Citrate Phosphate Dextrose Research Articles

Whole Blood Storage in CPDA1 Blood Bags Alters Erythrocyte Membrane Proteome.

Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived “red cell storage lesions.” The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.

Mesenchymal stromal cells can be applied to red blood cells storage as a kind of cellular additive.

During storage in blood banks, red blood cells (RBCs) undergo the mechanical and metabolic damage, which may lead to the diminished capacity to deliver oxygen. At high altitude regions, the above-mentioned damage may get worse. Thus, more attention should be paid to preserve RBCs when these components need transfer from plain to plateau regions. Recently, we found that mesenchymal stromal cells (MSCs) could rescue from anemia, and MSCs have been demonstrated in hematopoietic stem cells (HSCs) transplantation to reconstitute hematopoiesis in vivo by us. Considering the functions and advantages of MSCs mentioned above, we are trying to find out whether they are helpful to RBCs in storage duration at high altitudes. In the present study, we first found that mice MSCs could be preserved in citrate phosphate dextrose adenine-1 (CPDA-1) at 4 ± 2°C for 14 days, and still maintained great viability, even at plateau region. Thus, we attempted to use MSCs as an available supplement to decrease RBCs lesion during storage. We found that MSCs were helpful to support RBCs to maintain biochemical parameters and kept RBCs function well on relieving anemia in an acute hemolytic murine model. Therefore, our investigation developed a method to get a better storage of RBCs through adding MSCs, which may be applied in RBCs storage as a kind of cellular additive into preservation solution.

Characteristics of resveratrol and serotonin on antioxidant capacity and susceptibility to oxidation of red blood cells in stored human blood in a time-dependent manner.

ObjectiveIn stored red blood cells (RBCs), which are used in diseases (e.g., acute blood loss and leukaemia), storage lesions arise by oxidative stress and other factors over time. This study investigated the protective effects of resveratrol and serotonin on stored RBCs.MethodsBlood from each donor (n = 10) was placed in different bags containing 70 mL of citrate phosphate dextrose (total volume: 500 mL) and divided into three groups (n = 30): control, 60 µg/mL resveratrol, and 60 µg/mL serotonin. Malondialdehyde (MDA) and glutathione (GSH) levels, activity of glutathione peroxidase (GSH-Px), catalase, and carbonic anhydrase (CA), and susceptibility to oxidation in RBCs, and pH in whole blood were measured at baseline and on days 7, 14, 21, and 28.ResultsMDA levels and susceptibility to oxidation were increased in all three groups time-dependently, but this increase was greater in the serotonin group than in the other groups. Activity of GSH-Px, CAT, and CA, as well as GSH levels, were decreased in the control and serotonin groups time-dependently, but were significantly preserved in the resveratrol group. The pH was decreased in all groups time-dependently.ConclusionOur study shows that resveratrol attenuates susceptibility to oxidation of RBCs and protects their antioxidant capacity, and partially preserves CA activity time-dependently.

Label-Free Sensing and Classification of Old Stored Blood.

Transfusion is crucial in surgical operation and anemia treatment. However, several hemorheological properties of blood are adversely altered during blood storage. After transfusion, these adverse alterations are related with decrease of oxygen and ion transport in blood circulation, which increase the mortality of transfused patients. Therefore, accurate sensing of whether a blood supply is still viable for transfusion or not is extremely important. In this study, a H-shaped microfluidic device and digital in-line holographic microscopy were employed to measure temporal variations of blood viscosity and the optical focusing property of erythrocytes during blood storage. Stored rat blood samples separately preserved in citrate phosphate dextrose adenine-1 (CPDA-1) and ethylenediaminetetraacetic acid (EDTA) underwent considerable changes in their biophysical parameters after 2weeks. Compared with EDTA, CPDA-1 preserves the hemorheological properties of stored blood more effectively. We propose new criteria for depository period of stored blood and indexes, such as viscosity and focal length of erythrocytes, to determine its appropriateness for transfusion. These criteria and indexes can be effectively used for high-throughput prescreening to reduce the risk of transfusion of aged blood or diagnose hematological diseases.

