Microrheological characteristics of reticulocyte in vivo

Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticulocytes, which synchronously grow, were got in vivo. The measurements of deformation index, orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of reticulocytes turning into red blood cells in vivo. There were obvious changes in the micro- rheological characteristics of reticulocytes in the course of turning into erythrocytes. The present study is significant in clinic for studying erythrocytes' microrheological characteristics when there are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes microrheological characteristics. It makes up a deficiency in the study on microrheological characteristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.     

Microrheological characteristics of reticulocyte in vivo

Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticulocytes, which synchronously grow, were got in vivo. The measurements of deformation index, orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of reticulocytes turning into red blood cells in vivo. There were obvious changes in the microrheological characteristics of reticulocytes in the course of turning into erythrocytes. The present study is significant in clinic for studying erythrocytes’ microrheological characteristics when there are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes microrheological characteristics. It makes up a deficiency in the study on microrheological characteristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.     

60Co辐射对网织红细胞微观流变学特性的影响

采用 ⁶⁰Co大剂量全身均匀急性辐射的方法,造成一种辐射贫血的动物模型.以便在几天内连续研究 ⁶⁰Co辐射对新生的网织红细胞及红细胞流变学特性的影响.采用一种在低粘切变流场中能将红细胞变形指数DI分解为取向指数（DI）_or和小变形指数（DI）_d的新型激光衍射法,对网织红细胞及红细胞的变形指数、取向指数、综合变形指数（IDI）等血液流变学特性参数进行测量,发现在 ⁶⁰Co大剂量辐射后,新生的网织红细胞及红细胞流变学特性存在明显异常.将这种 ⁶⁰Co辐射造成的贫血模型与文宗曜等提出的用抗体诱导的大量同步化的球形红细胞贫血模型相比较,后者更具有明显的优点.同时为研究辐射对血液流变特性的影响及正确地挑选红细胞衰老模型提供了理论与实验的基础.     

苯肼对红细胞在体衰老过程中微观流变特性的影响

在Brunara等人用苯肼使动物造成急性溶血性贫血的方法基础上,建立一种由急性溶血性贫血后,而诱发家兔幼红细胞增多的非正常生理状态的红细胞在体衰老模型,继而研究新生红细胞从产生到死亡死亡过程,即衰老过程的流变学特性的变化规律。通过对新生红细胞的压积、变形、取向及与之相应的全血的粘度、血沉等指标的连续60多天的监测,发现红细胞在衰老过程中的微观流变学特性确实有明显改变。红细胞在体衰老过程中微观流变特性逐渐变差。     

小鼠网织红细胞微观流变学特性的研究

按Bishop方法,在小鼠血液里诱导生成大量网织红细胞,然后提取网织红细胞,对其电泳率、渗透脆性、膜的流动性、细胞的变形能力和取向性进行了系统研究。研究结果表明网织红细胞在转变为成熟红细胞的短短时间内,其微观流变学特性发生了明显的变化：电泳率变小、渗透脆性变好、膜的流动性变大、细胞的变形能力变强、取向性变好,最终发育成具有全面功能的成熟红细胞。     

网织红细胞成熟过程中膜剪切弹性模量及表面粘度的改变

用鸡抗兔血清的抗体使兔造成急性溶血性贫血的方法,诱发兔体内同步生长的新生网织红细胞,用文宗曜等提出的一种测量红细胞膜剪切弹性模量及表面粘度的新方法——新型激光衍射法,连续72h监测经过不同发育阶段的网织红细胞的小变形指数和变形恢复过程(即松弛过程)中变形恢复到最大值一半的时间(即变形恢复半时间,t0.5),将测得的结果分别代入红细胞膜的剪切弹性模量公式和表面粘度公式。计算出不同发育阶段的网织红细胞的膜剪切弹性模量和表面粘度,发现网织红细胞在转变为成熟红细胞的过程中,其膜剪切弹性模量和表面粘度有明显改变。这对研究由于贫血等原因造成的网织红细胞增多情况下全血的微观流变学特性有重要的-临床意义,同时对新生网织红细胞在转化过程中膜的剪切弹性模量和表面粘度的变化规律加以系统研究,具有重要的基础理论研究价值。     

