal preparation In total, 10 healthy beagles were used for this study (6 females and 4 males, 7 to 11 kg, 1- to 2-years-old). They were fed dry food twice a day and water was provided ad libitum. Before the study, the dogs were deprived of food for 12 h in the hospital. This experiment was approved by the Chonbuk University Animal Care and Use Committee (CBU 2011-0005, CNU IACUC-YB-2013-47).
Experimental protocols A 22-gauge intravenous catheter was inserted into the cephalic vein of all the dogs. The 3 dogs in the control group were injected with 5 mL of normal saline, while the 7 dogs in the experimental group were slowly injected with a sublethal dose of lipopolysaccharide [LPS, 1 mg/kg body weight (BW), from Escherichia coli serotype O111:B4; Sigma, St. Louis, Missouri, USA], diluted in 5 mL of normal
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saline. Clinical examinations were carried out periodically, based on our preliminary study. These examinations consisted of rectal temperature, heart rate, respiratory rate, and blood collection from the jugular vein. In detail, complete blood (cell) count (CBC) and biochemistries were measured before and 1, 3, 6, 9, 12, 18, 24, 36, and 48 h after LPS injection; coagulation studies were measured before and 12, 18, 24, 36, and 48 h after LPS injection; and flow cytometry was conducted before and 1, 3, 6, 12, 18, 24, 36, and 48 h after LPS injection. A 21-gauge scalp vein set and 2 syringes were used to collect blood samples. The first 2 to 3 mL of blood collected were used to measure CBC (in an EDTA tube, 1 mL) and biochemistries (in a lithium heparin tube, 1 mL). The next 3 mL of blood were collected in a sodium citrate tube (Greiner Bio-one, Frickenhausen, Germany) and used for coagulation studies and flow cytometry analysis.
General laboratory analysis Hematologic analysis, including white blood cell (WBC) count (consisting of counts of monocytes, lymphocytes, and granulocytes) and platelet count, was carried out by an impedance cell counter (Vet ABC Blood Counter; ABX Diagnostics, Montpellier, France). Plasma was analyzed to verify organ dysfunction due to endotoxin using a dry chemistry analyzer (SPOTCHEM SP-4410; Arkray, Kyoto, Japan) and included alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), and creatinine.
Hemostatic parameters The D-dimer concentration, prothrombin time (PT), and activated partial thromboplastin time (aPTT) were evaluated for the possibility of thromboembolic disease or DIC. D-dimer concentrations were determined using the point-of-care NycoCard D-Dimer test (Axis-Shield PoC; Abbott Park, Illinois, USA) (11). Values below the D-dimer detection limit were assigned the default values of 0.1 mg/L for statistical analysis. In order to evaluate secondary hemostatic disorder, PT and aPTT were measured by IDEXX Coag Dx Analyzer (Westbrook, Maine, USA).
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Prothrombin time (s)
Platelet counts (3 1000/mL)
Time (hours)
D-dimer (mg/L)
Activated partial thromboplastin time (s)
Time (hours)
Time (hours)
Time (hours)
Figure 2. Alterations in coagulation tests in control and in dogs given LPS. Dogs were slowly injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, grey line) and blood was periodically collected from the jugular vein. (a) Platelet counts; (b) Prothrombin time; (c) Activated partial thromboplastin time; and (d) D-dimer concentrations (all values from the controls were below detection limits). * P , 0.05, ** P , 0.01 versus control at each time point.
Flow cytometry analysis of platelet activation and aggregation Flow cytometry analysis was always conducted within 30 min of blood collection. Quantification of activated platelet surface antigen expression and leukocyte-platelet aggregation was done by flow cytometry, as previously reported, with a modification (9,12,13). To evaluate CD62P expression on platelets, citrated anti- coagulated whole blood diluted 1:2 in HEPES-Tyrode’s buffer [0.137 M sodium chloride (NaCl), 2.8 mM potassium chloride (KCl), 1 mM magnesium chloride (MgCl2), 12 mM sodium bicarbonate (NaHCO 3), 0.4 mM disodium hydrogen phosphate (Na 2HPO4), 0.35% bovine serum albumin (BSA), 5.5 mM glucose, 10 mM HEPES, pH 7.4] and then incubated with 98 mL of antibody and an agonist cocktail containing CD62P-FITC (IE3 Clone; Santa Cruz Biotech, Santa Cruz, California, USA) and CD61-PE (a platelet marker, VI-PL2
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Clone; BD Biosciences, San Jose, California, USA) for 20 min. To stop incubation, 600 mL of 1% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS) was added. Platelets were gated by a combination of light scattering and CD61-PE fluorescence: a threshold was set in the CD61-PE channel, so that only CD61-positive events were included in the analysis. Isotype control (FITC-conjugated mouse IgG; Santa Cruz Biotech) was used in every analysis. Data were acquired from a minimum of 10 000 CD61-positive cells. To detect platelet-leukocyte aggregation, 20 mL of the citrated anti-coagulated whole blood diluted 1:2 with HEPES-Tyrode’s buffer was incubated with 20 mL of anti-CD61-PE (a platelet marker) and 20 mL of anti-CD14-FITC (M5E2 Clone, BD Biosciences) in Tyrode’s buffer, to a total of 100 mL at 22°C for 30 min. FACS lysing buffer (BD Biosciences) was added to stop incubation and to disrupt red blood cells. Granulocytes (neutrophil)/monocytes were gated by CD14 antibody (14,15). CD14bright1/CD611 cells were regarded as
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Group
experimental control
Results Clinical observations
CD62P expression (%)
All animals that participated in this study survived the experiment. Injection of LPS resulted in fever, vomiting, diarrhea, and hypotension within an hour, all of which are common symptoms of endotoxemia (data not shown).
