Plastic Blood Bags Research Articles

A Simple, Safe and Effective Approach for Personalized Precision Ex Vivo Gene Therapy Based on Autoinfusion of Gene-Modified Leucoconcentrate (GML) Prepared from Routine Unit of Patient's Peripheral Blood

Nowadays gene and cell therapy become the basic methods in regenerative medicine. However only few gene and cell products are currently approved for clinical usage. Biosafety problems, complexity of cell and gene technologies and high cost of manufacturing are the main reasons for the slow introduction of such approaches in practical medicine. Treatment of hereditary diseases of the immune system based on the correction of the mutant gene by delivering functional recombinant gene into WBC is the first successfully employed in the clinical practice approach of cell-mediated or ex vivo gene therapy. Earlier we have reported the strategy of the cell-mediated gene therapy based on umbilical cord blood mononuclear cells transduced with adenoviral vectors carrying recombinant genes encoding neurotrophic factors for treatment neurodegenerative diseases, neurotrauma and stroke. Significant disadvantage of this method is the usage of the umbilical cord blood mononuclear cells as a cell carrier for the therapeutic genes. Considering immunodeficiency treatment and our own data we developed a new approach of recombinant gene delivery for personalized ex vivo gene therapy. The method is based on autoinfusion of patient's WBC transduced with recombinant therapeutic genes for correction of certain pathological conditions. In the present study for the first time the human gene-modified leucoconcentrate (GML) producing recombinant reporter gene encoding green fluorescent protein (GFP) was obtained without culturing WBC in vitro. The routine unit of peripheral blood (450 ml) was collected into the plastic blood bag and the leucocyte- and platelet-rich concentrates (50 ml) were obtained by standard method using Macopress Smart (Macopharma, France). Afterwards the equal volume of hydroxyethyl starch 6% was added into the plastic blood bag which was centrifuged (DP-2065 R PLUS, Centrifugal Presvac RV; Presvac, Buenos Aires, Argentina) at 350 rpm for 10 min at 10°C. The obtained supernatant was transferred into the new plastic blood bag using manual plasma extractor FK-01 (Leadcore, Russia) and 200 ml of saline was added into the bag which was centrifuged at 1300 rpm for 10 min at 10°C and the supernatant was expressed out of the bag so that the remaining solution in the bag (30 ml) contained leucoconcentrate (WBC - 45.56 ± 23.93 × 106/ml and RBC - 1.76 ± 3.33 × 109/ml). Transduction of WBC with chimeric adenoviral vector (Ad5/35) carrying GFP gene was performed in the plastic bag with MOI 5 according to the count of WBC in the leucoconcentrate. After transduction for 12 hours, 200 ml of saline was added to the bag with leucoconcentrate, the mixture was centrifuged at 1000 rpm for 10 min at 10°C and the supernatant was squeezed out of the bag. The remained in the bag solution (30 ml) was considered as gene-modified leucoconcentrate carrying GFP gen (WBC - 22.63 ± 8.90 × 106/ml and RBC - 1.77 ± 1.21 × 109/ml). For in vitro study of GFP gene expression the samples of GML-GFP were cultivated for 60 hours after GML-GFP preparation. Fluorescent microscopy in the cytoplasm of the transduced WBC showed specific intensive green fluorescence. Flow cytometry analysis demonstrated that 2.5% of WBC from the GML-GFP efficiently expressed GFP. Thus leucoconcentrate after 72 h of transduction with Ad5/35-GFP with MOI 5 resulted in 2.5% of the GFP-positive cells. Thus the results of this study represent a simple, safe and effective approach for preparation of GML for personalized ex vivo gene therapy aimed at temporary production of the specific recombinant biologically active molecules for pathogenetic therapy of the varied nosological form, such as trauma, ischemic, degenerative, autoimmune, infection and other diseases. This study was supported by the grant of Russian Science Foundation 19-75-10030. Disclosures No relevant conflicts of interest to declare.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury.

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.

Non-invasive spectroscopy of transfusable red blood cells stored inside sealed plastic blood-bags.

