Effect of leukoreduction on the metabolism of equine packed red blood cells during refrigerated storage.

BACKGROUND: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited. OBJECTIVE: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC). ANIMALS: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks. METHODS: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses. RESULTS: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC. CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC.

Delayed Leukoreduction of whole blood with a platelet-sparing filter to increase low-titer group O whole blood production in the United States.

BACKGROUND: Demand for low-titer Group O whole blood (LTOWB) is increasing for trauma. The whole blood (WB) platelet-sparing (WB-SP) filter enables leukoreduction (LR) while retaining platelet quantity and function; however, in the United States WB must be filtered and placed in the cold within 8 h of collection. A longer processing window would facilitate improved logistics and supply of LR-WB to meet the growing medical need. This study evaluated the impact of increasing filtration timing from <8 h to <12 h on the quality of LR-WB. STUDY DESIGN AND METHODS: Thirty WB units were collected from healthy donors. Control units were filtered within 8 h and test units within 12 h of collection. WB was tested throughout 21 days of storage. Hemolysis, WBC content, component recovery, and 25 additional markers of WB quality were tested including hematologic and metabolic markers, RBC morphology, aggregometry, thromboelastography, and p-selectin. RESULTS: There were 0 failures for residual WBC content, hemolysis, or pH, and no differences in component recovery between arms. Few differences in metabolic parameters were observed, but the small effect size suggests these are not clinically significant. Trends throughout storage were similar and filtration timing did not impact hematological parameters, platelet activation and aggregation, or hemostatic capacity. CONCLUSION: Our studies showed that extending filtration timing from 8 to 12 h from the collection does not significantly impact the quality of LR-WB. Characterization of the platelets demonstrated that storage lesions were not exacerbated. Extending the time from collection to filtration will improve LTOWB inventory in the United States.

Sickle cell trait results in a high leukoreduction quality control failure rate for whole blood donations.

BACKGROUND: Prior studies have shown that sickle cell trait (SCT) is the most common reason attributed to leukoreduction (LR) filter failure due to physical blockage. However, current Food and Drug Administration (FDA) guidelines do not require blood collectors to take a specific action to mitigate inadequate LR that may occur among donors with SCT. We sought to determine the scope of inadequate LR among whole blood (WB) donations collected from individuals with SCT and processed under standard manufacturing conditions. STUDY DESIGN AND METHODS: Between 8/2021 and 2/2022, a total of 40 red blood cells units (RBCs) manufactured from WB donations collected from donors historically positive for SCT had residual leukocyte testing performed. All 40 of the units had appeared to successfully complete leukofiltration. RESULTS: Out of the 40 units tested, 22 failed routine residual leukocyte quality control testing (55% failure rate, 95% confidence interval 40%-70%). Nine out of the 22 failures resulted in more than 100 residual leukocytes per microliter of product. CONCLUSION: Even when leukofiltration appears to have been completed successfully, WB units collected from donors with SCT have a high (55% in aggregate) rate of inadequate leukoreduction. Correlating this result with previous studies showing that of up to 50% of WB units collected from donors with SCT fail to pass through the leukoreduction filter, we estimate that only 25% of WB donations collected from individuals with SCT will result in a leukoreduced RBC unit that meets all FDA requirements. Blood centers should encourage individuals with SCT to donate platelets or plasma, rather than WB.

Minimal impact of anticoagulant on in vitro whole blood quality throughout a 35-day cold-storage regardless of leukoreduction timing.

BACKGROUND: There is increasing interest in leukoreduced whole blood (WB) as a transfusion product for trauma patients. In some jurisdictions, few leukoreduced filters are approved or appropriate for WB leukoreduction and quality information is therefore limited. This study assessed the impact of filtration timing of WB collected in CPDA-1 versus CPD on in vitro quality. STUDY DESIGN AND METHODS: WB was collected in CPDA-1 or CPD and leukoreduction filtered either after 3-8 h (early) or 18-24 h (late) from stop bleed time. In vitro quality was assessed after filtration and throughout 5 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation. RESULTS: Although there were some data points which showed statistically significant differences associated with anticoagulant choices or the filtration timing, no general trend in inferiority/performance could be discerned. After 35 days' storage, only clotting time, alpha angle and factor II in the early filtration arm comparing anticoagulants and prothrombin time and factor II in the CPDA-1 study arm comparing filtration timing showed a significant difference. CONCLUSION: In vitro WB quality seems to be independent on the choice of anticoagulant and filtration timing supporting WB hold-times to up to 24 h, increasing operational flexibility for transfusion services.