General laboratory tests
Time (hours) Figure 3. Alterations in CD62P (P-selectin) expression on platelets in control and in dogs given LPS. Dogs were injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, dashed line). Platelets were selected by sorting out CD61-PEpositive cells by flow cytometry. The percentage of CD62P-FITC positive cells compared to isotype control was calculated by histogram. The same letters designate a significant difference in the positive of CD62Ppositive cells within or between groups.
The mean white blood cell (WBC) count of the control group during the experiment was 8.9 6 1.5 3 103/mL. In the experimental group, the total WBC changed significantly after LPS injection (P , 0.01): neutrophils decreased very rapidly, while monocytes and lymphocytes were relatively constant in differential count (data not shown). Platelet counts also changed significantly (P , 0.01), gradually decreasing to 180 3 103/mL 48 h after LPS injection (P , 0.01) (Figure 2a). Alanine aminotransferase (ALT) increased significantly in 3 and 6 h each and then decreased (P , 0.05), whereas alkaline phosphatase (ALP) gradually increased and peaked at 36 h (P , 0.01). Total bilirubin, blood urea nitrogen (BUN), and creatinine increased, but were not significantly different from controls (data not shown).
Change in hemostatic parameters CD14dim1/CD611
representing monocyte-platelet aggregation, while cells were regarded as representing granulocyte-platelet aggregation (Figure 1). The percentage of positive events for both platelets and monocytes was also determined per region. For test validation, canine blood incubated with phorbol myristate acetate (PMA, 10 mM) at 37°C for 20 min in vitro, was used as positive control. To calculate platelet-leukocyte aggregation, data were acquired on a minimum of 1000 CD14bright1 cells. Flow cytometry analysis was done using FACS Calibur flow cytometer (Becton Dickinson, San José, California, USA). Data was analyzed by FCS Express software Version 3 (De Novo Software, Los Angeles, California, USA).
Statistical analysis The statistical package for social sciences software (SPSS Version 17.0; SPSS, Chicago, Illinois, USA) was used for all statistical analyses. Data normality was analyzed using the Shapiro-Wilk test. To detect significant differences from baseline measurements within the experimental group, repeated 1-way analysis of variance (ANOVA) tests were used. The homogeneity of variance across groups in repeated-measures ANOVA was assessed by Mauchly’s sphericity test. When data sets significantly violated Mauchly’s test requirements, the Greenhouse-Geisser (GG) epsilon correction (GG , 0.75) or Huynh-Feldt correction (GG $ 0.75) parameter for degrees of freedom was used to calculate a more conservative P value for each F ratio. For post-hoc test, the Bonferroni adjustment was used. For comparison between experimental and control groups, the paired t-test (parametric) or Wilcoxon Signed Ranks test (non-parametric) was used at each time point. The significance level for all statistical tests was set at P , 0.05. All data represent the mean 6 standard deviation (SD) (parametric), or median (non-parametric).
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In the control group, overall PT and aPTT values were 13.3 6 0.9 and 90.2 6 6.9 s, respectively, and D-dimer level was below the detection limitPT, aPTT, and D-dimer all changed significantly within the experimental group (P , 0.05, Figure 2b,c). PT peaked at 24 h, then decreased gradually. The aPTT increased continuously through the end of the test at 48 h (mean 104.9 s at 24 h and 103.7 s at 48 h). The D-dimer concentration increased to its peak of 0.93 mg/L at 18 h (P , 0.05), then decreased gradually (Figure 2d).
