A comprehensive study of ovine haemostasis to assess suitability to model human coagulation Samuel R. Foley, Connie Solano, Gabriela Simonova, Michelle M. Spanevello, Robert J. Bird, John W. Semple, Denise E. Jackson, Andreas Schibler, John F. Fraser, Yoke Lin Fung PII: DOI: Reference:
S0049-3848(14)00292-8 doi: 10.1016/j.thromres.2014.05.026 TR 5537
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
31 March 2014 17 May 2014 17 May 2014
Please cite this article as: Foley Samuel R., Solano Connie, Simonova Gabriela, Spanevello Michelle M., Bird Robert J., Semple John W., Jackson Denise E., Schibler Andreas, Fraser John F., Fung Yoke Lin, A comprehensive study of ovine haemostasis to assess suitability to model human coagulation, Thrombosis Research (2014), doi: 10.1016/j.thromres.2014.05.026
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Article
ACCEPTED MANUSCRIPT
A comprehensive study of ovine haemostasis to assess suitability to model human coagulation
PT
Samuel R. Foleya, Connie Solanob, Gabriela Simonovaa, c, Michelle M. Spanevellob, Robert J. Birdb, d, John W. Semplee, Denise E. Jacksonf, Andreas Schiblerg, John F Frasera, Yoke Lin Funga,h.
SC RI
The experiments were carried out at the Critical Care Research Group, The Prince Charles Hospital, Lvl 3 Clinical Sciences Bld, Rode Rd, Chermside, QLD 4032. a
MA
NU
Critical Care Research Group, The University of Queensland and The Prince Charles Hospital, Brisbane, QLD, Australia. b Pathology Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia. c Research and Development, Australian Red Cross Blood Service, Brisbane, QLD, Australia. d School of Medicine, Griffith University, QLD, Australia. e Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON, Canada. f School of Medical Sciences, RMIT University, Bundoora, VIC, Australia. g Mater Children's Hospital Mater, The University of Queensland, Brisbane, QLD, Australia. h School of Health and Sports Science, University of Sunshine Coast, Sippy Downs, QLD, Australia.
AC
CE
PT
ED
Correspondence to: Samuel R. Foley Critical Care Research Group, The Prince Charles Hospital Lvl 3 Clinical Sciences Bld, Rode Road, Chermside, QLD 4032 E-mail: [email protected] Tel.: + 61 410037406 Fax: +61 3139 6120
1
ACCEPTED MANUSCRIPT
Abstract
Introduction: Similarities in size, anatomy and physiology have supported the use of sheep to model a wide range of human diseases, including coagulopathy. However, coagulation studies involving sheep are limited by the absence of high quality data defining normal ovine coagulation and fibrinolysis.
PT
Materials and Methods: Full blood examination, routine and specialised coagulation tests, rotational thromboelastometry and whole blood platelet aggregometry was performed on 50
SC RI
healthy Samm & Border Leicester Cross ewes and compared to corresponding human ranges. Intraspecies breed and gender variability was investigated by comparison to a smaller population of 13 healthy Merino wethers.
Results: Ovine coagulation was similar to human according to routine coagulation methods
NU
(PT, aPTT, TCT, Fib(C)) and some specialised coagulation tests (vWF, AT, Plasmin Inh). Despite these similarities, ovine secondary haemostasis demonstrated substantial differences to
MA
that of human. Rapid initiation of the contact activation pathway, high levels of FVIII, low Protein C, greater overall clot firmness and a reduced capacity for clot lysis was documented in sheep. In addition, ADP and collagen agonists precipitated a reduced primary haemostatic
ED
response in sheep relative to human. Intraspecies differences in whole blood platelet aggregometry between the cohorts of sheep indicate the need for breed-specific normal ranges.
PT
Conclusions: The application of a board spectrum of coagulation assays has enabled elucidation of the similarities as well as differences between ovine and human coagulation. The
CE
new knowledge generated from this study will guide the design of future translational
Keywords
AC
coagulation studies in ovine models.
Animal, coagulation parameters, normal values, platelets, sheep, thromboelastometry.
Abbreviations PLT - platelet count; PT - prothrombin time; aPTT - activated partial thrombin time; TCTthrombin clotting time; Fib(C) – fibrinogen (clauss); vWF - von Willebrand factor, FVIII factor VIII; FXII - factor XII; Pl.Inh. - plasmin inhibitor; Prot.C - protein C; AT antithrombin; MP – Multiplate® whole blood platelet aggregometry; ADP – Adenosine Diphosphate; COL – Collagen; TEG – rotational thromboelastography®; ROTEM - rotational thromboelastometry®.
