Timely administration of tocilizumab improves outcome of hospitalized COVID-19 patients.

INTRODUCTION: The aim of this study was to determine the efficacy of early tocilizumab treatment for hospitalized patients with COVID-19 disease. METHODS: Open-label randomized phase II clinical trial investigating tocilizumab in patients with proven COVID-19 admitted to the general ward and in need of supplemental oxygen. The primary endpoint of the study was 30-day mortality with a prespecified 2-sided significance level of α = 0.10. A post-hoc analysis was performed for a combined endpoint of mechanical ventilation or death at 30 days. Secondary objectives included comparing the duration of hospital stay, ICU admittance and duration of ICU stay and the duration of mechanical ventilation. RESULTS: A total of 354 patients (67% men; median age 66 years) were enrolled of whom 88% received dexamethasone. Thirty-day mortality was 19% (95% CI 14%-26%) in the standard arm versus 12% (95% CI: 8%-18%) in the tocilizumab arm, hazard ratio (HR) = 0.62 (90% CI 0.39-0.98; p = 0.086). 17% of patients were admitted to the ICU in each arm (p = 0.89). The median stay in the ICU was 14 days (IQR 9-28) in the standard arm versus 9 days (IQR 5-14) in the tocilizumab arm (p = 0.014). Mechanical ventilation or death at thirty days was 31% (95% CI 24%-38%) in the standard arm versus 21% (95% CI 16%-28%) in the tocilizumab arm, HR = 0.65 (95% CI 0.42-0.98; p = 0.042). CONCLUSIONS: This randomized phase II study supports efficacy for tocilizumab when given early in the disease course in hospitalized patients who need oxygen support, especially when concomitantly treated with dexamethasone. TRIAL REGISTRATION: https://www.trialregister.nl/trial/8504.

Local and Systemic Immunity against Respiratory Syncytial Virus Induced by a Novel Intranasal Vaccine. A Randomized, Double-Blind, Placebo-controlled Clinical Trial.

Rationale: Needle-free intranasal vaccines offer major potential advantages, especially against pathogens entering via mucosal surfaces. As yet, there is no effective vaccine against respiratory syncytial virus (RSV), a ubiquitous pathogen of global importance that preferentially infects respiratory epithelial cells; new strategies are urgently required.Objectives: Here, we report the safety and immunogenicity of a novel mucosal RSV F protein vaccine linked to an immunostimulatory bacterium-like particle (BLP).Methods: In this phase I, randomized, double-blind, placebo-controlled trial, 48 healthy volunteers, aged 18-49 years, were randomly assigned to receive placebo or SynGEM (low or high dose) intranasally by prime-boost administration. The primary outcome was safety and tolerability, with secondary objectives assessing virus-specific immunogenicity.Measurements and Main Results: There were no significant differences in adverse events between placebo and vaccinated groups. SynGEM induced systemic plasmablast responses and significant, durable increases in RSV-specific serum antibody in healthy, seropositive adults. Volunteers given low-dose SynGEM (140 µg F, 2 mg BLP) required a boost at Day 28 to achieve plateau responses with a maximum fold change of 2.4, whereas high-dose recipients (350 µg F, 5 mg BLP) achieved plateau responses with a fold change of 1.5 after first vaccination that remained elevated up to 180 days after vaccination, irrespective of further boosting. Palivizumab-like antibodies were consistently induced, but F protein site ∅-specific antibodies were not detected, and virus-specific nasal IgA responses were heterogeneous, with the strongest responses in individuals with lower pre-existing antibody levels.Conclusions: SynGEM is thus the first nonreplicating intranasal RSV subunit vaccine to induce persistent antibody responses in human volunteers.Clinical trials registered with www.clinicaltrials.gov (NCT02958540).

Comparison of cryopreservation bags for hematopoietic progenitor cells using a WBC-enriched product.