CD62P expression on platelet and plateletleukocyte aggregation The positive percentage of CD62P (P-selectin) expression in CD61positive cells was significantly different after LPS injection (P , 0.05, Figure 3). Expression of CD62P increased, persisted higher than the baseline after LPS injection, and peaked at 36 h. Platelets and leukocytes aggregated after LPS injection. The aggregation increased in monocytes at 1 and 6 h after LPS injection (P , 0.05), whereas platelet-neutrophil significantly increased 3 h after LPS injection (P , 0.05) (Figure 4).
Discussion In this study, we observed thrombocytopenia; prolonged concentrations of aPTT, PT, and D-dimer; increased CD62P expression on platelets; and increased platelet-leukocyte aggregation by flow cytometry analysis in dogs injected with LPS. We found that platelet-leukocyte aggregation was the earliest detectable change among the markers measured, while increased CD62P expression on platelets was sustained relatively longer (approximately 24 h) than the changes in aPTT, PT, D-dimer, and platelet-leukocyte aggregation.
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experimental control
Platelet-monocyte aggregation (% in flow cytometry analysis)
Platelet-neutrophil aggregation (% in flow cytometry analysis)
Group
Time (hours)
Time (hours)
Figure 4. Percentage of platelet-leukocyte aggregation by flow cytometry in control and LPS dogs. Platelet-neutrophil aggregation increased 3 h after lipopolysaccharide (LPS) injection (a), whereas platelet-leukocyte aggregation increased in monocytes from 3 to 6 h after LPS injection (b). The same letters designate a significant difference in platelet-leukocyte aggregation between groups.
CD62P is present in the alpha (a) granules of inactive platelets and is expressed on the platelet’s surface when a granules are secreted. As measured by flow cytometry, CD62P was activated soon after LPS injection. The responsiveness of platelets to LPS is supported by earlier studies describing CD62P activation after in-vitro LPS stimulation of platelets (16). Our data show that the other coagulation tests (aPTT, PT, and D-dimer) took longer to significantly increase. In addition, the elevation of CD62P was sustained for longer than the other tests, indicating a wide diagnostic window. Our data suggest that CD62P can provide earlier information about hemostatic dysfunction in dogs with DIC. Further clinical study is required to investigate the usefulness of these biomarkers. We also observed increased platelet-leukocyte aggregation from 1 to 6 h after LPS injection. Platelet-leukocyte aggregation is considered to be a more sensitive marker of in-vivo platelet activation than circulating P-selectin-positive platelets in the clinical settings of stable coronary artery disease and venous insufficiency and circulating monocyte–platelet aggregates (17). We also found early changes of platelet aggregation with monocytes (, 6 h) and granulocytes (, 3 h). Early detection of the platelet-leukocyte aggregation might help to predict DIC and the prothrombotic state, especially in dogs with nonovert DIC, which could facilitate early treatment. We hypothesize that the detection of platelet-leukocyte aggregation using flow cytometry may enable preventive treatment for DIC before further problems arise in the coagulation cascade. Compared to controls, we found that experimental dogs had a non-significant increase in PT and significant increases in aPTT and D-dimer levels. Recently, a similar study that observed hemostatic changes using thromboelastography (TEG) in dogs injected with low dose LPS (0.02 mg/kg BW) reported that aPTT prolongation peaked at 4 h and D-dimer peaked at 2 and 24 h after injection (18). In contrast, we observed a sustained prolongation of aPTT through 48 h and of PT through 24 h. A potential explanation for this discrepancy in results was the difference in sampling time points: 1, 3, 6, 9, 12, 24,
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36, and 48 h in the current study and 0, 1, 2, 4, and 24 h in the cited study. Our more detailed kinetics data may more accurately track hemostatic changes in dogs with endotoxemia. One of the limitations of this study was the inability to show an association between biochemical markers of organ dysfunction and DIC, as DIC usually causes organ dysfunction due to microvascular occlusion by thrombin formation. Despite increases in ALT and ALP levels, neither bilirubin, BUN, nor creatinine met the criteria (19) for organ dysfunction. It may be that organ dysfunction took longer than 48 h to manifest biochemically, given that thrombocytopenia and increased aPTT and PT were observed 24 to 36 h after LPS injection. Another possible explanation is that, although the LPS injection may have caused sepsis, the insult is rapidly reversible, preventing overt organ dysfunction in this study. Further studies to confirm the presence of organ dysfunction in severe sepsis are warranted. In conclusion, we suggest that increased CD62P expression with prolonged PT and D-dimer reflects thromboembolic formation in late-stage endotoxemia. Our data also suggest that platelet-leukocyte aggregation is an early indicator of DIC and that CD62P expression has a wide diagnostic window. Future studies should evaluate the clinical usefulness of these parameters in naturally occurring canine sepsis.
