Pathogen inactivation techniques.

The desire to rid the blood supply of pathogens of all types has led to the development of many technologies aimed at the same goal--eradication of the pathogen(s) without harming the blood cells or generating toxic chemical agents. This is a very ambitious goal, and one that has yet to be achieved. One approach is to shun the 'one size fits all' concept and to target pathogen-reduction agents at the Individual component types. This permits the development of technologies that might be compatible with, for example, plasma products but that would be cytocidal and thus incompatible with platelet concentrates or red blood cell units. The technologies to be discussed include solvent detergent and methylene blue treatments--designed to inactivate plasma components and derivatives; psoralens (S-59--amotosalen) designed to pathogen-reduce units of platelets; and two products aimed at red blood cells, S-303 (a Frale--frangible anchor-linker effector compound) and Inactine (a binary ethyleneimine). A final pathogen-reduction material that might actually allow one material to inactivate all three blood components--riboflavin (vitamin B2)--is also under development. The sites of action of the amotosalen (S-59), the S-303 Frale, Inactine, and riboflavin are all localized in the nucleic acid part of the pathogen. Solvent detergent materials act by dissolving the plasma envelope, thus compromising the integrity of the pathogen membrane and rendering it non-infectious. By disrupting the pathogen's ability to replicate or survive, its infectivity is removed. The degree to which bacteria and viruses are affected by a particular pathogen-reducing technology relates to its Gram-positive or Gram-negative status, to the sporulation characteristics for bacteria, and the presence of lipid or protein envelopes for viruses. Concerns related to photoproducts and other breakdown products of these technologies remain, and the toxicology of pathogen-reduction treatments is a major ongoing area of investigation. Clearly, regulatory agencies have a major role to play in the evaluation of these new technologies. This chapter will cover the several types of pathogen-reduction systems, mechanisms of action, the inactivation efficacy for specific types of pathogens, toxicology of the various systems and the published research and clinical trial data supporting their potential usefulness. Due to the nature of the field, pathogen reduction is a work in progress and this review should be considered as a snapshot in time rather than a clear picture of what the future will bring.

Universal pre-storage leukoreduction--A defensible use of hospital resources: the Yale-New Haven Hospital experience.

In conclusion, as of 2004, it appears that in the United States in some hospitals, the use of LR blood products will probably remain as SLR rather than PSULR, due primarily to economic pressures. While some blood centres are slowly converting to ULR, there remains a mix of negative and positive feelings among physicians that make adoption of a national PSULR Standard of Care difficult. What is clear is that leukoreduction filters will cost more than the 170 um screen ("clot") filter. The use of PSULR to decrease the incidence of FNHTR, to decrease the incidence of HLA alloimmunization, and its use in lieu of CMV-seronegative blood products is well supported in the medical literature. However, this issue will probably continue to be revisited and debated for some time before a national standard policy for PSULR is adopted. Finally, we believe that despite increasing economic pressures and worsening budgetary constraints, the decision to adopt PSULR should rest primarily on medical reasons: as a means of improving patient care. In the view of the authors, pre-storage universal leukoreduction qualifies as a significant and medically justifiable improvement in the care of all hospital patients.

Reducing the risk of blood transfusion.

There are continuing concerns over the safety of the nation's and the world's blood supply. The allogeneic blood supply is tested for antibodies to HIV1/2, HTLVI/II, hepatitis B, hepatitis C (HCV) and syphilis. Testing is also performed for donor ALT (SGOT) levels, for the presence of hepatitis B surface antigen, human immunodeficiency virus (HIV) p24 antigen and, using nucleic acid amplification testing (NAT), for HIV and HCV nucleic acids. Still, there are concerns regarding other pathogenic agents. Dr. Roger Dodd addresses a series of pathogens that are already known to be transmissible by transfusion. These include malaria, Chagas' disease, babesiosis, bacteria and some viral agents. The need for new donor screening assays to protect the integrity and purity of the blood supply must be balanced against the loss of potential donors and the cost of developing and implementing these new screening assays. This issue will be highlighted. Dr. Edward Snyder reviews the status of research into development of systems for pathogen inactivation (PI) of blood and its components. A proactive technology wherein PI reagents such as psoralen, riboflavin, dimethylmethylene blue or inactine are added to blood collection bags could assure multiple log reduction of a variety of pathogens including viruses, bacteria, protozoa and fungi without the need to initially pre-screen the blood for a specific pathogen. Such a program could also cover new pathogens as they enter the blood supply. As a key issue relates to the toxicology of these agents, Dr. Snyder provides data on a novel carcinogenicity assay that uses a heterozygous p53 knock-out mouse model. The criteria likely to be needed for PI technology to be adopted by the transfusion community are summarized.

hADA3 is required for p53 activity.

