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Activation of neutrophils and monocytes by a leukocyte-depleting filter used throughout cardiopulmonary bypass.

OBJECTIVE: Cardiopulmonary bypass elicits systemic inflammation. Depletion of circulating leukocytes might alleviate inflammatory response. We studied the effects of a leukocyte-depleting filter on phagocyte activation during cardiopulmonary bypass. METHODS: Fifty patients undergoing coronary artery bypass grafting were randomly allocated into an arterial line leukocyte filter group (n = 25) with a Pall LeukoGuard 6 leukocyte-depleting filter (LG6; Pall Biomedical, Portsmouth, United Kingdom) and a control group without any filter (n = 25). Blood sampling took place from arterial line at predetermined time points. In the filter group, the sample was taken immediately before the filter; to evaluate activation at the site, an additional sample was taken immediately after the filter. CD11b/CD18 and L-selectin expressions and basal production of hydrogen peroxide were determined with whole-blood flow cytometry, and plasma lactoferrin level was determined with enzyme-linked immunosorbent assay. RESULTS: Neutrophil CD11b expression was higher in the filter group than in the control group (P < .001). Likewise, monocyte CD11b expression, neutrophil hydrogen peroxide production, and lactoferrin plasma levels were all significantly higher, whereas neutrophil and monocyte counts and neutrophil L-selectin expression were all significantly lower in the filter group (all P < .001). At 5 minutes of CPB, CD11b expression increased across the filter on neutrophils (median difference 197 relative fluorescence units, range 45-431 relative fluorescence units, P < .001) and monocytes (median difference 26 relative fluorescence units, range -68-111 relative fluorescence units, P < .001). CONCLUSION: The LG6 arterial line leukocyte filter is ineffective in its principal task of diminishing phagocyte activation during cardiopulmonary bypass.

Aged↗

Fludarabine phosphate may be useful in the treatment of graft-versus-host disease.

The increasing number of allogeneic stem cell transplantations has made the management of graft-versus-host disease (GVHD) a continuing problem for transplantation professionals. GVHD is a complicated disease the treatment of which requires an equally multifaceted approach. Despite therapeutic efforts to decrease its distressing and potentially lethal clinical manifestations, treatment is still not optimal. Fludarabine phosphate is a purine analogue, which is known to cause immunosuppression and long-lasting T-cell lymphopenia it is commonly employed in the therapy of hematological malignancies and non-myeloablative stem cell transplantation conditioning regimens. Myelosuppression, especially leuko- and lymphopenia is the major dose-limiting toxicity of fludarabine. However, a prolonged reduction in CD4+ T-cell count may be a desired effect for the treatment of GVHD. Clinical observations, preclinical data on the management of GVHD and well-known immunosuppressive properties suggest that fludarabine should be tested in clinical grounds for GVHD prophylaxis and treatment.

Animals↗

Transfusion-transmitted tick-borne infections: a cornucopia of threats.

Over the past several decades, the frequency of contact between humans and ticks has increased dramatically. Concomitantly, several newly recognized tick-borne pathogens have emerged joining those already known to be transmitted by ticks. Together these factors have led to an enhanced public health awareness of ticks, tick-borne agents, and their associated diseases. Reports that several of these agents are transmitted by blood transfusion have raised concerns about blood safety. The primary agents of interest are members of the genus Babesia, but Anaplasma phagocytophilum, Rickettsia rickettsii, Colorado tick fever virus, and tick-borne encephalitis virus also have been transmitted by transfusion. In many cases, these agents and their diseases share common features including vectors, symptoms, and diagnosis. Unfortunately, they also share the common problem of insufficient epidemiologic and transmissibility data necessary for making informed decisions regarding potential blood safety interventions. Although further surveillance and epidemiologic studies of tick-borne agents are clearly needed, at present only the Babesia warrant consideration for active intervention; because donor management strategies based on risk-factor questions are inadequate, leukoreduction not effective for agents found in red cells and pathogen inactivation remains problematic for red cell products. Despite the present unavailability of screening assays, some form of serologic and nucleic acid testing may be justified for the Babesia. Given that interactions between humans and ticks are likely to increase in the future, vigilance is required as new and extant tick-borne agents pose potential threats to transfusion safety.