Leukocyte reduction filters as an alternative source of peripheral blood leukocytes for research

ABSTRACT Introduction: Peripheral blood leukocytes are a suitable cell model for science research. However, blood samples from healthy volunteers are limited in volume and difficult to obtain due to the complexity of volunteer recruitment. Objective: Therefore, it is urgent to find an alternative source of peripheral blood leukocytes. Method: One of the possibilities is the use of leukocyte reduction filters (LRFs) in blood banks that is used for preparation of leukoreduced blood products. More than 90% of the leukocytes are trapped in the leukofilters allowing the desired blood product to pass through. Results: It has been reported that the biological function of leukocytes collected from the filters are no different from those isolated from buffy coats, leukapheresis products and whole blood (WB) cells. Moreover, LRFs are waste products that are discarded after leukoreduction. Conclusion: Thus, leukofilters represent an economic source of human cell populations that can be used for a variety of investigative purposes, with no cost. In the present study, we reviewed the different usage of LRFs in the research, clinical and commercial applications.

Guideline development for prevention of transfusion-associated graft-versus-host disease: reduction of indications for irradiated blood components after prestorage leukodepletion of blood components.

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, commonly fatal complication of transfusion preventable by irradiation of blood units. The revision of the Dutch transfusion guideline addressed the question whether irradiation is still necessary if blood components are prestorage leukodepleted. We searched for published cases of TA-GVHD following transfusion of prestorage leukodepleted blood and through contacting haemovigilance systems. Six presumed cases were found, dating from 1998 to 2013. Four out of six patients had received one or more non-irradiated units despite recognised indications for irradiated blood components. In the countries providing information, over 50 million prestorage leukodepleted, non-irradiated, non-pathogen-reduced cellular components were transfused in a 10-year period. Potential benefits of lifting indications for irradiation were considered. These include reduced irradiation costs (€ 1.5 million annually in the Netherlands) and less donor exposure for neonates. Findings were presented in an invitational expert meeting. Recommendations linked to human leukocyte antigen similarity between donor and recipient or intra-uterine transfusion were left unchanged. Indications linked to long-lasting deep T-cell suppression were defined with durations of 6 or 12 months after end of treatment (e.g. autologous or allogeneic stem cell transplantation). Need for continued alertness to TA-GVHD and haemovigilance reporting of erroneous non-irradiated transfusions was emphasised.

Supplemental findings of the 2019 National Blood Collection and Utilization Survey.

INTRODUCTION: Supplemental data from the 2019 National Blood Collection and Utilization Survey (NBCUS) are presented and include findings on donor characteristics, autologous and directed donations and transfusions, platelets (PLTs), plasma and granulocyte transfusions, pediatric transfusions, transfusion-associated adverse events, cost of blood units, hospital policies and practices, and implementation of blood safety measures, including pathogen reduction technology (PRT). METHODS: National estimates were produced using weighting and imputation methods for a number of donors, donations, donor deferrals, autologous and directed donations and transfusions, PLT and plasma collections and transfusions, a number of crossmatch procedures, a number of units irradiated and leukoreduced, pediatric transfusions, and transfusion-associated adverse events. RESULTS: Between 2017 and 2019, there was a slight decrease in successful donations by 1.1%. Donations by persons aged 16-18 decreased by 10.1% while donations among donors >65 years increased by 10.5%. From 2017 to 2019, the median price paid for blood components by hospitals for leukoreduced red blood cell units, leukoreduced apheresis PLT units, and for fresh frozen plasma units continued to decrease. The rate of life-threatening transfusion-related adverse reactions continued to decrease. Most whole blood/red blood cell units (97%) and PLT units (97%) were leukoreduced. CONCLUSION: Blood donations decreased between 2017 and 2019. Donations from younger donors continued to decline while donations among older donors have steadily increased. Prices paid for blood products by hospitals decreased. Implementation of PRT among blood centers and hospitals is slowly expanding.

Effect of leukoreduction and temperature on risk of bacterial growth in CPDA-1 whole blood: A study of Escherichia coli.

