Leukoreduction Of Blood Products Research Articles

Effect of Leukoreduction by Pre-Storage Filtration on Coagulation Activity of Canine Plasma Collected for Transfusion.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions and is extensively used in human, but rarely in veterinary patients. The concentration of some coagulation proteins can be affected by the processing steps used for the preparation of leuko-reduced plasma units. In this study, we assessed the effect of leukoreduction on coagulation activity of canine plasma collected for transfusion. Ten plasma units, five obtained from non-leuko-reduced (non-LR) whole blood (WB) units and five from leuko-reduced (LR) WB units were evaluated. Prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factor activities of factors (F) V, VIII, X, XI, and von Willebrand (vWF), fibrinogen and D-dimers content were assessed at collection (baseline value, D0) and after 7 days of frozen storage at −18 °C (D7). Compared to non-LR plasma units, LR units showed a statistically significant prolonged aPTT and reduced FXI activity. Filtration had no significant effect on the other factors and parameters evaluated. Filtration-dependent changes appear to have no impact on the therapeutic quality of plasma obtained from leuko-reduced whole blood, other than for FXI activity. Further studies on a larger sample size comparing the same unit before and after leukoreduction are needed to confirm these findings.

Effects of platelet-sparing leukocyte reduction and agitation methods on in vitro measures of hemostatic function in cold-stored whole blood.

Agitation of platelet units stored at room temperature is performed routinely to maintain platelet function, and leukoreduction of blood products is the standard of care in many countries to reduce immune consequences of transfusion. The effect of agitation and leukoreduction on whole blood stored at 4°C requires investigation, as reductions in hemostatic capacity of whole blood may reduce its efficacy in treating trauma-induced coagulopathy and platelet dysfunction. We hypothesize that agitation of whole blood will not affect hemostatic function and that leukoreduction will reduce hemostatic function of whole blood. In this in vitro randomized controlled study, 21 units of leukoreduced and 20 nonleukoreduced whole blood units were each randomly assigned into four agitation groups. Hemostatic parameters were measured using viscoelastic assays (rotational thromboelastometry-Extrinsic Screening Test (ROTEM-EXTEM) and thromboelastography (TEG) platelet mapping), impedance aggregometry (agonists-adenosine phosphate, arachidonic acid, thrombin receptor activating peptide, and collagen), and a thrombin generation assay from these whole blood units before and after filtration and on 0, 5, 10, and 15 days of storage at 4°C. Leukoreduction compared to nonleukoreduction reduced platelet concentration on Day 0. Viscoelastic measures and thrombin generation parameters revealed significant reduction in hemostatic function between the leukoreduced units and the nonleukoreduced units at a few time points. Leukoreduced units consistently demonstrated reduced platelet aggregation compared to the nonleukoreduced units. Agitation methods did not significantly affect any of the hemostatic parameters examined. Leukoreduction of whole blood with a platelet-sparing filter caused a moderate but significant reduction in some measures of whole blood hemostatic function most evident early in storage. The benefits of leukoreduction should be weighed against the potential reduced hemostatic function of leukoreduced units. Agitation of whole blood is not required to maintain hemostatic function. In vitro randomized controlled trial, level 1.

Real‐time amplification of glyceraldehyde‐3‐phosphate dehydrogenase gene for quality control of leukopoor platelets

Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L. The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.

Approaches to Minimize Infection Risk in Blood Banking and Transfusion Practice

The use of blood donor history and state-of-the-art FDA-licensed serological and nucleic acid testing (NAT) assays have greatly reduced the "infectious window" for several transfusion-transmitted pathogens. Currently transmission of human immunodeficiency virus (HIV), Human T-cell Lymphotropic Virus (HTLV), hepatitis viruses and West Nile Virus are rare events. The seroprevalence of cytomegalovirus in the donor population is high and cytomegalovirus infection can cause significant complications for immunocompromised recipients of blood transfusion. Careful use of CMV seronegative blood resources and leukoreduction of blood products are able to prevent most CMV infections in these patients. Currently, bacterial contamination of platelet concentrates is the greatest remaining infectious disease risk in blood transfusion. Specialized donor collection procedures reduce the risk of bacterial contamination of blood products; blood culture and surrogate testing procedures are used to detect potential bacterially contaminated platelet products prior to transfusion. A rapid quantitative immunoassay is now available to test for the presence of lipotechoic acid and lipopolysaccharide bacterial products prior to platelet transfusion. Attention has now turned to emerging infectious diseases including variant Creutzfeldt-Jakob disease, dengue, babesiosis, Chagas' disease and malaria. Challenges are presented to identify and prevent transmission of these agents. Several methods are being used or in development to reduce infectivity of blood products, including solvent-detergent processing of plasma and nucleic acid cross-linking via photochemical reactions with methylene blue, riboflavin, psoralen and alkylating agents. Several opportunities exist to further improve blood safety through advances in infectious disease screening and pathogen inactivation methods.

