Blood Bank Storage Conditions Research Articles

Mechanism investigation of platelet apoptosis inhibition by N-Arachidonoylethanolamine

To investigate the mechanism of N- Arachidonoylethanolamine (ANA) on inhibiting platelets (PLT) apoptosis under standard blood bank storage conditions. Samples taken from collected apheresis PLT by the Amicus instrument were split into three parts. An aliquot of 0.5 μmol/L ANA were added to one part of storage PLT as the ANA group; an aliquot of 0.5 μmol/L ANA and 1 μmol/L SR141716 was added to the another part as the ANA + SR141716 group; and the third part without ANA and SR141716 as the control group. These samples were stored on a flat-bed shaker at (22 ± 2) ⁰C for 7 days. The expression of phosphatidyl serine (PS) positive, phospho (p)-Akt, Akt, p-Bad, Bad, caspase-3, caspase-9, cytochrome C (Cyt-C) and BCL-XL interaction with Bak were detected. The rate of PLT PS positive in ANA group decreased significantly than that in control group[ (8.29 ± 1.44) % vs (14.24 ± 2.47) %, P<0.05]. The release of Cyt-C from mitochondria to cytosol in ANA group decreased significantly compared with control group[ (3.29 ± 1.44) % vs (15.24 ± 3.40) %, P<0.05]. Also the expressions of p-Akt and p-Bad in ANA group increased significantly than those in control group[ (71.33 ± 10.26) % vs (35.00 ± 6.00) %, P<0.05; (39.00 ± 9.64) % vs (10.33 ± 1.53) %, P<0.05, respectively]. Higher amounts of Bak protein were co-precipitated with BCL-XL in ANA group than that in control group (about 2.6 fold, P<0.05). The expressions of cleaved caspase- 9 and caspase- 3 in ANA group decreased significantly than those in control group[ (9.63 ± 1.47) % vs (23.24 ± 2.47) %, P<0.05; (6.30 ± 1.40) % vs (13.20 ± 2.50) %, P<0.05, respectively]. There were no significantly changes between ANA+SR141716 and control groups (P>0.05). ANA protected PLTs from apoptosis as a result of inhibiting the release of Cyt-C from mitochondria to cytosol by modifying the expressions of apoptosis-relative proteins.

34. The effects of biochemical rejuvenation on red blood cell microvesiculation, phosphatidylserine and CD47 expression during hypothermic storage

Red blood cells (RBCs) under blood bank storage conditions undergo biophysical and biochemical alterations, including biochemical depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), as well as cholesterol and phospholipid loss through microvesiculation. Microvesiculation is the process by which microparticles (MPs), phospholipid vesicles that are less than 1 μm in diameter, are released in response to different stimuli and conditions. RBC microparticles (RMPs) have been shown to be harmful effectors of inflammation and coagulation post transfusion, as well as in vitro indicators of RBC function and viability throughout ex vivo storage. Recent studies suggested that microvesiculation during storage results from biochemical depletion and/or cell senescence (Chernitskii˘ et al. 38 (8) (1993)18–21). Rejuvenation solutions have been shown to be able to restore ATP and 2,3-DPG levels in hypothermically stored RBCs. The purpose of this study was to examine the effects of the biochemical rejuvenation on red blood cell microvesiculation, as well as phosphatidylserine (PS) and CD47 expression during hypothermic storage. Leukoreduced packed RBC units (n = 14) were produced using the standard buffy coat method from whole blood in citrate–phosphate–dextrose (CPD) and stored in saline–adenine–glucose–mannitol (SAGM) at 1–6 °C for up to 49 days. Units were divided into three different experimental groups: untreated control, sham control and rejuvenated group. On days 28, 35, and 42 of hypothermic storage the rejuvenated group was incubated for one hour with 50 mL of rejuvenation solution containing pyruvate, inosine, phosphate and adenine (PIPA), while the sham control group was incubated for 1 h with 50 mL of 1× phosphate buffered saline. RBC units were tested weekly for ATP concentration, quantity of RMPs, and PS and CD47 expression. Microvesiculation was assessed using multicolor flow cytometric (FC) analysis and a spectrophotometric assay was used to determine ATP concentration. The results indicated that ATP significantly decreased throughout the hypothermic storage in untreated and sham RBC units (p

