An international review of the characteristics of viral nucleic acid-amplification testing (NAT) reveals a trend towards the use of smaller pool sizes and individual donation NAT.

BACKGROUND AND OBJECTIVES: Nucleic acid-amplification testing (NAT) is used for screening blood donations/donors for blood-borne viruses. We reviewed global viral NAT characteristics and NAT-yield confirmatory testing used by blood operators. MATERIALS AND METHODS: NAT characteristics and NAT-yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion-Transmitted Infectious Diseases. Reported characteristics are presented herein. RESULTS: NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini-pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT-yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow-up sample. CONCLUSION: In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT-yields, potentially benefitting blood operators seeking to implement NAT.

Intraoperative cell salvage: The impact on immune cell numbers.

BACKGROUND: Patient outcomes are influenced by many confounding factors peri-operatively, including the type of surgery, anaesthesia, transfusion, and immune competence. We have previously demonstrated (in-vitro) that compared to allogeneic blood transfusion (ABT), intraoperative cell salvage (ICS) improves immune competence. The peri-operative immune response is complex. Altered or impaired immune responses may predispose patients to develop adverse outcomes (i.e., post-operative wound infection, pneumonia, urinary tract infection etc.) Surgical patients may develop infection, even without the confirmed presence of a definite microbiological pathogen. With all these factors in mind it is important to consider changes in immune cell numbers (and sub-populations) and functional capacity during peri-operative transfusion. METHODS: In this TRIMICS-Cell (Transfusion Related Immune Modulation and Intraoperative Cell Salvage-Cell numbers) study (n = 17, October 2018-November 2019) we prioritized and analysed peri-operative changes in the number and proportions of immune cell populations and sub-populations (B cells (CD20+), NK (natural killer) cells (CD56+), monocytes (CD14+), T cells (total CD3+ and sub-populations: T helper cells (CD4+), cytotoxic T cells (CD8+), effector T cells (CD4+ CD127+), activated effector T cells (CD4+ CD25+ CD127+) and regulatory T cells (CD4+ CD25+ CD127-)), plasmacytoid dendritic cells (pDC; Lineage-, HLA-DR+, CD11c-, CD123+), classical dendritic cell (cDC) (Lineage-, HLA-DR+, CD11c+), and cDC activation (Lineage-, HLA-DR+, CD11c+), co-stimulatory/adhesion molecules and pDC (CD9+, CD38+, CD80+, CD83+, CD86+, CD123+). Firstly we analysed the whole cohort of study patients and secondly according to the relevant transfusion modality (i.e., three study groups: those who received no transfusion, received ICS only (ICS), or both ICS and allogeneic packed red blood cells (pRBC) (ICS&RBC)), during major orthopaedic surgery. RESULTS: For the whole study cohort (all patients), changes in immune cell populations were significant: leucocytes and specifically neutrophils increased post-operatively, returning towards pre-operative numbers by 48h post-operatively (48h), and lymphocytes reduced post-operatively returning to pre-operative numbers by 48h. When considering transfusion modalities, there were no significant peri-operative changes in the no transfusion group for all immune cell populations studied (cell numbers and proportions (%)). Significant changes in cell population numbers (i.e., leucocytes, neutrophils and lymphocytes) were identified in both transfused groups (ICS and ICS&RBC). Considering all patients, changes in immune cell sub-populations (NK cells, monocytes, B cells, T cells and DCs) and functional characteristics (e.g., co-stimulation markers, adhesion, activation, and regulation) were significant peri-operatively and when considering transfusion modalities. Interestingly DC numbers and functional capacity were specifically altered following ICS compared to ICS&RBC and pDCs were relatively preserved post-operatively following ICS. CONCLUSION: A transient peri-operative alteration with recovery towards pre-operative numbers by 48h post-surgery was demonstrated for many immune cell populations and sub-populations throughout. Immune cell sub-populations and functional characteristics were similar peri-operatively in those who received no transfusion but changed significantly following ICS and ICS&RBC. Interesting changes that require future study are a post-operative monocyte increase in the ICS&RBC group, changes in cDC considering transfusion modalities, and possibly preserved pDC numbers post-operatively following ICS. Future studies to assess changes in immune cell sub-populations, especially during peri-operative transfusion, while considering post-operative adverse outcomes, is recommended.

