ELP protocol: an original approach for the mitigation of anti-CD38 interference.

BACKGROUND: Transfusion medicine is facing new challenges from therapies which interfere with pre-transfusional tests, such as monoclonal antibodies targeting blood-cell antigens. Anti-CD38 monoclonal antibodies, widely used to treat multiple myeloma, cause panreactivity of indirect antiglobulin test; this can be resolved by treating cells with dithiothreitol to disrupt the CD38 disulphide bonds expressed on red blood cell surfaces. Interference mitigation strategy with dithiothreitol, however, has some drawbacks: it entails losing the traceability of results and the denaturation of blood group systems sensitive to reducing agents; it takes time to perform and quality controls are lost. MATERIALS AND METHODS: Panels were treated with 0.2 mol/L dithiothreitol and stored for 30 days with a commercial preservative solution. On day 30, we measured the hemolysis indices and ability to eliminate daratumumab and isatuximab interference in the treated cells using indirect antiglobulin test. We also tested the stability of erythrocyte antigenic structure by screening 42 samples with known antibodies; tests were repeated on day 1, 7, 15 and 30. All indirect antiglobulin testing was performed on gel card. RESULTS: After 30 days from treatment, panels preserved in preservative solution showed hemolysis indices comparable to untreated panels: all cases of interference by anti-CD38 in pre-transfusional tests were successfully mitigated. All antibodies were detected after 30 days, except for KEL system antibodies, as expected, although there was a detectability of anti-Kell antibodies in high titer samples (the first detection in dithiothreitol-treated cells since 1983). DISCUSSION: We propose the Extended Lifetime Protocol; a simple card-based method which is cheap and traceable, that combines the strengths of anti-CD38 mitigation strategies. It makes it possible to treat and store, at the same time, a sufficient volume of red blood cells, that can be used for the following 30 days, to avoid any delay in transfusional requests.

Bilirubin Removal by Plasmafiltration-Adsorption: Ex vivo Adsorption Kinetics Model and Single Case Report.

BACKGROUND: Extracorporeal removal of bilirubin in patients with severe liver dysfunction is a key blood purification strategy. We conducted an ex vivo study to assess the quantitative capacity to remove bilirubin from plasma of a novel adsorptive cartridge. METHODS: We studied a downscaled module of the BS330 Plasma Bilirubin Adsorption Column Cartridge (Jafron Biomedical, Zhuhai City, China) to minimize the plasma requirement in an ex vivo circulation using a solution of hyperbilirubinemic plasma. We measured the bilirubin concentration gap (ΔC) between inlet (Cpin) and outlet (Cpout) of the unit and we calculated the removal ratio (RR) as mass adsorbed at different time points. Moreover, we compared the ex vivo model with the bilirubin adsorption kinetics in a patient with acute on chronic liver failure treated with the BS330 cartridge. RESULTS: Bilirubin concentration change across the cartridge at 30 min was 16.5%, and cartridge saturation was reached at 750 min. We used a minimodule downscaled to 1:3 and containing approximately 131 g of BS330 sorbent beads: the device retained 759 mg of bilirubin with a RR of 78.1% and a RR of 42.6% at 120 min. Thus, the adsorption capacity was 5.76 mg of bilirubin per gram of sorbent. Bilirubin adsorption kinetics in our clinical case with a full-scale unit shows a coherent trend with a total bilirubin mass adsorbed after 180 min of 470 mg. DISCUSSION: Our findings provide the first assessment of bilirubin adsorption in an ex vivo model of plasma perfusion and can be used to design interventional studies in humans, providing guidance for an adequate prescription of treatment frequency and duration.

Outcome of SARS CoV-2 inpatients treated with convalescent plasma: One-year of data from the Veneto region (Italy) Registry.