BACKGROUND: Collection of non-leukoreduced citrate-phosphate-dextrose-adenine (CPDA-1) whole blood is performed in walking blood banks. Blood collected under field conditions may have increased risk of bacterial contamination. This study was conducted to examine the effects of WBC reduction and storage temperature on growth of Escherichia coli (ATCC® 25922™) in CPDA-1 whole blood. METHODS: CPDA-1 whole blood of 450 ml from 10 group O donors was inoculated with E. coli. Two hours after inoculation, the test bags were leukoreduced with a platelet-sparing filter. The control bags remained unfiltered. Each whole blood bag was then split into three smaller bags for further storage at 2-6°C, 20-24°C, or 33-37°C. Bacterial growth was quantified immediately, 2 and 3 h after inoculation, on days 1, 3, 7, and 14 for all storage temperatures, and on days 21 and 35 for storage at 2-6°C. RESULTS: Whole blood was inoculated with a median of 19.5 (range 12.0-32.0) colony-forming units per ml (CFU/ml) E. coli. After leukoreduction, a median of 3.3 CFU/ml (range 0.0-33.3) E. coli remained. In the control arm, the WBCs phagocytized E. coli within 24 h at 20-24°C and 33-37°C in 9 of 10 bags. During storage at 2-6°C, a slow self-sterilization occurred over time with and without leukoreduction. CONCLUSIONS: Storage at 20-24°C and 33-37°C for up to 24 h before leukoreduction reduces the risk of E. coli-contamination in CPDA-1 whole blood. Subsequent storage at 2-6°C will further reduce the growth of E. coli.

Cold-stored leukoreduced whole blood: Extending the time between donation and filtration has minimal impact on in vitro quality.

BACKGROUND: Leukoreduced whole blood (LR-WB) has received renewed attention as alternative to component-based transfusion in trauma. According to the manufacturer's instructions, leukoreduction should be carried out within 8 h after collection. This study assessed impact of (1) WB collection bag, (2) LR filtration, and (3) timing of filtration on in vitro quality. STUDY DESIGN AND METHODS: WB collected into different vendor bags was held at room temperature for <8 h or >16 h but <24 h prior to LR. In vitro quality was assessed before and after filtration, and throughout 3 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation, and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation analyzer or quantitative immunoblotting. RESULTS: Bag type had no impact on WB in vitro quality. LR by filtration had some impact, but is aligned with data in the literature. The time between donation and filtration resulted in some statistically significant differences in metabolic activity, platelet yield, platelet activation, and factor protein activity initially; however, these differences in in vitro quality attributes decreased throughout 21-day cold storage. CONCLUSION: WB hold time showed only a minor impact on WB in vitro quality, so it may be possible for blood processing facilities to explore extended hold times prior to filtration in order to provide greater operational flexibility.

Platelet aggregation in whole blood is not impaired by a platelet-sparing leukoreduction filter and instead depends upon the presence of leukocytes.

BACKGROUND: Recent studies characterizing in vitro hemostatic properties of whole blood (WB) leukoreduced (LR) with a platelet-sparing filter have described subtle, if any, changes to viscoelastic clotting; however, reductions in platelet (PLT) content and impedance aggregometry (IA) responses have been noted. The effects of filtration of WB (i.e., filter-contact effects, reduction in platelet and leukocyte count) have not been rigorously investigated as to their individual impacts on platelet IA responses. STUDY DESIGN AND METHODS: WB units from healthy donors were collected and characterized to assess the effects of platelet-sparing leukoreduction (LR) upon the in vitro hemostatic measures of platelet IA and thromboelastometry. Further characterization of platelet IA responses was carried out in WB samples to delineate the effects of platelet count and leukocyte presence/absence upon the response. RESULTS: WB filtration reduced the platelet count and IA responses but had no impact on viscoelastic clotting measures in fresh WB. Experiments revealed that IA responses have a linear correlation with platelet count in both apheresis platelets and WB and that passage of platelets through the WB-LR filter has no impact upon the strength of this response. Further experiments in LR WB showed that addition of autologous leukocytes back to the platelets fully restored the platelet aggregation response to pre-filtration levels. CONCLUSION: WB filtration results in platelet count reduction and leukocyte removal; however, platelet IA is not degraded by passage through the filter. Apparent declines in platelet IA responses can be fully attributed to the reduction in platelet count and the removal of leukocytes.