Human neutrophil antigen 3 genotype impacts neutrophil-mediated endothelial cell cytotoxicity in a two-event model of TRALI.

BACKGROUND: Antibodies against human neutrophil antigen (HNA)-3a are associated with severe cases of transfusion-related acute lung injury (TRALI). The HNA-3 system is located on choline transporter-like 2 (CTL-2) protein. CTL-2 is encoded by the gene SLC44A2 and a single-nucleotide polymorphism c.461G>A results in two antigens: HNA-3a and HNA-3b. Three HNA-3 genotypes/ phenotypes exist: HNA-3aa, HNA-3bb, and HNA-3ab. Two different pathways of anti-HNA-3a TRALI have been described: a two-hit neutrophil-dependent pathway and a one-hit neutrophil-independent pathway. However, it is not clear whether HNA-3ab heterozygous patients have a lower risk of anti-HNA-3a-mediated TRALI compared to HNA-3aa homozygous patients. MATERIALS AND METHODS: Healthy volunteers were genotyped for HNA-3 by real-time polymerase chain reaction, and phenotyped for HNA-3a by granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) against two donor sera containing anti-HNA-3a antibodies. The two sera were also used in in vitro models of human pulmonary microvascular endothelial cell (HLMVEC) cytotoxicity to investigate pathways of TRALI development. RESULTS: For both anti-HNA-3a sera, GIFT results matched the genotype, with a lower GIFT ratio for HNA-3ab neutrophils compared to HNA-3aa neutrophils, whereas GAT results showed no difference in agglutination. HLMVEC cytotoxicity was not observed in a one-hit neutrophil-independent model but was observed in a two-hit neutrophil-dependent model. Differences in cytotoxicity were observed between the two anti-HNA-3a sera used. Consistent with reduced HNA-3a antigen density as measured by GIFT, HNA-3ab neutrophils mediated less HLMVEC cytotoxicity than HNA-3aa neutrophils. CONCLUSION: HNA-3 genotype and HNA-3a antigen expression impacted the severity of anti-HNA-3a-mediated HLMVEC cytotoxicity in a two-hit neutrophil-dependent model of TRALI. Different HNA-3a antibodies might also impact the magnitude of HLMVEC cytotoxicity.

Transfusion-related acute lung injury (TRALI): Potential pathways of development, strategies for prevention and treatment, and future research directions.

Transfusion-related acute lung injury (TRALI) can occur during or after a transfusion, and remains a leading cause of transfusion-associated morbidity and mortality. TRALI is caused by the transfusion of either anti-leukocyte antibodies or biological response modifiers (BRMs). Experimental evidence suggests at least six different pathways that antibody-mediated TRALI might follow: (i) two hit neutrophil activation; (ii) monocyte and neutrophil dependent; (iii) endothelial cell, neutrophil Fc receptor, platelet and neutrophil extracellular trap dependent; (iv) direct monocyte activation; (v) direct endothelial cell activation; and (vi) endothelial cell, complement and monocyte dependent. Two of these pathways (i and v) also apply to BRM-mediated TRALI. Different antibodies or BRMs might initiate different pathways. Even though six pathways are described, these might not be distinct, and might instead be interlinked or proceed concurrently. The different pathways converge upon reactive oxygen species release which damages pulmonary endothelium, precipitating fluid leakage and the clinical symptoms of TRALI. Additional pathways to the six described are likely to also contribute to TRALI pathogenesis, and this requires further investigation. This review also discusses evidence of protective mechanisms and their implications for clinical TRALI treatment. Finally, it suggests directions for future research to support the translation of these findings into strategies to prevent and treat clinical TRALI.

Intraoperative Cell Salvage as an Alternative to Allogeneic (Donated) Blood Transfusion: A Prospective Observational Evaluation of the Immune Response Profile.

