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Red blood cell storage and adhesion to vascular endothelium under normal or stress conditions

An in vitro microfluidic study

Diebel, Lawrence N. MD; Liberati, David M. MS

Journal of Trauma and Acute Care Surgery: June 2019 - Volume 86 - Issue 6 - p 943–951
doi: 10.1097/TA.0000000000002239
AAST 2018 PODIUM PAPER
Free
Editor's Choice

BACKGROUND Observational studies have identified an association between duration of red blood cell (RBC) storage and adverse outcomes in trauma. Hemorrhagic shock (HS) leads to impaired tissue perfusion which is associated with endothelial cell glycocalyx (eGC) shedding. Adhesion of stored RBC to the vascular endothelium has been shown to lead to impaired perfusion in the microcirculation and contribute to organ failure and poor outcome. The role of either or both of the EC and RBC glycocalyx in this process is unknown and was studied in an in vitro model.

METHODS Human umbilical vein endothelial cells were perfused in a microfluidic device with RBC solutions from fresh, less than 14-day or longer than 21-day storage. In some experiments, the HS microenvironment was simulated by hypoxia-reoxygenation (H/R) and epinephrine (Epi) in the perfusion experiments. Measurements obtained included endothelial cell (EC) and RBC glycocalyx and RBC adherence to human umbilical vein endothelial cell monolayers at variable shear rates.

RESULTS Endothelial cell glycocalyx and RBC glycocalyx dimensions were reduced by H/R and Epi and storage duration respectively. Red blood cell adherence to the endothelium was increased by H/R + Epi treatment and duration of RBC storage.

CONCLUSION Our data may help explain some of the remaining discrepancies regarding the impact of RBC storage duration on outcomes in the trauma population. Consideration of the integrity of the EC and RBC glycocalyx may guide future transfusion strategies in the trauma population. The microfluidic device system platform may offer a high throughput modality to study emerging therapies to mitigate adverse consequence of RBC storage duration on the perfused endothelium in the trauma setting.

From the Michael and Marian Ilitch Department of Surgery (L.N.D., D.M.L.), Wayne State University, Detroit, Michigan.

Submitted: November 9, 2018, Revised: January 29, 2019, Accepted: February 14, 2019, Published online: March 1, 2019.

This study was presented at the 77th annual meeting of the American Association for the Surgery of Trauma, San Diego, CA September 26-29, 2018.

Address for reprints: Lawrence N. Diebel, MD, Michael and Marian Ilitch Department of Surgery, 6C University Health Center, 4201 St. Antoine, Detroit, MI 48201; email: ldiebel@med.wayne.edu.

Trauma patients are frequently given blood transfusions either during the resuscitation phase or within the first few days following injury. A number of reviews have addressed the impact of stored blood in the trauma population.1–3 The effect of the age of stored blood used has been carefully analyzed in a number of large studies and meta analyses. These studies suggest that red blood cell (RBC) storage time does not affect mortality.4,5 However, there is continued debate regarding other outcomes, including organ failure and infectious complications. Moreover, the trauma population has been underrepresented in recent randomized trials that attempted to study the effect of the duration of blood storage on outcome.2,6 There are also a number of confounding variables in the trauma population which complicates analysis of the age of transfused allogeneic RBCs. These include number of units of blood transfused and the timing of transfusion in relation to initial injury and the mixing of old and relatively “fresh” units of packed RBCs (PRBC) administered.7,8

Disturbances in the microcirculation have been described in trauma and other critically ill patients. This is relevant because impaired perfusion in the microvasculature has been shown to impact the response to RBC transfusion.9 In addition, the endothelial glycocalyx (eGC) is now recognized for its importance in the vascular barrier and promoting homogeneous blood flow distribution in the microcirculation.10–13 The eGC regulates vascular permeability, coagulation, and interactions between the endothelial cells and the blood and acts as a mechanotransducer of fluid shear stress on vascular tone via endothelial nitric oxide (NO) activity. As such, the eGC has been referred to as the “helmet” of the microcirculation in trauma.14

