Standard Red Blood Cell Research Articles

Lost in the Ds: Unexplained Loss of Rh(D) Antigen Expression By Serological Testing In Two Patients

Abstract Introduction/Objective While the loss of ABOH antigens has been well documented, reduced-to-absent expression of the RHD antigen is comparatively rare. Here, we describe two cases with D antigen loss presenting associated clinical settings, smear results, and red blood cell (RBC) genotyping findings to elucidate this unusual phenomenon. Methods/Case Report Cases (n=2) were retrospectively reviewed based on discovery during routine serological testing in our Blood Bank. RhD blood typing was performed via gel methods [Grifols, Los Angeles, CA]. Clinical information and laboratory data were extracted via the electronic medical record. Case 1 is a 79-year-old female with metastatic breast cancer. She initially typed as D positive (4+) in at least two separate analyses, but her D front type progressively became weaker until turning entirely negative after several weeks. Spherocytes and stomatocytes were present on her peripheral blood smear. This patient received 10 D+ red blood cell (RBC) units without anti-D formation. RBC genotyping revealed traditional “dce” by standard polymerase chain reaction (PCR). Case 2 is a 61-year-old male with a history of pre-B acute lymphocytic leukemia post-stem cell transplant, myelodysplastic syndrome, and progressive loss of D antigen expression (4+ to 0). Standard RBC genotyping revealed “Dce” by PCR. The peripheral blood smear was significant for RBC anisopoikilocytosis with increased stomatocytes, spherocytes, and ovalocytes. Results (if a Case Study enter NA) NA Conclusion The disappearance of the D antigen is rare, and our experience suggests an association between malignancies (both hematopoietic and non-hematopoietic) and D antigenic loss. Beyond these disease associations, however, loss of RhD is not otherwise readily explained in either case. Further analysis to test for other RHAG and RHCE mutational variants is an ongoing area of investigation in these cases, as more sensitive testing may reconcile the traditional PCR results with our initial detection of the D antigen.

Transfusion of female blood in a rat model is associated with red blood cells entrapment in organs.

Transfusion of red blood cells (RBCs) has been associated with adverse outcomes. Mechanisms may be related to donor sex and biological age of RBC. This study hypothesized that receipt of female blood is associated with decreased post-transfusion recovery (PTR) and a concomitant increased organ entrapment in rats, related to young age of donor RBCs. Donor rats underwent bloodletting to stimulate production of new, young RBCs, followed by Percoll fractionation for further enrichment of young RBCs based on their low density. Control donors did not undergo these procedures. Male rats received either a (biotinylated) standard RBC product or a product enriched for young RBCs, derived from either male or female donors. Controls received saline. Organs and blood samples were harvested after 24 hours. This study found no difference in PTR between groups, although only the group receiving young RBCs from females failed to reach a PTR of 75%. Receipt of both standard RBCs and young RBCs from females was associated with increased entrapment of donor RBCs in the lung, liver, and spleen compared to receiving blood from male donors. Soluble ICAM-1 and markers of hemolysis were higher in recipients of female blood compared to control. In conclusion, transfusing RBCs from female donors, but not from male donors, is associated with trapping of donor RBCs in organs, accompanied by endothelial activation and hemolysis.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Polymorphism of ABO*O alleles and its clinical significance

