Rheological properties and function of blood cells in stored bank blood and salvaged blood

The technique involving filtration of diluted blood enables the separate analysis of the flow properties of different cell subpopulations. This study was designed to assess the changes occurring in the flow properties and function of blood cells in stored bank blood and salvaged blood compared to patient blood in a given clinical situation. We measured hydrogen peroxide production by neutrophils and the filterability, through 5 μm Nucleopore filters, of isolated red blood cells and of diluted blood. Samples were obtained from patients undergoing aortic surgery and blood intended for transfusion: either salvaged during surgery or stored bank blood. Both salvaged and bank blood were much less filterable than patient blood, with reduced deformability of both red and white blood cells. However, salvaged blood contained highly activated neutrophils with a prolonged transit time of the 'fast-flowing' cells in the analysis compared to bank blood. Bank blood contained significantly more particles which acted as pore-blockers. Cells in bank and salvaged blood therefore have markedly abnormal flow and biochemical properties compared to patient blood.     

Characteristics of leucocyte adhesion directly observed in flowing whole blood in vitro

Use of whole blood in adhesion assays allows analysis of the rheological and haematological factors that may influence adhesion, and avoids the need for isolation procedures that may modify the properties of leucocytes. We have adapted an in vitro flow model to allow videomicroscopy of leucocytes fluorescently labelled with rhodamine 6G (20 microg/ml) in anticoagulated whole blood. Blood was perfused at a range of wall shear rates (35-280/s) through a vertical glass capillary with a rectangular cross-section (microslide) that had been coated with P-selectin (10 microg/ml). Nearly all adherent cells were rolling in blood that had been anticoagulated with buffered citrate, but 40-50% became immobilized when heparin or thrombin inhibitor (PPACK) were used. The efficiency of leucocyte adhesion decreased steadily during 1-4 h of blood storage. Smaller fluorescent cells (lymphocytes) adhered less efficiently than larger cells (granulocytes) and rolled faster. Adhesion decreased monotonically with increasing wall shear rate or stress, but the velocity of rolling varied little. Among healthy volunteer donors, adhesion correlated with blood leucocyte count, but did not vary significantly with natural variation in haematocrit, blood viscosity or red cell aggregation. In conclusion, we have characterized adhesion of leucocytes in flowing whole blood, identified key experimental variables and demonstrated that physical environmental factors can markedly influence adhesive behaviour.     

Lovastatin therapy in heterozygous familial hypercholesterolaemic patients: effect on blood rheology and fibrinogen levels

Thirteen heterozygous familial hypercholesterolaemic patients were treated with lovastatin for 1 year, and were investigated for the effect on lipid profile, blood rheology and fibrinogen levels. A significant dose-dependent reduction in serum levels of total and LDL-cholesterol, Apo B and the ratio of total cholesterol to HDL-cholesterol was noted. Improvement in red blood cell filterability and an increase in fibrinogen levels were also observed. We conclude that the hypocholesterolaemic effect of lovastatin in familial hypercholesterolaemia is accompanied by changes in blood rheology. While some of these haemorheological changes may be considered beneficial, others may be regarded as unfavourable. The net effect of lovastatin therapy on the coronary risk of familial hypercholesterolaemic patients warrants further investigation.     

Allogeneic cord blood red blood cells: assessing cord blood unit fractionation and validation

BackgroundFor neonates and preterm infants, in whom a transfusion dose is low, the use of red blood cells (RBC) from cord blood appears to be feasible. Standardisation of fractionation and identification and assessment of quality control parameters for such RBC are still lacking.Materials and methodsWe describe the process used to obtain RBC from cord blood for transfusion purposes, including quality controls to evaluate fractionation performance and the effects of storage. The cord RBC, to which SAG-M was added, were sampled on the day of fractionation, and 7 and 14 days (end of storage) later in order to measure the complete blood count, biochemical parameters and residual white blood cells. We also assessed microbial contamination.ResultsData relative to 279 cord blood units were evaluated. The median gestational age at collection was 40 weeks (interquartile range [IQR] 39.1–40.7) and the median volume was 90 mL (IQR 81–103). Units were subjected to automated fractionation with Compomat, and packed RBC were suspended in SAG-M solution. The median volume of the SAG-M-suspended units was 31 mL (IQR 24.0–38.1) and the median haematocrit was 54.2% (IQR 49.4–59.5). The median volume after leukoreduction was 22 mL (IQR 17–28), with the volume decrease being similar in units leukoreduced before (n=75) or after (n=204) storage. The haematocrit of leukoreduced units was higher than that of buffy coat-depleted units. Storage at 2–6 °C for 14 days was accompanied by an increase of potassium levels and percentage of haemolysis. Microbial cultures were positive for 2.9% of the collected units.DiscussionFractionation of whole cord blood can provide RBC concentrates with similar baseline characteristics as units from adults. The transfusion dose and quality of the units appear safe and suitable for clinical use in neonates, with a satisfactory haematocrit and residual white blood cell content, despite a very variable collection volume.     

