Thrombin Generation and Thromboelastography in Non-overt DIC
Thrombin Generation and Thromboelastography in Non-overt DIC
Sepsis is the 13th most common cause of death in the United States, causing approximately
 210,000 deaths per year. Once DIC has developed, irreversible organ injury has already
 occurred and the mortality rate is 70%. Inhibition of systemic coagulation with activated
 protein C concentrate has been the only therapy for sepsis introduced in the past several
 decades which has improved outcomes. Elucidation of the coagulopathic mechanisms early in the
 development of DIC may give rise to targeted therapies and strategies for early intervention.
 We hypothesize that an increase in endogenous thrombin potential precedes the development of
 overt DIC by a clinically significant time period. Our primary objective is to determine if
 endogenous thrombin potential (ETP) measured at first diagnosis of sepsis prior to the onset
 of DIC and organ failure is predictive of overt DIC and/or poor outcome. We will compare ETP
 to standard coagulation assays and the clinical assessment of DIC using the ISTH criteria for
 overt DIC. A secondary objective of this study is to determine if host coagulation variables
 predispose to the development of DIC and poor clinical outcome during sepsis.
Activation of the coagulation system occurs early in patients with sepsis, although
 clinically overt disseminated intravascular coagulation (DIC) is identified in only a
 minority of patients with severe sepsis. Uncontrolled activation of the coagulation system
 may contribute to the pathophysiology of multiple organ failure and the subsequent morbidity
 and mortality of sepsis. Identification of risk factors predicting progression to severe
 sepsis and DIC has been elusive. We propose that whole blood and cell-rich coagulation assays
 will offer improved sensitivity to both the procoagulant and anti-coagulant changes which
 occur early in sepsis and will improve recognition of non-overt DIC. Future studies will
 address whether these assays have sufficiently high predictive value to identify subgroups of
 patients who could benefit from early intervention.

 Specific Aims:

 1. We hypothesize that increased thrombin generation will precede development of overt DIC
 by a clinically significant time period. Our primary objective is to determine if
 endogenous thrombin potential measured at first diagnosis of sepsis prior to the onset
 of DIC and organ failure is predictive of overt DIC.

 2. There is significant individual variation among the healthy population in endogenous
 thrombin generation due to known and unknown polymorphisms within coagulation proteins.
 We predict that host variables in thrombin generation will contribute to susceptibility
 to DIC and poor outcome during sepsis. A secondary objective of this study is to
 determine if host coagulation variables predispose to the morbidity and mortality
 associated with sepsis.

 Experimental Design and Methods

 The study design will be a prospective observational study of patients presenting to the
 Memorial Hermann Hospital Emergency Department with sepsis. Criteria for sepsis include
 evidence of systemic inflammatory response syndrome as defined by the ACCP/SCCM Consensus
 Conference, and a known or suspected infection. Exclusion criteria include signs of severe
 sepsis or septic shock, as defined by the ACCP/SCCM Consensus Conference, at presentation,
 including: organ dysfunction, hypoperfusion and perfusion abnormalities. Chronic medical
 conditions associated with immune suppression or coagulopathies, such as neutropenia and
 sickle cell disease, and use of medications leading to immune dysfunction or coagulopathies,
 such as chronic steroid use or anti-coagulation will also be reasons for exclusion. In
 addition, because of the volume of blood required from each patient for laboratory studies,
 only patients 25 kg body weight or more will be enrolled. Withdrawal from the study will be
 at the discretion of the treating physician or subject. All efforts will be made to collect
 clinical information from patients who have withdrawn.

 Inclusion criteria

 -  Known or suspected infection as determined by the treating physician Patient to be
 admitted to the hospital

 -  Systemic Inflammatory Response Syndrome: 3 of the following 4 criteria:

 -  Temperature > 38 C or < 35 C

 -  Heart rate > 90 beats/min, except in patients with a medical condition known to increase
 the heart rate or those receiving treatment that would prevent tachycardia.

 -  Respiratory rate > 20 breaths/min or PaC02 < 32 mmHg, or on mechanical ventilation for
 an acute respiratory process.