Background. 62 ABO*O alleles of the ABO system are known. Some ABO*O alleles may be accompanied by the presence of residual A-glycosyltransferase activity in people of group O, which may lead to errors in determining the blood group. This confirms the important clinical significance of the ABO*O allele polymorphism. Knowledge of ABO*O gene polymorphisms and their prevalence contributes to the prevention of errors in determining the blood group of the ABO system.Objective: to study allele variants of the ABO*O gene in Russians.Materials and methods. The blood samples of 14,000 people were examined. The blood group was determined using anti-A, anti-Aweak, anti-B, lectin (anti-A1) and gel cards, as well as by cross-sectional method using standard red blood cells of O, A, and B groups. In one patient, the method of adsorption-elution with cold elution was used to identify a weak variant of antigen A, and the method of thermal elution was used to eliminate antigen- blocking plasma factors. Molecular determination of ABO*O alleles was performed in 130 individuals by polymerase chain reaction with sequence- specific primers and Sanger direct sequencing.Results. 13 allelic variants of the ABO*O gene were identified (10 with a typical deletion of c.261delG / N and 3 nondeletional alleles with polymorphism c.802G>A). Deletion alleles of ABO*O.01 were found in 92.85 % of the examined patients, nondeletion alleles of АВО*О.02 group – in 7.15 % of cases. The ABO*O.01.01 allele was detected with a frequency of 67.14 %, other deletion alleles – much less frequently: ABO*O.01.02 and ABO*O.01.11 – 5.71 %, ABO*O.01.26 – 5.00 %, ABO*O.01.12 – 4.30 %, ABO*O.01.13 and ABO*O.01.44 – 1.43 %, ABO*O.01.05, ABO*O.01.46, ABO*O.01.68 – 0.71 % each. Non-deletional alleles were found with the following frequencies: ABO*O.02.01 – 4.3 %, ABO*O.02.03 allele – 2.14 %, ABO*O.02.02 – 0.71 %. All individuals with the O group with the nondeletional allele had the Oαβ group, except for one patient (with the ABO*O.01.02 O.02.02 genotype), who had the Oβ group.Conclusion. For the first time, the immunogenetic characteristics of Russians are given according to ABO*O genes. Erythrocyte genomics helps to resolve the ambiguity of serological methods results and allows understanding mechanisms of different phenotypes formation. For the correct definition of natural isohemagglutinins and weak antigens variants should be used at least two different serological methods.

Single-Unit Transfusion Is Non Inferior to Double Unit Transfusion in Patients with Hematological Disorders Receiving Allogeneic or Autologous Bone Marrow Transplant or Induction Chemotherapy for Acute Leukemia: The 1versus2 Prospective Multicentric Randomized Clinical Trial

IntroductionAnemia is a common complication of hematological chemotherapy for acute leukemia and following hematopoietic stem cell transplantation (HSCT). Exposure to allogeneic blood transfusions has been associated with unfavorable outcome in several studies in a non-surgical settings. Retrospective studies in hematological intensive unit have suggested that single red blood cell (RBC) unit transfusion policy may reduce the number of RBC used in comparison with a classical double RBC unit transfusion policy, without clinical impact. The aim of the study was to demonstrate that single RBC transfusion was non inferior to the standard double RBC transfusion arm in terms of severe complication or mortality for inpatient with hematological malignancies. Key secondary endpoint, was the comparison of the numbers of RBC units transfused in each arm.MethodIn a phase 3 multicenter randomized trial, 245 adults' patients with acute leukemia requiring intensive chemotherapy or patients receiving autologous or allogeneic HSCT were randomly assigned (1:1) to receive either single RBC unit (1 RBC arm, n=125) per transfusion or double RBC (2 RBC arm, n=120) per transfusion when hemoglobin level was below 8g/dL.The primary composite endpoint was the percentage of patients who developed a grade ≥3 complications defined as stroke, transient ischemic attack, acute coronary syndrome, heart failure, elevated troponin level, intensive care unit transfer, death, new pulmonary infiltrates, and/or transfusion-related infections during hospital stays. The secondary endpoint was the number of red cell units transfused per patient per hospital stay. The primary endpoint was compared between groups by non-inferiority analysis for the proportion risk difference using Farrington-Manning method with a non-inferiority margin of 0.1, in ITT dataset.ResultsHematological disease were as followed: AML (59%), ALL (13.1%), Lymphoma (16.4%), others (11.5%). The median age was 55 years. Baseline characteristics were well balanced between the 2 arms (Figure 1A). A total of 981 and 592 transfusions have been necessary in the 1 RBC arm and 2 RBC arm, respectively. The median of RBC unit per transfusion was 1(1-1) and 2(2-2) in the 1 RBC and 2 RBC arm, respectively. The mean pre transfusion hemoglobin level was 7.49 +/- 0.83 g/dL in the 1 RBC arm and 7.46 +/- 0.67 g/dL in the 2 RBC arm (p=0.275). Hemoglobin level at discharge was 9.35 +/-1.14 g/dL in the 1 RBC arm and 9.58 +/-1.13 g/dL in the 2 RBC arm (p=0.118).The median (IQR) of red-cell units transfused per patient was 7 (4-12) in the single arm and 8 (4-12) in the double arm (p=0.65). The median number of platelet transfusion event was 7 (3.5-11.5) in the 1 RBC arm and 7 (3-13) in the 2 RBC arm (p=0.69). The median (IQR) number of red cell unit transfused per cycle and per day was 7 (3-9) and 0.28 (0.17-0.37) in the 1RBC arm and 6 (4-10) and 0.27 (0.20-0.38) in the 2 RBC arm (p=0.61 and p=0.47).The predefined non-inferiority criteria was achieved with 28 patients developing a serious complication in the 1 CGR arm (22.4%) and 28 patients in the 2 RBC arm (23.3%) (Risk difference 0.009; 95% Confidence interval [-0.0791- 0.0978] (Figure 1B).Conclusion: Single RBC transfusion policy is non inferior to double RBC transfusion policy in hematological intensive care unit for patient receiving a bone marrow transplant or intensive chemotherapy. Single RBC unit transfusion can be used safely in daily clinical practice. The single RBC transfusion policy does not reduce the number of RBC transfusion. [Display omitted] DisclosuresJardin: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. .

