The effect of cell hydration on the deformability of normal and sickle erythrocytes

The deformability of the erythrocyte (RBC) is greatly influenced by its state of hydration. The purpose of this investigation is to quantitate this relationship by measuring the deformability of an RBC population over a broad range of cell water content. By manipulation of the ion content of the RBC, we performed all of the experiments in media which were isotonic with plasma. To raise ion and water content, RBC were incubated in a Li₂CO₃ medium. To lower cell ion and water content, RBC were exposed to the K ionophore, valinomycin. The range of cell water content achieved during the entire experiment was 900–3200 g/kg cell solid (normal in vivo cell water content being 1800–1950 g/kg cell solid). By using the Ektacytometer, an automated cylindrical viscometer, we were able to measure deformability of the RBC sampled at various points along this range of cell water content. We found that optimal rheologic behavior was exhibited by normal RBC when their water content was in the normal range. A rise or a fall in cell hydration resulted in a decrease in cell deformability. By contrast, the deformability of freshly drawn, well-oxygenated sickle RBC was well below that found for normal RBC. Upon volume expansion, however, the deformability of these sickle RBC improved markedly. This observation suggests that sickle RBC are suboptimally hydrated and that their abnormal rheology is at least in part a consequence of cell dehydration.     

Influence of calcium permeabilization and membrane-attached hemoglobin on erythrocyte deformability.

The present study was designed to evaluate the influence of intracellular calcium [Ca]i regulated membrane attached hemoglobin (Hbm) on the deformability of human RBC and ghosts. [Ca]i of RBC was elevated via the ionophore A23187 (10 microM); the deformability of RBC and resealed ghosts was determined via measuring RBC and ghost transit times through 5 microns diameter pores with the Cell Transit Analyzer (CTA). Salient results included: (1) significantly increased RBC levels of Hbm following ionophore treatment; (2) elevated Hbm with increasing lysing medium calcium concentration (0-5 mM); (3) decreased deformability of both intact RBC and ghosts with increasing Hbm and significant (P less than 0.02 or better) linear relationships between Hbm and RBC or ghost transit times; and (4) an increased sensitivity to ionophore treatment/membrane attached hemoglobin for the higher percentiles of the CTA transit time distribution (i.e., for more rigid subpopulations). Our results thus indicate that calcium-induced interaction of hemoglobin with the RBC membrane produces cellular rheological changes; in addition, they demonstrate the usefulness of the CTA system in measuring both average RBC rheologic behavior and the distribution of cellular rheologic properties within an erythrocyte population.     

Studies on the sequestration of chemically and enzymatically modified erythrocytes

We have developed an experimental animal model to establish the patterns of sequestration of untreated, as well as chemically and enzymatically modified red blood cells (RBC). The intraperitoneal route of transfusion provides a useful way of transferring large numbers of untreated RBC into small animals and assuring their introduction into the circulation within 24 hr. Moreover, this route "filters" some types of modified erythrocytes, eg, glutaraldehyde treated RBC. From the pattern of sequestration, the RBC in the peritoneal cavity then pass through the liver where other types of modified RBC are sequestered, eg, after trypsin, pronase, protease, or sialidase treatment. Some modified RBC show a preference for the spleen as the site of sequestration, eg, galactose oxidase or N-ethylmaleimide treated RBC. These appear in the spleen despite intraperitoneal transfusion. Relevant to this study is the observation that in the rat old RBC are sequestered both by liver and spleen, while asialoerythrocytes are sequestered by liver only. A possible reason for this difference is discussed in the text.     

A detailed study of time-dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence

The use of microfabrication technology in the study of biological systems continues to grow rapidly in both prevalence and ascendancy. Customised microdevices that provide superior results than traditional macroscopic methods can be designed in order to investigate specific cell types and cellular processes. This study showed the benefit of this approach in precisely characterising the progressive losses of surface area and haemoglobin (Hb) content by the human red blood cell (RBC), from newborn reticulocyte to senescent erythrocyte. The high-throughput, multiparametric measurements made on individual cells with a specialised microdevice enabled, for the first time, delineation and quantification of the losses that occur during the two stages of the human RBC lifespan. Data acquired on tens of thousands of red cells showed that nearly as much membrane area is lost during the 1-2 d of reticulocyte maturation (c. 10-14%) as in the subsequent 4 months of erythrocyte ageing (c. 16-17%). The total decrease in Hb over the red cell lifespan is also estimated (c. 15%) and a model describing the complete time-course of diminishing mean RBC area and Hb is proposed. The relationship between the losses of Hb and area, and their possible influence on red cell lifespan, are discussed.     

