Mother-newborn ABO group discrepancy caused by a rare BW.17 variant.

Several ABO gene mutations are known to determine rare subgroups: these ABO variants are often responsible for weak or null phenotypes and may cause an incorrect determination of the serotype. Here we describe for the first time the phenotypic discrepancy of a rare B allele within the same Caucasian family that depends on the co-inheritance with A or H antigen. Blood samples from newborns, mothers, and grandmothers were analysed through routine serotype and genotype testing. Blood compatibility test was performed for red blood cells or serum of the grandmother. ABO exons were investigated through PCR and sanger sequencing. According to serology, the phenotype of the mother was AB, while it was O for the newborn. Genotype analysis confirmed that the mother was AB, while the newborn was found to be B. Sanger sequencing revealed the presence of a rare mutation in both individuals (784 G>A, D262N), corresponding to the ABO*BW.17 allele. The grandmother was found to have the same genotype/serotype of the newborn. Crossmatch testing suggested that subjects with this genotype/serotype might be considered O donors and recipients.

Cryopreservation affects platelet macromolecular composition over time after thawing and differently impacts on cancer cells behavior in vitro.

Cryopreservation affects platelets' function, questioning their use for cancer patients. We aimed to investigate the biochemical events that occur over time after thawing to optimize transfusion timing and evaluate the effect of platelet supernatants on tumor cell behavior in vitro. We compared fresh (Fresh-PLT) with Cryopreserved platelets (Cryo-PLT) at 1 h, 3 h and 6 h after thawing. MCF-7 and HL-60 cells were cultured with Fresh- or 1 h Cryo-PLT supernatants to investigate cell proliferation, migration, and PLT-cell adhesion. We noticed a significant impairment of hemostatic activity accompanied by a post-thaw decrease of CD42b+ , which identifies the CD62P--population. FTIR spectroscopy revealed a decrease in the total protein content together with changes in their conformational structure, which identified two sub-groups: 1) Fresh and 1 h Cryo-PLT; 2) 3 h and 6 h cryo-PLT. Extracellular vesicle shedding and phosphatidylserine externalization (PS) increased after thawing. Cryo-PLT supernatants inhibited cell proliferation, impaired MCF-7 cell migration, and reduced ability to adhere to tumor cells. Within the first 3 hours after thawing, irreversible alterations of biomolecular structure occur in Cryo-PLT. Nevertheless, Cryo-PLT should be considered safe for the transfusion of cancer patients because of their insufficient capability to promote cancer cell proliferation, adhesion, or migration.

What is the context? Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients.Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency.It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients.It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior.What is new? In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro.The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding.After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time.Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells.WHAT is the impact? Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients.It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition.

Effect of Plasma Unit Weight and Donor Sex on Post-Donation Citrate Level: An Experimental Study on Plasmapheresis Donors.

Introduction: Plasmapheresis donation is considered safe and well tolerated, although long-term effects need to be clarified. The volumes of anticoagulant (ACD-A) used are variable and depend primarily on hematocrit (HCT), total blood processed, amount of plasma collected, and donor characteristics. To elucidate the effect of the plasma unit weight setting on plasmapheresis efficiency and ACD-A distribution, we enrolled male donors undergoing a controlled apheresis process donating 700 g and 720 g in two different sessions. In parallel, we investigated a possible effect of sex, recruiting women donating 700 g of plasma. Methods: The study was conducted on men donating 720 g and (12 months later) 700 g of plasma, and on women donating 700 g of plasma. The main outcomes were pre-/post-donation delta (Δ) citrate concentration in donor plasma and ACD-A reinfused to the donor. Information concerning the annual check-up and the procedure was also collected. Intergroup comparisons (men donating 720 g vs. men donating 700 g and men vs. women both donating 700 g) and intragroup associations with donor and procedural characteristics were reported. Results: With the procedure set at 720 g, the machine processed around 44 mL more whole blood to collect 20 g more plasma, and 720 g donors received around 12 mL more anticoagulant than 700 g donors. Accordingly, Δ citrate concentration was 1.5 times higher (12 µm), with a greater variability observed for 720 g donations. Citrate concentration in the plasma unit was lower in the 720 g group, although not significantly. Comparing outcomes between women and men donating 700 g, we observed higher (and highly variable) Δ citrate and reinfused ACD-A in women, accompanied by lower anticoagulant levels in the unit. Increased Δ citrate is inversely associated with HCT and age in men and with HCT and triglycerides in women. Reinfused ACD-A correlates with HCT in women but not in men. Conclusion: Unit weight setting and sex influence an ACD-A shift from the estimated values toward an increased reinfusion to donor. In parallel, we observed an impact of age and sex on post-donation citrate metabolism. Altogether, these elements should be taken into account for the development of tailored approaches aimed at maintaining similar safety profiles for all donors using different plasmapheresis settings.

