Residual Leukocytes Research Articles

Surface modification of PBT nonwoven fabrics used for blood filtration and their blood compatibility study

It is necessary to remove residual leukocytes to prevent the blood transfusion-related adverse reactions. This paper describes a facile approach for the surface modification of commercial PBT nonwoven fabrics (PBTNF), used for blood filtration, followed by immobilizing polyvinylpyrrolidone (PVP). The whole blood filtration results revealed that the five types of PBTNF-PVPs' leucocytes retention rates and erythrocyte recovery rates increased to 96% and 92% compared with the untreated PBTNF. The blood compatibilities results indicated that PVP modified PBTNFs have good blood compatibility, suggesting that PVP-modified PBTNF is a very promising blood filter for selective removal of leukocytes.

Microcirculation and blood transfusion: effects of three different types of concentrated red blood cells - preliminary results

Red blood cell (RBC) transfusions are used to increase oxygen delivery; however, a restrictive transfusion strategy (predefined hemoglobin threshold of 7 g/dl) was demonstrated to be associated with lower mortality and incidence of nosocomial infections than a liberal one [1,2]. This may be related to the storage process, which could affect the ability of RBCs to transport and delivery oxygen, or to immunomodulating effects of cytokines from residual leukocytes [2]. The aim of the study is to evaluate the effects, on microcirculation of septic patient, of three types of RBCs.

Evaluation of the performance of Trima Accel® v5.2 for the collection of concentrated high‐dose platelet products and concurrent plasma from high platelet count donors, in germany

This study was undertaken to test the ability of Trima Accel® version 5.2 to simultaneously collect concentrated high-dose leukoreduced platelet products and double doses of plasma. Random volunteers (18-65 years of age) with preprocedure platelet counts above 270 × 10(3) /μl were recruited among the blood center's apheresis donors. All complied with the center's donor selection criteria. One hundred fourteen (114) collections were performed. Depending on which definition of single platelet dose is used (2.0 × 10(11) as prevalent standard in most European countries, and 3.0 × 10(11) as prevalent standard in the United States and Canada) in 107/114 (single dose = 2.0 × 10(11) ) and 39/114 (single dose = 3.0 × 10(11) ) instances, a triple platelet product was obtained. In 87 cases (76%), a double plasmaproduct (>430 ml) was collected, and in seven cases (6%), a single plasma product (>220 ml) was collected. In 20 procedures, only platelets without concurrent plasma were collected (18%). Overall procedure time was 87 ± 13 min and average platelet yield per procedure was 8.5 ± 1.4 × 10(11) (final storage concentration, 1,279 ± 153 × 10(3) /μl). The median residual leukocyte content per transfusion dose was 0.13 × 10(6) (0.02-0.98 × 10(6) ) for a single dose of 2.0 × 10(11) and 0.14 × 10(6) (0.02-0.98 × 10(6) ) for a single dose of 3.0 × 10(11) . Trima Accel® version 5.2 allows for collection of concentrated high yield platelet products. It offers high productivity and reliably achieves the configured yield targets. Leukoreduction performance complied with both US and EU legal requirements. Collection as hyperconcentrates furthermore allowed for concurrent collection of double dose plasma in the majority of the procedures.

Release of Procoagulant and Proinflammatory Cell-Derived Microparticles (MP) During Blood Storage: Effects of Storage Time, Leukoreduction, and Residual Platelets,

