Labeling Of Red Blood Cells Research Articles

Detection and clinical application of red blood cell survival.

There are 2 techniques for detecting red blood cell survival (RBCS) detection techniques: red blood cell labeling test and carbon monoxide (CO) breath test. The former has disadvantages such as long measurement times and complicated procedures, while the latter is simple, convenient, moderately priced, and capable of dynamically monitoring changes in RBCS before and after treatment. Currently, the CO breath test is gradually being implemented in clinical practice. RBCS is not only applied to hematologic diseases such as multiple myeloma, myelodysplastic syndromes, lymphoma, and thalassemia, but also to non-hematologic diseases like type 2 diabetes and chronic kidney disease. It can assist in diagnosis, guide treatment, evaluate drug treatment efficacy, and predict disease progression.

Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies.

The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a “hole” is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

UHAS-MIDA Software Package: Mass Isotopomer Distribution Analysis-Applying the Inverse Matrix for the Determination of Stable Isotopes of Chromium Distribution during Red Blood Cell Labelling

Clinical assessment of fluid volume status in children during malaria can be taxing and often inaccurate. During malaria, changes in fluid volume are rather multifarious and estimating this parameter, especially in sick children is very challenging for clinicians who frequently rely on indices such as long capillary refill times, tachycardia, central venous pressure and decreased urine volume as guides. Here, we present the UHAS-MIDA, an open-source software tool that calculates the red blood cell (RBC) concentration and blood volume during malaria in children determined using a stable isotope of chromium (53Cr as the label) by gas chromatography-mass spectrometry in selective ion monitoring (GC/MS-SIM) analysis. A key component involves the determination of the compositions of the most abundant naturally occurring isotopes of Cr (50Cr, 52Cr, 53Cr), and converting the proportions into a 3 × 3 matrix. To estimate unknown proportions of chromium isotopic mixtures from the measured abundances of three ions, an inverse matrix was calculated. The inverse together with several inputs is then used to calculate the corrected MS ion abundances. Thus, we constructed the software tool UHAS- MIDA using HTML, CSS/Bootstrap, JavaScript, and PHP scripting languages. The tool enables the user to efficiently determine RBC concentration and fluid volume. The source code, binary packages and associated materials for UHAS-MIDA are freely available at https://github.com/bentil078/Abaye-et-al_UHASmida

Abstract 10086: Cardiac Intravital Microscopy Enables Quantitative Mapping of Capillary-Level Blood Flow in the Murine Heart

Introduction: Coronary microvascular disease (CMD) is increasingly appreciated as a contributor to adverse cardiovascular outcomes, but underlying mechanisms of CMD remain poorly understood. Investigation of CMD in animal models is limited by a lack of techniques to quantify flow in the heart. We assessed the hypothesis that intravital microscopy (IVM) can enable in vivo imaging of red blood cell (RBC) flow profiles in the coronary microcirculation at cellular resolution. Methods: C57BL/6 mice (n=20) were used to develop cardiac IVM protocols. Cardiomyocyte membrane staining was achieved using intravenous injection of a voltage dye (di-2-anepeq). RBC labeling was done by ex-vivo staining of cells with Ter119 followed by transfusion. IVM was performed through a thoracotomy with animals anesthetized and ventilated. Confocal and two-photon microscopy was done using a custom tissue stabilizer and an Olympus FV1000MPE microscope synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols. Images were analyzed in MATLAB to extract myocyte contractile properties, capillary diameter, and capillary flow properties. Results: Using cardiac and respiratory gating, images with cellular resolution could be measured in the beating heart at all phases of the cardiac cycle. This enabled quantification of cardiomyocyte contractile function and capillary diameter and widefield imaging by stitching of several images (Fig. 1A). Tracking of RBCs over serial images allowed quantification of RBC velocity across multiple capillaries simultaneously (Fig. 1B). Conclusion: Cardiac IVM can provide quantification of cardiac function and blood flow at the single cell scale and will provide an exciting tool for investigating CMD in models of heart disease.