Allogeneic blood transfusion (ABT) is associated with transfusion-related immune modulation (TRIM) and subsequent poorer patient outcomes including perioperative infection, multiple organ failure, and mortality. The precise mechanism(s) underlying TRIM remain largely unknown. During intraoperative cell salvage (ICS) a patient's own (autologous) blood is collected, anticoagulated, processed, and reinfused. One impediment to understanding the influence of the immune system on transfusion-related adverse outcomes has been the inability to characterize immune profile changes induced by blood transfusion, including ICS. Dendritic cells and monocytes play a central role in regulation of immune responses, and dysfunction may contribute to adverse outcomes. During a prospective observational study (n = 19), an in vitro model was used to assess dendritic cell and monocyte immune responses and the overall immune response following ABT or ICS exposure. Exposure to both ABT and ICS suppressed dendritic cell and monocyte function. This suppression was, however, significantly less marked following ICS. ICS presented an improved immune competence. This assessment of immune competence through the study of intracellular cytokine production, co-stimulatory and adhesion molecules expressed on dendritic cells and monocytes, and modulation of the overall leukocyte response may predict a reduction of adverse outcomes ( i.e., infection) following ICS.

Soluble mediators in packed red blood cells augment lipopolysaccharide-induced monocyte interleukin-1ß production.

BACKGROUND AND OBJECTIVES: Soluble mediators in packed red-blood-cell (PRBC) units have been hypothesized as a mechanism associated with transfusion-related immune modulation. Soluble mediators including damage-associated molecular patterns (DAMPs) are known to activate inflammasomes. Inflammasome complexes maturate caspase-1 and interleukin (IL)-1ß. We assessed whether PRBC supernatants (SN) modulated IL-1ß driven inflammation and whether macrophage migration inhibitory factor (MIF) was a contributing factor. MATERIALS AND METHODS: Isolated monocytes were incubated with PRBC-SN in an in vitro transfusion model. Lipopolysaccharide (LPS) was added in parallel to model a bacterial infection. Separately, recombinant MIF was used in the model to assess its role in IL-1ß driven inflammation. IL-1ß and caspase-1 were quantified in the PRBC-SN and culture SN from the in vitro model. RESULTS: PRBC-SN alone did not induce IL-1ß production from monocytes. However, PRBC-SN alone increased caspase-1 production. LPS alone induced both IL-1ß and caspase-1 production. PRBC-SN augmented LPS-driven IL-1ß and caspase-1 production. Recombinant MIF did not modulate IL-1ß production in our model. CONCLUSIONS: Soluble mediators in PRBC modulate monocyte IL-1ß inflammation, which may be a contributing factor to adverse effects of transfusion associated with poor patient outcomes. While MIF was present in PRBC-SN, we found no evidence that MIF was responsible for IL-1ß associated immune modulation.

Inverse Relationship Between Lipopolysaccharide Concentration and Monocyte and Dendritic Cells Inflammatory Response.

Dendritic cells (DCs) and monocytes are key immunoregulatory cells that link the innate and adaptive immune response. However, understanding of human cell-specific responses to different doses of stimuli including lipopolysaccharide (LPS) is limited. This study investigated the monocyte and classical DC (cDC)-specific, as well as the overall inflammatory response after exposure to varying doses of LPS. Fresh peripheral whole blood (n = 8) was used in an in vitro peripheral blood culture model to assess cDC and monocyte responses in coculture with varying doses of LPS (0.25, 0.5, 0.75, 1 µg/mL). cDC and monocyte cytokine responses were measured through flow cytometry. Supernatants collected from the in vitro model were used in a cytometric bead array to assess the overall inflammatory response. Exposure to all doses of LPS tested increased monocyte, cDC, and the overall leukocyte response. A dose-dependent reduction in cDC and monocyte cytokine production was also evident with higher LPS doses. This study demonstrates that cell-subset-specific responses are more susceptible to LPS exposure compared with the overall inflammatory response. Therefore, assays that assess cell-specific immune responses may be more beneficial to identify underlying pathophysiology of infection and inflammation.

Coronary artery bypass grafting is associated with immunoparalysis of monocytes and dendritic cells.