The impact of COVID-19 outbreak on the Transfusion Medicine Unit of a Northern Italy Hospital and Cancer Centre.

BACKGROUND AND OBJECTIVES: The first wave of coronavirus disease-2019 (COVID-19) dramatically affected the Transfusion Medicine Unit of the Azienda Unità Sanitari Locale - Istituto di Ricovero e Cura a Carattere Scientifico (AUSL-IRCCS) di Reggio Emilia, which faced a total rearrangement of the procedures for donors and patients. This study aims to assess the major implications of COVID-19 on our department, focusing on the blood transfusion chain and therapies, in order to support transfusion specialists in seeking efficient ways to face similar future emergencies. MATERIALS AND METHODS: This retrospective study compares our Transfusion Medicine Unit data collected between February and May 2020 with the same period in 2017-2019. Data on red blood cells and platelets donations, transfusions and clinical procedures were collected as aggregates from our internal electronic database. RESULTS: During the lockdown, donor centres were re-organized to reduce the risk of contagion and avoid unnecessary blood collection. Blood donations were re-scheduled to meet the decrease in elective surgery; consequently, plateletapheresis was implemented to supply the reduction of buffycoat-derived platelets. Transfusions significantly decreased together with orthopaedic and vascular surgery, while they were only marginally diminished for both cancer and onco-haematological patients. Reduced procedures for inpatients and outpatients were matched by remote medicine, addressing the need of a constant healthcare support for patients with chronic diseases. CONCLUSIONS: The described measures were adopted to avoid excessive blood collection and expiration, guarantee the safety of our ward (for both patients and staff) and supply the necessary transfusion therapies. These measures may support the development of appropriate risk management plans and safety procedures for other hospitals and transfusion services that have to face similar events.

Associations of urinary and dietary cadmium with urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and blood biochemical parameters.

Cadmium is a heavy metal with established adverse effects on human health, namely on bone, liver and kidney function and the cardiovascular system. We assessed cadmium exposure and its correlation with biomarkers of toxicity. We recruited 137 non-smoking blood donors without a history of chronic disease or cancer who resided in the Northern Italy province of Reggio Emilia (mean age 47 years, range 30-60 years) in the 2017-2019 period. We used a semi-quantitative food frequency questionnaire to estimate dietary cadmium intake and urine samples to assess concentrations of urinary cadmium and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Median urinary cadmium and 8-oxodG concentrations were 0.21 µg/L (interquartile range (IQR): 0.11-0.34 µg/L) and 3.21 µg/g creatinine (IQR: 2.21-4.80 µg/g creatinine), respectively, while median dietary cadmium intake was 6.16 µg/day (IQR: 5.22-7.93 µg/day). We used multivariable linear and spline regression models to estimate mean differences exposure concentrations. Dietary and urinary cadmium were positively correlated, and both were positively and linearly correlated with 8-oxodG. We found a positive association of urinary cadmium with blood alanine aminotransferase (ALT), total cholesterol, low-density lipoprotein (LDL)-cholesterol and thyroid-stimulating hormone (TSH) concentrations. We also observed a positive association with triglycerides, in both linear (beta regression coefficient = 77.03, 95% confidence interval 32.27-121.78) and non-linear spline regression analyses. Despite the positive correlation between dietary and urinary cadmium estimates, dietary cadmium intake showed inconsistent results with the study endpoints and generally weaker associations, suggesting a decreased capacity to reflect actual cadmium exposure. Overall, these findings suggest that even low levels of cadmium exposure may adversely alter hematological and biochemical variables and induce oxidative stress.

