Apoptotic Microrna Profiling of Packed Red Blood Cells During Storage.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3145-3145
Author(s):  
Meganathan Kannan ◽  
Sandhya Kulkarni ◽  
Chintamani D Atreya
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Storage Period ◽  
Storage Condition ◽  
Packed Red Blood Cells ◽  
Cellular Expression ◽  
Underlying Mechanisms ◽  
Packed Rbcs

Abstract Abstract 3145 Poster Board III-82 Introduction During storage, RBCs undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo, following transfusion. MicroRNAs (miRs), the negative regulators of cellular mRNAs, control cellular expression of genes relevant to differentiation and apoptosis via mRNA degradation or inhibition of translation. Recent reports indicate that matured red blood cells (RBC) contain diverse population of miRs in abundance. Understanding the role of miRs in RBC during storage would perhaps provide insights into the mechanisms associated with storage lesions. Methods In this study, we utilized a membrane-based array to obtain differential miR profiles of 52-apoptosis-associated miRs in packed RBCs during storage. The packed RBCs were obtained from the National Institutes of Health (NIH) blood bank and stored at appropriate storage condition (4-8°C) for up to 40 days. Samples were collected at days 0, 10, 20, 30 and day 40 and subjected to miR analysis. Our rationale is that since miRs are regulators of apoptosis, profiling of apoptosis associated miRs in packed RBCs during storage would provide the first step towards understanding the underlying mechanisms associated with storage lesions. Results Our miR analysis identified perturbation of six miRNAs during packed RBC storage. Two miRs remain at high levels throughout the RBC storage period studied while four miRs demonstrated an upward trend from day 0 to day 40 of storage. TarMir bioinformatics-based target gene identification for miR-96 identified CASPN1 mRNA as its target. The presence of CASPN1 mRNA was confirmed by RT-PCR. Although this observation tempts us to speculate that an interaction of CASPN1 with miR-96 in stored RBC is possible, further experimental verification of this interaction is warranted. Conclusion The differential microarray analysis presented here suggest that further refinement of miR profiling of stored red blood cells could be of value as predictive markers of ‘cellular status of RBC’ during storage. Future experimental analysis of selected mRNA-miR interactions in packed RBC during storage would provide insights into the mechanisms of storage lesions The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures No relevant conflicts of interest to declare.

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Spatially Organized Structure of Coronary Thrombus in Acute Myocardial Infarction

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 716-716
Author(s):  
Jan E. Dyr ◽  
Tomas Riedel ◽  
Jana Stikarova ◽  
Jiri Suttnar ◽  
Jaroslav Cermak ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Red Blood Cells ◽  
Symptom Onset ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Freeze Fracture ◽  
Ischemic Time ◽  
Internal Structures