Plasmodium vivax: in vitro growth and reinvasion in red blood cells of Aotus nancymai

Plasmodium vivax was maintained in experimentally infected Aotus nancymai. Positive monkeys were used as donors for culture material. After leucocyte removal with two different methods, including the classic CF11 method and a commercially available filter, parasites were grown under continuous shaking conditions in standard RPMI 1640, containing 20% human AB + serum. When mature schizonts were present, artificially induced reticulocytes from monkeys pretreated with the hemolytic drug phenylhydrazine HCl were added. Addition of reticulocytes and shaking were both necessary to realize a significant reinvasion under in vitro conditions. A strong positive correlation between the percentage of reticulocytes and in vitro invasion was demonstrated, and a preferential invasion into reticulocytes was demonstrated in vivo and in vitro using blood films stained with brilliant cresyl blue and counterstained with Giemsa.     

Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.     

Preferential invasion of malarial merozoites into young red blood cells

The preferential invasion of malarial merozoites into subpopulations of red blood cells (RBCs) in vivo and in vitro has been the subject of repeated discussions. In this paper, an attempt is made to summarize these discussions and to pinpoint the mechanism by which this preference could arise. The available data suggest that a relatively simple mechanism, related to the capability of the merozoite to rearrange the proteins of the cytoskeleton of the RBC may determine the invasion rate into mature versus very young RBCs (reticulocytes). There is no evidence for significant differences between mature RBCs and reticulocytes in the presence of membrane proteins which might play a role in receptor-ligand binding of merozoites to their host cell. Consequently, the concept of "reticulocyte preference" is left and the ability of penetrating both mature and immature RBCs, versus immature RBCs only, is given as an explanation for the presence of ringforms exclusively in reticulocytes as observed for several species of vivax-type malaria parasites. The possible consequences of preferential invasion for the infection (in vivo) and the culture (in vitro) of different plasmodial species are discussed.     

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.     

Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHuEPO) analogues was developed. The assay took four days, normal mice were used and radioactive compounds were not needed. EPO activity was measured by the increased number of some part of reticulocytes which increased specifically and dose-dependently by the injection of rHuEPO. They were considered to be mostly immature reticulocytes and were counted as the residual particles from blood cells after treatment with a hemolysing reagent. These particles could be counted by conventional automated microcell counters. The assay procedure was simple and easy. The sensitivity, reliability and reproducibility of this method were acceptable for routine in vivo bioassay of rHuEPOs. This method was economical, and can be used instead of the existing bioassays for rHuEPOs.     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

Silica nanoparticles administered at the maximum tolerated dose induce genotoxic effects through an inflammatory reaction while gold nanoparticles do not

While the collection of genotoxicity data and insights into potential mechanisms of action for nano-sized particulate materials (NPs) are steadily increasing, there is great uncertainty whether current standard assays are suitable to appropriately characterize potential risks. We investigated the effects of NPs in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. We also incorporated additional endpoints into the in vivo study in an effort to delineate primary from secondary mechanisms. Amorphous silica NPs (15 and 55 nm) were chosen for their known reactivity, while gold nano/microparticles (2, 20, and 200 nm) were selected for their wide size range and lower reactivity. DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes were measured after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4h before sacrifice. Gold nano/microparticles were negative for MN induction in vitro and in vivo, and for the induction of DNA damage in all tissues. Silica particles, however, caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose (MTD). No genotoxic effects were observed at lower doses, and the in vitro MN assay was also negative. We hypothesize that silica NPs initiate secondary genotoxic effects through release of inflammatory cell-derived oxidants, similar to that described for crystalline silica (quartz). Such a mechanism is supported by the occurrence of increased neutrophilic infiltration, necrosis, and apoptotic cells in the liver, and induction of inflammatory markers TNF-α and IL-6 in plasma at the MTDs. These results were fairly consistent between silica NPs and the quartz control, thereby strengthening the argument that silica NPs may act in a similar, thresholded manner. The observed profile is supportive of a secondary genotoxicity mechanism that is driven by inflammation.     

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.     

