Preparation Of Red Blood Cells Research Articles

Preparation and preservation of red cells.

The production of blood components has undergone several changes during the last decades. Red blood cells will have slightly different properties depending on the way of preparation: whether a hard or soft spin has been used, whether platelets and/or leukocytes have been removed or not, and whether the red cells have been suspended in part of the original plasma or in an additive solution. Automated techniques are now often used for the separation of buffy coats, red cells and plasma. Recently, apheresis techniques have been applied for the preparation of red cells, mostly in combination with plasma or platelets. Continuous addition of the anticoagulant during collection reduces the delay between collection and separation, but the cost is higher and donor time longer. Most of the methods for the preparation and storage of red cells allow 35-42 days of storage with a mean in vivo recovery of > 75%. However, the content of erythrocyte 2,3-DPG is commonly lost within 1-2 weeks, caused by the accumulation of acid metabolites, but can be maintained longer with new systems of storage. Leukodepletion of red cells by filtration is used increasingly, but its importance in the majority of transfusions is still unclear. New options for the preparation and storage of red blood cells are available and undergo continuous evaluation.

Oxidative stress of vanadium-mediated oxygen free radical generation stimulated by aluminium on human erythrocytes.

It has been suggested that aluminium stimulates vanadium-mediated superoxide radical generation. The oxidative stress of generated superoxide radicals on antioxidant enzyme activity, oxidation of NADH and NADPH, membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC) was investigated. RBC were incubated with varying concentrations of vanadium and aluminium ions at 37 degrees C for 2 h. RBC incubated with vanadium ions showed significantly increased superoxide dismutase and catalase activities, and oxidized NADH and NADPH concentrations compared with control RBC preparations. Erythrocyte lipid peroxidation was assessed by measuring thiobarbituric acid reactivity. RBC incubated with elevated levels of vanadium showed significantly increased membrane lipid peroxidation when compared with control RBC; it increased further on addition of aluminium. A significant positive correlation was observed between the extent of vanadium induced membrane lipid peroxidation and the osmotic fragility of treated RBC. In the presence of vanadium, aluminium stimulates superoxide dismutase and catalase activities. NADH and NADPH oxidation and membrane lipid peroxidation, as well as increasing osmotic fragility of human erythrocytes. The stimulatory effect of aluminium was dependent on concentration. These results may have implications for the mechanism of toxicity of aluminium and vanadium in haemodialysis patients.

Preparation of red blood cells containing exogenous hemoglobin.

A technique is described for opening the membrane of a red blood cell by electroporation in a manner which permits free exchange of the native hemoglobin with exogenous hemoglobin in the surrounding medium. After resealing the RBC's demonstrate near normal size and hemoglobin content and retain an effective methemoglobin reduction system. This method can be used to introduce natural or genetically engineered hemoglobins with altered oxygen binding characteristics. Allosteric effectors and other non-diffusible small molecules can be encapsulated during the same procedure. A fish Root effect hemoglobin exchanged into rat RBC's produced oxygen transport characteristics, unloading at high pressure at acidic pH, which should be useful to treat tissue hypoxia from a variety of causes.

Hemoglobin polymorphism in the Atlantic croaker, Micropogon undulatus

Ten hemoglobins from the teleost Micropogon undulatus were separated in native polyacrylamide gel electrophoresis. In a large sample taken from a tributary of the Chesapeake Bay in Virginia, the distribution of these hemoglobins confirmed a polymorphic condition, which was previously known in North Carolina animals. The intraspecific variation was unrelated to body length, indicating that it is an adult phenomenon. The 10 hemoglobins were expressed in seven different phenotypic banding patterns. Although several morphs were infrequent, no single phenotype comprised a majority of the sample, indicating that the variation is quite great. Several phenotypes differed from those in North Carolina animals and, conversely, several North Carolina phenotypes were not recovered from the Chesapeake Bay sample. This finding suggests that the polymorphism is more complex than observed in either investigation alone. The O 2 binding of hemolysates stripped of co-factors by dialysis revealed functional differences between some, but not all, Chesapeake Bay morphs. The most variable property was O 2 affinity, although a few differences in pH dependence and cooperativity were also observed. The O 2 binding of red blood cell preparations also differed. These differences were not identical to those between dialyzed hemolysates, suggesting that organic PO 4 co-factors may be involved as well.

Detection of sarin hydrolysis products from sarin-like organophosphorus agent-exposed human erythrocytes

A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID 50 and LD 50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarin's hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.

