Autologous thrombin: intraoperative production from whole blood

Thrombin is routinely combined in surgical practice with a fibrinogen source to prepare fibrin sealant to promote hemostasis or with platelet concentrates to prepare platelet gels to enhance wound healing. The purpose of this study was to evaluate the robustness and reproducibility of a new sterile handheld disposable thrombin-processing device (TPD) to generate autologous human thrombin in the intraoperative setting, using whole blood as the starting source material. By using whole blood instead of plasma as the starting material, it is possible to eliminate the plasma separation step from whole blood and reduce the thrombin production time and increase its availability to the surgical team intraoperatively. Active thrombin was prepared by combining 4 mL of thrombin reagent (a mixture of calcium chloride and ethanol) to 11 mL of blood in a reaction chamber containing negatively charged particles. The whole blood, reagent and particle mixture was incubated for 25 minutes at either 18 degrees C or 24 degrees C (n = 25/group) to assess stability of the thrombin activity. The mean activity of the thrombin produced at 18 degrees C and 24 degrees C was 52 +/- 14 (n = 25) and 61 +/- 12.2 IU/mL (n = 25), respectively. The average volume of thrombin harvested from each aliquot of blood at 18 degrees C and 24 degrees C was 10 +/- 0.4 and 9 +/- 0.6 mL, respectively. The thrombin concentration generated was shown to rapidly (<5 seconds) coagulate fibrinogen concentrate and retained clotting activity for 1 hour at room temperature (18-26 degrees C) and up to 4 hours when stored on ice. The results show that the TPD is able to consistently generate high thrombin activity from human whole blood. The device offers a robust and rapid approach for preparing active thrombin from whole blood.     

Stability of human thrombin produced from 11 ml of plasma using the thrombin processing device

Autologous thrombin can be produced by activating the patient's own plasma. By adding calcium chloride (CaCl2) to the anticoagulated plasma, the coagulation cascade will be initiated, and active thrombin will be produced. However, thrombin obtained by this method degrades very quickly and is not practical for use during surgery. The aim of this study was to investigate the stability of the thrombin produced using the thrombin processing device (TPD; Thermogenesis Corporation). The TPD consists of a tubular chamber containing a negatively charged surface for activation. Plasma (11 ml) and reagent (CaCl2 and ethanol, 3.75 ml) were added to the TPD, and active thrombin was harvested after a 20-minute incubation. The production of thrombin was done at 18 degrees C (64 degrees F), 24 degrees C (75 degrees F), and 27 degrees C (81 degrees F) (n = 4/group). The produced thrombin was stored at the production temperature, 4 degrees C (39 degrees F), and 35 degrees C (95 degrees F). The thrombin activity was assessed by time to clot formation, using a fibrinogen concentrate as substrate, after 2, 4, and 6 hours of storage. Thrombin produced at 18 degrees C had clot times of less than 5 seconds for 2 hours (4.42 +/- 1.3 seconds) when stored at 4 degrees C, but 4 hours (4.1 +/- 1.3 seconds) when stored at 35 degrees C. In contrast, when thrombin was produced at 24 degrees C, the clot times were 4.3 +/- 0.7 and 4.6 +/- 1.6 seconds at 4 degrees C and 35 degrees C, respectively, for up to 6 hours. Similar results were obtained for thrombin produced at 27 degrees C. Active thrombin produced by the TPD is dependent on both the production temperature and the storage temperature. Autologous human thrombin with a stability of up to 6 hours can be obtained using the TPD when produced at 24 degrees C or 27 degrees C and stored at 4 degrees C.     

Quality of thrombin produced from the patient's own plasma using the TPD, a new Thrombin-processing Device

Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.     

