Removal Of Red Cells Research Articles

Abstract 2927: Optimization of sample preparation for single-cell RNA-sequencing using accumulated collection of preclinical and clinical specimens

Abstract Background: The heterogeneity of tumors became apparent with the introduction of single-cell RNA-sequencing technology, enabling the discovery of rare and novel cell types that were not attainable with bulk RNA-sequencing. Numerous papers have been published, and the data from single-cell RNA-sequencing are publicly available. While current literatures provide detailed guidance on effectively analyzing single-cell RNA sequencing data, documentation for sample preparations is less frequently reported in easily accessible protocols. Additionally, researchers hold diverse opinions on sample handling and practical information is scattered across multiple sources. Method: A total of 233 cases were included in the study, comprising 150 cases of clinical and 83 cases of preclinical samples. Among the 150 clinical samples, 58 were freshly obtained (32 biopsies and 26 surgical resections), and 92 were frozen. Of the 83 preclinical samples, 70 were fresh, and 13 were frozen. Sample preparations for 10X Genomics Chromium single-cell RNA-sequencing were carried out using the methods of 'Red blood cell lysis', 'Straining', and 'Dead cell removal using magnetic beads' with clinical specimens. Each method was applied based on specific situations involving the presence of red blood cells, cell aggregates, dead cells and debris. Results: There was a variation in the usage of red blood cell lysis, straining, and dead cell removal methods across different sample types. Specifically, 40.6% of clinical biopsy samples were primarily conducted with red blood cell lysis and/or straining without the removal of dead cells. Whereas, 96.2% of clinical surgical resections and 100% of clinical frozen samples predominantly utilized dead cell removal. The frequency of a double application of dead cell removal was higher in frozen samples, at 34.8% due to low viability as expected. For surgical resections, in comparison, only 15.4% required a second round of dead cell removal. In preclinical samples, both fresh and frozen, removal of dead cells was essential and implemented in 100% of cases. Among these, 62.9% of the fresh preclinical samples and 76.9% of the frozen preclinical samples underwent a second round of dead cell removal. Conclusions: The utilization of the methods red blood cell lysis, straining, and dead cell removal proved to be adequate in meeting the stipulated criteria of the 10x Genomics guidelines, demonstrating efficiency in terms of time. We have outlined a workflow for each sample type, accompanied by images illustrating pre- and post-conditions for plausible occurrences. Additionally, approximate cell numbers required, or percentages of cell loss are provided for each situation. We anticipate that this protocol will be beneficial for those new to the field, enabling them to generate samples for single-cell RNA-sequencing independently. Citation Format: Kyumin Lim, JuHyeon Lee, Kyoung-Ho Pyo, Byoung Chul Cho. Optimization of sample preparation for single-cell RNA-sequencing using accumulated collection of preclinical and clinical specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2927.

Single Needle Option: An Alternative to Dual-Needle Access in Erythrocytapheresis in Patients with Sickle Cell Disease