Transfection of Human Platelets with Short Interfering RNA

Platelets contain mRNAs and are capable of translating mRNA into protein, and it has been previously demonstrated that platelets increase their levels of integrin β3 overtime while in blood bank storage conditions. We are unaware of prior attempts to introduce nucleic acids into platelets. Considering the potential clinical and research utility of manipulating platelet gene expression, we tested whether small interfering RNAs (siRNAs) could be transfected into normal human platelets. Multiple conditions were tested, including lipofectamine versus electroporation, different amounts of siRNA, the effect of different buffers and the presence of plasma during transfection, and the time for optimal siRNA incorporation after transfection. Using flow cytometry to assess transfection efficiency, we found that optimal transfection was obtained using lipofectamine, washed platelets, and 400 pmoles siRNA. Cell sorting of transfected platelets suggested that the incorporated siRNA was able to knockdown the level of a targeted mRNA. This is the first ever demonstration that nucleic acids can be introduced directly into platelets, and offers proof of concept for manipulating gene expression in platelets by nonviral methods. Future technical improvements may permit improving the quality and/or lifespan of stored human platelets.

Transfection of Human Platelets Down-Regulates Endogenous mRNA.

Abstract 4026Poster Board III-962Platelets are anucleate and, as such, lack the capacity for transcribing and expressing exogenously introduced genes or cDNAs. However, platelets contain megakaryocyte-transcribed mRNAs and respond to physiological stimuli by translating mRNA into protein. Compared to the megakaryocyte, the relative contribution of platelet translation to the platelet proteome is unknown. However, it is noteworthy that platelets in Blood Bank storage conditions increase their levels of integrin β3 over time and that the half-life of some mRNA is longer in platelets than in nucleated cells (Thon et al, Transfusion 2009). Considering the potential clinical and research utility of manipulating platelet gene expression, we tested the feasibility of transfecting platelets in-vitro with a fluorescently (FAM)-labeled siRNA for GAPDH as assessed by flow cytometry. We tested both electroporation and Lipofectamine with both washed platelets and platelet-rich plasma (PRP) using a variety of conditions. Our studies demonstrated optimal transfection efficiency at 24 hours using 2 × 108/mL washed platelets, 6 μL Lipofectamine and 400 pmoles siRNA. These initial studies demonstrated that 8.4% of platelets were transfected with the FAM-labeled siRNA compared to control platelets without Lipofectamine. Transfected platelets were then isolated by cell sorting. Changes in GAPDH mRNA levels were determined by TaqMan gene expression assays using18S RNA as an endogenous control. Two independent experiments demonstrated a 40% reduction in GAPDH mRNA in platelets transfected with GAPDH siRNA as compared to a FAM-labeled scrambled siRNA. These results offer proof-of-concept that nucleic acids can be introduced into platelets by non-viral methods and that these nucleic acids can modify gene expression. Future studies to increase platelet transfection efficiency may be valuable for manipulating platelet qualities in stored platelet products or to use platelets as vehicles for delivering products to the sites of vascular damage. Disclosures:No relevant conflicts of interest to declare.

Apoptotic activity in stored human platelets

Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated. Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation. As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression. Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.

Development of Optimal Techniques for Cryopreservation of Human Platelets: I. Platelet Activation during Cold Storage (at 22 and 8°C) and Cryopreservation

Using the current blood bank storage conditions at 22°C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8°C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at −1°C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8°C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22°C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to −10°C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (−10, −30, −60, and −196°C). In conclusion, under the present experimental conditions, a storage temperature of 8°C appeared to be much better than 22°C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.

Lack of efficacy for conventional gamma irradiation of platelet concentrates to abrogate bacterial growth.