Detection of Melanoma Cells in Whole Blood Samples Using Spectral Imaging and Optical Clearing

Most cancer deaths are associated with metastases resulting from the spread of circulating tumor cells (CTCs) from the primary tumor to vital organs. The existing methods for detection of CTCs as markers of metastasis progression are time consuming with several steps of sample processing, including red blood cell removal, labeling, immunomagnetic capture and isolation, which can lead to loss of CTCs. Here we introduce a method for detection and identification of CTCs using spectral absorption imaging of melanoma cells and optical clearing of whole blood samples. Verification of this approach was performed using phantoms of human melanoma cells and suspensions of mouse melanoma cells of line B16F10 alone and in mixture with blood. A method for improving detection sensitivity has been demonstrated applying optical clearing of mouse blood using biocompatible chemical agents. The findings suggest that the proposed diagnostic platform has the potential to detect quickly CTCs in whole blood samples from patients with melanoma.

Reprogramming the rapid clearance of thrombolytics by nanoparticle encapsulation and anchoring to circulating red blood cells

Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.

Chromosome aberrations during in-vitro labeling of red blood cells with 99mTc: a study with cytokinesis block micronucleus assay in human peripheral blood lymphocytes.

Radiopharmaceuticals are special medicines composed by a radionuclide and a non-radioactive compound characterized by non-pharmacodynamic effects, low prevalence of side effects, and a possible risk of oncogenesis, since its administration to patients supposes a radiation dose to organism. Over these years, radiation damage induced by diagnosis radiopharmaceuticals has been evaluated, including the radiolabeled autologous cells, a group of radiopharmaceuticals where blood cells extracted from patients are labeled in-vitro and readministered for diagnosis. There is not a consensus about the possibility of increasement of risk for malignancies associated with the radiolabeled blood cells, so for a more accurate evaluation of the potential oncogenic risk related to the administration of [Tc]Tc labeled red blood cells, radiation dose received by the cells during the labeling process is studied by means of the cytokinesis-blocked micronucleus (CBMN) assay and a dose-response curve constructed by in-vitro external irradiation of blood samples. Our work enables to establish the range of activity to be added during the in-vitro labeling of red blood cells with [Tc]Tc pertechnetate to avoid radiation damage to cells. Activities recommended for blood volume determination and angiography do not increase the risk of malignancies, whilst activities of 370 MBq show chromosome aberrations in lymphocytes. Evaluation of the radiation damage related to the in-vitro labeling is recommended to estimate the potential oncogenic risk and minimize it.

Left ventricular ejection fraction determined with the simulation of a very low-dose CZT-SPECT protocol and an additional count-calibration on planar radionuclide angiographic data

PurposeTo determine whether the left ventricular ejection fractions (EFs), measured on a high-sensitivity CZT single photon emission computed tomography (SPECT)-camera with a 70% reduction in recording times and a prevention of EF overestimation through an additional count-calibration, are concordant with reference EF from planar radionuclide angiography (2D-RNA). MethodsAn additional 10-minute CZT-SPECT recording was performed in patients referred to 2D-RNA for cardiomyopathy (n = 23) or chemotherapy monitoring (n = 50) with an in vivo red blood cell labeling with 850 MBq 99mTcO4-. The EF, obtained from CZT-SPECT with 100% (SPECT100) or 30% (SPECT30) projection times and with a SPECT-count calibration on the 2D-RNA counts of corresponding cavity volumes, were compared to EF from 2D-RNA. ResultsStrong and equivalent relationships were documented between the EF from 2D-RNA and the calibrated EF from SPECT100 (y = 0.89x + 6.62; R2 = 0.87) and SPECT30 (y = 0.87x + 8.40; R2 = 0.85), and the mean EF from SPECT100 (54% ± 15%) and SPECT30 (53% ± 16%) were close to that from 2D-RNA (55% ± 15%). However, upward shifts in these mean values were documented in the absence of count calibration for both SPECT100 (60% ± 18%) and SPECT30 (60% ± 18%). ConclusionLeft ventricular EF may be determined on a high-sensitivity CZT-camera, a 70% reduction in injected activities, and an additional count-calibration for further enhancing the concordance with 2D-RNA values.

Use of Sonographic Contrast-enhanced Agents as an Alternative to Damaged Red Blood Cells for Imaging in Scintigraphy