Hemorrhagic shock (HS) has been shown to cause glycocalyx degradation and vascular barrier injury, and their magnitude is related to the severity of the insult.15–17 Because the eGC is the first layer of the vascular endothelium to come in contact with blood cells, it is likely that damage to the eGC would impact the flow properties of blood cells, especially RBCs. Red blood cell-vascular endothelial interactions have a causal relationship to the pathology of several disease states including sickle cell anemia, malaria and diabetes. A previous study by Chin-Lee and colleagues18 demonstrated that stored RBCs showed increased adherence of stored RBCs compared with fresh RBCs in the rat microvasculature. Anniss et al.19 demonstrated that storage duration increases adhesion of stored RBC to the vascular endothelium in an in vitro model. In another study by this group, a variable effect on RBC adhesion to activated vascular endothelium was noted.20 However, the role of the eGC in effecting RBC adhesion was not addressed in these studies.

Red blood cells are also covered by a glycocalyx. Aging of RBCs in circulation is associated with a decrease in thickness of its glycocalyx. The thickness of the RBC glycocalyx is also diminished in certain forms of hypertension, obesity, and diabetes.21 Oxidative stress is common in clinical conditions associated with glycocalyx degradation.22,23 Because oxidative stress may occur during RBC storage, we postulated that storage duration would impact the integrity of the RBC glycocalyx. The importance of both the RBC glycocalyx and the eGC in promoting blood flow in the microcirculation has been recognized.24 Under normal circumstances, the eGC with its negatively charged components (especially heparin sulfate) prevents RBCs (also negatively charged) from becoming attached to the endothelial surface which may have important consequences. Loss of the glycocalyx layer from either the endothelium or the RBC surface could increase the interaction of RBC with the endothelial surface of blood cells. Although the primary objective of RBC transfusion is the restoration of oxygen-carrying capacity, it is now apparent that RBC transfusion has an important role in the restoration of microvascular function. Loss or damage to either glycocalyx layer may impair microvascular perfusion due to adhesion of RBCs to the vascular endothelium. This may be more likely if there are perturbations in both glycocalyx layers. We, therefore, postulated that trauma/HS (T/HS) with subsequent eGC degradation and “older” RBC with a diminished glycocalyx layer would have the most adverse effect on blood flow in the microcirculation.

Microfluidics is a technology that has been used to study endothelial cell biology and stored RBC under in vitro flow conditions. This high throughput system allows study of the endothelial-RBC interaction under tightly controlled conditions and was used to evaluate the impact of eGC and RBC glycocalyx on RBC-endothelial interaction in vitro.

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METHODS

Human Umbilical Vein Endothelial Cell Culture

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza Walkersville, Inc. (Walkersville, MD). Cells were grown in a 75-cm2 flask using complete media (EGM-2 BulletKit; Lonza). Time to subculture is 5 days to 9 days, with media changes every 2 days. Cells are subcultured at 85% confluence using 2 mL of 0.5% trypsin-ethylenediaminetetraacetic acid (Life Technologies, Carlsbad, CA). A new culture flask is prepared and the remaining cells are used to seed the microfluidic channels of a BioFlux 48-well plate (Fluxion Bio) that has been primed and coated with 100 μg/mL fibronectin (Fisher Scientific) for 1 hour at room temperature. Monolayers were formed within the microfluidic channels after overnight perfusion of the cells with complete media at a shear force of 1 dyne/cm2. Human umbilical vein endothelial cell at passages 4 to 7 were used for all experiments.

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Well Plate Microfluidic Device System

The main components of the microfluidic device system (MDS) include BioFlux plates, a pressure interface device, a controller instrument, and software for the instrument control and image analysis. The BioFlux plates contain an array of microfluidic flow channels on a well plate format which are connected to inlet and outlet wells. The pressure interface device covers the top of the well plate and applies a controlled pneumatic pressure from the control instrument. This serves to drive the fluid or perfusate through the microfluidic channels at a user-defined flow rate. An observation window in the bottom of the microfluidic channels allows imaging by microscopy. The MDS software allows control of the flow settings, as well as other parameters including image analysis. Thus, the microfluidic device allows the study of the glycocalyx barrier function under flow-induced shear stress under controlled experimental conditions.