Hematopoietic progenitor cells (HPC) are stored in cryopreservation bags that are resistant to liquid nitrogen. Since Cryocyte bags of Baxter (B-bags) are no longer available, an alternative bag was sought. Also, the influence of freezing volume was studied. Miltenyi Biotec (MB)- and MacoPharma (MP)-bags passed the integrity tests without failure. Comparing MB- and MP-bags with B-bags, no difference in WBC recovery or viability was found when using a WBC-enriched product as a "dummy" HPC product. Further, a freezing volume of 30 mL resulted in better WBC recovery and viability than 60 mL. Additonal studies using real HPC might be necessary.

Is hydroxyethyl starch necessary for sedimentation of bone marrow?

Hydroxyethyl starch (HES) is used to separate hematopoietic progenitor cells after bone marrow (BM) collection from red blood cells. The aims were to study alternatives for HAES-steril (200 kDa; not available anymore) and to optimize the sedimentation process. Using WBC-enriched product (10 × 10(9) WBC/L), instead of BM, sedimentation at 10% hematocrit using final 0.6 or 0.39% Voluven (130 kDa) or without HES appeared to be good alternatives for 0.6% HAES-steril. MNC recovery >80% and RBC depletion >90% was reached. Optimal sedimentation was reached using 110-140 mL volume. Centrifugation appeared not suitable for sedimentation. Additional testing with BM might be necessary to confirm these results.

Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality.

BACKGROUND: Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. STUDY DESIGN AND METHODS: WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing ("fast-rate" or "slow-rate") and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. RESULTS: Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p < 0.05) and also for 50% compared to 20% D/P (two test variations). Effect of precooling D/P or WBC-enriched product and of storage in vapor-phase or liquid nitrogen was marginal. CONCLUSION: Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary.

Noninvasive pH monitoring of platelet concentrates: a large field test.

BACKGROUND: Developing new quality control methods for platelet concentrates (PCs) can contribute to increasing transfusion safety and efficiency. The aim of this study was to investigate in a large field test the quality of expired PCs and whether 100% noninvasive pH monitoring can be used to predict PC quality. STUDY DESIGN AND METHODS: The pH of 13,693 PCs produced for transfusion was monitored daily using Blood Storage, Inc.'s pH sterile, automated fluoroscopic evaluation technology. Upon indication of compromised quality or expiration, PCs were returned and in vitro tests were performed. RESULTS: A total of 998 PCs were returned, of which 962 outdated, 26 had a positive BacT/ALERT reaction, seven had aggregates, one was without swirl, one had low pH, and one had high pH. BacT/ALERT was faster in identifying bacterial contamination than pH measurements. The pH at the end of the storage period was significantly lower than at the beginning. In vitro tests indicated that while the PC quality was acceptable upon expiration, it rapidly declined after expiration. CONCLUSION: In this setting where the vast majority of PCs were of good quality and within acceptable pH limits, daily, noninvasive routine pH measurement has limited added value in identifying quality-compromised PCs.

Hemolysis of red blood cells during processing and storage.

BACKGROUND: During processing and storage, red blood cells (RBCs) undergo changes and cell injury resulting in hemolysis. Mostly, the separation of whole blood in top-and-bottom quadruple bag systems with break openings takes less than 4 minutes. However, longer separation times are not uncommon. The aims were to investigate whether hemolysis is increased in RBCs with longer separation time (RBCs(>6 min)) compared to regular RBCs (RBCs(reg)), to measure hemolysis increase during storage and to study whether frequency of hemolytic donations is donor dependent. STUDY DESIGN AND METHODS: RBCs(>6 min) (n = 172) and 172 matched controls were tested for hemolysis on Days 1 and 21 RBC units from each group were stored at 4 ± 2°C and tested again after 5 weeks. Donor dependency was retrospectively investigated for 100 hemolytic RBC units. RESULTS: RBCs(>6 min) exhibited a higher mean hemolysis rate than RBCs(reg) (0.058% vs. 0.033%). Four RBC units were hemolytic (>0.8%), all RBCs(>6 min) (2.36%). During storage, hemolysis in both groups increased with 0.24%. Hemolysis frequency did not seem to be donor dependent. CONCLUSIONS: Increased separation time is a useful screening tool for potentially increased hemolysis rate in RBCs. Hemolysis rate increased during storage equally in both groups. Hemolysis frequency appears donor independent.