Rejuvenated RBCs Increase Oxygen Consumption in a Simulated Exchange Transfusion Model of Sickle Cell Disease Patients

Introduction: Exchange transfusion of red blood cells (RBCs) in patients with Sickle Cell Disease (SCD) may require 6 to 8 units to reduce sickle hemoglobin (Hb-S) to less than 30 percent of total hemoglobin (NIH No. 02-2117). Low oxygen affinity (high p50) in patients with SCD is manifested by impaired blood viscosities and reduced hemoglobin concentration. Well characterized RBC metabolic changes during liquid storage of donor RBC include an increase in the affinity of hemoglobin for oxygen (low p50) by depletion of 2,3-diphosphoglycerate (2,3-DPG). Transfusion of stored RBCs (Standard RBC) suggests inefficient and impaired O2 delivery to tissues compared to fresh circulating RBCs (Hasan 1994, Scott 2016) or after exchange transfusion in SCD (Uchida 1998). Extracorporeal manipulation of p50 through RBC rejuvenation increases the level of 2,3-DPG and increases the p50 of stored RBCs by right-shifting the Oxyhemoglobin Dissociation Curve (ODC) (Dennis 1979). Rejuvenation occurs by incubating RBCs at 37 °C for 60 minutes with a rejuvenation solution (Citra Labs). An exchange transfusion simulation model was developed to evaluate the changes in oxygen release capacity of rejuvenated RBCs and the potential impact on estimated oxygen consumption (VO2) compared to Standard RBCs.Methods: Oxyhemoglobin dissociation curves were generated from (5) RBC units (leukocyte-reduced), CPD/AS-1, on Day 0, after 21 days of storage (Standard RBC), and after rejuvenation on day 21 (REJ RBC) which were used to estimate p50 values of the donor/recipient mixtures after each exchange transfusion and to determine VO2 as previously described (Li 2016, Srinivasan 2017). An elevated p50 of 30.4 mm Hg, typical for SCD patients (Uchida 1998), was used to estimate the baseline VO2 (200 mL O2/min). VO2 was determined after each simulated RBC exchange with Standard or REJ RBCs assuming a constant blood volume of 5L, cardiac output of 5 L/min, total hemoglobin concentration of 10 g/dL. The model assumed an exchange rate of one apheresis unit of circulating RBCs to transfusion of one unit of Standard or REJ RBCs.Results: Donor RBC p50 was 26.9 ± 2.0 mm Hg on Day 0 and declined with storage to 17.6 ± 1.5 mm Hg at 21 days (p < 0.001, t-test). Rejuvenation restored p50 to 34.8 ± 2.7 mm Hg which was significantly higher than Day 0 (p < 0.001, t-test). The model predicted a reduction in Hb-S from 70 % to 22%, a decrease in p50 after the 8 unit exchange transfusion to 21.6 mm Hg with Standard RBC, but an increase in p50 to 33.3 mm Hg with REJ RBC (Figures 1a & 1b). Similarly, VO2 increased in the REJ RBC group but decreased sequentially with Standard RBC upon each additional exchange transfusion (Figure 1c). The model predicted a decrease in VO2 to 118 mL O2/min for the Standard RBC group and an increase to 233 mL O2/min with REJ RBC group after the exchange transfusion.Conclusions: The model predicted a baseline VO2 of 200 mL O2/min; however, in fresh whole blood from healthy volunteers with Hb-A, normal VO2 was estimated at 244 mL O2/min (Srinivasan 2017). This simulation suggested that performing an exchange transfusion with REJ RBCs could achieve 95% of normal oxygen consumption assuming a constant cardiac output even though the hemoglobin was maintained at 10 g/dL. While exchange transfusion has been a successful therapy to reduce the Hb-S percentage in chronically transfused SCD patients, improving tissue oxygen exchange may not yet be optimized as evidenced by post transfusion declines in p50 (Miller 1980, Uchida 1998). The model is limited because it does not account for improved rheological properties and blood viscosity following exchange transfusion, which was previously reported to improve exercise capacity even though p50 declined (Miller 1980). The model also did not account for any influence from irreversibly sickled cells. Transfusion practices to correct anemia and improve tissue oxygenation may be less effective than intended due to the diminished oxygen release capacity of standard stored RBC units. Transfusion strategies should consider whether the use of RBCs with increased p50 may be physiologically advantageous.Figure 1: Predicted ODC (a) before and after the simulated exchange transfusion with Standard or REJ RBCs and predicted Hb-A vs Hb-S ratios (b) and VO2 (c) after each unit of the exchange transfusion with Standard or rejuvenated RBCs. [Display omitted] DisclosuresKausch:Zimmer Biomet: Employment, Research Funding. Gray:Zimmer Biomet: Employment, Research Funding. Landrigan:Zimmer Biomet: Employment, Research Funding.