Lymphocyte Survival and Activation in Stored Platelet Concentrates After Gamma-Irradiation or Pathogen Reduction Technology Treatment.

Abstract 2113Poster Board II-90The presence of white blood cells, particularly lymphocytes, in blood products has been shown to contribute to the development of a variety of adverse events including both donor anti-recipient and recipient anti-donor responses. Therefore leukoreduction of blood products or inactivation of leukocytes is necessary. We are currently using gamma-irradiation (Radiator Gammacell 3000 Elan) to inactivate leukocytes in blood components. This study shows the comparison of irradiation and PRT treatment (Mirasol*®) on lymphocyte survival and inactivation in non-leukoreduced platelet concentrates (PCs). PRT treatment is a pathogen reduction technology that targets nucleic acids after exposure to riboflavin and UV-light. We analyzed 7 untreated (C), 7 PRT treated (M) and 7 irradiated PCs (RD) in our study. Non-leukoreduced buffy coats (mean volume 65 ml) were obtained from the Regional Blood Center, Warsaw. PCs were prepared by pooling 15 buffy coats (ABO identical) suspended in 3 plasma units, and dividing the pool into 3 equal-weight units in bags made of the same material. Following addition of 35 ml of riboflavin solution, M units were illuminated. The same volume of saline solution, was added to the C group and RD units. All PCs were then stored at 22°C with agitation for 5 days. Samples were removed on days 1, 3 and 6 for analysis. The lymphocyte survival rate was determined by 7AAD (7-amino-actinomycin D staining of dead cells, Becton Dickinson) and their activation by anti-CD69-APC staining (Becton Dickinson). Samples were also stained with anti-CD45-PE antibodies to identify and gate on lymphocytes. Samples were analyzed on the Becton Dickinson Cytometer FACSCanto I.No increase in the number of dead cells was observed during 6 days of storage in the C group. After 3 days of storage however, in the M group the percentage of dead cells was significant higher than in C and RD groups (Student t-test, p=0.004 and p=0.03, respectively). After 6 days, the percentage of dead cells in the M samples was 72% vs. 30% following irradiation. The percentage of 7AAD-positive cells was significantly higher compare to C samples, both in M (p=0,001) and in RD samples (p=0,004). The percentage of dead lymphocytes was also observed to be statistically higher in M samples than in RD (p= 0,001).Analysis of lymphocyte activation was performed on live (7AAD-negative, CD45-positive) cells only. CD69 expression ranged between 20% and 40% in all tested samples (C, M, RD) during 6 days of storage. On days 1 and 3 of storage, Mirasol treatment significantly reduced lymphocyte activation as shown by the ratio (test/control) of %CD69-positive cells (p=0,004 and p=0,001, respectively) and mean fluorescence expression intensity of CD69. Interestingly, after 6 days of storage, RD samples showed significant higher lymphocyte activation then C and M samples (p=0,03 and p=0,02, respectively).In summary, a significant increase of dead lymphocytes after 6 days of storage was observed in PRT-treated PCs. This increase was two-fold higher than in gamma-irradiated PCs. At the same time a decrease in lymphocyte activation during 6 days of storage was observed in PRT-treated PCs. Overall, the use of PRT achieves better leukocyte inactivation than gamma-irradiation. Disclosures:No relevant conflicts of interest to declare.

Microchimerism.