Blood storage alters mechanical stress responses of erythrocytes

BACKGROUND: Erythrocytes undergo irreversible morphological and biochemical changes during storage. Reduced levels of deformability have been reported for stored erythrocytes. Erythrocyte deformability is essential for healthy microcirculation.OBJECTIVE: The aim of this study is to evaluate shear stress (SS) induced improvements of erythrocyte deformability in stored blood.METHODS: Deformability changes were evaluated by applying physiological levels of SS (5 and 10 Pa) in metabolically depleted blood for 48 hours and stored blood for 35 days with citrate phosphate dextrose adenine-1 (CPDA-1). Laser diffractometry was used to measure erythrocyte deformability before and after application of SS.RESULTS: Erythrocyte deformability, as a response to continuous SS, was significantly improved in metabolically depleted blood, whereas it was significantly impaired in the blood stored for 35 days with CPDA-1 (p≤0.05). The SS-induced improvements of deformability were deteriorated due to storage and relatively impaired according to the storage time. However, deformability of stored blood after exposure to mechanical stress tends to increase at low levels of shear while decreasing at high SS levels.CONCLUSION: Impairment of erythrocyte deformability after storage may contribute to impairments in the recipient’s microcirculation after blood transfusion. The period of the storage should be considered to prevent microcirculatory problems and insufficient oxygen delivery to the tissues.

In Vitro Evaluation of the Fresenius Kabi CATSmart Autotransfusion System

Use of autotransfusion systems to collect, wash, and concentrate shed blood during surgical procedures is a widely used method for reducing postoperative anemia and the need for blood transfusions. The aim of this study was to evaluate the CATSmart Continuous Autotransfusion System wash program performance with small (200 or 700 mL) and large volumes (1,000 mL) of shed blood and to determine non-inferiority of the CATSmart to the C.A.T.Splussystem. Human whole blood was collected in citrate phosphate dextrose, diluted, and divided into two aliquots to be processed as a pair using the C.A.T.Splusand CATSmart systems with their corresponding wash programs: low-volume, high quality/smart, or emergency wash. Final packed red cell product was analyzed for red blood cell (RBC), white blood cell, and platelet counts; hemoglobin; hemolysis; RBC recovery rates; and elimination of albumin, total protein, and potassium. The mean hematocrit (HCT) after processing with CATSmart and C.A.T.Splussystems were 59.63% and 57.71%, respectively. The calculated overall RBC recovery rates on the CATSmart and C.A.T.Splussystems were 85.41% and 84.99%, respectively. Elimination of albumin (97.5%, 98.0%), total proteins (97.1%, 97.5%), and potassium (92.1%, 91.9%) were also calculated for the CATSmart and C.A.T.Splussystems. The CATSmart and C.A.T.Splussystems both provided a high-quality product in terms of HCT, protein elimination, and hemolysis rates across the range of tested shed blood volumes and all wash programs. The study was able to confirm the CATSmart is non-inferior to the C.A.T.Splussystem.

Influence of L-carnitine on Stored Rat Blood: A study on Plasma

Objective: Plasma acts as a good indicator of oxidative stress in blood. L-Carnitine is an antioxidant that reduces metabolic stress in cells, thereby providing a protective effect against oxidative stress (OS). L-Carnitine as an additive in storage has not been explored. Thus, this study attempts to analyze the role of L-carnitine in blood storage solution, citrate phosphate dextrose adenine (CPDA)-1, through OS markers including antioxidant enzymes, lipid peroxidation, and protein oxidation.Materials and Methods:Blood was collected from male Wistar rats and stored in CPDA-1 solution with L-carnitine (10 mM, 30 mM, and 60 mM: groups LC 10, LC 30, and LC 60, respectively) and without L-carnitine (control group). Plasma was isolated every 5th day and the OS markers were analyzed.Results:Superoxide dismutase (SOD) and sulfhydryl (SH) increased over storage in controls, LC 30, and LC 60. Catalase increased in LC 30 and LC 60 during storage. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PrC) levels in all groups increased initially and reduced towards the end of storage. SOD and SH levels were maintained while TBARS and PrC levels increased in LC 10.Conclusion:L-Carnitine was beneficial in terms of increased antioxidant capacity and SH and decreased lipid peroxidation. This forms the basis for further studies on L-carnitine as a constituent in storage solutions.

Improving the Diet for the Rearing of Glossina brevipalpis Newstead and Glossina austeni Newstead: Blood Source and Collection - Processing - Feeding Procedures.

One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10−4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way.

Measuring cell surface area and deformability of individual human red blood cells over blood storage using quantitative phase imaging.