Abstract 3373 IntroductionSeveral studies have indicated that transfusion with older blood carries more risk of adverse reactions than transfusion with younger blood, but this remains controversial. It is not clear why older blood may carry increased risks, or what the “safe age” of stored blood is. It is known that multiple bioactive substances are generated from blood during storage, and one or more of these substances may be involved in transfusion-related complications. Among them, MP are a recognized marker of the storage lesion, and their involvement in transfusion-related complications has been postulated. However, questions such as MP species, quantity, biological activity, and factors affecting their release are not well elucidated. The purpose of this study was to quantify MP species and their activity in stored RBC as a function of storage time, and to evaluate the impact of leukoreduction and residual platelets on MP release. Methods(I) MP generation and functional activity Thirty-four bags of packed RBC (16 non-leukoreduced, 18 leukoreduced) of known blood types (A+, B+, AB+, O+) were obtained from the blood bank within 2–4 days of drawing, and then stored at 4°C. Time of receipt was defined as day 0. At days 0, 10, 20, 30, and 40, 40 mL samples were centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for: (1) subtypes of MP by flow cytometry comprising (a) red cell MP (RMP) assessed by CD235a, (b) leukocyte MP (LMP) by CD45, (c) platelet MP (PMP) by CD41, (d) endothelial MP (EMP) by CD144, and (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV); (2) MP-mediated thrombin generation assay (TGA); (3) MP-mediated inflammatory activity by CD 11b expression in neutrophils following incubation with MP. (II) Reconstitution of increasing platelet counts in leukoreduced RBC. To investigate the effect of residual platelets on RMP generation, we mixed a constant amount of RBC with increasing amounts of type-matched platelets (0 to 250,000/μL f.c.) in standard storage bags and measured time-dependent MP release. Results(A) Time-course of MP generation (i) Non-leukoreduced. RMP, PMP and LMP all increased with time, but with different patterns. RMP increased little to day 10 but then rose exponentially, and by day 40 they were 4–6 fold higher than at day 0. PMP counts rose steadily from day 0 and peaked at day 20, being 2–3 fold higher than at day 0. LMP showed no significant change until day 20 when they started to increase, and then increased sharply after day 30, and by day 40 were 1.5–2 fold higher than at day 0. Levels of PMP (days 0 to 20) and RMP (days 20 to 40) correlated with increasing MP-mediated procoagulant and inflammatory markers. (ii) Leukoreduced. Pre-storage leukoreduction decreased RMP generation by 20–40%, completely suppressed PMP and LMP generation, and reduced total MP-mediated procoagulant and inflammatory markers by 40–60%. CBC showed that leukoreduction not only removed >99% WBC but also reduced residual platelets by >95% (from 90 ±30 ×103/μL to 3.5 ±1.3 ×103/μL). This suggests that residual leukocytes and platelets potentiate RMP generation. (B) Effects of residual platelets on RMP generation. To further study the effects of platelets on RMP generation, we mixed known counts of platelets with leukoreduced RBC, and then evaluated RMP generation over time. We found that RMP levels released were proportional to the platelet counts, as were the procoagulant and inflammatory markers. These results show that platelets in stored RBC play a key role in RMP generation. ConclusionMultiple MP types (PMP, LMP, RMP) are released during storage, and their levels increase over time but their patterns of change are different. Procoagulant and inflammatory markers increase in parallel with PMP and RMP. Our data support the hypothesis that age of stored blood could be important in transfusion-related complications, via MP production. Leukoreduction sharply reduces MP generation and procoagulant and inflammatory markers, suggesting that known benefits of leukoreduction may be attributable to reduced MP production. The finding that residual platelets in stored RBC can potentiate RMP generation suggests that minimizing platelets in non-leukoreduced packed cells could reduce the risk of transfusion-related complications. (Supported by NIH grant 1R01HL098031). Disclosures:No relevant conflicts of interest to declare.

Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with β-thalassaemia.

The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content. Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 μm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan). Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50-70%), percentage haemolysis (<0.8/unit), white cell count (<1×10(6)) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%. The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some "low dose" normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units.