Enrichment of leukocytes in peripheral blood using 3D printed tubes.

Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.

Laboratory and clinical comparison of the efficacy of prestorage leukoreduction of red cells at cold versus room temperature.

BACKGROUND: The temperature at which filtration takes place has been reported to influence the efficacy of leukoreduction. We aimed to compare the residual leukocyte count (RLC) in red cell units (RCUs) filtered at cold (CT) versus room temperature (RT) and to assess whether this correlates clinically with a difference in the incidence of acute transfusion reactions (ATRs). METHODS AND MATERIALS: In the first part of the study, whole blood units collected were randomly allocated for subsequent filtration at CT and RT, respectively. RLC postfiltration was assessed using flow cytometry. The second part of the study was a nonrandomized clinical trial in which incidence of ATR was compared between RCUs filtered at RT and CT for 6 months each. RESULTS: Thirty-five RCUs each underwent leukofiltration at CT and RT, respectively. The median RLCs in the filtered units at CT and RT were 0.02 × 106 and 0.1 × 106 leukocytes/unit, respectively (p = .0001), with no difference in red blood cell (RBC) recovery (p = .41). During the second part, 3455 RCUs filtered at RT and 3539 RCUs filtered at CT were transfused to patients. The rate of febrile non-hemolytic transfusion reaction (FNHTR) among transfused patients was less with units filtered at CT (1 per 2000 transfusions) in comparison to RT (1 per 588 transfusions). The difference was, however, not significant (p = .14). CONCLUSION: If change in temperature alone can cause significant reduction in leukocytes, then it is a simple way to curtail the rate of this common yet unpleasant reaction and reduce the reaction rate at minimal cost.

Impact of the preparation method of red cell concentrates on transfusion indices in thalassemia patients: A randomized crossover clinical trial.

BACKGROUND: The average hemoglobin content of red cell concentrates (RCC) varies depending on the method of preparation. Surprisingly less data are available concerning the clinical impact of those differences. STUDY DESIGN AND METHODS: The effects of two types of RCC (RCC-A, RCC-B) on transfusion regime were compared in a non-blinded, prospective, randomized, two-period, and crossover clinical trial. RCC-A was obtained by whole blood leukoreduction and subsequent plasma removal, RCC-B removing plasma and buffy coat first, followed by leukoreduction. Eligible patients were adult, with transfusion-dependent thalassemia (TDT). RESULTS: RCC-A contained 63.9 (60.3-67.8) grams of hemoglobin per unit (median with 1st and 3rd quartile), RCC-B 54.5 (51.0-58.2) g/unit. Fifty-one patients completed the study. With RCC-B, the average pre-transfusion hemoglobin concentration was 9.3 ± 0.5 g/dl (mean ± SD), the average transfusion interval 14.2 (13.7-16.3) days, the number of RCC units transfused per year 39.3 (35.4-47.3), and the transfusion power index (a composite index) 258 ± 49. With RCC-A, the average pre-transfusion hemoglobin concentration was 9.6 ± 0.5 g/dl (+2.7%, effect size 0.792), the average transfusion interval 14.8 (14.0-18.5) days (+4.1%, effect size 0.800), the number of RCC units transfused per year 34.8 (32.1-42.5) (-11.4%, effect size -1.609), and the transfusion power index 272 ± 61 (+14.1%, effect size 0.997). All differences were statistically highly significant (p < .00001). The frequency of transfusion reactions was 0.59% with RCC-A and 0.56% with RCC-B (p = 1.000). CONCLUSION: To reduce the number of RCC units consumed per year and the number of transfusion episodes, TDT patients should receive RCC with the highest average hemoglobin content.

Is there a renoprotective value to leukodepletion during heart valve surgery? A randomized controlled trial (ROLO).