Optimising plasmapheresis procedure: The role of plasma unit weight setting.

The AUSL-IRCCS of Reggio Emilia Transfusion Unit operates in two blood donor centers. Plasmapheresis protocols and machines are identical in both centers, except for the final unit weight setting: 700 g in Center 1 and 720 g in Center 2. Within a wider study to assess the anticoagulant content in plasma units through proton nuclear magnetic resonance, we compared the efficiency of the two settings. We analyzed 215 and 100 consecutive samples from Centers 1 and 2, respectively. We collected processed blood volume, net plasma collected and anticoagulant volume in the plasma units. In our experience, setting the machine at 720 g instead of 700 g was associated with a small increase in plasma content of the final unit (only 4 mL), but implied an increase of more than 100 mL of the total processed blood and a higher amount of anticoagulant in the unit. On the contrary, the difference in donor's reinfused anticoagulant was negligible. Our findings come from an observational study suggesting that, in view of a minimal advantage in terms of collected net plasma, there might be relevant disadvantages for the donor in prolonging plasmapheresis over 700 g. Since observed differences may be attributed to confounding factors, we recommend always checking the marginal efficiency of the procedure when the balance target value of the setting is increased. Randomized cross-over studies are needed to find the optimal target weight for plasma units. These studies could also help defining personalized plasmapheresis procedures, thus further optimizing donor safety.

Metabolomics comparison of cord and peripheral blood-derived serum eye drops for the treatment of dry eye disease.

Allogeneic peripheral blood-derived (PBS) serum eye drops have been largely used in the treatment of dry eye disease (DED). Recently, cord blood has emerged as an effective alternative serum source (cord blood serum, CBS), containing a higher amount of growth factors than PBS, it holds the promise of a better capability to stimulate corneal healing. However, the lack of a standardized method for preparation, dispensation, storage and a poor biochemical characterization still hamper the establishment of a clinical consensus. Here the metabolomes of the two different serum eye drop preparations were compared using proton nuclear magnetic resonance spectroscopy. We found that both PBS and CBS contained several organic compounds, the majority of them already detected in human tears and may be thereby considered lacrimal substitutes. Metabolites having in the multivariate statistical analysis Partial least squares discriminant analysis (PLS-DA) a VIP scores > 1.0 were considered to be significantly different. All the metabolites identified were found to have a p < 0.05 in the univariate analysis. CBS, in particular, showed the highest amount of choline, myo-inositol, glutamine, creatine and ß-hydroxybutyrate. These evidences constitute relevant advances towards serum eye drops characterization and confirm that cord blood is a valid alternative source of serum eye drops.

Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential.

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects' treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-ß and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

Heparin-induced lipoprotein precipitation apheresis in dyslipidemic patients: A multiparametric assessment.

Low-density lipoprotein (LDL) apheresis (LA) selectively eliminates lipoproteins containing apolipoprotein B 100 (ApoB100) on patients affected by severe dyslipidemia. In addition to lowering lipids, LA is thought to exert pleiotropic effects altering a number of other compounds associated with atherosclerosis, such as pro- and anti-inflammatory cytokines or pro-thrombotic factors. More knowledge needs to be gathered on the effects of LA, and particularly on its ability to modify blood components other than lipids. We performed a multiparametric assessment of the inflammatory, metabolic and proteomic profile changes after Heparin-induced lipoprotein precipitation (H.E.L.P.) apheresis on serum samples from nine dyslipidemic patients evaluating cholesterol and lipoproteins, plasma viscosity and density, metabolites, cytokines, PCSK9 levels and other proteins selectively removed after the treatment. Our results show that H.E.L.P. apheresis is effective in lowering lipoprotein and PCSK9 levels. Although not significantly, complement and inflammation-related proteins are also affected, indicating a possible transient epiphenomenon induced by the extracorporeal procedure.

