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Red blood cell (RBC) transfusion is a key element of modern medical care. Fetal medicine, trauma, surgery, the treatment of heart ailments and cancer require blood transfusion. It also provides comfort when other treatments are no longer appropriate. The goal of blood storage is to make transfusion blood available, safe, effective and cheap1.
In the modern RBC storage systems, whole blood is collected into an anticoagulant-nutrient solution such as citrate-phosphate-dextrose (CPD). The RBCs, plasma and platelets are separated into satellite bags and once the plasma is removed, the red cell additive solution (AS) is added to the RBC fraction to support the nutrient needs of RBCs. This system allows for RBCs to be stored for 35-42 days at 4Â°C.
Unfortunately, this storage is not perfect as RBCs undergo storage lesions, which include metabolic effects, shape change, membrane loss, oxidative injury to lipid and proteins, changes in oxygen affinity and delivery, shedding of active proteins, lipids and microvesicles and reduced RBC lifespan2. In order to determine the condition of stored RBCs, the storage systems are tested on their ability to prevent hemolysis and maintain RBC 24-hour in vivo recovery in a clinical setting. However, some preclinical testing can be used as quality control for the experimental storage system in the laboratory. These include the measurements of RBC ATP as a surrogate for recovery, 2, 3-DPG as a surrogate for oxygen affinity, and free hemoglobin, which is indicative of RBC hemolysis3.
The RBC is a highly specialized cell without a nucleus and devoid of protein synthesis. This means that it has to rely on its original enzymes and the metabolism of glucose to lactate and ATP. The composition of the anticoagulant-nutrient is shown as below: sodium citrate 89.4 mM, citric acid 15.6 mM, dextrose 128.8 mM, monobasic sodium phosphate 16.1 mM; and additive solutions: dextrose 111.0 mM, adenine 1.8 mM, mannitol 41.2 mM, sodium chloride 154.0 mM. They not only contain glucose, but in a high concentration compared in vivo concentrations. Consider for example 500 mL of whole blood collected into 70 mL CPD solution after plasma removal and AS-1 addition, there are about 180 mL RBCs, 40 mL plasma with CPD and 100 mL AS-1. Assuming volume of a RBC to be 87fL, the number of RBCs in this system is estimated to be 2.07Ã-1012. Also, the glucose contained by CPD and AS is 16 mmol, which is 7.9 Ã- 10-15 moles per RBC. However, in vivo, the blood glucose level in a healthy individual is 5.5 mM and 40% of the blood is RBCs, we can estimate that is 1.2Ã-10-15 moles glucose per RBC. This means the RBCs are exposed to glucose levels that are about 6-fold that of conditions found in vivo and is much higher than those found in diabetic patients (Fasting plasma glucose level â‰¥Â 7.0Â mM, 1.5 Ã-10-15 moles per RBC).
Interestingly, some properties of RBCs obtained from people with diabetes are similar to the hallmark features of the RBC lesions. Specifically, it is well established that diabetic RBCs are less deformable4, suffer from oxidative stress5, possess abnormal lipid compositions6, and exhibit increased advanced glycation endproducts (AGE)7. Although this does not suggest that the diabetic blood stream and stored RBCs have the same problem, we propose they are similar as both are exposed to an increased concentration of glucose in their extracellular environment. Some studies indicate that glycolysis occurs ten times more slowly at 3Â°C than at 25Â°C and only about a third of the original glucose in the storage system would be used by the end of the 42-day storage8. As a result, we hypothesize that the hyperglycemic condition of anticoagulant CPD and preservative AS solutions contribute to the storage lesions during RBC concentrates storage.
To test this hypothesis and improve the preservation solutions used in RBC storage, I want to prepare reduced glucose versions of CPD and AS, which have the other nutrients at the original levels. Through a series of preclinical tests and property measurements, I will try to find a better environment and method for stored RBCs.
Hypothesis: The hyperglycemic condition of anticoagulant citrate-phosphate-dextrose solution and additive solution contribute to storage lesions during RBC concentrates storage.
Develop a miniature RBC storage system, which can store a small volume of RBCs for use in laboratory studies.
To quantitatively determine and compare the various metabolic properties of RBCs collected and stored in licensed and experimental versions of CPD and AS.
To determine the membrane alterations and oxidation damage of both versions of stored RBCs.