Rapid quantitation of mixed red cell populations using flow cytometry

Methods are described for the identification and quantitation of mixed red cell populations using flow cytometry. Antibodies specific for a wide range of blood group antigens have been used and examples are given in which these analyses have proved to be of clinical use. These examples include monitoring of erythropoiesis following engraftment in allogeneic bone marrow transplant recipients and the detection of chimaeric states months or years after transplantation. The techniques involved are fast, simple and inexpensive.     

甲状腺功能低下大鼠血浆激素和脑红细胞膜神经鞘磷脂变化

目的为了了解大鼠实验甲低条件下，血浆激素、脑、红细胞膜神经鞘磷脂的变化。方法应用薄层色谱扫描法，观察了甲基硫脲嘧啶（ＭＴＵ）复制甲状腺功能低下大鼠甲状腺重量、血浆Ｔ３、Ｔ４及脑和红细胞膜神经鞘磷脂（ＳＭ）变化。结果持续用ＭＴＵ组甲状腺重量及脑和红细胞膜ＳＭ明显增加，血浆Ｔ３、Ｔ４水平下降，随着停ＭＴＵ时间不同，其变化不同。结论甲状腺激素不足，可能通过脑、红细胞ＳＭ改变使神经系统功能紊乱和结构障碍     

Restoration of red blood cell volume following 2-unit red blood cell apheresis

BACKGROUND AND OBJECTIVES: Blood donors who weigh at least 130 lbs (59 kg) and have a haematocrit of at least 40 V per cent can donate 2 units of blood, from which a 360-ml volume of red blood cells (RBC) can be isolated. This study was carried out in seven healthy male blood donors to assess the restoration of the RBC volume 1 month following a 2-unit RBC apheresis procedure. MATERIALS AND METHODS: RBC volumes were measured prior to donation and 4 weeks after the 2-unit RBC apheresis procedure without oral iron supplementation. RESULTS: Four weeks after the removal of 2 units of RBC from the male donors not supplemented with oral iron, the RBC volume was restored to 92% of the precollection value. The 360-ml volume of RBC collected represented 12-19% of the donor's original RBC volume. CONCLUSIONS: Male donors can safely donate 2 units of RBC and will restore a mean of 92% of their RBC volume within 1 month without iron supplementation.     

单采血小板献血者血液因素对采集冲红影响的分析

目的:了解血小板献血者血液因素对采集冲红的影响。方法:检测捐献时出现冲红者和正常捐献者血小板采集前各血液因素状况,并计算本次捐献前半年捐献血小板次数,进行统计分析。结果:以RBC等11个因素为自变量进行单因素Logistic回归分析,RBC、HGB、HCT、MCV、MCHC、PDW、MPV、P-LCR、RDW-CV、半年内献血次数均不具有意义,而MCH异常者冲红的概率平均是正常者的9.726倍。拟合方程:P=e-0.429+2.275x/(1+e-0.429+2.275x)。结论:献血者单采前MCH是影响是否出现冲红现象的重要因素。     

Spontaneous endocytosis in human neonatal and adult red blood cells: comparison to drug-induced endocytosis and to receptor-mediated endocytosis

Neonatal RBC contain many more spontaneous endocytic vacuoles than do adult RBC. It is not known if this difference is a result of an increase in production of vacuoles in the neonatal RBC (as is the case in drug-induced endocytosis), or is the result of a less effective neonatal macrophagic "pitting" process. Using an in vitro model of spontaneous endocytosis, we compared the rate and quantity of vacuoles and the shape of cord and adult RBC containing pits, visible by interference contrast microscopy (Nomarski method). The mechanism of the spontaneous endocytosis was explored using different inhibitors: sodium vanadate an inhibitor of ATPases, sodium fluoride which inhibits the generation of ATP and sodium cyanide a potent inhibitor of oxidative phosphorylation. We then compared spontaneous endocytosis with two other forms of RBC endocytosis: drug-induced endocytosis and receptor-mediated endocytosis. Spontaneous endocytosis is in fact increased in neonatal RBC initially but the increase in number of RBC containing pits after 144 hr of incubation is almost the same in adult RBC and neonatal RBC. Comparing spontaneous endocytosis with drug-induced endocytosis, it appears that their mechanisms are different in that spontaneous endocytosis is not preceded by stomatocytic shape change and is not inhibited by sodium vanadate or sodium fluoride as is the case for drug-induced endocytosis. Spontaneous endocytosis is different than transferrin receptor-mediated endocytosis because it occurs in many RBC, not only in the motile R1 reticulocytes and is not inhibited by sodium cyanide as is receptor-mediated endocytosis. Thus spontaneous endocytosis appears to be different than drug-induced endocytosis and transferrin receptor-mediated endocytosis. The increase in spontaneous endocytosis in cord RBC seen in vivo is probably a consequence of an immaturity of the neonatal macrophage pitting process.     