Background: Erythrocytapheresis or red cell exchange (RCE) is an invaluable treatment option for patients with complications related sickle cell disease, including acute stroke, stroke prevention, acute chest syndrome, and recurrent pain crisis. The procedure entails the removal of each patient's red blood cells containing the abnormal sickle hemoglobin and replacing them with normal red blood cells carrying non-sickled hemoglobin. Adequate vascular access is essential for erythrocytapheresis to allow for high flow rates and various forms of access are used including peripheral veins and central venous access devices. Our center typically uses a single vortex port (Angiodynamics, Walnut Creek, CA) with placement of a peripheral IV at time of procedure in order to maintain a circuit for exchange. Using peripheral access reliably becomes particularly difficult in young patients and those that require multiple access over time due to scaring. To ensure a successful procedure in patients with poor peripheral access, a single-needle (SN) option for TPE (SN-TPE) that is available on Spectra Optia (Terumo BCT, Lakewood, CO) was used. The single-needle procedure utilizes intermittent, rather than continuous, flow, and thus requires extra procedure run time. One discontinuous cycle consists of “exchanging red cells,” which is the drawing of blood and removal of the red cells, and “adjusting the volume in the reservoir,” which is the returning of blood. These cycles continue until the procedure is complete. This procedure allows us to continue RCE in a select number of patients with poor vascular access.Methods: We evaluated our institutional experience on patients treated using single-needle RCE. In addition, information regarding each RCE session was collected including duration of procedure and inlet flow rate.Results: An average of 45 RCE procedures are performed each month. Patients are scheduled every 3 to 8 weeks, with an average of every 4-5 week frequency. We started the Single Needle option in July of 2019 on 3 patients: one adult aged patient and 2 pediatric patients. By the end of 2019 we had perform a total of 27 SN procedures. In 2020, we performed a total of 112 SN procedures, average of 9 procedures each month. As of the first 6 months of 2021, we have completed 35 SN procedures, averages 6 a month.In patients undergoing single needle exchange we were able to increase inlet flow rates from an average of 30-50ml/min to 60-80ml/min. This decreased the duration of run times from 120-198 min to 77- 119 min. Pre and post hemoglobin S% was comparable between dual and single exchange patients and there was no change in the interval between RCE sessions. Conclusion: With our increasing experience with single-needle RCE, our findings suggest that RCE can be successfully completed using the single-needle option with no impact on pre- and post-exchange hemoglobin S% levels. There was a reduction in the total length of procedure due to ability to maintain higher inlet rates and decreased time to obtain access for RCE. The single needle option also improved patient satisfaction due to more reliable access and negating need for peripheral IV access. DisclosuresMunson: Terumo Medical Corporation: Consultancy, Honoraria, Speakers Bureau. Raj: Forma therapeutics: Consultancy; Terumo Medical Corporation: Honoraria, Speakers Bureau; Global biotherapeutics: Speakers Bureau.

Blood group systems

Blood group systems

Increasing Post-Processing Total Nucleated Cell (TNC) Recovery in Cord Blood Banking: Hespan Addition in cGMP Environment