2
Introduction
ACCEPTED MANUSCRIPT
Comparative studies of human, sheep, pig, rabbit, rat and dog using routine coagulation tests, clotting factor assays and rotational thromboelastometry indicate that the human coagulation system exhibits greatest similarity with that of sheep (1, 2). Recently, a line of sheep with hemophilia A (HA) was re-established (3) and recombinant ovine FVIII (oFVIII) developed to Surprisingly, despite the utility of ovine models in haemostasis
PT
support this model (4).
research, data defining ‘normal’ parameters for both classical and newer modes of coagulation
SC RI
assessment is deficient. Studies to date are limited by small sample size (1, 2, 5) and many define only a small range of coagulation parameters (6, 7). Table 1 summarises published coagulation parameters of sheep and reveals variability in results (e.g. FVIII) between studies.
NU
The aim of this study was to define a robust and comprehensive normal ovine coagulation profile. To do this we studied a large cohort (n=50) of healthy Samm & Border Leicester Cross
MA
(SBL) ewes to define full blood examination (FBE), routine and specialised coagulation tests, as well as the newer whole blood coagulation measures of rotational thromboelastometry (ROTEM®) and whole blood platelet aggregometry (Multiplate®). To investigate inter- and
ED
intraspecies differences in coagulation, this data was compared to corresponding human ranges and another population of healthy Merino wethers (n=13). Data from this comprehensive range
PT
of coagulation assays has enabled a thorough assessment of the benefits as well as the
CE
limitations of using ovine models for translational coagulation studies.
Material and Methods Animals
AC
SBL ewes (female sheep) (n=50) and Merino wethers (castrated male sheep) (n=13) were evaluated for this study. The sheep were housed in a purpose built animal facility for at least 1 week prior to testing, fed pellets and had access to water ad libitum. All animals were 2-3yr old, weighed 45-55 kg and physically assessed to be healthy by a veterinarian prior to blood sampling. This study was approved by the Animal Research Ethics Committee of the Queensland University of Technology and University of Queensland (QUT UAEC Approval number 1100000053 and 1000000025) and conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 7th Edition 2004 (The Code) of the National Health and Medical Research Council (NHMRC).
3
ACCEPTED MANUSCRIPT
Blood sampling protocol
Blood samples were obtained from the left internal jugular vein of the sheep through a central venous catheter. Whole blood samples for ROTEM® analysis was collected in 3.2% sodium citrate tubes (Greiner Bio-One, Amata Nakorn, Chonburi, Thailand), EDTA tubes (Greiner Bio-One, Kremsmünster, Austria) for FBE, and hirudin tubes (Verum Diagnostica, GmbH,
PT
Munich, Germany) for Multiplate® whole blood platelet aggregometry. FBE, ROTEM® and Multiplate® analysis was conducted within 2 hours of collection. Citrated blood for the routine
SC RI
and specialised coagulation tests was centrifuged for 15 minutes at 3000g, this was repeated on separated plasma and stored at -80°C prior to analysis.
Full blood examination, routine and specialised coagulation tests
NU
FBE was evaluated using the veterinary mode of the Act diff™ haematology analyser (Beckman Coulter Australia Pty Ltd, NSW, Australia) and consisted of white cell count
MA
(WCC), red cell count (RCC), haemoglobin (Hb), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and platelet count (PLT). Routine coagulation tests (prothrombin time (PT), activated partial
ED
thromboplastin time (aPTT), Clauss fibrinogen [Fib(C)], thrombin clotting time (TCT) and Ddimer) and specialised coagulation tests (Factor VIII (FVIII), Factor XII (FXII), von
PT
Willebrand Factor (vWF), Protein C, antithrombin (AT), plasmin inhibitor (Plasmin Inh)) were performed on the ACL TOP® analyser (Instrumentation Laboratory, Werfen Australia, NSW,
CE
Australia) as per the manufacturer’s instructions.
Whole blood platelet aggregometry (Multiplate®)
AC
Tests were performed on the Multiplate® 5.0 - Platelet Function Analyser (Haemoview Diagnostics, Brisbane, Australia) (8) using Multiplate® Mini test cells in accordance with the manufacturer’s instructions. Hirudin-anticoagulated blood was tested using ADP (6.4μmol/L) and collagen (3.2 μg/ml) agonists.