IMMUNE ANTIBODIES IN MOKSHA AND ERZYA ETHNIC GROUPS OF THE REPUBLIC OF MORDOVIA

ABO system's immune antibodies are common, but not as persistent as natural anti-A and anti-B antibodies. They are the result of hyper immunization of an iso- or heteroimmune nature and belong to the IgG class. The study of serum for the presence of immune antibodies of the ABO, Rh system is necessary for the diagnosis of hemolytic disease cases of newborns with ABO, Rh-conflict pregnancy. Clinically significant immune antibodies of erythrocyte systems capable to cause in vivo destruction of red blood cells are of great importance in transfusiology and immunohematology. The most common cause of their occurrence is alloimmunization with red blood cell antigens during childbirth. The aim of this study was to determine the characteristics of the immune anti-erythrocyte antibodies in residents of the Republic of Mordovia belonging to the ethnic groups of Moksha and Erzya. Methods. The cross-sectional study involved 294 people from the Moksha and 387 people and Erzya ethnic groups, randomly selected in 7 districts of the Republic of Mordovia. Detection of IgG anti-A, anti-B ABO systems was performed by inactivation of disulfide bonds in IgM molecules using a 5 % unitiol solution and then by direct agglutination method at indoor temperature with standard red blood cells of group O(I), A(II), and B(III). The study of incomplete anti-erythrocyte antibodies of other systems was performed by indirect Coombs' test using gel technology. Results. The study of immune antibodies of the ABO system revealed from 10.61 to 16.00 % of anti-A antibodies and from 2.27 to 6.38 % of anti-B antibodies in both men and women Moksha and Erzya. We studied immune anti-erythrocyte antibodies: anti-D 0.34 % in Moksha and anti-D 0.26% and anti-Kell 0.26 % antibodies in Erzya. The scale of immunogenicity of red blood cell antigens in the studied groups: A&gt;, B&gt;, D&gt; and K&gt;. The alloimmunization index of transfusion-hazardous antigens was calculated as 0.34 for Moksha and 0.52 for Erzya. Conclusions. Physiologically, the immune system in Moksha and Erzya actively reacts with antibody's production and it is sensitive to immunization by the group of antigens.

Effects of repleting organic phosphates in banked erythrocytes on plasma metabolites and vasoactive mediators after red cell exchange transfusion in sickle cell disease.

Red blood cell (RBC) exchange (RCE) transfusion therapy is indicated for certain patients with sickle cell disease (SCD). Although beneficial, this therapy is costly and inconvenient to patients, who may require it monthly or more often. Identification of blood and plasma biomarkers that could improve or help individualise RCE therapy is of interest. Here we examined relevant blood and plasma metabolites and biomarkers of vasoactivity and RBC fragility in a pilot study of SCD patients undergoing RCE using either standard RBC units or RBC units treated with a US Food and Drug Administration (FDA)-approved additive solution containing phosphate, inosine, pyruvate, and adenine ("PIPA"). In this prospective, single-blind, cross-over pilot clinical trial, patients were randomised to receive either standard RBC exchange or PIPA-treated RBC exchange transfusion with each RCE session over a 6-month treatment period. Pre- and post-transfusion blood samples were obtained and analysed for RBC O2 affinity, ATP, purine metabolites, RBC microparticles, and cell free haemoglobin. Red blood cell O2 affinity was maintained after PIPA-RCE in contrast to standard RCE, after which P50 fell (net O2 affinity rose). Plasma ATP did not change significantly after RCE using either of the RBC unit types. Exchange transfusion with PIPA-treated RBC units led to modest increases in plasma inosine and hypoxanthine. Plasma cell free haemoglobin fell after either standard or PIPA-treated RBC exchange transfusion (novel findings), and to a similar extent. RBC-derived microparticles in the plasma fell significantly and similarly after both standard and PIPA-treated RCE transfusion. In summary, treatment of RBCs with PIPA prior to RCE elicited favourable or neutral changes in key metabolic and vascular biomarkers. Further study of its efficacy and safety is recommended and could ultimately serve to improve outcomes in chronically transfused SCD patients.