α- Tocopherol Binding Activity of Red Blood Cells in Smokers

Despite high plasma levels of vitamin E, red blood cell membranes contain relatively low levels of vitamin E. This suggests the existence of a selective vitamin E uptake/regeneration system in human red blood cell membranes. α-Tocopherol binding sites on human red blood cells are thought to be involved in the uptake of α-tocopherol from the plasma. To understand the role of the uptake system we have compared the α-toco-pherol content and binding activity of red blood cells from smokers and non-smokers. The specific binding of [3H]α-tocopherol to pure red blood cell preparations from smokers (n = 7, 28.4 ± 2.8 years) was 30.6 ± 3.2 fmoles per 3 × 108 red blood cells and for non-smokers (n = 17, 27.9 ± 1.3 years) was 41.7 ± 3.7 fmoles per 3 × 108 red blood cells. Thus a-tocopherol uptake activity was significantly lower in smokers (P = 0.05). Red blood cells from smokers contained less (1.8 ± 0.4 μg/gHb) α-tocopherol than non-smokers (2.8 ± 0.3 μg/gHb), (P < 0.05), despite plasma levels of α-toco-pherol being similar: 12.9 ± 0.8 μM in non-smokers vs. 12.7 ± 0.5 μM in smokers. However, adjusting plasma α-tocopherol for total plasma cholesterol plus triacyl-glycerols showed α-tocopherol levels were higher (P < 0.01) in non-smokers (2.84 ± 0.10 μmol α-tocopherol/ mmol [cholesterol+triacylglycerol]) than in smokers (2.36 ± 0.11 μmol α-tocopherol/mmol [cholesterol+triacylglycerol]). The reduced α-tocopherol levels in red blood cells from smokers may be due to impairment of α-tocopherol uptake activity. The reduced levels of α-tocopherol in smokers red blood cells was not associated with any changes in cell membrane fluidity. At present it is not known whether supplementation of smokers with vitamin E would normalise the α-tocopherol uptake activity of red blood cells.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10 3.2 egg lethal doses (ELD 50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40°C) was indicative to the likelihood of detectable antigenaemia. After day 7 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

Influence of the red blood cell preparation method on the efficacy of a leukocyte reduction filter.

The performance of a leukocyte reduction bedside filter with different types of RBC concentrates was analyzed. Three types of RBCs were prepared: buffycoat-depleted RBCs suspended in saline-adenine-glucose-mannitol (SAGM)-additive solution (BC-RBCs; n = 20), RBCs suspended in SAGM-additive solution without buffy coat removal (SAGM-RBCs; n = 20), and RBCs drawn in CPDA-I conservative solution and processed for component preparation by the platelet-rich plasma method (CPDA-RBCs; n = 20). The units were filtered within 8 h of collection. One filter was used for every 2 units. High numbers of residual WBCs were found even in the units filtered first. Filtration of CPDA-RBCs resulted in a higher residual WBC content than SAGM-RBCs or BC-RBCs (p = 0.0032 and p = 0.0002, respectively). The filter performance strikingly decreased when the WBC load per filter exceeded 4 x 10(9) or the platelet load was less than 100 x 10(9). We conclude that filter performance varies with the WBC and platelet content of the RBC concentrates. Under the experimental conditions assayed in this study CPDA-RBCs are the least appropriate ones to be used for bedside leukocyte reduction.

Influence of the Red Blood Cell Preparation Method on the Efficacy of a Leukocyte Reduction Filter

Influence of the Red Blood Cell Preparation Method on the Efficacy of a Leukocyte Reduction Filter

Optimal volume of anticoagulant citrate dextrose solution in the preparation of technetium Tc 99m-labeled red blood cells

Optimal volume of anticoagulant citrate dextrose solution in the preparation of technetium Tc 99m-labeled red blood cells

Transfusion-transmitted Yersinia enterocolitica infection. Protection through buffy coat removal and failure of the bacteria to grow in platelet-rich or platelet-poor plasma.