OBJECTIVES AND BACKGROUND: Convalescent plasma (CP) has been used worldwide to contrast SARS-CoV-2 infection. Since April 2020, it has also been used in the treatment of patients with COVID-19 in the Veneto region (Italy), along with all the other available drugs and therapeutic tools. Here we report data analysis and clinical results in 1,517 COVID-19 inpatients treated with CP containing high-titre neutralizing anti-SARS-CoV-2 antibodies (CCP). Mortality after 30 days of hospitalization has been considered primary outcome, by comparing patients treated with CCP vs all COVID-19 patients admitted to hospitals of the Veneto region in a one-year period (from April 2020 to April 2021). PATIENTS AND METHODS: Adult inpatients with a severe form of COVID-19 have been enrolled, with at least one of the following inclusion criteria: 1) tachypnea with respiratory rate (RR) ≥ 30 breaths/min; 2) oxygen saturation (SpO2) ≤ 93% at rest and in room air; 3) partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ≤ 200 mmHg, 4) radiological picture and/or chest CT scan showing signs of interstitial disease and/or rapid progression of lung involvement. Patients received a maximum of three therapeutic fractions (TFs) of CCP with a neutralizing antibody titre of ≥ 1:160, administered over a period of 3-5 days. If TFs of CCP with titre ≥ 1:160 were unavailable, 2 with antibody titre of ≥ 1:80 have been administered. RESULTS: Of the 1,517 patients treated with CCP, 209 deceased at the 30-day follow-up (14%). Death was significantly associated with an older age (p<0.001), a longer time of hospitalization before CCP infusion (p<0.001), a greater number of inclusion criteria (p<0.001) and associated comorbidities (p<0.001). Conditions significantly associated with an increased frequency of death were PaO2/FiO2 ≤ 200 (p<0.001) and tachypnea with RR>30 (p<0.05) at entry, concurrent arterial hypertension (p<0.001), cardiovascular disease (p<0.001), chronic kidney disease (p<0.001), dyslipidemia (p<0.05) and cancer (p<0.05). Moreover, factors leading to an unfavorable prognosis were a life-threatening disease (p<0.001), admission to Intensive Care Unit (p<0.001), high flow oxygen therapy or mechanical ventilation (p<0.05) and a chest X-ray showing consolidation area (p<0.001). By analyzing the regional report of hospitalized patients, a comparison of mortality by age group, with respect to our series of patients treated with CCP, has been made. Mortality was altogether lower in patients treated with CCP (14% v. 25%), especially in the group of the elderly patients (23% vs 40%,), with a strong significance (p<0.001). As regards the safety of CCP administration, 16 adverse events were recorded out of a total of 3,937 transfused TFs (0,4%). CONCLUSIONS: To overcome the difficulties of setting up a randomized controlled study in an emergency period, a data collection from a large series of patients with severe COVID-19 admitted to CCP therapy with well-defined inclusion criteria has been implemented in the Veneto region. Our results have shown that in patients with severe COVID-19 early treatment with CCP might contribute to a favourable outcome, with a reduced mortality, in absence of relevant adverse events.

Flow Dynamic Analysis by Contrast-Enhanced Imaging Techniques of Medium Cutoff Membrane Hemodialyzer.

INTRODUCTION: Medium cutoff (MCO) membranes represent an interesting innovation in the field of hemodialysis. Given the correlation between large (PM >25 kDa) middle molecules (LMM) and clinical outcomes, the possibility to broaden the spectrum of solutes removed in hemodialysis with MCO membranes introduces a new perspective for end-stage kidney disease patients. Due to low diffusion coefficients of LMM, the use of convection is required to maximize extracorporeal clearance. High convective rates are achieved with high-flux membranes in hemodiafiltration, a technique not available in the US. In case of the MCO membrane, remarkable clearances of LMM are achieved combining the permeability of the membrane with a significant amount of internal convection. The mechanism of filtration-backfiltration inside the dialyzer enables effective removal of LMM in a technique called expanded hemodialysis (HDx). Given such theoretical explanation, it is important to demonstrate the blood and ultrafiltration rheology inside the MCO dialyzer. METHOD: This study for the first time describes flow dynamic parameters and internal cross-filtration, thanks to specific radiology and nuclear imaging techniques. RESULTS: Flow dynamic analysis of the blood and dialysate compartment confirms excellent distribution of velocities and an excellent matching of blood and dialysate. Average blood flow velocity allows for wall shear rates adequate to avoid protein stagnation at the blood membrane interface and increase in blood viscosity. Cross-filtration analysis demonstrates a remarkable filtration/backfiltration flux reaching values >30 mL/min at a blood flow of 300 mL/min and zero net filtration. CONCLUSION: The MCO dialyzer Theranova 400 appears to have a design optimized to perform expanded hemodialysis (HDx).