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Blood Preparation

Blood was collected from healthy volunteer donors (N = 4) in citrated vacuum tubes containing ethylenediaminetetraacetic acid present in the collection tubes. The blood samples were centrifuged for 10 minutes at 1,400g. After the first centrifugation, the buffy coat is removed and the plasma layer leaving the RBC in the bottom of the tube. Add 3 mL of phosphate-buffered saline (PBS) (no calcium or magnesium) to the tube containing the RBC and centrifuge for a second time for 10 minutes at 1,400g. Discard the PBS and repeat above a third time to yield clean RBCs.25 The RBC segments (<14-day storage [N = 6] and >21-day storage [N = 6]) were also obtained from the blood bank at Harper University Hospital Detroit, MI. The blood bank samples were obtained from saved segments of blood tubes which are routinely attached to the blood unit in blood banks. All RBC samples were diluted in PBS at 1.5% or 23%.

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Experimental Design

Once confluent HUVEC monolayers are formed, RBC obtained from volunteers (fresh) or from the blood bank (<14-day storage or >21-day storage) at 1.5% and 23% cell suspension were added to the perfusate at different flow rates. In some experiments, HUVEC are exposed to 10−3 μM epinephrine (epi) and/or hypoxia for 60 minutes at 37°C and 95% N2/5% O2. Following this, standard culture conditions are reinstated (37°C with 5% CO2; reoxygenation). The RBC adhesion to the endothelial cell monolayer under the microfluidic device under constant flow conditions was determined by light microscopy. The RBC adherence strength was determined by progressively increasing the shear rate from 0.5 dyne/cm2 to 5 dyne/cm2. Glycocalyx shedding in the endothelial monolayer and in fresh RBCs or those stored less than 14 days and greater than 21 days was assessed by measuring both syndecan-1 (Syn-1) release and hyaluronic acid (HLA) present in cell supernatants. Glycocalyx injury was also assessed in HUVEC and RBC by staining with Fluorescein isothiocyante conjugated wheat germ agglutinin (FITC-WGA; Sigma Aldrich, St. Louis, MO) antibody which binds to N-acetyl neuraminic acid and N-acetyl glucosamine residues of proteoglycans and glycoproteins present in the glycocalyx and visualizing the glycocalyx using a fluorescent microscope. Image analysis for quantification of glycocalyx thickness was accomplished using Volocity software.

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RBC Adhesion

Fresh RBC, less than 14-day stored and longer than 21-day stored RBC were added to the perfusate and allowed to flow through the microfluidic device at sequentially increasing shear rates of 0.5 dyne/cm2 to 5 dyne/cm2. Flow of the RBC through the microfluidic device at each increasing shear rate was stopped after 5 minutes, and adherent cells were photographed and counted using the Zeiss Observer Spinning Disk Confocal Microscope (Microscopy, Imaging and Cytometry core facility at Wayne State University, Detroit, MI). Nonadherent RBCs were removed by washing the monolayer with PBS 2× (shear rate of 0.5 dyne/cm2 for 1 minute to wash).

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Syn-1 and HLA Analysis

Quantitative measurement of syndecan protein and HLA shed by HUVEC into the supernatants was accomplished using the Syn-1 human ELISA kit (Abcam, Cambridge, MA) and the hyaluronic immunoassay kit (from R & D Systems, Inc., Minneapolis, MN), respectively. Standards and unknown samples are added to the microplate wells, and assay procedures were followed. The optical density is determined using a microplate reader set to 450 nm, and the concentration of Syn-1 and HLA in the supernatants is calculated using a standard curve. The sensitivity of the Syn-1 and HLA ELISAs are 5 pg/mL and 10 pg/mL, respectively.