After being separated from (donated) whole blood, red blood cells are suspended in specially formulated additive solutions and stored (at 4 °C) in polyvinyl chloride (PVC) blood-bags until they are needed for transfusion. With time, the prepared red cell concentrate (RCC) is known to undergo biochemical changes that lower effectiveness of the transfusion, and thus regulations are in place that limit the storage period to 42 days. At present, RCC is not subjected to analytical testing prior to transfusion. In this study, we use Spatially Offset Raman Spectroscopy (SORS) to probe, non-invasively, the biochemistry of RCC inside sealed blood-bags. The retrieved spectra compare well with conventional Raman spectra (of sampled aliquots) and are dominated by features associated with hemoglobin. In addition to the analytical demonstration that SORS can be used to retrieve RCC spectra from standard clinical blood-bags without breaking the sterility of the system, the data reveal interesting detail about the oxygenation-state of the stored cells themselves, namely that some blood-bags unexpectedly contain measurable amounts of deoxygenated hemoglobin after weeks of storage. The demonstration that chemical information can be obtained non-invasively using spectroscopy will enable new studies of RCC degeneration, and points the way to a Raman-based instrument for quality-control in a blood-bank or hospital setting.

The caspase-3 inhibitor (peptide Z-DEVD-FMK) affects the survival and function of platelets in platelet concentrate during storage

BackgroundAlthough apoptosis occurs in nucleated cells, studies show that this event also occurs in some anucleated cells such as platelets. During storage of platelets, the viability of platelets decreased, storage lesions were observed, and cells underwent apoptosis. We investigated the effects of caspase-3 inhibitor on the survival and function of platelets after different periods of storage.MethodsPlatelet concentrates were obtained from the Iranian Blood Transfusion Organization in plastic blood bags. Caspase-3 inhibitor (Z-DEVD-FMK) was added to the bags. These bags along with control bags to which no inhibitor was added were stored in a shaking incubator at 22℃ for 7 days. The effects of Z-DEVD-FMK on the functionality of platelets were analyzed by assessing their ability to bind to von Willebrand factor (vWF) and to aggregate in the presence of arachidonic acid and ristocetin. Cell survival was surveyed by MTT assay.ResultsAt day 4 of storage, ristocetin-induced platelet aggregation was significantly higher in the inhibitor-treated (test) than in control samples; the difference was not significant at day 7. There was no significant difference in arachidonic acid-induced platelet aggregation between test and control samples. However, at day 7 of storage, the binding of platelets to vWF was significantly higher in test than in control samples. The MTT assay revealed significantly higher viability in test than in control samples at both days of study.ConclusionTreatment of platelets with caspase-3 inhibitor could increase their functionality and survival.

Commercially available blood storage containers

Plastic blood bags improve the safety and effectiveness of blood component separation and storage. Progress towards optimal storage systems is driven by medical, scientific, business and environmental concerns and is limited by available materials, consumer acceptance and manufacturing and regulatory concerns. Blood bag manufacturers were invited to submit lists of the bags they manufacture. The lists were combined and sorted by planned use. The lists were analysed by experts to assess the degree to which the products attend to scientific problems. Specific issues addressed included the use of di-ethylhexyl phthalate (DEHP) as plasticizer for polyvinyl chloride (PVC) blood bags, the size, material and thickness of platelet bags, and the fracture resistance of plasma bags. Alternatives to DEHP for red blood cell (RBC) storage exist, but are mostly in a developmental stage. Plastic bags (DEHP-free, PVC-free) for platelet storage with better gas diffusion capabilities are widely available. Alternatives for plasma storage with better fracture resistance at low temperatures exist. Most RBC products are stored in DEHP-plasticized PVC as no fully satisfactory alternative exists that ensures adequate storage with low haemolysis. A variety of alternative platelet storage systems are available, but their significance - other than improved oxygen transport - is poorly understood. The necessity to remove DEHP from blood bags still needs to be determined.

Thrombocytopenia and bleeding in pediatric oncology patients.

Prophylactic platelet transfusions are the standard of care for patients with hypoproliferative thrombocytopenia after receiving chemotherapy or radiation for the treatment of malignancy, for BM replacement by leukemia or solid tumor, or in preparation for a hematopoietic stem cell transplantation.(1) During this time of thrombocytopenia, these patients may receive both prophylactic platelet transfusions, which are given to prevent potentially life-threatening bleeding when a patient's platelet count drops below a predetermined threshold, and therapeutic platelet transfusions, which are given to treat active or recurrent bleeding. In the 1950s, the invention of the plastic blood bag allowed for the production and storage of platelet concentrates,(2) and in the 1960s, it was recognized that prophylactic platelet transfusions effectively reduced hemorrhagic death in patients with newly diagnosed leukemia.(3,4) In 1962, Gaydos published the paper that is frequently credited with the inception of the 20 000/μL platelet transfusion threshold.(5) Despite a half-century of experience with prophylactic platelet transfusions, there are still insufficient data to provide clinicians with evidence-based guidelines specific to pediatric oncology and hematopoietic stem cell transplantation (HSCT) patients.