Scintigraphy imaging is based on the uptake of radiopharmaceuticals by a tissue or organ. Hepatosplenic scintigraphy involves the uptake of labelled colloid by the mononuclear phagocyte system. The radiocolloid distribution in the body, where 80–90% is taken up by the liver, 5–10% by the spleen and 5% by the bone marrow, has the disadvantage of obscuring the rim of the spleen in patients with liver hypertrophy. However, the spleen takes up proportionally more particles if they are larger. While only 10–15% of 99mTc-sulfur colloid particles of 0.4 µ diameter are taken up by the spleen, about 90% of denatured red blood cells (RBCs), approximately 7 µm in diameter, are trapped in the splenic tissue. Because of this, labelled denatured RBCs are used for selective spleen imaging. RBCs present many disadvantages that preclude their use in clinical practice, as the steps involved in efficient labelling and denaturation of RBCs are labor-intensive, time-consuming, and not widely available. Therefore, an alternative method to denatured RBCs is highly desirable. Microbubbles, ultrasound-enhanced agents, are promising candidates for use as an alternative to damaged RBCs. Labelling of microbubbles is potentially simpler, safer, and less expensive. Furthermore, denatured RBCs and ultrasound contrast agents share certain characteristics, such as their size, shape, membrane composition and pharmacokinetics. Based on these similarities, the latter should be tested as an alternative carrier in scintillation scanning. Aside from the potential application of labelled microbubbles as a carrier alternative to denatured RBCs, they could also be used instead of natural RBCs for applications including the investigation of gastrointestinal bleeding, cardiovascular imaging studies, and to localize hemangiomas. Finally, they could be used in renal transplant recipients as a marker of rejection and as an adjunct to the diagnosis in certain diseases in which the immune response includes phagocytic cells.

On-cartridge preparation and evaluation of 68Ga-, 89Zr- and 64Cu-precursors for cell radiolabelling

IntroductionIndium-111 when formulated as indium-111 oxine remains the gold standard for long term cell tracking, whereas radiometals for improved PET applications still have to be established. We here describe the on-cartridge formation of gallium-68, zirconium-89 and copper-64 complexes in small volumes suitable for cell labelling, including labelling of red blood cells (RBC) and white blood cells (WBC) and their biological evaluation in vivo. MethodsSmall volumes (1–2 mL) of tracers (oxine, tropolone) were directly prepared on an anion exchange cartridge (Sep-Pak QMA). Cells were radiolabelled and the labelling efficiency and efflux were evaluated. The in vivo biodistribution of copper-64-labelled WBC using [64Cu][Cu(oxinate)2] and [64Cu][Cu(tropolonate)2] was monitored in an infection and inflammation animal model using BALB/c mice. ResultsOn-cartridge concentration of gallium-68, zirconium-89 and copper-64 enabled formation of oxine and tropolone tracers in small volumes with good yields (≥50%) and quality (extraction ≥90%). Prepared tracers radiolabelled the RBC comparable to indium-111 tracers and in vivo biodistribution of copper-64 labelled WBC showed clear accumulation of cells at the site of infection and inflammation. ConclusionsThis on-cartridge preparation method enables simple formation of various PET tracers for cell radiolabelling. Zirconium-89 and copper-64 tracers radiolabelled cells with sufficient stability. Due to their longer half-life this approach could be promising for routine applications where longer evaluation periods for cell tracking are needed. Advances in knowledge and implications for patient careThis novel approach for on-cartridge concentration and preparation of oxine and tropolone precursors with different positron emitters, in small volume and suitable pH, offers a versatile tool towards cell labelling for preclinical and clinical PET applications.

Tissue Physiology of Cynomolgus Monkeys: Cross-Species Comparison and Implications for Translational Pharmacology

We previously performed a comparative assessment of tissue-level vascular physiological parameters in mice and rats, two of the most commonly utilized species in translational drug development. The present work extends this effort to non-human primates by measuring tissue- and organ-level vascular volumes (Vv), interstitial volumes (Vi), and blood flow rates (Q) in cynomolgus monkeys. These measurements were accomplished by red blood cell labeling, extracellular marker infusion, and rubidium chloride bolus distribution, respectively, the same methods used in previous rodent measurements. In addition, whole-body blood volumes (BV) were determined across species. The results demonstrate that Vv, Vi, and Q, measured using our methods scale approximately by body weight across mouse, rat, and monkey in the tissues considered here, where allometric analysis allowed extrapolation to human parameters. Significant differences were observed between the values determined in this study and those reported in the literature, including Vv in muscle, brain, and skin and Q in muscle, adipose, heart, thymus, and spleen. The impact of these differences for selected tissues was evaluated via sensitivity analysis using a physiologically based pharmacokinetic model. The blood-brain barrier in monkeys was shown to be more impervious to an infused radioactive tracer, indium-111-pentetate, than in mice or rats. The body weight-normalized total BV measured in monkey agreed well with previously measured value in rats but was lower than that in mice. These findings have important implications for the common practice of scaling physiological parameters from rodents to primates in translational pharmacology.