In a previous study, removal of white blood cells (WBC), by filtration 5 h after deliberate contamination of whole blood with a type 0:3 strain of Yersinia enterocolitica, was shown to be an effective way of avoiding bacterial growth in red blood cells (RBC) during storage. In the present study the Opti-System technique was used to remove the buffy coat from whole blood, leaving 10-20% of the original number of WBC in the RBC preparation. In one series of experiments, all of 4 units of RBC suspension, from which buffy coats were removed 2 h after inoculation of 112 colony-forming units (cfu) per ml of Y. enterocolitica, became Yersinia-free, while abundant bacterial growth occurred in all of 4 units where RBC suspension and buffy coat had been recombined. In a second series of 10 experiments, with an inoculum of 80 cfu/ml, no growth was found in platelet-poor plasma stored for 42 days at 4 degrees C. Five out of 10 RBC suspensions in SAGM additive solution remained Yersinia-free throughout a 6-week storage period; 4 of these 10 units showing growth of Yersinia after 4 weeks and 5 after 6 weeks. In the buffy coats bacterial growth was found in 1 out of 10 units after 1 week, 4 after 2 weeks, and in all of 10 units after 4 weeks. In 2 control experiments with WBC-reduced RBC inoculated with the same bacterial dose, growth started within 24 h and was abundant after 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfusion-Transmitted Yersinia enterocolitica Infection

In a previous study, removal of white blood cells (WBC), by filtration 5 h after deliberate contamination of whole blood with a type 0:3 strain of Yersinia enterocolitica, was shown to be an effective way of avoiding bacterial growth in red blood cells (RBC) during storage. In the present study the Opti-System technique was used to remove the buffy coat from whole blood, leaving 10- 20% of the original number of WBC in the RBC preparation. In one series of experiments, all of 4 units of RBC suspension, from which buffy coats were removed 2 h after inoculation of 112 colony-forming units (cfu) per ml of Y. enterocolitica, became Yersinia-iree, while abundant bacterial growth occurred in all of 4 units where RBC suspension and buffy coat had been recombined. In a second series of 10 experiments, with an inoculum of 80 cfu/ml, no growth was found in platelet-poor plasma stored for 42 days at 4°C. Five out of 10 RBC suspensions in SAGM additive solution remained Yersinia-free throughout a 6-week storage period; 4 of these 10 units showing growth of Yersinia after 4 weeks and 5 after 6 weeks. In the buffy coats bacterial growth was found in 1 out of 10 units after 1 week, 4 after 2 weeks, and in all of 10 units after 4 weeks. In 2 control experiments with WBC-reduced RBC inoculated with the same bacterial dose, growth started within 24 h and was abundant after 1 week. In platelet-rich plasma (PRP), prepared from 4 units of whole blood that had been inoculated with Y. enterocolitica (106 cfu/ml), no bacterial growth was found during 10 days of storage at 22°C. Also, after Y. enterocolitica inoculation (114 cfu/ml) directly into PRP, bacteria failed to grow. We conclude that removal of the buffy coat from whole blood that had been previously contaminated with a moderate inoculation of Y. enterocolitica partly protects the red cells from bacterial growth during storage. The risk of transmitting Y. enterocolitica with platelet-poor plasma, liquid stored at 4°C, seems minimal. This bacterial species shows very poor capacity to grow in PRP at 22 °C.

Red blood cells from patients with sickle cell disease exhibit an increased adherence to cultured endothelium pretreated with tumour necrosis factor (TNF).

Red blood cells (RBCs) from 24 patients with sickle cell disease were more adherent to cultured endothelium pretreated with the inflammatory cytokine, tumour necrosis factor (TNF) than RBCs from 22 healthy subjects. The enhanced sticking was apparent in RBC preparations from patients who were in crisis (mean 190% increase from controls) and out of crisis (mean 220% increase) and was not related to the number of circulating RBCs, reticulocytes, platelets, leucocytes or haemoglobin levels. When irreversibly sickled RBCs, enriched by centrifugation on density gradients, were added to TNF-treated endothelium they were found to be significantly more adherent (mean 411% increase; P < 0.001) than the unfractionated RBCs from the same patients. There was no difference between the adherent properties of sickle RBCs and normal RBCs for untreated endothelium. Contributing factors to the enhanced adhesion to TNF-treated endothelium may be the low surface change of sickle RBCs, and increased levels of fibrinogen and von Willebrand's factor (vWF) in the patients' plasma. By acting on vascular endothelium to increase its adhesiveness for sickled RBCs, it is concluded that inflammatory cytokines such as TNF may have a prominent role in mediating the events that lead to microvascular occlusions in sickle cell disease.

Clustering of erythrocytes by fibrinogen is inhibited by carnitine: Evidence that sulfhydryl groups on red blood cell membranes are involved in carnitine actions

Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde-fixed human erythrocytes in a non-saturable, temperature-dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.

Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: A photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.

