Effect of temperature dependent changes in mechanical stability of red cell aggregates on relative apparent whole blood viscosity

As the temperature dependence of relative apparent whole blood viscosity eta rel is still controversial, the relation between the temperature dependence of red cell aggregation (RCA) and that of eta rel was examined in normal donors and in patients with venous ulcers of the leg. Apparent whole blood viscosity was measured in the DEER-rheometer (0.01 Pa less than tau less than 2.9 Pa) at 10 degrees C, 20 degrees C, 30 degrees C and 37 degrees C. The instrument was calibrated for each temperature to correct for changes in viscometer geometry. Simultaneously the minimal shear stress tau Tmin to keep RCA dispersed was determined by photometric aggregometry. eta rel was found to increase with decreasing temperature. By basing the relative cold induced increase in eta rel on the state of RCA as defined by the ratio of tau/tau Tmin the relation between both features is verified: With increasing RCA the cold induced increase in eta rel is progressively enhanced.     

Theoretical and experimental study of the time dependent flow of red blood cell suspension through narrow pores

The Hemorheometer has been adapted to allow the recording of the flow rate during the filtration process. For newtonian fluids, the flow rate variation versus time through the pores is well approximated by Poiseuille's law. For dilute red blood cell suspensions, the same analysis can be applied by introducing the concept of "apparent filtration viscosity" which is higher than the usual viscosity measured by Couette viscometry. The apparent filtration viscosity parameter is related to the deformations undergone by red blood cells as they pass through the narrow pores. Apparent filtration viscosity can be used to obtain a precise determination of the erythrocyte deformability. Measurements performed, for a given blood sample, with pores of different diameters (5 microns, 8 microns and 12 microns) show that the error on the value of apparent filtration viscosity is less than 3%. As a result, the sensitivity of the filtration method allows to discriminate among normal blood samples. High concentrations of erythrocytes or leucocytes are found to modify the apparent filtration viscosity. These factors are apparent in the recorded filtration curves. Their effects on filtration measurements can be easily estimated.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Hemorheology and oxygen transport in vertebrates. A role in thermoregulation?

We studied the effect of temperature on blood rheology in three vertebrate species with different thermoregulation and erythrocyte characteristics. Higher fibrinogen proportion to total plasma protein was found in turtles (20%) than in pigeons (5.6%) and rats (4.2%). Higher plasma viscosity at room temperature than at homeotherm body temperature was observed in rats (1.69 mPa x s at 20 degrees C vs. 1.33 mPa x s at 37 degrees C), pigeons (3.40 mPa x s at 20 degrees C vs. 1.75 mPa x s at 40 degrees C), and turtles (1.74 mPa x s at 20 degrees C vs. 1.32 mPa x s at 37 degrees C). This fact allow us to hypothesize that thermal changes in protein structure may account for an adjustment of the plasma viscosity. Blood viscosity was dependent on shear rate, temperature and hematocrit in the three species. A different behaviour in apparent and relative viscosities between rat and pigeon at environmental temperature was found. Moreover, the blood oxygen transport capacity seems more affected by a reduction of temperature in rats than in pigeons. Both findings indicate a greater influence of temperature on mammalian erythrocyte than on nucleated red cells, possibly as a consequence of differences in thermal sensitivity and mechanical stability between them. A comparison between the three species revealed that apparent blood viscosity measured at homeotherm physiological temperature was linearly related to the hematocrit level of each species. However, when measured at environmental temperature, rat blood showed a higher apparent viscosity than those found in species with non-nucleated red cells, thus indicating a higher impact of temperature decrease on blood viscosity in mammals. This suggest that regional hypothermia caused by cold exposure may affect mammalian blood rheological behaviour in a higher extent than in other vertebrate species having nucleated red cells and, consequently, influencing circulatory function and oxygen transport.     

Induction of Tk polyagglutination by Bacteroides fragilis culture supernatants. Associated modifications of A B H and I i antigens

Tk transformed red blood cells were obtained in vitro by treatment with supernatants from cultures of three different Bacteroides fragilis strains. The reactions of these cells with AB sera show that Tk is different from other known types of polyagglutination. Beside the already known modifications of A B H antigens, we found that Tk activated cells have an important modification of I and i antigens: both are reduced, and can even be completely destroyed.     

Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.

Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.     

血浆是引起血沉增快的主要因素

目的：阐明血沉增快的原因是血浆还是红细胞。方法：收集72例血沉异常的抗凝血标本,同时收集血型相对应的72例血沉正常标本,组成血型相同的血沉异常和正常标本72对,每对互换血浆后重新测定血沉,与原始血沉结果比较,并通过多元线性回归分析血沉与血浆蛋白及血脂浓度的关系。结果：血沉异常标本的红细胞加入血沉正常标本的血浆后,72例（100%）血沉均减慢,其中30例血沉下降90%以上,35例下降70%-90%,7例小于70%。血沉正常标本的红细胞加入血沉异常标本的血浆后,67例（93%）血沉加快,其中58例（81%）变为异常（18例血沉加快10倍以上,40例加快5-10倍）。球蛋白、白蛋白和纤维蛋白原与血沉具有线性关系,球蛋白（r=0.420,P〈0.001）和纤维蛋白原（r=0.673,P〈0.001）与血沉呈正相关,而白蛋白（r=-0.558,P〈0.001）与血沉呈负相关。结论：血沉增快主要与血浆因素相关,红细胞对于血沉的影响作用很小。     

