Leukocyte filtration and leukocyte modulation therapy during extracorporeal cardiopulmonary resuscitation in a porcine model of prolonged cardiac arrest.

Extracorporeal cardiopulmonary resuscitation (ECPR) is emerging as a feasible and effective rescue strategy for prolonged cardiac arrest (CA). However, prolonged total body ischemia and reperfusion can cause microvascular occlusion that prevents organ reperfusion and recovery of function. One hypothesized mechanism of microvascular "no-reflow" is leukocyte adhesion and formation of neutrophil extracellular traps. In this study we tested the hypothesis that a leukocyte filter (LF) or leukocyte modulation device (L-MOD) could reduce NETosis and improve recovery of heart and brain function in a swine model of prolonged cardiac arrest treated with ECPR. Thirty-six swine (45.5 ± 2.5 kg, evenly distributed sex) underwent 8 min of untreated ventricular fibrillation CA followed by 30 min of mechanical CPR with subsequent 8 h of ECPR. Two females were later excluded from analysis due to CPR complications. Swine were randomized to standard care (Control group), LF, or L-MOD at the onset of CPR. NET formation was quantified by serum dsDNA and citrullinated histone as well as immunofluorescence staining of the heart and brain for citrullinated histone in the microvasculature. Primary outcomes included recovery of cardiac function based on cardiac resuscitability score (CRS) and recovery of neurologic function based on the somatosensory evoked potential (SSEP) N20 cortical response. In this model of prolonged CA treated with ECPR we observed significant increases in serum biomarkers of NETosis and immunohistochemical evidence of microvascular NET formation in the heart and brain that were not reduced by LF or L-MOD therapy. Correspondingly, there were no significant differences in CRS and SSEP recovery between Control, LF, and L-MOD groups 8 h after ECPR onset (CRS = 3.1 ± 2.7, 3.7 ± 2.6, and 2.6 ± 2.6 respectively; p = 0.606; and SSEP = 27.9 ± 13.0%, 36.7 ± 10.5%, and 31.2 ± 9.8% respectively, p = 0.194). In this model of prolonged CA treated with ECPR, the use of LF or L-MOD therapy during ECPR did not reduce microvascular NETosis or improve recovery of myocardial or brain function. The causal relationship between microvascular NETosis, no-reflow, and recovery of organ function after prolonged cardiac arrest treated with ECPR requires further investigation.

A randomized control trial to compare mortality in recipients of leucoreduced and non-leucoreduced whole blood transfusion in patients with cancer in Uganda.

BACKGROUND: Mortality benefit of transfusion with leucoreduced whole blood has not been demonstrated in the sub-Saharan Africa (SSA). We compared mortality in patients with cancer transfused with leucoreduced and non-leucoreduced whole blood in a SSA setting. METHODS: An open-label randomized controlled trial was conducted at the Uganda Cancer Institute where participants were randomized in a 1:1 ratio into the leucoreduced and non-leucoreduced whole blood transfusion arms. Leucocyte filtration of whole blood was performed within 72 h of blood collection. Patients aged ≥ 15 years who were prescribed blood transfusion by the primary physicians were eligible for study enrolment. Mortality difference was analyzed using intention-to-treat survival analysis and cox proportional hazard model was used to analyze factors associated with mortality. RESULTS: There were 137 participants randomized to the leucoreduced and 140 to the non-leucoreduced arms. Baseline characteristics were similar between the two arms. The median number of blood transfusions received was 1 (IQR, 1-3) unit and 2 (IQR, 1-3) units in the leucoreduced and non-leucoreduced arms respectively, p = 0.07. The 30-day mortality rate in the leucoreduced arm was 4.6% (95% CI, 2.1-10) and was 6.2% (95% CI, 3.2-12.1) in the non-leucoreduced arm (p = 0.57), representing an absolute effect size of only 1.6%. Increasing age (HR = 0.92, 95% CI, 0.86-0.98, p = 0.02) and Eastern Co-operative Oncology Group (ECOG) performance score of 1 (HR = 0.03, 95% CI, 0.00-0.31, p < 0.01) were associated with reduced 30-day mortality. CONCLUSIONS: The study failed to demonstrate mortality difference between cancer patients transfused with leucoreduced and non-leucoreduced whole blood. Although this study does not support nor refute universal leucoreduction to reduce mortality in patients with cancer in SSA, it demonstrates the feasibility of doing transfusion RCTs in Uganda, where a multi-center trial with an appropriate sample size is needed. TRIAL REGISTRATION: Pan African Clinical Trial Registry, https://pactr.samrc.ac.za/ (PACTR202302787440132). Registered on 06/02/2023.