The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusions. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called citrate phosphate dextrose adenine-1 (CPDA-1). With 3-D quantitative phase imaging techniques, the optical measurements for 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and progressive alterations of stored RBCs. Our results show that stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within two weeks which was accompanied by significant decreases in cell deformability and cell surface area, and increases in sphericity. However, the stored RBCs with CPDA-1 maintained their morphology and deformability for up to 6 weeks.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Change in haematological and selected biochemical parameters measured in feline blood donors and feline whole blood donated units.

Objectives The quality of whole blood (WB) units is influenced by many factors, starting with selection of donors and the method of blood collection. The aim of this study was to investigate the changes that occur in haematological and selected biochemical parameters in blood transferred from a feline blood donor to feline WB unit. Methods Data from 27 feline blood donations were used in this study. Cats were anaesthetised with a combination of tiletamine and zolazepam. Blood (10 ml/kg body weight to a maximum of 60 ml/cat) was collected in citrate-phosphate-dextrose-adenine (CPDA) anticoagulant. Lactated Ringer's solution (10 ml/kg) was administered intravenously starting halfway through the donation. Haematological and selected biochemical parameters (complete blood count, free haemoglobin, % haemolysis, glucose, sodium, potassium, pH) were measured in the blood donor before donation and in the corresponding donated WB unit soon after collection. Results Significant decreases occurred between blood donor and WB unit in red blood cells (mean difference -1.06 × 1012/l; P <0.0001), haemoglobin (mean difference -1.6 g/dl; P <0.0001), haematocrit (mean difference -4.6%; P <0.0001), red cell distribution width (mean difference -0.9%; P = 0.0003), white blood cells (mean difference -2.17 × 109/l; P <0.0001), pH (mean difference -0.5; P <0.0001) and potassium (mean difference -1.4 mmol/l; P <0.0001). Significant increases occurred between blood donor and WB unit in platelets (mean difference +87.00 ×109/l; P = 0.0039), glucose (mean difference +25.42 mmol/l; P <0.0001) and sodium (mean difference +20 mmol/l; P <0.0001). Conclusions and relevance When using a blood collection protocol with intravenous fluid administration midway through the donation and a CPDA:blood ratio of 1:7, there were significant changes in both the haematological and biochemical characteristics between the blood donors and WB units. The majority of these changes may be the result of the anticoagulants used for storage. Understanding these changes may assist selection of blood donors and help prediction of the characteristics of the donated WB unit.

Customized Platelet-Rich Plasma for Skeletal Muscle Injuries

Objectives:Skeletal muscle injuries are among the most common sports-related trauma. Current treatment strategies result in formation of fibrous tissue that hinders the healing process before complete recovery. Incomplete recovery impairs muscle function and predisposes to re-injury. Platelet-Rich-Plasma (PRP) contains a multitude of growth factors and is an autologous source of growth factors for various tissue repairs. It is well established that PRP contains beneficial growth factors for muscle repair; however, it also contains high concentrations of deleterious growth factors for optimal muscle healing, such as transforming growth factor-beta 1 (TGF-β1). TGF-β1 leads to increased fibrosis impeding muscle healing. We therefore hypothesized that neutralization of TGF-β1’s action within PRP could improve PRP’s beneficial effect on skeletal muscle repair.Methods:Sixteen week old in-bred Fisher rats were used. Three rats were used for PRP isolation. 10 ml of blood were extracted from abdominal aorta and mixed with citrate phosphate dextrose solution. PRP were isolated by twice centrifugation. 24 rats were randomly assigned to four groups. A small incision was made along the tibialis anterior (TA) muscle; 50 µl cardiotoxin (CTX) (0.15ug/ul) was injected intramuscularly to the TA. One day after CTX injection, the animals were treated with PBS (control), plain PRP (PRP group), customized PRP+Ab-1x, and PRP+Ab-5x. Animals were sacrificed, and TA muscles were dissected on week 1 and 2 for assessment of muscle regeneration, fibrosis, macrophage infiltration, and satellite cell activation.Results:We observed significantly more regenerative myofibers in the PRP and customized PRP groups compared to control (Fig 1A-C). Collagen deposition (fibrosis) was detected in all groups at week 1 and week 2 after injury; while customized PRP group showed significantly decreased collagen deposition at week 1 and week 2 when compare to control and PRP groups (Fig. 1D-F). PRP was also shown to activate satellite cells at day 7 while customized PRP extended satellite cell activation through day 14 (Fig. 1G, H). Macrophage infiltration was significantly decreased in PRP group when compared to control at week 1 after injury; however the macrophage infiltration was increased in customized PRP group when compare with PRP group, specifically, we observed an increased in the percentage of the M2 macrophage subtype in the customized PRP group at week 1 but not week 2(Fig. 1I, J).Conclusion:This study demonstrates that substantial fibrosis occurs in skeletal muscle following CTX injury. PRP treatment significantly increases muscle regeneration by promoting angiogenesis, satellite cell activation, and reducing the infiltration of inflammatory macrophages. However, augmented fibrosis was also observed. Neutralizing TGF-β1 within PRP also significantly promotes muscle regeneration while significantly reducing fibrosis. It has been suggested that M2 macrophages are involved in the suppression, resolution and reversal of fibrosis. Hence, the increase in M2 macrophages in the customized PRP group may have contributed to the observed decrease in fibrosis. Our results demonstrate that neutralization of TGF-β1 within PRP is a promising approach to improve PRP’s beneficial effect on skeletal muscle repair while decreasing deleterious fibrosis.