Abstract Introduction The use of thromboaspiration in primary percutaneous intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) has offered a unique opportunity to study thrombus composition, its dynamic formation, and architecture in vivo. There has been, however, several limitations, not least the fact that the technique has not yet allowed a precise transversal analysis from one side of the artery to the other, as is done in histological analysis. The dynamic process of intracoronary thrombus formation in STEMI patients is thus still not well understood. Ischemic time was hypothesized to be among the strongest independent correlates of thrombus architecture. In time the platelets are decreasing its proportion and fibrin proportion is increasing (J Silvain, J-P Collet, JW Weisel et al, J Am Coll Cardiol 2011; 57:1359). However, no real report on the internal structures of the in vivo formed thrombi has been shown so far. Therefore, we investigated both the surface and the composition of longitudinally freeze-fractured thrombi. Methods Thrombi were collected by PCI from 119 STEMI patients. Out of the patients there were "early comers " (˃12 h from symptom onset; 23 patients) and "late comers" (more than 720 min; 29 patients). The mean age of all patients was 64 years, 70% of patients were males, 51% were smokers, 50% had arterial hypertension, 20% were diabetics and 23% had chronic renal insufficiency. Scanning electron microscopy; collected thrombi obtained by PCI were thoroughly washed in saline solution and stored in 4% formaldehyde prior dehydration. To reveal the internal structures of the thrombi selected samples were longitudinally freeze fractured in liquid nitrogen and coated with platinum. Samples were examined in SEM Vega Plus TS 5135 (Tescan s.r.o., Brno, Czech Republic). Whole areas of the freeze-fractured thrombi were scanned. Results and discussion The thrombus composition of longitudinally freeze-fractured thrombi was compared between groups of "early-comers" and "late-comers. The distribution of the components in the "early comers" thrombi freeze-fracture seemed to be uniform. Platelets were far the main component (about 75 % in proportion) of the "early comers" thrombus, followed by fibrin and other compounds. The amount of red blood cells was negligible (about 2 - 8 %). We did not observe any significant differences between the thrombi in the group of early comers. Thrombi of the "late-comers" group were composed mainly of red blood cells; platelets and fibrin formed only minority of the thrombi. In contrast to the "early comers" the distribution of the main thrombus components in the "late comers" thrombi was dramatically different between individual parts of the thrombus. The number of platelets and red blood cells varied from 0% to almost 99% and vice versa. It was possible to estimate the initiating place of the thrombus as well as the direction of the growth. Each thrombus could be divided into parts formed mainly either by platelets or by red blood cells. It seems that thrombus develops a regional architecture defined by the extent of platelet activation and packing density. It has been reported that in contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface demonstrating how contracted clots form an impermeable barrier important for hemostasis and wound healing (D Cines, T Lebedeva, J Weisel et al, Blood 2014; 123:1596). Our investigation of the composition of the in vivo formed thrombi supports these results and helps to explain how fibrinolysis is greatly retarded as clots grow and contract. We have found that on the surfaces of late-comers thrombi fibrin thick fibrils were present. It has been shown that the association of soluble fibrinogen with the fibrin clot results in the reduced adhesiveness of such fibrinogen/fibrin matrices toward leukocytes and platelets (VK Lishko, T Burke, T Ugarova, Blood 2007; 109:1541). Fibrinopeptides A are less accessible for thrombin in surface bound fibrinogen which thus provides additional level of protection of thrombi from premature dissolution (T Riedel, L Medved, JE Dyr, Blood 2011; 117:1700). These findings may have great impact on our knowledge of pathophysiology of the thrombus growth and possible therapeutic consequences related to the time of symptom onset. Disclosures No relevant conflicts of interest to declare.

Donor Variation In Biological Mediators During Storage Of Packed Red Blood Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3655-3655 ◽  
Author(s):  
Melinda M Dean ◽  
Luke D Samson ◽  
Kelly Rooks ◽  
Jesse Fryk ◽  
Shoma Baidya ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Immune Modulation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Biological Response ◽  
Storage Lesion ◽  
Packed Red Blood Cells ◽  
Donor Variation ◽  
Time Point

Abstract Introduction During routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. Methods Cytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). Results Of the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P<0.001, ANOVA). Of particular interest, the concentrations of these nine biological mediators varied greatly between the individual PRBC units. ICAM-1, VCAM-1 and IL-1α concentrations each varied 10 fold between units (range 1000 – 10 000 pg/mL for each), sCD14 varied 5 fold (range 20 000 - 100 000 pg/mL), sCD62L varied 4.4 fold (range 9000 – 40 000 pg/mL), and sCD62P varied 6.5 fold (range 200 -1300 pg/ml). In addition, it was apparent that a sub population (3/8) of the units assessed consistently had the highest levels of ICAM-1, sCD106 (VCAM-1), sCD14, sCD62L, IL-1α, sCD62P and angiogenin. For sCD62P, in particular, this “high” sub population had significantly different levels of sCD62P at each time point compared to the other five units (P<0.05 at each time point). The remaining BRMs studied were at the limits of detection (<20 pg/mL) for every unit at each time point, and no storage related changes were evident. Conclusions There was minimal change in the BRMs studied relevant to storage duration of the PRBC units. The most notable differences in the levels of biological mediators present in PRBC SN were due to donor-to-donor variation. These data suggest high levels of BRMs and potential immune modulation in transfusion recipients may be the result of donor-associated differences rather than storage-associated differences in blood components. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Quality control in the different stages of producing red blood cell concentrate from dogs