Features of interpretation of the results of studies of ABО and Rhesus antigens and antibodies in patients with hematological diseases

Aim. To assess the aspects of interpretation of pre-transfusion tests in patients with hematological diseases. Material and Methods. We performed an analysis of the results of serological studies of ABО, Rh blood groups in blood samples of 857 patients with oncohematological diseases. ABO blood group determination and typing of D, C, c, E, e, К antigens were carried out using a gel agglutination test. Results. The decrease in strength of the agglutination of standard red blood cells by the patient’s anti-A and/or anti-B antibodies was observed in 112 patients (13.07% of the total number of patients). Abnormal agglutination strength in ABO and Rh antigens testing was observed in 17 patients (1.98% of the total number of patients), among them were 7 patients with acute myeloid leukemia (AML), 6 - with сhronic myeloid leukemia (CML), 2 – myelodysplastic syndrome (MDS), 2 – polycythemia vera (PV). Double populations of red blood cells were mainly detected in patients with MDS (45.61 %), aplastic anemia (AA) (27.27 %), primary myelofibrosis (PMF) (22.73 %), acute leukemia (AL) (22.2 %). In most cases double populations were associated with previous transfusions of blood products, meanwhile, three patients from this group (two patients with CML and one patient with PV) had never received blood transfusions before. Conclusion. Differences in anti-A and anti-B antibodies content were much more common in patients with lymphoproliferative disorders (LPDs) than in patients with myeloproliferative neoplasms (MPNs) (85.71% and 8.04%, respectively), while decrease of expression of red blood cell antigens was more typical for MPNs and did not occur in patients with LPDs.

The Impact of Red Blood Cell Transfusion on Mortality and Treatment Efficacy in Patients Treated with Radiation: A Systematic Review of the Literature

Introduction: Oncology patients are generally transfused to maintain a hemoglobin (Hb) above 7 g/dL (Holst et. al. BMJ 2015). Some centers use higher Hb thresholds before radiation therapy (RT) because pre-clinical data suggest that oncology patients with low hemoglobin levels may respond sub-optimally to radiation therapy due to hypoxia-induced radio-resistance (Pitson et. al. Int J Radiat Oncol Biol Phys 2001; Tatum et. al. Int J Radiat Biol 2006). It is hypothesized that higher hemoglobin levels improve tissue oxygenation, which facilitates formation of reactive oxygen species that optimize cancer cell death. No standard red blood cell transfusion thresholds have been established using interventional study data for patients with malignancies treated with radiation. This systematic review aimed to investigate the role of red blood cell (RBC) transfusion in radiation oncology patients and whether maintaining a higher Hb leads to improved overall survival, efficacy of RT on disease control and transfusion-related morbidity. Methods: We performed a literature search of PubMed, EMBASE and the Cochrane Library for randomized controlled trials, cohort studies and large case series comparing RBC transfusion thresholds in radiation oncology patients. We hand-searched references of identified articles. Eligibility criteria were studies that assessed patients receiving any RT modality for malignancy of any diagnosis and stage. Exclusion criteria were studies that did not evaluate hemoglobin or transfusion as an intervention or outcome. The primary outcome was overall survival. Secondary outcomes included locoregional tumor control, number of transfusions and adverse events associated with either radiation or transfusion. Screening and data extraction were done in duplicate. A meta-analysis was planned with outcomes presented as odds ratios with 95% confidence intervals. Results: Our search yielded 6172 titles. After exclusions, 39 articles underwent full text review (Figure 1). Only one study met the pre-specified inclusion criteria; therefore, a meta-analysis was not performed. The identified study pooled results from two Danish randomized controlled trials (DAHANCA 5 and 7) that stratified patients with head and neck squamous cell carcinoma with low pre-radiation hemoglobin levels (females &lt;13 g/dL and males &lt;14.5 g/dL) to RBC transfusion (235 patients) versus no transfusion (230 patients) (Hoff et. al. Acta Oncol 2011). The study found no significant differences between groups in locoregional disease control or overall survival after five years of follow up, despite increased hemoglobin levels in the transfused group. Given the search results, we expanded the review to extract data from other relevant trials not meeting inclusion criteria to conduct a narrative review of the available literature. The extracted studies involved a variety of malignancies and were primarily single-center retrospective studies assessing outcomes of local transfusion policies. Some studies featured univariate and multivariate analyses, but had conflicting results on the effects of transfusion on disease control and mortality. A major criticism of these studies was the lack of comparison between different transfusion thresholds in anemic patients. Conclusions: This systematic review illustrates the lack of data on the effect of RBC transfusion strategies in oncology patients undergoing radiation. Well-designed prospective studies are urgently needed in this area due to variability in practice. Disclosures No relevant conflicts of interest to declare.