Oxidative stress in erythrocytes of banked ABO blood

Objective: To understand the responses of A, B, and O blood groups to oxidative stress (OS) induced through storage.Methods: A, B, and O blood units were obtained from the blood bank at KIMS Hospital, Bangalore, and stored for 35 days at 4°C in citrate phosphate dextrose adenine-1 solution. Every fifth day, hemoglobin (Hb) was assessed in whole blood and erythrocytes were isolated from each group. OS markers such as (i) antioxidant enzymes [superoxide dismutase and catalase] and superoxides were assessed in hemolysate; (ii) lipid peroxidation product – malondialdehyde (MDA) and protein oxidation products [protein carbonyls, advanced oxidation protein products (AOPP), and protein sulfhydryls] were assessed in membrane ghosts.Results: Antioxidant enzymes and Hb were similar in all groups. Superoxides increased in blood group O. MDA and AOPP differed between the groups, where levels in blood group O were lower than blood groups A and B. Sulfhydryls were maintained throughout storage.Discussion: The antioxidant defense in A, B, and O groups were similar as evident from our results of Hb, antioxidant enzymes and sulfhydryls. However, the response of blood group O diverged from that of A and B, substantiated by the results of MDA, AOPP, and superoxides. Thus blood group O endured oxidative insult more efficiently than A and B. This study forms the basis for future studies on erythrocyte membrane and exploring blood group O as a potential candidate for prolonging storage.

Comparative Study on Some Biochemical Parameters in Stored Whole Blood in Standard Blood Bank and Traditional Refrigerator

Whole blood or any of its components is stored for the purpose of future transfusion. To ensure therapeutic relevance of the product, strict adherence to instructions and procedures on the operation of a blood bank is crucial. Storage outside the stipulated temperatures could lead to biochemical changes that may reduce the therapeutic value. This study aims at evaluating the in vitro effect of storage on selected biochemical parameters (Na + , K + , Cl - , HCO3 - , Total protein, Albumin, Ca 2+ , pH, Glucose and Hemoglobin concentration) in citrate phosphate dextrose adenine (CPDA-1) whole blood stored in a Standard Blood Bank (SBB) and compare values with those stored in a Traditional Refrigerator (TR). A total of 37 apparently healthy volunteer donor subjects were used for the study. Twenty donors donated 450mL of whole blood each into CPDA-1 blood bags and were stored in a standard blood bank, while 17 units were collected from 17 donors into the same type of anticoagulant/preservatives but instead stored in a traditional refrigerator. Both refrigerators and standard blood bank were allowed the same relatively stable power supply for 35 days. Five milliliters of blood was taken at intervals of 7 days (1, 7, 14, 21, 28, and 35) from each of the bags for both SBB and TR methods of refrigeration and analyzed for ten parameters. It was observed that for both SBB and TR, K + levels increased from the 1 st to the 35 th day. K + levels for TR were significantly higher than those of SBB for days 7 (F=17.256, p= 0.000), 14 (F= 10.358, p= 0.000), 21 (F= 14.381, p= 0.000), 28 (F= 4.810, p= 0.000) and 35 (F=0.499, p=0.000). This statistically shows that the rise in K + level was more in TR refrigeration. Plasma Na + levels generally decreased for both groups. Comparison showed that Na + values for SBB were significantly higher than those of TR refrigeration for Days 7 (F= 1.684, p= 0.027) and 14 (F= 1.623, p= 0.009). Bicarbonate, albumin, pH, chloride and glucose levels for both groups were observed to decrease with storage time. Significant decreases were observed for Cl - and HCO3 - for Day 7 (F= 17.019, p= 0.000 and F= 0.404, p= 0.035 respectively), Cl - only for Day 14 and 21 (F= 3.253, p= 0.000 and F= 2.112, p= 0.000 respectively), albumin and glucose for Day 35 (F= 2.541, p= 0.036 and F= 1.272, p= 0.039 respectively) when mean values for SBB and TR were compared. Other parameters for the different days were not significant. Mean total protein levels for SBB and TR were significant for Days 7 (F= 13.136, p= 0.011); 14 (F= 9.842, p= 0.011); 21 (F= 6.344, p= 0.015) and 28 (F= 8.137, p= 0.035), while no significant difference was observed on the last day. The results show that there were significant changes in the levels of almost all ten parameters at different weeks of storage and the changes were more in the units stored in the traditional refrigerator and this could pose a very high health risk to the recipient on transfusion.

