Current transfusion practice and need for new blood products to ensure blood supply for patients with major hemorrhage in Europe.

BACKGROUND: New blood products are considered for treatment of patients with major hemorrhage. The aim of this report is to describe the current transfusion practices in Europe for patients with major hemorrhage and explore the need for new or modified blood products to ensure prehospital and in-hospital blood supply. STUDY DESIGN AND METHOD: The European Blood Alliance (EBA) Working Group on Innovation and New Blood Products' subgroup on major hemorrhage performed a survey among the EBA member states. RESULTS: The response rate was 58% (17 responses from 15 of the 26 EBA member states). Of these, sixteen (94%) provide massive transfusion packages (MTPs) with balanced ratio of red blood cells and plasma. Seven of the respondents included platelets from the start of treatment. Eleven (65%) provide prehospital blood products, mainly red cell concentrates or dried and/or thawed plasma with 5 days of extended storage. Two countries provide prehospital whole blood. Twelve respondents (71%) saw a need for implementation of new or modified blood components in their institution. The top three priorities were whole blood (12 of 12, 100%), dried plasma (8 of 12, 67%), and cold-stored platelets (7 of 12, 58%). DISCUSSION: Current national guidelines for use of blood products in patients with major hemorrhage in Europe agree on the use of balanced transfusion, however the timing and source of platelets differ. Blood products for prehospital transfusion are available in several European countries. An interest in new or modified blood products for patients with major hemorrhage was observed, especially for whole blood.

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

A comparison of the effect of X and gamma irradiation on red cell storage quality.

BACKGROUND AND OBJECTIVES: Irradiation of red cell components is indicated for recipients at risk of transfusion-associated graft vs. host disease. Current technologies available comprise of a gamma (γ) or an x source of radiation. The benefits of x vs. γ include non-radioactivity and hence no decay of the source. We aimed to compare the effect of the two technologies on red cell component storage quality post-irradiation. MATERIALS AND METHODS: Paired units of red cell concentrates (RCC), neonatal red cell splits (RCS), red cells for intra-uterine transfusion (IUT) or neonatal exchange transfusion (ExTx) were either γ- or x-irradiated. Units were sampled and tested for five storage parameters until the end of shelf life. Equivalence analysis of storage quality parameters was performed for pairs of the same components (RCC, RCS, IUT or ExTx) that were either γ- or x-irradiated. RESULTS: Nearly all component comparisons studied showed equivalence between γ and x irradiation for haemolysis, ATP, 2,3-DPG, potassium release and lactate production. The exceptions found that were deemed non-equivalent were higher haemolysis with x irradiation for ExTx, lower 2,3-DPG with x irradiation for RCS irradiated early and higher ATP with x irradiation for IUT. However, these differences were considered not clinically significant. CONCLUSION: This study has demonstrated that a range of red cell components for use in different age groups are of acceptable quality following x irradiation, with only small differences deemed clinically insignificant in a few of the measured parameters.

A comparison of platelet function in cold-stored whole blood and platelet concentrates.

BACKGROUND: There is renewed interest in the use of whole blood (WB) for the resuscitation of trauma patients. Platelet function in stored WB compared to platelet concentrates is not well established and was assessed in vitro in this study. METHODS: Leucocyte-depleted cold-stored WB (CS-WB) was prepared using a Terumo WB-SP Imuflex kit and held at 2-6°C alongside: (A) UK standard pooled platelets stored at 20-24°C (RT-PLTS), (B) pooled platelets stored at 2-6°C (CS-PLTS), and (C) platelet-rich plasma produced using the Terumo kit (CS-PRP), for 21 days. A series of in vitro assays were assessed platelet function. RESULTS: Platelet count was retained to 57 ± 14% of starting number at day 21 in CS-WB. Over time, CS-WB platelets become more activated, with increased CD62P expression (day 1: 7 ± 3.7% vs. day 21: 59 ± 17.1%) and annexin V binding (day 1: 2 ± 0.2% vs. day 21: 21 ± 15.1%). For comparison, 18.6 ± 6% of platelets in RT-PLTS demonstrated CD62P expression at day 7, whereas annexin V binding in RT-PLTS at day 7 was 2.6 ± 0.5%. Over storage, aggregatory response to agonists decreased in all arms. Functional platelet microparticles increased steadily in CS-WB throughout storage. CONCLUSION: During storage, platelet count reduced in CS-WB, whereas CD62P expression and annexin V binding increased. This was accompanied by a reduced aggregatory response, although compared to 7-day-old RT-PLTS, CS-WB maintained a maximal response to agonists for longer, suggesting that the shelf life for CS-WB can be considered for up to 21 days.

Effect of storage of plasma in the presence of red blood cells and platelets: re-evaluating the shelf life of whole blood.