Functional analysis of variant lysosomal acid glycosidases of Anderson‐Fabry and Pompe disease in a human embryonic kidney epithelial cell line (HEK 293 T)

The functional significance of missense mutations in genes encoding acid glycosidases of lysosomal storage disorders (LSDs) is not always clear. Here we describe a method of investigating functional properties of variant enzymes in vitro using a human embryonic kidney epithelial cell line. Site-directed mutagenesis was performed on the parental plasmids containing cDNA encoding for alpha-galactosidase A (α-Gal A) and acid maltase (α-Glu) to prepare plasmids encoding relevant point mutations. Mutant plasmids were transfected into HEK 293 T cells, and transient over-expression of variant enzymes was measured after 3days. We have illustrated the method by examining enzymatic activities of four unknown α-Gal A and one α-Glu variants identified in our patients with Anderson-Fabry disease and Pompe diseases respectively. Comparison with control variants known to be either pathogenic or non-pathogenic together with over-expression of wild-type enzyme allowed determination of the pathogenicity of the mutation. One leader sequence novel variant of α-Gal A (p.A15T) was shown not to significantly reduce enzyme activity, whereas three other novel α-Gal A variants (p.D93Y, p.L372P and p.T410I) were shown to be pathogenic as they resulted in significant reduction of enzyme activity. A novel α-Glu variant (p.L72R) was shown to be pathogenic as this significantly reduced enzyme activity. Certain acid glycosidase variants that have been described in association with late-onset LSDs and which are known to have variable residual plasma and leukocyte enzyme activity in patients appear to show intermediate to low enzyme activity (p.N215S and p.Q279E α-Gal A respectively) in the over-expression system.

Effect of pathogen inactivation on the storage lesion in red cells and platelet concentrates

A primary function of blood transfusion services is to ensure the safety of the community’s blood supply. Multilayer strategies of safety need to be incorporated into the processing of blood components in order to minimize untoward transfusion events related to infections or the blood storage lesion. While there have been considerable technical advances over the past few decades to advance blood component safety, there is a need for continued improvement. One significant problem area is transfusion transmitted bacterial infections. In infectious disease, blood borne bacteria are the major cause of morbidity in transfusion medicine. Proactive implementation of pathogen inactivation technologies, PIT can help to eliminate bacterial contamination of blood. This is accomplished by inactivation of various known and emerging bacterial pathogens, which have been identified through their nucleic acid sequences. The current PIT has additional positive impacts on transfusion safety by reducing adverse clinical events associated with viable residual leukocytes in the blood. Given the valuable benefits of pathogen inactivation, there is a practicable need to advance this technology. In order for a particular pathogen inactivation strategy to gain widespread acceptance, it must satisfy a number of important criteria. The technology shall be: (1) Effective to eliminate pathogens. (2) Uncomplicated and cost-effective to implement in the preparation of blood components. (3) Minimally toxic while maintaining the quality of blood storage products for transfusion purposes. (4) Safe. Currently, PIT is not suitable for all blood components. Future advances in PIT for whole blood and red cell components should expand the application of this technology across a broader range of blood products. Based on available laboratory and clinical data, current pathogen inactivation procedures appear to leave the cell components sufficiently viable to achieve an effective transfusion. Novel technologies continue to evolve for the inactivation of pathogens in the blood supply. We need to ensure these procedures do not worsen the storage lesion causing potential harm to patients. Quality research and development supporting multi-centre clinical trials are necessary. One purpose of such trials is to determine whether this new generation of procedures for processing blood yields bioequivalency regarding the quality of the products. Validated haematological assays should be utilized to test the extent of the blood storage lesion. Post-marketing surveillance involving active haemovigilance is necessary regarding possible adverse outcomes of transfusion.

CD4+ CD28+ lymphocytes on day 5 after high-dose melphalan for multiple myeloma predict a low risk of infections during severe neutropenia and are associated with the number of reinfused T lymphocytes of the autologous stem cell graft

Neutropenia following high-dose chemotherapy is associated with a substantial risk of infectious complications. The aim of this study was to identify variables in residual leukocyte subsets during neutropenia that are predictive for neutropenic fever. Residual leukocytes in the peripheral blood on day 5 after autologous blood stem cell transplantation were analyzed by three-color flow cytometry in 55 consecutive patients with multiple myeloma. Furthermore, the number of T cells transfused with the autografts was determined. Neutrophil counts at day 5 and neutrophil engraftment were similar in patients with and without neutropenic fever. Low absolute lymphocyte, CD4(+) CD28(+) and CD45RO(+) CD28(+) counts at day 5 were associated with neutropenic fever. T-cell counts at day 5 correlated with the CD3(+) cell number in the graft. Our data show that the absolute lymphocyte, CD4(+) CD28(+) and CD45RO(+) CD28(+) counts play a role in host defense during severe neutropenia. The T-cell number in the graft may help to identify patients at high risk of neutropenic infections.