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Fluorescent Imaging and Thickness of Glycocalyx

Human umbilical vein endothelial cells were cultured in endothelial cell growth medium supplemented by growth factors (EGM-2 BulletKit) Lonza. Before cell seeding, micro channels were coated with human fibronectin (100 μg/mL; Fisher Scientific) for 1 hour at room temperature. The HUVEC suspensions were seeded into the outlet wells of a 48-well BioFlux plate and infused into the microchannel network using the BioFlux 200 system (Fluxion Biosciences, Inc.). Cells were cultured for 72 hours with complete media under flow conditions (shear force of 1 dyne/cm2) or static conditions. Live cell staining was performed inside the microfluidic channels using WGA conjugated with FITC (Sigma). Briefly, endothelial cell cultures treated with epinephrine + HR or standard media had FITC-WGA infused into the microchannel network using the BioFlux 200 system, and cells were allowed to incubate for 30 minutes. The cells were washed two times, and fresh culture medium was added, and cells were examined under a fluorescent microscope. In a separate series of experiments, RBC glycocalyx thickness and fluorescence intensity measurements of the glycocalyx were also determined. Briefly, RBC were resuspended and incubated in PBS/1% bovine serum albumin containing FITC-WGA for 30 minutes at 37°C. The RBC were subsequently centrifuged (1,400g for 10 minutes), and the supernatant removed. RBCs were resuspended in PBS and centrifuged two more times to remove excess FITC-WGA antibody. Red blood cells were subsequently mounted on a slide with coverslip and imaged using a Leica TCS SP5 inverted fluorescent microscope with a 63× oil objective. Further image analysis was performed using Volocity software at the Microscopy, Imaging and Cytometry core facility at Wayne State University (Detroit, MI). Measurement of glycocalyx thickness was achieved by XYZ image stacks of the endothelial cell layer using a Leica TCS SP5 microscope and a 20× objective.

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Statistical Analysis

An analysis of variance with a post hoc Tukey test was used to analyze the data. Statistical significance was inferred at p values less than 0.05. All data are expressed as mean ± SD.

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RESULTS

The composition of the anticoagulant-preservative solution for RBCs obtained from the blood bank was citrate phosphate dextrose. Red blood cell additive solutions included AS-1 and AS-3. The range of days in storage for the less than 14-day storage group was 7 days to 13 days. The range for the longer than 21-day storage group was 25 days to 38 days.

Endothelial glycocalyx visualized using FITC-labeled WGA demonstrated decreased thickness in the eGC layer after exposure to hypoxia-reoxygenation (H/R) and Epi. Glycocalyx thickness was 41.2 ± 4.6 in the control group and 13.9 ± 2.3 in the H/R + Epi group (p < 0.05) (Figs. 1A and B). Fluorescent intensity of the EC glycocalyx was also measured and was reduced from 265.3 ± 19.6 in the HUVEC control to 143.4 ± 18.5 in HUVEC exposed to both H/R and Epi (p < 0.05).

Figure 1

Figure 1

The effect of storage on the RBC glycocalyx is shown in Figures 2A and B. Fluorescence intensity of the RBC glycocalyx is related to its thickness. The highest intensity was noted in the fresh RBC group with progressive decline in the less than 14-day and longer than 21-day RBC storage groups (p < 0.05).

Figure 2

Figure 2

Endothelial glycocalyx degradation or “shedding” was indexed by the recovery of Syn-1 and HLA glycocalyx components in the microfluidic device perfusate. The Syn-1 and HLA concentrations in the perfusate of HUVEC exposed to H/R and Epi were increased 3.6-fold and 4.5-fold versus the control HUVEC group (p < 0.05) (Fig. 3A). Data for RBC glycocalyx shedding during storage are shown in Figure 3B. The Syn-1 concentration in the RBC solution increased from 28.6 ± 3.6 in fresh RBC to 43.8 ± 3.1 and 55.9 ± 4.5 in the less than 14-day and longer than 21-day RBC storage groups, respectively (p < 0.05 vs. Fresh RBC). Similarly, HLA concentrations in the RBC solution increased from 22.5 ± 2.9 (fresh RBC) to 51.7 ± 5.2 and 66.8 ± 5.8 in the less than 14-day and longer than 21-day RBC groups (p < 0.05 vs. fresh RBC).