手工分少白细胞红细胞和悬浮红细胞输注疗效观察

目的:了解并比较手工分少白细胞红细胞和悬浮红细胞的治疗效果。方法:将2 582例患者分为输注悬浮红细胞组(1 450例)和输注手工分少白细胞红细胞组(1 132例),输注前测定Hb值,复查患者及献血者ABO和RH(D)血型并作凝聚胺法交叉配血试验,输血后给每例患者进行血液学分析,检查Hb值,然后再进行输血不良反应发生率统计。结果:输注手工分少白细胞红细胞效果好于普通悬浮红细胞,并且输注手工分少白细胞红细胞的输血不良反应发生率明显减少。结论:输注手工分少白细胞红细胞可以使输血后非溶血性输血反应发生率降低。     

Disturbed flow promotes deposition of leucocytes from flowing whole blood in a model of a damaged vessel wall

Departure from simple laminar flow in arteries may promote the local attachment of leucocytes either to intact endothelium or platelet thrombi. We perfused blood through a chamber with a backward facing step, to observe whether adhesion from whole blood to P-selectin was indeed localized to a region of recirculating flow, and whether platelets binding to collagen in such a region could capture leucocytes. Blood flowing over the step established a stable vortex, a reattachment point where forward and backward flow separated, and a simple laminar flow with wall shear rate c. 400/s further downstream. Fluorescently labelled leucocytes were observed to attach to P-selectin immediately upstream or downstream of the reattachment point, and to roll back towards the step or away from it, respectively. There was negligible adhesion further downstream. When a P-selectin-Fc chimaera was used to coat the chamber, stable attachment occurred, again preferentially in the disturbed flow region. Numerous platelets adhered to a collagen coating throughout the chamber, although there were local maxima either side of the reattachment point. The adherent platelets captured flowing leucocytes in these regions alone. Leucocytes may adhere from flowing blood in vessels with high shear rate if the flow is disturbed. While platelets can adhere over a wider range of shear rates, their ability to capture leucocytes may be restricted to regions of disturbed flow.     

Assessing quality of blood components derived from whole blood treated with riboflavin and ultraviolet light and separated with a fully automated device

BackgroundCombining pathogen reduction and automated separation of whole blood (WB), together with the use of improved additive solutions, may increase reproducibility and extend shelf-life of blood components.Materials and methodsForty WB units were collected from volunteer donors and randomised 1:1 into two groups: 1) pathogen reduction with riboflavin and ultraviolet light (PRT); or 2) no treatment (Control). After two hours (h) at room temperature, all units underwent fully automated separation into red blood cell concentrate (RBCC), plasma and leukopack components. RBCCs were leukoreduced and stored in phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) solution while plasma units were shock frozen within 8 h of collection and stored at ≤ −25°C. RBCCs were sampled on day 1 and weekly thereafter until day 42, while plasma was sampled on days 1 and 30. The main study objective was to assess the in vitro quality of separated RBCCs using biochemical and haematological parameters. Plasma protein content after one cycle of freeze-thaw was also analysed.ResultsThe quality of RBCCs was largely comparable between the PRT and Control groups, except for a significantly higher degree of haemolysis and extracellular potassium levels in the PRT group after 35 days of storage. While potassium concentration was significantly higher in the PRT group at all timepoints, the degree of haemolysis exceeded the accepted European threshold (i.e., <0.8% of red cell mass in ≥ 90.0% of tested units) after day 35. Most plasma protein levels were significantly lower in the PRT than the Control group at both day 1 and day 30.DiscussionPathogen reduction with riboflavin and ultraviolet light treatment of WB can be combined with fully automated separation to obtain RBCCs that may be stored for up to 35 days in PAGGSM solution with acceptable quality, comparable to that of RBCCs from untreated blood. The relative differences between factor concentrations in plasma from the PRT and the Control groups were similar during the 30-day storage.     