Humans↗

David B. Pall, PhD (1914-2004).

In this edition of the Pioneers and Pathfinders Series, the contributions of David B. Pall, PhD, to transfusion medicine are discussed. With the aid of Dr Pall's unpublished personal history and assistance from family members and the company he founded, we are able to provide perspective to several remarkable scientific advances. For those of us in transfusion medicine, the discovery of the world's first leukoreduction filter prevails as his most significant invention. However, to the rest of the world, Dr Pall pioneered filtration with applications in aerospace, microelectronics, general industry, and most recently, contamination control. The almost 60-year-old Pall Corporation continues to preserve his legacy.

Blood Component Transfusion↗

An overview of prion biology and the role of blood filtration in reducing the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease.

Prions are infectious proteins believed to be responsible for a variety of progressive and fatal neurodegenerative diseases, collectively referred to as transmissible spongiform encephalopathies (TSE). By 1996, it was recognized that ingestion of beef from cattle afflicted with a TSE known as bovine spongiform encephalopathy, could result in a devastating human TSE known as variant Creutzfeldt-Jakob disease (vCJD). Two recent reports of probable transfusion-transmitted vCJD have raised concerns about the safety of the blood supply. The relatively long asymptomatic latency of vCJD, as well as the lack of sensitive and specific antemortem tests, increase the risk that asymptomatic, infected individuals may become blood donors. To this point, donor deferral has been a strategy used to reduce this risk. Nevertheless, this strategy may be unreliable and, furthermore, may threaten blood availability. Leukoreduction has also been helpful in reducing cell-associated infectious prion, which has been reported to reduce up to 42% of the infectivity in blood. Proprietary prion affinity surface modifications have been developed and applied to filters, which exploit an understanding of the unique chemical characteristics of prion surfaces. These have been successfully adapted to existing high-efficiency blood filter matrices for the reduction of prions present in blood components for transfusion.

Chromatography, Affinity↗

[Residual leukocyte counting in plasma by a real-time genomic amplification assay].

BACKGROUND: Systematic plasma leukoreduction, which was introduced in France in April 1st 2001, has given rise to more sensitive methods for residual leukocytes counting. The technologies in application at this moment (Nageotte hemocytometers and flow cytometry methods) have been modified by a thirty fold sample concentration prior to analysis, inducing frequent downgrading of the plasma unit. So, in order to improve the detection threshold, we developed a more sensitive assay using "Real-Time Polymerase Chain Reaction" technology. MATERIALS AND METHODS: Real-time polymerase chain reaction was performed on a highly conserved HLADQalpha1 gene sequence. In order to determine the analytical performances of the method (accuracy, sensitivity, linearity and specificity) serial dilutions series ranging from 10(4) to 1 cells/ml were performed. A total of 18 series were prepared from three leukocyte stock solutions, by 1 in 10 serial dilutions in three plasmas completely devoided of white cells (called negative plasmas). To examine the specificity of the assay two negative controls were analyzed in each run. RESULTS: a sensitivity of 10 cells/ml (10(4) leukocytes/l) was achieved and the assay was linear between 10 and 10(4) cells/ml. The slope (-3.72) of the average standard curve calculated from all series, showed an amplification yield of 92.85%. CONCLUSION: we developed a quantitative assay for residual leukocytes in leukodepleted plasma, that agreed with the quality control requirement specifications.

Benzothiazoles↗

Leukoreduction and acute rejection in liver transplantation: an interim analysis.

BACKGROUND: Little is known about the effect of blood transfusions and leukoreduction on acute rejection in liver transplantation. The purpose of this study was to assess the impact of leukoreduction on the occurrence of early rejection episodes in liver transplantation. METHODS: In 1999, mandatory leukoreduction was implemented in our program. Data from 339 consecutive liver transplant recipients were analyzed with attention to the time period as a proxy for leukoreduction, the number of transfusions, the wait list status, the hepatitis B or C status, the recipient age, and the type of immunosuppression. RESULTS: Using an early (6-month) rejection-free graft survival model, we observed that introduction of leukoreduction was independently associated with fewer rejection episodes (P =.001). Despite the lower rejection rate, due to a regimen of tacrolimus and antithymocyte globulin, the effect of implementation of leukoreduction remained significant (P =.021). CONCLUSION: The use of leukoreduction is associated with fewer early rejections, irrespective of the type of immunosuppression. These data support an exploration of the immunomodulatory effect of leukoreduction.