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, p53 protein is stabilized and activated by post-translational modifications that enable it to induce either apoptosis or cell cycle arrest. Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity. p53 and hADA3 physically interact in human cells. This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N-terminus. Proper hADA3 function is essential for full transcriptional activity of p53 and p53-mediated apoptosis.

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Non-infectious complications of transfusion therapy.

Blood transfusion is considered safe when the infused blood is tested using state of the art viral assays developed over the past several decades. Only rarely are known viruses like HIV and hepatitis C transmitted by transfusion when blood donors are screened using these sensitive laboratory tests. However, there are a variety of transfusion risks which still remain that cannot be entirely eliminated, many of which are non-infectious in nature. Predominantly immune-mediated complications include the rapid intravascular or slow extravascular destruction (hemolysis) of transfused red cells or extravascular removal of platelets by pre-formed antibodies carried by the transfusion recipient. Alternatively, red cells can be damaged when exposed to excessive heat or incompatible intravenous fluids before or during the transfusion. Common complications of blood transfusion that at least partly involve the immune system include febrile non-hemolytic and allergic reactions. While these are usually not life-threatening, they can hamper efforts to transfuse a patient. Other complications include circulatory overload, hypothermia and metabolic disturbances. Profound hypotensive episodes have been described in patients on angiotensin-converting enzyme (ACE) inhibitors who receive platelet transfusions through bedside leukoreduction filters. These curious reactions appear to involve dysmetabolism of the vasoactive substance bradykinin. Products contaminated by bacteria during blood collection and transfused can cause life-threatening septic reactions. A long-term complication of blood transfusion therapy unique to chronically transfused patients is iron overload. Less common - but serious - reactions more specific to blood transfusion include transfusion-associated graft-versus-host disease and transfusion-associated acute lung injury. Many of these complications of transfusion therapy can be prevented by adhering to well-established practice guidelines. In addition, individuals who administer blood transfusions should recognize these complications in order to be able to quickly provide appropriate treatment.

Protein/peptide transduction domains: potential to deliver large DNA molecules into cells.

In vivo gene delivery can be achieved by direct injection of plasmid DNA. However, inefficient cellular uptake and nuclear import of plasmid DNA result in much lower levels of gene expression than observed when viral vectors are used as gene delivery agents. Recent studies have shown that transducing peptides, such as the HIV Tat protein, can carry large biomolecules from the extracellular environment directly into the cytoplasm and the nucleus of cells, both in vitro and in vivo. Thus, TAT-mediated transduction has the potential to increase the delivery of plasmid DNA to the nuclei of cells in vivo and thereby increase gene expression.

Breast tumor contamination of PBSC harvests: tumor depletion by positive selection of CD34(+) cells.

BACKGROUND: Positive selection of CD34(+) cells may reduce or eliminate tumor cells contaminating PBSC harvests of breast cancer (BrCa) patients. However, to assess tumor purging accurately methods may be needed that are of higher sensitivity than standard immunocytochemistry (ICC) assays. METHODS: BrCa-cell depletion, resulting from CD34(+) cell selection, was evaluated using a novel, highly sensitive assay based upon immunomagnetic enrichment with ICC detection in 36 BrCa patients undergoing highdose chemotherapy with autologous PBSC support. RESULTS: The prevalence of BrCa-cell contamination was significantly lower (P = 0.0078) in selected CD34(+) cell fractions (17/35, 49%) from apheresis collections compared with CD34(-) cell fractions (25/35, 71%). In 8/34 (24%) patients, BrCa cells were detected in CD34(-) cell fractions, but not in paired CD34(+) cell fractions. Significantly lower total numbers (P < 0.0005) of BrCa cells were enumerable in CD34(+) cell fractions compared with corresponding apheresis harvests. The median total BrCa-cell content of selected CD34(+) cell fractions with measurable contamination was 22 BrCa cells (range, 6-73 BrCa cells), compared with 3297 BrCa cells (range, 10-98 400 BrCa cells) in apheresis harvests. The median log depletion of BrCa cells achieved by positive CD34(+) cell selection in specimens with detectable contamination both before and after selection was 2.2 (range, 1.7-4.0). Total pre-selection BrCa cell number was significantly predictive (P = 0.004) of residual detectable post-selection contamination. DISCUSSION: Positive CD34(+) cell selection is an effective tumor purging strategy. The prevalence of PBSC contamination in BrCa patients is substantially higher than formerly appreciated.