Which groups of patients are transfused? A study of red cell usage in London and southeast England

BACKGROUND AND OBJECTIVES: There are few published data on the use of red blood cells (RBC) by specialty in the United Kingdom. The aim of this survey was to identify major specialty users of blood to target for audit of transfusion practice with a view to minimizing inappropriate use and to support planning of blood supply needs for the future. MATERIALS AND METHODS: Data were collected retrospectively of RBC units transfused at 62 hospitals/trusts in London and southeast England between April 1997 and March 1998. RESULTS: A total of 594 810 transfused RBC units were successfully traced to their respective clinical specialties, representing 91.9% of all RBC units issued to the study hospitals. Of the RBC units transfused, 51.2% were transfused in surgical, 36.0% in medical and 12.8% in "combined" specialties. CONCLUSIONS: This large study has accurately documented the clinical disciplines that are significant users of RBCs in our service area. It has heightened general understanding of RBC usage within hospitals. It has also raised questions on future strategies for reducing allogeneic blood transfusion that will be important if anticipated major reductions in blood supply occur in the near future.     

Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes

The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.     

甲状腺功能减退症患者的红细胞与血红蛋白变化及临床意义

目的:探讨红细胞(RBC)与血红蛋白(Hb)在甲状腺功能减退症(简称甲减)患者中的影响变化及其临床意义.方法:采用血细胞计数仪测定甲减患者48例、亚临床甲减患者44例与正常对照组50例的RBC和Hb水平.结果:甲减组与对照组比较,RBC与Hb均明显降低,差异有统计学意义(P<0.01);亚临床甲减组与对照组比较,RBC与Hb差异均无统计学意义(P>0.05).结论:甲减常易引发贫血,全血细胞分析对甲减患者尤其是甲减性贫血患者有较高的临床辅助诊疗价值.     

The effects of polyvinyl chloride and polyolefin blood bags on red blood cells stored in a new additive solution.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) must be stored in polyvinyl chloride (PVC) bags plasticized with di-2-ethylhexyl phthalate or a similar plasticizer to achieve their full storage life with conventional storage solutions. Improved storage solutions might remove this requirement and allow blood storage in other plastics. Experimental Additive Solution-61 (EAS-61), which maintains RBCs for 9 weeks with reduced haemolysis and satisfactory 51Cr 24-h recovery, is an appropriate candidate improved RBC storage solution. MATERIALS AND METHODS: Twenty-four units of packed RBCs were pooled in groups of four units, each pool was realiquoted into four units and stored, six pooled units per arm, in one of the following: 100 ml of EAS-61 in PVC; 200 ml of EAS-61 in PVC; 100 ml of EAS-61 in polyolefin (PO); and 200 ml of EAS-61 in PO. Haemolysis, RBC morphology indices, RBC ATP concentrations, and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC haemolysis exceeded 1% by 7 weeks in PO bags containing 100 ml or 200 ml of EAS-61. In PVC bags, haemolysis was less than 1% at 11 weeks. RBC ATP concentrations were 1 mol/g of haemoglobin (Hb) higher at 2 weeks in the PVC-stored units. CONCLUSIONS: RBCs stored in PVC had markedly less haemolysis and higher RBC ATP concentrations than those stored in PO. Haemolysis would limit RBC storage in PO bags to a duration of 6 weeks, even with EAS-61.     