Trend towards reduction of transfusion reactions using prestorage leukocyte-reduced and pooled whole blood-derived platelets and cost savings compared with poststorage whole blood-derived random platelets as evidenced by real-time hemovigilance.

BACKGROUND: Due to chemotherapy-induced neutropenia or hematologic malignancies, immunocompromised cancer patients may have higher incidence of febrile nonhemolytic transfusion reactions compared with the general population and frequently require platelet transfusions. This quality improvement project compared the safety of transfusion using prestorage leukocyte-reduced and pooled whole blood-derived platelets (Acrodose/WBD) with conventionally produced poststorage WBD platelets (RDP) using an active hemovigilance system. METHODS: Every patient receiving a blood product at the hospital was virtually monitored in real time by trained nurses from a remote hemovigilance unit. These nurses monitor a digital dashboard, which populates a watch list of patients from the time blood product administration is initiated until 12 hours posttransfusion. Over the course of 6 months, 371 patients receiving 792 RDP transfusions and 423 patients receiving 780 Acrodose/WBD platelets transfusions were monitored for transfusion reactions. RESULTS: We identified 26 transfusion reactions in RDP but only 12 transfusion reactions in the Acrodose/WBD platelet group. CONCLUSION: Acrodose platelet transfusion was associated with fewer transfusion reactions, which resulted in significant cost savings.

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.

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.

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.

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.

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.

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.

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.

Missed irradiation of cellular blood components for vulnerable patients: Insights from 10 years of SHOT data.

BACKGROUND: Irradiation of cellular blood components is recommended for patients at risk of transfusion-associated graft-vs-host disease (TA-GvHD). Prestorage leucodepletion (LD) of blood components is standard in the UK since 1999. STUDY DESIGN AND METHODS: Analysis of 10 years' reports from UK national hemovigilance scheme, Serious Hazards of Transfusion (2010-2019), where patients failed to receive irradiated components when indicated according to British Society for Haematology guidelines (2011). RESULTS: There were 956 incidents of failure to receive irradiated components all due to errors. One hundred and seventy two incidents were excluded from analysis, 125 of 172 (72.7%) because of missing essential information. No cases of TA-GvHD were reported in this cohort. The 784 patients received 2809 components (number unknown for 67 incidents). Most failures occurred in patients treated with purine analogues (365) or alemtuzumab (69), or with a history of Hodgkin lymphoma (HL) (192). Together these make up 626 of 784 (79.9%). Poor communication is an important cause of errors. CONCLUSION: Leucodepletion appears to reduce the risk for TA-GvHD. None of 12 cases of TA-GvHD reported to SHOT prior to introduction of LD occurred in patients with conditions recommended for irradiated components by current guidelines. Irradiation indefinitely for all stages of HL is not based on good evidence and is a difficult guideline to follow. Further research on long-term immune function in HL is required. Variation between different national guidelines reflects the very limited evidence.

Higher donor body mass index is associated with increased hemolysis of red blood cells at 42-days of storage: A retrospective analysis of routine quality control data.