Importance of stoichiometry in cells science: iPSC, CNS leukocytes, and more

the hope of becoming the source and basis of sideeffect–free ‘‘medication’’ and repair of deficits in our body,stem cells have been, for a few decades, of interest in basic cellresearch, regenerative medicine, and public opinion. Cytome-try was from the beginning a guiding technology in thisendeavor. The substantial past and present scientific interest isreflected by the fact that publications on stem cells and theirunequivocal identification are the ‘‘best sellers’’ regarding cita-tion and download rates also in the cytometry-focused litera-ture (1). Nevertheless, stem cell therapy still plays a minor rolein clinical practice. The main stem cell therapy is allogenic orautologous bone marrow transplantation in cancer or insevere life-threatening autoimmune diseases such as rheuma-toid arthritis. This transplantation is either done by crudebone marrow allografting or by using purified (i.e., sorted orotherwise enriched) material.Another far less frequent application is transplantation offetal brain tissue in Parkinson’s disease, rendering (temporary)relief from the symptoms. Fetal brain transplant in Hunting-ton’s disease was far less successful as indicated by clinicaltrials enrolling a limited number of patients. In addition,transplantation of parts of the fetal brain is a continuing issueof ethical concern. One of the reasons for the limited applica-tion of stem cell therapy in regenerative medicine is the limita-tion of appropriate cell sources. Mesenchymal stem cells fromfatty tissue (2), as one example, can be relatively easily har-vested but may be limited in their regenerative abilities.Consequently, the discovery of embryonic pluripotentstem cells has led to both a great deal of activity and hype inresearch for novel cell-based therapies. As ethical issuesinvolved in using embryonic stem cells exist, only a limitednumber of countries equipped with appropriate high-endresearch knowledge and technology can embrace these cells fortherapy development.Therefore, the discovery of inducible pluripotent stemcells (iPSCs)—somatic cells (of in principle any type) that canbe reprogrammed by the transfection of a limited number oftranscription factors to become pluripotent—was an impor-tant breakthrough. By their creative and diligent work, thegroup of Shinya Yamanaka from Kyoto University in Japandiscovered that the transfection of only four genes was suffi-cient to reprogram somatic cells to become iPSCs and formany possible cell type of an organism (3).How to optimize the process of transfection to producemaximum numbers of reprogrammed cells and how to per-form appropriate quality control of yield and cell type remainsa central issue. Tiemann and coworkers from Hannover andM€unster in Germany (this issue, page 426) developed a poly-chromatic flow cytometry approach by attaching each of thefour transfected transcription factors (Oct4, c-Myc, Sox2, andKlf4) to four different fluorescent proteins. Quadruplet posi-tive cells contain the reprogrammed iPSCs that can then besorted and cloned. By testing different stoichiometric combi-nations of these transcription factor expression levels,the authors were able to optimize iPSC yield and quantitateoptimal transcription factor levels.The importance of polychromatic cytometry has beenapplied to identify unusual biological material in the pri-mate brain. Bischoff from the University of G€ottingen,Germany and colleagues (this issue, page 436) focusedtheir interest on cells of the immune system infiltratingthe brain. The quantity and composition of these cells canrender information on inflammation processes in neuro-logical diseases and also in stem cell transplantation,

Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPase

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Comparative study of predeposit and bedside leucodepletion filters