Endogenous lithium and boron red cell-plasma ratios: normal subjects versus bipolar patients not on lithium therapy

This study was undertaken to compare endogenous lithium concentrations in human blood and its components from normal donors versus bipolar patients. The patients were not on lithium therapy at the time that the blood samples were donated and had not received any lithium therapy for at least 2 yr. Blood components were separated by centrifugation. The analytical method for lithium as developed in this laboratory consists of thermal-neutron activation of freeze-dried samples. 3H is produced via the reaction 6Li + n = 3H + 4He, and high-sensitivity rare gas mass spectrometry is used to measure 3He formed from beta-decay of 3H. Boron measurements are made concurrently using 4He from the reaction 10B + n = 4He + 7Li. Seven normal donors and seven patients with a diagnosis of bipolar disorder participated in this study. Measurements of lithium and boron were made in whole blood, plasma, and red cells. Red cell-plasma ratios R(Li) and R(B) were calculated after corrections were made for trapped plasma in the red cells. The results show that bipolar patients may have higher concentrations of lithium in blood, plasma, and red cells (p = 0.08, 0.02, and 0.02, respectively) and may have higher R(Li) values than normal donors (p = 0.01). No evidence was found for bipolar-normal differences in these four parameters for boron. Although our sample size is admittedly very small, the results clearly show that the endogenous red cell ratio R(Li) and plasma or red cell lithium concentrations may become useful diagnostic indicators for bipolar illness if the analytical methods are further developed.     

Tentative application of the tangent simple system method to the study of the viscoelastic behaviour of blood

The method of the tangent simple systems is applied to the study of the viscoelastic behaviour of human blood in unstationary flow for rectangular steps and triangular ramps of shear rate. The tangent systems we utilize, Maxwell liquids, enable us to determine, at every point of the rheograms, apparent instantaneous values of retardation or relaxation time, viscosity coefficient and elasticity modulus of the studied blood samples, and to plot the curves of variation of these parameters as a function of flow duration. A qualitative interpretation of the results is proposed from data on the aggregation-disaggregation kinetics of red blood cells. Examples are given for samples of normal and pathological bloods.     

Studies with dextran 40 in cryopreservation of blood

Whole blood from healthy donors was washed twice with phosphate-buffered saline (PBS) and then resuspended in sufficient PBS to give a final concentration of 2 × 10⁹/cells/ml. Aliquots were combined with equal volumes of the required diluents to give final dextran 40 concentrations of 0, 5, 10, 15, and 20% in PBS. Fifty-lambda samples in 50-lambda Micropets (Clay Adams) were frozen in alcohol baths at temperatures ranging from ?10 to ?80 °C. The specimens were frozen either for 1 min or 16 min, rapidly thawed, and resuspended in PBS or PBS plus dextran. Percentage of hemolysis was determined colorimetrically. Results indicate that concentraitons as low as 5% dextran exert a cryoprotective effect. Increased dextran concentration increases cryoprotection at high subzero bath temperatures (?10 ° and ?20 °C). Dextran concentrations beyond 12% have a damaging effect at low subzero bath temperatures (below ?30 °C). Based on this a two-factor hypothesis for cryopreservation is proposed. Apparent partial recovery of red blood cells without dextran or with 5% dextran during subzero storage was demonstrated.     

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.     

华南虎血常规两种测定方法的比较应用

通过全自动血细胞分析仪与人工镜检法分别对华南虎(Panthera tigris amoyensis)血常规进行分析,并对获得结果做比较分析,探究建立适合华南虎血细胞分析的方法。收集临床华南虎血液标本40份,分别使用全自动血液分析仪和人工显微镜检法对红细胞、白细胞进行计数检测,并对白细胞进行分类分析。如人工计数和仪器计数获得的数据均符合正态分布,则应用配对样本T检验比较差异性,否则应用非参数检验(两个相关样本:Wilcoxon带符号秩检验)。相关性采用Spearman分析,以P <0.05为差异有统计学意义。结果表明,通过两种方法获得的红细胞计数、白细胞计数、中性粒细胞比率、嗜酸性粒细胞比率结果差异均不显著(P> 0.05);淋巴细胞比率、单核细胞比率和嗜碱性粒细胞比率,两种方法检测结果具有显著性差异(P <0.05)。红细胞计数(r=0.915)、白细胞计数(r=0.832)、淋巴细胞比率(r=0.832)、中性粒细胞比率(r=0.481)应用两种方法获得的结果相关性均较好。单核细胞比率(r=0.283)、嗜酸性粒细胞比率(r=0.309)、嗜碱性粒细胞比率(r=0....     

Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.     

Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10⁴∶10²∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm² track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.     