Thrombocytopenia and bleeding in pediatric oncology patients

Abstract Prophylactic platelet transfusions are the standard of care for patients with hypoproliferative thrombocytopenia after receiving chemotherapy or radiation for the treatment of malignancy, for BM replacement by leukemia or solid tumor, or in preparation for a hematopoietic stem cell transplantation.1 During this time of thrombocytopenia, these patients may receive both prophylactic platelet transfusions, which are given to prevent potentially life-threatening bleeding when a patient's platelet count drops below a predetermined threshold, and therapeutic platelet transfusions, which are given to treat active or recurrent bleeding. In the 1950s, the invention of the plastic blood bag allowed for the production and storage of platelet concentrates,2 and in the 1960s, it was recognized that prophylactic platelet transfusions effectively reduced hemorrhagic death in patients with newly diagnosed leukemia.3,4 In 1962, Gaydos published the paper that is frequently credited with the inception of the 20 000/μL platelet transfusion threshold.5 Despite a half-century of experience with prophylactic platelet transfusions, there are still insufficient data to provide clinicians with evidence-based guidelines specific to pediatric oncology and hematopoietic stem cell transplantation (HSCT) patients.

The plasma wars: a history

The plasma wars: a history

Blood processing

Blood processing

Factor VIII preservation following collection into commercial plasmapheresis systems.

The two-stage method of Pool and Robinson was used in determining plasma Factor VIII activity following contact with four commercially available plastic blood and pooling bag films. Employing a two-arm double plasmapheresis technique, it was shown that Factor VIII activity in whole blood initially drawn into BB-69 and PL-130 blood bags decreased only slightly from in vivo levels for activity in PL-130 blood bags (t equal 3.2317, p. less than 0.01). Factor VIII activity fell to 98 and 90 per cent preservation levels from an initial 104 per cent in vivo activity, for BB-69 and PL-130 respectively. Factor VIII activity in plasma collected in Ellay and PL-146 pooling bags, both decreased significantly to 85 and 86 per cent, respectively, after three hours storage at room temperature (24 to 25.5 C). Following five weeks storage (-30 C) in Ellay and PL-146 pooling bags, Factor VIII activity significantly (p less than 0.005) fell to 79 and 77 per cent, respectively. No significant differences were observed when Factor VIII activity levels were compared in BB-69 versus PL-130 or Ellay versus PL-146 plastic film systems.

Migration of a phthalate ester plasticizer from polyvinyl chloride blood bags into stored human blood and its localization in human tissues

Abstract A plasticizer, di-2-ethylexyl phthalates, was found to be extracted from polyvinyl chloride plastic blood bags by both human and dog blood at 4°C at a rate of 0.25±0.03 mg per 100 ml per day. Plasticizer was found in both the lipid-containing and lipid-free fractions of plasma whereas red cells contained only minor amounts. Seven of 12 lung-tissue samples taken at autopsy from patients who had received transfusions of stored blood contained detectable amounts of plasticizer. One patients, after open-heart surgery, excreted a urinary metabolite of the plasticizer in excess of the amount calculated to have been received in the transfused blood. The additional metabolite possibly came from plasticizer extracted from the polyvinyl chloride tubing of the cardiopulmonary unit. Although the toxicology implications of these observations are not currently known, it is suggested that tractable materials to blood or its fractions be developed.

A Simplified Method for Purification, Concentration and Freezing of Bone Marrow Cells for Autologous Transplantation

Autologous bone marrow (BM) transplantation is being increasingly applied in hematological and oncological patients. However, because of the need to purify and preserve BM requiring high technology, such treatments are virtually concentrated in the "developed" countries. This paper examines methods of BM purification and freezing that could make the technique potentially applicable in developing countries. Hemapheresis is routinely applied for BM purification in our Dutch Centre, where the buffy coats obtained from routine blood donations were utilised in experimental settings. Using DMSO as cryoprotectant, semi-purified white cells were frozen in liquid N2 (LN2), by mechanical freezer or snap-frozen at -55 degrees C. Different types of containers were compared including plastic tubes and ordinary blood bags. After thawing the results show that snap-freezing had a deleterious effect but the cell yields and viability were similar in LN2 or the mechanical freezer where the tubes and the bag were equally effective as containers (86% cell recovery with 90% viability). In the purification/concentration stage, reduction of the volume of the material by extra centrifugation, thus requiring less DMSO, produced better results--96% cell yield and 90% viability after thawing. This simplified method was applied in a general hospital in Sao Paulo where four oncology patients underwent BM collection. BM was purified and concentrated within a blood bank facility. Hydroxyethyl starch sedimentation and centrifugation of the material in plastic blood bags gave 80% BM cell harvest. After thawing from the mechanical freezer 1 x 10(8) BM cells/kg were available for reinfusion to patients. There was no immediate untoward reaction. Three of the patients showed signs of bone marrow regeneration by three weeks, but one patient died 16 days after transplantation, of septicemia. We conclude that certain high-technology procedures for ABMT can be adapted for existing facilities in developing countries.

