Untreated Whole Blood Research Articles

Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients' Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays.

Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients' blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients' clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients' blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive-(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.

Pathogen reduction of whole blood: Supplementing fibrinogen partly corrects clot formation in a massive transfusion model.

The use of whole blood (WB) to treat trauma patients is becoming more common. Similar to the treatment of individual components, pathogen inactivation (PI) technologies are available to treat WB. The impact of PI on WB function is not well understood. This study investigated the impact of PI of WB with riboflavin/ultraviolet (UV) light on its hemostatic function by modeling transfusion scenarios for trauma patients and assessing transfusion efficacy by rotational thromboelastometry (ROTEM). As fibrinogen is affected by PI of WB, the effect of fibrinogen supplementation commonly used in trauma patients was also analyzed in this model. Trauma transfusion scenarios were simulated by mixing untreated WB or WB treated with the Mirasol PI technology (riboflavin/UV) in different ratios with hemodiluted blood, and the thromboelasticity was monitored by ROTEM. The impact of supplementation with the fibrinogen concentrate RiaSTAP was investigated in this model. Pathogen-inactivated WB (PI-WB) showed decreased activity in the hemostatic profile compared to the untreated control. Hemodiluted blood at a hematocrit (hct) of 20%, which was reconstituted with PI-WB or untreated WB, exhibited increased alpha values, maximum clot firmness, and clot formation time. Simulating transfusion scenarios by blood replacement with PI-WB resulted in a significant difference in ROTEM parameters between reconstituted PI-treated and -untreated WB (p≥ .05). The effect of PI treatment waned when PI-WB was enriched with fibrinogen. ROTEM investigations suggest that PI treatment has a negative impact on WB clot formation unless fibrinogen supplementation is used.

Effect of leukoreduction and pathogen reduction on the hemostatic function of whole blood.

There is renewed interest in the use of whole blood (WB) for resuscitation of patients in hemorrhagic shock. Leukoreduction with platelet-sparing filters and pathogen reduction may be used to improve the safety profile of WB, yet the effects of leukoreduction and pathogen reduction on WB hemostatic function are not well characterized. Blood from 32 healthy group O donors was divided into treatment groups (n = 8 for each group): untreated, pathogen reduced (PR+ ), leukoreduced using an in-line filter (LR+ ), or PR+ LR+ . Units were stored without agitation for 21 days between 1° and 6°C, with sampling on days 0 (pre- and post-treatments), 1, 3, 5, 10, 15, and 21 for hemostatic function as assessed by thromboelastometry, thrombin generation, platelet activation factors, and platelet impedance aggregometry. From day 3 (D3) to D15 of storage, platelet count was reduced in PR+ /LR+ units compared to PR- /LR- units. From D10 to D21 of storage, maximum clot firmness (MCF) was reduced in PR+ /LR+ units compared to PR- /LR- units. From D3 to D21 of storage, platelet aggregation was reduced in PR+ /LR+ units compared to PR- /LR- units. Total thrombin generation was similar in all groups from D0 to D21. The combination of LR with a platelet-sparing filter and PR significantly reduces hemostatic function compared to either treatment alone or untreated WB. The clinical consequences of LR and PR of WB in patients with severe bleeding should be examined in trials before both are used in combination in patients.

Red blood cells derived from whole blood treated with riboflavin and ultraviolet light maintain adequate survival in vivo after 21 days of storage.