Purpose. Evaluate the effects of HESPAN (HES) addition on indices of cord blood unit (CBU) potency, stability and safety after automated volume reduction (reduction of erythrocyte bulk and plasma volume) using the AXP AutoXpressTM (AXP) processing system.Background and Methods. TNC recovery varies significantly and unpredictably after volume reduction during CBU processing. However, prompt engraftment of CBU allotransplants correlates with their TNC including hematopoietic stem/progenitor cells (HPC). As a result, TNC is important in selecting CBU for transplantation: for example, 75% of the National Cord Blood Program (NCBP) CBUs shipped during the period 2010-2013 had TNC >120 x 107. Therefore, minimizing TNC losses during processing improves the chances that a CBU will be useful for clinical transplantation.The first method to increase post-processing TNC recovery was the addition of HES to the centrifugal method for CB volume reduction [Rubinstein et al, PNAS (USA), 92:10119-10122, 1995]. Volume reduction consists of concentrating the buffy-coat (containing leukocytes and HPC) by centrifugal stratification and removal of bulk red cells and platelet-containing plasma. NCBP processed manually over 30,000 CBUs using HES during the period 1995-2006. In 2006, the AXP, a closed, automated and FDA-approved processing method, without HES, was implemented. AXP was designed to enhance the MNC and CD34+ cell recoveries but not those of granulocytes. This resulted in lower post- processing TNC counts. Additionally, CBUs with larger volume and TNC content have somewhat higher TNC losses during automated processing. Using the AXP method, NCBP’s HEMACORD® obtained FDA License approval on November 10, 2011. However, since TNC (not MNC or CD34+ count) remains the most commonly used indicator of CBU potency and engraftment ability, we describe here implementation of HES in AXP processing to augment TNC recovery, by adding HES to the CBU to a 1-2% final concentration.Results.The results of manual CBU processing showed that HES addition improves yield, without changes in cell viability after cryopreservation, freezing at -196°C and thawing, after 20 years (from NCBP continuing stability study). HES addition also preserved CFU numbers and CD34+ cell counts after thawing.1. Comparison of TNC recoveries without and with HES addition in the same CBU: A total of 25 CBUs were initially processed with the AXP platform without HES, as per routine procedure, and the TNC recoveries were calculated. Each CBU was then reconstituted after its initial processing into a new AXP bag set. HES was added aseptically to the reconstituted product and each CBU was processed in the same conditions as first time (same AXP device, centrifuge, etc.). The TNC recoveries after the second volume reduction process (AXP with HES) were ~20% higher on average than after the first (AXP without HES).2. Comparison of TNC recoveries in different cohorts of CBUs: Thirty clinical-grade CBUs, with volumes 80-156 mL and TNC counts 111-290 x 107, were processed with HES in the AXP system and the results were compared with those of AXP-processed CBUs without HES over an earlier six month period. TNC and CD45+ cell recoveries improved by 16 - 20% maintaining mean post-processing hematocrit at 30.6% (SD ± 2). CD34+ and CD45+ cell viabilities were unchanged: 99% (SD ± 0.7) and 96% (SD ± 2.7), respectively, while their mean recoveries were 95% (SD ± 18) and 94% (SD ± 6). In addition, the same consistent post-processing volume was obtained (mean 20.8 mL, SD ± 0.1). CFU - CD34+ correlation (R2) after processing with HES was 0.788 (not different than what was observed in the CBUs without HES). Six CBUs AXP-processed with HES, were thawed and tested, with very minor losses in cell count and viability, similar to results of thawed AXP-processed CBU without HES. Finally, HES addition did not result in microbial contamination in any of these AXP-processed CBUs.Conclusion. Adding HES to CBUs before automated AXP processing increased substantially the TNC and CD45+ recoveries without loss of viability, while the CD34+ recoveries remained basically unchanged, with a mean of 95%. The post-processing hematocrit was consistent and low. AXP-automated CB processing with HES addition can be performed in the GMP environment, results in higher post-processing TNC and therefore, increases the CB bank’s ability to store larger CBUs that are most useful to patients. DisclosuresNo relevant conflicts of interest to declare.

