Thrombin Generation and Platelet Activation in CRS/HIPEC
- Conditions
- Peritoneal CarcinomatosisMesothelioma; PeritoneumPseudomyxoma Peritonei
- Interventions
- Procedure: CRS/HIPEC
- Registration Number
- NCT03034850
- Lead Sponsor
- Ziekenhuis Oost-Limburg
- Brief Summary
Cytoreductive surgery (CRS) with hyperthermic intraperitoneal peroperative chemotherapy (HIPEC), indicated for patients with peritoneal metastases from digestive or gynecological malignancies alike, demonstrates a considerable impact on hemostatic metabolism, both on platelet and on coagulation level. The potential hemostatic interference in CRS and HIPEC is phase dependent. This study demonstrates the combined use of ROTEM (rotational thromboelastometry), PACT (platelet activation test) and CAT (thrombin generation test) assays during CRS and HIPEC with a follow-up of 7 days postoperative.
- Detailed Description
The purpose of this study was to quantitatively assess the impact of CRS and HIPEC, on various components of hemostasis. Routine laboratory assays such as activated clotting time, activated partial thromboplastin time, prothrombin time, or platelet count might, as demonstrated previously, insufficiently provide specificity and/or sensitivity to assess coagulation and platelet disorders. Therefore, additionally thrombin generation (TG) was analyzed by the calibrated automated thrombogram assay (CAT). Also, platelet function was quantitatively assessed by the PAC-t-UB assay and rotational thromboelastometry (ROTEM) was used to elucidate the contribution of platelets, intrinsic and extrinsic coagulation pathways in peri-operative bleeding. The hypothesis of this study was that the procedure exposed an increased thrombotic risk, resulting in a faster and increased TG and hyper platelet function?
Recruitment & Eligibility
- Status
- COMPLETED
- Sex
- All
- Target Recruitment
- 27
- a confirmed histological diagnosis of peritoneal disease (e.g., mesothelioma; pseudomyxoma peritonei; colorectal, ovarian, or gastric peritoneal carcinomatosis of colorectal, ovarian, or gastric cancer origin; or abdominal sarcomatosis); and
- age <80 years; and
- a cardiac, renal, hepatic, and bone marrow function compatible with surgery; and
- informed written consent to participate in the study
(or)
- inherited coagulation abnormalities,
- active systemic infections,
- interstitial lung disease,
- serious cardiac dysrhythmia or condition, New York Heart Association classification of III or IV, congestive cardiac failure, uncontrolled hypertension (diastolic blood pressure constantly >100 mm Hg, systolic blood pressure constantly > 180 mm Hg).
- inadequate bone marrow function at the beginning of the trial, defined as platelet count less than <150 GPT/L or neutrophil granulocyte count less than <1.5 GPT/L.
- inadequate renal function at the beginning of the trial, defined as GFR less than <60 ml/min,
- inadequate liver function at the beginning of the trial, defined as bilirubin >1.5 times ULN (upper limit of normal), active hepatitis B or C infection,
- female patients who are pregnant or breast feeding
- participation in another therapeutic clinical trial.
Study & Design
- Study Type
- OBSERVATIONAL
- Study Design
- Not specified
- Arm && Interventions
Group Intervention Description CRS/HIPEC CRS/HIPEC Patients with a confirmed histological diagnosis of peritoneal disease treated by cytoreductive surgery (CRS) with hyperthermic intraperitoneal peroperative chemotherapy (HIPEC).
- Primary Outcome Measures
Name Time Method Blood loss From surgical incision to 7 days postoperative Blood loss and administration of red blood cells, fresh frozen plasma and platelets. Blood loss is quantitatively assessed based on surgical drainage volume measurements, recorded every hour. Once the surgical drains are removed (average 7 days), blood loss is quantified by hemodynamic instability and abrupt, significant decrease of hemoglobin concentration. Blood loss is assessed from the date of CRS/HIPEC surgery until 7 days postoperative or date of death from any cause, whichever came first.
- Secondary Outcome Measures
Name Time Method Coagulation Time CT EXTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Coagulation Time (CT): test start until a clot firmness amplitude of 2 mm is reached; sec.
Coagulation Time CT FIBTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands), All samples were measured within 1 h after blood collection. Coagulation Time (CT): test start until a clot firmness amplitude of 2 mm is reached; sec.
Activated Partial Thromboplastin Time (aPTT) From surgical incision to 7 days postoperative Activated Partial Thromboplastin Time was measured using an ACL-9000 coagulation analyser (sec).