Lactate-transport activity in RBCs of trained and untrained individuals from four racing species

In red blood cells (RBC) of horses, both lactate-transport activity and lactate accumulation during races vary interindividually. To study whether similar variation in lactate transport is apparent also in RBCs of other racing species, blood samples were collected from 21 reindeer, 40 horses, 31 humans, and 38 dogs. Total lactate-transport activity was measured at 10 and 30 mM concentrations, and the roles of the monocarboxylate-transporter (MCT) and the inorganic anion-exchange transporter (band-3 protein) were studied with inhibitors. In the reindeer and in one-third of the horses, lactate transport was low and mediated mainly by band-3 protein and nonionic diffusion. In the humans, dogs, and the remaining two-thirds of the horses, lactate transport was high and MCT was the main transporter. No correlation existed between MCT activity and the athleticism of the species. In the horses and humans, training had no effect on lactate transport, but in the reindeer and sled dogs, training increased total lactate transport. These results show that among the racing species studied, only in horses was the distribution of lactate-transport activity bimodal, and the possible connection between RBC lactate and performance capacity, especially in this species, warrants further studies.     

Blood's critical Taylor number and its flow behavior at supercritical Taylor numbers

When the inner cylinder of a fluid-filled Couette viscometer is rotated rapidly, a vortical flow pattern develops when a dimensionless value referred to as the critical Taylor number (Tc) is reached. We have determined its magnitude in our viscometer for three Newtonian fluids and for blood at 37 degrees C, using the inflection point of torque/RPM vs. RPM (sudden rise in apparent viscosity). Its position was identified by least squares line fitting. Because blood was studied, the viscosity used in Tc calculation was the apparent bob shear stress/shear rate ratio at the inflection marking vortical flow onset. For glycerol-water mixtures Tc was 41.8 +/- 0.3 (N = 11), for propylene glycol 42.0 +/- 0.2 (N = 14), for silicone oil 41.8 +/- 0.2 (N = 11). For healthy blood Tc was 40.7 +/- 0.9 (N = 140). This evidence against blood's increased resistance to flow instability was accompanied by a slower rate of rise in torque both above and below Tc compared to the three Newtonian fluids. Newtonian fluids and blood both developed wavy vortical flow at a rotation rate moderately higher than Tc. Blood resisted this unstable flow behavior more than the Newtonian fluids but it also experienced a slower rate of rise in torque with increasing rotation rate above the critical Taylor number. Shear-thinning is the simplest explanation for blood's mildly altered Taylor vortex behavior; blood's resistance to flow instability is otherwise not found to be sufficient to affect its flow stability in man.     

Erythrocyte volume in man and various animals after the addition of glucose]

The optimal transmission of very diluted blood samples from 11 vertebrate species and man were measured. The red cells of 10 species reduced their volume when glucose or equi-osmotic amounts of sodium chloride were added. Only the erythrocytes of man and monkey (Japanese macaques) did not reduce their volume after addition of glucose or renormalized or overcompensated minor transitory changes. This increase of the volume of human red cells is, however, too low for noticeable viscosity changes of whole blood to be caused, if any. The different response of red cells to glucose makes a simple differentiation between animal and human blood samples possible, provided that primates other than man are excluded.     

Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents

Human umbilical cord blood has been shown to be an effective source of stem cells for marrow reconstitution in pediatric patients. Unfortunately, the quantity of stem cells obtained from an individual donor can be quite limited in both the total volume and the numbers of stem cells per ml of cord blood. HLA matching further limits the availability, but recent publications indicate close matching may be unnecessary. Therefore, if cord blood from different donors can be combined, larger numbers of stem cells can be available for clinical use provided pooling does not produce a negative effect. Storage of single cord blood specimens at 4 degrees C for 10-21 days in gas permeable bags produced an apparent increase in the percentage of immature cells (CD34, CD117, GPA) and mitotic activity (S+G2/M cells) over day 1. With similar storage of pooled specimens there was a further increase in the number of immature colonies cultured, CD34, CD117, GPA, S+G2/M cells. In addition, nucleated red blood cells increased over the mean values obtained from single cord blood samples. Our previous studies have indicated that large numbers of human mononuclear cells are necessary to reconstitute an irradiated animal model. By combining multiple samples of human cord blood, adequate numbers of stem cells could be pooled for use in adults and would provide cells for megadose therapy, including those patients that had accidentally received lethal irradiation.     

天花粉蛋白通过抗原加工提呈调节T细胞免疫应答

用三种抗原加工阻断剂预处理抗原提呈细胞（ＡＰＨ）１ｈ后再用天花粉蛋白（Ｔｋ）脉冲处理,观察经由ＡＰＣ递呈的Ｔｋ对ＰＭＡ和Ａ２３１８７诱发Ｔ细胞增殖的作用。结果表明,这些药物均能不同程度地阻断Ｔｋ的抑制效应,以氯喹和Ｌｅｕｐｅｐｔｉｎ的作用更为明显。用胶体金标记Ｔｋ（Ｔｋ－Ｇ）,电镜下观察Ｔｋ在Ｔ细胞和ＡＰＣ内的定位,发现Ｔｋ－Ｇ仅与ＡＰＣ发生细胞表面粘附,继而内化,先后出现在ＡＰＣ的内体和溶酶体中     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.