Background: Removal of leucocytes from cellular blood components is associated with reduction of several transfusion associated adverse reactions. Methods: A total of 400 units of packed red blood cells (RBCs) were subjected to leucodepletion at room temperature and 4°C using different commercially available prestorage and bedside filters (Terumo Penpol Immugard III and Pall Medical BPF-4). Prefiltration and post-filtration parameters were compared to assess the efficacy of prestorage leucodepletion vis-à-vis bedside leucodepletion and the requirement of universal leucodepletion. Result: Mean post-filtration red cell recovery ranged from 88.49-93.49% with all bags showing more than 85% red cell recovery. Mean post-filtration residual leucocyte count ranged from 0.205 x 10 6-0.338 x 10 6/bag with all bags showing more than log 3 leucoreduction. Prestorage leucoreduction achieved by the polyurethane filter was better than that achieved by the polyester filter. Red cell recovery with the bedside filters at room temperature was significantly less than that with prestorage filters at either temperature. Conclusion: This study suggests that prestorage leucoreduction is preferable over bedside leucoreduction and that polyurethane filters are better than polyester filters since leucodepletion achieved with the former is higher. We recommend selective log 3 leucodepletion using polyurethane prestorage filters for patients with specific indications.

Buffy-coat-derived pooled platelet concentrates and apheresis platelet concentrates: which product type should be preferred?

There is an ongoing debate whether platelet concentrates (PCs) prepared from either whole-blood donations or by plateletpheresis are superior. Usage of these two product types varies greatly between countries and individual institutions. Some use mainly apheresis PCs; others prefer pooled PCs which are produced from whole-blood donations. This review summarizes the existing information on these product types. In the first part data on quality, efficacy and safety are reviewed. It is important to note that the issue cannot be answered just by comparing 'the' apheresis platelet concentrate versus 'the' pooled platelet concentrate. Other factors which determine the quality of a product, e.g. residual leukocyte count, plasma content, additive solution or storage period may be even more important. The focus of the debate should be shifted. It is much more needed to further improve the overall quality of PCs and to optimize treatment of thrombocytopenic patients than to concentrate on a single-edged view on just the preparation method. In the second part of this review we compare the product types from the donor's point of view. If PCs which are equally safe and effective can be obtained by various methods, ethics and the safety of the healthy volunteer donor tips the scales. The decision on the use of a particular product type should take into account all aspects of efficacy, side effects and availability of the product as well as the donor's perspective and the commitment to maximize the use of the valuable whole-blood donation.

Inactivation of Human Leukocytes in Platelet Products After Pathogen Reduction Technology Treatment in the Presence of Platelet Additive Solution.

Abstract 2116Poster Board II-93During the transfusion of blood or blood products, a recipient can receive a large number of allogeneic leukocytes. This can lead to leukocyte-mediated adverse reactions in the recipient and include donor anti-recipient responses such as the life-threatening transfusion-associated graft versus host disease (TA-GVHD) and cytokine production; or recipient anti-donor responses that are induced by direct presentation of foreign antigen by donor leukocytes or indirectly after processing of the donor cells by recipient antigen-presenting cells. To avoid or minimize leukocyte mediated reactions, the leukocytes present in blood products are inactivated or depleted prior to administration. Nucleic acid targeted pathogen reduction processes (PRT) are well suited for leukocyte inactivation. The Mirasol® PRT System uses riboflavin (Vitamin B2) and ultraviolet (UV) light to reduce the active pathogen load and inactivate residual leukocytes in blood products used for transfusion. To make the PRT System more widely applicable, the effect of treating leukocytes in the presence of platelet additive solution (PAS) was tested. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation and placed in 350 ml of storage solution consisting of 65% PAS (SSP+) and 35% plasma. An untreated control sample was removed before addition of 35 ml of riboflavin (500 μM) and exposure to UV light (9.1 J/ml). PBMNC were recovered after treatment and tested for their ability to proliferate in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Treatment was found to inhibit proliferation as well as T cell activation as measured by the upregulation of CD69 expression when incubated with phorbol 12-myristate 13-acetate. Treated PBMNC were unable to produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. Levels of cytokines that are released in the absence of activation, such as IL-6, IL-8 and IL1β, were reduced below levels of detection of the assay after PRT-treatment. Quantitation of the degree of inactivation using limiting dilution assays showed that 5.2 log inactivation could be achieved at the specified energy doses. These treatment conditions resulted in acceptable platelet cell quality over 8 days in storage. In summary, PRT treatment was able to functionally inactivate leukocytes in the presence of PAS to the levels seen with gamma-irradiation without adversely affecting the quality of the platelets. Disclosures:Fast:CaridianBCT Biotechnologies: Research Funding. Marschner:CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment.