BACKGROUND: There is renewed interest in administering whole blood (WB) for the resuscitation of patients with bleeding trauma. The shelf life of WB was established decades ago based on the viability of red blood cells. However, plasma quality during WB storage is not established. STUDY DESIGN AND METHODS: White blood cell- and platelet-reduced WB (WB-PLT) was prepared using standard processes and compared to WB processed using a platelet-sparing WBC reduction (WB + PLT) filter. WB (± PLT) was held at 2 to 6°C for 35 days alongside control units of red blood cells (RBCs) in saline, adenine, glucose, and mannitol and liquid plasma. A series of assays explored the coagulation potential and RBC quality. RESULTS: While fibrinogen and α2-antiplasmin remained unaffected by storage, other factors varied between components or over time at 2 to 6°C. At 14 days factor V, factor VII, α2 -antiplasmin and free protein S antigen remained on average greater than 0.50 IU/mL or 50%, as appropriate, in WB ± PLT. Factor VIII was on average 0.49 IU/mL in WB+PLT, and 0.56 IU/mL for WB-PLT. Free protein S activity decreased significantly in all arms but remained on average greater than 40% at Day 14. Contact activation was not demonstrated before Day 14. Thrombin generation in plasma remained relatively stable to Day 35 in all arms. CONCLUSIONS: Clotting factor activity remained at or above a mean of 0.5 IU/mL, or 50%, at Day 14 for factor V, factor VII, factor VIII, free protein S, fibrinogen, and α2-antiplasmin in all arms. Further data on platelet function in WB+PLT is needed to inform its shelf life.

A Comparison of Red Cell Rejuvenation versus Mechanical Washing for the Prevention of Transfusion-associated Organ Injury in Swine.

BACKGROUND: We evaluated the effects of two interventions that modify the red cell storage lesion on kidney and lung injury in experimental models of transfusion. METHODS: White-landrace pigs (n = 32) were allocated to receive sham transfusion (crystalloid), 14-day stored allogeneic red cells, 14-day red cells washed using the red cells washing/salvage system (CATS; Fresenius, Germany), or 14-day red cells rejuvenated using the inosine solution (Rejuvesol solution; Zimmer Biomet, USA) and washed using the CATS device. Functional, biochemical, and histologic markers of organ injury were assessed for up to 24 h posttransfusion. RESULTS: Transfusion of 14 day red cells resulted in lung injury (lung injury score vs. sham, mean difference -0.3 (95% CI, -0.6 to -0.1; P = 0.02), pulmonary endothelial dysfunction, and tissue leukocyte sequestration. Mechanical washing reduced red cell-derived microvesicles but increased cell-free hemoglobin in 14-day red cell units. Transfusion of washed red cells reduced leukocyte sequestration but did not reduce the lung injury score (mean difference -0.2; 95% CI, -0.5 to 0.1; P = 0.19) relative to 14-day cells. Transfusion of washed red cells also increased endothelial activation and kidney injury. Rejuvenation restored adenosine triphosphate to that of fresh red cells and reduced microvesicle concentrations without increasing cell-free hemoglobin release. Transfusion of rejuvenated red cells reduced plasma cell-free hemoglobin, leukocyte sequestration, and endothelial dysfunction in recipients and reduced lung and kidney injury relative to 14-day or washed 14-day cells. CONCLUSIONS: Reversal of the red cell storage lesion by rejuvenation reduces transfusion-associated organ injury in swine.

Investigation of the quality of stored red blood cells after simulated air drop in the maritime environment.

BACKGROUND: Maritime medical capability may be compromised by blood resupply. Air-dropped red blood cells (RBCs) is a possible mitigation factor. This study set out to evaluate RBC storage variables after a simulated parachute air drop into the sea, as limited data exist. STUDY DESIGN AND METHODS: The air load construction for the air drop of blood was subject to static drop assessment to simulate a worst-case parachute drop scenario. One control and two test Golden Hour shipping containers were each packaged with 10 RBC units. The control box was not dropped; Test Boxes 1 and 2 were further reinforced with waterproof boxes and underwent a simulated air drop on Day 7 or Day 8 postdonation, respectively. One day after the drop and once a week thereafter until Day 43 of storage, RBCs from each box were sampled and tested for full blood counts, hemolysis, adenosine triphosphate, 2,3-diphosphoglycerate, pH, extracellular potassium, glucose, lactate, deformability, and RBC microvesicles. RESULTS: The packaging configuration completed the air drop with no water ingress or physical damage. All units met UK specifications for volume, hemoglobin, and hemolysis. There were no significant differences for any of the variables studied between RBCs in the control box compared to RBCs in Test Boxes 1 and 2 combined over storage. CONCLUSION: The test proved that the packaging solution and the impact of a maritime air drop as performed in this study, on Day 7 or Day 8 postdonation, did not affect the in vitro quality of RBCs in SAGM over storage for 35 days.