Disease-Free Survival↗

Multiple sclerosis: MHC associations and therapeutic implications.

Multiple sclerosis (MS) is an autoimmune disease with an important genetic component. The strongest genetic association is with the major histocompatibility complex (MHC) region. Several MHC alleles predispose to the disease, the most prominent of which are certain alleles in the HLA-DR2 haplotype. Functional and structural studies have helped to explain the molecular basis of these associations. Although there is currently no curative treatment for MS, an increased understanding of the disease has aided the design of immunotherapies that act on the immune system more specifically than the longstanding drugs. Many of these therapies work at the antigen-specific level, disrupting the interaction between T-cell receptors and MHC molecules that leads to disease.

Animals↗

B-cell targeted therapies in rheumatoid arthritis and systemic lupus erythematosus.

B cells appear to have a central role in the immunopathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE); both autoantibody production and B-cell anomalies are characteristic of these diseases. With the recent availability of biologic agents that can deplete B cells or block their function in vivo, it has become possible to target B cells therapeutically. Evidence strongly suggests that novel B-cell targeting agents are effective. In addition, the mechanistic specificity of B-cell targeted approaches, combined with the ability to test them in large randomized controlled trials, will provide an unprecedented opportunity to study the precise roles of B cells in the immunopathogenesis of RA and SLE. The largest volume of information is available for rituximab, a chimeric monoclonal antibody that depletes B cells by binding to the CD20 cell-surface antigen. Information from multiple investigator-sponsored trials and from off-label use suggests efficacy of this antibody in RA, SLE, and other autoimmune syndromes. Randomized controlled trials have also provided solid evidence for the efficacy of rituximab in RA and are ongoing in SLE. Other therapeutic agents supported by controlled data include cytotoxic T-lymphocyte-associated protein 4 immunoglobulin and antibodies against the interleukin-6 receptor and the B-cell survival molecule BLyS. Additional agents and targets are in earlier stages of development. The concerns about infectious complications have so far not proven to be justified. We can reasonably expect important advances in the understanding and treatment of RA and SLE in the next 5-10 years, as B-cell targeting methods become more widespread and sophisticated.

Animals↗

Transfusion of leukoreduced cellular blood components from cytomegalovirus-unscreened donors in allogeneic hematopoietic transplant recipients: analysis of 72 recipients.

Leukoreduction of blood components has been considered a safe alternative to screening donors for CMV. The objective of this study is to analyze the effectiveness of bedside leukoreduction in preventing CMV transmission. We retrospectively studied 72 transplant recipients and donors who were CMV-seronegative pairs. All patients were transfused with CMV-unscreened cellular blood products leukoreduced at the bedside using leukoreduction filters. Quality control measures performed monthly in our leukoreduced blood components consistently demonstrated that at least 95% of the units sampled meet the leukoreduction criterion established by the American Association of Blood Banks standards. The CMV status of the recipients and donors was determined before transplantation by the latex agglutination assay. Recipients were observed for at least 100 days after transplantation. CMV cultures of urine, buffy coat, bone marrow, and bronchial washings were done weekly when indicated. CMV antigenemia testing was performed twice weekly: 11 transplant recipients seroconverted after transplantation. One patient was positive for CMV antigenemia 4 months after transplantation, but did not have CMV infection. Two of 61 patients who were not seroconverted were CMV antigenemia positive and did not have CMV infection: leukoreduction of cellular blood products is an efficient method of preventing CMV infection.

Adolescent↗

Cytokine load in prestorage leukoreduced PRBC transfusions in premature infants.

