Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes.

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display the cis geometrical configuration due to desaturase enzymatic activity and they are known to be involved in the peroxidation process, whereas the reactivity through cis-trans double bond isomerization has not yet been identified. Using 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (C18 plasm-20:4 PC) as a representative molecule, we showed that the cis-trans isomerization can occur at both plasmalogen unsaturated moieties, and the product has characteristic analytical signatures useful for omics applications. Using plasmalogen-containing liposomes and red blood cell (RBC) ghosts under biomimetic Fenton-like conditions, in the presence or absence of thiols, peroxidation, and isomerization processes were found to occur with different reaction outcomes due to the particular liposome compositions. These results allow gaining a full scenario of plasmalogen reactivity under free radical conditions. Moreover, clarification of the plasmalogen reactivity under acidic and alkaline conditions was carried out, identifying the best protocol for RBC membrane fatty acid analysis due to their plasmalogen content of 15-20%. These results are important for lipidomic applications and for achieving a full scenario of radical stress in living organisms.

Diagnostic value of plasma p-tau181, NfL, and GFAP in a clinical setting cohort of prevalent neurodegenerative dementias.

BACKGROUND: Increasing evidence supports the use of plasma biomarkers of neurodegeneration and neuroinflammation to screen and diagnose patients with dementia. However, confirmatory studies are required to demonstrate their usefulness in the clinical setting. METHODS: We evaluated plasma and cerebrospinal fluid (CSF) samples from consecutive patients with frontotemporal dementia (FTD) (n = 59), progressive supranuclear palsy (PSP) (n = 31), corticobasal syndrome (CBS) (n = 29), dementia with Lewy bodies (DLB) (n = 49), Alzheimer disease (AD) (n = 97), and suspected non-AD physiopathology (n = 51), as well as plasma samples from 60 healthy controls (HC). We measured neurofilament light chain (NfL), phospho-tau181 (p-tau181), and glial fibrillary acid protein (GFAP) using Simoa (all plasma biomarkers and CSF GFAP), CLEIA (CSF p-tau181), and ELISA (CSF NfL) assays. Additionally, we stratified patients according to the A/T/N classification scheme and the CSF α-synuclein real-time quaking-induced conversion assay (RT-QuIC) results. RESULTS: We found good correlations between CSF and plasma biomarkers for NfL (rho = 0.668, p < 0.001) and p-tau181 (rho = 0.619, p < 0.001). Plasma NfL was significantly higher in disease groups than in HC and showed a greater increase in FTD than in AD [44.9 (28.1-68.6) vs. 21.9 (17.0-27.9) pg/ml, p < 0.001]. Conversely, plasma p-tau181 and GFAP levels were significantly higher in AD than in FTD [3.2 (2.4-4.3) vs. 1.1 (0.7-1.6) pg/ml, p < 0.001; 404.7 (279.7-503.0) vs. 198.2 (143.9-316.8) pg/ml, p < 0.001]. GFAP also allowed discriminating disease groups from HC. In the distinction between FTD and AD, plasma p-tau181 showed better accuracy (AUC 0.964) than NfL (AUC 0.791) and GFAP (AUC 0.818). In DLB and CBS, CSF amyloid positive (A+) subjects had higher plasma p-tau181 and GFAP levels than A- individuals. CSF RT-QuIC showed positive α-synuclein seeding activity in 96% DLB and 15% AD patients with no differences in plasma biomarker levels in those stratified by RT-QuIC result. CONCLUSIONS: In a single-center clinical cohort, we confirm the high diagnostic value of plasma p-tau181 for distinguishing FTD from AD and plasma NfL for discriminating degenerative dementias from HC. Plasma GFAP alone differentiates AD from FTD and neurodegenerative dementias from HC but with lower accuracy than p-tau181 and NfL. In CBS and DLB, plasma p-tau181 and GFAP levels are significantly influenced by beta-amyloid pathology.

Fibrinogen Depletion Coagulopathy Predicts Major Bleeding After Thrombolysis for Ischemic Stroke: A Multicenter Study.