Allogeneic single-donor cryoseal produced from fresh-frozen quarantine apheresis plasma as alternative for multidonor or autologous fibrin sealants.

BACKGROUND: Fibrin sealant is a human blood product consisting of two components: cryoprecipitate and thrombin. Commercial fibrin sealants are produced from multidonors, increasing the viral risk, and contain fibrinolytic inhibitors such as tranexamic acid or bovine aprotinin. Autologous fibrin sealants reduce the viral risk and are mostly produced during a surgical procedure or well in advance. Alternatively, the allogeneic single-donor fibrin sealant cryoseal can be used. In this study cryoseal was characterized and the manufacturing consistency of the production process was investigated. STUDY DESIGN AND METHODS: Cryoseal was produced from plasma collected on apheresis machines using a commercial device. In a research setting the protein composition and recovery were determined. Also, the manufacturing consistency of the production process was tested in a research setting as well as in a routine setting. RESULTS: In the research setting all produced cryoseal met the quality control requirements of a clotting time of less than 10 seconds and the presence of Factor (F)XIII (qualitative). In the routine setting, one procedure per year did not meet these requirements. The protein composition showed the following mean ± standard deviation (%recovery) results: thrombin 25.7 ± 11.1 IU/mL, fibrinogen 19.9 ± 4.6 (15%) mg/mL, FVIII 15.6 ± 5.4 (44%) IU/mL, FXIII 2.7 ± 0.7 (6%) IU/mL, and plasminogen 1.8 ± 0.2 (4%) U/mL. In both research and routine settings the production process resulted in a consistent product. CONCLUSION: The cryoseal manufacturing process resulted in a consistent product, which meets the predetermined specifications. The single-donor origin and the absence of fibrinolytic inhibitors make cryoseal a good alternative for multidonor and autologous fibrin sealants.

Is red blood cell rheology preserved during routine blood bank storage?

BACKGROUND: Red blood cell (RBC) units stored for more than 2 weeks at 4 degrees C are currently considered of impaired quality. This opinion has primarily been based on altered RBC rheologic properties (i.e., enhanced aggregability, reduced deformability, and elevated endothelial cell interaction), during prolonged storage of nonleukoreduced RBC units. In this study, the rheologic properties and cell variables of leukoreduced RBC units, during routine blood bank storage in saline-adenine-glucose-mannitol, were investigated. STUDY DESIGN AND METHODS: Ten leukoreduced RBC units were stored at the blood bank for 7 weeks at 4 degrees C. RBCs were tested weekly for aggregability, deformability, and other relevant variables. RESULTS: RBC aggregability was significantly reduced after the first week of storage but recovered during the following weeks. After 7 weeks aggregability was slightly, but significantly, reduced (46.9 + or - 2.4-44.3 + or - 2.2 aggregation index). During storage the osmotic fragility was not significantly enhanced (0.47 + or - 0.01% phosphate-buffered saline) and the deformability at shear stress of 3.9 Pa was not significantly reduced (0.36 + or - 0.01 elongation index [EI]). The deformability at 50 Pa was reduced (0.58 + or - 0.01-0.54 + or - 0.01 EI) but remained within reference values (0.53 + or - 0.04). During 5 weeks of storage, adenosine triphosphate was reduced by 54% whereas mean cell volume, pH, and mean cell hemoglobin concentration were minimally affected. CONCLUSIONS: RBC biochemical and physical alterations during storage minimally affected the RBC ability to aggregate and deform, even after prolonged storage. The rheologic properties of leukoreduced RBC units were well preserved during 7 weeks of routine blood bank storage.

Counting platelets in platelet concentrates on hematology analyzers: a multicenter comparative study.

