Autologous thrombin preparations: Biocompatibility and growth factor release.

Platelet-rich plasma (PRP) has been investigated to promote wound healing in a variety of tissues. Thrombin, another essential component of wound healing, is sometimes combined with PRP to generate a fibrin clot in order to retain the sample at the delivery site and to stimulate growth factor release. Using a fully autologous approach, autologous serum (AS) with thrombin activity can be prepared using a one-step procedure by supplementing with ethanol (E+ AS) to prolong room temperature stability or prepared ethanol free (E- AS) by utilizing a two-step procedure to prolong stability. The objective of this study was to evaluate potential wound healing mechanisms of these two preparations using commercially available devices. A variety of tests were conducted to assess biocompatibility and growth factor release from PRP at various ratios. It was found that E- AS contained greater leukocyte viability in the product (97.1 ± 2.0% compared to 41.8 ± 11.5%), supported greater bone marrow mesenchymal stem cell proliferation (3.7× vs 0.8× at a 1:4 ratio and 3.6× vs 1.6× at a 1:10 ratio), and stimulated release of growth factors and cytokines from PRP to a greater extent than E+ AS. Of the 36 growth factors and cytokines evaluated, release of 27 of them were significantly reduced by the presence of ethanol in at least one of the tested configurations. It is concluded that the high concentrations of ethanol needed to stabilize point of care autologous thrombin preparations could be detrimental to normal wound healing processes.

An Ethanol-Free Autologous Thrombin System.

Thrombin is a coagulation protein of central importance to hemostasis and wound healing that can be sourced from human blood, bovine blood, and engineered cell lines. Only autologous thrombin lacks the risks of transmitting emergent pathogens or eliciting an immunogenic response. Previous commercial autologous thrombin devices require the use of high concentrations of ethanol to achieve thrombin stability, introducing cytotoxicity risks. A new point of care device for preparing an ethanol-free autologous thrombin serum was investigated. The ethanol-free autologous serum (AS) was prepared using the Thrombinator™ System (Arthrex, Inc., Naples, FL). A total of 120 devices were tested with the blood of 30 healthy donors to determine the reliability and flexibility of the procedure. AS was prepared from both whole blood (WB) and platelet-poor plasma (PPP). Study endpoints were thrombin activity determined using a coagulation analyzer and formation of cohesive bone graft composites objectively measured using a durometer. The average thrombin activity produced by this system from 24 donors was 20.6 ± 2.7 IU/mL for WB and 13.4 ± 3.8 IU/mL for PPP which correlated to clot times of 3.9 and 5.9 seconds, respectively. The device tolerated use of varying volumes of blood to prepare AS. In addition, the system was able to generate four successive and comparable AS productions. When combined with platelet-rich plasma and bone graft material, cohesive scaffolds were always formed. A new device and method for preparing single donor, ethanol-free, AS with thrombin activity was demonstrated.

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.

Exposure of hematopoietic stem cells to ethylene oxide during processing represents a potential carcinogenic risk for transplant recipients.

Stem cells for transplantation are obtained from bone marrow, umbilical cord blood, and peripheral blood. A rare complication of hematopoietic stem cell transplantation is donor cell-derived leukemia (DCL). The donors remain cancer free and the causes of these DCL are unknown. Stem cells must repopulate the bone marrow and then give rise to all hematopoietic cells for the rest of the transplant recipient's life. No procedure is acceptable that might introduce precancerous or cancerous mutations in cells performing such a critical function. Medical disposable sets consisting of bags, tubing sets and freezing containers are used to collect, purify and store stem cells. Sterilization of disposables with ethylene oxide is widespread, even though those sets unavoidably retain residual amounts of ethylene oxide which is a potent, direct-acting mutagen and clastogen that has been demonstrated to induce hematopoietic cancer in mice, rats and human beings. Potential exposure levels to ethylene oxide during processing under proposed US FDA guidelines for residual ethylene oxide would be biologically active and present a significant risk factor for DCL. For direct-acting mutagens, there is no recognized "no effect" dose using currently accepted cancer risk assessment models. The safety concerns with ethylene oxide can be eliminated by the use of alternative technologies including electron beam, gamma irradiation, or steam for the sterilization of all products used for stem cell processing and storage.

Whole blood thrombin: development of a process for intra-operative production of human thrombin.