Develop a novel slow-release glucose source for maintenance of a healthy glucose level during RBCs storage.
Background and significance
Blood is often called "fluid of life" because of its importance for living organisms. Transfusion is indeed vital and applied in numerous clinical situations. Typically, red blood cell (RBC) concentrates are administered to sustain the oxygenation of tissues, hemostasis imbalance or disorders under conditions such as severe haemorrhage, anaemia or hypovolemia9.
Since the beginning of transfusion medicine, numerous efforts have been made to secure blood products. The first RBC storage solution was developed in 191510, which was a mixture of citrate and glucose for storing rabbit RBCs. Citrate is used to fully anticoagulate plasma while glucose feeds the RBCs, which allows separating the donor and recipient in space and time. Therefore, storage solutions make blood banking possible. The history of the development of blood storage includes heat sterilization in the 1940s, phosphate in the 1950s, plastic bags in the 1960s, adenine in the 1970s, additive solutions in the 1980s, and leucoreduction in the 1990s2.
Nowadays, in a typical whole-blood collection system, blood is drained into the primary collection bag containing an anticoagulant solution such as citrate-phosphate-dextrose (CPD). As mentioned before, citrate is used to chelate the calcium ion in the plasma and thus anticoagulate the blood; phosphate supports the synthesis of new RBC ATP; dextrose, or D-glucose, provides the nutrients for RBCs. After blood collection is complete, the primary bag is centrifuged to separate RBCs and the platelet-rich-plasma. The additive solution (AS) is then added to the packed RBCs for storage. There are several kinds of AS which only differ modestly in the concentration of salt, sugar and mannitol2. The one widely used in the USA is AS-1, containing dextrose, adenine, mannitol and sodium chloride. Sodium chloride is added to keep the osmosis. Mannitol works as a free radical scavenger, but also as a membrane stabilizer11. Adenine is added for maintaining the ATP production and dextrose for feeding the RBCs. Under this condition, the RBCs can be stored for 35-42 days at 4 Â°C.
During storage, RBCs lose 2,3-diphosphoglycerate (2,3-DPG), ATP stores, lipids and membrane symmetry, while becoming more rigid. Also the suspending fluid has higher concentrations of free hemoglobin and biologically active lipids. These changes during storage are known as RBC storage lesions12. Among these lesions, maintaining normal ATP concentrations are important and necessary. People have tried many ways to maintain normal ATP concentration and correct the storage lesion such as adjusting pH, volume and adding more nutrients in the solution2. However, if we consider the pathway of ATP production, we may find the glucose plays an important role in the glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi â†’ 2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O
It has been noticed that both the CPD and AS contain dextrose which is the nutrient for the metabolism of stored RBCs. As mentioned in the introduction, the glucose concentration in both CPD and AS-1 are much higher than the need of stored RBCs. Therefore, the focus will be on adjusting the glucose concentration in the storage solution to maintain the ATP concentration and try to prevent the loss of viability, function and membrane during storage.
To determine the viability of stored RBCs, the storage systems are tested on their ability to prevent hemolysis and maintain RBC 24-hour in vivo recovery in clinical. The general criteria are free Hemoglobin (Hb) no higher than 1% of total Hb and the presence of at least 75% of the transfused cells still circulationg 24 hours after transfusion2. However, some preclinical testing can be used as quality control for the experimental storage system in laboratory. These include the measurements of intracellular ATP as a surrogate for recovery, 2,3-DPG as a surrogate for oxygen affinity, and free hemoglobin, which is indicative of RBC hemolysis3.
The major function of RBCs is to transport oxygen in vivo. In addition to oxygen transport, it has been reported that RBCs have the capacity to sense oxygen need and effect changes in oxygen supply to meet that need13. RBCs contain millimolar amounts of ATP and have been shown to release micromolar amounts of ATP when subjected to low levels of oxygen (hypoxia). The extracelluar ATP is known as a stimulus of endothelium-derived nitric oxide (NO) which relaxes the smooth muscle cells surrounding circulatory vessels14 thus, increasing blood flow and oxygen delivery to hypoxic tissue.