Quantitative assessment of the anticoagulant in plasma units collected by plasmapheresis.

BACKGROUND: To date, the quantification of the anticoagulant (ACD-A) in plasma units has been based on theoretical calculations. An accurate quantification could help minimize the risks associated with plasmapheresis, given that the total ACD-A used during the procedure is distributed between the donor and the plasma unit. Our aim was to experimentally quantify the volume of ACD-A in units collected by plasmapheresis. STUDY DESIGN AND METHODS: We used proton nuclear magnetic resonance spectroscopy to measure the ACD-A volume in 295 plasma units collected by the Azienda USL-IRCCS of Reggio Emilia, Italy. We analyzed the determinants of the differences between estimated and measured ACD-A through multivariate regression models. RESULTS: The experimentally measured ACD-A in plasma units was variable, with 45% of the samples showing a discrepancy of more than 15 mL compared to the manufacturer's estimate. ACD-A was underestimated for higher density of the units (p < 0.0005); a weak association was also observed with triglycerides (underestimated for higher levels, p = 0.015) and sex (overestimated in females, p = 0.008), but our model explained only 35% of the individual variability. CONCLUSION: The manufacturer's algorithms do not accurately estimate the ACD-A in units collected by plasmapheresis. Donor-related characteristics may affect ACD-A distribution between donor and plasma unit, thereby explaining the discrepancies between estimate and measurement. Errors in the estimate of the ACD-A actually received by donors could hamper studies on dose-response relationship between anticoagulant and adverse reactions. Our work should stimulate research on tailored procedures aimed at minimizing the anticoagulant received by donors and increasing plasmapheresis safety.

Red blood cells metabolome changes upon treatment with different X-ray irradiation doses.

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.

Homologous platelet gel on radiation-induced dermatitis in a patient receiving head and neck radiotherapy plus cetuximab: A case report.

INTRODUCTION: Acute radiodermatitis is a significant complication of cancer radiotherapy, and platelet-based therapies are emerging as potential new treatments. MAIN SYMPTOMS AND IMPORTANT CLINICAL FINDINGS: In this report, we present the case of a patient with head and neck cancer undergoing radiotherapy combined with the monoclonal antibody cetuximab. After 4 weeks of this treatment, the patient developed cutaneous radiation dermatitis. Despite receiving standard treatment with corticosteroids and emollient cream, the lesion did not improve. MAIN DIAGNOSIS: cutaneous radiation dermatitis on head and neck cancer patient. THERAPEUTIC INTERVENTIONS: Topical application of platelet gel was initiated on the wound. From the second week of radiotherapy to the 4th week, homologous platelet-rich plasma was applied on the dermatitis using a bandage, 4 times a day. OUTCOMES: The topical treatment with homologous platelet gel resulted in complete healing of the radiodermatitis, including restoration of the epidermis, reepithelialization, and reduction in associated pain. CONCLUSION: homologous platelet gel might be an alternative to standard treatment of radiation dermatitis.

A cohort study on the biochemical and haematological parameters of Italian blood donors as possible risk factors of COVID-19 infection and severe disease in the pre- and post-Omicron period.

To investigate the association between biochemical and blood parameters collected before the pandemic in a large cohort of Italian blood donors with the risk of infection and severe disease. We also focused on the differences between the pre- and post-Omicron spread in Italy (i.e., pre- and post-January 01, 2022) on the observed associations. We conducted an observational cohort study on 13750 blood donors was conducted using data archived up to 5 years before the pandemic. A t-test or chi-squared test was used to compare differences between groups. Hazard ratios with 95% confidence intervals for SARS-CoV-2 infection and severe disease were estimated using Cox proportional hazards models. Subgroup analyses stratified by sex, age and epidemic phase of first infection (pre- and post-Omicron spread) were examined. We confirmed a protective effect of groups B and O, while groups A and AB had a higher likelihood of infection and severe disease. However, these associations were only significant in the pre-Omicron period. We found an opposite behavior after Omicron spread, with the O phenotype having a higher probability of infection. When stratified by variant, A antigen appeared to protect against Omicron infection, whereas it was associated with an increased risk of infection by earlier variants. We were able to stratify for the SARS CoV-2 dominant variant, which revealed a causal association between blood group and probability of infection, as evidenced by the strong effect modification observed between the pre- and post-Omicron spread. The mechanism by which group A acts on the probability of infection should consider this strong effect modification.