Effect of stress actions on some hematological and biochemical parameters of rat blood and on energetic intermolecular interactions in lipid extract under effect of light radiation

Comparative study has been carried out on effects of the three-day long starvation, running, and their combination on morphological parameters of rat blood, lipid metabolism, and activity of blood Na,K-ATPase. Different effects of these stress factors on the blood erythrocyte composition have been shown. Starvation is accompanied by the most pronounced release of stored erythrocytes into blood, which results in a significant decrease of both the total amount of reticulocytes and in the complete absence of reticulocytes of I stage of maturity (the youngest). The running on a treadmill led to a significant increase in the total amount of blood reticulocytes and to a multifold increase of immature reticulocytes (RC-1 and RC-2), which can indicate some stress of the bone marrow erythroid stem line. The curve of acid resistance of blood reticulocytes has shown the animals to experience the greatest stress at a combination of starvation and running. Starvation and running also produced different effects on blood lipid characteristics. The content of triacylglycerides (TAG) in blood rose by 40% at starvation and decreased by 30% at running, a similar tendency being found for the atherogeneity index. The fatty acid composition of blood phospholipids at running and its combination with starvation practically did not differ from control. A change of Na,K-ATPase activity, which is so typical for reactions of various kinds of stress sharply fell at starvation (by 22%), but increased at running (by 13%) and decreased markedly at combination of these actions. The absorption spectra of the whole blood lipid extracts of the rats subjected to various stress actions showed that organic substance with coupled bonds, which absorbs light within diapason of 360–620 nm, is extracted from the blood (at different amount depending on the kind of action). The absorption of light in diapason of 400–410 nm has been found to belong to the Soret band of ferroheme and ferriheme. The shift of Soret band indicates an electron transitions in the iron cation. By the change and disappearance of Soret band it is possible to judge about the processes occurring in the lipid extract. Disappearance of the Soret band from the lipid extract indicates formation in it of the steady radicals as a result of the ferriheme disintegration due to accumulation of energy in porphyrin, which does not seem to occur in the blood cell membranes. The iron atom in the ferriheme molecule is known to accept electron and to yield a part of energy, probably to porphyrin. Then ferroheme yields electron and becomes ferriheme with an excess of the energy in porphyrin. Hence, at admission of the next electron to the iron atom the porphyrin molecule is to get rid of the energy obtained earlier to prevent its disintegration. The heme is possible to be an accumulator and distributor of energy in tissue.     

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.     

Optimization of flow-cytometric discrimination between reticulocytes and erythrocytes

An automatized technique to count reticulocytes by means of flow cytometry is described. Blood samples were stained by the fluorescent dye acridine orange without the use of fixative. Scatter and red fluorescence of the blood cells were measured in a flow cytometer. A discrimination between reticulocytes and erythrocytes was only achieved by using logarithmic amplification. The discrimination was better in peak mode than in area mode. The optimum dye concentration was 0.5 mg/liter acridine orange. At lower dye concentrations, not all reticulocytes were measured, whereas at higher dye concentrations the degree of discrimination between reticulocytes and erythrocytes decreased. There was a suitable discrimination between reticulocytes and erythrocytes. The reticulocyte numbers were scored by flow cytometry as well as by microscope for blood samples with 0.1-14% reticulocytes. The correlation between both methods was close.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

A relative morphological evaluation of hemoglobin biosynthesis in peripheral blood reticulocytes of normal and anemic rabbits

1. Peripheral blood reiculocytes of normal and bled rabbits and of rabbits with phenylhydrazine-induced anemia, were morphologically analysed, through silver sections, for a relative evaluation of hemoglobin (Hb) biosynthesis activity. 2. Reticulocytes of maturation degrees within the range of 35-60 polysomes/microns2, were compared as to their mean numbers of hemosomes (sites of heme integration into the globin chains), and mitochondria (indirect precursors for hemosome formation). 3. The results on the mean numbers of hemosomes per reticulocyte section, correlated to several physiological data under those three conditions, suggested a close relationship between Hb biosynthesis activity and hemosome frequency. 4. In bled rabbits, reticulocytes showing a low mean number of hemosomes (means hB/section = 0.32), as compared to reticulocytes of normal rabbits (means hN/section = 0.70) and to reticulocytes of rabbits with hemolytic anemia (means hH/section = 2.10), gave rise to a new erythrocyte population characterized by a low Hb content. 5. Hb concentration differences were verified by confronting hematological data before bleeding with those obtained after the regression of anemia.