Coronary artery bypass grafting (CABG) triggers a systemic inflammatory response that may contribute to adverse outcomes. Dendritic cells (DC) and monocytes are immunoregulatory cells potentially affected by CABG, contributing to an altered immune state. This study investigated changes in DC and monocyte responses in CABG patients at 5 time-points: admission, peri-operative, ICU, day 3 and day 5. Whole blood from 49 CABG patients was used in an ex vivo whole blood culture model to prospectively assess DC and monocyte responses. Lipopolysaccharide (LPS) was added in parallel to model responses to an infectious complication. Co-stimulatory and adhesion molecule expression and intracellular mediator production was measured by flow cytometry. CABG modulated monocyte and DC responses. In addition, DC and monocytes were immunoparalysed, evidenced by failure of co-stimulatory and adhesion molecules (eg HLA-DR), and intracellular mediators (eg IL-6) to respond to LPS stimulation. DC and monocyte modulation was associated with prolonged ICU length of stay and post-operative atrial fibrillation. DC and monocyte cytokine production did not recover by day 5 post-surgery. This study provides evidence that CABG modulates DC and monocyte responses. Using an ex vivo model to assess immune competency of CABG patients may help identify biomarkers to predict adverse outcomes.

Understanding occult hepatitis C infection.

BACKGROUND: Occult hepatitis C infection (OCI) is a type of hepatitis C virus (HCV) infection, defined as the presence of HCV RNA in hepatocytes or peripheral blood mononuclear cells (PBMCs) and the absence of HCV RNA in serum. STUDY DESIGN AND METHODS: A literature review was conducted to identify articles that characterized OCI as a disease, including its epidemiology, mode of transmission, pattern of infection, progression, and treatment. RESULTS: OCI patients experience a milder degree of inflammatory and cirrhotic changes than patients with chronic hepatitis C. OCI is transmissible parenterally both in vivo and in vitro, however the duration and outcome of OCI remains unclear. OCI is most consistently found in patients with previous hepatitis C disease and hemodialysis patients. Beyond the at-risk populations, OCI has also been demonstrated among healthy individuals and blood donors. CONCLUSIONS: This review summarises our current understanding of OCI and suggests areas for further research to improve our understanding of this phenomenon, including a better understanding of its epidemiology and full clinical course. The current understanding of OCI and its clinical implications remain limited. Further standardized detection methods, ongoing surveillance, and investigation of its potential transmissions are required.

Genetic Variants Within the Erythroid Transcription Factor, KLF1, and Reduction of the Expression of Lutheran and Other Blood Group Antigens: Review of the In(Lu) Phenotype.

Erythroid-specific Krüppel-like factor 1, or KLF1, is an integral transcriptional activator for erythropoiesis. Genetic variants within KLF1 can result in a range of erythropoietic clinical phenotypes from benign to significant. The In(Lu) phenotype refers to changes in the quantitative expression of blood group-associated red cell surface molecules due to KLF1 variants which are otherwise clinically benign. These clinically benign KLF1 variants are associated with a reduced expression of 1 or more red cell membrane proteins/carbohydrates that carry blood group antigens for the LU (Lutheran), IN (Indian), P1PK, LW (Landsteiner-Wiener), KN (Knops), OK, RAPH, and I blood group systems. This is of significance during routine serologic blood typing when expression falls below the test sensitivity and therefore impacts on the ability to accurately detect the presence of affected blood group antigens. This is of clinical importance because the transfusion requirements for individuals with the In(Lu) phenotype differ from those of individuals that have a true Lunull phenotype. With this review, we summarize the current body of knowledge with regard to the In(Lu) phenotype and associated KLF1 variants. Our review also highlights discordant reports and provides insights for future research and management strategies. Serological heterogeneity in blood group expression of In(Lu) individuals has been shown, but studies are limited by the low prevalence of the phenotype and therefore the small numbers of samples. They are further limited by availability and inconsistent application of serological reagents and varying test algorithms. With the advent of genome sequence-based testing, an increasing list of In(Lu)-associated KLF1 variants is being revealed. The spectrum of effects on blood group expression due to these variants warrants further attention, and a consistent methodological approach of studies in larger cohorts is required. We propose that a recently reported testing framework of standardized serological studies, flow cytometry, and variant analysis be adopted; and that the international databases be curated to document KLF1 variability and the resultant In(Lu) red cell blood group expression. This will provide better classification of KLF1 variants affecting blood group expression and allow for phenotype prediction from genotype, accurate typing of In(Lu) individuals, and better transfusion management of related challenging transfusion scenarios.