A novel network analysis tool to identify relationships between disease states and risks for red blood cell alloimmunization

We hypothesized that diagnoses may be associated with alloantibody ‘responder’ status and examined associations between disease states and alloimmunization. Patients with ≥1 alloantibody and non‐alloimmunized controls were analysed. Pearson's coefficients were calculated to determine associations between alloimmunization and diseases; significant correlations were selected to construct a network. Inflammatory disorders and diseases requiring chronic transfusion support were associated with responder status. Mitigation steps may be considered in patients with these disorders.     

洗涤红细胞的临床应用分析

目的:探讨洗涤红细胞的适应症及临床意义。方法:统计应用洗涤红细胞治疗患者72例(治疗组)和应用全血治疗患者66例(对照组)的临床资料,进行统计分析。结果:应用全血和应用洗涤红细胞对于贫血患者红细胞和血红蛋白的提升无显著差别,而2者引起的不良反应发生率明显不同,具有统计学意义。结论:应用洗涤红细胞安全、有效、不良反应少,具有重要的临床意义。     

Sustained increase in haemoglobin and RBC following long-term administration of recombinant human erythropoietin to patients with homozygous beta-thalassaemia

Summary. Recombinant human erythropoietin (rHuEPO) was given subcutaneously three times per week in an escalating dose from 500u/kg to 950u/kg together with ferrous fumarate 305 mg and folic acid 5mg/d, to 10 patients from four unrelated Arab families with homozygous /3-thaIassaemia. Six splenectomized patients showed a mean (istandard error) increase in haemoglobin from 7-1 – 0-1 to 9-3 – 0-1 g/dl (P = 0-0001), in RBC from 4-0 to 5-0 × 10¹²/1 (P = 0-0001) and in nucleated RBC from 32 – 7 × 10¹⁰/1 to 82–6 × 10¹⁰/1 while receiving 750u/kg three times per week which persisted for 4-11 months. In two patients there was no need for further blood transfusions. In three out of four unsplenectomized patients there were no changes in Hb and RBC despite dose escalation. There were no significant changes in MVC, MCH and reticulocyte count, serum bilirubin, LDH, malonyldialdehyde (MDA) and vitamin E levels. After 13 weeks of rHuEPO there was a mean increase in the percentage of F cells from 31 –10% to 86 –6% (P< 0-003) in three splenectomized patients and in one unsplenectomized patient from 56-4% to 80% without changes in the levels of Hb F. Globin chain synthesis ratios did not change in four responding patients. Mean serum iron and transferrin saturation index did not change, whereas mean serum ferritin increased from 299–45/ng/I to 480–20/ig/l (P< 0-001). In seven responding patients an accelerated linear growth was indicated by positive changes in height standard deviation score for chronological age. Side-effects were minimal throughout the treatment period.     

牛磺酸对糖尿病患者糖耐量曲线的影响

观察口服牛磺酸对糖尿病患者口服葡萄糖耐量试验（ＯＧＴＴ）的影响。连续两次的ＯＧＴＴ结果显示牛磺酸可降低ＯＧＴＴ曲线的峰值水平，此作用不伴有胰岛素水平的升高。在牛磺酸的慢性降糖实验中，糖尿病患者每天口服牛磺酸１２．０ｇ，测定给药前后的空腹血糖（ＦＢＧ）、血浆丙二醛（ＭＤＡ）及红细胞内果糖水平。用药２周后ＦＢＧ水平与治疗前相比无明显差异（Ｐ＞０．０５），但停药后血糖明显升高，同时牛磺酸治疗可使２４小时的尿糖排出量明显减少（Ｐ＜０．０１），牛磺酸治疗可使红细胞内果糖水平和ＭＤＡ水平显著下降。提示牛磺酸可能对降低糖尿病患者的餐后血糖负荷有利，对于慢性并发症同样有治疗作用     

实验性甲状腺功能低下大鼠脑、红细胞膜神经磷脂与磷脂酰胆碱分子比变化的研究

应用薄层色谱扫描法，动态观察了实验性甲低大鼠脑，红细胞膜神经磷脂（ＳＭ）／磷脂酰胆碱（ＰＣ）分子比变化，结果表明持续用甲基硫脲嘧啶（ＭＴＵ）和脑发育临界期后停ＭＴＵ组，ＳＭ／ＰＣ分子比明显升高（Ｐ＜０．０５）。提示脑发育临界期内甲状腺激素缺乏，可导致生物膜的明显老化，膜磷脂分布破坏造成一系列结构和功能的改变，进而使中枢神经系统发育异常和功能障碍。