Donor Iron Deficiency Study (DIDS): protocol of a study to test whether iron deficiency in blood donors affects red blood cell recovery after transfusion.

Despite fulfilling all requirements for blood donation, a large proportion of regular blood donors are iron deficient. Red blood cells (RBC) from iron-deficient donors may be particularly susceptible to damage induced by standard refrigerated storage. Herein, we present a study protocol for testing whether correcting iron deficiency in donors with iron-deficient erythropoiesis will improve the quality of their refrigerator-stored RBC. This is a randomised, controlled, double-blind clinical trial. Sixty healthy regular donors who meet donation standards, while exhibiting iron-deficient erythropoiesis by laboratory testing criteria, will donate a single standard RBC unit that will be leucoreduced and stored in a refrigerator under standard conditions for 40-42 days. A 51Cr-radiolabelled 24-hour RBC recovery study will be performed and then these donors will be randomised to receive, in a double-blinded fashion, either intravenous saline, as a control, or low-molecular weight iron dextran (1 g), to provide total iron repletion. Four to six months later, they will donate a second RBC unit, which will be similarly stored, and autologous 51Cr-labelled 24-hour post-transfusion RBC recovery will again be determined. The primary endpoint will be the change in 24-hour post-transfusion recovery from the first to the second donation. The primary outcome will be the group mean difference in the primary endpoints between the group receiving intravenous saline and the group receiving intravenous iron dextran. Secondary outcomes will be quality of life, fatigue, and emotional health, assessed by surveys. This study will provide definitive evidence as to whether donor iron deficiency affects the quality of the blood supply and will assess the severity of symptoms affecting iron-deficient blood donors.

Influence of blood storage age on immune and coagulation parameters in critically ill transfused patients.

Several retrospective studies have suggested that transfusion with red blood cells (RBCs) stored for longer periods is associated with increased mortality. The Age of Blood Evaluation (ABLE) study randomized subjects to receive fresh vs. standard issue RBC units and showed no difference in the primary or secondary endpoints of mortality or change in multi-organ dysfunction syndrome (MODS) score. In this study a subset of 100 ABLE subjects were enrolled to measure coagulation and immune parameters. Samples were collected pre-transfusion and on days 2, 6, 28, and 180 post-transfusion. Levels of 16 coagulation parameters, regulatory and functional T cells, 25 cytokines, and 16 markers of extracellular vesicles (EVs) were determined. Changes from baseline in levels of protein C, factor V, and EVs expressing phosphatidyl serine and CTLA-4 (CD152) differed between recipients of fresh and standard storage age RBC units, with the vast majority of coagulation and EV markers and all cytokines tested showing no difference between study arms. Although most analytes showed no difference between subjects in the fresh and standard arms of the study, 6 coagulation parameters, 15 cytokines, and 7 EV parameters changed significantly in the period post-transfusion. Transfusion of fresh vs. standard issue RBC units does not result in substantial changes in coagulation or immune parameters, up to day 35 of RBC storage. Furthermore, significant changes in multiple coagulation and immune parameters are detectable post-transfusion, though causality cannot be determined based on the current study.

Method for ABO Blood Group Testing Using a General-Purpose Automated Biochemical Analyzer.