OBJECTIVE: Despite the high frequency of packed red blood cell (PRBC) transfusions given to premature neonates, there has been no previous investigation in this population to determine whether small-volume PRBC transfusions using prestorage leukoreduction techniques (1) provide a cytokine load in the transfusate and (2) if there is a load, whether that load alters serum cytokine levels after transfusion. STUDY DESIGN: In all, 27 PRBC units, which were leukoreduced at the time of donation, were followed for cytokine analysis for the duration of the unit's shelf life (1 to 42 days). Infants who received transfusion from these units had cytokines measured pre and post-transfusion. RESULTS: There were no significant levels of interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1 beta (IL-1beta), or human tumor necrosis factor alpha (TNF-alpha) detected during the storage time period. Nine premature infants who received transfusions from these units had serum cytokines levels measured pre- vs post-PRBC transfusion, with no evidence of alterations (IL-6 p=0.51, IL-10 p=0.10, IL-1beta p=0.44, TNF-alpha p=0.86). CONCLUSIONS: The determination of a nondetectable or very low level of a cytokine load contained within the PRBC transfusate, combined with the absence of evidence of an in vivo cytokine effect, is important in establishing the safety profile for PRBC blood-banking methods used with premature neonates.

Blood Preservation↗

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.

Animals↗

Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery.

Monocytes are circulating precursors for tissue macrophages and dendritic cells (DCs) but are not recognized to directly participate in antigen presentation. We developed techniques to label mouse monocyte subsets with particulate tracers in vivo. Gr-1lo but not Gr-1hi monocytes were stably labeled by intravenous injection of 0.5-microm microspheres. Gr-1hi monocytes could be labeled when the microspheres were injected after systemic depletion of blood monocytes and spleen macrophages. In this condition, the phagocytic tracer was transferred to immature bone marrow monocytes by neutrophils and B cells that first carried the particles to the bone marrow. Moreover, antigens from B cells or proteins conjugated to the tracer particles were processed for presentation by monocytes and could induce T cell responses in the periphery. Cell-associated antigen taken up by bone marrow monocytes was retained intracellularly for presentation of the antigen days later when monocyte-derived DCs migrated to lymph nodes or in vitro after differentiation with granulocyte/macrophage colony-stimulating factor. These data reveal that immature monocytes unexpectedly sample antigen from the bone marrow environment and that they can present these antigens after they leave the bone marrow.

Animals↗

Nationwide outbreak of red eye syndrome associated with transfusion of leukocyte-reduced red blood cell units.

OBJECTIVE: To characterize red eye reactions occurring within 24 hours after receipt of units of leukocyte-reduced red blood cells, determine their etiology, and investigate their potential link to transfusion. METHODS: We conducted a survey of transfusion facilities nationwide to determine the scope and magnitude of the reactions; performed case-control and cohort studies among transfused patients at the facility where most reactions occurred; and performed animal experiments, using cellulose acetate derivatives extracted from leukocyte-reduction filters and filter precursors, to reproduce reactions. RESULTS: From January 1, 1997, through January 15, 1998, we identified 159 reactions in 117 patients from 17 states. Reactions were characterized by conjunctival erythema or hemorrhage (in 100% of patients), eye pain (in 62%), photophobia (in 46%), and decreased visual acuity (in 32%). Symptom onset occurred 1-24 hours after initiation of transfusion and resolved within a median of 5 days. Reactions were associated with transfusion sessions that included units of red blood cells filtered with a specific brand of filter, the LeukoNet filter (HemaSure) (odds ratio, 100.4; P<.001). There was a dose-response relationship between the number of LeukoNet-filtered units transfused and the attack rate for reactions, ranging from 0.8% among sessions in which 1 unit was transfused to 27.3% among sessions in which 3 or more units were transfused (P<.001). A similar ocular syndrome was elicited in rabbits injected with cellulose acetate derivatives extracted from unused LeukoNet filters or filter precursors. No reactions were reported after LeukoNet filters were withdrawn from the market. CONCLUSIONS: This transfusion-associated red eye syndrome was linked to a specific brand of leukocyte-reduction filter and likely resulted from cellulose acetate derivatives leached from the filter membrane.

Animals↗