Magdy El-Ekiaby

Blood-derived biomaterials and platelet growth factors in regenerative medicine

Several biomaterials can be obtained from human blood. Some are used for clinical indications requiring a high content in fibrinogen, while others are used because they contain multiple platelet growth factors. Mimicking thrombin-induced physiological events of coagulation leading to fibrino-formation and platelet activation, blood biomaterials have critical advantages of being devoid of tissue necrotic effects and of being biodegradable by body enzymes. Fibrin-based biomaterials, known as fibrin glues or fibrin sealants, have been used for more than 30 years as surgical hemostatic and sealing agents, demonstrating benefits in essentially all surgical fields, including reconstructive plastic surgery and wound treatment. Clinical interest in platelet growth factor-rich biomaterials (often known as platelet gels or platelet-rich-plasma) has emerged more recently. Platelet gels are used in clinical situations to achieve wound healing and repair soft and hard tissues. Applications include the healing of recalcitrant ulcers and burns, and stimulation of osseous tissue regeneration in dentistry, implantology, and maxillofacial and plastic surgery. They were evaluated recently in knee osteoarthritis and for the repair of musculoskeletal tissue lesions in sports medicine. Platelet lysates are now used as a substitute for fetal bovine serum and for ex vivo clinical-scale expansion of stem cells, opening new perspectives in regenerative medicine. We present the scientific rationale that prevailed in the development of blood biomaterials, describe their modes of production and biochemical and functional characteristics, and present clinical applications in regenerative medicine.

The platelet-cancer loop

The relationship between cancer and thrombosis has been established since 1865 when Armand Trousseau described superficial thrombophlebitis as forewarning sign of occult visceral malignancy. Platelets are the primary hemostatic tool and play a primordial role in cancer-induced thrombosis. Tumor-induced numerical and functional platelet abnormalities have been described in conjunction to changes in coagulation. Such changes are reported even in the absence of clinically detectable thrombosis and correlate with tumor progression and metastasis. Reciprocally, platelets seem to interplay with the tumors and the immune system, both directly and indirectly favoring tumor progressions, tethering and distant spread. A number of growth factors supporting tumor growth, angiogenesis and metastasis are released from the platelets. A reciprocating loop of tumor-induced platelet activation/platelet-induced tumor growth and dissemination is initiated, acting as a thrombosis trigger/tumor amplifier. Recent studies have demonstrated that the use of anti-platelet agents can break this loop resulting in a reduction of short-term risk for incident cancer, cancer mortality and metastasis. The beneficial effect in reduction in cancer-induced thrombosis remains to be established. The current review aims at shedding the light on the intimate reciprocal cross-talk between platelets and cancer and on exploring the potential beneficial effect of anti-platelet agents in breaking the deadly loop of tumor amplification.

A process for solvent/detergent treatment of plasma for transfusion at blood centers that use a disposable-bag system

BACKGROUND: Solvent/detergent (S/D) inactivates enveloped viruses in plasma. The current technology requires a plasma fractionation facility and is applied to large plasma pools, which increases the cost and risks of exposure to S/D‐resistant pathogens and lowers the content of protein S and α2‐antiplasmin. Two S/D treatment procedures for single donations or minipools of plasma have been developed with a single‐use bag system.

A minipool process for solvent–detergent treatment of cryoprecipitate at blood centres using a disposable bag system

Background and Objectives  Single‐donor or small‐pool cryoprecipitates are produced by blood establishments, mostly in developing countries, for substitute therapy in haemophilia A, von Willebrand disease and fibrinogen deficiency, as well as for the manufacture of fibrin sealant. As cryoprecipitate may be contaminated with pathogenic plasma‐borne viruses, there is an urgent need to develop a simple method for the viral inactivation of cryoprecipitate.

Nanofiltration of single plasma donations: Feasibility study

Background and Objectives Major technical developments have been made in recent years to improve the quality and safety of human plasma for transfusion and fractionation. The present study was performed to assess, for the first time, the feasibility of applying a nanofiltration process, using 75‐nm and 35‐nm mean pore size membranes (Planova® 75N and Planova® 35N), to human plasma.