Unique Requirements for Lipoproteins Derived From Human Plasma for Maximal Expansion of Human Erythroid Cells Ex-Vivo Under Humanized Conditions

Abstract 1257Ex-vivo expanded erythroid cells (EBs) have been proposed as alternative transfusion products. We have recently formulated humanized media (HEMAdef) that sustain amplification of EBs from cord blood as efficiently as media containing fetal bovine serum (HEMAser) and 10-times greater from adult blood (Migliaccio et al, Cell Transplantation, 2010; 19:453–469). However, while a minority (14.1±5.3%) of EBs generated under HEMAser conditions contain alterations of their membrane which produce cell protrusions (see Figure), membrane abnormalities were observed in 22.7±7.1% of EBs generated under HEMAdef conditions. [Display omitted] The membrane of the red cells consists of a lipid-bilayer attached to the cytoskeleton by integral membrane proteins. The physical-chemical properties of these membranes rely on maintenance of the integrity of the lipid bilayer as disruption of this layer leads to shorten red cell survival or cell removal from the circulation. EBs internalize apolipoprotein-lipid complexes (lipoproteins) present in the environment. Lipoproteins are classified on the basis of floating density into high density lipoproteins [HDL, 1.063<ρ<1.210, ∼50:50 (in dry weight) complex of apoliprotein A-I (apoAI; 32%) and apoAII (10%) with cholesterol esters (15%)], low density lipoproteins [LDL, 1.019<ρ<1.063, ∼20:80 complex of apoB100 (18%) with cholesterol esters (40%)] and very low density lipoproteins [VLDL, 0.950<ρ<1.006, ∼8:82 complex of apoB100 (4%), apoE and apoCI, CII and CIII (4%) with triglycerides (55–65%)]. In addition, all lipoproteins contain 20% of phospholipids. In culture, lipids are provided by lipoproteins present in fetal bovine serum or by synthetic vesicles containing animal (egg cholesterol) and/or vegetal (soybean lecithin) lipids conjugated with albumin (bovine or human). In spite of emerging associations between genetic apolipoprotein gene deficiency and red cell membrane abnormalities, the role played by individual lipoproteins in erythroid cell differentiation is unknown.The aim of this study was to analyze the effect of lipoproteins purified from human plasma on the growth and maturation of EBs generated by mononuclear cells (MNC) from adult blood. HDL, LDL and VLDL were purified according to published methods (Havel RJ et al, J Clin Inv 1955; 34:1345) and their purity assessed on the basis of apoprotein/lipid content analyses. HDL, LDL, VLDL and an artificial mixture of the three fractions (total plasma lipoprotein fraction, TPLF) were used at a concentration of 20 mM/mL which had been previously determined to induce optimal EBs proliferation in preliminary concentration/MTT proliferation curves both under HEMAser and HEMAdef conditions.In semisolid assay, none of the fractions had an effect on the growth of CFU-GM-derived colonies. By contrast, LDL and VLDL both increased by 0.5 and 2-fold, respectively, the numbers of BFU-E derived colonies generated by adult blood MNC in serum-supplemented cultures (78±8 and 113±15 vs 57±9, p<0.01) while only LDL increased the numbers of BFU-E-derived colonies under serum-deprived conditions (64±8 vs 39±5, p<0.05). The various lipoprotein fractions did not improve the overall FI in HEMAser cultures (FI ∼20 by day 14 in all conditions) but increased the frequency of EBs with membrane abnormalities (from 14% to 20–27%). LDL and VLDL also increased by 2-fold the total numbers of progenitor cells observed at day 10 (total progenitor cells/culture 756±104 and 1064±292 vs 542±54, p<0.01–0.05). Under HEMAdef conditions, LDL and VLDL increased by 3-2-fold the FI (95±10 and 64±14 vs 27±6, p<0.01–0.05) by day 14 but did not affect the frequency of EBs with membrane abnormalities which remained high (∼30%). By contrast TPLF modestly increased the FI over those observed in HEMAser cultures (40±12 vs 20±14 in HEMAdef + TPLF vs HEMAser, p<0.05) but greatly reduced the frequency of EBs with membrane abnormalities (14±6% vs 28±7%, p<0.05). The frequency of mature CD36posCD235ahigh EBs observed by day 14 was ∼38–50% in HEMAser and 8–16% in HEMAdef.Human LDL and VLDL increase cloning efficiency and ex-vivo expansion of human adult erythroid progenitors in liquid culture but impair membrane assembly which is improved by TPLF. These results suggest that a balanced combination of lipoproteins, in concentrations still to be identified, may be required to observe maximal expansion of morphologically normal human EBs under HEMAdef conditions. Disclosures:No relevant conflicts of interest to declare.

LeukoCatch, a quick and efficient tool for the preparation of leukocyte extracts from blood

BackgroundWhole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin.MethodsA filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples.ResultsThis study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method.ConclusionWe conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.

Stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS)

We present a self-powered integrated microfluidic blood analysis system (SIMBAS) that does not require any external connections, tethers, or tubing to deliver and analyze a raw whole-blood sample. SIMBAS only requires the user to place a 5 μL droplet of whole-blood at the inlet port of the device, whereupon the stand-alone SIMBAS performs on-chip removal of red and white cells, without external valving or pumping mechanisms, followed by analyte detection in platelet-containing plasma. Five complete biotin-streptavidin sample-to-answer assays are performed in 10 min; the limit of detection is 1.5 pM. Red and white blood cells are removed by trapping them in an integral trench structure. Simulations and experimental data show 99.9% to 100% blood cell retention in the passive structure. Powered by pre-evacuation of its PDMS substrate, SIMBAS' guiding design principle is the integration of the minimal number of components without sacrificing effectiveness in performing rapid complete bioassays, a critical step towards point-of-care molecular diagnostics.