BACKGROUND: Symptomatic intracerebral hemorrhage (sICH) and major bleeding can be fatal complications of intravenous thrombolysis (IVT) for acute ischemic stroke. We investigated the impact of early fibrinogen depletion after IVT on major bleeding events. METHODS: This multicenter observational prospective cohort study enrolled 1678 consecutive patients receiving IVT for acute ischemic stroke at 6 Italian centers, undergoing fibrinogen concentration assessment at baseline, 2 hours and 6 hours after IVT. Fibrinogen depletion was defined as a reduction below 200 mg/dL after 2 hours from IVT, or as a reduction below 50% of baseline fibrinogen levels after 2 hours from IVT. Main outcomes were (1) sICH (National Institute of Neurological Disorders and Stroke criteria) and (2) major bleeding defined as fatal bleeding, decrease in the hemoglobin level>2 g/dL/>1 unit transfusion, or bleeding at critical site. Additional outcomes were (1) any ICH, (2) any bleeding, (3) fatal ICH, and (4) sICH according to ECASSII definition. Good functional recovery was defined as modified Rankin Scale score 0 to 2 at 3 months. RESULTS: Overall, 1678 patients were included (mean age 72 years, 46% female). sICH (n=116) and major bleeding (n=297) were associated with lower rate of good functional recovery (P<0.001). Despite similar fibrinogen levels at admission, fibrinogen depletion after 2 hours from IVT was more common in people with sICH, major bleeding and all additional bleeding outcomes. In the backward stepwise multivariable logistic regression model, fibrinogen depletion remained a significant predictor of sICH (OR, 1.55 [95% CI, 1.04-2.32]) and major bleeding (OR, 1.36 [95% CI, 1.03-1.8]). Thirty-one percent of sICH could be attributable to fibrinogen depletion. The association between fibrinogen depletion and worse clinical outcome at 3 months after stroke (P=0.012) was attributable to the higher risk of major bleeding/sICH. CONCLUSIONS: Fibrinogen depletion significantly increases the risk of sICH and major bleeding after IVT for acute ischemic stroke. Fibrinogen depletion represents an independent risk factor for bleeding, and routine assessment could be considered to stratify the risk of ICH. Trials on early fibrinogen repletion are needed to investigate mitigation of bleeding risk.

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing.

BACKGROUND: We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. MATERIALS AND METHODS: Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. RESULTS: All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. DISCUSSION: Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.

Immune thrombotic thrombocytopenic purpura: Personalized therapy using ADAMTS-13 activity and autoantibodies.

Recently, treatment of immune-mediated thrombotic thrombocytopenic purpura (ITTP) has changed with the advent of caplacizumab in clinical practice. The International Working Group (IWG) has recently integrated the ADAMTS-13 activity/autoantibody monitoring in consensus outcome definitions. We report three ITTP cases during the coronavirus disease 2019 pandemic, that received a systematic evaluation of ADAMTS-13 activity and autoantibodies. We describe how the introduction of caplacizumab and ADAMTS-13 monitoring could change the management of ITTP patients and discuss whether therapeutic choices should be based on the clinical response alone. ADAMTS-13 activity/antibodies were assessed every 5 days. Responses were evaluated according to updated IWG outcome definitions. These kinetics, rather than clinical remission, guided the therapy, allowing early and safe caplacizumab discontinuation and sensible administration of rituximab. Caplacizumab was cautiously discontinued after achieving ADAMTS-13 complete remission. These cases illustrate that prospective ADAMTS-13 evaluation and use of updated IWG definitions may improve real-life patients' management in the caplacizumab era.

Emilia-Romagna Regional Blood System accreditation as an example of improvement through application of specific requirements: a retrospective analysis.

BACKGROUND: Institutional accreditation in Italy represents the license given by a region to a public or private facility to provide services in the name and on behalf of the National Health Service. This study aims to evaluate the improvement of the Emilia-Romagna Regional Blood System and to highlight its unresolved issues, analysing non-conformities observed during accreditation and maintenance inspections between 2013 and 2018. METHODS: All the Emilia-Romagna Regional Blood facilities were invited to participate in this study voluntarily and anonymously. Participants had to access a web application that we developed specifically. For each of the three inspections evaluated in this study, they had to enter data about the state of their organisation branches and non-conformities observed by regional inspectors. All data entered were finally exported from the web application database and analysed with spreadsheets. Statistical analysis was performed using Wilcoxon signed-rank test with continuity correction. RESULTS: 17 structures took part in the study, with a total of 174 organisation branches. The number of branches changed over the years because of new openings and closures due to reorganisations or non-conformities that were too difficult to correct. Inspectors observed 2381 non-conformities (291 structural, 611 technological and 1479 organisational). As a result of accreditation inspections and consequent improvement actions, non-conformities were reduced by 88%. The most frequent non-conformities concerned the management software and the transportation of blood and blood components. CONCLUSION: An improvement in the Emilia-Romagna Regional Blood System over time is evident: institutional accreditation certainly pushed it to change and overcome its problems to comply with specific requirements. The remaining non-conformities after the three inspections were mostly organisational and management software was the most critical issue. Despite these non-conformities, all currently active structures are accredited and guarantee high standards of quality and safety of products and services.