The maximum storage time for platelet concentrates is 5 days, owing to the higher risk bacterial contamination with longer storage. The expiration date could potentially be extended if a rapid system to detect microbial contamination or a safe sterilization technique could be developed and easily implemented. Gamma irradiation has decreased bacterial contamination in food products. Conventional doses of gamma irradiation were tested for their efficacy in decreasing bacterial growth during the 5-day platelet shelf life. An initial pilot study determined that bacteria suspended in normal saline at concentrations of 1 to 2 x 10(7) colony-forming units per milliliter showed a dose-related susceptibility to gamma irradiation. Subsequently, four platelet concentrates were pooled, inoculated with a known concentration of Staphylococcus autreus or Serratia marcescens, and divided. The concentrates were exposed to varying amounts of gamma irradiation, ie, no irradiation (control), 25, 50, and 75 Gy, and subjected to typical blood bank storage conditions. The platelet concentrates were sampled daily for 7 consecutive days to monitor bacterial growth by quantitative cultures. An inverse linear dose-related extinction of bacteria was evident in the pilot study with an extrapolated total kill in the 100 to 150 Gy range. There is no difference in bacterial growth with S aureus using irradiation levels from 0 to 75 Gy. A 1-day delay in bacterial growth at 75 Gy was found with S marcescens compared with units irradiated with 0 through 50 Gy. Exposure of bacteria-contaminated platelet concentrates on storage day zero to gamma irradiation at levels up to 75 Gy is ineffective at sterilizing the platelet concentrates. Higher levels of irradiation may be effective in sterilizing platelet concentrates. Function, survival, and sterility after higher than conventional levels of irradiation need further study.

Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsola. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol®. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A 2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol® than in EAS 25 (44.3% ± 11.7 vs. 25.3% ± 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by ∼ 50% and glutathione levels decreased by ≈ 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P > 0.01) in EAS 25 than in Adsole. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol®. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.

Survivability and Infectivity of Viscerotropic Leishmania tropica from Operation Desert Storm Participants in Human Blood Products Maintained Under Blood Bank Conditions

To assess the potential for leishmaniasis being transmitted through blood transfusion, we studied the survival of Leishmania in blood products under blood bank storage conditions. We report that L. tropica- or L. donovani-contaminated transfusable blood products are a risk to the blood supply for at least 25 days postdonation under blood bank general conditions. The blood components that have been implicated are whole blood, packed red blood cells, platelet concentrate, and frozen-deglycerolized red blood cells, but not, as would be expected, fresh frozen plasma. Blood units containing four infected monocytes per milliliter of blood with a mean of three amastigotes per monocyte contain viable parasites for 15 days under blood bank storage conditions. Furthermore, animal studies showed the presence of parasites in the blood of cutaneously infected animals and the possibility of transmitting the disease to healthy experimental animals by blood transfusion from infected animal donors. Three of three BALB/C mice showed metastasis to the lower extremities and face after they received 0.25 ml of blood from a CPDA-1 bag seeded with 1.5 x 10(5) amastigotes per ml of blood kept under blood bank conditions for 30 days. This proves that Leishmania not only survives blood banking procedures and storage conditions but that the parasite retains its infectivity. The results of this study and the recent demonstration of L. tropica-infected monocytes in the blood of a patient returning from Southwest Asia suggests that transfusion-associated leishmaniasis can occur.

Survival of Cytomegalovirus in Viremic Blood under Blood Bank Storage Conditions

Survival of Cytomegalovirus in Viremic Blood under Blood Bank Storage Conditions

Survival of Borrelia burgdorferi in blood products.

The incidence of Lyme disease is rapidly increasing in the United States. To assess the potential of transmission of the disease through blood transfusion, we studied the survival of Borrelia burgdorferi in blood products under blood bank storage conditions. Two units of whole blood, separated into red cells (RBCs), fresh-frozen plasma (FFP), and platelet concentrates (PCs), were inoculated with B. burgdorferi (strain B31) in concentrations of approximately 3000 organisms per mL of RBCs and FFP and 200 organisms per mL of PCs. Products were then stored under blood banking conditions and sampled at several storage times. The viability of the spirochete in blood components was determined by darkfield microscopic examination of cultures in modified Kelly's medium. The organism was shown to survive in RBCs (4 degrees C) and FFP (below -18 degrees C) for 45 days and in PCs (20-24 degrees C) for 6 days. The results of this study do not exclude the possibility of transmission of Lyme disease through blood transfusion.