BACKGROUND: For reasons unclear, some stored red blood cells (RBCs) have low hemolysis, while others have high hemolysis, which impacts quality consistency. To identify variables that influence hemolysis, routine quality control (QC) data for 42-days-stored RBCs with corresponding donor information were analyzed. STUDY DESIGN AND METHODS: RBC QC and donor data were obtained from a national blood supplier. Regression models and analyses were performed on total cohort stratified by donor sex and by high hemolysis (≥90th percentile) vs control (<90th percentile) samples, including matching. RESULTS: Data included 1734 leukoreduced RBCs (822 female, 912 male), processed by buffy coat-poor or whole blood filtration methods. Male RBCs had larger volume, hemoglobin content, and higher hemolysis than female RBCs (median hemolysis, 0.24% vs 0.21%; all P < .0001). Multivariable regression identified increased body mass index (BMI) and RBC variables were associated with higher hemolysis (P < .0001), along with older female age and buffy coat-poor processing method (P < .002). Logistic regression models comparing the high and control hemolysis subsets, matched for RBC component variables and processing method, identified overweight-obese BMI (>27 kg/m2 ) in males remained the single donor-related variable associated with higher hemolysis (P < .0001); odds ratio, 3 (95% confidence interval [CI], 1.3-6.7), increasing to 4 (95% CI, 1.8-8.6) for obese males (BMI > 30 kg/m2 ). Female donor obesity and older age trended toward higher hemolysis. CONCLUSION: Donor BMI, sex, and female age influence the level of hemolysis of 42-days-stored RBCs. Other factors, not identified in this study, also influence the level of hemolysis.

Improving blood transfusion safety in resource-poor countries: a case study of using leucocyte reduced blood in Uganda.

BACKGROUND: The majority of blood transfusion safety strategies recommended by the WHO for resource-poor countries focus mainly on reducing the risk of transfusion-transmitted infections (TTIs). Other technologies such as leucocyte reduction may represent complementary strategies for improving transfusion safety. OBJECTIVE: To evaluate the role of using leucocyte reduced blood in a resource-poor country. METHODS: Pre-storage leucocyte reduced (LR) red blood cells (RBCs) were specially prepared for the Tissue Oxygenation by Transfusion in severe Anaemia and Lactic acidosis (TOTAL) study, at the Uganda Blood Transfusion Services from February 2013 through May 2015. Quality control tests were performed to evaluate the procedure, and the incremental cost of an LR-RBC unit was estimated. RESULTS: A total of 608 RBCs units were leucocyte reduced. Quality control tests were performed on 55 random RBCs units. The median (IQR) residual leucocyte count was 4 (0·5-10) WBC/uL, equivalent to 1·8x106 WBC per unit. The estimated incremental unit cost of leucocyte reduction was $37 USD per LR RBC unit. CONCLUSION: Leucocyte reduction of blood in a resource-poor country is doable although relatively costly. As such, its value in resource-poor countries should be weighed against other transfusion safety propositions.

Cold-stored leukoreduced CPDA-1 whole blood: in vitro quality and hemostatic properties.

BACKGROUND: Some jurisdictions require leukoreduction of cellular blood components. The only whole blood collection set with a platelet-saving filter uses citrate-phosphate-dextrose (CPD) as storage solution. Substituting CPD with citrate-phosphate-dextrose-adenine (CPDA-1) increases shelf life from 21 to 35 days. This would simplify prehospital and rural resupply and reduce wastage. We investigated in vitro quality and hemostatic properties of CPDA-1 whole blood leukoreduced with a platelet-saving filter. STUDY DESIGN AND METHODS: CPDA-1 whole blood was leukoreduced using a platelet-saving filter and stored 35 days. EDQM requirements, hematology, metabolic parameters, thromboelastography, light transmission aggregometry, fibrinogen, factor VIII, and interleukin-6 were measured on Days 0, 1, 14, 21, and 35 and compared to non-leukoreduced blood. RESULTS: All units met EDQM requirements. Leukoreduction yielded residual white blood cell count <1 × 106 and 87% platelet recovery on Day 1. It caused reduction in thromboelastography parameters, but not aggregometry response. No hemolysis >0.8% was observed. Factor VIII was higher on Day 35 in the leukoreduced group, 37.9 (95% CI: 26.0, 49.8) versus 13.8 (9.4, 18.2) IU/dL. In both groups, aggregation was significantly reduced by Day 14. Thromboelastography showed remaining platelet activity on Day 35, MA 46.9 (42.1, 51.7) in the leukoreduced and 44.3 (39.6, 49.0) mm in the non-leukoreduced group. Fibrinogen was within reference ranges at Day 35 (>2 g/dL). Interleukin-6 was not detectable. CONCLUSION: Leukoreducing CPDA-1 whole blood with a platelet-saving filter did not compromise hemostatic properties. We encourage development of a single bag CPDA-1 whole blood collection set with in-line platelet-saving filter.