South american rattlesnake venom: its hemolytic power

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage of positivity for hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly prepared for the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

４００ｍｌ採血由来白血球除去赤血球の調製に用いるフィルターの検討

Patients who have antileukocyte antibody production should be transfused with Leukocyte-Poor Red Blood Cells to prevent non-hemolytic febrile transfusion reactions.We prepared Leukocyte-Poor Red Blood Cells by four kinds of leukocyte removal filters in accordance with the Technical Manual of the Japanese Red Cross Society and have discussed respective functions of the filters.The removal of Leukocytes, removal of platelets and the recovery of erythrocytes after filtration were as follows: Imugard IG-400Y filtration were 98.5±0.7%, 68.1±9.7%, 95.2±1.6%, respectively, Erypur filtration were 96.3±1.5%, 89.9±5.7%, 85.3±3.0%, respectively, Miropore filtration were 91.2±2.3%, 85.0±3.1%, 95.0±1.7%, respectively, Sepacell filtration were 97.7±1.1%, 94.6±2.2%, 97.4±1.6%, respectively.No significant changes of erythrocytes were observed after filtration by four kinds of filters with respect to ATP content, 2, 3-DPG content and osmotic resistance value of erythrocytes.

The influence of ascorbic acid on the free-radical metabolism of xenobiotics: the example of diaziquone.

Diaziquone (AZQ) is a quinone-containing alkylating agent undergoing trials as an antitumor drug. The quinone moiety of this compound places it among a group of compounds whose activity is believed to be modulated by a redox cycle that activates the compounds to their free radicals (e.g., adriamycin). AZQ is unique among these compounds in that it can be reduced to its free radical (AZQH) by a variety of cells in culture, including human and murine cancer cells. Red blood cells (RBC) were also observed to reduce AZQ to its free radical. Using electron spin resonance (ESR), we observed that soon after the AZQ free radical appeared, it decayed and was replaced by a doublet with ESR parameters that suggested the presence of the ascorbyl radical (AH). The identity of AH was confirmed by adding exogenous ascorbic acid to AZQ free radicals generated by a suspension of L1210 murine leukemia cells. The endogenous ascorbic acid was shown to arise mostly from the "buffy coat" of an RBC preparation which contained leukocytes. Leukocytes are second only to the adrenals in level of ascorbic acid in humans. Cyclic voltammetry of ascorbic acid, AZQ, and adriamycin in Hank's Balanced Salt Solution (HBSS pH 7.5), the buffer used for biological measurements, showed that the oxidation peak potential for ascorbic acid (Eap = +0.43 V) is closer to the reduction peak potential for AZQ (Ecp = -0.36 V) than that for adriamycin (Ecp = -0.67 V). This may explain why the ascorbic acid redox system interacts with that of AZQ but not with that of ADR.

Separation of red blood cell-depleted mononuclear marrow cells by continuous-flow centrifugation using the IBM 2997 blood cell separator—Results of a preclinical study in a dog model

As an alternative technique to density-gradient separation, we studied the preparation of red blood cell (RBC)-depleted marrow mononuclear cells (MNC) by continuous-flow centrifugation (CFC) using the IBM 2997 cell separator in a dog model. Mean recoveries of nucleated and mononuclear cells obtained by this separation technique were 43.1% and 77.7% , respectively, and the mean residual RBC volume was 1.8 mL (1.2% of the RBC volume prior to separation). A mean of 91.4% of marrow progenitor cells were regained in the RBC-depleted fraction. Transplantation of the separated marrow MNC into seven lethally irradiated beagle littermates resulted in complete and durable hematopoietic chimerism. Our results indicate that separation of marrow MNC using an IBM 2997 cell separator is an effective and technically uncomplicated alternative to gradient medium separation.

Monitoring of platelet contamination in filtered red blood cell concentrates. Use of a radioimmunoassay detecting the platelet glycoprotein IIb-IIIa complex.

Monitoring of contaminating platelets, granulocytes, and lymphocytes in leukocyte-poor red blood cell concentrates is usually done by counting in an electronic particle counter. Sensitivity and specificity of this technique are compromised by the contamination of the preparations with other cell types and particles thereof. In this report we studied platelet contamination in filtered red blood cell concentrates by use of a radioimmunoassay for detection of the platelet glycoprotein complex IIb-IIIa. Our results indicate that platelets and/or fragments thereof, not detectable for particle counters, are present in blood cell concentrates. This finding might have important implications for the preparation of pure red blood cell concentrates to avoid unwanted immunization after transfusion.