A nationwide survey of clinical use of blood in Italy.

BACKGROUND: In Italy, as in many developed countries, a decline in blood component (BC) consumption, especially red blood cells (RBC), has been described, but not studied at a national level. We, therefore, designed a nationwide survey to determine the main features of BC recipients and obtain a picture of the clinical use of BC for the first time in Italy, in order to understand BC demands better and identify specific fields in which to apply Patient Blood Management (PBM). MATERIAL AND METHODS: A nationwide survey of all Italian Blood Establishments working as Hospital Blood Banks (HBB) was performed. Data were collected through an online report form recording information on the number and geographic area of the responding HBB, year of birth and sex of the BC recipients, and clinical indications for every unit of RBC, platelets and plasma transfused in a 24-hour period. RESULTS: On the survey date, 153/237 (64.5%) of the HBB returned 4,356 forms, reporting 7,523 transfusions. The median and mean ages of the recipients were 73 (range, 0-106) and 67.5 years, respectively; 33.0% were >80 years old and 64.9% >65 years old, with no relevant differences between males and females. Overall, 6,309 RBC units were transfused to 3,850 recipients, 66.7% of them for a medical indication and 32.4% for a surgical indication. The commonest medical indication was acquired, non-cancer-related anaemia and more than 30% of the transfusions occurred in the Emergency Department. Five hundred and sixty platelet transfusions were given to 520 recipients, 62.7% of them for prophylaxis and 28.6% for bleeding before or during an invasive procedure or surgery or for thrombocytopenia. One hundred and ninety-four patients received 654 units of plasma, 71.1% of which were for therapeutic purposes in bleeding patients. DISCUSSION: We collected a picture of blood transfusion epidemiology in Italy. The data suggest that future PBM plans should be focused on medical anaemia especially in the setting of Emergency Departments.

Preparedness and activities of the anti-SARS-CoV-2 convalescent plasma bank in the Veneto region (Italy): An organizational model for future emergencies.

BACKGROUND: Convalescent plasma (CP) has been used in the past in various pandemics, in particular in H1N1, SARS and MERS infections. In Spring 2020, when ongoing the SARS-CoV-2 pandemics, the Veneto Region (V-R) has proposed setting-up an anti-SARS-CoV-2 CP (CCP) Bank, with the aim of preparing a supply of CCP immediately available in case of subsequest epidemic waves. MATERIALS AND METHODS: Key-points to be developed for a quick set-up of the V-R CCP Bank have been recruitment of donors recovered from COVID-19 infection, laboratory analysis for the biological qualification of the CCP units, including titre of neutralizing antibodies and reduction of pathogens, according to National Blood Centre (CNS) Directives, adaptation of the V-R Information Technology systems and cost analysis. Some activities, including diagnostic and viral inactivation processes, have been centralized in 2 or 3 sites. Laboratory analysis upon preliminary admission of the donor included all tests required by the Italian laws and the CNS directives. RESULTS: From April to August 2020, 3,298 people have contacted the V-R Blood Transfusion Services: of these, 1,632 have been evaluated and examined as first time donors and those found to be suitable have carried out 955 donations, from which 2,626 therapeutic fractions have been obtained, at a cost around 215,00 Euro. Since October 2020, the number of COVID-19 inpatients has had a surge with a heavy hospital overload. Moreover, the high request of CCP therapy by clinicians has been just as unexpected, showing a wide therapeutic use. CONCLUSIONS: The organizational model here presented, which has allowed the rapid collection of a large amount of CCP, could be useful when facing new pandemic outbreaks, especially in low and middle income countries, with generally acceptable costs.

Vancomycin Adsorption During in vitro Model of Hemoperfusion with HA380 Cartridge.