In addition to hypoxia-induced ATP release from RBCs, there are other mechanisms, both pharmacological and physiological, of inducing ATP release. Physiologically, RBCs are stimulated to release of ATP when they are under shear stress of mechanical deformation15. The deformability of RBCs is to adapt their shape to minimize their resistance to flow under dynamically changing flow conditions. Reduced deformability will result in impaired perfusion and rigid RBCs might directly block capillaries16. Initial studies of ATP release from RBCs in response to mechanical deformation were done by filtering the RBCs through filters with calibrated pores17. However, the disadvantage of this technique is it does not allow for ATP to be measured as it is released from the RBCs. The method developed by Spence group allows a continuous flow system using fused-silica tubing to determine the amount of ATP release from RBCs in near-real time18.
NO is well known as the relaxing factor, which is produced by endothelial cells and RBCs. Besides the endothelium-derived NO, a major fraction of NO in the blood is bound to thiols of hemoglobin (Hb), forming S-nitrosohemoglobin (SNO-Hb), which releases the NO group under hypoxic conditions. It is reported that both the SNO-Hb and extracellular NO inhibit platelet aggregation19. Additionally, dysregulation of the formation, export, or actions of RBC-derived SNOs has been implicated in human diseases including sepsis, sickle cell anemia, pulmonary arterial hypertension, and diabetes mellitus20.
As a result, the ATP release under deformation and hypoxic conditions, hypoxia-induced NO release are the indicators of the biological function of RBCs, which will be helpful in characterizing RBCs.
Phosphatidylserine (PS) is a type of membrane phospholipid, which is normally situated in the inner layer of the plasma membrane by means of energy-dependent transfer21. Since the depletion of ATP during storage, the PS is translocated to the outer leaflet. This is a also marker indicative of red cell storage lesion, senescence in RBCs and influences the function of transfused RBCs. Exposure of PS is a signal for phagocytosis and the removal of RBCs from circulation22, which may result in a relative low posttransfusion recovery of the stored RBCs.
During storage, RBCs slowly lose their smooth biconcave discoid shape and become spiculated (echinocytes). This shape change is generally reversible to a great extent with increasing ATP concentrations and regeneration of DPG23. However, RBCs actually lose membrane lipids through the budding of microparticles from the spicules, which is irreversible. Microparticles are heterogeneous and vary in size, concentration, phospholipid composition, surface antigens and protein content9. Some of them also expose PS which originally resides on the inner surface of the membrane. Thus the exposed negatively-charged lipids on them can make microparticles proinflammatory and prothrombotic. Although their functions are still largely unknown, an increase in the concentration of microparticles in plasma has been demonstrated under various pathological conditions such as thrombocytopenic disorder, cardio vascular disease and diabetes24.
Some studies showed that RBCs exposed to high levels of plasma glucose acquire membrane damage25. There are several molecular and cellular modifications associated with glucose-related reactions such as glucose autoxidation, protein glycation, and formation of advanced glycation end-products (AGEs). This nonenzymatic glycation is the formation of stable ketoamine adducts from glucose and free amino groups in proteins. And AGEs is a heterogeneous group of chemically active compounds (i.e. NÎµ- (carboxymethyl)lysine (NÎµ -CML)) formed as the result of a chain of chemical reactions after initial glycation reaction26. Consider to the hyperglycaemia condition of CPD and AS-1 for RBCs storage, it may be a good point to investigate the relationship between the high glucose level and membrane alterations. Overall, the oxygen transport function of RBCs requires the membrane being deformable and all the components of membrane, such as protein and lipid, play important roles in their function.
At the beginning of the project, the experimental CPD (CPD-N) and AS-1 (AS-1N) solutions was made with the glucose concentration as 5.5 mM, which is normal glucose level in vivo. As described before, RBCs were collected and stored RBCs in both the original and experimental CPD and AS-1 solutions. And then the following data was collected.
Miniature RBCs storage system
The setup of the storage system is described in the experimental approach (Protocol 1). Preliminary studies showed this miniature storage system would keep percent hemolysis of RBCs less than 1% after 35 days, which indicated this system is relatively good.
Flow-induced ATP release from stored RBCs
Figure 1. The ability of RBCs to release ATP in fresponse to flow-induced deformation. The data represent the averages and S.E.M. of n=4 human samples analyzed on various days for up to 16 days.At various time points, aliquots of each of the samples were removed from storage and analyzed for flow-induced ATP release. The experimental method is described in Protocol 2C. The results are shown in Fig.1. As shown, the RBCs collected and stored in the CPD-N and AS-1N, which is the reduced glucose forms, released a significantly higher level of ATP. These values were significantly different even on 16 days of storage (p<0.001). (Longer storage data will be shown later.)