Pathogen reduction (PR) of whole blood (WB) may increase blood safety when applied before component separation. This study evaluates the in vivo performance of red blood cells (RBCs) derived from WB treated with the riboflavin and ultraviolet (UV) light PR (Mirasol) system. This was a prospective, two-center, single-blind, randomized, two-period, crossover clinical trial designed to evaluate autologous 51 Cr/99m Tc-radiolabeled recovery and survival of RBCs derived from Mirasol-treated WB compared to untreated WB. RBCs were stored in AS-3 for 21 days at 1 to 6°C. In vitro RBC variables were characterized. Frequency and severity of treatment-emergent adverse event (TEAE) and neoantigenicity were determined. Twenty-four healthy adult volunteers (n=12 per site) were evaluated. The Mirasol 24-hr RBC recoveries were 82.5±3.9% with one-sided 95% lower confidence limit of 80.9%, meeting US Food and Drug Administration acceptance criteria, albeit at lower level than controls (91.7±6.8%, p<0.001). Mean RBC survival and T50 were reduced in the Mirasol group (61 and 23 days, respectively) versus controls (82 and 36 days, respectively; p<0.001) with a mean area under the curve survival of treated RBCs of 83% of untreated controls. End-of-storage hemolysis in the Mirasol group was 0.22±0.1% (control, 0.15±0.1%; p<0.001). No neoantigenicity or differences in TEAEs were found. RBCs derived from Mirasol WB and stored for up to 21 days in AS-3 maintained acceptable cell quality and recovery, albeit modestly reduced compared with untreated RBCs. Mirasol WB may represent a valid single WB PR platform that allows manufacture of RBC for storage for up to 21 days.

Pathogen inactivation by riboflavin and ultraviolet light illumination accelerates the red blood cell storage lesion and promotes eryptosis.

Pathogen reduction treatment using riboflavin and ultraviolet light illumination (Mirasol) effectively reduces the risk of transfusion-transmitted infections. This treatment is currently licensed for only platelets and plasma products, while its application to whole blood (WB) to generate pathogen-inactivated red blood cells (RBCs) is under development. RBC storage lesion, constituting numerous morphologic and biochemical changes, influences RBC quality and limits shelf life. Stored RBCs further show enhanced susceptibility to RBC programmed cell death (eryptosis) characterized by increased cytosolic Ca2+ -provoked membrane phosphatidylserine (PS) externalization. Using a "pool-and-split" approach, we examined multiple variables of RBC storage lesion and eryptosis in RBC units, derived from Mirasol-treated or untreated WB, after 4 to 42 days of storage, under blood bank conditions. In comparison to untreated RBC units, Mirasol treatment significantly altered membrane microvesiculation, supernatant hemoglobin, osmotic fragility, and intracellular adenosine triphosphate levels but did not influence membrane CD47 expression and 2,3-diphosphoglycerate levels. Mirasol-treated RBCs showed significantly higher PS exposure after 42, but not after not more than 21, days of storage, which was accompanied by enhanced cytosolic Ca2+ activity, ceramide abundance, and oxidative stress, but not p38 kinase activation. Mirasol treatment significantly augmented PS exposure, Ca2+ entry, and protein kinase C activation after energy depletion, a pathophysiologic cell stressor. Mirasol-treated RBCs were, however, more resistant to cell shrinkage. Prolonged storage of Mirasol-treated RBCs significantly increases the proportion of eryptotic RBCs, while even short-term storage enhances the susceptibility of RBCs to stress-induced eryptosis, which could reduce posttransfusion RBC recovery in patients.

Prevention of Transfusion-Transmitted Malaria By Treatment of Whole Blood with the Mirasol® PRT System