INTRODUCTION: A critical point for using blood purification during sepsis may be the potential interaction with antimicrobial therapy, the mainstay of sepsis treatment. The aim of our study was to investigate the vancomycin removal during hemoperfusion (HP) using HA380 cartridge. METHODS: This is an experimental study, in which 500 mL of solution was circulated in a closed-circuit (blood flow of 250 mL/min) simulating HP ran using HA380. Vancomycin was added to reach a through concentration or a very high concentration to evaluate the removal ratio (RR) during 120 min of HP. Comparison between blood-crystalloid solution and balanced solution was performed by using Kruskal-Wallis test. The kinetics of vancomycin removal and the adsorption isotherm were evaluated. RESULTS: We found a complete removal of vancomycin at baseline through concentration of 23.0 ± 7.4 mg/L. Using extremely high concentration (baseline 777.0 ± 62.2 mg/L), RR was 90.1 ± 0.6% at 5 min and 99.2 ± 0.6% at 120 min. No difference in terms of RR was found between blood-crystalloid mixture and balanced solution. The kinetics of the vancomycin reduction followed an exponential decay. Repeated boluses (total amount of 2,000 mg) resulted in cumulative adsorption of 1,919.4 mg with RR of 96.6 ± 1.4%, regardless of the amount injected (100 vs. 500 mg). Vancomycin adsorption onto HA380 followed the Langmuir isotherm model. CONCLUSIONS: A considerable amount of vancomycin was rapidly removed during in vitro HP with HA380. Clinical studies are needed to determine whether this may lead to underdosing. Drug therapeutic monitoring is highly recommended when using HA380 for blood purification in patients receiving vancomycin.

The use of telemedicine in Italian Blood Banks: a nationwide survey.

BACKGROUND: Telemedicine is defined as the use of electronic information and communication technologies to provide health care between distant people. Many activities in transfusion medicine could benefit from the application of telemedicine. To map the spread of the use of telemedicine in transfusion medicine in Italy, the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) performed a nationwide survey: the results are presented in this paper. MATERIALS AND METHODS: A survey, dealing with different aspects of the use of telemedicine, was performed by sending a questionnaire to 280 Italian Blood Centres. The survey was designed to evaluate the diffusion of telemedicine and the features of the systems, with special attention to the systems' safety and legal adequacy. One section of the questionnaire was designed to identify the features of the systems considered essential by the respondents. RESULTS: Out of 280 Blood Services contacted, 196 (70%) filled in at least one of the questions of the online questionnaire. Globally the use of some form of telemedicine was reported by 70% of the respondents. Telemedicine is used for remote validation of laboratory tests by 32% of the Centres that responded, for remote biological validation of blood units by 34% and for assignment of blood components by 29%. Less frequently, telemedicine is used to control electronic refrigerators, for electronic blood requests and for bed-side identification of patients. DISCUSSION: The use of telemedicine is widespread in Italian Blood Services. There appears to be some heterogeneity between structures with regards to the evaluation of the systems' safety and their legal adequacy. No telemedicine system should be introduced into practice until it has proven to have the same standards of safety as the corresponding "on site" activity.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

BACKGROUND: Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals. MATERIALS AND METHODS: Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene. RESULTS: We found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity. DISCUSSION: These findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.

Institutional authorisation and accreditation of Transfusion Services and Blood Donation Sites: results of a national survey.

INTRODUCTION: The aim of the survey described in this article was to determine decisional and strategic factors useful for redefining minimum structural, technological and organisational requisites for transfusion structures, as well as for the production of guidelines for accreditation of transfusion structures by the National Blood Centre. MATERIALS AND METHODS: A structured questionnaire containing 65 questions was sent to all Transfusion Services in Italy. The questions covered: management of the quality system, accreditation, conformity with professional standards, structural and technological requisites, as well as potential to supply transfusion medicine-related health care services. All the questionnaires returned underwent statistical analysis. RESULTS: Replies were received from 64.7% of the Transfusion Services. Thirty-nine percent of these had an ISO 9001 certificate, with marked differences according to geographical location; location-related differences were also present for responses to other questions and were confirmed by multivariate statistical analysis. Over half of the Transfusion Services (53.6%) had blood donation sites run by donor associations. The statistical analysis revealed only one statistically significant difference between these donation sites: those connected to certified Transfusion Services were more likely themselves to have ISO 9001 certification than those connected to services who did not have such certification. CONCLUSIONS: The data collected in this survey are representative of the Italian national transfusion system. A re-definition of the authorisation and accreditation requisites for transfusion activities must take into account European and national legislation when determining these requisites in order to facilitate their effective applicability, promote their efficient fulfilment and enhance the development of homogeneous and transparent quality systems.