Ebola: a call for blood transfusion strategy in sub-Saharan Africa

WHO has stated that convalescent blood or plasma is an option in the treatment of Ebola. In 1999, transfusion of locally collected convalescent blood helped decrease Ebola mortality. WHO recommends collection of convalescent plasma to treat patients in the fi ght against the Ebola outbreak. As there is an estimated 70% mortality, a randomised clinical evaluation involving 50 patients, receiving convalescent and control normal plasma, would be suffi cient to confi rm the usefulness of this approach in treatment strategies. Capacity building for the collection and testing of suffi cient convalescent blood or plasma from recovered Ebola patients is crucial. However, paradoxically, the outbreaks are occurring in the countries that have the least capability for blood and plasma collection or viral screening, and which lack infrastructure, equipment, and trained personnel. To ensure collection of safe convalescent plasma, donors must be clinically and virally free of Ebola Virus Disease (EVD) and other relevant viruses. Convalescent plasma is the preferred product, either fresh or fresh-frozen, collected by plasmapheresis with out compromising the donor’s haemoglobin level. Plasmapheresis provides large volumes (500 ml) and can usually be repeated at 2–3 day intervals. Donors selected from the same geographical area are recommended and can provide treatment for many patients. In most African countries there is a paucity of expertise, infrastructure and equipment; however, plasmapheresis equipment can be donated and training provided in-country or at regional Blood Services. Portable generators can provide power for equipment and refrigerators. The Ebola emergency shows the importance of strengthening the technical capacity and infrastructure of local transfusion systems, to respond to present and future infectious outbreaks. The importance of ensuring adequate, accessible, and safe blood—in all countries—is a global priority. Whole blood and labile blood components are now on the WHO’s Essential Medicines List (EML), emphasising the crucial role of transfusions in public health. In sub-Saharan Africa, whole blood, when available, is a life-saving product for emergency use that, together with convalescent plasma, might be the only available clinical option in the treatment of Ebola patients at present. National governments should develop sustainable local blood services for an adequate supply of safe blood as a priority. WHO’s urgent appeal, supporting the use of convalescent blood products to fight Ebola, is a timely reminder of the many World Health Assembly resolutions supporting such actions, particularly in low resource countries.

Properties of a concentrated minipool solvent‐detergent treated cryoprecipitate processed in single‐use bag systems

Summary.  Cryoprecipitate is still used to treat factor VIII (FVIII), von Willebrand factor (VWF) and/or fibrinogen deficiency. Recently a solvent‐detergent (S/D) process of minipools of cryoprecipitate performed in a closed bag system has been designed to improve its viral safety. Still, cryoprecipitate has other drawbacks, including low concentration in active proteins, and presence of haemolytic isoagglutinins. We report here the biochemical evaluation of S/D‐treated minipools of cryoprecipitates depleted of cryo‐poor plasma. Cryoprecipitates were solubilized by 8 mL of a sterile glucose/saline solution, pooled in batches of 40 donations and subjected to S/D treatment in a plastic bag system using either 2% TnBP or 1% TnBP‐1%Triton X‐45, followed by oil extractions (n = 10). Mean (±SD) FVIII and fibrinogen content was 8.86 (±1.29) IU mL−1 and 16.02 (±1.98) mg mL−1, and 8.92 (±1.05) IU mL−1 in cryoprecipitate minipools treated with 2% TnBP, and 17.26 (±1.71) mg mL−1, in those treated by TnBP‐Triton X‐45, respectively. The WWF antigen, ristocetin cofactor and collagen binding activities were close to 10, 7 and 8 IU mL−1, respectively, and were not affected by either SD treatment. VWF multimeric pattern of SD‐treated cryoprecipitates were similar to that of normal plasma, and the >15 mers and >10 mers content was identical to that of the starting cryoprecipitates. The anti‐A and anti‐B titre was 0–1 and 0–1/8, respectively. Therefore, it is possible to prepare virally inactivated cryoprecipitate minipools depleted of isoagglutinins and enriched in functional FVIII, VWF and clottable fibrinogen.