Hypoxia-induced NO release from RBCs
Figure 2. The ability of RBCs to release NO in fresponse to hypoxia. The data represent the averages and S.E.M. of n=4 human samples analyzed on various days for up to 16 days.The amount of nitric oxide (NO) released from flowing RBCs exposed to a hypoxic buffer was measured using a fluorescein-based probe, diaminofluorofluorescein (DAF-FM). The hypoxic RBCs, stored in CPD/AS-1 or CPD-N/AS-1N, were pumped through channels in one layer of the PDMS device. NO released from these RBCs flowed through a porous polycarbonate membrane (2 micron in diameter) to the probe. The device was then placed into a standard microtiter plate reader for measurement. By calibrating with NO standards of known concentration, data are shown in Fig.2 that the NO release from RBCs in CPD/AS-1 and CPD-N/AS-1N. As shown, the NO release from the RBCs stored in reduced glucose form is nearly 100% higher on the first day. Importantly, there is a significant difference in the signals even out to day 16 of storage. These data strongly suggest that the ability of the experimental stored RBCs to release NO is significantly increased.
The ATP release and NO release study showed that the low glucose experimentally stored RBCs had relatively better properties within 16 storage days. However, to determine whether the reduced amount of glucose in the AS-1N could be enough for the 35-42 days storage. So the lactate accumulation measurement was performed.
Figure 3. Lactate accumulation in stored RBCs. The data represent the averages and S.E.M of n=4 human samples analyzed on various days for up to 16 days. As shown in Fig.3, the lactate levels in both stored RBCs are initially statistically equivalent, regardless of the storage strategy. However, after 8 days, the RBCs stored in experimental CPD-N/AS-1N cease to produce increasing levels of lactate beyond day 8; while the ones in CPD/AS-1 continue to produce lactate, indicating that metabolism is still active.
After calculations, it was found that the AS-1N (5.5 mM glucose) provided 0.37Ã-10-15 moles per RBC instead of 1.2Ã-10-15 moles per RBC (the healthy glucose level in vivo) when the volume of RBCs was taken into account. Also the lactate accumulation study suggested that each stored RBC would consume about 0.37Ã-10-15 moles glucose after 8 days. Increasing the initial glucose level in the experimental AS-1 solution and also developing a method of adding glucose to the stored RBC without increasing the glucose concentrations above the physiological levels would be beneficial for long term storage. As a result, the glucose concentration of the experimental AS-1 solution was raised to 22. 6 mM (named AS-1M), which would provide 1.2Ã-10-15 moles glucose for each RBC in the storage conditions. Then a small volume of high glucose concentrated saline solution was added into the storage bag every 15 days. Specifically, 0.922 g dextrose was dissolved in saline and diluted to 10 mL. Each time 10 ÂµL of this glucose saline was added into 1.2 mL of the RBCs stored in CPD-N/AS-1M. This means that each RBC is given extra 0.74Ã-10-15 moles glucose every 15 days and 2.68Ã-10-15 moles glucose total after 35-42 days storage. The amount of glucose is still much lower than the CPD/AS-1 system and added over time to keep it under the physiological levels.
The following data was collected under the conditions described above.
To ensure the survival of the RBCs during the storage, the percentage hemolysis was monitored every week.
PS exposure study
More data will be collected on 04/07
Experimental approach to specific aims
Aim 1 - Develop a miniature RBC storage system which can store a small volume of RBCs for use in laboratory studies.
Rationale - Clinically, RBC concentrates are stored as a unit which is approximately 320 mL. However it is inconvenient to collect the required volume of whole blood for this amount of RBCs, which is not needed in the lab study. Thus, a miniature version of the blood storage bags was devised for every test day.
Protocol 1A - Shrink polyvinyl chloride (PVC) tubing is used as storage bag - Uline shrink tubing was chosen as the experimental bag as this tubing is made of PVC, which is used in the clinical setting. Actually, the PVC was shown that it can help to increase RBC survival, reduce hemolysis and microvesicle formation. Also, this tubing can be heat sealed on both sides and size is easily controlled.