Reduction of EpCAM-Positive Cells from a Cell Salvage Product Is Achieved by Leucocyte Depletion Filters Alone.

Intraoperative cell salvage reduces the need for allogeneic blood transfusion in complex cancer surgery, but concerns about the possibility of it re-infusing cancer cells have hindered its application in oncology. We monitored the presence of cancer cells on patient-salvaged blood by means of flow cytometry; next, we simulated cell salvage, followed by leucodepletion and irradiation on blood contaminated with a known amount of EpCAM-expressing cancer cells, assessing also residual cancer cell proliferation as well as the quality of salvaged red blood cell concentrates (RBCs). We observed a significant reduction of EpCAM-positive cells in both cancer patients and contaminated blood, which was comparable to the negative control after leucodepletion. The washing, leucodepletion and leucodepletion plus irradiation steps of cell salvage were shown to preserve the quality of RBCs in terms of haemolysis, membrane integrity and osmotic resistance. Finally, cancer cells isolated from salvaged blood lose their ability to proliferate. Our results confirm that cell salvage does not concentrate proliferating cancer cells, and that leucodepletion allows for the reduction of residual nucleated cells, making irradiation unnecessary. Our study gathers pieces of evidence on the feasibility of this procedure in complex cancer surgery. Nevertheless, it highlights the necessity of finding a definitive consensus through prospective trials.

The adoption of clinical pathways and the inclusion of likelihood ratios in the report: a controlled before and after study.

BACKGROUND: The aim of this study was to evaluate the consequence of the introduction of Clinical Pathways (CPs) and the inclusion of Likelihood Ratios (LRs) in test results instead of the reference intervals in the workload for the laboratory and in-patients. METHODS: A controlled before (2003) and after (2004, 2005, and 2006) study compared the exams requested using CPs instead of single exams for three pathologies (pulmonary embolism, acute myocardial infarction, and bile-pancreatic pathologies) in order to evaluate if this procedure is followed in clinical care and if it provides useful and accurate investigation for clinical care. Data were collected from the laboratory computer archive system over 3 years (2004 - 2006). RESULTS: The total number of requests decreased between the "before" and "after" 2004 and 2005. We found an increase (30% to 40%) in the requests of CPs. The percentage for each of the considered CPs showed a significant increase for the pulmonary embolism (EP) and bile-pancreatic pathologies: during the semesters monitored, we found an 80% increase in the request of these CPs. The percentage of acute myocardial infarction (AMI) CPs did not show a difference between the semesters compared. We also found an improvement in the diagnostic effectiveness: the concordance between the emergency department (ED) and ward diagnosis for EP increased. CONCLUSIONS: We demonstrated that this procedure is followed in clinical care for the three pathologies studied and that the use of CPs and LRs provide useful tools in making a more appropriate diagnosis. We demonstrated also that clinical audit is very important for monitoring. Audit is integral to good clinical practice and, in fact, aims to influence activity on a local level and is useful in maintaining best practice.

Using pathology-specific laboratory profiles in clinical pathology to reduce inappropriate test requesting: two completed audit cycles.