In vitro collection and posttransfusion engraftment characteristics of MNCs obtained by using a new separator for autologous PBPC transplantation.

BACKGROUND: A clinical study was performed to evaluate the peripheral blood progenitor cell (PBPC) collection, transfusion, and engraftment characteristics associated with use of a blood cell separator (Amicus, Baxter Healthcare). STUDY DESIGN AND METHODS: Oncology patients (n = 31) scheduled for an autologous PBPC transplant following myeloablative therapy were studied. PBPCs were mobilized by a variety of chemotherapeutic regimens and the use of G-CSF. As no prior studies evaluated whether PBPCs collected on the Amicus separator would be viable after transfusion, to ensure patient safety, PBPCs were first collected on another cell separator (CS-3000 Plus, Baxter) and stored as backup. The day after the CS-3000 Plus collections were completed, PBPC collections intended for transfusion were performed using the Amicus instrument. For each transplant, >2.5 x 10(6) CD34+ PBPCs per kg of body weight were transfused. RESULTS: Clinical data collected on the donors immediately before and after PBPC collection with the Amicus device were comparable to donor data similarly obtained for the CS-3000 Plus collections. While the number of CD34+ cells and the RBC volume in the collected products were equivalent for the two devices, the platelet content of the Amicus collections was significantly lower than that of the CS-3000 Plus collections (4.35 x 10(10) platelets/bag vs. 6.61 x 10(10) platelets/bag, p<0.05). Collection efficiencies for CD34+ cells were 64 +/- 23 percent for the Amicus device and 43 +/- 14 percent for the CS-3000 Plus device (p<0.05). The mean time to engraftment for cells collected via the Amicus device was 8.7 +/- 0.7 days for >500 PMNs per microL and 9.7 +/- 1.5 days to attain a platelet count of >20,000 per microL-equivalent to data in the literature. No CS-3000 Plus backup cells were transfused and no serious adverse events attributable to the Amicus device were encountered. CONCLUSIONS: The mean Amicus CD34+ cell collection efficiency was better (p<0.05) than that of the CS-3000 Plus collection. Short-term engraftment was durable. The PBPCs collected with the Amicus separator are safe and effective for use for autologous transplant patients requiring PBPC rescue from high-dose myeloablative chemotherapy.

Purified JC virus T and T' proteins differentially interact with the retinoblastoma family of tumor suppressor proteins.

The amino termini of polyomavirus T antigens contain LXCXE and J domains, which are necessary for binding and inactivating the retinoblastoma family of tumor suppressors. Both of these motifs are found in the JC virus (JCV) early proteins T'(135), T'(136), and T'(165), leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating function of the JCV large T antigen (TAg). To investigate this hypothesis, the three JCV T' proteins were produced in a baculovirus expression system and purified by immunoaffinity chromatography. To facilitate purification, hybridomas that secrete antibodies recognizing amino-terminal epitopes of JCV early proteins were produced. Potential interactions between the early viral proteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAg and T' proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosphorylated species of the cellular proteins and exhibited the highest binding affinity to p107 and the lowest affinity to pRB. TAg and T'(165) bound more pRB and less p107 than did T'(135) and T'(136); T'(165) also bound less p130 than the other three early proteins. Results of these in vitro interactions were compared to those obtained in vivo using POJ cells, a transformed human glial cell line that expresses JCV early proteins, relatively high levels of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylated p130 is detected in the transformed cells, and most of this protein was found in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins.

Isolation and flow cytometric analysis of T-cell-depleted CD34+ PBPCs.