Background: Across sub-Saharan Africa, blood supplies are threatened by numerous pathogens. In some locations, Plasmodium parasitemia prevalence in donor blood is nearly 50%. Donor testing for malaria in these areas is not effective and the risk of transfusion-transmitted malaria (TTM) is high. The Mirasol® PRT System for Whole Blood (WB) is a medical device intended for extracorporeal pathogen reduction of WB. The current clinical study evaluated the ability of Mirasol-treated WB to reduce the incidence of TTM.Study Design/Methods: This was a prospective, randomized, double-blind, controlled, single-center study in Ghana, which is hyperendemic for malaria. The study had 90% power to demonstrate a 90% reduction in TTM. Hospitalized patients requiring WB transfusions were randomly allocated to receive ≤ 2 transfusions of standard (untreated) or Mirasol-treated WB. The primary endpoint was the incidence of TTM as measured by quantitative polymerase chain reaction and Plasmodium alleleic sequence homology between transfused and patient WB during 28 days of follow-up. Patient safety was assessed by monitoring treatment-emergent adverse events (TEAEs) and transfusion reactions.Clinical outcomes related to hemoglobin increments, hemostatic parameters, and clinical chemistries were monitored for 28 days post-transfusion.Results: Overall, 226 subjects (113 Mirasol, 113 Untreated) were enrolled; 223 subjects were included in the safety analysis. Sixty-five (65) subjects were non-parasitemic at pre-transfusion (28 Mirasol, 37 Untreated) and received at least 1 parasitemic WB transfusion. Of 16 cases of suspected TTM (3 Mirasol, 13 Untreated) with 2 consecutive days of parasitemia, 9 were confirmed by alleleic homology (1 Mirasol, 8 Untreated). Incidence of TTM was significantly reduced in patients receiving treated products. Hemoglobin (mean [standard deviation]) was similar between groups at baseline (6.71 g/dL; p = 1.0), and Day 1 following 1 transfusion (8.53 [2.0] vs 8.49 [1.5] g/dL; p = 0.93) or 2 transfusions (7.09 [1.5] vs 7.38 [1.6] g/dL; p = 0.33). Ninety-two subjects (48 Mirasol, 44 Untreated) reported 145 TEAEs (75 Mirasol, 70 Untreated). Transfusion reactions were observed in 8.1% and 13.4% of subjects receiving Mirasol-treated and untreated WB, respectively.TableIncidence of TTMMirasol n (%); 95% CIUntreated n (%); 95% CIP-Value2 Consecutive days of ParasitemiaN = 653 (10.7); 2.3, 28.2 n = 2813 (35.1); 20.2, 52.2n = 37< 0.052 Consecutive days of Parasitemia and >2 Allele match by PCRN = 651 (3.6); 0.1, 18.3n = 288 (21.6); 9.8, 38.2n = 37< 0.05ITT PopulationN = 2231 (0.9); 0.0, 4.9n = 1118 (7.1); 3.1, 13.6n = 112< 0.05Abbreviation: CI = confidence interval, ITT = intent-to-treat, PCR = polymerase chain reaction.Conclusions: The primary endpoint of the study was met. Mirasol treatment of WB clinically and statistically reduced TTM infections in the study population. This was the first human clinical study demonstrating that a PRT system can reduce transmission of a bloodborne pathogen. No safety issues were related to the device or device-treated WB. Transfusion reactions did not differ between patients receiving Mirasol-treated or untreated WB. Hemoglobin increments and transfusion outcome parameters in transfused patients did not differ between the treatment groups. DisclosuresAllain:Terumo BCT: Consultancy. Owusu-Ofori:Terumo BCT: Other: Clinical Study Sub-Investigator. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment. Owusu-Ofori:Terumo BCT: Other: Clinical Study Investigator.

Whole blood treated with riboflavin and ultraviolet light: quality assessment of all blood components produced by the buffy coat method.

Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported. In a "pool-and-split" study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods. PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors. Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.

Antiaggregatorische und anticoagulante Eigenschaften von Oligoaminen, 13. Mitt.: Verbesserung der Lagerfähigkeit von Vollblut und Erythrocytensuspensionen durch Oligoamine

The impairment of the functions of red blood cells or their destruction during storage can be delayed or even inhibited by oligoamines especially RE 1492 (N,N',N''-Tris-(4-phenylbutyl)benzene-1,3,5-trimethanamine). When citrated whole blood (WB) is stored at 4 degrees C for 7 d half of the red blood cells (RBC) have lost their ability to form rouleaux. Addition of 100 mumols/L RE 1492 maintains 50% reaggregability up to day 28th of storage. When citrated WB is stored at 37 degrees C the reaggregability has declined to 40 percent after 10 h. With 100 mumols/L RE 1492 no reduction of this property is observed up to 48 h. These results are correlated with the maintainance of the discocyte form of RBC and a persistent filtrability of RBC suspensions through a 5 microns microporous membrane. With 100 mumols/L RE 1492 only one fifth of the haemolysis of untreated WB occurs. The efflux of potassium ions from RBC into the blood plasma during a 72 h storage is bisected by RE 1492. The binding of oxygen to RBC remains unchanged.