４種類の赤血球保存液の比較試験 ７施設での検討結果

Four red cell additive solutions, in which 3 are licenced in the U. S. A. and the last is an experimental additive were evaluated in 7 Blood Centers with the same experimental protocol. The ACD-A-anticoagulated red cells were suspended with one of additives (ADSOL, Baxter; SAGM, Terumo; OPTISOL, Terumo; MAP developed by Dr. S. Sasagawa of Japanese Red Cross) with a hematocrit of 55-65%. Then they were stored in PVC plastic blood bags for 49 days at 4°C. The ATP, 2, 3-DPG levels, morphologic scores, and glucose consumption of red cells were similar in 4 additives. The degree of %hemolysis was the least in MAP. But, the pH levels of RC suspended with MAP were lower by 0.1 unit than those of remaining 3 additives. The resistance to low osmolarity was slightly better in MAP than those of 3 remaining additives. The overall results suggest that MAP and OPTISOL caused a slightly better results in preventing hemolysis, although the data from another parameters indicate that there were no significant differences between 4 additives. Therefore, we should introduce MAP or OPTISOL into the Japanese Blood Program because of the low levels of hemolysis.

The effects of plastic bag extracts prepared in either mouse serum or phosphate‐buffered saline with ethanol on mouse splenic cells

Chemicals leached from plastic blood bags are potentially immunogenic when introduced parenterally into humans. The experiments reported here represent an attempt to sensitize B6C3F1 mice to parenteral injections of solutions used to extract potentially immunogenic material from plastic blood bags. Mice were given intraperitoneal injections: blood bag extracts prepared from phosphate-buffered saline and ethanol; mouse red cells stored in extract solution; or blood bag extracts prepared from mouse serum. None elicited a significant in vitro immunologic reaction as determined by 3H-thymidine or 3H-leucine uptake into spleen cells. In contrast, intraperitoneal injections in mice of large amounts (0.5 ml) of the phosphate-buffered saline and ethanol extracts decreased spleen cellularity to 59 percent of the control value and reduced spleen cell blastogenic activity in vitro. When solutions of either mouse serum extract or mouse cells stored in extract were given intraperitoneally to mice, no spleen toxicity or increased blastogenic activity was observed. In addition, the extracts obtained from plastic blood bags by using either allogenic serum or buffered saline and ethanol did not elicit an immunologic response when they were administered parenterally to mice.

Incorporation of plasticizer into red cells during storage.

The development of flexible plastic blood bags has permitted effective blood component production and therapy. However, the plasticizer di(2-ethylhexyl)phthalate (DEHP), whose toxicity in humans is still undefined, is known to leach from the plastic into stored blood. Despite the availability of bags made of plastics not using DEHP, the collection and storage of red cells is still done in DEHP plasticized packs, and in fact the storage life for red cells has recently been increased up to 49 days using new anticoagulant-preservative solutions. We examined the relationship between DEHP and stored red cells. We found that 28 percent of available 14C-DEHP binds immediately to sites in both the membrane and cytosol fractions of the red cells, and that the total amount and distribution of 14C-DEHP does not change significantly over 7 days. When red cell concentrates were stored with or without DEHP, using either plastic (polyolefin) bags not containing DEHP or glass, definite reduction in the osmotic stability of the red cells was found in the absence of DEHP. Plasma-free hemoglobin levels were 90.3 mg per dl after 35 days of storage in plastic packs containing DEHP and 181.7 mg per dl in the polyolefin bags. The advantages of improved in vitro stability of red cells stored in plastics containing DEHP must be weighed against the potential hazards of patient exposure to DEHP.

Quantitative assessment of platelet morphology by light scattering: a potential method for the evaluation of platelets for transfusion.

Normal fresh platelets are discoid, and platelets that have been stored become spherical. Discoid morphology correlates with normal viability and imparts to a platelet suspension a "streaming" appearance when agitated. We developed a device that quantitates the percent of discoid cells in a suspension by means of laser light scattering at low angles. It performs this assay within 3 minutes on platelets contained in a plastic blood bag. The ability of the device to quantitate discoid platelets was demonstrated in the following ways: (1) comparison of percentage discs calculated from light-scattering data with visually observed optical streaming; (2) correlation of percentage discs from light scattering with the percentage determined by phase microscopy; and (3) observation of changes during room temperature storage. Platelet suspensions containing extreme (dendritic and balloon) forms were examined, and we conclude that these forms do not significantly affect the light-scattering assay. The optical measurements were also used to accurately approximate the total platelet counts of the suspensions. This device may have a role in the evaluation of platelets for transfusion.