Labelling of Red Blood Cells with Technetium-99m for Nuclear Medicine Studies

ABSTRACT Autologous red blood cells can be labelled with the gamma emitting radionuclide, technetium-99m (Tc-99m) and used for nuclear medicine imaging procedures. Over the last 15 years, the use of Tc-99m red blood cells has ranged from placental and spleen imaging studies to gastrointestinal bleeding and ventricular radiography studies. In vitro, in vivo and modified in vivo methods have been described by several authors to maximize the efficiency of the Tc-99m binding to red cells. The mechanism of radiolabelling likely involves the binding of a reduced form of Tc-99m to intracellular components in the red cell. The amount of stannous ion used as a reducing agent is important in providing maximal labelling. Interactions with various drugs including heparin, doxorubicin, iodinated contrast media, methyldopa, quinidine and digoxin have been reported to interfere with the labelling efficiency. RESUME Le gamma radionucleide emis par le technetium 99M ( 99 Tc) peut etre etiquete avec les globules rouges autologues pour l’utilisation de la visualisation de la medecine nucleaire. Durant les quinze dernieres annees, l’utilisation des globules rouges fixes au 99 Tc a demontre une bonne visualisation de la rate de placenta ainsi que des saignements gastro-intestinaux et ventriculaires. Certains auteurs ont decrit les methodes in vitro , in vivo et in vitro modifiees maximisant l’efficacite de la fixation du 99 Tc reduit aux elements intracellulaires des globules rouges. Il est important de fournir l’etiquetage au maximum a cause du montant d’ions stanneux utilises comme agent de reduction. Quelques interactions medicamenteuses ont ete rapporte avec certaines agents dont l’heparine, la doxorubicine, les produits de contraste iodes, la methyldolpa, la quinidine et la digoxine qui ont interfere avec l’efficacite de l’etiquetage.

Development, validation, and potential applications of biotinylated red blood cells for posttransfusion kinetics and other physiological studies: evidenced-based analysis and recommendations.

The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-51 (51 Cr) RBC labeling for determining RBC volume, 24-hour posttransfusion RBC recovery, and long-term RBC survival. Here we provide evidence supporting adoption of a method for kinetics that uses the biotin-labeled RBCs (BioRBCs) as a superior, versatile method for both regulatory and investigational purposes. RBC kinetic analysis using BioRBCs has important methodologic, analytical, and safety advantages over 51 Cr-labeled RBCs. We critically review recent advances in labeling human RBCs at multiple and progressively lower biotin label densities for concurrent, accurate, and sensitive determination of both autologous and allogeneic RBC population kinetics. BioRBC methods valid for RBC kinetic studies, including successful variations used by the authors, are presented along with pharmacokinetic modeling approaches for the accurate determination of RBC pharmacokinetic variables in health and disease. The advantages and limitations of the BioRBC method-including its capability of determining multiple BioRBC densities simultaneously in the same individual throughout the entire RBC life span-are presented and compared with the 51 Cr method. Finally, potential applications and limitations of kinetic BioRBC determinations are discussed.

Impact d’une réduction d’activité sur la mesure de la fraction d’éjection du ventricule gauche sur caméra D-SPECT

IntroductionRadionuclide ventriculography provides a reproducible measurement of the left ventricular fraction ejection (LVEF) but with a significant body radiation (effective dose of 5,9 mSv for the injection of 850 Mbq of 99mTc). The highly sensitive semi-conductor (CZT) cameras could allow decreasing the injected activity by a factor 3, similarly to that of myocardial perfusion imaging. Our study was aimed to determine whether the LVEF measurement provided by radionuclide ventriculography on the CZT D-SPECT camera is impacted by a 70% reduction in recorded counts. Materials and methodsAfter the in vivo labeling of red blood cells with 850 MBq of 99mTc, 49 patients completed a conventional 2D recording (Conv-2D) on Anger camera followed by a 3D recording on the D-SPECT camera (3D-100%). The CZT recordings of all projections were subsequently shortened to 30% of their initial durations (3D-30%) in order to assess the LVEF measured with a 70% reduction in recorded counts. ResultsMean LVEF values were 62.7±11.1% on Conv-2D and higher on both 3D-100% (66.8±14.8%, P<0.001) and 3D-30% (66.3±15.7%, P<0.001). The correlation coefficients with the LVEF determined with the reference Conv-2D method were equivalents for 3D-100% (r2=0.73) and 3D-30% (r2=0.70) and with a similar level of overestimation for the highest LEVF values. ConclusionA 70% reduction in recorded counts does not significantly impact the LVEF measured with radionuclide ventriculography on the CZT D-SPECT camera. These values are coherent with those obtained with the reference 2D method but with a clear overestimation for the highest LVEF values.