BACKGROUND: Hematology analyzers are designed to count whole blood samples, but are also used by blood centers to perform quality control on blood components. In platelet (PLT) concentrates, the number of PLTs is approximately fivefold higher and red blood cells are absent, causing variable PLT counting results. It was our aim to compare currently used hematology analyzers for counting PLTs in PLT concentrates using fixed human PLTs. STUDY DESIGN AND METHODS: PLT samples were fixed, diluted into seven concentration levels (plus one blank), aliquoted, and shipped to 68 centers. Evaluable data were obtained for 89 hematology analyzers. All samples were counted six times, and results were reported to the coordinating center. The overall group mean was calculated, and the percentage deviation from this mean was calculated for each analyzer. RESULTS: At PLT levels relevant for blood centers, 750 x 10(9) to 2000 x 10(9) per L, analyzers gave results that were between 35 percent lower and 16 percent higher than the overall group mean. Within a group of analyzers, results were comparable with coefficient of variations usually below 10 percent, indicating that the observed differences were caused by instrument characteristics. A smaller study with fresh, unfixed PLT samples showed that analyzers behaved similarly for fixed and fresh PLTs. CONCLUSION: With a wide array of currently used hematology analyzers, a marked difference was determined for the PLT counts of fixed human-based identical samples provided to 68 laboratories by a centralized facility. A gold standard method is needed to allow for more valid interlaboratory comparisons between hematology analyzers.

Platelet capacity of various platelet pooling systems for buffy coat-derived platelet concentrates.

BACKGROUND: Platelet (PLT) storage lesions might depend on the total PLT count in the storage container and also on the PLT pooling system, especially the storage container, that is used for preparation of PLT concentrates (PCs). In this study, the PLT capacity of four commercially available PLT pooling systems was studied. MATERIALS AND METHODS: Four PCs were prepared in pooling systems of Baxter, Fresenius, Terumo, or Pall. The PCs were pooled and divided with various total PLT counts over the four storage containers (<225 x 10(9), 225 x 10(9)-324 x 10(9), 325 x 10(9)-424 x 10(9), and >424 x 10(9) PLTs). Volumes were kept equal by adding plasma to PCs with less than 425 x 10(9) PLTs until a same volume as for PCs with more than 424 x 10(9) PLTs was reached. PCs were stored at room temperature and tested for various in vitro variables on Days 1, 3, 5, 7, and 9. Paired experiments were repeated for each system five times. RESULTS: In vitro variables remained good for 9 days, that is, swirling score of 2 or more, pH value of 6.8 or more, glucose level of 10 mmol per L or more, lactate level of less than 25 mmol per L, and CD62p expression of less than 50 percent, for PCs in Baxter systems with more than 225 x 10(9) PLTs, for PCs in Fresenius and Terumo systems with 225 x 10(9) to 424 x 10(9) PLTs, and for PCs in Pall systems with fewer than 425 x 10(9) PLTs. CONCLUSION: PLT capacity depended on the PLT pooling systems used. All systems provide acceptable storage conditions. The Baxter system was the only system with capacity for more than 424 x 10(9) PLTs per PC.

Overnight or fresh buffy coat-derived platelet concentrates prepared with various platelet pooling systems.