 -  White blood cell count > 12,000/mm3, < 4,000/mm3, or > 10% bands

 Exclusion criteria

 -  - Use of the following medications: unfractionated heparin to treat an active thrombotic
 event within 8 hours before the infusion; low-molecular-weight heparin at a higher dose
 than recommended for prophylactic use within 12 hours before the infusion, warfarin used
 within 7 days of study entry, aspirin use at a dose of more than 650 mg/day within 3
 days before the study, thrombolytic therapy within 3 days before the study, glycoprotein
 IIb/IIIa antagonists within 7 days before the study entry, administration of activated
 protein C (Xigris ) in the 24 hours before study entry.

 -  Neutropenia

 -  Hemophilia

 -  Diabetic ketoacidosis

 -  Weight < 25 kg

 Patients will be enrolled in the emergency department within 2 hours of meeting eligibility.
 Written informed consent will be obtained from the patients or their authorized
 representatives. Blood samples will be collected by peripheral venipuncture during the first
 hour after enrollment and then once daily for 7 days (see Appendix A).

 Patients will be withdrawn from the study if blood sampling is not performed within 2 hours
 of obtaining informed consent.

 Clinical data collection Clinical data forms (see Appendix B) will be completed at the time
 of enrollment and daily for 7 days , at hospital discharge, and at 28 days. Clinicians will
 be blinded to the results of the research laboratory tests.

 Laboratory Methods

 Blood and plasma Each patient will have 15 ml blood collected at enrollment. Specimens will
 be obtained through antecubital venipuncture. Free flow or minimal suction will be employed;
 vacuum containers will be avoided. Specimens for platelet-poor plasma (PPP) and whole blood
 analysis will be collected into siliconized glass tubes with 0.105M tri-sodium citrate in the
 ratio of 1 part anticoagulant to 9 parts whole blood. PPP will be separated within 60 minutes
 and analyzed immediately. Platelet-rich plasma (PRP) specimens will be separated from the
 upper ¾ volume of plasma supernatant after centrifugation at 265g for 10 minutes at room
 temperature. The platelets will be counted on a Beckman Coulter counter and adjusted to 150 X
 109 platelets/l with autologous PPP. PRP will be used within 60 minutes. Cbc, PT/PTT,
 Fibrinogen, d-dimer, Protein C activity, Protein S activity, ATIII activity, Factor V Leiden
 mutation, Prothrombin G20210A mutation analysis will be performed in Memorial Herman Hospital
 clinical laboratories.

 RoTEG

 Whole blood specimens will be drawn as described above. Measurements will be performed on two
 roTEG Coagulation Analyzers from Pentapharm. RoTEG all-plastic reaction cups are procured
 from the manufacturer. Polypropylene and polyethylene pipettes are used to handle reagents
 and blood. Citrated whole blood will be re-calcified by 20 mcl 0.2 mol/L CaCl2 and activated
 by 20 mcl solution of recombinant human TF (Innovin) diluted 1:1000 using a sodium barbital
 buffer. The measured parameters have been defined by the manufacturer as follows: The
 clotting time(CT) is the time in seconds from Ca2+ activation of coagulation and until an
 increase in elasticity corresponding to 2 graphical mm is obtained on the ordinate. The clot
 formation time (CFT) is the time in seconds passing while the elasticity increases from 2 mm
 to 20 mm on the ordinate. The maximum clot formation (MCF) expresses the maximum strength in
 millimeters of the final clot. Several additional parameters representing the continuous
 registration of clot formation have been defined by Sorensen et al 28: the maximum velocity
 (MaxVel) of clot formation, the time to maximum velocity (T,MaxVel) of clot formation, and
 the area under the velocity curve (AUC).