BACKGROUND: To extend allogeneic HPC transplantation to a greater range of patients, the use of partially matched related donors is under development. Because of the inherently higher degree of histoincompatibility in such transplants, there is increased risk of GVHD as well as of graft failure. Ex vivo depletion of donor-derived T-lymphocytes from PBPCs is one of the most effective methods of preventing GVHD. Thus far, individual centers have used custom-developed procedures to deplete the graft of T cells that are responsible for alloreactivity, often employing relatively impure, nonstandardized reagents such as soybean agglutinin and complement. In addition, with improved methods of T-cell depletion, it has been difficult to accurately assess the number of T cells remaining. Because different centers have used different protocols to assay T cells, it has been difficult to reproduce and validate the results between institutions, and this has limited direct comparison of data between centers. STUDY DESIGN AND METHODS: A standardized approach for T-cell depletion was developed by using a Good Manufacturing Practice-manufactured magnetic cell separator (Isolex 300i, Nexell Therapeutics) and commercially available OKT3 antibody. T-cell depletion was performed on PBPCs from six haploidentical donors. RESULTS: CD34+ cell recovery was 47 percent (range, 31-63%) with a median purity of 94 percent (range, 75-99%) and median T-cell log depletion of 4.72 (range, 3.90-5.83). Because this high degree of depletion makes it challenging to accurately quantitate the remaining T cells, two highly sensitive flow cytometric protocols were developed, each of which accurately detects T cells with a sensitivity of 2 per 10,000 (0.02%). The purified CD34+ cells administered to the patients (dose range, 6.13-13.50 x 10(6)/kg) provided rapid neutrophil and platelet engraftment. CONCLUSION: With the Isolex 300i and a MoAb directed against T cells, a high degree of T-cell depletion is obtained. Sensitive, accurate, and reproducible assays have now been developed for T-cell enumeration in these highly purified cell populations.

Optimal dosing and triggers for prophylactic use of platelet transfusions.

Despite the reliance on platelet transfusion support in patients receiving myeloablative therapy, controversies surround platelet transfusion practices. These include the appropriate platelet dose and the threshold at which prophylactic platelet transfusions will be most effective. These issues bear directly on patient outcome (donor exposure and bleeding complications), cost effectiveness of transfusion, and maintenance of adequate platelet inventories. This review examines the recent studies that have taken on the task of resolving these questions in order to provide optimal platelet transfusion guidelines. Studies now have convincingly demonstrated that a 10,000/microL threshold for prophylactic platelet transfusion is safe and effective in uncomplicated thrombocytopenic patients. Although platelet dosages vary, in general, smaller doses are both effective and inventory-sparing in the more complicated inpatient setting, while larger platelet doses allow for an increased transfusion interval for chronic outpatient support.

Effects of methylene blue-treated plasma on red cells and stored platelet concentrates.

BACKGROUND: Photochemical methods can effectively inactivate extracellular viruses and bacteria found in blood components. Treatment of plasma with methylene blue (MB), a phenothiazine dye, and visible light inactivates enveloped viruses including HIV-1. The effects of MB-treated plasma on cellular components stored in vitro have not been well characterized. STUDY DESIGN AND METHODS: MB-treated plasma (83 microg MB/250 mL plasma) was added to single-donor platelets, stored AS-1 red cells (RBCs), irradiated RBCs, and frozen-deglycerolized RBCs. In vitro platelet assays performed after 1 and 5 days of storage in MB-treated plasma included pH, pO2, pCO2, HCO3, platelet number, lactate dehydrogenase, glucose, osmotic recovery, and CD62 expression. RBC components were examined at specific intervals for leakage of potassium, plasma hemoglobin level, and percentage of hemolysis. Direct antiglobulin tests, osmotic fragilities, and RBC antigen stability tests were also performed on RBCs stored in MB-treated plasma. Components stored with autologous plasma or nontreated allogeneic plasma served as controls. RESULTS: Similar storage-induced changes in pH, glucose, and platelet numbers, as well as increases in lactate dehydrogenase, CD62 expression, and lactate were seen in single-donor platelets stored with MB-treated and control plasma. Platelet morphology scores and osmotic recoveries were not altered. Plasma hemoglobin and potassium and percentage of hemolysis increased equally in the various RBC components stored with MB-treated or nontreated plasma. Osmotic fragility and RBC antigen stability were not appreciably altered by MB-treated plasma. CONCLUSION: Plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets or RBCs, because of the lack of influence of MB-treated plasma on a variety of in vitro platelet and RBC assays.