BACKGROUND: Acute Kidney Injury (AKI) adversely affects outcomes after cardiac surgery. A major mediator of AKI is the activation of leukocytes through exposure to the cardiopulmonary bypass circuit. We evaluate the use of leukodepletion filters throughout bypass to protect against post-operative AKI by removing activated leukocytes during cardiac surgery. METHODS: This is a single-centre, double-blind, randomized controlled trial comparing the use of leukodepletion versus a standard arterial filter throughout bypass. Elective adult patients undergoing heart valve surgery with or without concomitant procedures were investigated. The primary clinical outcome measured was the development of AKI according to the KDIGO criteria. Secondary measures included biomarkers of renal tubular damage (urinary Retinol Binding Protein and Kidney Injury Molecule-1), glomerular kidney injury (urinary Micro Albumin and serum Cystatin C) and urinary Neutrophil Gelatinase Associated Lipocalin, as well as the length of hospital stay and quality of life measures through EQ-5D-5L questionnaires. RESULTS: The ROLO trial randomized 64 participants with a rate of recruitment higher than anticipated (57% achieved, 40% anticipated). The incidence of AKI was greater in the leukodepletion filter group (44% versus 23%, risk difference 21, 95% CI - 2 to 44%). This clinical finding was supported by biomarker levels especially by a tendency toward glomerular insult at 48 h, demonstrated by a raised serum Cystatin C (mean difference 0.11, 95% CI 0.00 to 0.23, p = 0.068) in the leukodepleted group. There was however no clear association between the incidence or severity of AKI and length of hospital stay. On average, health related quality of life returned to pre-operative levels in both groups within 3 months of surgery. CONCLUSIONS: Leukocyte depletion during cardiopulmonary bypass does not significantly reduce the incidence of AKI after valvular heart surgery. Other methods to ameliorate renal dysfunction after cardiac surgery need to be investigated. TRIAL REGISTRATION: The trial was registered by the International Standard Randomized Controlled Trial Number Registry ISRCTN42121335 . Registered on the 18 February 2014. The trial was run by the Bristol Clinical Trials and Evaluation Unit. This trial was financially supported by the National Institute of Health Research (Research for Patient Benefit), award ID: PB-PG-0711-25,090.

IL-33-mediated Eosinophilia Protects against Acute Lung Injury.

Pneumonia-induced lung injury and acute respiratory distress syndrome can develop because of an inappropriate inflammatory response to acute infections, leading to a compromised alveolar barrier. Recent work suggests that hospitalized patients with allergies/asthma are less likely to die of pulmonary infections and that there is a correlation between survival from acute respiratory distress syndrome and higher eosinophil counts; thus, we hypothesized that eosinophils associated with a type 2 immune response may protect against pneumonia-induced acute lung injury. To test this hypothesis, mice were treated with the type 2-initiating cytokine IL-33 intratracheally 3 days before induction of pneumonia with airway administration of a lethal dose of Staphylococcus aureus. Interestingly, IL-33 pretreatment promoted survival by inhibiting acute lung injury: amount of BAL fluid proinflammatory cytokines and pulmonary edema were both reduced, with an associated increase in oxygen saturation. Pulmonary neutrophilia was also reduced, whereas eosinophilia was strongly increased. This eosinophilia was key to protection; eosinophil reduction eliminated both IL-33-mediated protection against mortality and inhibition of neutrophilia and pulmonary edema. Together, these data reveal a novel role for eosinophils in protection against lung injury and suggest that modulation of pulmonary type 2 immunity may represent a novel therapeutic strategy.

Hypothermic storage of leukoreduced red blood cells for greater than 21 days is a safe alternative to irradiation.

BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

Mechanical Reperfusion Following Prolonged Global Cerebral Ischemia Attenuates Brain Injury.

Previous experiments demonstrated improved outcome following prolonged cerebral ischemia given controlled brain reperfusion using extracorporeal circulation. The current study further investigates this. Young adult pigs were exposed to 30 min of global normothermic cerebral ischemia, achieved through intrathoracic clamping of cerebral arteries, followed by 20 min of isolated mechanical brain reperfusion. Leukocyte-filtered blood was delivered by a roller-pump at fixed pressure and flow. One experimental group additionally had a custom-made buffer solution delivered at 1:8 ratio with the blood. Hemodynamics including intracranial pressure were monitored. Blood gases were from peripheral arteries and the sagittal sinus, and intraparenchymal brain microdialysis was performed. The brains were examined by a neuropathologist. The group with the added buffer showed lower intracranial pressure as well as decreased intraparenchymal glycerol and less signs of excitotoxicity and ischemia, although histology revealed similar degrees of injury. A customized mechanical reperfusion improves multiple parameters after prolonged normothermic global cerebral ischemia. Graphical Abstract The current study investigates if it possible to improve neurological outcomes following prolonged global brain ischemia. The results indicate that a customized mechanical reperfusion protocol can attenuate neurological injury.