Protocol 1B - Whole blood collection from human subjects - Whole blood is collected from volunteer donors by venipuncture and collected into untreated vacutainer containing either CPD or CPD-N at a ratio of blood to CPD as 7.1 to 1. Allow 30 minutes to anticoagulant and then centrifuge at 5000 g for 10 min to separate the plasma and RBCs. Next, the remaining RBCs will have AS-1 or experimental AS added to them at a ratio of packed RBCs to AS as 1.8 to 1. The RBCs in AS will be separated in the storage bags and immediately stored at 4 Â°C.
Protocol 1C - Sterile condition is created for storage - To avoid bacterial contamination, all the solutions are autoclaved at 10 bars, 121 Â°C. The PVC bags are sterilized under UV light overnight. All the processes of blood collection and storage are under sterile condition. Each bag contains about 1.2 mL RBC concentrates, which is enough for the tests of each day. This allows the other bags to be maintained under sterile conditions.
Anticipated results and problems
The current result is shown in the preliminary studies. Though this shrink tubing is made of PVC as the clinical ones, there are several different types of PVC materials. Materials with different properties such as the thickness, the plasticizer of PVC and the permeability of gas to mimic the clinical storage container will be also considered. Another problem is that with storage days increasing, the volume of RBC concentrates is difficult to draw out of the bag completely, so it requires a larger volume of stored RBCs of tests at the beginning of storage.
Aim 2 - To quantitatively determine and compare the various metabolic properties of RBCs collected and stored in licensed and experimental versions of CPD and AS.
Rationale - Since the application of the experimental solutions to collect and store RBCs, several properties of RBCs should be determined and compared to show whether the modification is beneficial. These parameters, such as ATP concentration and percent hemolysis, are common indicators of quality of stored RBCs. These measurements will help demonstrate the functionality of the miniature storage of Aim 1 and the measurement of glucose concentration will provide helpful information for Aim 4.
Protocol 2A - The determination of the intracellular ATP and glucose levels27 - 0.6 mL of stored RBCs is diluted with 0.9 mL of phosphate-buffered saline (PBS, pH 7.4). 60 ÂµL of perchloric acid (70%, wt/vol) is added to acidify the cell mixture on ice for 10 min. Then the mixture is centrifuged in the cold for 5 minutes at 6000 Ã- g to obtain the protein-free supernatant. 1 mL of this supernatant is neutralized with 56 ÂµL of K2CO3 (5N) and centrifuged to remove KClO4 precipitate after being thawed. The clear supernatants are ready for ATP and glucose measurement.
For ATP measurement, the enzyme solution is prepared with L glucose (10 mM), glucose-6-dehydrogenase (0.7U/mL), and NADP+ (0.5 mM). The mixture of supernatant and enzyme solution is added in a 96-wells microtiter plate and read the adsorption at 340 nm at first. Then ATP conversion is initialed by addition of hexokinase (5 U / mL) and last for 10 min. The adsorption reading is taken again and the changes are converted to ATP content with the molecular extinction coefficient for NADPH. For glucose measurement, the same enzymatic reaction is used; instead of glucose 2 mM ATP is added.
Protocol 2B - The percent hemolysis of stored RBCs is determined by Harboe method28 - Percent hemolysis is determined by the ratio of the free hemoglobin (Hb) released into the surrounding media to the total Hb contained in the unit. Harboe direct spectrophotometric method is used to determine the supernatant Hb and the total Hb. Hb concentration is quantified by measuring absorbance at 415 nm. In addition to this, the other two absorbencies (at 380 nm and 450 nm) are measured as 'Allen correction'. Then the following formula can be used to convert absorbance measurements directly into Hb concentration.