Leukocytospermia and sperm preparation - a flow cytometric study

BackgroundLeukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.MethodsSemen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm.ResultsBoth sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters) after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical microscopy was satisfactory, the sensitivity of peroxidase test for the detection of leukocytospermia resulted low.ConclusionSeminal leukocytes do not seem to influence sperm preparation results. However, for assisted conception, semen samples containing leukocytes should be processed using swim-up method. Although peroxidase-test is recommended by WHO as the standard method for determining semen leukocytes, it should not be used in clinical research study.

An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation

AbstractNeutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin–mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin–mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1–deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1–deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1–deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1–independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.

Sporadic leukoreduction filter failure during red blood cell component preparation: beware of rapid filtration

Sporadic leukoreduction filter failure during red blood cell component preparation: beware of rapid filtration

National French observatory of the quality of blood components for transfusion

Purpose Since 1998, prestorage leucoreduction of cellular blood components (BC) is mandatory in France. The French Blood Service needs to follow the data on the quality of the BC prepared by blood centers. This article gives an overview of the quality control (QC) data from 2001 to 2006. Material and methods QC data are submitted to a central data bank by each centre. The data are stratified according to preparation process for analysis of key performance criteria – residual leukocytes and haemoglobin or platelet content. BC preparation processes, methods for measuring haemoglobin and platelet content, and for counting residual leukocytes are those routinely employed by centers. Results The preparation process of red cell concentrates (RCC) influences the haemoglobin content: 57.6 ± 6.8 g per unit versus 50.9 ± 5.4 g per unit for whole blood or RCC filtration, respectively. Apheresis RCC exhibits a reduced variability (51.2 ± 3.4 g per unit). For apheresis platelet concentrates, the median residual leukocyte count remains low for all separators (0.019–0.044 × 10 6 leukocytes per unit, in 2006). However, the percentage of units exceeding 1 × 10 6 leukocytes per unit is significantly higher with one separator (1.8% versus 0.8%, in 2006). For pooled buffy-coat derived platelets, we observed a significant increase in platelet recovery throughout the years (0.66–0.77 × 10 11 platelets per buffy-coat in 2001 and 2006, respectively). Conclusion Our QC data show an overall compliance with the requirements for cellular BC. Our data bank is useful to inform on the performance of leucoreduced BC preparation processes carried out with market available devices.

Effects of leukoreduced blood on acute lung injury after trauma: A randomized controlled trial*