We investigated a high-throughput and high-precision forward ABO blood typing screening method that utilizes a general-purpose biochemical analyzer to perform direct red blood cell sampling. The blood group antisera used were Ortho® BioClone® Anti-A Serum and Ortho® BioClone® Anti-B Se-rum. AFFIRMAGEN® Reagent Red Blood Cells (Ortho Clinical Diagnostics) were used for AB standard red blood cells. The general-purpose biochemical analyzer employed was the TBATM-120FR HbA1c measurement unit (Canon Medical Systems). ABO blood group of patient samples was determined based on values relative to amount of change in the AFFIRMAGEN® response. Repeatability was CV5% or lower, and testing of 1,112 patient samples showed 100% agreement between the results obtained using the proposed method and those obtained using the tube test method. The proposed method allows ABO blood typing to be performed simply, quickly, and with a high degree of precision.

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12–15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

Impact of different standard red blood cell storage temperatures on human and canine RBC hemolysis and chromium survival.

Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

Rapid bedside rejuvenation of red blood cell with an autologous cell salvage device.

During storage, red blood cells (RBCs) undergo physicochemical changes which affect the quality, function, and invivo survival of transfused packed RBCs (pRBC). Changes include decreased 2,3-diphosphoglycerate (2,3-DPG) levels, decreased ATP, changes in mechanical properties and oxidative injury. RBC rejuvenation is a method used to increase levels of 2,3-DPG and ATP in pRBCs. This process requires incubating the pRBCs with a rejuvenation solution and subsequent washing. Standard blood bank protocols using the COBE 2991 Cell Processor require several hours of preparation. The objective of this study was to verify if a bedside protocol for rejuvenating pRBC and washing with the Sorin Xtra autologous cell salvage system could be used. Outdated pRBC units were obtained and rejuvenated in a model operating suite using a dry air incubator for 1h at 37°C. Six units of pRBCs were pre-diluted with saline (1000ml) and six units were not pre-diluted with saline. All units were washed with normal saline (1000ml) using an apheresis-design cell salvage device in manual mode and wash volume set to 3000ml. Samples were collected and analyzed for standard RBC quality parameters at baseline and post-wash. Total pRBC wash efficiency was 94% ± 12% at a final hematocrit of 67.7±5.9% while maintaining post-wash hemolysis 0.24±0.12 %. Pre-dilution prior to washing did not confer statistically significant differences in final RBC quality parameters with the notable exceptions of calculated hemolysis and supernatant potassium levels (P<0.05). The washing process can be completed within 10min. The post-wash RBC parameters are appropriate for immediate transfusion to patients.

In vitro kinetics of reticulocyte subtypes: maturation after red blood cell storage in additive solution-1 (AS-1)

BackgroundReticulocytes are immature red blood cells containing RNA remnants. Their population kinetics has been documented under various in vivo and in vitro conditions, including after storage of red blood cells in blood banks. The purpose of this study was to describe the influence of blood bank storage on the kinetics of reticulocyte disappearance by in vitro culturing. MethodSamples of reticulocyte-enriched fractions (Percoll density-gradient) were obtained over different storage times from six red blood cell units stored in additive solution-1 (AS-1). Reticulocyte fractions were then cultured in enriched media at 37°C and analyzed by flow cytometry with thiazole orange taking into account hemolysis. ResultsDensity-gradient enriched reticulocyte fractions were obtain from standard red blood cell units with <1% of reticulocytes. An exponential drop of reticulocytes was observed in cultures. The time taken for reticulocyte disappearance in cultures was shorter with increased blood bank storage time (144±46h at 0.5 weeks of storage and 15±14h in the sixth week). High fluorescence reticulocytes disappeared completely in 42.5±8.5h, medium fluorescence reticulocytes in 73.4±20.8h and low fluorescence reticulocytes in 269.9±98.8h in red blood cell units stored for half a week. These times significantly decreased in red blood cell units stored for more time. ConclusionIn vitro reticulocyte disappearance was significantly faster after prolonged storage of red blood cell units at 4°C. The in vitro half-life at 0.5 weeks of storage was not significantly different from the values reported for fresh venous blood, but after the sixth week of storage, the half-lives were shorter. The possible explanation is that blood bank storage does not cause irreversible damage to the human reticulocyte maturational machinery.

Effect of age of red cells for transfusion on patient outcomes: a systematic review and meta-analysis.