Acknowledgment This study was financially supported by Chonnam National University, Korea in 2013.
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2. Bentley AM, Otto CM, Shofer FS. Comparison of dogs with septic peritonitis: 1988–1993 versus 1999–2003. J Vet Emerg Crit Care (San Antonio) 2007;17:391–398. 3. Yu D-H, Nho D-H, Song R-H, et al. High-mobility group box 1 as a surrogate prognostic marker in dogs with systemic inflammatory response syndrome. J Vet Emerg Crit Care (San Antonio) 2010;20:298–302. 4. Rau S, Kohn B, Richter C, et al. Plasma interleukin-6 response is predictive for severity and mortality in canine systemic inflammatory response syndrome and sepsis. Vet Clin Pathol 2007 Sep;36:253–260. 5. Luschini MA, Fletcher DJ, Schoeffler GL. Retrospective Study: Incidence of ionized hypocalcemia in septic dogs and its association with morbidity and mortality: 58 cases (2006–2007). J Vet Emerg Crit Care 2010;20:406–412. 6. Ralph AG, Brainard BM. Update on disseminated intravascular coagulation: When to consider it, when to expect it, when to treat it. Top Companion Anim Med 2012;27:65–72. 7. Levi M, ten Cate H, van der Poll T, van Deventer SJ. Pathogenesis of disseminated intravascular coagulation in sepsis. JAMA 1993 Aug 25;270:975–979. 8. Stokol T. Laboratory diagnosis of disseminated intravascular coagulation in dogs and cats: The past, the present, and the future. Vet Clin North Am Small Anim Prac 2012;42:189–202. 9. Russwurm S, Vickers J, Meier-Hellmann A, et al. Platelet and leukocyte activation correlate with the severity of septic organ dysfunction. Shock 2002;17:263–268. 10. Kalsch T, Elmas E, Nguyen XD, et al. Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation. Basic Res Cardiol 2007;102:460–466. 11. Dewhurst E, Cue S, Crawford E, Papasouliotis K. A retrospective study of canine D-dimer concentrations measured using an
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immunometric “Point-of-Care” test. J Small Anim Pract 2008;49: 344–348. 12. Tarnow I, Kristensen AT, Krogh AK, Frelinger AL, 3rd, Barnard MR, Michelson AD. Effects of physiologic agonists on canine whole blood flow cytometry assays of leukocyte-platelet aggregation and platelet activation. Vet Immunol Immunopathol 2008; 123:345–352. 13. Schmitz G, Rothe G, Ruf A, et al. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998;79:885–896. 14. Schuberth HJ, Kucinskiene G, Chu RM, Faldyna M. Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes. Vet Immunol Immunopathol 2007;119:47–55. 15. Yu D-H, Kim B, Park J. Pathophysiologic and Immunologic changes in a canine endotoxemia over a period of 24 hours. J Vet Med Sci 2012;74:537–544. 16. Mirlashari MR, Hagberg IA, Lyberg T. Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis. Platelets 2002;13:91–99. 17. Michelson AD, Barnard MR, Krueger LA, Frelinger AL, Furman MI. Evaluation of platelet function by flow cytometry. Methods 2000;21:259–270. 18. Eralp O, Yilmaz Z, Failing K, Moritz A, Bauer N. Effect of experimental endotoxemia on thrombelastography parameters, secondary and tertiary hemostasis in dogs. J Vet Inter Med 2011;25: 524–531. 19. Kenney EM, Rozanski EA, Rush JE, et al. Association between outcome and organ system dysfunction in dogs with sepsis: 114 cases (2003–2007). J Am Vet Med Assoc 2010;236:83–87.
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Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model Dohyeon Yu, Dongho Noh, Jinho Park
Abstract Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and , 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.