Donor white blood cell survival and cytokine profiles following red blood cell transfusion in Australian major trauma patients.

BACKGROUND: The potential for the co-existence of genetically disparate cells (microchimerism) and associated cytokine profiles following red blood cell (RBC) transfusion in trauma patients has not been well characterized to date. This study investigated the incidence of surviving donor white blood cells (known as transfused-associated microchimerism (TAM)) and cytokine changes following blood transfusion in trauma patients. STUDY DESIGN AND METHODS: Trauma patients with an injury severity score (ISS) >12 who had been transfused between 2012-2016 with at least 5 units of RBC units over a 4 h period were recruited. Trauma patients with ISS > 12 who did not require blood transfusion were recruited as controls. The incidence of TAM was determined using a panel of insertion/deletion (InDel) bi-allelic polymorphisms. Selected pro- and anti-inflammatory cytokine profiles were analyzed using cytometric bead array. RESULTS: The transfused cohort (n = 40) had median ISS of 28 [12-66], received a median of 11 RBC units [4-114] and had median hospital length of stay of 35 days [1-152]. Only 11 (27.5%) patients returned for follow-up blood sampling after discharge. Of these, one patient showed an InDel pattern indicating the presence of TAM. No patients in the control cohort (n = 49) showed TAM. Cytokines IL-10 and IL-6 were found to be elevated in the transfused trauma patients. CONCLUSION: In this cohort, TAM was found to occur in one patient of the 11 who received a blood transfusion. Elevated IL-6 and IL-10 cytokines were detected in those patients who were transfused. However, the incidence of TAM could not be correlated with the elevated cytokine profiles for this cohort.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

INTRODUCTION: KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. STUDY DESIGN AND METHODS: In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. RESULTS: Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. CONCLUSION: Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

Packed Red Blood Cell Transfusion Modulates Myeloid Dendritic Cell Activation and Inflammatory Response In Vitro.

Transfusion of packed red blood cells (PRBCs) modulates patients' immune responses and clinical outcomes; however, the underpinning mechanism(s) remain unknown. The potential for PRBC to modulate myeloid dendritic cells (mDC) and blood DC antigen 3 was assessed using an in vitro transfusion model. In parallel, to model processes activated by viral or bacterial infection, toll-like receptor agonists polyinosinic:polycytidylic acid or lipopolysaccharide were added. Exposure to PRBC upregulated expression of CD83 and downregulated CD40 and CD80 on both DC subsets, and it suppressed production of interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor-α, and interferon-gamma-inducible protein-10 by these cells. Similar effects were observed when modeling processes activated by concurrent infection. Furthermore, exposure to PRBC at date of expiry was associated with more pronounced effects in all assays. Our study suggests PRBC have an impact on recipient DC function, which may result in failure to establish an appropriate immune response, particularly in patients with underlying infection.

Platelet concentrates modulate myeloid dendritic cell immune responses.