Detection of Specific Antibodies against Toscana Virus among Blood Donors in Northeastern Italy and Correlation with Sand Fly Abundance in 2014.

Toscana virus (TOSV) is a Phlebovirus transmitted by phlebotomine sand flies and is an important etiological agent of summer meningitis in the Mediterranean basin. Since TOSV infection is often asymptomatic, we evaluated the seroprevalence in blood donors (BDs) in the Bologna and Ferrara provinces (Northeastern Italy)-the areas with the highest and lowest numbers of TOSV neuroinvasive cases in the region, respectively. A total of 1208 serum samples from BDs were collected in April-June 2014 and evaluated for the presence of specific TOSV-IgG by ELISA. The IgG-reactive samples were confirmed by indirect immunofluorescence assay (IIF) and by microneutralization test (MN). Serum samples were defined as positive for anti-TOSV IgG when reactive by ELISA and by at least one second-level test; TOSV seroprevalence was 6.8% in the Bologna province, while no circulation of TOSV was detected in the Ferrara province. Sand fly abundance in 2014 was also estimated by a geographic information system using a generalized linear model applied to a series of explanatory variables. TOSV seroprevalence rate was strongly associated with the sand fly abundance index in each municipality, pointing out the strong association between sand fly abundance and human exposure to TOSV.

Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy.

OBJECTIVES: Human leishmaniasis can be severe and fatal, yet in the Mediterranean region only a small percentage of infections progress to clinical disease. We evaluated the percentage of asymptomatic Leishmania infection in the Bologna province, northeastern Italy. METHODS: We examined the presence of specific antibodies by Western Blot (WB) and parasitic DNA by real time PCR in peripheral blood of 240 blood donors residing in the Bologna province. RESULTS: Anti-Leishmania IgG were detected by WB in 27 subjects (11.2%, 95% CI 7%-15%), while Leishmania kinetoplast DNA was detected in peripheral blood specimens of 4 out of 240 donors (1.7%, 95% CI 0.2%-3.2%). Overall, the prevalence of Leishmania infection in the blood donor cohort was 12.5%, thus indicating an elevated cumulative exposure to the Leishmania parasite in the examined municipality. CONCLUSIONS: Our results suggest that a surveillance system for monitoring Leishmania infection in blood donors and/or strategies of protozoan inactivation in whole blood should be taken into consideration in areas with circulation of the Leishmania parasite.

Blood derived treatment from two allogeneic sources for severe dry eye associated to keratopathy: a multicentre randomised cross over clinical trial.

AIM: To compare the efficacy of cord blood and peripheral adult donor blood serum eyedrops, controlled for growth factor content, in the treatment of severe dry eye diseases (DED) resistant to conventional therapy. METHODS: This was a multicentre randomised, double-masked, cross-over clinical trial. Sixty patients diagnosed as severe DED, associated to persistent corneal epithelial defects were randomised and equally assigned to group A (treated with cord blood serum (CBS)) or group B (treated with PBS), eyedrops administered eight times/day for 1 month. Primary outcome was the pretreatment and post-treatment change in corneal fluorescein staining. Secondary outcomes included the pretreatment and post-treatment change in Ocular Surface Disease Index (OSDI) questionnaire and Visual Analogue Score (VAS) of subjective symptoms, Schirmer I test, tear break-up time and conjunctival staining. Patients with relapse in signs or symptoms after further 2 months switched to the remaining group for one additional month. Data were statistically analysed (p<0.05). RESULTS: Corneal staining was more significantly reduced after the CBS treatment, both VAS and OSDI score reduction was observed in both groups, but group A reported significantly less grittiness and pain. Nineteen patients shifted in the crossover period, the within individual comparison confirmed a better recovery in the CBS treatment period. Reduction in epithelial damage was positively associated with epidermal growth factor, transforming growth factorα and platelet-derived growth factor content. Levels of interleukins (IL-13) were positively associated with symptom decrease. CONCLUSIONS: Overall, DED signs improved after both CBS and PBS treatments, with potential advantages of CBS for subjective symptoms and corneal damage reduction. CLINICAL TRIAL REGISTRATION: NCT03064984.