Résumé Chez les chiens la septicémie est associée à une morbidité et une mortalité élevée. Les modifications de l’hémostase par une inflammation systémique jouent un rôle important dans la pathophysiologie de la septicémie. Afin d’évaluer les changements hémostatiques fonctionnels lors de septicémie, une évaluation fut faite des profils de coagulation et des mesures par cytométrie en flux de l’expression de P-sélectine (CD62) sur les plaquettes, ainsi que de l’agrégation plaquettes-leucocytes dans un modèle d’endotoxémie induite par le lipopolysaccharide (LPS) chez des chiens (n = 7). Une dose sub-léthale de LPS [1 mg/kg de poids corporel] induisit une thrombocytopénie et augmenta le temps de thromboplastine partielle activée (aPTT), le temps de prothrombine (PT), et les concentrations de dimère-D. L’analyse par cytométrie en flux a montré une augmentation significative de l’expression de P-sélectine sur les plaquettes entre 1 et 24 h du total de 48 h que dura l’expérience. De plus, l’agrégation plaquettes-leucocytes était augmentée de manière significative dans les stages initiaux de l’endotoxémie (à 1 et , 6 h pour l’agrégation plaquettes-monocytes et 3 h pour l’agrégation plaquettes-neutrophiles). Nos résultats suggèrent que l’expression de CD62P sur les plaquettes et l’agrégation plaquettes-leucocytes, telle que mesurée par cytométrie en flux, peuvent être des biomarqueurs utiles de la coagulation intravasculaire disséminée (DIC) lors de septicémie canine. Ces changements fonctionnels contribuent à notre compréhension de la pathophysiologie de l’hémostase lors d’endotoxémie. (Traduit par Docteur Serge Messier)
Introduction Sepsis is defined as a systemic inflammatory response to infection and is associated with a high morbidity and mortality rate in both humans and dogs (1–5). In a state of severe sepsis, inflammatory cytokines and tissue factors lead to acute inflammation and the coagulation cascade becomes activated, with an active consumption of both coagulation factors and platelets. If this systemic inflammation progresses with a continual activation of the blood coagulation system, the systemic hypercoagulable state of the blood may progress toward the hypocoagulable state, with the fulminant clinical signs of hemorrhage, which is a condition known as disseminated intravascular coagulation (DIC). Although DIC is most often caused
by over-activated inflammatory responses such as sepsis, other diseases, such as neoplasia, infections, and immune-mediated diseases, can also trigger DIC in small animals (6). Disseminated intravascular coagulation (DIC) can cause thrombotic occlusion of small blood vessels and is believed to contribute to the development of multiple organ dysfunction syndrome (7). Diagnosis of DIC can be complicated in veterinary clinics, however, especially in dogs with nonovert DIC (6). Disseminated intravascular coagulation (DIC) is a syndrome defined by alterations in primary hemostasis and a secondary hemostasis. Traditionally, DIC has been diagnosed in dogs with an underlying clinical condition capable of inciting DIC, as well as 2 or more of the following laboratory abnormalities: thrombocytopenia; prolonged activated partial
College of Veterinary Medicine, Chonnam National University, Gwangju, 500-757 (Yu), and Chonbuk National University, Jeonju, 561-756 (Noh, Park), Korea. Address all correspondence to Dr. Jinho Park; telephone: 182-63-270-2557; fax: 182-63-270-3780; e-mail: [email protected] The authors declare that there are no conflicts of interest involved in this study. Received August 5, 2013. Accepted January 28, 2014. 52
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Figure 1. Measurement of platelet-leukocyte aggregation from PMA stimulated blood. Canine blood was incubated with phorbol myristate acetate (PMA, 10 mM) at 37°C for 20 min in vitro and platelet-leukocyte aggregation was measured using flow cytometry. CD14 antibody was used as a neutrophil/ monocyte surface marker and CD61 was used as a platelet surface marker. Neutrophils/monocytes were logically gated based on forward/side scatter in flow cytometry (a) and discriminated by CD14 (b). Among the gated cell population shown in Figure 1(a) and (b), CD14 bright1/CD611 double positive cells (upper right quadrant population) represented monocyte-platelet aggregation and CD14 dim1/CD611-double-positive cells (upper left quadrant population) represented granulocyte-platelet aggregation (c).
thromboplastin time (aPTT), prothrombin time (PT), or thrombin clot time; hypofibrinogenemia; decreased antithrombin; elevated markers of fibrinolysis [fibrin(ogen) degradation products or D-dimers]; or evidence of erythrocyte fragmentation on a peripheral blood smear (including schistocytes, keratocytes, and acanthocytes) (8). This approach is aimed at markers of consumption, however, and does not reliably identify nonovert cases of DIC, which highlights the importance of new tools for diagnosing DIC. In order to develop novel diagnostic methods, platelet activation is assessed by a flow cytometry analysis of P-selectin (CD62P) or by a detection of increased numbers of platelet-leukocyte aggregations in human medicine (9,10). In canine sepsis, however, there is a lack of study on the flow cytometric evaluation of hemostatic changes. The purpose of this study was, therefore, to measure CD62P expression and platelet-leukocyte aggregation as indicators of DIC in an endotoxemia model emulating canine sepsis.
Materials and methods Animal preparation In total, 10 healthy beagles were used for this study (6 females and 4 males, 7 to 11 kg, 1- to 2-years-old). They were fed dry food twice a day and water was provided ad libitum. Before the study, the dogs were deprived of food for 12 h in the hospital. This experiment was approved by the Chonbuk University Animal Care and Use Committee (CBU 2011-0005, CNU IACUC-YB-2013-47).