Estimation of adult and neonatal RBC lifespans in anemic neonates using RBCs labeled at several discrete biotin densities.

BackgroundPrior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifespans than allogeneic adult RBCs were not based on direct comparison of autologous neonatal vs. allogeneic adult RBCs performed concurrently in the same infant. Biotin labeling of autologous neonatal RBCs and allogeneic adult donor RBCs permits concurrent direct comparison of autologous vs. allogeneic RBC lifespan.MethodsRBCs from 15 allogeneic adult donors and from 15 very low birth weight (VLBW) neonates were labeled at separate biotin densities and transfused simultaneously into the 15 neonates. Two mathematical models that account for the RBC differences were employed to estimate lifespans for the two RBC populations.ResultsMean±SD lifespan for adult allogeneic RBC was 70.1±19.1 d, which is substantially shorter than the 120 d lifespan of both autologous and adult allogeneic RBC in healthy adults. Mean±SD lifespan for neonatal RBC was 54.2±11.3 d, which is only about 30% shorter than that of the adult allogeneic RBCs.ConclusionThis study provides evidence that extrinsic environmental factors primarily determine RBC survival (e.g., small bore of the capillaries of neonates, rate of oxygenation/deoxygenation cycles) rather than factors intrinsic to RBC.

Biochemical Surrogate Markers of Hemolysis Do Not Correlate with Directly Measured Erythrocyte Survival in Sickle Cell Anemia

Introduction: Hemolysis has been proposed as a cause of several complications of sickle cell anemia (HbSS), including pulmonary hypertension, priapism, leg ulcers, and stroke. No human study of the role of hemolysis in these complications has directly quantified hemolysis; all used surrogate markers. We applied a recently validated stable isotope glycine red blood cell (RBC) labeling method (Am J Hematol. 2015;90:50-5) to measure RBC survival, a direct measure of the rate of hemolysis, to determine whether commonly used surrogate markers of hemolysis accurately estimate RBC survival in HbSS.Methods: Orally administered stable isotope-labeled glycine (15N-glycine), a metabolic precursor of heme, was used to track an age cohort of RBCs in participants with HbSS. The atomic excess of 15N in heme extracted from blood over time was monitored by mass spectrometry to calculate mean RBC survival. We also measured complete blood counts, reticulocyte counts, fetal hemoglobin (HbF), blood levels of biochemical surrogates of hemolysis (LDH, AST, bilirubin, and plasma free Hb), alpha-globin genotype, and tricuspid jet regurgitant velocity (TRV) by echocardiography.Results: There were 5 males and 6 females; mean age was 23 years (range 16-41). Two participants completed the study twice for a total of 13 labeling studies. Mean RBC survival was 31.9 days (S.D. 12.0; range 14.1-53.6). Mean RBC survival was inversely correlated with absolute reticulocyte count (ARC; r = -0.84, P < 0.001; Panel A) and percentage of reticulocytes (r = -0.78, P = 0.002). There was also a positive correlation between mean RBC survival and percent HbF (r = 0.58, P = 0.038; Panel B). The commonly used biochemical surrogate markers of hemolysis, AST, LDH, total bilirubin, indirect bilirubin, and plasma free Hb, were not correlated with directly measured RBC survival (Panels C & D). The "hemolytic index" or "hemolytic component" [derived from the combination of LDH, AST, total bilirubin and reticulocyte count (Blood. 2009;114:4639-44)] was not more strongly correlated with mean RBC survival than ARC (r = -0.73, P = 0.004). That is, the hemolytic component provided no more information about mean RBC survival than ARC alone. We found a correlation between TRV and LDH (r = 0.62, P = 0.03; Panel E), as previously described, but no correlation between TRV and directly measured RBC survival in the same participants (r = 0.09, P = 0.79; Panel F). This indicates that the relationship between TRV and LDH, while valid and reproducible, is not readily explained by hemolytic rate.Conclusions: RBC labeling with orally administered 15N-glycine is a safe and practical method to measure RBC survival directly, and thereby quantify hemolysis. Commonly used biochemical surrogate markers of hemolytic rate (LDH, AST, bilirubin, and plasma free Hb) did not correlate with directly measured RBC survival. These markers should be interpreted cautiously in HbSS. Only reticulocyte count and HbF level correlated with directly measured RBC survival. Although ARC appears to be a reasonable surrogate marker of hemolysis, it is indirect and explains only 70% of the variation in RBC survival. If greater accuracy is required for physiological studies or clinical trials, 15N-glycine RBC labeling can directly and accurately quantify hemolysis. [Display omitted] DisclosuresQuinn:Amgen: Research Funding; Eli Lilly: Research Funding; Silver Lake Research Corporation: Consultancy. Joiner:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Reduced cardiac volumes in chronic fatigue syndrome associate with plasma volume but not length of disease: a cohort study