Utilization of a topical autologous blood clot for treatment of pressure ulcers

Management and treatment of pressure ulcers (PUs) are met with great difficulty due to various factors that cause vulnerability of the soft tissue such as location, limited mobility, increased friction and shearing forces, as well as other comorbidities that may delay or halt wound healing. The topical autologous blood clot therapy (TABCT) is a point-of-care treatment used as a blood clot to assist in recreating and repairing the extracellular matrix (ECM). The mechanism of action consists of reconstruction of the ECM by incorporating into the ulcer, providing protection from further external destruction, while assisting in advancement through the wound healing phases via interaction of necessary growth factors, mediators, and chemokines. This study aims to assess the efficacy of the TABCT in the treatment of PUs in comparison to standard of care (SOC) treatment. Twenty-four patients, 18 years or older, with PUs ranging from stage 1 to 4, were included in this study. TABCT was created by using the patient's own peripheral blood in a point of care setting. Efficacy in percent area reduction (PAR) on weeks 4 and 12 with TABCT over SOC was assessed. Treatment using TABCT in PUs resulted in 77.9% of the patients achieving a 50% PAR on week 4. The mean PAR on week 12 was 96.23% with 45% of the wounds treated with TABCT achieving complete wound closure. TABCT exhibited efficacy in PAR of PUs. In addition, TABCT use prompted granulation tissue formation over vital structures, such as bone, which is often present in later stage PUs. The potential of bringing an affordable, cost-effective, advanced biologic bedside treatment that is efficacious in resolution of these complex wounds has the potential to drastically reduce the burden of treatment on the health system.     

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.     

Prospective randomized evaluation of a collagen/thrombin and autologous platelet hemostatic agent during total knee arthroplasty

The purpose of this study was to evaluate the effectiveness of a collagen/thrombin and autologous platelet hemostatic agent in preventing blood loss during primary total knee arthroplasty. This prospective, double-blinded, randomized study was designed to enroll a total of 100 patients. Patients were randomized 1:1 to either the treatment arm (standard hemostasis plus study product) or the control arm (standard hemostasis alone). Transfusion requirements, as determined by a blinded investigator using standardized criteria, were significantly lower in the treatment group (no blood transfusions) compared with the control group (5 transfusions; P = .007). These data support the addition of the study product to prevent blood transfusions after primary total knee arthroplasty.     

The role of intra-operative cell salvage in patient blood management for revision hip arthroplasty: a prospective cohort study

Cell salvage is an important component of blood management in patients undergoing revision hip arthroplasty surgery. However concerns regarding efficacy and patient selection remain. The aims of this study were to describe intra-operative blood loss, cell salvage re-infusion volumes and red blood cell transfusion rates for revision hip procedures and to identify factors associated with the ability to salvage sufficient blood intra-operatively to permit processing and re-infusion. Data were collected from a prospective cohort of 664 consecutive patients undergoing revision hip surgery at a single tertiary centre from 31 March 2015 to 1 April 2018. Indications for revision surgery were aseptic (n = 393 (59%)) fracture (n = 160 (24%)) and infection (n = 111 (17%)). Salvaged blood was processed and re-infused when blood loss exceeded 500 ml. Mean (SD) intra-operative blood loss was 1038 (778) ml across all procedures. Salvaged blood was re-infused in 505 of 664 (76%) patients. Mean (SD) re-infusion volume was 253 (169) ml. In total, 246 of 664 (37%) patients received an allogeneic red blood cell transfusion within 72 h of surgery. Patients undergoing femoral component revision only (OR (95%CI) 0.41 (0.23–0.73)) or acetabular component revision only (0.53 (0.32–0.87)) were less likely to generate sufficient blood salvage volume for re-infusion compared with revision of both components. Compared with aseptic indications, patients undergoing revision surgery for infection (1.87 (1.04–3.36)) or fracture (4.43 (2.30–8.55)) were more likely to generate sufficient blood salvage volume for re-infusion. Our data suggest that cell salvage is efficacious in this population. Cases where the indication is infection or fracture and where both femoral and acetabular components are to be revised should be prioritised.     

Inhibition of neointimal hyperplasia by a specific thrombin inhibitor

Objective—Restenosis secondary to neointimal hyperplasia remains the major limiting factor after vascular interventions. Thrombin generated in high concentrations at the site of vascular injury plays a central role in thrombosis and hemostasis. Thrombin has also been implicated as a mitogen for smooth muscle cell proliferation that contributes to restenosis. This study was designed to determine the effects of a specific thrombin inhibitor on neointimal hyperplasia after balloon injury in a rat carotid artery model.

Design—A total of 47 male Sprague–Dawley rats were divided into five groups. All groups underwent balloon injury of the left carotid artery. A specific thrombin inhibitor, inogatran, was given in four different regimens: low and high dose injections, short‐term infusion for 3?h, and long‐term infusion for 1 week. After 2 weeks the animals were killed and the carotid neointima/media area ratio and the luminal narrowing were calculated.