Metabolomic Evaluation of Normal, In Vivo, Red Cell Aging

AbstractAbstract 2035Red cell lifespan follows a well-defined, species-specific trajectory approaching 120 days in humans and 50 days in the mouse. At the end of this period, ‘senescent’ red cells are removed from the circulation by phagocytes, upon recognition of a surface change, or changes in the physical properties of the aged RBC. Because mature red cells have no capacity for synthesis of new proteins, and have little ability to repair damage—limitations on red cell survival are likely to relate to functional decline secondary to accumulation of damage to cell constituents. Among key targets are glycolytic enzymes, proteins involved in redox metabolism, and structural proteins, as inherited defects in these proteins can shorten red cell survival and cause hemolytic anemia. In order to further evaluate metabolic changes in normal RBC over the course of RBC lifespan, we used an in vivo biotinylation method followed by magnetic bead separation to isolate murine red cells of defined age at intervals. Purified RBC were assessed for protein oxidation, reactive oxygen species production, surface antigen profile, and the abundance of metabolic intermediates using a multiplatform mass spectrometry approach. 167 metabolites were detected in RBC extracts collected at 8, 15 and 35 days after labeling, using 6 biologic replicates for each time point. Multiple metabolites showed statistically significant changes in abundance as a function of red cell age. The figure below presents representative examples of patterns of change observed—where each line corresponds to either the biotin – (younger) or the biotin + (older) fraction. Panel A was the most common pattern observed—where a steep initial decline indicates a metabolite that may be specific for reticulocytes, found at high concentration only in the youngest cell fraction. Panel B indicates a metabolite that didn't change, panel C is an example of a metabolite with a more linear pattern of decay, and panel D is an example of a metabolite that increased with increasing red cell age. Older cells had higher levels of glucose, and lower levels of downstream metabolites from both the glycolytic and pentose phosphate pathways—consistent with a decline in hexokinase activity when comparing young and old red cells. Older cells had evidence of cumulative oxidative damage as demonstrated by increased protein carbonyl content, and had higher levels of reactive oxygen species. Surprisingly, despite this evidence of oxidative stress, reduced glutathione levels increased with cell age, and content of oxidized glutathione was unchanged. However, accumulation of alternate products of red cell gamma glutamylcysteine synthetase suggests substrate deprivation. The pattern of glutathione-related metabolites we observed in aged red cells has been described in tissues subjected to acute oxidant stress. The observed changes in metabolite concentrations support the hypothesis that metabolic decline during red cell aging impairs energy production, generation of reducing equivalents, and capacity to reverse protein oxidation. How (or whether) these changes in metabolic equilibrium are converted into a signal that results in removal of senescent red cells from the circulation remains speculative. However, we observe red cell age-dependent changes in additional metabolites, including specific lipids, that may help to bridge this gap in understanding. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

How do I collect and process stem cells?

How do I collect and process stem cells?

Current knowledge about the functional roles of phosphorylative changes of membrane proteins in normal and diseased red cells

With the advent of proteomic techniques the number of known post-translational modifications (PTMs) affecting red cell membrane proteins is rapidly growing but the understanding of their role under physiological and pathological conditions is incompletely established. The wide range of hereditary diseases affecting different red cell membrane functions and the membrane modifications induced by malaria parasite intracellular growth represent a unique opportunity to study PTMs in response to variable cellular stresses. In the present review, some of the major areas of interest in red cell membrane research have been considered as modifications of erythrocyte deformability and maintenance of the surface area, membrane transport alterations, and removal of diseased and senescent red cells. In all mentioned research areas the functional roles of PTMs are prevalently restricted to the phosphorylative changes of the more abundant membrane proteins. The insufficient information about the PTMs occurring in a large majority of the red membrane proteins and the general lack of mass spectrometry data evidence the need of new comprehensive, proteomic approaches to improve the understanding of the red cell membrane physiology.