Blood-derived biomaterials: fibrin sealant, platelet gel and platelet fibrin glue

Blood‐derived biomaterials include fibrin sealant (FS) (also called fibrin glue), platelet gel (PG), and platelet fibrin glue. They are used in many surgical fields because of their functional properties and unique physical advantages compared to synthetic products. FS can be made industrially by the fractionation of large plasma pools, or from single plasma donations. Thanks to a high content in fibrinogen, FS exhibits, after activation by thrombin and formation of a strong fibrin clot, tissue sealing and haemostatic properties. PG and platelet fibrin glue are made from single blood donations (platelet concentrates combined or not with cryoprecipitate). Owing to their richness in platelet, PG and PFG can release, upon thrombin activation, a myriad of growth factors that can stimulate cell growth and differentiation, generating much interest for hard and soft tissues regeneration and healing, as well as, increasingly, cell therapy protocols to replace fetal bovine serum. Blood‐derived biomaterials have the advantages, over synthetic glues and other biomaterials, of being physiologically compatible with human tissues, and of not inducing tissue necrosis or other tissue reactions. They can be readily colonized by cells and are totally biodegradable in a matter of days to weeks. These blood‐derived biomaterials are used increasingly as tissue engineering tools, allowing surgeons to influence and improve the in vitro or in vivo cellular environment to enhance the success of tissue grafting. We review here the three main types of biomaterials that can be made from human blood and describe their biochemical and physiological properties as well as their clinical applications.

Minipool Caprylic Acid Fractionation of Plasma Using Disposable Equipment: A Practical Method to Enhance Immunoglobulin Supply in Developing Countries

Background Immunoglobulin G (IgG) is an essential plasma-derived medicine that is lacking in developing countries. IgG shortages leave immunodeficient patients without treatment, exposing them to devastating recurrent infections from local pathogens. A simple and practical method for producing IgG from normal or convalescent plasma collected in developing countries is needed to provide better, faster access to IgG for patients in need. Methodology/Principal Findings IgG was purified from 10 consecutive minipools of 20 plasma donations collected in Egypt using single-use equipment. Plasma donations in their collection bags were subjected to 5%-pH5.5 caprylic acid treatment for 90 min at 31°C, and centrifuged to remove the precipitate. Supernatants were pooled, then dialyzed and concentrated using a commercial disposable hemodialyzer. The final preparation was filtered online by gravity, aseptically dispensed into storage transfusion bags, and frozen at <-20°C. The resulting preparation had a mean protein content of 60.5 g/L, 90.2% immunoglobulins, including 83.2% IgG, 12.4% IgA, and 4.4% IgM, and residual albumin. There was fourfold to sixfold enrichment of anti-hepatitis B and anti-rubella antibodies. Analyses of aggregates (<3%), prekallicrein (5-7 IU/mL), plasmin (26.3 mU/mL), thrombin (2.5 mU/mL), thrombin-like activity (0.011 U/g), thrombin generation capacity (< 223 nM), and Factor XI (<0.01 U/mL) activity, Factor XI/XIa antigen (2.4 ng/g) endotoxin (<0.5 EU/mL), and general safety test in rats showed the in vitro safety profile. Viral validation revealed >5 logs reduction of HIV, BVDV, and PRV infectivity in less than 15 min of caprylic acid treatment. Conclusions/Significance 90% pure, virally-inactivated immunoglobulins can be prepared from plasma minipools using simple disposable equipment and bag systems. This easy-to-implement process could be used to produce immunoglobulins from local plasma in developing countries to treat immunodeficient patients. It is also relevant for preparing hyperimmune IgG from convalescent plasma during infectious outbreaks such as the current Ebola virus episode.

Impact of Transfusion on Cancer Growth and Outcome

For many years, transfusion of allogeneic red blood cells, platelet concentrates, and plasma units has been part of the standard therapeutic arsenal used along the surgical and nonsurgical treatment of patients with malignancies. Although the benefits of these blood products are not a matter of debate in specific pathological conditions associated with life-threatening low blood cell counts or bleeding, increasing clinical evidence is nevertheless suggesting that deliberate transfusion of these blood components may actually lead to negative clinical outcomes by affecting patient’s immune defense, stimulating tumor growth, tethering, and dissemination. Rigorous preclinical and clinical studies are needed to dimension the clinical relevance, benefits, and risks of transfusion of blood components in cancer patients and understand the amplitude of problems. There is also a need to consider validating preparation methods of blood components for so far ignored biological markers, such as microparticles and biological response modifiers. Meanwhile, blood component transfusions should be regarded as a personalized medicine, taking into careful consideration the status and specificities of the patient, rather than as a routine hospital procedure.