Hb (g/l) = (167.2 Ã- A415 -83.6 Ã- A380 -83.6 Ã- A450) Ã- 1/1000 Ã- 1/dilution in dH2O
Supernatants are prepared from RBCs samples through first centrifugation at 2000 g for 10 min and second centrifugation at 15000 g for 15 min with the supernatant in first step. This supernatant is diluted 1/30 in DDW. Similarly, the total hemoglobin is prepared by diluting RBCs samples 1/1000 in DDW. Hematocrit is determined manually by collecting RBCs in microcapillary tubes, spinning in microhematocrit centrifuge and visually quantifying the percentage of packed red cells using microcapillary reader. So hemolysis is calculated according to the following formula:
Protocol 2C - Flow-induced ATP release from stored RBCs29 - A 1 mM ATP stock solution was prepared by dissolving ATP (0.0055g) in DDW and diluting to 10 mL in DDW. ATP standards (0.0-1ÂµM) were prepared by diluting aliquots of the stock ATP solution in AS-1 or AS-1N. The RBCs were diluted to 7% hematocrit with AS-1 or AS-1N, which was suitable for this measurement. To prepare the luciferin/luciferase mixture required for chemiluminescence determination of ATP, luciferin (2 mg, Sigma) was dissolved in 5 mL of DDW and then added to a vial containing firefly tail extract (F6303-15VL, Sigma) which was used as the source for luciferase. The reaction between luciferin and ATP which is catalyzed by luciferase is shown in two steps as below:
luciferin +ATP â†’ luciferyl adenylate + PPi
luciferyl adenylate + O2 â†’ oxyluciferin + AMP + CO2 + light
To measure the ATP release, the luciferin/luciferase mixture was placed in a 500 ÂµL syringe (Hamilton, Fisher Scientific). ATP standards or 7% of RBCs were placed in the second syringe and both solutions were pumped through 30 cm sections of microbore tubing having an internal diameter of 50 Âµm (Polymicro Technologies, Phoenix, Z) at a rate of 6.7 ÂµL min-1 using the dual syringe pump (Harvard Apparatus, Boston, MA). The streams containing the luciferin/luciferase mixture and ATP standard/ RBCs were combined at the mixing T-junction. The combined stream flowed through a segment of microbore tubing having an internal diameter of 75 Âµm, allowing the detection of resultant chemiluminescence from the reaction of ATP (either in standard solution or that released from RBCs) using a photomultiplier tube (PMT, Hamamatsu Corporation, Hamamatsu, Japan) placed in a light excluding box. The polymide coating was removed from the microbore tubing on the segement over the PMT to facilitate light transport through the tubing and to the PMT. At various time points, aliquots of each of the samples were removed from storage and analyzed for ATP release.
Protocol 2D - Hypoxia-induced ATP release from stored RBCs - Samples of 7% RBCs are prepared in either hypoxic AS-1 or AS-1M. Hypoxic buffer is prepared by diluting the original Oxyrase (Oxyrase Inc, Mansfield, OH) (to consume oxygen) 1/10 with AS and to incubate for 30 min. This 30 min incubation allows the concentration of dissolved oxygen in the solution decrease to 3% of the saturated concentration30. RBCs samples are then incubated in the hypoxic buffer for more than 10 min to become hypoxic and release ATP. 1000 ÂµM of the ATP stock solution is diluted to 0.25, 0.5, 0.75 and 1 ÂµM with hypoxic buffer to make the working solutions. Prepared RBCs and standard ATP solutions are pumped through the device.
Since the ATP release of hypoxic RBCs is based on the reaction of luciferin/luciferase for chemiluminescence determination which requires oxygen to react, a novel poly(dimethrylsiloxane) (PDMS) microfluidic device is designed for this measurement. In this device, there are three PDMS layers from top to bottom: a Y-shape channel layer, a very thin PDMS membrane and a Z-shape channel layer. The master of wafer was made by the group design. Different ratios of bulk polymer to curing agent are used in the fabrication to achieve different polymer properties, such as rigidity and adhesive quality. Typically, 5:1, 10:1 and 20:1 ratio of polymer to curing agent are used here. The mixtures are then degassed under vacuum to eliminate bubbles. The middle layer is fabricated by spin coating mixture of 10:1 ratio on a cleaned silicon wafer at 500 rpm for 15 s and then 1000 rpm for 30 s, producing feature that is 100 Âµm tall. After half-cured for 10 min at 75 Â°C in a convection oven, the mixture of 5:1 ratio is poured on the edge to add structural integrity and then baked for another 10 min. For the channel layers, the ratios of mixtures used are 20:1 on the surface to seal to other surfaces and 5:1 as an overcoat to add structural integrity around inlets. The features of Y-shape channel are 50 Âµm wide and 1 cm long on braches, 100 Âµm wide and 2 cm long on the main part as well as 100 Âµm tall. The features of Z-shape channel are simpler as 200 Âµm wide, 5 cm long and 100 Âµm tall. Inlets are punched using 20 gauge tubing and waste wells are punched using a 7/32 in. Securely sealing of the three PDMS layers together is achieved by placing the two clean surfaces toward the middle layer and heating at 75 Â°C for 20 min.
