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.
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?
Study Type
OBSERVATIONAL
Enrollment
27
The generic surgical approach involved peritonectomy procedures and visceral resections called CRS as described by Sugarbaker (1995). Peritoneal disease burden was assessed using the perito- neal cancer index (PCI), which scores 13 intra-abdominal sites on a scale of 0 (no disease) to 3 (lesion size \> 5 cm), thus giving a range of possible scores from 0 to 39. The same team performed the surgical procedure of all included patients. Before connection to the patient, the circuit was filled with dextrose 5% (2 L/m2 body surface area) and warmed to 37°C.
Blood loss
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.
Time frame: From surgical incision to 7 days postoperative
Red blood cell count
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 frame: From surgical incision to 7 days postoperative
White blood cell count
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)
Time frame: From surgical incision to 7 days postoperative
Platelet count
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)
Time frame: From surgical incision to 7 days postoperative
Fibrinogen levels
Fibrinogen levels were determined with an ACL-9000 (Diamond Diagnostics, Holliston, MA) coagulation analyser. (g/dL)
Time frame: From surgical incision to 7 days postoperative
Prothrombin Time (PT)
Prothrombin time was measured using an ACL-9000 coagulation analyser (sec).
Time frame: From surgical incision to 7 days postoperative
Activated Partial Thromboplastin Time (aPTT)
Activated Partial Thromboplastin Time was measured using an ACL-9000 coagulation analyser (sec).
Time frame: From surgical incision to 7 days postoperative
Endogenous Thrombin Potential (Thrombin generation assay (CAT))
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)
Time frame: From surgical incision to 7 days postoperative
Lag Time (Thrombin generation assay (CAT))
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)
Time frame: From surgical incision to 7 days postoperative
Time-to-Thrombin Peak (Thrombin generation assay (CAT))
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)
Time frame: From surgical incision to 7 days postoperative
Thrombin Peak (TP) (Thrombin generation assay (CAT))
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)
Time frame: From surgical incision to 7 days postoperative
P-selectin expression (Platelet activation test (PACT))
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)
Time frame: From surgical incision to 7 days postoperative
αIIbβ3 activation (Platelet activation test (PACT))
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)
Time frame: From surgical incision to 7 days postoperative
A5 EXTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
A5 FIBTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
A5 HEPTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
A30 EXTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
A30 FIBTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
A30 HEPTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
Alpha EXTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
Alpha FIBTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
Alpha HEPTEM (Rotational thromboelastometry (ROTEM))
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)
Time frame: From surgical incision to 7 days postoperative
Coagulation Time CT EXTEM (Rotational thromboelastometry (ROTEM))
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.
Time frame: From surgical incision to 7 days postoperative
Coagulation Time CT FIBTEM (Rotational thromboelastometry (ROTEM))
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.
Time frame: From surgical incision to 7 days postoperative
Coagulation Time CT HEPTEM (Rotational thromboelastometry (ROTEM))
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.
Time frame: From surgical incision to 7 days postoperative
Clot Formation Time CFT EXTEM (Rotational thromboelastometry (ROTEM)
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)
Time frame: From surgical incision to 7 days postoperative
Clot Formation Time CFT FIBTEM (Rotational thromboelastometry (ROTEM)
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)
Time frame: From surgical incision to 7 days postoperative
Clot Formation Time CFT HEPTEM (Rotational thromboelastometry (ROTEM)
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)
Time frame: From surgical incision to 7 days postoperative
Maximum Lysis (ML) EXTEM Rotational thromboelastometry (ROTEM)
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
Time frame: From surgical incision to 7 days postoperative
Maximum Lysis (ML) FIBTEM Rotational thromboelastometry (ROTEM)
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
Time frame: From surgical incision to 7 days postoperative
Maximum Lysis (ML) HEPTEM Rotational thromboelastometry (ROTEM)
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
Time frame: From surgical incision to 7 days postoperative
This platform is for informational purposes only and does not constitute medical advice. Always consult a qualified healthcare professional.