Results—All treatments significantly reduced the neointimal hyperplasia. Inogatran given as a long‐term infusion for 1 week had the lowest neointima/media ratio compared with the other groups. The percentage of lumen narrowing was also significantly lower in all treatment groups compared with the control group.

Conclusion—A specific direct thrombin inhibitor, inogatran, reduces neointimal hyperplasia after arterial injury in rats. A more prolonged administration of the thrombin inhibitor gave a further reduction of the neointimal hyperplasia. It seems that inhibition of thrombin activity is not only important early after injury, but also later. This could have clinical implications in the treatment of restenosis and needs to be further evaluated.     

Optimized thrombin dilution protocol for a slowly setting fibrin sealant in surgery

Summary BACKGROUND: Fibrin sealants find applications in many surgical fields (e.g., plastic and reconstructive surgery) where a slower setting time of about 30 to 60 s is beneficial. This can be achieved by diluting the thrombin component of the fibrin sealant to a concentration of about 5 IU/ml. This work was carried out to define an optimal dilution medium for thrombin which maintains all relevant functional parameters of the fibrin sealant and which is easy to prepare with components available in most operating theaters. METHODS: Different performance criteria of a fibrin sealant were tested in vitro by the following methods: adhesive strength in a rat skin adhesion model, turbidimetry and scanning electron microscopy to determine the fibrin clots structure, tensile strength of the fibrin clots by measuring the force at break. The cross-linking of the fibrin sealant by factor XIII (FXIII) as a function of the calcium concentration was assessed by quantitative electrophoresis. RESULTS: A dilution medium containing only calcium chloride in a concentration of about 20 mM performed best in most of the applied test systems. Dilution media with high ionic strength resulted in clots with low adhesive and tensile strength, thin fibrin fibers, and low clot elasticity. Dilution media without calcium prevented proper cross-linking of the resulting fibrin clots and led to clots with low tensile and adhesive strength. CONCLUSIONS: We present an optimized simple dilution protocol for the dilution of the thrombin component for a slowly setting fibrin sealant.     

The effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic response after cardiac surgery

Background: Thrombin formation during cardiac surgery could result in disordered hemostasis and thrombosis. The aim of the study was to examine the effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic activity in patients undergoing cardiac surgery. Methods: Data were collected prospectively from 60 patients undergoing coronary artery bypass grafting using cardiopulmonary bypass (CPB). In a randomized sequence, 20 patients received aprotinin, 20 patients received tranexamic acid, and in 20 patients placebo was used. Results: Significant thrombin activity was found in all the studied patients. Thrombin generation was less in the aprotinin group than in the tranexamic acid and the placebo group (thrombin/anti‐thrombin III complexes 33.7 ± 3.6, 53.6 ± 7.0 and 44.2 ± 5.3 µg/l 2 h after CPB and F1 + 2 fragment 1.50 ± 0.10, 2.37 ± 0.37 and 2.04 ± 0.20 nmol/l 6 h after surgery, respectively). The inhibition of fibrinolysis was significant with both anti‐fibrinolytic drugs (d ‐dimers 0.427 ± 0.032, 0.394 ± 0.039 and 2.808 ± 0.037 mg/l 2 h after CPB, respectively). The generation of d ‐dimers was inhibited until 16 h after CPB in the aprotinin group. The plasminogen activation was significantly less in the aprotinin group (plasmin/anti‐plasmin complexes 0.884 ± 0.095, 2.764 ± 0.254 and 1.574 ± 0.185 mg/l 2 h after CPB, respectively). Conclusion: Thrombin formation is inevitable in coronary artery bypass surgery when CPB is used. The suppression of fibrinolytic activity, either with aprotinin or with tranexamic acid interferes with the hemostatic balance as evaluated by biochemical markers. Further investigations are needed to define the role of hemostatic activation in ischemic complications associated with cardiac surgery.     