Alternative treatment paradigm for thalassemia using iron chelators

beta-thalassemia major, or Cooley's anemia, is a red blood cell disorder requiring lifelong blood transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction in high numbers. We hypothesized that removing this proximate iron compartment as a primary treatment, using standard and alternative orally active iron chelators, could prevent hastened red cell removal and, clinically, perhaps alleviate the need for transfusion. Iron chelators of the pyridoxal isonicotinoyl hydrazone family (pyridoxal isonicotinoyl hydrazone and its analog pyridoxal ortho-chlorobenzoyl hydrazone) were evaluated in addition to the present mainstay, desferrioxamine and deferiprone, in vitro and in vivo. Treatment of human beta-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron and reduction of oxidative stress, as evaluated by methemoglobin levels. When administered to beta-thalassemic mice, iron chelators mobilized erythrocyte membrane iron, reduced cellular oxidation, and prolonged erythrocyte half-life. The treated thalassemic mice also showed improved hematological abnormalities. Remarkably, a beneficial effect as early as the erythroid precursor stage was manifested by normalized proportions of mature vs immature reticulocytes. All four compounds were also found to mitigate iron accumulation in target organs, a critical determinant for patient survival. In this respect, pyridoxal ortho-chlorobenzoyl hydrazone displayed higher activity relative to other chelators tested, further diminishing iron in liver and spleen by up to approximately fivefold and twofold, respectively. Our study demonstrates the ability of iron chelators to improve several of the fundamental pathological disturbances of thalassemia, and reveals their potential for clinical use in diminishing requirement for transfusion when administered early in disease development.

Heme degradation and oxidative stress in murine models for hemoglobinopathies: Thalassemia, sickle cell disease and hemoglobin C disease

Red blood cells with abnormal hemoglobins (Hb) are frequently associated with increased hemoglobin autoxidation, accumulation of iron in membranes, increased membrane damage and a shorter red cell life span. The mechanisms for many of these changes have not been elucidated. We have shown in our previous studies that hydrogen peroxide formed in association with hemoglobin autoxidation reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent emission bands and the release of iron. Heme degradation was assessed by measuring the fluorescent band at ex 321 nm. A 5.6 fold increase in fluorescence was found in red cells from sickle transgenic mice that expressed exclusively human globins when compared to red cells from control mice. When sickle transgenic mice co-express the γM transgene, that expresses HbF and inhibits polymerization, heme degradation is decreased. Mice expressing exclusively hemoglobin C had a 6.9 fold increase in fluorescence compared to control. Heme degradation was also increased 3.5 fold in β-thalassemic mice generated by deletion of murine β major. Membrane bound IgG and red cell metHb were highly correlated with the intensity of the fluorescent heme degradation band. These results suggest that degradation of the heme moiety in intact hemoglobin and/or degradation of free heme by peroxides are higher in pathological RBCs. Concomitant release of iron appears to be responsible for the membrane damage that leads to IgG binding and the removal of red cells from circulation.

Human cord blood CD133+ cells immunoselected by a clinical‐grade apparatus differentiate in vitro into endothelial‐ and cardiomyocyte‐like cells

Recent findings on human hematopoietic stem cell (HSC) properties suggest a possible therapeutic role of human umbilical cord blood (UCB) HSC-based cellular therapies in the treatment of myocardial infarction. Nine UCB units were subjected to sequential red cell removal, freezing, and postthawing CD133+ HSC immunoselection by a clinical-grade, CE-approved, magnetic apparatus and microbead-coated anti-CD133 monoclonal antibody. Selected UCB CD133+ cells were cultured in vitro in medium supporting either endothelial or cardiomyocytic differentiation in parallel experiments. Immunoselection allowed recovery of 79 percent of initial CD133+ cells with a CD133+ cell purity of 81 percent, on average. Parallel cultures showed the appearance of endothelial markers (VE-cadherin, CD146, and KDR and bright expression of CD105), morphofunctional features of endothelium in endothelial-supporting cultures, of cardiac muscle proteins (troponin I and myosin ventricular heavy chain alpha/beta; MYHC) and specific gene expression (GATA4, NKX2.5, troponin I, and MYHC) in cardiomyocyte-oriented cultures. The appearance of both endothelial- and cardiomyocyte-like cells from parallel cultures of frozen-thawed-immunoselected UCB CD133+ cells by a clinical-grade method and previously reported data on lack of major signs of rejection of these cells in immunocompetent rats subjected to experimental liver damage suggest a possible role of these allogeneic HSCs in cell therapies designed for regenerative treatments of ischemic diseases of human myocardium.