A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays.

BackgroundAvian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.—infected wild avian blood and it is reliable at a parasitaemia of at least 1 %. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1 %. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤0.0005 %) has been explored and validated.MethodsTo obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.—infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays.ResultsStorage of Plasmodium spp.—infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium.ConclusionsA simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤0.0005 %) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.

Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells.

Umbilical cord blood (CB) is considered one of the youngest available sources of adult stem cells. Besides hematopoietic stem cells, CB has been shown to contain endothelial progenitor cells as well as mesenchymal stromal/stem cells (MSC). To isolate MSC from cord blood, CB is collected into a sterile bag containing the anticoagulant citrate-phosphate-dextrose (CPD). The CB is then processed by density-gradient centrifugation to obtain mononuclear cells (MNC). These are cultured until the outgrowth of fibroblastoid cell colonies appears. After reaching a subconfluent stage, cells are harvested, expanded, and characterized as cord blood mesenchymal stromal cells (CB-MSC) according to standard criteria: plastic adherence, fibroblast morphology, CFU-f assay, proliferation potential, immune phenotype, and differentiation potential.Apparently, the frequency of MSC in CB is extremely low. Thus, not every CB unit will provide adequate MSC isolation yields. Different strategies have been proposed aiming to optimize the isolation success by selecting CB units of optimal quality. It is commonly agreed on that a high CB volume, a high cellular content, and a short time frame between birth and MSC isolation are criteria that will enhance the MSC isolation success.The procedures in this chapter are standardized protocols that were established and optimized in the authors' research laboratory; however, various modifications of the protocols are possible.

Gender and chronological age affect erythrocyte membrane oxidative indices in citrate phosphate dextrose adenine-formula 1 (CPDA-1) blood bank storage condition.

It is well known that in vitro storage lesions lead to membrane dysfunction and decreased number of functional erythrocytes. As erythrocytes get older, in storage media as well as in peripheral circulation, they undergo a variety of biochemical changes. In our study, the erythrocytes with different age groups in citrate phosphate dextrose adenine-formula 1 (CPDA-1) storage solution were used in order to investigate the possible effect of gender factor on oxidative damage. Oxidative damage biomarkers in erythrocyte membranes such as ferric reducing antioxidant power, pro-oxidant-antioxidant balance, protein-bound advance glycation end products, and sialic acid were analyzed. Current study reveals that change in membrane redox status during blood-bank storage condition also depends on both gender depended homeostatic factors and the presence of CPDA-1. During the storage period in CPDA-1, erythrocytes from the male donors are mostly affected by free radical-mediated oxidative stress but erythrocytes obtained from females are severely affected by glyoxidative stress.