Platelet transfusion has been reported to modulate the recipients' immune system. To date, the precise mechanism(s) driving poor patient outcomes (e.g., increased rate of mortality, morbidity, infectious complications and prolonged hospital stays) following platelet transfusion are largely undefined. To determine the potential for platelet concentrates (PC) to modulate responses of crucial immune regulatory cells, a human in vitro whole blood model of transfusion was established. Maturation and activation of human myeloid dendritic cells (mDC) and the specialized subset blood DC antigen (BDCA)3+ DC were assessed following exposure to buffy-coat derived PC at day (D)2 (fresh) and D5 (date-of-expiry). In parallel, to model recipients with underlying viral or bacterial infection, polyinosinic:polycytidylic acid or lipopolysaccharide was added. Exposure to PC had less of an impact on mDC responses than BDCA3+ DC responses. PC alone downregulated BDCA3+ DC expression of co-stimulatory molecules CD40 and CD80. In the model of viral infection, PC downregulated expression of CD83, and in the bacterial model of infection, PC downregulated CD80, CD83, and CD86. PC alone suppressed mDC production of interleukin (IL)-8, IL-12 and tumor necrosis factor (TNF)-α and BDCA3+ DC production of IL-8, IL-12, and IL-6. In the model of viral infection, production of IL-12 and interferon-gamma inducible protein (IP)-10 was reduced in both DC subsets, and IL-8 was reduced in BDCA3+ DC following PC exposure. When modeling bacterial infection, PC suppressed mDC and BDCA3+ DC production of IL-6 and IL-10 with a reduction in TNF-α evident in mDC. This study assessed the impact of PC "transfusion" on DC surface antigen expression and inflammatory mediator production and provided the first evidence that PC transfusion modulates blood mDC and BDCA3+ DC maturation and activation, particularly in the models of infection. Results of this study suggest that patients who receive PC, particularly those with underlying infectious complications, may fail to establish an appropriate immune response precipitating poor patient outcomes.

Immunomodulatory effect of cryopreserved platelets: altered BDCA3+ dendritic cell maturation and activation in vitro.

BACKGROUND: Cryopreservation of platelets (PLTs) is useful in remote areas to overcome logistic problems associated with supply and can extend the shelf life to 2 years. During cryopreservation, properties of PLTs are modified. Whether changes in the cryopreserved PLT (CPP) product are associated with modulation of recipients' immune function is unknown. We aimed to characterize the immune profile of myeloid dendritic cells (mDCs) and the specialized blood DC antigen (BDCA)3+ subset after exposure to CPPs. STUDY DESIGN AND METHODS: Using an in vitro whole blood model of transfusion, the effect of CPPs on mDC and BDCA3+ DC surface antigen expression and inflammatory mediator production was examined using flow cytometry. In parallel, polyinosinic:polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) was utilized to model processes activated in viral or bacterial infection, respectively. RESULTS: Cryopreserved PLTs had minimal impact on mDC responses but significantly modulated BDCA3+ DC responses in vitro. Exposure to CPPs alone up regulated BDCA3+ DC CD86 expression and suppressed interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon-γ inducible protein (IP)-10 production. In both models of infection-related processes, exposure to CPPs down regulated BDCA3+ DC expression of CD40, CD80, and CD83 and suppressed BDCA3+ DC production of IL-8, IL-12, and TNF-α. CPPs suppressed CD86 expression in the presence of LPS and IP-10 and IL-6 production with poly(I:C). CONCLUSION: Cryopreserved PLTs may be immunosuppressive, and this effect is more evident when processes associated with infection are concurrently activated, especially for BDCA3+ DCs. This suggests that transfusion of CPPs in patients with infection may result in impaired BDCA3+ DC responses.

Procoagulant role of microparticles in routine storage of packed red blood cells: potential risk for prothrombotic post-transfusion complications.

During routine storage, packed red blood cells (PRBC) undergo biochemical and morphological changes including loss of red blood cell (RBC) membrane asymmetry and release of microparticles (MPs) bearing phosphatidylserine (PS), a procoagulant phospholipid. This study investigated the association between PRBC storage duration, MP profile and procoagulant activity. Leukodepleted PRBC-supernatant (PRBC-SN; n=13) was prepared at weekly intervals throughout storage. Phospholipid-dependent procoagulant activity, assessed using a factor X-activated clotting time (XACT) assay, decreased throughout storage (p<0.0001), corresponding with increased procoagulant phospholipid content. As determined by flow cytometry, total numbers of MPs and of PS-bearing MPs increased by Day 28 of storage (p<0.01 and p<0.05, respectively, versus D1), and these MPs were predominantly RBC-derived (CD235+). Depletion of MPs from stored (Day 42) PRBC-SN using 0.22 µm filters reduced the number of PS-bearing MPs (p<0.01) but did not increase XACT clotting times. Furthermore, the reduction in procoagulant activity when lactadherin was used to block PS was not altered pre- or post-filtration of PRBC-SN. In conclusion, routine PRBC storage was associated with accumulation of MPs (particularly RBC-derived PS-bearing MPs) and of procoagulant phospholipids; however, depletion of PS-bearing MPs by 0.22 µm filtration did not reduce phospholipid-dependent procoagulant activity.