Experimental basilar artery spasm caused by autologous blood application: Effects of clot removal and topical nicardipine

Summary In 24 albino New-Zealand rabbits spasm of the basilar artery was produced by local application of autologous blood. The effects of blood and bloot clot removal or topical nicardipine, each alone and in combination of both methods, were studied and compared to untreated controls.As well clot removal alone as topical application of nicardipine without clot removal resulted in some, but statistically not significant, enlargement of the spastically narrowed basilar artery. Only the combination of both methods caused a significant vasodilatation, up to almost normal diameter range.     

Hemostatic consequences of a non-fresh or reconstituted whole blood small volume cardiopulmonary bypass prime in neonates and infants

Objectives: Despite aggressive measures to miniaturize the cardiopulmonary bypass (CPB) circuit in neonates and infants, the CPB prime volume is often at least as large as the patients’ blood volume. We conducted an observational study to characterize the hemostatic consequences of a CPB prime consisting of either non‐fresh or reconstituted whole blood. Methods: Hematocrit, fibrinogen, platelet count, plasminogen, anti‐thrombin III (AT‐III), and factors (F) II, V, VII, IX, and X of 30 neonates and infants undergoing cardiac surgery with CPB utilizing either a non‐fresh or reconstituted whole blood prime were prospectively evaluated at eight time points. Following protamine administration, microvascular bleeding was treated by protocol. Results: The hemostatic composition of the CPB prime was the same following the use of either non‐fresh or reconstituted whole blood. The CPB prime platelet count (mean ± sd ) was 5.87 ± 2.84 × 10³ μl^?1 when compared to a preoperative platelet count of 298 ± 142 × 10³ μl^?1 (P < 0.0001). Twenty patients received 17.3 ± 9.2 ml·kg^?1 (0.86 ± 0.46 units·kg^?1) of platelets with significant improvement in platelet count. Nine patients received 16.7 ± 13.4 ml·kg^?1 (0.84 ± 0.67 units·kg^?1) of cryoprecipitate with significant improvements in FVIII and fibrinogen. Conclusions: Non‐fresh or reconstituted whole blood as a component of a small volume CPB prime in neonates and infants induces clinically significant dilutional thrombocytopenia in conjunction with less significant reductions in fibrinogen, FII, FV, FVII, FVIII, FIX, FX, plasminogen, and AT‐III.     

Removal of subarachnoid blood clots after subarachnoid hemorrhage

The difficulty in removing subarachnoid blood clots was evaluated in terms of the interval after subarachnoid hemorrhage. Subarachnoid blood clots were removed from a total of 30 cisterns with a Hounsfield unit of more than 70. In 20 cisterns, removal was performed within 24 hours, and in 10 between 24 and 72 hours after subarachnoid hemorrhage. In 16 of the 20 cisterns (80%) and in 4 of the 10 cisterns (40%), the density was reduced to a Hounsfield unit of less than 60 after removal of subarachnoid blood clots. Two typical cases are presented.     

重组人生长激素对化疗大鼠外周血象的影响

目的探讨重组人生长激素（rhGH）对化疗大鼠外周血细胞学指标的影响。方法实验分空白对照组、生理盐水组、rhGH组、化疗组和化疗＋rhGH组。给药后第7天、14天、21天和28天应用血液自动分析仪检测实验各组大鼠外周血中各系细胞的变化。结果给药后第7天rhGH组网织红细胞绝对数较其他各组明显增加（P〈0．05）；化疗组和化疗＋rhGH组外周血液中的白细胞（WBC）、血红蛋白（Hb）和网织红细胞数较其他各组明显降低（P〈0．01），但化疗＋rhGH组WBC数和网织红细胞绝对数减少程度明显低于化疗组（P〈0．05）。给药后第14天化疗组和化疗＋rhGH组外周血中红细胞数（RBC）和Hb与其他各组比较明显降低（P〈0．05），但化疗＋rhGH组减少程度明显低于化疗组（P〈0．05）；化疗＋rhGH组网织红细胞绝对数较其他各组明显增加（P〈0．05）；各组间WBC比较差异无显著性（P〉0．05）。给药后第21天，化疗组RBC、Hb计数低于对照组（P〈0．05），而化疗＋rhGH组WBC、RBC、Hb计数与对照组比较差异无显著性（P〉0．05），网织红细胞绝对数高于其他各组（P〈0．05）。结论rhGH对化疗大鼠的造血功能有一定的保护作用。