Protecting the blood supply from emerging pathogens: the role of pathogen inactivation.

As a consequence of the many blood-safety interventions introduced since the mid-1980s, the major causes of transfusion-associated mortality have shifted from being mainly due to transfusion-transmitted infections (TTIs) to being mainly due to non-infectious serious events such as TRALI, hemolytic reactions, transfusion overload, and graft versus host disease. Thus, TTIs now account for only 10 to 15% of all transfusion associated mortalities! Relevantly, manufacturers of purified plasma protein fractions have, over the same time period, shown that pathogen inactivation technologies can be successfully implemented resulting in little or no transmission of HIV, HCV or HBV since the late 1980s. These technologies, however, cannot be applied to cellular blood components. Thus, new technologies have evolved over the past decade to treat cellular blood components as well as plasma. These technologies, particularly those involving plasma and platelets, have begun to be used in Europe and this proactive paradigm has evolved to become a potential pre-emptive approach for ridding the blood supply of most TTIs (virus, bacteria, and protozoa). However, in order for pathogen inactivation technologies to become widely accepted, they must be shown to be both cost effective and not associated with new risks to recipients!

The clinical benefits of the leukoreduction of blood products.

Many adverse events associated with the transfusion of allogeneic blood products have been shown to be related to the presence of allogeneic leukocytes in the blood product transfused. Until recently little attention has been paid to the leukocytes present in various blood components, however, over the past two decades it has been shown that the removal of such "passenger" leukocytes is associated with improved clinical outcomes. These include: the reduction in the incidence and severity of febrile transfusion reactions; reducing the CMV transfusion transmission risk; reducing the risk of alloimmune platelet refractoriness; the possible avoidance of vCJD transmission; as well as reducing the risk of mortality and organ dysfunction in cardiac surgery patients, and possibly in other categories of patients.

Transfusion immunomodulation or TRIM: what does it mean clinically?

Evidence from a variety of sources indicate that allogeneic blood transfusions can induce clinically significant immunosuppression, as well as other effects, in recipients. This clinical syndrome is generally referred to in the Transfusion Medicine literature as transfusion-associated immunomodulation, or TRIM. TRIM has been linked to an improved clinical outcome in the setting of renal allograft transplantation. Possible deleterious TRIM-associated effects include an increased rate of cancer recurrence and of post-operative bacterial infection. The recognition that TRIM can increase morbidity and mortality in allogeneically transfused individuals has become a major concern for those involved in Transfusion Medicine. However, based on available randomized controlled trials, whether TRIM predisposes recipients to increased risk for cancer recurrence and/or bacterial infection is still unproven. In contrast, data from experimental animal studies suggest that TRIM is an immunologically mediated biological effect, associated with the transfusion of allogeneic leukocytes; an effect, which can be completely ameliorated by the pre-storage leukoreduction of blood products. Relevantly, several (n = 5) recent large observational trials have provided important evidence for the existence of deleterious TRIM and related effects (mortality and organ dysfunction) of leukocyte-containing allogeneic cellular blood products. These latter data suggest that allogeneic blood product transfusions, containing leukocytes, are associated with an increased risk both for mortality, and organ dysfunction in recipients.

Assessing the effectiveness of whole blood-derived platelets stored as a pool: a randomized block noninferiority trial.

BACKGROUND: Prestorage pooling of whole blood-derived platelets (PLTs) would simplify bacterial detection. This study evaluated the in vivo effect of the prestorage pooling of PLTs stored for up to 5 days, by assessing the corrected count increment (CCI) 18 to 24 hours after transfusion of the product. STUDY DESIGN AND METHODS: A randomized block noninferiority design was used. Eligible patients had chemotherapy-induced thrombocytopenia and were considered likely to need at least six PLT transfusions. For every block of two transfusion events, one consisted of PLTs stored individually and then pooled before transfusion, and the other was a product pooled before storage. The primary outcome was categorized as a successful (>4.5) or unsuccessful (

In vitro evaluation of prestorage pooled leukoreduced whole blood-derived platelets stored for up to 7 days.

BACKGROUND: Advantages to storing whole blood-derived platelets (PLTs) as a pool for 7 days would include operational efficiencies and facilitation of bacterial testing and pathogen inactivation. The in vitro quality of pre-storage pooled PLTs stored for up to 7 days was assessed. STUDY DESIGN AND METHODS: Leukoreduced PLTs were pooled before storage (5 units/pool) and stored for either 5 or 7 days. Samples were collected at the time of pooling and either on Day 5 (n=16-29) or on Day 7 (n=4-30) and tested for biochemical and activation markers and morphology and/or shape change. Control PLTs were stored individually for 5 or 7 days and then tested as indicated above. RESULTS: The mean PLT counts (x10(9)/L) were similar: control PLTs, 1344 (464 SD); and prestorage pooled PLTs, 1327 (220 SD; p=0.93). On Day 5, the pH value was significantly lower (p

Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII.

The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non-conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we 'rabbitized' the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nM compared with 7.5 nM for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF.