A paired comparison of thawed and liquid plasma.

BACKGROUND: To make plasma readily available to treat major hemorrhage, some centers are internationally using either thawed plasma (TP) or "never-frozen" liquid plasma (LP). Despite the routine use of both, there are limited data comparing the two. The hemostatic properties of LP were evaluated and compared to TP in a paired study. STUDY DESIGN AND METHODS: Two ABO-matched plasma units were pooled and split to produce 1 unit for LP and 1 unit for TP. Samples of TP and LP, stored at 2 to 6°C, were tested for a range of coagulation factors, thrombin generation, and rotational thromboelastometry. An additional 119 units of LP were collected and analyzed for markers of contact activation (S-2302 cleavage) and cellular content. RESULTS: LP and TP were compared, up to 7 days of storage, with results showing no difference in the rate of change over time for any variable measured. When compared to Day 5, LP on Day 7 showed no difference for any factors measured; however, on Day 11 Factor (F)II, FV, FVII, and protein S (activity) were lower. Analysis of 119 LP units showed that 26 of 119 (22%) exhibited cold-induced contact activation by Day 28. CONCLUSION: LP and TP were comparable in terms of hemostatic variables up to 7 days of storage. Decreasing coagulation factor activity along with an increased activation risk during storage of LP needs to be balanced against availability to supply and clinical need when considering using LP with more than 7 days of storage.

Effects of riboflavin and amotosalen photoactivation systems for pathogen inactivation of fresh-frozen plasma on fibrin clot structure.

BACKGROUND: Fresh-frozen plasma (FFP) transfusion carries a risk of viral transmission from donor to recipient. Riboflavin (Mirasol) and amotosalen (Intercept) are two pathogen inactivation (PI) methods that may enhance the safety of FFP for transfusion. Our study investigated the effects of Mirasol and Intercept treatment on fibrin formation and clot structure. STUDY DESIGN AND METHODS: FFP underwent either Mirasol or Intercept treatment, and aliquots were taken before addition of the compound, before illumination (after addition of compound only), and after treatment (addition of compound plus illumination). All samples underwent turbidimetric analysis, lysis analysis, assessment of clot permeation, and analysis by laser scanning confocal microscopy. RESULTS: After treatment, there was a decrease in optical density of the fibrin network for Mirasol and Intercept, lag time to fibrin formation was prolonged for Mirasol and lysis time for Intercept, clot permeability was significantly decreased, and clot density was increased for both. CONCLUSIONS: Our study shows that plasma treated with Mirasol and Intercept produces denser clots consisting of thinner fibers and warrants further studies to evaluate the clinical significance of these structural changes in fibrin clot formation.

Familial pseudohyperkalemia in blood donors: a novel mutation with implications for transfusion practice.

BACKGROUND: Familial pseudohyperkalemia (FP) is a dominantly inherited condition in which red blood cells (RBCs) have an increased cold-induced permeability to monovalent cations. Potassium leaks into the supernatant of all stored blood with time, but FP RBCs leak potassium more rapidly. We investigated two unrelated blood donors whose RBC donations demonstrated unexpectedly high potassium after 5 and 6 days' storage. We matched the observed pattern of RBC cation leak to a previously recognized family with FP (FP-Cardiff) and investigated the likely cause with targeted DNA analysis. STUDY DESIGN AND METHODS: Cation leakage from the donor RBCs and from standard donor units was measured. DNA analysis of donors and family members with FP-Cardiff was performed. Allele frequencies were obtained from human variation databases. RESULTS: Both implicated donors were found to have increased cold-induced potassium leak identical in pattern to affected members of the family with FP-Cardiff. We found a heterozygous substitution Arg723Gln in the ATP-binding cassette, Subfamily B, Member 6 protein that segregated with FP in the Cardiff family and was also present in both blood donors. Arg723Gln is listed in human variation databases with an allele frequency of approximately 1:1000. CONCLUSIONS: We describe a novel FP mutation that may affect 1:500 European blood donors and causes rapid loss of potassium from stored RBCs. This finding has implications for neonates and infants receiving large-volume RBC transfusions. Genomic screening of donors could be used to identify donors with this mutation and potentially improve the quality and safety of donor units.

Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets.

BACKGROUND: We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study. STUDY DESIGN AND METHODS: For in vitro studies, pooled or single buffy coat-derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two-arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC-treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor. RESULTS: There were no significant differences on Day 7 of storage between paired UVC-treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC-1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC-treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088). CONCLUSION: UVC-treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.

On-chip digital microfluidic architectures for enhanced actuation and sensing.