Thrombin-based clotting agents currently used for topical hemostasis with absorbable sponges, fibrin sealants, and platelet gels are primarily derived from bovine or pooled human plasma sources. Autologous thrombin has important safety advantages in that it does not carry the same safety concerns as pooled plasma-derived products and it avoids exposure to risks associated with bovine-derived proteins. The goal of our research was to develop a rapid, reliable, and simple to perform process to generate autologous human thrombin in the intraoperative setting, from patient whole blood as the starting source material. Using whole blood instead of plasma as the starting material, it is possible to avoid the inherent delay in thrombin availability associated with a primary step of plasma isolation. In this study, we varied several key processing parameters to maximize thrombin production, reproducibility and stability. Autologous thrombin production was generated using a dedicated, single use disposable with a sterile reagent. The disposable consists of a tubular reaction chamber containing glass microsphere beads to activate the alternative pathway of the coagulation cascade. At the end of the process, thrombin-activated serum was harvested from the reaction chamber. The average activity of the thrombin produced at room temperature by this system was 82.8 +/- 15.9 IU/mL. The total processing time was < 30 minutes. The system was compatible with Anticoagulant Citrate Dextrose-Solution A (ACD-A) (8%-12%). The average volume of thrombin harvested from each aliquot of blood was 7.0 +/- 0.3 mL, and the stability of thrombin was observed to be temperature dependent, with cold storage better preserving thrombin activity. Clot times with platelet concentrates at 1:4.3 and 1:11 ratios (thrombin to platelet concentrate) were < 10 and 20 seconds, respectively. A process for the preparation of thrombin from whole blood, under conditions compatible with the resources of an operating room, has been developed. The device is simple to use, requires 30 minutes, and can consistently produce thrombin solutions that achieve rapid clotting of platelet concentrates, plasma, and fibrinogen concentrates even when mixed at thrombin to blood product ratios of 1:11.

Development of a sensitive PCR inhibition method to demonstrate HBV nucleic acid inactivation.

BACKGROUND: The evaluation of pathogen reduction technologies with relevant viruses currently contaminating the blood supply is limited by the availability of high-titer virus inocula and sensitive in vitro or in vivo infectivity assays. Because HBV infectivity can only be assessed by in vivo studies with chimpanzees, a sensitive PCR inhibition assay was developed to measure PEN110 inactivation of HBV. STUDY DESIGN AND METHODS: PCR amplification of 1.1 kb of HBV genome was optimized to determine DNA damage introduced by treatment with PEN110 in RBCs. Inactivation of duck HBV (DHBV) in RBCs, with measurement of the in vitro infectivity, was performed to validate the PCR assay. RESULTS: The PCR was highly specific and sensitive for amplification of the HBV genome and used to demonstrate a reduction of at least 7.2 and 8.1 log geq per mL within the first 18 hours of PEN110 treatment. PEN110 inactivation of DHBV was also achieved within the first 18 hours with a reduction factor of at least 5.0 log tissue culture infectious dose 50 percent per mL, suggesting that PCR inhibition is an alternative to infectivity assays. CONCLUSION: This study establishes PCR inhibition as a reasonable approach to assess the efficiency of PEN110 inactivation of human pathogens with human plasma donations that have been found to contain high titers of relevant agents during different stages of infection.

Pathogen inactivation of RBCs: PEN110 reproductive toxicology studies.

BACKGROUND: The novel PEN110 chemistry (INACTINE, V.I. Technologies) process for the purification of blood for transfusions involves treating WBC-reduced RBCs with PEN110 to inactivate a wide spectrum of pathogens. The washed RBC preparation has a residual PEN110 level of less than 0.00005 mg per mL. It is important to verify that the trace amounts of residual PEN110 in blood prepared for transfusions will not produce adverse effects on reproduction, fertility, or fetal development. STUDY DESIGN AND METHODS: A fertility and early embryonic development study was conducted in male and female Sprague-Dawley rats at IV doses of up to 0.5 mg PEN110 per kg of body weight following standard regulatory protocols. A fetal developmental study was conducted in Hra:(NZW)SPF pregnant rabbits at IV doses of up to 1.0 mg per kg of body weight following standard regulatory protocols. In both cases the highest dose was shown to be a maximum tolerated dose in pregnant animals based on body weight gain during pregnancy. RESULTS: In the fertility and early embryonic development study, no treatment-related effects were noted on estrous cycles, pregnancy rate, implantation sites, corpora lutea, number of resorptions, and live embryos in female rats or sperm motility, sperm morphology, and sperm counts in male rats. In the fetal developmental study, PEN110 had no effect on embryofetal viability and growth. This is consistent with other data indicating that PEN110 is rapidly cleared by urinary excretion. On a mg per kg of body weight dose basis, the no-observed-effect level doses for rats in the fertility study and rabbits in the developmental study were 2,000 and 4,000 (320 and 1,300 scaled to dose per unit body surface area [DBSA]) times that which a person would receive given 1 unit of treated blood. Considering the cumulative animal dosages, the safety factor values increase to 48,000- and 60,000-fold (7,700 and 19,400 scaled to dose per unit body surface area). CONCLUSION: These results indicate that the trace amount of residual PEN110 in the purified blood component is well below the level that could present a risk of reproductive toxicity to the patient.