2019 ◽  
Vol 71 (1) ◽  
pp. 93-101
Author(s):  
M.N.A. Marchi ◽  
P.E. Luz ◽  
R.R. Martins ◽  
S.M. Simonelli ◽  
U.P. Pereira ◽  
...  
Keyword(s):  
Quality Control ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Storage Period ◽  
Packed Red Blood Cells ◽  
Production Stages ◽  
The Mean ◽  
Selection Of

ABSTRACT The objective of this study was to perform a quality control assessment of red blood cells after standardization of the blood production stages. For this purpose, separation of the blood components to obtain red blood cells, the storage of the blood packets and an evaluation of blood quality were performed. The mean (± SD) volume, globular volume, hemoglobin and hemolysis percentage of the red blood cell concentrate were 299.77±30.08mL, 60.87±2.60%, 20.57±0.93g/DL and 0.09±0.07%, respectively. The means (± SD) of the volume, globular volume, total hemoglobin percentage of hemolysis and hemoglobin per unit of packed red blood cells after the storage period (8.83±6.73 days) were 57.55±3.01%, 20.30±0.89 0, 20±0.12%, and 60.90±7.65. The red blood cell packets were within the parameters of quality control established by Health Ministry legislation in humans and allow us to conclude that the standardization of blood production stages involves the selection of donors until the end of storage and is necessary to produce quality red blood cells. Quality control aims to find possible flaws in the procedures to be repaired, increasing transfusion safety.

Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2313-2313
Author(s):  
Christopher G. Skipwith ◽  
Juan (Jenny) Xiao ◽  
John W. Weisel ◽  
X. Long Zheng
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Full Length ◽  
Apparent Difference ◽  
Cell Accumulation ◽  
Carboxyl Terminal ◽  
Normal Hemostasis

Abstract Abstract 2313 Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity. Disclosures: No relevant conflicts of interest to declare.

Incidents Related to the Transfusion By Hemosiderosis Notified to the National Haemovigilancy System: Results from Last Two YEARS in Our Hospital

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4745-4745
Author(s):  
Maria Almudena Garcia Ruiz ◽  
Romero Martinez Francisco Jose ◽  
Maria Pilar Garrido Collado ◽  
Estefania Morente Constantin ◽  
Manuel Jurado
Keyword(s):  
Iron Overload ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
World Health ◽  
Near Misses ◽  
Packed Red Blood Cells ◽  
History Of ◽  
Iron Overdose ◽  
Health Organization

Abstract OBJECTIVES According to the World Health Organization, "Haemovigilance is required to identify and prevent occurrence or recurrence of transfusion related unwanted events, to increase the safety, efficacy and efficiency of blood transfusion, covering all activities of the transfusion chain from donor to recipient." The system should include monitoring, identification, reporting, investigation and analysis of adverse events near-misses and reactions related to transfusion and manufacturing. Transfusion-dependent patients receive iron overdose in each transfusion: one packed red blood cells containing 200-250 mg iron (1 mg/ml), which accumulates gradually in different tissues. Transfusions of packed red blood cells in a volume ≥120 mL / kg can cause iron overload, which correlates with ferritin levels in serum equal to or greater than 1000 g/L. The iron overload may be detected after 10 to 20 transfused packed red blood cells, increasing the risk of morbidity and mortality. PATIENTS, MATERIAL AND METHODS We studied the hematological patients with iron overload in 2013 and 2014, analyzing theirs levels of ferritin. The database of the transfusion service was also used, as well as the transfusion history of patients who had received more than 10 packed red blood cells, correlating with greater than 1000 mg/L ferritin. In 2013, 57 cases transfused with packed red blood cells were reported: 32 men and 25 women between 20 and 87 years (average: 56). Results vary between 10 and 73 concentrates (average: 25). Posttransfusion ferritin levels exceeded 1.000 mg/L, with an average of 2869 mg/L. The accountability and severity was recorded as non-assessable. In 2014, 76 cases were reported between the ages of 24 and 83 years (average: 56 years), with 50 men and 26 women. The number of packed red blood cells transfused ranged from 10 to 130 (average: 31). The ferritin posttransfusion quantities ranged from 1.041 to 15.190 mg/L. Regarding accountability, 23 were grade 2, 43 were grade 1 and 10 were not assessable. The severity was recorded as non-assessable. Table. Cases reported in 2013 and 2014 Year Cases (nº) Average Packed red blood cells average per patient Posttransfusion ferritin levels 2.013 57 56 25 2.869 μg/L (average) 2.014 76 56 31 Values between 1041-15190 μg/L CONCLUSIONS It is very important to keep track of ferritin levels in polytransfused patients. In order to reduce the risk of hemosiderosis, it is essential the optimal use of component (safe, efficient and clinically effective) and that the transfusion is performed when the patient needs it. Chelation reduce morbidity in patients with transfusional dependency and iron overload. In the annual report of Andalusian Health Service, the incorporation of Haemovigilance system of notifications of post-transfusion hemosiderosis stands out, although only 4 hospitals shared the information, therefore working in this field is necessary. We believe it is essential to establish protocols to improve the reporting of incidents by hemosiderosis Haemovigilance System. Disclosures No relevant conflicts of interest to declare.

