BBI Contributing Writer

TYSONS CORNER, Virginia – More than 30% of the U.S. population will receive a blood transfusion some time during their lives. According to Bernard Horowitz, PhD, a private consultant and a director of V.I. Technologies (Melville, New York), 1 in 50 blood units is contaminated with the hepatitis G virus, and 1 in 300 to 1 in 6,000 blood units is contaminated with parvovirus. Bacterial and parasitic contamination is also of increasing concern, given recent reports which indicate that 1.3% of red blood cell concentrates and approximately 2.5% of platelet concentrates are culture positive for bacteria. Although major progress has been made in improving the safety of the blood supply with respect to HIV, HBV, and HCV, the transmission of these and other viruses has not been eliminated completely. In the U.S., the risk of transmission of HIV, HBV, or HCV is estimated at 1 in 7,000, assuming that a patient receives five units of blood.

A conference sponsored by the Cambridge Healthcare Research Institute, held here earlier this year, focused on methods for improving blood product safety. Such methods fall into two categories: pathogen screening, which allows for the elimination of contaminated blood or blood products, and pathogen clearance, which assures that no transmissible pathogens reside in blood or blood products to be transfused.

Pathogen screening systems

Anthony DiMarco, PhD, of Abbott Diagnostics (North Chicago, Illinois), spoke about automated, high-volume blood screening using the Abbott PRISM system. The PRISM system is fully automated and uses microparticle capture and chemilluminescent detection. PRISM assays include three microtiter assays (Hepatitis B surface antigen [HbsAg], HBc, HCV), and two bead assays (HTLV-I/HTLV-II, and HIV-1/HIV-2). The system has a tamper-resistant design and features redundant process controls as the sample moves through nine stations, ensuring the integrity of each of the three assay steps. Operator interactions are minimized in order to eliminate manual and visual verification errors and to decrease record maintenance and review.

Larry Mimms, PhD, vice president of product development at GenProbe (San Diego, California), talked at length about in vitro nucleic acid amplification (transcription-mediated amplification, or TMA) assays for virus detection in blood and plasma. In order to carry out these assays, Mimms said that GenProbe is developing a semi-automated system (SAS) and a fully automated system (TIGRIS). Both are based on the same assay. The assay starts with a 500-microliter sample in which virus is lysed. The sample is then processed, allowing hybrids to form between viral RNA and a capture oligomer or oligomeric sequence, thus binding the viral RNA to a magnetic particle. Plasma and other potentially inhibitory substances are then washed away, readying the sample for amplification. The sample-preparation process can be automated to allow for the processing of 100 specimens per hour. Amplification uses two enzymes, reverse transcriptase and T7 RNA polymerase, and yields more than a billion new RNA copies for each original RNA molecule in less than one hour.

Pathogen clearance systems

There are two ways of clearing pathogens: inactivation and removal. Inactivation methods involve physical methods (light, heat, radiation) or chemical methods (formaldehyde, solvents, detergents). Removal methods involve filtration, chromatography, or partitioning. The oldest method for removing viruses from blood involves filtration. Usha Varadarajan, PhD, of Pall (Greenvale, New York), discussed the use of filters in the removal of human B19 parvovirus. This is a major source of contamination for biologicals, such as plasma derivatives, and is a source of some serious illnesses. Although it is an extremely challenging virus to deal with because of its small size and difficulty in detection, it has been successfully filtered using Ultipor VF grade DV 20 membrane direct-flow filters made by Pall. These filters contain a hydrophillic modified polyvinylidene fluoride (PVDF) membrane capable of removing small viruses without binding proteins such as albumin and immunoglobulins.

Among physical methods for pathogen inactivation, the use of gamma irradiation is the oldest technique. Tom Busby, MD, director of biomedical services for the American Red Cross (Washington), spoke about the use of gamma irradiation for the treatment of human plasma fibrinogen. Busby focused on the efficacy of gamma irradiation in reducing the titer of active parvovirus by four logs while maintaining the biological activity of the fibrinogen. He noted that gamma irradiation could be used for terminal sterilization of products in sealed containers and that gamma irradiation is easily scaled up for commercial applications. The Red Cross is now looking at the use of gamma irradiation for pathogen inactivation with proteins other than fibrinogen.

Ultraviolet light of wavelengths between 240 nm and 290 nm (UVC) also has been used for pathogen inactivation. Kathryn Remington, PhD, who is involved with pathogen safety research for Bayer (Pittsburgh, Pennsylvania), presented such a method using UVC. Although she did not describe the reactor in detail, she noted that UVC is well-suited in inactivating non-enveloped viruses that are not targeted by chemical methods such as solvent/detergent usage and low pH techniques. UVC targets the viral DNA and causes the production of photo dimers and photo hydrates, arresting viral replication.