Substitutes and alternatives to platelet transfusions in thrombocytopenic patients.

Over the past decade, there have been many improvements in both the safety profile and quality of liquid-stored allogeneic platelet concentrates. However, significant problems with the clinical use of such products remain. Efforts to overcome some of these have resulted in the development of an array of novel therapeutic strategies for the manufacture of platelet products and platelet substitutes, as well as other approaches using alternatives to platelet concentrates. These various products or procedures are at various stages of clinical development. This review summarizes some recent advancements in the preparation of liquid and frozen stored platelets, as well as approaches used for the pathogen inactivation of platelets. Thus, the status of lyophilized platelets, infusible platelet membranes, red blood cells (RBCs) bearing RGD ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents are discussed. Pre-clinical studies and phase 1-3 clinical trials have been encouraging for several of these; however, to date, very few have been licensed for clinical use. Potential alternatives to allogeneic platelet transfusions including correction of anemia by RBC transfusions, recombinant activated factor VII and HLA-reduced platelets are also reviewed. With the ongoing technical and scientific development of such diverse products, those properties that may be necessary for such agents to have hemostatic efficacy will become apparent. However, safety and efficacy must be demonstrable in preclinical studies and clinical trials, before novel platelet concentrates, platelet substitutes and alternatives to platelets can be used in patients with thrombocytopenia.

Incidence and significance of the bacterial contamination of blood components.

A septic reaction occurring during or following the transfusion of cellular blood components was one of the earliest recognized complications of allogeneic blood transfusions. The presence of bacteria in blood products has been a problem for many decades and currently it is probably the most common microbiological cause of transfusion-associated morbidity and mortality. Transfusion-associated sepsis due to contaminated platelet concentrates appears to be much more common than those due to red cells. The overall prevalence of contaminated cellular blood products (red cells and platelets) is approximately one in 3000; however, the transfusion to a recipient of a contaminated blood product may not necessarily be associated with clinically evident morbidity. This is because the majority of contaminated blood product units contain only few bacteria. In other instances, contaminated units may contain large numbers of virulent bacteria as well as endotoxins, and their transfusion may be associated with significant morbidity and even be lethal to the recipient. The prevalence of severe episodes of transfusion-associated sepsis has not been clearly established, but is probably of the order of one in 50,000 per platelet unit and one in 500,000 per red cell unit transfused. As a result of the increased recognition that such transfusion-associated episodes can occur, a variety of measures have been proposed to try to prevent and/or control the risk of transfusion-associated septic reactions.

Bacterial contamination of platelet concentrates: incidence, significance, and prevention.

Severe transfusion reactions associated with bacteria and/or their products, during or following a blood transfusion, were one of the earliest recognized complications of allogeneic blood transfusions. Bacterial contamination of blood products has thus been a problem for many decades and at present is likely the most common microbiological cause of transfusion-associated morbidity and mortality. Transfusion-associated sepsis due to contaminated platelet concentrates appears to be much more common than that due to contaminated red blood cells. The overall incidence of contaminated cellular blood products is approximately 1 in 3,000. However, transfusion to a recipient of a contaminated platelet unit may not necessarily be associated with clinically apparent morbidity, because the majority of contaminated platelet units contain relatively few organisms. In a minority of instances, contaminated units contain large numbers of potentially virulent bacteria, as well as endotoxins, and their transfusion is often associated with significant recipient morbidity and mortality. The incidence of severe septic episodes has not been clearly established, but is probably of the order of 1 per 50,000 platelet units transfused. With heightened awareness in recent years of the possibility that platelet transfusion-associated septic episodes can occur, a variety of measures have been proposed, and in some cases implemented, to try to prevent and control this transfusion risk.

Red blood cell transfusion strategies.

Although the hemoglobin level of 100 g/L has been used for many years as the allogeneic red blood cell (RBC) transfusion trigger, current evidence indicates that for most patients a more restrictive transfusion strategy is at least as effective as and possibly superior to a liberal transfusion strategy. Moreover, the available data indicate that the use of smaller volumes of allogeneic RBCs may be associated with decreased risk of morbidity and mortality. Thus several recent studies indicate that the use of more restrictive triggers than 100 g/L does not appear to adversely affect patient outcomes. Indeed, the majority of recently published RBC transfusion guidelines recommend a more conservative and cautious approach to allogeneic RBC transfusion practice, primarily to reduce the risk of transfusion-related adverse effects. However, the available transfusion trigger studies do not provide sufficient data to allow the claim that the improved outcomes observed are the sole result of the transfusion strategy used. It is possible that the results are the consequence of effects yet to be defined clearly. Additional studies will be necessary to determine the effects of RBC storage time and the presence of allogeneic leukocytes in allogeneic RBC transfusion practice. Nonetheless, the available data, together with detailed information about alternatives to blood product transfusions, will enable physicians to improve outcomes in transfused patients.

Novel platelet products, substitutes and alternatives.

