Susanne M. Picker

Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B2) for pathogen-reduction.

Background  To examine if different pathogen‐reduction technologies (PRTs) induce different degrees of platelet (PLT) storage lesion.

Functional characteristics of buffy‐coat PLTs photochemically treated with amotosalen‐HCl for pathogen inactivation

BACKGROUND: One blood system for PLTs (INTERCEPT, Baxter Transfusion Therapies) is based on photochemical treatment (PCT) with small molecules that target cross‐link nucleic acids (Helinx technology, Cerus Corp.) with amotosalen‐HCl (S‐59) and UVA light (320‐400 nm) to inactivate pathogens and WBCs.

Effects of Mirasol PRT treatment on storage lesion development in plasma-stored apheresis-derived platelets compared to untreated and irradiated units

BACKGROUND: The aim of this study was to examine the effects of a new riboflavin‐based pathogen reduction technology (PRT), the Mirasol PRT process (Navigant Biotechnologies) on platelet (PLT) storage lesion development.

In-vitro assessment of platelet function

Platelets (PLTs), play a key role in hemostasis, clot stability and retraction as well as in vascular repair and anti-microbial host defense. Upon vessel wall damage, PLTs undergo a highly regulated set including adhesion, spreading, aggregation, release reactions as well as exposure of procoagulant surfaces to rapidly form a hemostatic plug that occludes the site of damage. When PLT function is impaired, the bleeding risk increases, but (hyperreactive) PLTs are also involved in many pathophysiological events like thrombosis, vessel constriction, atherogenesis, tumor growth and metastasis, inflammation including atherosclerosis and the subsequent formation of arterial thrombi resulting in stroke and myocardial infarction. While hereditary PLT function disorders are very rare, acquired PLT function abnormalities occur in the course of many diseases and can be associated with many drugs, i.e., non-steroidal anti-inflammatorics, antibiotics or heparin. Therefore, apart from disease diagnosis, severity, and prognosis, assessment of PLT function also serves for identifying the efficacy of anti-PLT therapy and PLT hyperfunction as a possible predictor for thromboembolic events. Since PLTs undergo a lot of measurable changes during storage ex-vivo, one effort of transfusion medicine is the quality monitoring of PLT concentrates (PCs), but also the detection of donors with PLT dysfunction and the determination of patients in which PLT transfusions are effective. The majority of PLT tests focus only on PLT functions involved directly in hemostasis including adhesion/aggregation, coagulation, and clot retraction. Traditional tests, almost complex, time-consuming, and poorly specified, are meanwhile enriched by more user friendly and easy-to-use point-of-care tests on fully automated instruments within whole blood without the requirement of sample processing. These tests help identifying surgical patients at increased risk of post-operative bleeding or with resistance to anti-PLT therapy, therefore at increased risk of thromboembolism. However, up to now, no study shows real outcome benefits by including these tests into the disease management. To date, no function test is suitable to address all distinct steps of PLT activation or reliably predict PLT behavior in vivo following transfusion.

Cell viability during platelet storage in correlation to cellular metabolism after different pathogen reduction technologies.

BACKGROUND: The objective of this study was to evaluate if pathogen reduction technologies (PRTs) affect platelet (PLT) viability by alteration of PLT metabolism during storage.

Cell integrity and mitochondrial function after Mirasol-PRT treatment for pathogen reduction of apheresis-derived platelets: Results of a three-arm in vitro study

BACKGROUND Mirasol pathogen reduction technology (PRT) treatment uses riboflavin (vitamin B(2)) in combination with ultraviolet light (UV) to inactivate pathogens in platelet concentrates (PCs). This treatment has been reported to increase glycolytic flux, which could result from damage to mitochondria and/or increased ATP demand. DESIGN Triple-dose PCs were collected by the Trima Accel device. Immediately after splitting, single units were designated to Mirasol-PRT treatment (M), gamma irradiation (X) or remained untreated (C). Platelet (PLT) mitochondrial transmembrane potential (Deltapsi) was evaluated (JC-1 assay) as well as mitochondrial enzymatic activity (MTS assay). LDH release, p selectin expression, glucose/oxygen consumption and lactate production rates were quantified and compared among study groups during 7days of storage. RESULTS Immediately after PRT treatment, no significant changes were found in JC-1 signal, MTS activity, and LDH release indicating that PRT treatment did not alter functional/structural cell or mitochondrial integrity as evidenced by LDH release comparable to untreated study groups. In parallel to significantly higher p selectin expression, treated PLTs exhibited significantly accelerated oxygen and glucose consumption rates associated with increased acidity due to higher lactate production rates throughout storage. Despite larger cell populations with depolarized Deltapsi particularly at days 5 and 7, mitochondrial reduction activity of M units as measured by the MTS assay was maintained and appeared to be up-regulated relative to untreated and irradiated controls. CONCLUSION Mirasol-PRT treated PLTs increased both glycolytic flux as well as respiratory/enzymatic mitochondrial activity. An increased demand for ATP due to increased alpha granule degranulation may be the driving force for these observations.