Figure 3

Figure 3

The interaction between different RBC groups and the endothelium (normal or experimental group) is shown in Figures 4A and B. The RBC adherence to the control HUVEC monolayer was increased twofold in the less than 14-day storage group and 2.3-fold in the longer than 21-day storage group versus fresh RBCs. Adherence to the HUVEC monolayer exposed to HR and Epi (simulating HS) was increased in all RBC groups. Fresh RBC adherence to the HUVEC monolayer exposed to both H/R and Epi was 80 ± 1.5 while RBC adherence in the less than 14-day storage group increased to 189 ± 21 when perfused over the H/R and Epi HUVEC monolayer (p < 0.05). Notably, the absolute numbers of adherent RBC in the longer than 21-day storage group were 1.5 to 1.8 times greater than the values from the comparable HUVEC group after less than 14 days RBC storage. The RBC groups at 23% cell suspension showed a dramatic increase in adherence to the HUVEC monolayer exposed to HR and Epi versus 1.5% suspension. The number of adherent RBCs at 23% suspension increased twofold in the less than 14-day storage group (325 ± 52) and 2.3-fold in the longer than 21-day storage group (556 ± 68) versus RBC at 1.5% suspension. Fresh RBC adherence increased 1.8-fold in the 23% suspension group (143 ± 46) versus the 1.5% group. The shear rate was progressively increased from 0.5 dyne/cm2 to 5 dyne/cm2 to assess the firmness of vascular adhesion of the RBCs (Figs. 5A and B). There was a linear decrease in RBC adhesion to the vascular endothelium in the control HUVEC group. This result was less apparent in the less than 14-day RBC storage group following H/R and Epi exposure and virtually nonexistent in the longer than 21-day RBC storage group, indicating a much firmer adhesion of the relatively old RBC to the HUVEC layer exposed to conditions simulating hemorrhagic shock.

Figure 4

Figure 4

Figure 5

Figure 5

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DISCUSSION

Red blood cell flow properties include self-aggregation, deformability, and adherence to the vascular endothelium.26 All may adversely affect tissue perfusion. Microfluidic platforms have been used to study RBC aggregation and deformability and have shown significant differences between fresh RBC and stored RBC.27 The decline of these properties is related to the duration of RBC storage. There is minimal information on the effect of RBC storage on the adherence to the vascular endothelium, especially as it relates to the integrity of the glycocalyx layer. Previous studies using isolated endothelial cells to model the endothelial barrier function are limited by the dimensions of the glycocalyx established under standard conditions.28 Endothelial cells cultured within microchannels of microfluidic devices and subjected to physiologic fluid shear stress develop a glycocalyx layer with a thickness comparable to that found in micro capillaries in vivo. We have demonstrated the development of a hydrodynamically relevant glycocalyx layer with this platform in a previous study.29

It has been known for some time that major trauma is associated with endothelial glycocalyx injury as demonstrated by shedding of glycocalyx components into the systemic circulation.15 Naumann and colleagues30 have demonstrated that poor microcirculatory flow dynamics after T/HS are associated with endothelial cell damage and glycocalyx shedding. The impact of these changes in the microcirculation on fresh and stored RBC flow properties is unknown.