Prenatal non-invasive foetal RHD genotyping: diagnostic accuracy of a test as a guide for appropriate administration of antenatal anti-D immunoprophylaxis.

BACKGROUND: Foetal RHD genotyping can be predicted by real-time polymerase chain reaction (qPCR) using cell-free foetal DNA extracted from maternal plasma. The object of this study was to determine the diagnostic accuracy and feasibility of non-invasive RHD foetal genotyping, using a commercial multiple-exon assay, as a guide to appropriate administration of targeted antenatal immunoprophylaxis. MATERIAL AND METHODS: Cell-free foetal DNA was extracted from plasma of RhD-negative women between 11-30 weeks of pregnancy. The foetal RHD genotype was determined non-invasively by qPCR amplification of exons 5, 7 and 10 of the RHD gene using the Free DNA Fetal Kit® RhD. Results were compared with serological RhD cord blood typing at birth. The analysis of diagnostic accuracy was restricted to the period (24-28+6 weeks) during which foetal genotyping is usually performed for targeted antenatal immunoprophylaxis. RESULTS: RHD foetal genotyping was performed on 367 plasma samples (24-28+6 weeks). Neonatal RhD phenotype results were available for 284 pregnancies. Foetal RHD status was inconclusive in 9/284 (3.2%) samples, including four cases with RhD maternal variants. Two false-positive results were registered. The sensitivity was 100% and the specificity was 97.5% (95% CI: 94.0-100). The diagnostic accuracy was 99.3% (95% CI: 98.3-100), decreasing to 96.1% (95% CI: 93.9-98.4) when the inconclusive results were included. The negative and positive predictive values were 100% (95% CI: 100-100) and 99.0% (95% CI: 97.6-100), respectively. There was one false-negative result in a sample collected at 18 weeks. After inclusion of samples at early gestational age (<23+6 week), sensitivity and accuracy were 99.6% (95% CI: 98.7-100) and 95.5% (95% CI: 93.3-97.8), respectively. DISCUSSION: This study demonstrates that foetal RHD detection on maternal plasma using a commercial multiple-exon assay is a reliable and accurate tool to predict foetal RhD phenotype. It can be a safe guide for the appropriate administration of targeted prenatal immunoprophylaxis.

Adverse reactions to blood donations: the READ project.

BACKGROUND: In 2006 in Italy 2,404,267 donations of blood components were made by 1,539,454 donors; approximately 55% of the donations were collected directly by Transfusion Structures (TS), while about 45% were collected in Donation Centres managed by Associations and Federations of Donors. The aim of the READ (Rilevamento Eventi Avversi alla Donazione) project is to create a network of TS to test a standardised system for monitoring adverse events (AE) related to blood donations. MATERIALS AND METHODS: Shared, standardised data collection forms, compatible with the forms produced by the ISBT-EHN, were prepared. Two types of form were used: (i) a form to collect data on single events (READ 1), to be used at the individual collection sites; (ii) a form for processing the data collected by each TS (READ 2). RESULTS: Between February and August 2008 six TS collected data related to the donation of 89,332 units of blood. Overall, 523 AE were recorded. The AE occurred in 0.59% of the donations. The mean duration of the symptoms was 17 minutes. Fifteen percent of the symptoms were related to the venipuncture (mainly haematomas) and 77% to vasovagal AE. The AE were defined severe (grade C) in 47 cases. The donations in which AE were recorded were completed in 81% of the cases; 59% of the AE did not require treatment. Three donors were monitored briefly (for less than 4 hours) in hospital. CONCLUSIONS: The use of standardised forms enabled the collection of data that could be analysed. Some problems related to the performance of the haemovigilance programme did, however, emerge: (i) organisational problems, (ii) limited sensitivity, (iii) inadequate training, and (iv) poorly defined responsibilities. These problems must be resolved at various levels: local, regional and national.