scholarly journals Plasma from stored packed red blood cells and MHC class I antibodies causes acute lung injury in a 2-event in vivo rat model

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 2079-2087 ◽  
Author(s):  
Marguerite R. Kelher ◽  
Tomhiko Masuno ◽  
Ernest E. Moore ◽  
Sagar Damle ◽  
Xianzhong Meng ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Red Blood Cells ◽  
Mhc Class I ◽  
Rat Model ◽  
Blood Cells ◽  
Class I ◽  
Packed Red Blood Cells ◽  
Stored Blood

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.

scholarly journals The Entry of Sodium into Human Red Blood Cells in Vivo

1966 ◽  
Vol 50 (1) ◽  
pp. 75-88 ◽  
Author(s):  
L. J. Beilin ◽  
D. Eyeions ◽  
G. Hatcher ◽  
G. J. Knight ◽  
A. D. Munro-Faure ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cells ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Compartmental Analysis ◽  
Analogue Computer ◽  
Packed Red Blood Cells ◽  
Human Red Blood Cells ◽  
In Series

The kinetics of sodium, movement into human red blood cells has been studied in vivo with 24Na. When human serum albumin-131I is used to measure the percentage of plasma trapped in the packed red blood cells after centrifugation, approximately 30 % of red blood cell sodium is found to equilibrate immediately with plasma. It is concluded that this immediately exchangeable compartment of red blood cell sodium is an experimental artefact, associated with the use of labeled albumin for measuring plasma trapping. This immediately exchangeable fraction disappears when sucrose-14C is used to measure plasma trapping. The experimental results were examined by compartmental analysis, using an analogue computer. The results obtained, when plasma trapping was measured with sucrose-14C could be simulated by the use of models containing two compartments, arranged in series or in parallel. The errors of the techniques used and the possible physical basis for the results are discussed.

α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3355-3355
Author(s):  
Nicole Tucker ◽  
Monika Dzieciatkowska ◽  
Kirk Hansen ◽  
Samina Khan ◽  
Marguerite Kelher ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Injury Severity ◽  
Conflicts Of Interest ◽  
Injured Patient ◽  
Post Injury ◽  
Packed Red Blood Cells ◽  
Severity Scores ◽  
Moonlighting Proteins ◽  
Injured Patients

Abstract Abstract 3355 A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods: Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results: HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Ibrahim Mustafa ◽  
Asma Al Marwani ◽  
Khuloud Mamdouh Nasr ◽  
Noora Abdulla Kano ◽  
Tameem Hadwan
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Storage Time ◽  
Morphological Changes ◽  
Storage Period ◽  
Osmotic Fragility ◽  
Prolonged Storage ◽  
Morphological Alterations ◽  
Packed Red Blood Cells ◽  
Stored Blood

Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2–6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy.

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