An on-chip system is presented with integrated architectures for digital microfluidic actuation and sensing. Localized actuation is brought about by a digital microfluidic multiplexer layout that overcomes the challenges of multi-microdrop interference, and complete two-dimensional motion is shown for microdrops on a 14 × 14 grid with minimized complexity by way of 14+14 inputs. At the same time, microdrop sensing is demonstrated in a folded-cavity design for enhanced optical intensity probing of internal fluid refractive indices. The heightened intensities from this on-chip refractometer are shown to have a linear response to the underlying fluid refractive index. An electro-dispensing technique is used to fabricate the folded-cavity optical architecture in a format that is tuned for the desired refractive index range and sensitivity. The overall lab-on-a-chip system is successful in integrating localized microdrop actuation and sensing.

Manufacture of red cells in additive solution from whole blood refrigerated for 5 days or remanufactured from red cells stored in plasma.

BACKGROUND AND OBJECTIVES: To investigate methods for the production of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAG-M), from whole blood or red cells stored in plasma for 5 or 6 days and to provide evidence that exchange transfusion RCC in citrate phosphate dextrose (CPD) plasma or citrate, phosphate, dextrose, adenine (CPDA-1) plasma are of comparable quality. METHODS AND MATERIALS: Ten RCC in SAG-M were produced following the remanufacture of red cells in CPD plasma on day 5/6 or after 5 days hold as leucodepleted CPD whole blood. In addition, 10 RCC in CPD plasma and 9 in CPDA-1 plasma were stored without further processing. Units were assessed for red cell parameters including haemolysis, adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG) and extracellular potassium. RESULTS: Units in SAG-M produced by remanufacture of RCC in plasma or by delayed manufacture of whole blood had comparable levels of haemolysis, ATP and 2,3-DPG. Furthermore, these units underwent biochemical changes similar to reference SAG-M units, with the exception of haemolysis which was greater at the end of shelf life and supernatant potassium which was lower following remanufacture. As expected, the decline in ATP was greater in red cells stored in CPD plasma compared with CPDA-1 plasma. In general, units in CPD plasma were of similar quality at day 28 compared to those in CPDA-1 plasma at day 35. CONCLUSIONS: RCC produced following the remanufacture of RCC in plasma or the delayed manufacture of whole blood are of acceptable in vitro quality and should be assigned the same shelf life as standard RCC in SAG-M.

Prion reduction of red blood cells: impact on component quality.

BACKGROUND: A filter has been developed (P-Capt, MacoPharma) to remove infectious prions from red blood cells (RBCs). We sought to assess 1) its operational use, 2) the quality of filtered components, and 3) whether filtration resulted in any significant changes to blood group antigens. STUDY DESIGN AND METHODS: A total of 272 leukoreduced RBC units, including units processed using "top-and-top" (TAT) and "bottom-and-top" (BAT) methods, were prion reduced using the P-Capt filter. All RBCs were assessed using standard in vitro tests of RBC quality. Changes to blood group antigen expression were also investigated, including the exposure of cryptantigens and the ability of filtered RBCs to be crossmatched. RESULTS: Ninety-nine percent of TAT units and 58% of BAT units had a hemoglobin (Hb) content of more than 40 g. Hemolysis increased immediately after filtration, but units remained within UK specification throughout storage. Prion reduction resulted in the loss of 7 to 8 g of Hb and reductions in hematocrit of 6% to 9% due to the filter containing 40 mL of saline, adenine, glucose, and mannitol. Other RBC quality data, including extracellular potassium, 2,3-diphosphoglycerate, and adenosine triphosphate were similar to historical control data. There was no evidence of any immunologic changes of clinical relevance to the RBC membrane after filtration. CONCLUSIONS: Prion filtration does not appear to have a detrimental effect on basic in vitro measures of RBC quality or on blood group antigens as assessed by in vitro methods. However, prion filtration using the P-Capt filter results in loss of Hb.

PheP, a putative amino acid permease of Staphylococcus aureus, contributes to survival in vivo and during starvation.

PheP, a putative amino acid permease in Staphylococcus aureus, contributes to starvation survival under glucose-limiting conditions and virulence. A pheP mutation led to poor growth after microaerobic or anaerobic incubation on pig serum agar, which was recovered by phenylalanine addition. Genetic complementation of pheP restored growth and starvation survival.

IsdA of Staphylococcus aureus is a broad spectrum, iron-regulated adhesin.

As an important facet of host-pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad-spectrum ligand-binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron-regulated proteins of S. aureus and in other Gram-positive organisms. The isdA gene is only expressed in iron-limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron-limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.

Metamaterial endoscope for magnetic field transfer: near field imaging with magnetic wires.

We report on the development and use of a highly anisotropic magnetic metamaterial for near-field imaging. The material consists of an array of Swiss Roll structures, resonant near 21.3 MHz, with a peak value of relative permeability ~35. At this peak, the material transfers an input magnetic field pattern to the output face without loss of intensity and with a spatial resolution equal to the roll diameter. It behaves as a near-field imaging device consisting of a bundle of magnetic wires.