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.

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.

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.

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.

Postoperative patient blood management: transfusion appropriateness in cancer patients.

BACKGROUND: While patient blood management (PBM) principles are not specific to cancer patients, their application contains the pathophysiological premises that could also benefit this patient population. In this study, we assessed the effects of implementing a PBM bundle for cancer patients in the postoperative period. MATERIALS AND METHODS: The Azienda USL-IRCCS of Reggio Emilia implemented a two-step PBM bundle for the postoperative period of cancer patients hospitalised in the semi-intensive post-surgery (SIPO) ward. Step 1 included seminars and lessons specifically targeting SIPO personnel; Step 2 introduced Points of Care (POCs) for the continuous monitoring of haemoglobin (Radical7, Masimo Corp, Irvine, CA, USA). We conducted 3 audits on 600 cancer patients recruited between 2014 and 2017: Audit 1 on 200 patients before the application of our PBM bundle; Audit 2 after Step 1 on 200 patients; Audit 3 after Step 2 on 200 patients monitored with POCs. Red blood cell (RBC) transfusion appropriateness in the postoperative period was evaluated using the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) recommendations. RESULTS: RBC transfusion appropriateness in the postoperative period of cancer patients rose from 38% to 75% after seminars, and reached 79% after the introduction of POC. The mean number of RBC units each patient received remained unchanged after training sessions (1.8 units/patient) while the introduction of POCs saw a simultaneous decrease in the number of prescribed units (1.3 units/patient). DISCUSSION: Our PBM bundle positively impacted RBC transfusion appropriateness in postsurgical cancer patients, both in terms of quality and quantity. A structured PBM programme specifically dedicated to surgical oncology should cover the entire perioperative period and might further improve transfusion appropriateness in these patients. The publication of guidelines on the management of anaemia in surgical oncology should be a priority.

The effect of donor's characteristics on plasmapheresis products: insights for a personalised approach.

BACKGROUND: Anticoagulant concentration in plasma units is extremely variable. Understanding the underlying causes of this variability could help personalise plasmapheresis procedures in order to optimise the risk-benefit ratio. We studied the association between anticoagulant solution A (usually ACD-A, Citrate Dextrose Solution A) volume in plasma units and donor characteristics to build a model to determine the needed weight of the final plasma unit to have an 80% probability of reaching 600 mL net plasma. MATERIALS AND METHODS: We experimentally measured ACD-A in 296 plasma units from an Italian blood donor centre, where machines are set for the collection of 700 g of plasma. Next, we built a statistical model to predict how the final volume of the unit should be set to obtain 50%, 80% or 90% probability of having at least 600 mL net plasma. RESULTS: ACD-A volume was associated with haemoglobin, total proteins and triglycerides. Donors with low haemoglobin reach an 80% probability of at least 600 mL net plasma with units of approximately 690 g, while 720 g are needed for donors with high haemoglobin levels. For total proteins and triglycerides, plasma units may vary within a range of ±20 g. DISCUSSION: Our model, based on easily measurable individual characteristics, makes it possible to customise plasmapheresis procedures by determining the blood volume to be processed for each donor. Tailored plasma donations might result in both a reduction in adverse events and an increase in the quality of collected plasma.

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.

Patient Blood Management: transfusion appropriateness in the post-operative period.

BACKGROUND: Within the context of Patient Blood Management (PBM) policy for the peri-operative period, the transfusion medicine unit of our institution adopted a series of strategies to support and enhance red blood cell (RBC) transfusion best practices. This study aimed to evaluate the appropriateness of RBC transfusion therapy in the post-operative period, before and after starting a multifactorial PBM policy. MATERIALS AND METHODS: A 2-phase observational study was conducted on patients who underwent major surgery. The study was designed as follows: 3 months of preliminary audit, followed by multifactorial PBM policy, and a final audit. The policy comprised seminars, teaching lessons, periodic consultations and the insertion of Points of Care. RBC transfusion appropriateness was evaluated in both audits. RESULTS: The preliminary audit, performed on 168 patients, showed that 37.7% of the patients were appropriately transfused. The final audit, performed on 205 patients, indicated a significant increase of RBC transfusion appropriateness to 65.4%. DISCUSSION: In our experience, our multifactorial PBM policy improved the RBC transfusion appropriateness in the post-operative period. We believe that our multifactorial PBM policy, which comprises the insertion of Points of Care, supported the healthcare workers in the transfusion decision-making process. This enhancement of transfusion appropriateness implies clinical and managerial advantages, such as reduced transfusion-related risks, optimisation of health care resources, and reduction in costs.

Effect of adhesive properties of buffy coat on the quality of blood components produced with Top & Top and Top & Bottom bags.