Longer storage duration of red blood cell (RBC) units prior to transfusion has been associated with worse outcomes in observational studies. We performed a systematic review, including recently published randomized trials, to determine if storage age of RBCs is associated with mortality, morbidity or adverse events in patients. Searches were performed up to 21st July 2017 in Medline (OvidSP), 20 July in EMBASE (OvidSP) and June 2017 in Cochrane Library. Eligible studies were randomized controlled trials comparing transfusion of fresher or freshest available with older or standard issue RBCs. Human volunteer and autologous RBC transfusion studies were excluded. Data were extracted from published reports independently by 2 authors and strength of evidence assessed according to GRADE criteria. The primary outcome was latest-reported mortality. Sixteen trials randomizing 31,359 patients were identified. Transfusion with fresher compared with older RBC was not associated with risk of death (relative risk [RR] 1.04, 95% CI 0.98-1.09; P=.20, I2=0%, high quality evidence), but was associated with higher risk of transfusion reactions (RR 1.35, 95% CI 1.04-1.76; P=.02; I2=0%; high quality evidence) and infection (RR 1.08, 95% CI 1.00-1.17; P=.05; I2=0%, moderate evidence). Trial sequential analysis showed required information size has now been reached to exclude a 10% relative risk increase or decrease in mortality. Transfusion of fresher RBCs is not associated with decreased risk of death but is associated with higher rates of transfusion reactions and possibly infection. The current evidence does not support a change from current usual transfusion practice.

Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery

BackgroundExperimental studies suggest that mechanical cell washing to remove pro-inflammatory components that accumulate in the supernatant of stored donor red blood cells (RBCs) might reduce inflammation and organ injury in transfused patients. MethodsCardiac surgery patients at increased risk of large-volume RBC transfusion were eligible. Participants were randomized to receive either mechanically washed allogenic RBCs or standard care RBCs. The primary outcome was serum interleukin-8 measured at baseline and at four postsurgery time points. A mechanism substudy evaluated the effects of washing on stored RBCs in vitro and on markers of platelet, leucocyte, and endothelial activation in trial subjects. ResultsSixty adult cardiac surgery patients at three UK cardiac centres were enrolled between September 2013 and March 2015. Subjects received a median of 3.5 (interquartile range 2–5.5) RBC units, stored for a mean of 21 (sd 5.2) days, within 48 h of surgery. Mechanical washing reduced concentrations of RBC-derived microvesicles but increased cell-free haemoglobin concentrations in RBC supernatant relative to standard care RBC supernatant. There was no difference between groups with respect to perioperative serum interleukin-8 values [adjusted mean difference 0.239 (95% confidence intervals −0.231, 0.709), P=0.318] or concentrations of plasma RBC microvesicles, platelet and leucocyte activation, plasma cell-free haemoglobin, endothelial activation, or biomarkers of heart, lung, or kidney injury. ConclusionsThese results do not support a hypothesis that allogenic red blood cell washing has clinical benefits in cardiac surgery. Clinical trial registrationISRCTN 27076315.

Creating the hybridoma to produce monoclonal antibodies causing the agglutination of the human red blood cells containing A antigen

The determination of ABO blood group is obliged in many cases especially before blood transfusion, that is indicated at Point a, Clause 4, Article 14 Circular 26/2013/TT-BYT - Vietnam, date 09.16.2013. For this purpose, both standard sera (monoclonal antibodies) and standard red blood cells are common used but monoclonal antibodies are prefered. In Vietnam, monoclonal antibodies against ABO blood group are not available in domestic production. In this study, we succeeded in the generation of hybidoma cells secreting anti-A monoclonal antibody. Firstly, Balb/c mice were injected with Vietnamese human group A red blood cells to evoke B lymphocyte cells against A antigen present on the surface of the red blood cells. Afterward the lymphocytes were fused with sp2/0 myeloma cells in the presence of polyethylene glycol (PEG) to gain hybrid cells that were identified through ability to expand cells in a selective medium (hypoxanthine aminopterine thymine - HAT) at 37°C and 5% CO2. During screening and isolation process, the positive clones were identified by agglutination test with standard group A red blood cells. Of the 1440 wells, 12 monoclonal hybrid clones were selected. The hybrid cell line (designated A6G11C9) was the best one secreting the highest anti-A monoclonal antibody into culture with the antibody titer of 512. The antibody showed good intensity (+++), and the agglutination was visible by 10 seconds. This antibody is the promising for ABO-grouping kit development.