The requirement for a blood transfusion after trauma is associated with an increased risk of acute lung injury. Residual leukocytes contaminating red cells are potential mediators of this syndrome. The goal of this trial was to test our hypothesis that prestorage leukoreduction of blood would reduce rates of posttraumatic lung injury. Double blind, randomized, controlled clinical trial. University-affiliated level I trauma center in King County, Seattle, WA. Two hundred sixty-eight injured patients requiring red blood cell transfusion within 24 hrs of injury. Prestorage leukoreduced vs. standard allogeneic blood transfusions. We compared the incidence of acute lung injury and acute respiratory distress syndrome at early (< or = 72 hrs) and late (> 72 hrs) time points after injury. In a subset, we compared plasma levels of surfactant protein-D and von Willebrand factor antigen between intervention arms. Rates of acute lung injury (relative risk [RR] 1.06, 95% confidence interval [CI] .69-1.640) and acute respiratory distress syndrome (RR .96, 95% CI 0.48-1.91) were not statistically different between intervention arms early after injury. Similarly, no statistically significant effect of leukoreduced transfusion on rates of acute lung injury (RR .88, 95% CI .54-1.44) or acute respiratory distress syndrome (RR .95, 95% CI .58-1.57) was observed to occur late after injury. There was no significant difference in the number of ventilator-free days or in other ventilator parameters between intervention arms. No statistically significant effect of leukoreduced blood on plasma levels of surfactant protein-D or von Willebrand factor antigen was identified. Prestorage leukoreduction had no effect on the incidence or timing of lung injury or on plasma measures of systemic alveolar and endothelial inflammation in a population of trauma patients requiring transfusion. The relationship between transfusion and lung injury is not obviously explained by mechanistic pathways involving the presence of transfused leukocytes.

Regulatory T cells alleviate allergic airway hyper‐responsiveness by modulating angiogenesis of airway remodelling via Notch signaling pathway

Chronic inflammation and airway remodelling are two key features of asthma. Whilst CD4+CD25+ Treg cells have been shown to prevent allergen induced airway inflammation, the role of Treg cells in asthma with established airway remodelling has not been investigated. To address this issue, we induced airway remodelling in OVA‐sensitized mice by chronic aerosol OVA exposure, followed by adoptive transfers of OVA peptide‐specific Treg cells. Without further allergen challenges, residual leukocyte infiltrates in the lungs can be found regardless of the cell types transferred. Airway hyper‐responsiveness (AHR) and pulmonary vasculature were significantly reduced after Treg transfers (P&lt;0.001), suggesting a relationship between pulmonary vasculature and AHR and an anti‐angiogenic effect of Treg cells in airway remodelling. A direct anti‐angiogenic function of Treg cells was validated by both in vitro and in vivo angiogenesis assays which showed a significant reduction of vessel formation at the presence of Treg cells (P&lt;0.001). Notch signaling pathway is known to regulate both peripheral T cell differentiation and angiogenesis. We found that the anti‐angiogenic effect of Treg cells was abrogated by pre‐incubation with recombinant Notch‐Fc proteins. This study demonstrates a novel function of Treg cells in chronic airway inflammation where Treg inhibits angiogenic remodelling via Notch signaling pathway.The project was supported by grants from the Taiwan National Science Council and National Taiwan University Hospital.

Thrombin-induced interleukin 1β synthesis in platelet suspensions: Impact of contaminating leukocytes

A controversial discussion as to whether human platelets are capable of regulated protein synthesis has been ongoing for over half a century. A previous study has suggested that human platelets synthesize large amounts of interleukin 1β (IL-1β) in response to external cues and in a physiologically significant manner. However, cytokines such as IL-1β are generally considered to be products of leukocytes and it could not be completely excluded that contaminating leukocytes may have contributed to the IL-1β results in platelet preparations. It was therefore our intention to investigate whether residual leukocytes had an impact on thrombin-induced IL-1β synthesis. Using various methods to reduce the level of contaminating leukocytes, we found that IL-1β production in platelet-rich suspensions is dependent on the presence of leukocytes, as it was decreased by reducing the number of leukocytes. In addition, we found that thrombin-induced IL-1β synthesis was completely eliminated in leukocyte-free platelet preparations and could be restored by adding leukocytes. IL-1β synthesis could be detected in platelet suspensions contaminated with at least 1 leukocyte per 105 platelets. This study demonstrated that platelets are incapable of synthesizing detectable amounts of IL-1β on their own. We suggest that any IL-1β synthesis detected is a by-product of leukocytes contaminating the platelet preparations. Thus, the hypothesis that platelets producing IL-1β, provide a new link between thrombosis and inflammation needs to be reconsidered.