Rotational thromboelastometry (ROTEM®) Citrated blood samples were tested using rotational thromboelastometry (ROTEM®, Haemoview Diagnostics, Brisbane, Australia). The INTEM, EXTEM and FIBTEM tests were run for 35 minutes following clot initiation (until A30 recorded) according to manufacturer’s instructions (9). EXTEM clotting time (CT, seconds (s)), clot formation time (CFT, s), alpha angle (, °), clot lysis index (CLI30, %), clot amplitude at 10 minutes (A10, mm) and at 30 minutes (A30, mm), as well as FIBTEM-A10 was compared between the two sheep populations.
4
Statistical analysis
ACCEPTED MANUSCRIPT
Data analysis was performed using GraphPad Prism 6 statistical package. Descriptive statistics and histograms showed normal and non-normal distributions of results. Data was expressed as mean ± standard deviation (SD) and range (2.5-97.5%) with multigroup analysis using oneway ANOVA and Bonferroni’s multiple comparison tests for normal data and Kruskal–Wallis
PT
and Dunn’s multiple comparison tests for non-normal data. Spearman’s rank correlation coefficients were determined to assess for correlation between plasma-based coagulation
SC RI
parameters (PT, aPTT, Fib(C), FVIII, FXII, Plasmin Inh), platelet count (PLT) and corresponding ROTEM® measurements in healthy SBL ewes.
Results
NU
Haematological and coagulation parameters in sheep and human
The mean and standard deviation of all haematological and plasma-based coagulation
MA
parameters from 50 adult SBL ewes (female) are detailed and compared to corresponding female human ranges (Table 2). The range and upper limit of ovine WCC and PLT count were higher. Although sheep had higher RCC, they had a corresponding lower MCV and MCH, but
ED
MCHC was comparable. PT, aPTT, TCT, Fib(C), vWF, AT and Plasmin Inh levels were similar in sheep and human. Sheep had higher D-dimer, FVIII, and FXII, and lower Protein C
PT
levels.
CE
ROTEM® and Multiplate® parameters in sheep and human (i) Analysis of two distinct sheep populations No significant difference in INTEM- or EXTEM-CT, -CFT, -, -A10, -A30 or FIBTEM-A10
AC
was documented between the two populations of sheep (Table 3). No fibrinolysis, as measured by INTEM-CLI30 or EXTEM-CLI30 was detected in either subgroup of sheep. In contrast, ADP (p
S0049-3848(14)00292-8 doi: 10.1016/j.thromres.2014.05.026 TR 5537
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
31 March 2014 17 May 2014 17 May 2014
Please cite this article as: Foley Samuel R., Solano Connie, Simonova Gabriela, Spanevello Michelle M., Bird Robert J., Semple John W., Jackson Denise E., Schibler Andreas, Fraser John F., Fung Yoke Lin, A comprehensive study of ovine haemostasis to assess suitability to model human coagulation, Thrombosis Research (2014), doi: 10.1016/j.thromres.2014.05.026
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Article
ACCEPTED MANUSCRIPT
A comprehensive study of ovine haemostasis to assess suitability to model human coagulation
PT
Samuel R. Foleya, Connie Solanob, Gabriela Simonovaa, c, Michelle M. Spanevellob, Robert J. Birdb, d, John W. Semplee, Denise E. Jacksonf, Andreas Schiblerg, John F Frasera, Yoke Lin Funga,h.
SC RI
The experiments were carried out at the Critical Care Research Group, The Prince Charles Hospital, Lvl 3 Clinical Sciences Bld, Rode Rd, Chermside, QLD 4032. a
MA
NU
Critical Care Research Group, The University of Queensland and The Prince Charles Hospital, Brisbane, QLD, Australia. b Pathology Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia. c Research and Development, Australian Red Cross Blood Service, Brisbane, QLD, Australia. d School of Medicine, Griffith University, QLD, Australia. e Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON, Canada. f School of Medical Sciences, RMIT University, Bundoora, VIC, Australia. g Mater Children's Hospital Mater, The University of Queensland, Brisbane, QLD, Australia. h School of Health and Sports Science, University of Sunshine Coast, Sippy Downs, QLD, Australia.
AC
CE
PT
ED
Correspondence to: Samuel R. Foley Critical Care Research Group, The Prince Charles Hospital Lvl 3 Clinical Sciences Bld, Rode Road, Chermside, QLD 4032 E-mail: [email protected] Tel.: + 61 410037406 Fax: +61 3139 6120
1
ACCEPTED MANUSCRIPT
Abstract
Introduction: Similarities in size, anatomy and physiology have supported the use of sheep to model a wide range of human diseases, including coagulopathy. However, coagulation studies involving sheep are limited by the absence of high quality data defining normal ovine coagulation and fibrinolysis.