The device is placed in a light excluding box and the chemiluminescence from the reaction of ATP and luciferin/luciferase mixture is measured using a photomultiplier tube. ATP standards or 7% RBCs are placed in a 500 ÂµL syringe and the luciferin/luciferase mixture is placed in the second syring. Both solutions are pumped through tubing into the Y-shape channel at a rate of 1 ÂµL min-1 using the dual syringe pump. The streams are combined at the main part but without oxygen to react normally. So a third syringe is used to pump the oxygen-rich TBS buffer into the Z-shape layer at a rate of 3 ÂµL min-1, which is under the middle PDMS membrane and allowed oxygen transportation to the upper layer. After reoxygenation, the chemiluminescence of ATP from either the standards or the hypoxic RBCs can be measured and quantified.
Anticipated results and problems
The percent hemolysis should be less than 1% after 35-42 days storage, which is the current criterion of stored RBCs. However, the storage conditions such as storage bag and solution are still under development. So, the results may be more than 1%, but still can provide information on to improve the system. Both the intracellular ATP and induced ATP release should be higher in the EAS than the AS. The concentration of ATP may vary because of individual differences between blood donors. As a result, several parallel measurements should be done to obtain valid data.
Aim 3 - To determine the membrane alterations and oxidation damage of both versions of stored RBCs.
Rationale - In order to test the effect of the experimental storage solution, PS exposure, microparticle shedding, and oxidation damage, such as glycation protein and AGEs, will be determined. These alterations are common biological markers of storage lesions during RBC storage.
Protocol 3A - Flow cytometric method to determination of PS exposure31 - RBCs samples are washed twice with cold TBS (Tris-buffered saline, 25 mM Tris, 150 mM NaCl, 2 mM KCl, and pH 7.4) and then resuspended in binding buffer at a concentration of 1 Ã- 106 cells/mL. One hundred microliters of the solution (1Ã- 105 cells) is added to a culture tube, followed by 5 ÂµL of FITC Annexin V (556419, BD Pharmingen). The tubes are gently vortex and incubated for 15 min at room temperature in the dark. Then 400 ÂµL of binding buffer is added to each tube. The samples are analyzed by flow cytometry (Accuri C6 flow cytometer) within 1 hour. The percentage of positive RBCs is determined from the fluorescence signal in excess of the obtained with negative (unlabeled) control RBCs for each sample. The results are expressed as a percentage of the total events acquired and the geometry mean of fluorescence intensity.
Protocol 3B - Validation and determination of microparticles32 - Microparticles are separated with RBCs suspension by three centrifugation steps: at 4150 Ã- g for 10 minutes twice and ultracentrifugation at 34,000 Ã- g for 20 min. The ultracentrifuge sediments are resuspended in PBS to have a proper concentration for dual-color flow cytometry study. The mixture is stained with allophycocyanine-conjugated antiglycophorin A (GPA, CD 325) monoclonal antibody and a fluorescein isothiocyanate (FITC)-conjugated annexin V protein. The former staining is to identify the microparticles shedding from RBCs and the second is to determine the surface PS of microparticles. Then labeled microparticles are analyzed on a flow cytometer. The microparticles are gated based on their sizes, approximate 1 Âµm and smaller, which can be validated using commercially available beads. The unlabeled sample, isotype control, is also analyzed to determine the criteria of positive fluorescence. The microparticles which are of RBC origin and express PS, are shown as GPA+ Annexin V+ type. The results are expressed as a percentage of the total events acquired.
Protocol 3C - Colorimetry method to determine the glycation protein33 - Stored RBCs are lysed with 20 volumes of hypotonic buffer (5mM Tris-HCl, pH 7.4 plus 0.1 mM EDTA). The lysates are centrifuged at 20000 rpm for 20 min at 4 Â°C and then the supernatants are removed by aspiration. This process is repeated until the hemoglobin-free ghosts are obtained. The RBCs ghosts are used for protein glycation assay.