Increasing oxygen tension improves filtration of sickle trait donor blood

A major cause of filter failure of red cell (RBC) components from donors with sickle cell trait (HbAS) is the polymerization of haemoglobin. The oxygen saturation (sO2) of blood stored in various plastics and different volumes of air was assessed. Blood from 10 HbAS donors was collected and divided into two bags, one with air added, one without. Bags with added air had increased sO2 levels (from 49 +/- 10% to 76 +/- 6%). Filtration was successful for nine of 10 components with air, and one of 10 without air. Successful filtration of RBC components occurs when sO2 is increased.     

Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools

Background and Objectives Microparticles (MPs) are small phospholipid vesicles of less than 1 µm, shed in blood flow by various cell types. These MPs are involved in several biological processes and diseases. MPs have also been detected in blood products; however, their role in transfused patients is unknown. The purpose of this study was to characterize those MPs in blood bank conditions. Materials and Methods Qualitative and quantitative experiments using flow cytometry or proteomic techniques were performed on MPs derived from erythrocytes concentrates. In order to count MPs, they were either isolated by various centrifugation procedures or counted directly in erythrocyte concentrates. Results A 20-fold increase after 50 days of storage at 4°C was observed (from 3370 ± 1180 MPs/µl at day 5 to 64 850 ± 37 800 MPs/µl at day 50). Proteomic analysis revealed changes of protein expression comparing MPs to erythrocyte membranes. Finally, the expression of Rh blood group antigens was shown on MPs generated during erythrocyte storage. Conclusions Our work provides evidence that storage of red blood cell is associated with the generation of MPs characterized by particular proteomic profiles. These results contribute to fundamental knowledge of transfused blood products.     

A new density gradient system for the separation of human red blood cells

A new density system for the separation of human red blood cells by density-gradient centrifugation is described. The gradient medium is made with colloidal silica particles coated with polyvinylpyrrolidone suspended in aqueous solution of meglamine diatrizoate. By this method, more than 10 red-cell fractions can be separated. These show different ages (by creatine content and ⁵⁹Fe in vivo labelling) and different characteristics (ie, potassium content). A 40-fold enrichment in reticulocytes can be obtained in the top layers, with a great improvement of specific activity in labelling procedures of newly synthesized globin chains. The method is simple, rapid, inexpensive, reliable, nontoxic for erythrocytes, and is suitable for globin synthesis and other studies of erythrocyte metabolism.     

Aggregation of red blood cells in patients with Gaucher disease

Gaucher disease is associated with increased red blood cell (RBC) aggregation, but the pathophysiological significance of this phenomenon and its correlation with disease manifestations are unclear. RBC aggregation was evaluated in 43 patients with Gaucher disease and 53 healthy controls. Dynamic RBC aggregation was examined in a narrow-gap flow chamber at varying shear stress. Compared with the controls, RBC aggregation in Gaucher disease was increased by 25%. Comparison of RBC aggregation in autologous plasma and in dextran (500 kDa) showed an increase both in plasma-dependent (extrinsic) and -independent (intrinsic) RBC aggregation. Subgroup analysis revealed that increased RBC aggregation was limited to patients with an intact spleen. RBC aggregation in patients did not correlate with plasma fibrinogen concentration, disease severity, enzyme replacement therapy or genotype. We conclude that RBC aggregation is increased in patients with Gaucher disease and an intact spleen, possibly reflecting the accumulation of glucocerebroside and other substances in the plasma and RBC membranes of these patients. Our results do not support a role for RBC aggregation in the pathogenesis of vascular complications of Gaucher disease.     