Experimental protocols A 22-gauge intravenous catheter was inserted into the cephalic vein of all the dogs. The 3 dogs in the control group were injected with 5 mL of normal saline, while the 7 dogs in the experimental group were slowly injected with a sublethal dose of lipopolysaccharide [LPS, 1 mg/kg body weight (BW), from Escherichia coli serotype O111:B4; Sigma, St. Louis, Missouri, USA], diluted in 5 mL of normal
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saline. Clinical examinations were carried out periodically, based on our preliminary study. These examinations consisted of rectal temperature, heart rate, respiratory rate, and blood collection from the jugular vein. In detail, complete blood (cell) count (CBC) and biochemistries were measured before and 1, 3, 6, 9, 12, 18, 24, 36, and 48 h after LPS injection; coagulation studies were measured before and 12, 18, 24, 36, and 48 h after LPS injection; and flow cytometry was conducted before and 1, 3, 6, 12, 18, 24, 36, and 48 h after LPS injection. A 21-gauge scalp vein set and 2 syringes were used to collect blood samples. The first 2 to 3 mL of blood collected were used to measure CBC (in an EDTA tube, 1 mL) and biochemistries (in a lithium heparin tube, 1 mL). The next 3 mL of blood were collected in a sodium citrate tube (Greiner Bio-one, Frickenhausen, Germany) and used for coagulation studies and flow cytometry analysis.
General laboratory analysis Hematologic analysis, including white blood cell (WBC) count (consisting of counts of monocytes, lymphocytes, and granulocytes) and platelet count, was carried out by an impedance cell counter (Vet ABC Blood Counter; ABX Diagnostics, Montpellier, France). Plasma was analyzed to verify organ dysfunction due to endotoxin using a dry chemistry analyzer (SPOTCHEM SP-4410; Arkray, Kyoto, Japan) and included alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), and creatinine.
Hemostatic parameters The D-dimer concentration, prothrombin time (PT), and activated partial thromboplastin time (aPTT) were evaluated for the possibility of thromboembolic disease or DIC. D-dimer concentrations were determined using the point-of-care NycoCard D-Dimer test (Axis-Shield PoC; Abbott Park, Illinois, USA) (11). Values below the D-dimer detection limit were assigned the default values of 0.1 mg/L for statistical analysis. In order to evaluate secondary hemostatic disorder, PT and aPTT were measured by IDEXX Coag Dx Analyzer (Westbrook, Maine, USA).
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Prothrombin time (s)
Platelet counts (3 1000/mL)
Time (hours)
D-dimer (mg/L)
Activated partial thromboplastin time (s)
Time (hours)
Time (hours)
Time (hours)
Figure 2. Alterations in coagulation tests in control and in dogs given LPS. Dogs were slowly injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, grey line) and blood was periodically collected from the jugular vein. (a) Platelet counts; (b) Prothrombin time; (c) Activated partial thromboplastin time; and (d) D-dimer concentrations (all values from the controls were below detection limits). * P , 0.05, ** P , 0.01 versus control at each time point.
Flow cytometry analysis of platelet activation and aggregation Flow cytometry analysis was always conducted within 30 min of blood collection. Quantification of activated platelet surface antigen expression and leukocyte-platelet aggregation was done by flow cytometry, as previously reported, with a modification (9,12,13). To evaluate CD62P expression on platelets, citrated anti- coagulated whole blood diluted 1:2 in HEPES-Tyrode’s buffer [0.137 M sodium chloride (NaCl), 2.8 mM potassium chloride (KCl), 1 mM magnesium chloride (MgCl2), 12 mM sodium bicarbonate (NaHCO 3), 0.4 mM disodium hydrogen phosphate (Na 2HPO4), 0.35% bovine serum albumin (BSA), 5.5 mM glucose, 10 mM HEPES, pH 7.4] and then incubated with 98 mL of antibody and an agonist cocktail containing CD62P-FITC (IE3 Clone; Santa Cruz Biotech, Santa Cruz, California, USA) and CD61-PE (a platelet marker, VI-PL2
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Clone; BD Biosciences, San Jose, California, USA) for 20 min. To stop incubation, 600 mL of 1% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS) was added. Platelets were gated by a combination of light scattering and CD61-PE fluorescence: a threshold was set in the CD61-PE channel, so that only CD61-positive events were included in the analysis. Isotype control (FITC-conjugated mouse IgG; Santa Cruz Biotech) was used in every analysis. Data were acquired from a minimum of 10 000 CD61-positive cells. To detect platelet-leukocyte aggregation, 20 mL of the citrated anti-coagulated whole blood diluted 1:2 with HEPES-Tyrode’s buffer was incubated with 20 mL of anti-CD61-PE (a platelet marker) and 20 mL of anti-CD14-FITC (M5E2 Clone, BD Biosciences) in Tyrode’s buffer, to a total of 100 mL at 22°C for 30 min. FACS lysing buffer (BD Biosciences) was added to stop incubation and to disrupt red blood cells. Granulocytes (neutrophil)/monocytes were gated by CD14 antibody (14,15). CD14bright1/CD611 cells were regarded as
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Group
experimental control
Results Clinical observations
CD62P expression (%)
All animals that participated in this study survived the experiment. Injection of LPS resulted in fever, vomiting, diarrhea, and hypotension within an hour, all of which are common symptoms of endotoxemia (data not shown).