Glycated protein levels are evaluated as ketoamine equivalents using the hydrazine/phenylhydrazine method. The concentration of glycation protein is based on the colorimetry of 2-keto-glucose, which is easily released from the glycation protein (ketoamine) by heating with hydrane, and made to form the chemically stable glucose phenylosazone with phenyl hydrazine. Specifically, 100 ÂµL of RBCs ghost and aqueous solution of hydrazine monohydrate (4.0 M, adjusted to pH 9.4 with acetic acid) are heated at 100 Â°C for 30 min. Then 600 ÂµL of a 0.02 M solution of phenylhydrazine hydrochloride in 40% aqueous acetic acid is added to the reaction mixture, incubating at 60 Â°C for 1h. The reaction mixture is centrifuged at 1400 g for 10 min. The absorbance of the supernatant from this is measured at 390 nm. The working solutions of standard are prepared by diluting 1 M aqueous solution of N-p-tolyl-D-isoglucosamine to 100, 250, 500, 750 and 1000 ÂµM for the assay. The concentration of glycation protein is calculated from the absorbance of standard solutions and sample.
Protocol 3D - Immunoassay-based evaluation of advanced glycation end-products (AGEs)26 - 96-well plates are coated with NÎµ -CML bovine serum albumin (BSA) at a concentration of 500 ng/mL for 2 hours at 37 Â°C. After blocking (with 0.5% gelatin in PBS) and washing (with 0.05% Tween 20 in PBS), NÎµ -CML BSA standards or stored RBCs are added with mouse anti- NÎµ -CML (75 ng/mL). The bound antibody is determined using horseradish peroxidase-conjugated donkey anti-mouse antibody. After subsequent washes, substrate solution is added, allowing it reacts for 15 min until the stop solution (2 M H2SO4) added. Then the optical density is measured at 450nm with a multi-well plate reader.
Anticipated results and problems
The above protocols are based on recent publications, thus the execution of assays should not pose a problem. However, it remains to be seen if these assays are sensitive enough to distinguish between samples of different storage conditions as well as samples of stored RBCs and fresh cells.
Aim 4 - Develop a novel slow-release glucose source for maintenance of a healthy glucose level during RBCs storage.
Rationale - In order to provide a healthy glucose level for the stored RBCs during 35-42 days, it is necessary to develop a glucose source which can release glucose slowly according to the glucose metabolism rate of stored RBCs at 4 Â°C. Two techniques are employed here to achieve the goal. First, a porous membrane can be used, which has molecular channels and permits selective transportation of particles of a certain size, such as glucose molecule. Besides this, an idea of controlled release pellet is also applied. Because of the water soluble feature of the dextrose, sustained release pellets of glucose are achieved through double coated technique.
Protocol 4A - Application of a porous membrane to transport glucose34 - A porous membrane, such as polyethyleneterephthalate, is applied in this protocol. This membrane should allow glucose molecules (180 MW) pass through, but block larger proteins such as hemoglobin and microparticles. Two chambers with identical volumes are connected with the membrane, and then the AS with either high or low glucose concentration is placed in the chambe. The dynamics of glucose transportation from the high to low through the porous membrane is studied by determination of glucose concentration on both sides at various times. To mimic the storage condition, this study is done at 4 Â°C. The glucose concentration is measured by the method mentioned before.
Protocol 4B - develop a slow-release glucose pellet35 - A double coating technique is used to achieve the controlled release of glucose. The dextrose powder can be used as the core pellet and the size is selected by mesh sieve for further coating. Polymer mixtures of ethylcellulose and polyethylene glycol are employed as the first sealing coat. This helps to prevent migrating of the dextrose from the core pellet to the polymer films during coating. Methyl methacrylate copolymer is used as outer-coating material, which controls the release profile. To determine the release profile, varying numbers of pellets will be placed in AS under the storage conditions and the concentration of glucose will be determined at various time points. The glucose concentration is measured by the method mentioned before in protocol 2A.
Anticipated results and problems
Both of these techniques have not been employed as a long-term substance release, in this case, 40 days. Therefore, the properties of either the porous membrane or the polymer coat, such as thickness and the number of pore per unit area, should be determined based on the dynamic of glucose release. Also, the biological compatibilities need to be examined before applying to the stored RBCs.