Red-cell ICAM-4 is a ligand for the monocyte/macrophage integrin CD11c/CD18: characterization of the binding sites on ICAM-4

AbstractIntercellular adhesion molecule 4 (ICAM-4) is a unique member of the ICAM family because of its specific expression on erythroid cells and ability to interact with several types of integrins expressed on blood and endothelial cells. The first reported receptors for ICAM-4 were CD11a/CD18 and CD11b/CD18. In contrast to these 2, the cellular ligands and the functional role of the third β2 integrin, CD11c/CD18, have not been well defined. Here, we show that ICAM-4 functions as a ligand for the monocyte/macrophage-specific CD11c/CD18. Deletion of the individual immunoglobulin domains of ICAM-4 demonstrated that both its domains contain binding sites for CD11c/CD18. Analysis of a panel of ICAM-4 point mutants identified residues that affected binding to the integrin. By molecular modeling the important residues were predicted to cluster in 2 distinct but spatially close regions of the first domain with an extension to the second domain spatially distant from the other residues. We also identified 2 peptides derived from sequences of ICAM-4 that are capable of modulating the binding to CD11c/CD18. CD11c/CD18 is expressed on macrophages in spleen and bone marrow. Inhibition of erythrophagocytosis by anti–ICAM-4 and anti-integrin antibodies suggests a role for these interactions in removal of senescent red cells.

Immunoregulation of Cellular Life Span

Our current studies focus on the molecular changes induced by aging. During aging, changes in proteins occur that alter their function and render them immunogenic. These "neoantigens" are recognized by physiologic autoantibodies. Physiologic autoantibodies and their corresponding antigens offer therapeutic strategies for disease intervention through the innate immune response. Early studies done in the 1970s showed humans and animals to have physiologic antibodies that bind to a neoantigen called senescent cell antigen (SCA), which appears on senescent and damaged cells and initiates their removal by macrophages. These studies led to the discovery that oxidation can generate a new antigen in situ. Oxidation accelerated aging of red cells, generated SCA and IgG binding, and triggered removal of red cells by macrophages. Since then, a number of laboratories have found that oxidation can generate other neoantigens. For example, oxidized LDL (OxLDL) induces antibodies that can modify the natural progression of atherosclerosis. Apoptotic cells express oxidatively modified moieties on their surfaces that are involved in macrophage recognition and phagocytosis. Physiologic autoantibodies were used to isolate SCA from brain tissue. HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) of the isolated antigen suggested that the aging antigen is a subset of band 3, a family of proteins also called anion exchange proteins (AE1-3). FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2, and AE3) are detected in aging antigen fractions. Among the fragments identified with FAB-MS was a sequence corresponding to an aging epitope, human band 3 sequence LFKPPKYHPDVPYVKR, residue 812-830 in AE1; HHPDVTYVK, residue 1144-1152 in AE2; or HHPEQPYVTK, residue 1135-1144 in AE3. A residue that is close to that region was identified in mouse AE1 ASGPGAAAQIQEVK, residue 762-775. The potential for altering the natural progression of diseases using select peptide-defined epitopes within or overlapping the aging antigenic site (547-553 and 824-829) is discussed using the innate immune response to band 3 in malaria as an example.

Altered red cell turnover in diabetic mice

In diabetes, a subpopulation of red cells expose phosphatidylserine, and the size of this subpopulation of cells is related to blood glucose levels. Because phosphatidylserine exposure can lead to the recognition and removal of red cells we hypothesized that red cell survival would be altered. In this study, diabetic female mice (db/db) and heterozygous littermate controls were used to explore relationships between red cell characteristics, phosphatidylserine exposure, and red cell survival. Red cell turnover was assessed using random red cell labeling with biotin. To determine if phosphatidylserine exposure was related to age, red cells were double-labeled and analyzed by flow cytometry. Cellular characteristics were measured by flow cytometry and ektacytometry. While red cell removal was linear for the control mice, for the diabetic mice an exponential survival curve was noted with an extended survival of a subpopulation of cells. The subpopulation of red cells exposing phosphatidylserine was significantly increased in the diabetic mice relative to the control mice (0.9% ± 0.07%, n = 12, and 0.6% ± 0.05 %, n = 22, respectively; P =.007). This subpopulation increased during the life of the red cell, with a higher rate of increase in the diabetic mice. Red cell hemoglobin content and cell volume were abnormal, but no obvious signs of anemia were found in the diabetic animals. A normal hematocrit, low reticulocyte count, and a much reduced spleen in these animals were consistent with a low red cell turnover. The presence of a population of older phosphatidylserine exposing cells indicates altered red cell removal and suggests a role for older red cells in vascular damage. (J Lab Clin Med 2002;140:161-5)