Platelet Concentrates Stored at 22°C Need Oxygen

Platelet concentrates prepared by platelet apheresis were stored in plastic blood bags with different gas permeability properties. Inadequate oxygen supply gave an insufficient adenosine triphosphate (ATP) regeneration and a compensatory increase in glycolysis and lactic acid production, giving a rapidly falling pH. At pH below 6.0 the glycolysis was inhibited, oxygen consumption ceased, and ATP dropped towards depletion. Adequate oxygen supply kept the lactic acid production low with small pH changes only, and allowed a sufficient ATP regeneration. The release of α-granular platelet Factor 4 (PF4) was almost total at pH below 6.0, while at intact metabolic function there was a slow release of PF4. Platelet preservation is enhanced by the use of blood bags with adequate gas exchange properties. In our study one polyvinyl chloride plastic (PVC) bag gave poor results, while another PVC bag and a polyolefin bag showed intact metabolism for 5 days and a moderate release of PF4.

Leaching of chemicals from plastic intravenous fluid bags.

To the Editor.— We have previously reported 1 that Vitamin A could disappear rapidly from the solutions stored in the polyvinyl chloride plastic intravenous fluid bags that are marketed under the trade name, Viaflex, by Baxter-Travenol. In light of a recent study 2 showing that a rather significant amount of the plasticizer, diethylhexyl phthalate (DEH-phthalate), leached at a constant rate into the blood from the plastic blood bags also marketed by the same company, we have conducted some preliminary investigations as to the possible leaching of chemicals from the Viaflex fluid bags. In this preliminary phase of the study, the ultraviolet absorption spectra of solutions (200 ml) stored in 250-ml bags were recorded as a function of time. The samples were kept at room temperature (24 ± 2 C), and the solutions stored in volumetric flasks served as controls. The ultraviolet absorbencies of distilled water, normal saline, and 5% dextrose solution

Di-2-ethylhexyl phthalate, a plasticizer contaminant of platelet concentrates.

Di‐2‐ethylhexyl phthalate (DEHP), a plasticizer used in polyvinyl chloride plastic blood bags, is extracted by human blood during storage at 4 C. The rate of extraction (0.25 mg/100 ml/day) suggested that after only two days of storage, a concentration of 0.5 mg/100 ml might be found in human blood. In contrast to this, five platelet concentrates that were stored at 22 C for two days had a calculated DEHP concentration of approximately 19 mg/100 ml. The concentration of DEHP in platelet‐poor plasma was 16.7 mg/100 ml while the platelets were found to contain 37.7 mg/100 ml packed platelet volume. This result might suggest that platelets can preferentially accumulate DEHP. It is not known if such concentrations of DEHP are injurious to humans who receive platelet transfusions.

Effect of phthalate esters on reproduction in rats.

Numerous plastic devices and materials used for medical, dental, and food collection, processing, and packaging are made of polyvinyl chloride) and plasticized mainly with di-2-ethylhexyl phthalate (DEHP) to impart the desired physical and chemical characteristics. Some finished products may contain more than 40% plasticizer, which may be leached from the material by blood (1) or milk (2) or various other solutions. DEHP has generally not been considered to be a health hazard because of its low acute oral toxicity (3-10) and because the amount of DEHP leached from plastics is quite small. However, there is evidence that DEHP is not readily hydrolyzed but accumulates in body tissues. When two patients were infused with blood stored in plastic blood bags plasticized with DEHP, Jaeger and Rubin (11) found DEHP in the spleen, liver, lung, and abdominal fat in concentrations ranging from 2.5 to 27 mg-% (dry weight). The teratogenic effects of several phthalate esters when injected into the yolk sac or allantoic cavity or applied to the chorioallantoic membrane of developing chick embryos have been reported by Haberman and others (12-14). More recently, tDepartment of Dairy Science, Michigan State University, East Lansing, Michigan 48823. *To whom reprint requests should be sent. *Supported by the Michigan Agricultural Experiment Station (journal article 6113). Singh et al. (15) have reported the teratogenicity of phthalate esters in rats without regard to the effect these compounds might have on parturition. The present study was undertaken to determine the effects of dibutyl phthalate, dimethyl phthalate, and di-2-ethylhexyl phthalate on embryonic development and parturition.