Abstract SCI-46Microchimerism (MC) is the long-term persistence of an allogeneic cell population of <5% relative to host cells. MC may result from various allogeneic exposures, including transplantation, pregnancy, twinning and blood transfusion. Advances in PCR, particularly development of allele-specific, real-time PCR in closed systems, have facilitated detection/quantitation of MC. Though conceptually related to fetal-maternal MC, transfusion-associated MC (TA-MC) is distinct. Transient chimerism has been found in patients transfused for elective surgery, hemoglobinopathies, congenital heart disease and advanced HIV infection. In contrast, persistent TA-MC occurs frequently among trauma patients who received cellular blood products shortly after massive injury and hemorrhage. At hospital discharge, TA-MC is present in about half of severe trauma patients; most loose detectable chimeric cells, but approximately 1 in 5 (10% overall) develop persistent TA-MC which may approach 5% of all circulating leukocytes and persist for years. While TA-MC has been primarily studied in civilian trauma populations, recent studies of soldiers transfused from World War II to the Iraq war have confirmed its occurrence in military trauma settings. The severity of injury and freshness of blood are prominent determinants of the incidence and persistence of TA-MC. Of note, leukoreduction of blood products, widely implemented in the 1990s, did not affect the incidence of TA-MC. TA-MC appears to represent engraftment at the level of the hematopoietic stem cell (HSC), given long-term persistence of multiple lineages of donor leukocytes in humans and in murine models of TA-MC. As with multiple cord blood transplantation, in most cases, leukocytes from only a single donor persist. The presence of a focal deficit in the cellular immunologic repertoire following injury but prior to transfusion that matches a blood donor's HLA type appears to be an important predisposing factor. This focal deficit is superimposed on a generalized immune suppression that occurs following severe injury. Preliminary studies have also documented allelic-polymorphisms in immune response genes that predispose some patients to development of TA-MC. Transfusion studies using knock-out mice have demonstrated innate and adaptive immune functions that predispose to TA-MC, supporting these human data. In addition to ongoing studies of degree of engraftment and cellular immunity, we recently analyzed 41 soluble immune mediators in 56 trauma patients, both transfused (n = 39) and non-transfused (n = 17), and healthy blood donor controls. A number of these proteins fluctuate in response to trauma and transfusion, including those involved in immune modulation, inflammation, lymphocyte homeostasis, lymphocyte homing, and wound healing. Preliminary data suggest that failure to upregulate IL-10 immediately following trauma may be associated with the development of TA-MC. The phenomenon of TA-MC presents an opportunity to study the immunobiology of trauma, transfusion, tolerance, engraftment and alloresponsiveness. Our studies are now expanding to other populations (burn and peripartum recipients), and addressing clinical manifestations of TA-MC that may be either pathologic (autoimmunity, GVHD) or therapeutic (tolerance induction, cell therapies). DisclosuresNo relevant conflicts of interest to declare.

Use of a Prestorage Leukoreduction Filter Effectively Removes Leukocytes from Canine Whole Blood While Preserving Red Blood Cell Viability

Leukoreduction of blood products is a technique used to prevent leukocyte‐induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4°C) for 4 hours. Filtration efficacy was determined by measurement of pre‐ and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin‐streptavidin‐phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24‐hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Polymerase chain reaction based assessment of leukoreduction efficacy using a cytomegalovirus DNA transfected human T-cell line.

Detection of CMV DNA by PCR in seropositive blood donors is affected by nucleic acid extraction, amplification conditions and PCR product detection sensitivity. Clinical studies have shown that leukoreduced blood products are as effective as CMV-seronegative blood products in minimizing transfusion-associated CMV infection. We developed a PCR-based test model to assess the efficacy of leukoreduction on the removal of CMV DNA from blood donations. Whole blood units were spiked with a human Jurkat T-lymphocyte cell line which had been stably transfected with a CMV DNA immediate early sequence. All blood units were either CMV seronegative or shown to be CMV PCR negative. The amount of CMV DNA by PCR before, during and after filtration of blood units with third-generation leukocyte reduction filters were determined using a semi-quantitative adaptation of the Digene SHARP Signal System Assay (DSSSA). Whole blood units spiked with 1-2 x 10(6) CMV transfected Jurkat cells contained no detectable CMV DNA after leukoreduction. With a higher inoculum of 2.9 x 10(8) transfected Jurkat cells per unit, CMV DNA was detected after leukoreduction; however, there was an approximate 3 log decrease in the amount of CMV DNA detected. This CMV DNA transfected human Jurkat cell line in conjunction with semi-quantitative CMV PCR may be used to model leukoreduction efficacy and provides evidence that leukoreduction of blood products can decrease the amount of cellular CMV DNA by approximately 3 logs.