Forms of light energy other than UVC may prove to be more effective in pathogen inactivation than UVC. Bill Cover, PhD, director of clinical and regulatory affairs for PurePulse Technologies (San Diego, California), discussed his company's PureBright technology. This technology consists of light wavelengths from 200 nm to 1,100 nm, pulsed at rates up to five times per second. These light pulses are generated by applying a series of 300 microsecond high voltage pulses to either a quartz or sapphire lamp filled with xenon. The ability of the PureBright technology to inactivate pathogens was successfully tested against 12 specific microbes contained in blow-filled seal bottles containing 20 ml solutions of glucose, saline, and highly purified water. The technology has also been shown to successfully inactive four specific viruses: HIV-1 (human immunodeficiency virus), SV 40 (simian virus 40), BVD (bovine diarrhea virus), and CPV (canine parvovirus). Tests also have shown that proteins are not significantly harmed by PureBright technology. Solutions of immunoglobulin and albumin showed greater than 90% protein recovery after viral inactivation. Proteins that are sensitive to light, such as alkaline phosphotase, acid phosphotase, or lactate dehydrogenase, can be protected from degradation from the pulsed light by adding bovine serum albumin to the solution.

Light energy can also be used to activate a chemical that then can work to inactivate pathogens. Most of the work in this area has been done pertaining to a class of compounds called psoralens. Psoralens preferentially bind to nucleic acids in the dark, and upon exposure to UVA light, form adducts with pyrimidines, inhibiting nucleic acid replication, transcription, and translation. David Wages, MD, PhD, a senior clinical scientist in clinical affairs for Cerus (Concord, California), presented his company's experience with a specific psoralen, S-59. It was shown that more than six-log inactivation could be achieved with S-59 against HIV, HBV, HCV, and HCMV viruses. It was further shown by examining bleeding parameters in thrombocytopenic patients given platelets that were treated with S-59 and UVA light, that the hemostatic efficiency of administered platelets was not adversely affected by treatment with S-59 and UVA light.

The company is working with another group of compounds called FRALEs, which require light for viral inactivation. FRALEs are being developed for viral inactivation in units of red blood cells. One FRALE being developed by the company, S-303, has been used to treat red cells. Early clinical trials of red cells treated with S-303 have shown that S-303 does not significantly decrease red blood cell viability and that S-303 does not change antigens on the surface of red blood cells which would then lead to the formation of antibodies.

Raymond Goodrich, PhD, therapy scientist at Cobe BCT (Wheat Ridge, Colorado), spoke about the use of endogenous photosensitizers for pathogen inactivation in platelet and plasma transfusions. These agents exist in nature, are ingested in large quantities in normal diets, and have been extremely well characterized in terms of toxicology, in vivo behavior, and in vitro effects. Cobe has spent the last year studying the use of riboflavin as a photosensitizer aimed at pathogen inactivation. Riboflavin has been known to sensitize the photochemistry of nucleic acids. Several photo products are known to form upon the exposure of riboflavin to light in solution. The knowledge of these compounds and their toxicological profiles provides a major advantage in the use of this agent in the blood. Several studies, including feasibility studies using viruses (HIV, BVDV, pseudorabies, porcine parvovirus, vaccinia, HSV-1, and HSV-2) spiked into platelet and plasma samples, have been carried out to evaluate the effectiveness of riboflavin at inactivating pathogens in platelets and plasma. Treatment with riboflavin was shown to have more than 75% of activity recovery for a wide variety of plasma derivatives.

Thomas Montie, PhD, a professor of microbiology at the University of Tennessee (Knoxville, Tennessee), presented results on the use of a one-atmosphere uniform glow discharge plasma (OAUGDP) for the rapid inactivation of viruses in a lyophilized preparation of Factor VIII. OAUGDP operates in air and produces a uniform glow discharge plasma without filametary discharges at room temperature, which is advantageous for the sterilization of heat-sensitive biologicals. OAUGDP operates at a frequency where ion trapping occurs, provided that the electric field is at least 8.5 kV/cm. Viral simulants, PhiX174 and T2, were used to study viral inactivation of OAUGDP on different surfaces including glass, polypropylene, and agar.

Pressure cycling technology (PCT) uses pulses of high hydrostatic pressure in the range of 300 to 800 MPa to inactivate both enveloped and non-enveloped viruses. Robert Hess, PhD, of BBI-BioSeq, a subsidiary of Boston Biomedica (West Bridgewater, Massachusetts), discussed the use of PCT for the reduction of the viral infectivity of human plasma, plasma derivatives, biopharmaceuticals, and bovine serum for cell culture. Hess' work has demonstrated inactivation of HIV-1, HSV-1, lambda bacteriophage, and MS2 bacteriophage with the retention of biological activities for Factor X, immunoglobulin, IgM, and factor VIII. In addition, high titer MS2 bacteriophage, a non-enveloped, spherical virus, was diluted in normal human plasma and subjected to various conditions of pressure, temperature, and pulsation. More work is being done to optimize PCT for the inactivation of non-enveloped viruses.

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