PT
Materials and Methods: Full blood examination, routine and specialised coagulation tests, rotational thromboelastometry and whole blood platelet aggregometry was performed on 50
SC RI
healthy Samm & Border Leicester Cross ewes and compared to corresponding human ranges. Intraspecies breed and gender variability was investigated by comparison to a smaller population of 13 healthy Merino wethers.
Results: Ovine coagulation was similar to human according to routine coagulation methods
NU
(PT, aPTT, TCT, Fib(C)) and some specialised coagulation tests (vWF, AT, Plasmin Inh). Despite these similarities, ovine secondary haemostasis demonstrated substantial differences to
MA
that of human. Rapid initiation of the contact activation pathway, high levels of FVIII, low Protein C, greater overall clot firmness and a reduced capacity for clot lysis was documented in sheep. In addition, ADP and collagen agonists precipitated a reduced primary haemostatic
ED
response in sheep relative to human. Intraspecies differences in whole blood platelet aggregometry between the cohorts of sheep indicate the need for breed-specific normal ranges.
PT
Conclusions: The application of a board spectrum of coagulation assays has enabled elucidation of the similarities as well as differences between ovine and human coagulation. The
CE
new knowledge generated from this study will guide the design of future translational
Keywords
AC
coagulation studies in ovine models.
Animal, coagulation parameters, normal values, platelets, sheep, thromboelastometry.
Abbreviations PLT - platelet count; PT - prothrombin time; aPTT - activated partial thrombin time; TCTthrombin clotting time; Fib(C) – fibrinogen (clauss); vWF - von Willebrand factor, FVIII factor VIII; FXII - factor XII; Pl.Inh. - plasmin inhibitor; Prot.C - protein C; AT antithrombin; MP – Multiplate® whole blood platelet aggregometry; ADP – Adenosine Diphosphate; COL – Collagen; TEG – rotational thromboelastography®; ROTEM - rotational thromboelastometry®.
2
Introduction
ACCEPTED MANUSCRIPT
Comparative studies of human, sheep, pig, rabbit, rat and dog using routine coagulation tests, clotting factor assays and rotational thromboelastometry indicate that the human coagulation system exhibits greatest similarity with that of sheep (1, 2). Recently, a line of sheep with hemophilia A (HA) was re-established (3) and recombinant ovine FVIII (oFVIII) developed to Surprisingly, despite the utility of ovine models in haemostasis
PT
support this model (4).
research, data defining ‘normal’ parameters for both classical and newer modes of coagulation
SC RI
assessment is deficient. Studies to date are limited by small sample size (1, 2, 5) and many define only a small range of coagulation parameters (6, 7). Table 1 summarises published coagulation parameters of sheep and reveals variability in results (e.g. FVIII) between studies.
NU
The aim of this study was to define a robust and comprehensive normal ovine coagulation profile. To do this we studied a large cohort (n=50) of healthy Samm & Border Leicester Cross
MA
(SBL) ewes to define full blood examination (FBE), routine and specialised coagulation tests, as well as the newer whole blood coagulation measures of rotational thromboelastometry (ROTEM®) and whole blood platelet aggregometry (Multiplate®). To investigate inter- and
ED
intraspecies differences in coagulation, this data was compared to corresponding human ranges and another population of healthy Merino wethers (n=13). Data from this comprehensive range
PT
of coagulation assays has enabled a thorough assessment of the benefits as well as the
CE
limitations of using ovine models for translational coagulation studies.
Material and Methods Animals
AC
SBL ewes (female sheep) (n=50) and Merino wethers (castrated male sheep) (n=13) were evaluated for this study. The sheep were housed in a purpose built animal facility for at least 1 week prior to testing, fed pellets and had access to water ad libitum. All animals were 2-3yr old, weighed 45-55 kg and physically assessed to be healthy by a veterinarian prior to blood sampling. This study was approved by the Animal Research Ethics Committee of the Queensland University of Technology and University of Queensland (QUT UAEC Approval number 1100000053 and 1000000025) and conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 7th Edition 2004 (The Code) of the National Health and Medical Research Council (NHMRC).