 Thrombin Generation

 Calibrated automated thrombin generation measurements will be performed using the Hemker
 methodology. 29 Platelet rich plasma (PRP) will be prepared as described above. Fluorogenic
 substrate and chromogenic thrombin will be purchased through Thermolabsystems, and
 recombinant relipidated tissue factor(TF) through Dade Behring. alpha 2
 macroglobulin-thrombin complex used as a calibrator will be purchased through
 Thrombolabsystems. The thrombograms will be measured in a 96-well plate fluorometer (Ascent
 reader, Thrombolabsystems) and analyzed with software purchased through Thrombinoscope.
 Experiments will be carried out in quadruplicate and compared to a calibrator. To each well
 80 mcl of plasma will be added, followed by the calibrator or buffer, then the "trigger": 20
 mcl of 3 mM of TF. The plate will be placed in the fluorometer and allowed to warm to 37 C.
 The instrument dispenses 20 mcl of Fluorogenic substrate 2.5 mM and CaCl2 100 mM (FluCa) to
 all the wells to be measured, registers this as zero time, shakes them for 10 s and starts
 reading. During the measurement, the program compares the readings from the TG and the CL,
 calculates thrombin concentration and displays the thrombin concentration in time. The peak
 thrombin is the highest thrombin concentration reached during the time course of thrombin
 formation and inhibition. The thrombin potential is the amount of thrombin that is formed
 within 60 minutes (Area under the curve). The lag phase and the peak time refer to the start
 and velocity of thrombin formation, respectively.

 Data Management Data will be collected on paper forms which will be designed by the GCRC
 informatics core section. The GCRC informatics core will maintain data on a secure database
 (Access). Each subject will be assigned a unique ID reflecting the order in which enrollment
 took place.

 Data Analysis and Presentation

 This is a pilot study intended to provide the preliminary data for future clinical studies.
 Descriptive statistics will be used to characterize ETP and roTEG at initial presentation of
 sepsis. Means and ranges will be reported for each of the variables in the thrombin
 generation assay (lag time, slope of rise, peak value, and area under the curve) and roTEG
 (Clot formation time (CFT), maximum clot formation (MCF), maximum velocity (MaxVel) of clot
 formation, time to maximum velocity (T,MaxVel) of clot formation, and area under the velocity
 curve (AUC)). The primary objective of this study is to compare ETP at presentation to the
 development of DIC, as defined by a positive ISTH DIC score. The secondary objective will be
 to compare host coagulation variables, including ETP, roTEG, Pro C, Pro S, ATIII, FVL, and
 prothrombin G20210A mutation at presentation, with the secondary outcome measures of 28-day
 mortality and organ dysfunction. All statistics will be performed using NCSS/PASS.

 Sample size determination: There are no published reports of thrombin generation assays or
 thromboelastography in sepsis, or DIC. In our previous studies of patients taking an
 anticoagulant (warfarin), there was a 400% decrement of ETP (1719 to 404). In order to derive
 a mean ETP with a target width of 100 and 95% confidence interval, assuming a standard
 deviation of 259, we will need to enroll 100 patients.

 We will employ the following statistical methods for the analysis of data:

 Specific Aim #1: The endogenous thrombin potential (ETP) is the area under the curve of the
 thrombin generation assay. The values are continuous and range from 0 to 2,500. Maximum clot
 formation (MCF) from the roTEG will also be used to approximate ETP. The DIC score will be
 calculated and coded as a dichotomous variable: Yes (score > 5) or No (score < 5). A receiver
 operating curve will be derived for ETP and MCT and its sensitivity and specificity for
 predicting DIC. If the area under the curve is found to be significant, an optimal cut-off
 value with the highest degree of accuracy will be chosen. This value will be used to estimate
 the predictive value of the test. Positive predictive value will be calculated with the
 formula: # patients with ETP or MCT above the cut-off and development of DIC / # patients
 with positive DIC score. The predictive value of a negative DIC score will be calculated
 using the formula: # patients with ETP or MCT below the cut-off and no progression to DIC / #
 patients with negative DIC score.

 Specific Aim #2: A multivariate analysis will be done to determine which of the predictor
 variables (ETP, MCT, Pro C, Pro S, ATIII, FVL, ProG20210A, DIC) are associated with the
 secondary dichotomous outcome measures: organ dysfunction at 28 days (Any/None), 28-day
 mortality (Dead/Alive).
Inclusion Criteria:

 -  Systemic inflammatory response syndrome with known or suspected infection

 -  Patient to be admitted to the hospital

 Exclusion Criteria:

 -  Diabetic ketoacidosis, Hemophilia, weight < 25 kg, use of hemostatic agents prior to
 entry.