It has been shown that RBCs undergo progressive changes when stored for prolonged time. In our study, we demonstrate that the RBC glycocalyx undergoes degradation similar to the RBC cell membrane damage that has been well described. In vivo numerous structural changes of the RBC glycocalyx are known to occur as part of aging.32 These changes may contribute to removal of senescent cells from the circulation. The progressive loss of the RBC glycocalyx with storage may similarly lead to enhanced RBC aggregation and adhesion to the vascular endothelium. Under normal conditions, the anionic properties of the endothelial glycocalyx and the negatively charged RBC determine hydrodynamic resistance and effect the flow pattern of RBCs.23 Obertleiner demonstrated that endothelial and RBC glycocalyx may cause adverse effects when one or both are damaged. This interaction was also suggested by Yalcin et al.31 in a rat cremasteric muscle preparation. Stored but not fresh RBC led to microrheolgic disturbances which included a disruption of the RBC free layer and cell shear stress signals, both of which are related to a functional endothelial glycocalyx.

Our study further delineated the interrelationship of the respective eGC and RBC glycocalyx layers during flow conditions. Fresh versus stored RBC had significantly different glycocalyx layers and were perfused through microfluidic channels lined with control HUVEC with an intact glycocalyx or HUVEC exposed to H/R and Epi to mimic the microcirculation after T/HS. The H/R and Epi exposure resulted in a markedly diminished endothelial glycocalyx layer. We chose to study RBC adherence to the vascular endothelium rather than aggregation or deformability as this property is most reflective of a functional endothelial glycocalyx layer.33,34 Our study demonstrated that RBC endothelial adherence was more significant when both the RBC and endothelial glycocalyx layers were degraded.

The shear-dependent behavior of RBC adherence was demonstrated in our microfluidic system using a range of physiologically relevant shear rates. The effect of shear was most apparent in the HUVEC monolayer control group with either fresh or stored RBCs. However, RBC adherence to the endothelial monolayer subjected to H/R and Epi was significantly greater and relatively unaffected by increasing shear rates. This suggested a much firmer attachment to the HUVEC monolayer in these groups. In vivo, this would likely lead to prolonged compromise of the microvasculature. In summary, our model suggests that the microrheologic impact of stored RBCs is related to the status of the microcirculation and more importantly the endothelial glycocalyx. This may in part be due to the combined effects of increased NO scavenging following RBC storage and decreased endothelial production of NO due to shock induced endothelial glycocalyx degradation.35,36 Most clinical studies regarding detection of glycocalyx degradation from systemic blood is associated with adverse outcomes. However, little information exists regarding the actual glycocalyx thickness or integrity. Our previous study did show an association between endothelial glycocalyx thickness and concentrations of glycocalyx degradation products detected in the perfusate of our biomimetic model of hemorrhagic shock.29 The correlation of syndecan and other glycocalyx degradation products with microvascular glycocalyx thickness was also shown in a rodent model performed by Torres Filho and colleagues.37

There are several limitations to our study. Two dilute RBC solutions were used to quantitate RBC adherence to the endothelium. Viscosity has been shown to effect RBC flow properties which may not be significant when using dilute RBC solutions. Second, other blood elements, including leukocytes, platelets, and plasma, may impact RBC vascular adherence and were not included in the current study. Finally, the rectangular geometric design of the microchannels in our microfluidic plates do not recapitulate the complex branching pattern of vessels in the microcirculation, which may also effect RBC flow properties.

There are a number of other potential uses of our microfluidic system to study RBC-endothelial interactions under flow conditions. These include assessment of the efficacy of current and future RBC storage solutions and RBC rejuvenation strategies.38,39 In addition, it may provide useful information regarding the flow properties of cryopreserved RBC when exposed to both normal and perturbed endothelial surfaces.40 The potential role of RBC microparticles in our study is unknown. Although microparticles including those from RBCs are normally continuously shed into the circulation, they are also produced during processing and storage of blood for transfusion. Because they may adhere to vascular endothelium, the impact of RBC microparticles in normal and perturbed microvasculature is under investigation in our laboratory.41,42