General laboratory tests
Time (hours) Figure 3. Alterations in CD62P (P-selectin) expression on platelets in control and in dogs given LPS. Dogs were injected with lipopolysaccharide (LPS, 1 mg/kg BW from E. coli O111: B4, solid black line) or saline (control, dashed line). Platelets were selected by sorting out CD61-PEpositive cells by flow cytometry. The percentage of CD62P-FITC positive cells compared to isotype control was calculated by histogram. The same letters designate a significant difference in the positive of CD62Ppositive cells within or between groups.
The mean white blood cell (WBC) count of the control group during the experiment was 8.9 6 1.5 3 103/mL. In the experimental group, the total WBC changed significantly after LPS injection (P , 0.01): neutrophils decreased very rapidly, while monocytes and lymphocytes were relatively constant in differential count (data not shown). Platelet counts also changed significantly (P , 0.01), gradually decreasing to 180 3 103/mL 48 h after LPS injection (P , 0.01) (Figure 2a). Alanine aminotransferase (ALT) increased significantly in 3 and 6 h each and then decreased (P , 0.05), whereas alkaline phosphatase (ALP) gradually increased and peaked at 36 h (P , 0.01). Total bilirubin, blood urea nitrogen (BUN), and creatinine increased, but were not significantly different from controls (data not shown).
Change in hemostatic parameters CD14dim1/CD611
representing monocyte-platelet aggregation, while cells were regarded as representing granulocyte-platelet aggregation (Figure 1). The percentage of positive events for both platelets and monocytes was also determined per region. For test validation, canine blood incubated with phorbol myristate acetate (PMA, 10 mM) at 37°C for 20 min in vitro, was used as positive control. To calculate platelet-leukocyte aggregation, data were acquired on a minimum of 1000 CD14bright1 cells. Flow cytometry analysis was done using FACS Calibur flow cytometer (Becton Dickinson, San José, California, USA). Data was analyzed by FCS Express software Version 3 (De Novo Software, Los Angeles, California, USA).
Statistical analysis The statistical package for social sciences software (SPSS Version 17.0; SPSS, Chicago, Illinois, USA) was used for all statistical analyses. Data normality was analyzed using the Shapiro-Wilk test. To detect significant differences from baseline measurements within the experimental group, repeated 1-way analysis of variance (ANOVA) tests were used. The homogeneity of variance across groups in repeated-measures ANOVA was assessed by Mauchly’s sphericity test. When data sets significantly violated Mauchly’s test requirements, the Greenhouse-Geisser (GG) epsilon correction (GG , 0.75) or Huynh-Feldt correction (GG $ 0.75) parameter for degrees of freedom was used to calculate a more conservative P value for each F ratio. For post-hoc test, the Bonferroni adjustment was used. For comparison between experimental and control groups, the paired t-test (parametric) or Wilcoxon Signed Ranks test (non-parametric) was used at each time point. The significance level for all statistical tests was set at P , 0.05. All data represent the mean 6 standard deviation (SD) (parametric), or median (non-parametric).
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In the control group, overall PT and aPTT values were 13.3 6 0.9 and 90.2 6 6.9 s, respectively, and D-dimer level was below the detection limitPT, aPTT, and D-dimer all changed significantly within the experimental group (P , 0.05, Figure 2b,c). PT peaked at 24 h, then decreased gradually. The aPTT increased continuously through the end of the test at 48 h (mean 104.9 s at 24 h and 103.7 s at 48 h). The D-dimer concentration increased to its peak of 0.93 mg/L at 18 h (P , 0.05), then decreased gradually (Figure 2d).