Function, expression and localization of annexin A7 in platelets and red blood cells: Insights derived from an annexin A7 mutant mouse

BackgroundAnnexin A7 is a Ca2+- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca2+-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components.ResultsThe role of annexin A7 in red blood cells was addressed in erythrocytes from anxA7-/- mice. Interestingly, the Ca2+-mediated vesiculation process was not impaired. Also, the membrane organization appeared not to be disturbed as assessed using gradient fractionation studies. Instead, lack of annexin A7 led to an altered cell shape and increased osmotic resistance of red blood cells. Annexin A7 was also identified in platelets. In these cells its loss led to a slightly slower aggregation velocity which seems to be compensated by an increased number of platelets. The results appear to rule out an important role of annexin A7 in membrane fusion processes occurring in red blood cells. Instead the protein might be involved in the organization of the membrane cytoskeleton. Red blood cells may represent an appropriate model to study the role of annexin A7 in cellular processes.ConclusionWe have demonstrated the presence of both annexin A7 isoforms in red blood cells and the presence of the small isoform in platelets. In both cell types the loss of annexin A7 impairs cellular functions. The defects observed are however not compatible with a crucial role for annexin A7 in membrane fusion processes in these cell types.

Platelet function assessed by shear-induced deposition of split triple-dose apheresis concentrates treated with pathogen reduction technologies.

BACKGROUND: Pathogen inactivation/reduction technology (PRT) may alter quality of stored platelet (PLT) concentrates (PCs). Therefore, PLT adhesion and aggregation should be studied before transfusion of PRT‐treated PLTs.

Evaluation of White Blood Cell- and Platelet-Derived Cytokine Accumulation in MIRASOL-PRT-Treated Platelets

Background: Soluble mediators in platelet concentrates (PCs) released from contaminating white blood cells (WBCs) and platelets (PLTs) themselves are supposed to promote allergic and non-hemolytic febrile transfusion reactions in the recipient. Pathogen reduction technologies (PRTs) prevent replication and proliferation of pathogens as well as of WBCs, and may reduce cytokine accumulation in PCs during storage and prevent adverse events after PLT transfusion. On the other hand, such treatments may also lead to increased cytokine production by stimulation of WBCs or PLTs due to the photochemical or photodynamical process itself. Material and Methods: 12 triple-dose PLT apheresis collections were leukoreduced by the process-controlled leukoreduction system of the Trima Accel machine and split into 3 units undergoing Mirasol-PRT treatment (M) or gamma irradiation (X) or remaining untreated (C). During storage for up to 7 days, PLT activation, WBC-derived Th-1/2, and inflammatory as well as PLT-derived cytokines were measured by cytometric bead array and enzymelinked immunosorbent assay, respectively. Results: Independent of treatment, all PLT products exhibited low levels of WBC-associated cytokines near or below assay detection limits. WBC-associated cytokines were not elevated by Mirasol-PRT treatment. PLT-derived cytokines were detected at higher levels and increased significantly during storage in all units. Most likely due to higher PLT activation, M units showed significantly higher levels of PLT-derived cytokines compared to untreated and gamma-irradiated units on day 5 of storage. Conclusion: In all PCs, PLTs themselves were the main source of cytokine release. Mirasol-PRT treatment was associated with a significantly increased PLT activation and accumulation of PLT-derived cytokines during storage, without affecting WBC-derived cytokines relative to controls.

Leukocyte Depletion in Allogeneic Blood Transfusion Does Not Change the Negative Influence on Survival Following Transthoracic Resection for Esophageal Cancer

BackgroundPerioperative transfusion of allogeneic blood has been hypothesized to have an immunomodulatory effect and influence survival in several cancer types. This study evaluates the association between receipt of leucocyte-depleted and non-depleted allogeneic blood and survival following esophagectomy for cancer.MethodsA retrospective analysis was performed including 291 patients with esophageal cancers who underwent transthoracic en bloc esophagectomy and extended mediastinal lymphadenectomy. Neoadjuvant chemoradiation was administered in 152 (52.2%) patients. Perioperative blood transfusions were quantified and the potential prognostic cutoff for transfused units was calculated according to LeBlanc.ResultsThe median number of perioperative blood transfusions was 2 (0–24), and 106 patients (36.4%) received no transfusions. Patients with one or less blood transfusion showed a significantly improved survival compared to patients receiving more than one unit (p < 0.009). In multivariate analysis, blood transfusion categories showed significance (p < 0.015) next to pT, pN, pM category, and residual tumor categories (R-categories). Separate analysis of 183 patients treated after the mandatory introduction of leukocyte-depleted blood transfusions detected a strong tendency, but no significant difference in survival for patients getting one or less or more than one transfusion (p = 0.056). Receipt of leukocyte-depleted versus non-depleted units, however, had no influence on survival (p = 0.766).ConclusionsThe need for perioperative allogeneic blood transfusions is significantly associated with poorer survival following resection for esophageal cancer by univariate and multivariate analysis. Our data suggest that the reduction of leukocytes in allogeneic transfusions is not sufficient to overcome the negative influence on survival.