BACKGROUND: The Transfusion Medicine Unit of Reggio Emilia currently collects whole blood using conventional quadruple Fresenius Top & Top bags. In this study, new Fresenius Top & Bottom bags were assessed and compared to the routine method with regards to product quality and operational requirements. MATERIAL AND METHODS: Twenty-one whole blood units were collected with both the new and the traditional bags, and then separated. Quality control data were evaluated and compared in order to estimate yield and quality of final blood components obtained with the two systems. We collected other bags, not included in the ordinary quality control programme, for comparison of platelet concentrates produced by pools of buffy coat. RESULTS: Compared to the traditional system, the whole blood units processed with Top & Bottom bags yielded larger plasma volumes (+5.7%) and a similar amount of concentrated red blood cells, but with a much lower contamination of lymphocytes (-61.5%) and platelets (-86.6%). Consequently, the pooled platelets contained less plasma (-26.3%) and were significantly richer in platelets (+17.9%). DISCUSSION: This study investigated the effect of centrifugation on the adhesiveness of the buffy coat to the bag used for whole blood collection. We analysed the mechanism by which this undesirable phenomenon affects the quality of packed red blood cells in two types of bags. We also documented the incomparability of measurements on platelet concentrates performed with different principles of cell counting: this vexing problem has important implications for biomedical research and for the establishment of universal product standards. Our results support the conclusion that the Top & Bottom bags produce components of higher quality than our usual system, while having equal operational efficiency. Use of the new bags could result in an important quality improvement in blood components manufacturing.

Hematopoietic stem cell transplantation for paroxysmal nocturnal hemoglobinuria: long-term results of a retrospective study on behalf of the Gruppo Italiano Trapianto Midollo Osseo (GITMO).

BACKGROUND: Paroxysmal nocturnal hemoglobinuria is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. DESIGN AND METHODS: The aim of this retrospective study was to assess the long-term clinical and hematologic results in 26 paroxysmal nocturnal hemoglobinuria patients who received hematopoietic stem cell transplantation in Italy between 1988 and 2006. The patients were aged 22 to 60 years (median 32 years). Twenty-three donors were HLA-identical (22 siblings and one unrelated) and 3 were HLA-mismatched (2 related and one unrelated). RESULTS: Fifteen patients received a myeloablative conditioning consisting of busulfan and cyclophosphamide (in all cases from identical donor) and 11 were given a reduced intensity conditioning (8 from identical donor and 3 from mismatched donor). The cumulative incidence of graft failure was 8% (4% primary and 4% secondary graft failure). Transplant-related mortality for all patients was 42% (26% and 63% for patients transplanted following myeloablative or reduced intensity conditioning, respectively). As of October 31, 2009, 15 patients (11 in the myeloablative conditioning group and 4 in the reduced intensity conditioning group) are alive with complete hematologic recovery and no evidence of paroxysmal nocturnal hemoglobinuria following a median follow-up of 131 months (range 30-240). The 10-year Kaplan-Meier probability of disease-free survival was 57% for all patients: 65% for 23 patients transplanted from identical donor and 73% for 15 patients transplanted with myeloablative conditioning. No thromboembolic event nor recurrence of the disease were reported following transplant. CONCLUSIONS: The findings of this study confirm that most patients with paroxysmal nocturnal hemoglobinuria may be definitively cured with hematopoietic stem cell transplantation.

Graft versus host disease-related Hafnia alvei colonization and probable infection.

We describe the case of a graft versus host disease (GvHD) patient, in whom Hafnia alvei was cultured as a single organism, and at high bacterial counts from stool samples, from the onset of the disease until its resolution. This case is a further example of the contentious role of this species in causing human intestinal disease. Furthermore, it focuses on enteric damage by GvHD as a risk factor for acquiring H. alvei colonization, and probably infection.

Long-term results of survival in patients with thalassemia major treated with bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) is the only available curative approach for thalassemia major, although long-term morbidity and mortality are not established. The aim of this study was to assess the long-term clinical and hematological results in children and adults with thalassemia major treated with BMT. We analyzed the outcome of 115 patients (median age 9 years, range 11 months to 28 years) with thalassemia major undergoing BMT from a related donor between 1983 and 2006. All patients received the same protocol, consisting of busulfan and cyclophoshamide as conditioning therapy and cyclosporin (CSA) alone or CSA and methotrexate for graft-versus-host disease (GvHD) prophylaxis. The cumulative probability of graft rejection was 6.7%. The transplant-related mortality at 1 year was 8.7%. The 20-year Kaplan-Meier estimate of overall survival and disease-free survival was 89.2% and 85.7%, respectively. Ninety-nine patients out of 103 survivors were in excellent clinical and hematological conditions at last visit following a median follow-up of 15 years (range, 1-24 years) with the exception of two patients who had invalidating chronic GvHD. This study conducted with a large cohort of patients and covering a long period of observation time, showed BMT to be curative for the majority of patients with thalassemia major. The impact of long-term transplant-related sequelae was very limited.

Recombinant activated factor VII therapy in acquired hemophilia of the elderly.

Acquired hemophilia is a life-threatening condition associated with a high mortality, occurring mainly in elderly patients. We report on a 92-year-old male, administered two courses of 90 microg kg(-1) of recombinant activated factor VII (rFVIIa) due to failure of human factor VIII (FVIII) therapy, without side effects nor evidence of thrombotic complications. Despite the bleeding being completely under control, the patient died on day 11 of acute respiratory failure due to pneumonia. This case confirms previous reports on the complexity and severity of this disorder, showing that rFVIIa is an effective and safe agent for achieving hemostasis even in elderly patients with a FVIII inhibitor.