CD62P expression on platelet and plateletleukocyte aggregation The positive percentage of CD62P (P-selectin) expression in CD61positive cells was significantly different after LPS injection (P , 0.05, Figure 3). Expression of CD62P increased, persisted higher than the baseline after LPS injection, and peaked at 36 h. Platelets and leukocytes aggregated after LPS injection. The aggregation increased in monocytes at 1 and 6 h after LPS injection (P , 0.05), whereas platelet-neutrophil significantly increased 3 h after LPS injection (P , 0.05) (Figure 4).
Discussion In this study, we observed thrombocytopenia; prolonged concentrations of aPTT, PT, and D-dimer; increased CD62P expression on platelets; and increased platelet-leukocyte aggregation by flow cytometry analysis in dogs injected with LPS. We found that platelet-leukocyte aggregation was the earliest detectable change among the markers measured, while increased CD62P expression on platelets was sustained relatively longer (approximately 24 h) than the changes in aPTT, PT, D-dimer, and platelet-leukocyte aggregation.
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experimental control
Platelet-monocyte aggregation (% in flow cytometry analysis)
Platelet-neutrophil aggregation (% in flow cytometry analysis)
Group
Time (hours)
Time (hours)
Figure 4. Percentage of platelet-leukocyte aggregation by flow cytometry in control and LPS dogs. Platelet-neutrophil aggregation increased 3 h after lipopolysaccharide (LPS) injection (a), whereas platelet-leukocyte aggregation increased in monocytes from 3 to 6 h after LPS injection (b). The same letters designate a significant difference in platelet-leukocyte aggregation between groups.
CD62P is present in the alpha (a) granules of inactive platelets and is expressed on the platelet’s surface when a granules are secreted. As measured by flow cytometry, CD62P was activated soon after LPS injection. The responsiveness of platelets to LPS is supported by earlier studies describing CD62P activation after in-vitro LPS stimulation of platelets (16). Our data show that the other coagulation tests (aPTT, PT, and D-dimer) took longer to significantly increase. In addition, the elevation of CD62P was sustained for longer than the other tests, indicating a wide diagnostic window. Our data suggest that CD62P can provide earlier information about hemostatic dysfunction in dogs with DIC. Further clinical study is required to investigate the usefulness of these biomarkers. We also observed increased platelet-leukocyte aggregation from 1 to 6 h after LPS injection. Platelet-leukocyte aggregation is considered to be a more sensitive marker of in-vivo platelet activation than circulating P-selectin-positive platelets in the clinical settings of stable coronary artery disease and venous insufficiency and circulating monocyte–platelet aggregates (17). We also found early changes of platelet aggregation with monocytes (, 6 h) and granulocytes (, 3 h). Early detection of the platelet-leukocyte aggregation might help to predict DIC and the prothrombotic state, especially in dogs with nonovert DIC, which could facilitate early treatment. We hypothesize that the detection of platelet-leukocyte aggregation using flow cytometry may enable preventive treatment for DIC before further problems arise in the coagulation cascade. Compared to controls, we found that experimental dogs had a non-significant increase in PT and significant increases in aPTT and D-dimer levels. Recently, a similar study that observed hemostatic changes using thromboelastography (TEG) in dogs injected with low dose LPS (0.02 mg/kg BW) reported that aPTT prolongation peaked at 4 h and D-dimer peaked at 2 and 24 h after injection (18). In contrast, we observed a sustained prolongation of aPTT through 48 h and of PT through 24 h. A potential explanation for this discrepancy in results was the difference in sampling time points: 1, 3, 6, 9, 12, 24,
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36, and 48 h in the current study and 0, 1, 2, 4, and 24 h in the cited study. Our more detailed kinetics data may more accurately track hemostatic changes in dogs with endotoxemia. One of the limitations of this study was the inability to show an association between biochemical markers of organ dysfunction and DIC, as DIC usually causes organ dysfunction due to microvascular occlusion by thrombin formation. Despite increases in ALT and ALP levels, neither bilirubin, BUN, nor creatinine met the criteria (19) for organ dysfunction. It may be that organ dysfunction took longer than 48 h to manifest biochemically, given that thrombocytopenia and increased aPTT and PT were observed 24 to 36 h after LPS injection. Another possible explanation is that, although the LPS injection may have caused sepsis, the insult is rapidly reversible, preventing overt organ dysfunction in this study. Further studies to confirm the presence of organ dysfunction in severe sepsis are warranted. In conclusion, we suggest that increased CD62P expression with prolonged PT and D-dimer reflects thromboembolic formation in late-stage endotoxemia. Our data also suggest that platelet-leukocyte aggregation is an early indicator of DIC and that CD62P expression has a wide diagnostic window. Future studies should evaluate the clinical usefulness of these parameters in naturally occurring canine sepsis.
Acknowledgment This study was financially supported by Chonnam National University, Korea in 2013.
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