3
ACCEPTED MANUSCRIPT
Blood sampling protocol
Blood samples were obtained from the left internal jugular vein of the sheep through a central venous catheter. Whole blood samples for ROTEM® analysis was collected in 3.2% sodium citrate tubes (Greiner Bio-One, Amata Nakorn, Chonburi, Thailand), EDTA tubes (Greiner Bio-One, Kremsmünster, Austria) for FBE, and hirudin tubes (Verum Diagnostica, GmbH,
PT
Munich, Germany) for Multiplate® whole blood platelet aggregometry. FBE, ROTEM® and Multiplate® analysis was conducted within 2 hours of collection. Citrated blood for the routine
SC RI
and specialised coagulation tests was centrifuged for 15 minutes at 3000g, this was repeated on separated plasma and stored at -80°C prior to analysis.
Full blood examination, routine and specialised coagulation tests
NU
FBE was evaluated using the veterinary mode of the Act diff™ haematology analyser (Beckman Coulter Australia Pty Ltd, NSW, Australia) and consisted of white cell count
MA
(WCC), red cell count (RCC), haemoglobin (Hb), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and platelet count (PLT). Routine coagulation tests (prothrombin time (PT), activated partial
ED
thromboplastin time (aPTT), Clauss fibrinogen [Fib(C)], thrombin clotting time (TCT) and Ddimer) and specialised coagulation tests (Factor VIII (FVIII), Factor XII (FXII), von
PT
Willebrand Factor (vWF), Protein C, antithrombin (AT), plasmin inhibitor (Plasmin Inh)) were performed on the ACL TOP® analyser (Instrumentation Laboratory, Werfen Australia, NSW,
CE
Australia) as per the manufacturer’s instructions.
Whole blood platelet aggregometry (Multiplate®)
AC
Tests were performed on the Multiplate® 5.0 - Platelet Function Analyser (Haemoview Diagnostics, Brisbane, Australia) (8) using Multiplate® Mini test cells in accordance with the manufacturer’s instructions. Hirudin-anticoagulated blood was tested using ADP (6.4μmol/L) and collagen (3.2 μg/ml) agonists.
Rotational thromboelastometry (ROTEM®) Citrated blood samples were tested using rotational thromboelastometry (ROTEM®, Haemoview Diagnostics, Brisbane, Australia). The INTEM, EXTEM and FIBTEM tests were run for 35 minutes following clot initiation (until A30 recorded) according to manufacturer’s instructions (9). EXTEM clotting time (CT, seconds (s)), clot formation time (CFT, s), alpha angle (, °), clot lysis index (CLI30, %), clot amplitude at 10 minutes (A10, mm) and at 30 minutes (A30, mm), as well as FIBTEM-A10 was compared between the two sheep populations.
4
Statistical analysis
ACCEPTED MANUSCRIPT
Data analysis was performed using GraphPad Prism 6 statistical package. Descriptive statistics and histograms showed normal and non-normal distributions of results. Data was expressed as mean ± standard deviation (SD) and range (2.5-97.5%) with multigroup analysis using oneway ANOVA and Bonferroni’s multiple comparison tests for normal data and Kruskal–Wallis
PT
and Dunn’s multiple comparison tests for non-normal data. Spearman’s rank correlation coefficients were determined to assess for correlation between plasma-based coagulation
SC RI
parameters (PT, aPTT, Fib(C), FVIII, FXII, Plasmin Inh), platelet count (PLT) and corresponding ROTEM® measurements in healthy SBL ewes.
Results
NU
Haematological and coagulation parameters in sheep and human
The mean and standard deviation of all haematological and plasma-based coagulation
MA
parameters from 50 adult SBL ewes (female) are detailed and compared to corresponding female human ranges (Table 2). The range and upper limit of ovine WCC and PLT count were higher. Although sheep had higher RCC, they had a corresponding lower MCV and MCH, but
ED
MCHC was comparable. PT, aPTT, TCT, Fib(C), vWF, AT and Plasmin Inh levels were similar in sheep and human. Sheep had higher D-dimer, FVIII, and FXII, and lower Protein C
PT
levels.
CE
ROTEM® and Multiplate® parameters in sheep and human (i) Analysis of two distinct sheep populations No significant difference in INTEM- or EXTEM-CT, -CFT, -, -A10, -A30 or FIBTEM-A10
AC
was documented between the two populations of sheep (Table 3). No fibrinolysis, as measured by INTEM-CLI30 or EXTEM-CLI30 was detected in either subgroup of sheep. In contrast, ADP (p