Improved platelet compatiblity of water vapour glow discharge treated non-woven poly(ethylene terephthalate) leukocyte-reduction filters for different types of platelet concentrates.

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.

Concentration of Progenitor Cells Collected from Bone Marrow Fluid Using a Continuous Flow Cell Separator

In ABO major incompatibility on bone marrow transplantation (BMT), red cells must be removed from collected marrow fluid to prevent hemolysis. We report the concentration of progenitor cells collected using a continuous flow cell separator (Cobe Spectra). The average volume of concentrated bone marrow was 132 +/- 47 ml and that of red cells included was 5.1 +/- 2.4 ml. The red cell removal rate was 97.6%. The recovery rate was 40.6% for total nuclear cells, 77.9% for mononuclear cells, 100% for CD34+ cells, and 93.9% for colony forming unit granulocyte-macrophage. Eighteen patients undergoing allogeneic BMT showed no signs of fever or hemolysis during concentrated marrow fluid transfusion. The recovery rate of progenitor cells was high, indicating sufficient recovery of hemopoiesis. This technique is applicable in ABO-incompatible BMT and in frozen-storage stem cells.

Neutrophil Priming State Predicts Capillary Leak after Gut Ischemia in Rats

Background.Multiple organ failure after serious injury or illness is a major determinant of mortality. An initial insult is believed to “prime” circulating neutrophils and induce systemic inflammation. Thereafter, a second insult will precipitate distant organ injury. The aim of these studies was to evaluate circulating neutrophil function after mesenteric ischemia–reperfusion to determine the neutrophil “priming state,” a quantitative and clinically useful predictor of multiple organ failure.Materials and methods.Anesthetized Sprague–Dawley rats underwent superior mesenteric artery occlusion for 30 min or sham operation and were euthanized after 2, 6, or 24 h of reperfusion. Control animals had blood taken without any intervention. To determine changes in lung capillary permeability, another group of rats received Evan's blue, a dye that binds albumin, 1 h before sacrifice. Flow cytometric analysis was performed on 5 million white blood cells after removal of red cells by lysis and centrifugation. Neutrophil number, oxidative burst, and CD18 expression were measured.Results.The number of circulating neutrophils was elevated similarly in rats subjected to sham operation or ischemia–reperfusion. Oxidative burst potential was increased at 2 h, maximum at 6 h, and normal at 24 h after reperfusion, but not in sham rats. CD18 expression was similar in all groups. There was a significant temporal correlation between the “priming state” of the circulating neutrophil, defined as the product of the neutrophil number times oxidative burst, and lung leak.Conclusions.The neutrophil “priming state” may allow the clinician to better predict those patients at greatest risk for multiple organ failure.

Red cell deformability, splenic function and anaemia in thalassaemia.

Red cell deformability (RCD) was measured in 38 patients with alpha-thalassaemia and 48 patients with beta-thalassaemia, of whom 13 had undergone splenectomy. All splenectomized patients, but none of those with intact spleens, had very rigid erythrocytes with an elongation index <0.45 at a high shear stress of 30 Pa suggesting a splenic recognition threshold for removal of rigid red cells. At this shear stress RCD correlated strongly with the degree of anaemia in both the splenectomized (r = 0.81, P < 0.001) and non-splenectomized beta-thalassaemic patients (all patients r = 0.81, P < 0.001; homozygous beta-thalassaemic patients r = 0.51, P = 0. 01). These data suggest that reduced RCD is a major determinant of anaemia in thalassaemia.