BACKGROUND: Systematic reviews have shown that, although well prepared, the Consensus Guidelines have failed to change clinical practice. In the healthcare district of Castelnovo né Monti (Reggio Emilia, Italy), it became necessary for the GPs and Clinical Pathologists to work together to jointly define laboratory profiles. METHODS: Observational study with two cycles of retrospective audit on test request forms, in a primary care setting. Objectives of the study were to develop pathology-specific laboratory profiles and to increase the number of provisional diagnoses on laboratory test request forms. A Multiprofessional Multidisciplinary Inter-hospital Work Team developed pathology-specific laboratory profiles for more effective test requesting. After 8 training sessions that used a combined strategy with multifaceted interventions, the 23 General Practitioners (GPs) in the trial district (Castelnovo nè Monti) tested the profiles; the 21 GPs in the Puianello district were the control group; all GPs in both districts participated in the trial. All laboratory tests for both healthcare districts are performed at the Laboratory located in the trial district. A baseline and a 1-year audit were performed in both districts on the GPs' request forms. RESULTS: Seven pathology-specific laboratory profiles for outpatients were developed. In the year after the first audit cycle: 1) the number of tests requested in the trial district was distinctly lower than that in the previous year, with a decrease of about 5% (p < 0.001); 2) the provisional diagnosis on the request forms was 52.8% in the trial district and 42% in the control district (P < 0.001); 3) the decrease of the number of tests on each request form was much more marked in the trial district (8.73 vs. 10.77; p < 0.001). CONCLUSIONS: The first audit cycle showed a significant decrease in the number of tests ordered only in the trial district. The combined strategy used in this study improved the prescriptive compliance of most of the GPs involved. The presence of the clinical pathologist is seen as an added value.

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation.

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex® assay. RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Platelet-rich Plasma Lysate for Treatment of Eye Surface Diseases.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at -80 °C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at -15 °C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Imbalance of Mg Homeostasis as a Potential Biomarker in Colon Cancer.

BACKGROUND: Increasing evidences support a correlation between magnesium (Mg) homeostasis and colorectal cancer (CRC). Nevertheless, the role of Mg and its transporters as diagnostic markers in CRC is still a matter of debate. In this study we combined X-ray Fluorescence Microscopy and databases information to investigate the possible correlation between Mg imbalance and CRC. METHODS: CRC tissue samples and their non-tumoural counterpart from four patients were collected and analysed for total Mg level and distribution by X-Ray Fluorescence Microscopy. We also reviewed the scientific literature and the main tissue expression databases to collect data on Mg transporters expression in CRC. RESULTS: We found a significantly higher content of total Mg in CRC samples when compared to non-tumoural tissues. Mg distribution was also impaired in CRC. Conversely, we evidenced an uncertain correlation between Mg transporters expression and colon malignancies. DISCUSSION: Although further studies are necessary to determine the correlation between different cancer types and stages, this is the first report proposing the measurement of Mg tissue localisation as a marker in CRC. This study represents thus a proof-of-concept that paves the way for the design of a larger prospective investigation of Mg in CRC.

Comparison of Two Alternative Procedures to Obtain Packed Red Blood Cells for ß-Thalassemia Major Transfusion Therapy.

ß-thalassemia major (ßTM) patients require frequent blood transfusions, with consequences that span from allogenic reactions to iron overload. To minimize these effects, ßTM patients periodically receive leucodepleted packed red blood cells (P-RBCs) stored for maximum 14 days. The aim of this study was to compare two alternative routine procedures to prepare the optimal P-RBCs product, in order to identify differences in their content that may somehow affect patients' health and quality of life (QoL). In method 1, blood was leucodepleted and then separated to obtain P-RBCs, while in method 2 blood was separated and leucodepleted after removal of plasma and buffycoat. Forty blood donors were enrolled in two independent centers; couples of phenotypically matched whole blood units were pooled, divided in two identical bags and processed in parallel following the two methods. Biochemical properties, electrolytes and metabolic composition were tested after 2, 7 and 14 days of storage. Units prepared with both methods were confirmed to have all the requirements necessary for ßTM transfusion therapy. Nevertheless, RBCs count and Hb content were found to be higher in method-1, while P-RBCs obtained with method 2 contained less K+, iron and storage lesions markers. Based on these results, both methods should be tested in a clinical perspective study to determine a possible reduction of transfusion-related complications, improving the QoL of ßTM patients, which often need transfusions for the entire lifespan.