Analysis of Plasma Products for Cellular Contaminants: Comparing Standard Preparation Methods.

BACKGROUND: Recent reports suggest that component plasma products contain significant quantities of cellular contamination. We hypothesized that leukoreduction of whole blood before preparation of derived plasma is an effective method to prevent cellular contamination of stored plasma. STUDY DESIGN: Samples of never-frozen liquid plasma prepared by standard methods (n = 25) were obtained from 3 regional blood centers that supply 3 major trauma centers. Samples were analyzed for leukocyte and platelet contamination by flow cytometry. To determine if leukoreduction of whole blood before centrifugation and expression of plasma prevents cellular contamination of liquid plasma, 1 site generated 6 additional units of liquid plasma from leukoreduced whole blood, which were then compared with units of liquid plasma derived by standard processing. RESULTS: Across all centers, each unit of never-frozen liquid plasma contained a mean of 12.8 ± 3.0 million leukocytes and a mean of 4.6 ± 2 billion platelets. Introduction of whole blood leukoreduction (LR) before centrifugation and plasma extraction essentially eliminated all contaminating leukocytes (Non-LR: 12.3 ± 2.9 million vs LR: 0.05 ± 0.05 million leukocytes) and platelets (Non-LR: 4.2 ± 0.3 billion platelets vs LR: 0.00 ± 0.00 billion platelets). CONCLUSIONS: Despite widespread belief that stored plasma is functionally acellular, testing of liquid plasma from 3 regional blood banks revealed a significant amount of previously unrecognized cellular contamination. Introduction of a leukoreduction step before whole blood centrifugation essentially eliminated detectable leukocyte and platelet contaminants from plasma. Therefore, our study highlights a straightforward and cost-effective method to eliminate cellular contamination of stored plasma.

Performance of a Nageotte hemocytometer method and a flow cytometric assay for residual leukocyte quantification in leukoreduced canine packed red blood cells.

OBJECTIVE: To evaluate the performances of a manual Nageotte hemocytometer method and commercial fluorescent bead-based flow cytometric assay for quantifying [rWBC] in leukoreduced canine packed red blood cell (pRBC) units. DESIGN: Prospective study. Five, commercially purchased, double leukoreduced canine pRBC units were spiked with canine leukocytes to create 6 pRBC standards with the following [rWBC]: < 0.1, 0.375, 1.5, 3.0, 6.0, and 24.0 WBC/µL. [rWBC] of each pRBC standard was measured with the Nageotte hemocytometer and flow cytometric techniques. Limit of detection (LoD), linearity, and bias were determined for each method. For each standard, accuracy and precision were calculated; the cumulative accuracy and mean precision for measurements between the LoD and 24.0 WBC/µL were also determined. SETTING: University veterinary blood bank and clinical pathology laboratory. MEASUREMENTS AND MAIN RESULTS: The Nageotte hemocytometer method had an LoD = 1.48 WBC/µL, inadequate linearity (R2  = 0.92), and a significant negative proportional bias (slope best-fit line = 0.52 ± 0.03). Between [rWBC] 1.5-24 WBC/µL, the technique demonstrated poor cumulative accuracy (6.7%) but acceptable mean precision (17.3%). Relative to a 2 rWBC/µL threshold, at 1.5 WBC/µL the method was inaccurate (6.7%) with acceptable precision (16.6%). The flow cytometric assay had an LoD = 1.3 WBC/µL, acceptable linearity (R2  = 0.99), and a mild positive proportional bias (slope best-fit line = 1.11 ± 0.01). The technique had acceptable cumulative accuracy (80%) and mean precision (10.7%) for measuring [rWBC] between 1.5 and 24 WBC/µL. At 1.5 WBC/µL, this method was acceptably accurate (86.7%) and precise (16.0%). CONCLUSIONS: The flow cytometric assay demonstrated acceptable performance for quantification of [rWBC] in leukoreduced canine pRBC units. The Nageotte hemocytometer method should be used cautiously due to poor accuracy and significant negative bias.