Immunomodulation of inflammatory leukocyte markers during intravenous immunoglobulin treatment associated with clinical efficacy in chronic inflammatory demyelinating polyradiculoneuropathy.

OBJECTIVE: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. METHODS: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. RESULTS: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185+ follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16+ myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16+ dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. CONCLUSIONS: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome.

Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage.

The contribution of ex-vivo storage duration of packed red blood cells (PRBC) to patient outcomes and transfusion-related immunomodulation (TRIM) remains a broadly debated area in transfusion medicine. Kode™ Technology with fluorescein conjugated function-spacer-lipid (FSL-FLRO4) constructs is a tool that can aid in-vitro visualization and tracking of red blood cells (RBC) during routine storage. FSL-FLRO4 is incorporated into the RBC membrane without altering cell function. In this study, we explore the suitability of this technology to label clinical grade PRBC and to determine if the label would be retained during ex-vivo storage. Firstly, to confirm feasibility and assess the limit of detection of FSL-FLRO4 on PRBC at date of expiry (42 days post-collection), we tracked the binding of FSL-FLRO4 on PRBC at weekly intervals during routine storage. Over the time course, all cells remained labelled with FSL-FLRO4, although a decrease in the intensity of labelling was observed (P<0.0001). We then further investigated differences in FSL-FLRO4 labelling during RBC storage by labelling separated light-young and dense-old RBC from the same PRBC unit. There were no differences in the capacity of FSL-FLRO4 to label these different RBC subsets. Together, these data demonstrate that FSL-FLRO4 is a suitable reagent for labelling PRBC at any point during routine storage. This technology will facilitate the development of immunoassays and transfusion models focused on addressing the mechanisms involved in TRIM.

Soluble Mediators in Platelet Concentrates Modulate Dendritic Cell Inflammatory Responses in an Experimental Model of Transfusion.

The transfusion of platelet concentrates (PCs) is widely used to treat thrombocytopenia and severe trauma. Ex vivo storage of PCs is associated with a storage lesion characterized by partial platelet activation and the release of soluble mediators, such as soluble CD40 ligand (sCD40L), RANTES, and interleukin (IL)-8. An in vitro whole blood culture transfusion model was employed to assess whether mediators present in PC supernatants (PC-SNs) modulated dendritic cell (DC)-specific inflammatory responses (intracellular staining) and the overall inflammatory response (cytometric bead array). Lipopolysaccharide (LPS) was included in parallel cultures to model the impact of PC-SNs on cell responses following toll-like receptor-mediated pathogen recognition. The impact of both the PC dose (10%, 25%) and ex vivo storage period was investigated [day 2 (D2), day 5 (D5), day 7 (D7)]. PC-SNs alone had minimal impact on DC-specific inflammatory responses and the overall inflammatory response. However, in the presence of LPS, exposure to PC-SNs resulted in a significant dose-associated suppression of the production of DC IL-12, IL-6, IL-1α, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP)-1ß and storage-associated suppression of the production of DC IL-10, TNF-α, and IL-8. For the overall inflammatory response, IL-6, TNF-α, MIP-1α, MIP-1ß, and inflammatory protein (IP)-10 were significantly suppressed and IL-8, IL-10, and IL-1ß significantly increased following exposure to PC-SNs in the presence of LPS. These data suggest that soluble mediators present in PCs significantly suppress DC function and modulate the overall inflammatory response, particularly in the presence of an infectious stimulus. Given the central role of DCs in the initiation and regulation of the immune response, these results suggest that modulation of the DC inflammatory profile is a probable mechanism contributing to transfusion-related complications.