BACKGROUND: For logistic reasons, possibilities to produce both platelet (PLT) concentrates prepared from fresh or overnight-stored whole blood (fresh and o/n PCs, respectively) are convenient. The consequences of both possibilities are not well described. The PLT pooling system used might also influence the condition of PCs. Our aim was to compare fresh and o/n PCs with different PLT pooling systems. STUDY DESIGN AND METHODS: Fresh and o/n PCs were prepared from buffy coats and plasma in PLT pooling systems of Baxter, Fresenius, Terumo, or Pall (n = 5). PCs were stored for 9 days. The in vitro quality was determined by the PLT count, pH, glucose, lactate, pO(2), pCO(2), CD62P expression, and annexin V binding. RESULTS: The o/n PCs showed higher PLT count (approx. 460 x 10(9)/PC vs. approx. 310 x 10(9)/PC), pCO(2), and lactate concentration and lower pH, pO(2), glucose concentration, CD62P expression (until Day 5), and annexin V binding (until Day 7) compared with fresh PCs (p < 0.05). Only for o/n PCs in the Baxter and Fresenius systems did the pH and glucose concentration remain higher, and the lactate concentration and CD62P expression remained lower than that of o/n PCs in the Pall and Terumo systems. The pH for fresh PCs in the Baxter and Fresenius systems was more often greater than 7.4 than for fresh PCs in the Terumo or Pall systems. CONCLUSION: The quality of PCs depended on whether PCs were prepared from fresh or overnight-stored whole blood and on the used PLT pooling system. The main difference between fresh and o/n PCs was the PLT count.

Platelet counting in platelet concentrates with various automated hematology analyzers.

BACKGROUND: Hematology analyzers use impedance, optical, and/or immunologic techniques for counting platelets (PLTs). PLT counting in whole blood has been validated thoroughly; however, this is not the case for PLT counting in PLT concentrates (PCs), in which red cells (RBCs) are absent. Therefore, this study is focused on PLT counting in PCs to study use of ethylenediaminetetraacetate (EDTA), carryover, and accuracy of the analyzers. STUDY DESIGN AND METHODS: In total six hematology analyzers (AcT 8, Beckman Coulter; ADVIA 2,120, Bayer; Cell-Dyn 4,000, Abbott; Onyx, Beckman Coulter; K4,500, Sysmex; and XT 2,000i, Sysmex) were tested for PLT counting. PC samples with various PLT concentrations were made (0-1,700 x 10(9)/L) and measured 10 times. Carryover was determined five times. RESULTS: PC samples (1,000 x 10(9) PLTs/L) in EDTA tubes showed significantly higher PLT counts than samples in "dry" tubes for all analyzers except for the Cell-Dyn 4,000 with the impedance technique. Carryover was not more than 0.3 percent for all analyzers. The K4,500 showed the most accurate results, whereas the Cell-Dyn 4,000 with the impedance technique had low accuracy due to an overestimation of more than 20 percent. CONCLUSION: Most tested analyzers seemed to be suitable for counting PLTs in PCs. All hematology analyzers should be validated for counting PLTs in absence of RBCs as is the case in PCs, in addition to validation of PLT counting in whole blood.

Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells.

BACKGROUND: Flow cytometric methods can be used to count residual white blood cells (WBCs) in WBC-reduced blood products, which should contain fewer than 1 x 10(6) WBCs per unit (approximately 3.3 WBCs/ microL). In this study two flow cytometric methods for counting WBCs under routine conditions in nine laboratories were evaluated. STUDY DESIGN AND METHODS: Panels of red blood cells (RBCs), platelets (PLTs), and plasma were prepared containing 33.3, 10.0, 3.3, 1.0, and 0.3 WBCs per microL and counted with flow cytometric methods (either LeucoCOUNT, BD Biosciences, four laboratories; or LeukoSure, Beckman Coulter, five laboratories). Requirements were that at the level of 3.3 WBCs per microL, coefficient of variation was < or =20 percent and accuracy was > or =80 percent. Routine flow cytometric quality control (QC) data of WBC-reduced blood products from two laboratories were analyzed. RESULTS: At the level of 3.3 WBCs per microL, none of the laboratories met the requirements for all three blood products. The LeucoCOUNT method met requirements at more laboratories than the LeukoSure method for RBCs and PLTs, but the opposite was true for plasma. Routine QC data showed that >99 percent of the flow cytometric measurements for WBC-reduced products was below the 95 percent prediction interval at 3.3 WBCs per microL. CONCLUSION: None of the laboratories met the requirements for accuracy and precision for all three blood products. Nevertheless, routine results showed that in >99 percent of the products, WBC counts were below guideline limits. Therefore, both flow cytometric methods are suitable for QC with pass-fail criterion.