Anticoagulants on yield of bone marrow-derived mononuclear cells harvested from dogs

Cell therapy with bone marrow-derived mononuclear cells is an alternative to therapy with mesenchymal stem cell cultures. The aim of the present research was the comparison of the yield of bone marrow-derived mononuclear cells harvested from dogs with two different anticoagulants. Bone marrow was harvested from the iliac crest of five healthy dogs aged between 15 and 30 months, and the effect of two anticoagulant solutions, CPDA-1 (citrate phosphate dextrose adenine-1) and heparin, on the isolation of mononuclear cells was compared. Mononuclear cells were isolated in a density gradient and stained for CD9 and CD44 for characterization by flow cytometry. Means were compared using Student's paired t-test. Samples harvested with CPDA-1 yielded an average of 5.16x106 (±1.76x106) to 20.20x106 (±1.55x106) mononuclear cells/mL, whereas the yield of samples harvested with heparin varied between 4.56x106 (±0.69x106) and 24.30x106 (±2.12x106) mononuclear cells mL-1. By flow cytometry, mean percentage of double-stained cells varied from 1.96% (±0.64%) to 5.01% (±0.73%) for CPDA-1 and from 2.23% (±0.70%) to 7.27% (±0.97%) for heparin. No significant statistical differences were observed on yield or CD9 and CD44 expression. Further studies are recommended to assess efficacy of CPDA on mononuclear cell isolation.

Survival Time of Cross-Match Incompatible Red Blood Cells in Adult Horses.

BackgroundThere is a markedly reduced half‐life of transfused RBCs when donor and recipient cats or humans are cross‐match incompatible. Only 10–20% of horses have naturally occurring alloantibodies. Therefore, cross‐match testing before blood transfusion is not always performed.HypothesisCross‐match incompatibility predicts shortened RBC survival time as compared to that of compatible or autologous blood.AnimalsTwenty healthy adult horses.MethodsProspective trial. Blood type, anti‐RBC antibody screen (before and 1 month after transfusion) and major and minor cross‐match determined 10 donor‐recipient pairs. Two pairs were cross‐match compatible, the remainder incompatible. Donor blood (4 L) was collected into citrate phosphate dextrose adenine‐1, labeled with NHS‐biotin, and transfused into recipients. Samples were collected at 1 hour and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after transfusion, and biotinylated RBCs were detected by flow cytometry. Horses were monitored for transfusion reaction during transfusion and daily for 5 days.ResultsCross‐match incompatibility was significantly associated with decreased RBC survival time (P < .001). The half‐life of transfused incompatible (cross‐match >1+) allogenic equine RBCs was 4.7 (95% CI, 3.2–6.2) days versus 33.5 (24–43) days for compatible pairings. Cross‐match incompatibility was associated with acute febrile transfusion reaction (P = .0083). At day 30, only 1 horse had developed novel anti‐RBC antibodies.Conclusions and Clinical ImportanceCross‐match incompatibility was predictive of febrile transfusion reaction and shortened transfused RBC survival, but did not result in production of anti‐RBC antibodies at 30 days. Cross‐match testing before transfusion is recommended.

Evaluation of an immunochromatographic test for feline AB system blood typing

To determine theaccuracy of an immunochromatographic cartridge (IC) test for blood typing feline type A, B, and AB blood samples. Prospective observational study. University teaching hospital. Fifty-one nonanemic and 19 anemic feline blood samples. Samples were blood typed by both card agglutination (CA) and IC techniques. Discordant results were analyzed using a back-typing technique for the presence of alloantibodies. Repeatability and reproducibility of the IC method were evaluated. Accuracy of the IC method was determined for feline whole blood anticoagulated with EDTA and citrate phosphate dextrose adenine (CPDA1), for feline-packed RBCs with CPDA1 and saline adenine glucose-mannitol, and for autoagglutinated and hemolytic samples. Accuracy of IC testing was determined for feline blood after room temperature, 4 ± 2, -20, and -80°C storage following 24, 48, 72, and 96 hours, and after 1, 2, 3, and 4 weeks of storage. In anemic and nonanemic samples the IC technique had a specificity, sensitivity, positive predictive value, and negative predictive value of 100% in detecting feline blood types A, B, and AB, outperforming a CA test. Results were repeatable and reproducible. Using IC it was possible to blood type samples anticoagulated with EDTA and CPDA1, packed RBC samples with CPDA1 and saline adenine glucose-mannitol, autoagglutinated and hemolytic samples, and samples stored at 4 ± 2°C and at room temperature for up to 1 month. The IC technique is an accurate assay for the identification of A, B, and AB blood types in anemic and nonanemic feline blood. It has a higher sensitivity and specificity than the CA test, and can be used in samples stored with common anticoagulants or preservative solutions used in feline transfusion medicine.