Glycine integrated zwitterionic hemocompatible electrospun poly(ethylene-co-vinyl alcohol) membranes for leukodepletion.

This paper describes a novel strategy for the hemocompatibility improvement of poly(ethylene-co-vinyl alcohol) (EVAL) membranes by incorporation of a naturally occurring zwitterion, glycine. Crystalline glycine was directly integrated to the EVAL fibers via electrospinning. The membranes were characterized by Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), Water Contact Angle measurements (WCA) and measurement of Critical Wetting Surface Tension (CWST). The impact of glycine integration on the membrane parameters was assessed by variations in fiber diameter, pore size and percentage porosity. The release of glycine from the membranes was also quantitatively evaluated by ninhydrin assay. The interplay of zwitterion structural features on the blood compatibility was studied by in vitro hemocompatibility evaluation and blood filtration studies. The outcomes of these investigations highlight that glycine incorporated membranes offer greater hemocompatibility than virgin EVAL membranes in terms of reduced hemolysis, increased RBC retention, decreased adhesion and activation of platelets. The type of membrane modification can be considered in future for the development of leukodepletion filter membranes.

Characterization of peripheral blood mononuclear cells isolated using two kinds of leukocyte filters.

OBJECTIVE: The objective of this study was to compare the activity and biological function of leukocytes isolated using apheresis platelet leukoreduction system chambers (LRSC), whole blood leukoreduction filters (LRF), and leukocytes in unfiltered peripheral whole blood (WB). METHODS: Peripheral blood mononuclear cells (PBMCs) and granulocytes were obtained by density gradient centrifugation using recovery filters and WB. Flow cytometry was used to detect the activity, phenotype, and apoptosis ratio of each cell subtype. RESULTS: The proportion of lymphocytes obtained from PBMCs was similar when using the two different filters as compared to traditional isolation; however, there were significant differences between the monocytes and granulocytes. The phenotypic frequency of lymphocytes was similar, but the apoptosis rate of lymphocytes from the two filters was slightly higher. Additionally, monocytes isolated via the three sources were able to be induced into dendritic cells expressing specific molecules; Granulocytes isolated from the LRF showed a lower purity and a higher level of apoptosis than granulocytes isolated from the WB. CONCLUSION: Compared with WB, the PBMCs isolated from the filters used in our blood center had no statistical difference in their activity and biological function, but they did differ in the proportion and quantity of monocytes and granulocytes. Our results show that the two filters can be used as an alternative method to collect leukocytes, which solves the problem of an insufficient blood supply for clinical and basic science research. Thus, these filters have significant value beyond their practical use in clinics.

A positive clinical experience of a long-term treatment pattern with leukocyte apheresis and Adacolumn in ulcerative proctitis.

Ulcerative proctitis (UP) is often involved in the diagnosis of inflammatory bowel disease (IBD). The increase of leukocytes and pro-inflammatory factors in peripheral blood and in the active forms, as well as the infiltration of neutrophils and monocytes/macrophages in the intestinal mucosa is known to occur in this entity. This infiltration of cells damages the mucosa due to the liberation of proteases, oxidation radicals and cytokines, among others. Apheresis techniques such as leukocyte apheresis may be used among the different therapeutic options such as steroids, sulfasalazine, 5-aminosalicylic, tacrolimus, azathioprine, cyclosporine, mycophenolate and biological agents.