Despite the many advances in the safety, processing, and storage of conventional 22 degrees C liquid-stored allogeneic platelet concentrates, there still are significant drawbacks to the use of such products. Efforts to overcome these shortcomings have resulted in an array of novel platelet products, substitutes, and alternatives; which are currently at various stages of development. This review summarizes the recent developments in the frozen and cold storage of platelets; their pathogen inactivation; as well as the status of lyophilized platelets, infusible platelet membranes (IPMs), red cells bearing arginine-glycine-aspartic acid (RGD) ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents; as potential alternatives to the use of conventional platelet transfusions. Pre-clinical studies have been encouraging for several of these novel products; however, to date, very few have entered human trials. Nonetheless, with the ongoing development of diverse products, those properties that may be necessary for their hemostatic effectiveness will become apparent. However, safety and efficacy must be demonstrated in pre-clinical and phase I to III clinical trials before these novel agents, substitutes and alternatives can be used clinically for patients with thrombocytopenia.

Metabolism of rabbit plasma-derived factor VII in relation to prothrombin in rabbits.

In the human circulation, factor VII is present in relatively low plasma concentration (0.01 microM) and has been reported to have a short half-life (t(1/2); 6 h). In contrast, prothrombin is present in a relatively high plasma concentration (2 microM) and has a relatively long catabolic half-life (t(1/2) = approximately 2-3 days). This report examines the metabolic characteristics of purified rabbit plasma factor VII and prothrombin, radiolabeled with (125)I and (131)I, respectively, in healthy young rabbits. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. Turnover of factor VII within the intravascular space (2.95 days) exceeded that of prothrombin (1.9 days). However, the whole body fractional catabolic rate of factor VII (0.34 days(-1); catabolic t(1/2) = 2.04 days) was significantly slower than that of prothrombin (0.53 days(-1); t(1/2) = 1.31 days). Furthermore, the fractional distributions of factor VII in the intravascular (0.14) and extravascular compartments (0.76) differed from those of prothrombin (0.29 and 0.53). Absolute quantities of factor VII and prothrombin catabolized by a 3-kg rabbit amounted to 0.18 and 24.0 mg/day, respectively (molar ratio of prothrombin to factor VII = 100). The molar ratio of catabolism was compared with the release rates of factor VII and prothrombin from rabbit livers perfused ex vivo. After correction for uptake of factor VII and prothrombin by the liver, the molar ratio of released prothrombin to factor VII in the perfusate was approximately 293:1 over a 0.25- to 3-h interval. These results indicate that, compared with prothrombin, factor VII in the healthy rabbit circulates as a relatively long-lived protein. This behavior does not reflect that reported for factor VII in the human circulation.

Do blood transfusions improve outcomes related to mechanical ventilation?

BACKGROUND: Correcting the decrease in oxygen delivery from anemia using allogeneic RBC transfusions has been hypothesized to help with increased oxygen demands during weaning from mechanical ventilation. However, it is also possible that transfusions hinder the process because RBCs may not be able to adequately increase oxygen delivery. In this study, we determined whether a liberal RBC transfusion strategy improved outcomes related to mechanical ventilation. METHODS: Seven hundred thirteen patients receiving mechanical ventilation, representing a subgroup of patients from a larger trial, were randomized to either a restrictive transfusion strategy, receiving allogeneic RBC transfusions at a hemoglobin concentration of 7.0 g/dL (and maintained between 7.0 g/dL and to 9.0 g/dL), or to a liberal transfusion strategy, receiving RBCs at 10.0 g/dL (and maintained between 10.0 g/dL and 12.0 g/dL). The larger trial was designed to evaluate transfusion practice rather than weaning per se. RESULTS: Baseline characteristics in the restrictive-strategy group (n = 357) and the liberal-strategy group (n = 356) were comparable. The average durations of mechanical ventilation were 8.3 +/- 8.1 days and 8.3 +/- 8.1 days (95% confidence interval [CI] around difference, - 0.79 to 1.68; p = 0.48), while ventilator-free days were 17.5 +/- 10.9 days and 16.1 +/- 11.4 days (95% CI around difference, - 3.07 to 0.21; p = 0.09) in the restrictive-strategy group vs the liberal-strategy group, respectively. Eighty-two percent of the patients in the restrictive-strategy group were considered successfully weaned and extubated for at least 24 h, compared to 78% for the liberal-strategy group (p = 0.19). The relative risk (RR) of extubation success in the restrictive-strategy group compared to the liberal-strategy group, adjusted for the confounding effects of age, APACHE (acute physiology and chronic health evaluation) II score, and comorbid illness, was 1.07 (95% CI, 0.96 to 1.26; p = 0.43). The adjusted RR of extubation success associated with restrictive transfusion in the 219 patients who received mechanical ventilation for > 7 days was 1.1 (95% CI, 0.84 to 1.45; p = 0.47). CONCLUSION: In this study, there was no evidence that a liberal RBC transfusion strategy decreased the duration of mechanical ventilation in a heterogeneous population of critically ill patients.