Trauma-related injury to the glycocalyx and endothelial vascular barrier occurs early after injury and is related to shock severity.17,43 The duration of the vascular barrier dysfunction is unknown. In vivo data suggest that recovery time of the glycocalyx endothelial barrier may require 5 days to 7 days.44 Spinella et al.45 have shown that use of fresh whole blood may be optimal for the early resuscitation of the patient with significant hemorrhage. This may in part be due to the improved perfusion offered by fresh RBCs as opposed to standard issue PRBC. Measurement of glycocalyx and endothelial parameters may help guide the use of “young” versus “old” banked blood during this vulnerable period.30

The microfluidic platform used in our in vitro study of the interaction of the vascular endothelium with RBCs from fresh and blood bank sources suggest that both entities are important in assessing the impact of the age of stored blood in the ability to restore microvascular perfusion in the transfusion therapy following severe trauma.

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AUTHORSHIP

L.N.D. and D.L. made substantial contributions to the conception or design of the work; they also conceived and designed the experiments. L.N.D. and D.L. analyzed the data. D.L. and L.N.D. drafted the article. L.N.D. and D.L. critically revised the article for intellectual content. All authors provided final approval of the version to be published.

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DISCLOSURE

The authors declare no conflicts of interest.

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DISCUSSION

ROSEMARY A. KOZAR, M.D., Ph.D. (Baltimore, Maryland): Good afternoon. The authors have continued their investigations into the endothelial cell glycocalyx using their in-vitro microfluidic system.

In the current study the authors examined the interactions of endothelial cells and red blood cells under stress conditions with the hypotheses that older red cells would adversely affect this interaction.

The authors confirmed changes in glycocalyx thickness as well as syndecan-1 and hyaluronic acid shedding in activated endothelial cells and, interestingly, demonstrated changes in red blood cell glycocalyx with age of red cells.

They then examined red cell adherence in their activated endothelial cells and found there to be an increase in RBC adherence as age of the red cells increased.

I must admit I was not aware that there was a glycocalyx in red blood cells. I was wondering how the glycocalyx in red cells differed from that of the endothelial cell? What is the function of the red cell glycocalyx?

Second, the authors demonstrated changes in endothelial cell glycocalyx when the cells were activated and in red cell glycocalyx with duration of storage. But do you have evidence that the increased adherence of the red cells is causally related to the changes in the glycocalyx?

Third, as a control, did you look at the red cell adherence when cells were stimulated with the fresh red blood cells?

You showed us data for the 14 days and the 21 days but as a control did you look at how the fresh red blood cells were affected in the stimulated endothelial cells?

Lastly, could you hypothesize if the degree of red cell adherence that you demonstrate is sufficient to cause microvascular thromboses and subsequent organ damage that we see in some of these clinical studies?

Thank you. I appreciate the privilege of the presentation.

Lawrence Diebel M.D. (Detroit, Michigan): The Glycocalyx is a part of the red blood cell membrane. Storage of red blood cells is known to cause compositional changes of the red blood cell membrane which increase with time in storage.

Thus the results of this experiment add new information to the existing data regarding the effects of storage on red blood cells. Also the loss of the red blood cell glycocalyx does occur with senescence in vivo, and contributes to capture and removal in the splenic vasculature.

Regarding metabolic effects of storage, this is an entirely different storys. What I can tell you in studies we are doing comparing red blood cells in the obese with the metabolic syndrome that there are differences compared to red blood cells from non-obese patients. So this is something we will look at in the future.

The three different blood groups, fresh blood, banked blood with storage less than 14 days, or greater than 21 days were all perfused with normal endothelium and endothelium subjected to biomimetic conditions of shock...so all group variations were performed.

We use a hematocrit of 1.5% in these studies and have recently completed studies at a hematocrit of 21%. The lower hematocrit makes it easier to accurately count individual red blood cells. The studies with the higher hematocrit lead to significantly higher number of red blood cells adherent to the endothelium. And each microfluidic channel may be thought of as representing a single microcirculatory unit in vivo, I believe these results would be clinically relevant.

Keywords:

Microfluidic device; red blood cell glycocalyx; red blood cell storage lesion

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