Lag Time (Thrombin generation assay (CAT)) From surgical incision to 7 days postoperative TG in plasma, measured with the calibrated automated thrombogram (CAT) . Briefly, 80 μl platelet poor plasma (PPP) was mixed with 20 μl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 μM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 μl of calibrator (α2macroglobulin- thrombin complex) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z- Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 μM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) \[20\]. lagtime (LT)(min)
Endogenous Thrombin Potential (Thrombin generation assay (CAT)) From surgical incision to 7 days postoperative TG in plasma, measured with the calibrated automated thrombogram (CAT) . Briefly, 80 μl platelet poor plasma (PPP) was mixed with 20 μl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 μM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 μl of calibrator (α2macroglobulin- thrombin complex) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z- Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 μM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) \[20\]. Endogenous thrombin potential (ETP) (nM\*min)
P-selectin expression (Platelet activation test (PACT)) From surgical incision to 7 days postoperative Platelet activation was quantitatively assessed in un-processed blood by the PACT (Platelet activation test). Addition of specific agonists to whole blood (granule release capacity and in the aggregation potential of platelets). (1) the protease activated receptor (PAR-1) agonist thrombin receptor activator peptide, (2) the glycoprotein VI (GPVI) agonist collagen-related peptide , and (3) the P2Y12 agonist ADP. The reaction mixtures also contain three antibodies directed against GPIb, activated αIIbβ3 and P-selectin. Flow cytometry was used to distinguish between platelets and other cells on forward and sideward scatter pattern and by gating on the CD42b positive cells. Fluorescent intensity in the FITC gate and PE gate was selected to determine activated αIIbβ3 and P-selectin density, respectively, and results are expressed as median fluorescent intensity (MFI). P-selectin expression(MFI, median fluorescent intensity)
Red blood cell count From surgical incision to 7 days postoperative EDTA-anticoagulated blood was used for cytometric analysis using a whole blood counter Sysmex XE 2100® (Sysmex,Kobe, Japan) to obtain a whole blood count. (million cells/mcL)
Time-to-Thrombin Peak (Thrombin generation assay (CAT)) From surgical incision to 7 days postoperative TG in plasma, measured with the calibrated automated thrombogram (CAT) . Briefly, 80 μl platelet poor plasma (PPP) was mixed with 20 μl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 μM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 μl of calibrator (α2macroglobulin- thrombin complex) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z- Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 μM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) \[20\]. Time-to-Thrombin Peak (TTP)(min)
Thrombin Peak (TP) (Thrombin generation assay (CAT)) From surgical incision to 7 days postoperative TG in plasma, measured with the calibrated automated thrombogram (CAT) . Briefly, 80 μl platelet poor plasma (PPP) was mixed with 20 μl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 μM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 μl of calibrator (α2macroglobulin- thrombin complex) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z- Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 μM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) \[20\]. Thrombin Peak (TP)(nM)
A5 EXTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands), A5: amplitude of clot firmness 5 min after CT (mm)
A5 FIBTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands), All samples were measured within 1 h after blood collection. A5: amplitude of clot firmness 5 min after CT (mm)
Maximum Lysis (ML) FIBTEM Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Maximum Lysis (ML; %): maximum lysis during runtime
White blood cell count From surgical incision to 7 days postoperative EDTA-anticoagulated blood was used for cytometric analysis using a whole blood counter Sysmex XE 2100® (Sysmex,Kobe, Japan) to obtain a whole blood count. (cells/mcL)
Platelet count From surgical incision to 7 days postoperative EDTA-anticoagulated blood was used for cytometric analysis using a whole blood counter Sysmex XE 2100® (Sysmex,Kobe, Japan) to obtain a whole blood count. (platelets/mcL)
Fibrinogen levels From surgical incision to 7 days postoperative Fibrinogen levels were determined with an ACL-9000 (Diamond Diagnostics, Holliston, MA) coagulation analyser. (g/dL)
Prothrombin Time (PT) From surgical incision to 7 days postoperative Prothrombin time was measured using an ACL-9000 coagulation analyser (sec).
A30 HEPTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. A30: amplitude of clot firmness 30 min after CT (mm)
Alpha EXTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Alpha (the angle between the baseline and a tangent to the clotting curve through the 2 mm point; degree)
Maximum Lysis (ML) EXTEM Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Maximum Lysis (ML; %): maximum lysis during runtime
αIIbβ3 activation (Platelet activation test (PACT)) From surgical incision to 7 days postoperative Platelet activation was quantitatively assessed in un-processed blood by the PACT (Platelet activation test). Addition of specific agonists to whole blood (granule release capacity and in the aggregation potential of platelets). (1) the protease activated receptor (PAR-1) agonist thrombin receptor activator peptide, (2) the glycoprotein VI (GPVI) agonist collagen-related peptide , and (3) the P2Y12 agonist ADP. The reaction mixtures also contain three antibodies directed against GPIb, activated αIIbβ3 and P-selectin. Flow cytometry was used to distinguish between platelets and other cells on forward and sideward scatter pattern and by gating on the CD42b positive cells. Fluorescent intensity in the FITC gate and PE gate was selected to determine activated αIIbβ3 and P-selectin density, respectively, and results are expressed as median fluorescent intensity (MFI). αIIbβ3 activation (MFI, median fluorescent intensity)
A5 HEPTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. A5: amplitude of clot firmness 5 min after CT (mm)
Alpha HEPTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Alpha (the angle between the baseline and a tangent to the clotting curve through the 2 mm point; degree)
Coagulation Time CT HEPTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Coagulation Time (CT): test start until a clot firmness amplitude of 2 mm is reached; sec.
Clot Formation Time CFT HEPTEM (Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. CFT: in seconds indicates the time between 2 and 20 mm clot firmness amplitude is achieved (sec)
Maximum Lysis (ML) HEPTEM Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: HEPTEM (ref.: 503-09, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Maximum Lysis (ML; %): maximum lysis during runtime
A30 EXTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. A30: amplitude of clot firmness 30 min after CT (mm)
A30 FIBTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. A30: amplitude of clot firmness 30 min after CT (mm)
Alpha FIBTEM (Rotational thromboelastometry (ROTEM)) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. Alpha (the angle between the baseline and a tangent to the clotting curve through the 2 mm point; degree)
Clot Formation Time CFT EXTEM (Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: EXTEM (ref.: 503-05, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands), All samples were measured within 1 h after blood collection. CFT: in seconds indicates the time between 2 and 20 mm clot firmness amplitude is achieved (sec)
Clot Formation Time CFT FIBTEM (Rotational thromboelastometry (ROTEM) From surgical incision to 7 days postoperative Thrombus formation was measured by ROTEM (Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). Standard assays were used according to the manufacturer's recommendations: FIBTEM (ref.: 503-06, Tem International GmbH c/o Dutch Affiliate, Tilburg, The Netherlands). All samples were measured within 1 h after blood collection. CFT: in seconds indicates the time between 2 and 20 mm clot firmness amplitude is achieved (sec)