Hypertonic cryohemolysis: a diagnostic test for hereditary spherocytosis

Red blood cells of subjects with hereditary spherocytosis are specifically susceptible to temperature changes while suspended in hypertonic solutions. Based on this property, a new diagnostic test for hereditary spherocytosis is presented. The suggested method is 100% sensitive in the diagnosis of all patients, including asymptomatic obligate carriers with hereditary spherocytosis, and is very specific in different control groups. Unlike other methods designed for the diagnosis of hereditary spherocytosis, this test does not depend on the cells' surface- area-to-volume ratio. Normal red blood cells that were induced to become spherocytes by different means (i.e., vinblastine, lysophosphatidyl choline, and heat) and showed increased osmotic fragility did not become susceptible to the hypertonic cryohemolysis conditions.     

Preferential localisation of red cell vacuoles to pathologically shaped human red blood cells

The percentage of pitted erythrocytes and Howell-Jolly bodies in peripheral blood samples of 51 individuals following posttraumatic splenectomy and 20 patients splenectomized because of various haematological diseases differed significantly from each other (p less than 0.001) and from that of healthy controls (p less than 0.001). The percentage of pitted erythrocytes was significantly higher in pathologically shaped red blood cells (RBCs) (acanthocytes, schizocytes, elliptocytes) than in normal discoid shaped RBCs (p less than 0.001). As the number of pits per RBC showed great individual variations, a scoring system for the evaluation of pitted RBCs is proposed.     

Antibodies against human neutrophil antigens in non-transfused women with red blood cell alloimmunisation induced by pregnancy

BackgroundAlloantibodies against human neutrophil antigens (HNA) resulting from allogeneic exposure may be associated with transfusion-related acute lung injury and immune neutropenia. Understanding the risk factors for the formation of such antibodies could have a great impact on the adoption of measures to prevent potentially fatal transfusion reactions. The aim of the study was to determine the prevalence of anti-HNA alloantibodies in non-transfused pregnant women with and without red blood cell (RBC) alloantibodies.Materials and methodsHNA alloantibodies were investigated in blood samples from 147 pregnant women with RBC alloimmunisation induced by pregnancy as the only allogeneic stimulus (group 1). The control group (group 2) consisted of 563 women with at least one pregnancy without RBC alloimmunisation. Both groups were investigated for the presence and identity of HNA alloantibodies using granulocyte agglutination tests, white blood cell immunofluorescence testing, and the bead-based LABScreen Multi Kit. Genotyping was performed to confirm the specificity of the HNA alloantibodies.ResultsGroup 1 women had a statistically higher number of HNA alloantibodies compared to group 2 women (9/147 [6.1%] vs 9/563 [1.6%]; p=0.005, OR=4.01; 95% CI 1.5–10.3). Considering only multiparous women, there was a higher statistical significance for the difference in the presence of HNA alloantibodies between the two groups (7/82 [8.5%] vs 9/493 [1.8%]; p=0.002, OR=5.02; 95% CI 1.8–13.9).DiscussionOur data show that RBC alloimmunisation is significantly associated with the development of anti-HNA alloantibodies, corroborating the hypothesis that some individuals are better immune responders and react strongly to allogeneic exposure. The presence of RBC alloantibodies can, therefore, facilitate the identification of individuals with a higher risk of alloimmunisation to antigens from other cells, also acting as a tool to avoid potentially fatal transfusion reactions.     

红细胞单采在真性红细胞增多症治疗中的应用

目的:观察分析红细胞单采在真性红细胞增多症(PV)治疗中的应用方法及效果。方法:对符合国内PV诊断标准的19例患者,运用血细胞分离机进行异常增生红细胞单采,配合临床药物治疗。比较治疗前后患者血常规中Hb、RBC、Hct、WBC、PLT均值变化,采用SPSS17.0统计学软件统计分析。观察依患者公斤体重和Htc设定红细胞采集量,单次采集后Hct降低情况及首次采集前后Hb均值变化与再次采集前后Hb均值变化的差异。结果:治疗后血液Hb、RBC、Hct均值明显低于治疗前,差异有统计学意义(P0.01)。8例完全缓解,7例临床缓解,4例好转,有效率为100%;单次采集Hct平均降低幅度为16.11%。结论:红细胞单采在PV的治疗中具有显著的疗效,单次采集应考虑PV患者血容量绝对增加的情况,根据患者病情和对红细胞去除的耐受情况适当增加采集量。