ObjectivesTo explore potential mechanisms that underpin the cardiac abnormalities seen in chronic fatigue syndrome (CFS) using non-invasive cardiac impedance, red cell mass and plasma volume measurements.MethodsCardiac MR (MR) examinations were...

Clearance of stored red blood cells is not increased compared with fresh red blood cells in a human endotoxemia model.

It is thought that the clearance of transfused red blood cells (RBCs) is related both to the storage time of the transfusion product and to the inflammatory status of the recipient. We investigated these effects in a randomized, "two-hit," healthy volunteer transfusion model, comparing autologous RBCs that were stored for 35 days with those that were stored for 2 days. Healthy male volunteers donated 1 unit of autologous RBCs either 2 days (2D) or 35 days (35D) before the study date. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). Two hours later, the stored RBCs ("second hit") were reinfused, followed by the labeling of RBCs with biotin. Clearance of biotin-labeled RBCs (BioRBCs) was measured during the 5-hour posttransfusion endotoxemia period along with measurements of phosphatidylserine (PS) exposure, lactadherin binding, and expression of CD47 (cluster of differentiation 47; a transmembrane protein encoded by the CD47 gene). In the 2D stored RBCs group, 1.5% ± 3.4% of infused BioRBCs were cleared from the circulation 5 hours posttransfusion versus 4.8% ± 4.0% in the 35D stored RBCs group (p = 0.1). There were no differences in PS exposure, lactadherin binding, or CD47 expression between fresh and stored RBCs or between pretransfusion and posttransfusion measurements. Our study shows a low clearance of RBCs even during endotoxemia. Furthermore, short-term clearance of BioRBCs during endotoxemia was not related to storage duration. Consistent with these observations, PS exposure, lactadherin binding, and CD47 expression did not differ between 2D and 35D stored cells before or after transfusion. We conclude that, in the presence of endotoxemia, clearance of 35D stored autologous RBCs is not increased compared with 2D stored fresh RBCs.

Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry

The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria.

Autologous Infant and Allogeneic Adult Red Cells Demonstrate Similar Concurrent Post-Transfusion Survival in Very Low Birth Weight Neonates

Based on the hypothesis that neonatal autologous red blood cell (RBC) survival (RCS) is substantially shorter than adult RBC, we concurrently tracked the survival of transfused biotin-labeled autologous neonatal and allogeneic adult RBC into ventilated, very low birth weight infants. RBC aliquots from the first clinically ordered, allogeneic adult RBC transfusion and from autologous infant blood were labeled at separate biotin densities (biotin-labeled RBC [BioRBC]) and transfused. Survival of these BioRBCs populations were concurrently followed over weeks by flow cytometric enumeration using leftover blood. Relative tracking of infant autologous and adult allogeneic BioRBC was analyzed by linear mixed modeling of batched weekly data. When possible, Kidd antigen (Jka and Jkb) mismatches between infant and donor RBCs were also used to track these 2 populations. Contrary to our hypothesis, concurrent tracking curves of RCS of neonatal and adult BioRBC in 15 study infants did not differ until week 7, after which neonatal RCS became shortened to 59%-79% of adult enumeration values for uncertain reasons. Analysis of mismatched Kidd antigen RBC showed similar results, thus, confirming that BioRBC tracking is not perturbed by biotin RBC labeling. This study illustrates the utility of multidensity BioRBC labeling for concurrent measurement of RCS of multiple RBC populations invivo. The similar RCS results observed for neonatal and adult BioRBCs transfused into very low birth weight infants provides strong evidence that the circulatory environment of the newborn infant, not intrinsic infant-adult RBC differences, is the primary determinant of erythrocyte survival. Clinicaltrials.gov: NCT00731588.