On the in vivo action of erythropoietin: a quantitative analysis

The composite response of the erythron to exogenous erythropoietin has been studied in normal, splenectomized, and polycythemic mice. After stimulation the normal animal doubled its marrow nucleated red cells by the 3rd day with little further change by the 5th. Nucleated red cells within the spleen began to increase sharply on the 2nd day and, by the 5th, exceeded those in the marrow. The total nucleated erythroid response represented a fourfold increase. Reticulocytes lagged behind the expansion of the nucleated red cell mass, but by the 5th day the original ratio was re-established. Hemoglobin synthesis was increased, but the ratio of hemoglobin synthesized in nucleated red cells and reticulocytes was basically unchanged. Early displacement of marrow reticulocytes into circulation and the production of a larger red cell also occurred. No evidence of a change in the number of erythroid mitoses was found; only a slight decrease in the average cell cycle time was demonstrated. Thus, whereas erythropoietin stimulation induced several changes in erythropoiesis, the increased number of cells entering into the maturing pool appeared to be of greatest quantitative significance.Splenectomy reduced the proliferative response of the erythron over 5 days stimulation to three-fourths that found in the normal animal. This difference, also reflected in a proportionately lower reticulocyte response and increment in circulating red cell mass, suggests that erythropoiesis within the mouse marrow is spatially or otherwise restricted and that the spleen provided a supplemental area of erythroid expansion.     

Use of reticulocyte cellular indices in the diagnosis and treatment of hematological disorders

Automated counting of reticulocytes has markedly increased the precision and accuracy of this assay compared with the traditional manual counts. In addition, several new reticulocyte parameters are now available to clinicians and pathologists. This review examines the potential role of these parameters in the diagnosis and management of anemias. Reticulocyte maturity can now be assessed based on the staining intensity of reticulocytes, which is proportional to their RNA content. However, the clinical value of the numerical estimate of the immature reticulocyte fraction has not been yet demonstrated. In the bone marrow transplant setting, there is no clear evidence that the use of this index results in improved care of these patients, and many studies have failed to show its superiority compared with the traditional white cell count, especially for autologous transplants. Direct measurement of reticulocyte volume, hemoglobin concentration, and hemoglobin content are now available. Studies have shown that these parameters, and hemoglobin content in particular, allow a real-time assessment of the functional state of the erythroid marrow. In the setting of recombinant human erythropoietin therapy, studies of hemoglobin content have shown that this index allows an early detection of functional iron deficiency. Preliminary studies have also shown that this index may be helpful in the diagnosis of iron deficiency and in the monitoring of iron replacement therapy.     

Preferential hemolysis of immature erythrocytes in experimental iron deficiency anemia: source of erythropoietic bilirubin formation

Bilirubin-(14)C production was measured in rats transfused with labeled erythrocytes from animals with iron deficiency anemia, a condition associated with ineffective erythropoiesis. With labeled reticulocytes harvested 1 day after the administration of glycine-2-(14)C, conversion of hemoglobin-(14)C to bilirubin averaged 47.3% over the 3 days of observation; the corresponding value for reticulocytes from normal rats was only 1.7%. Findings were not altered by splenectomy. Bilirubin-(14)C production fell to 35.8% with iron-deficient cells harvested 3 days after glycine-(14)C administration, and declined further to a plateau averaging 25% with cells labeled 5, 7, 10, or 15 days earlier. The latter values still far exceed those for mature erythrocytes from normal animals.The findings indicate that experimental iron deficiency anemia is associated with hemolysis of red cells of various ages, but with preferential destruction of the youngest cells. Degradation of hemoglobin from reticulocytes is sufficient to account for a major fraction of the increase in erythropoietic bilirubin production found in this disorder, as has also been shown for physiologically regulated erythroid hyperplasia. However, the defect is quantitatively much more striking in experimental iron deficiency, and this and perhaps a similar defect in bone marrow cells appear to explain the decrease in net hemoglobin production that is characteristic of pathologic ineffective erythropoiesis.     

Cytogenetic changes of the bone marrow in massive blood loss and their correction with mexidol

The citogenetic lesions were evaluated in the marrow erythroblasts of 45 anesthetized white nonlinear male rats, weight--200-300 g who were subjected to an acute blood loss with a 1-hour arterial hypotension (ABR = 40 mm Hg); the micronucleus tests was made use of. Two stages of the increase of polychromatophilic erythrocytes with micronuclei in the marrow of the animals, who underwent a massive blood loss, were registered: stage 1--an incomplete marrow ischemia with a subsequent arterial hypotension and with a reliably confirmed formation of cytogenetic lesions in the marrow polychromatophilic erythrocytes; stage 2--the reperfusion period contributed to a 1.7-fold increase of polychromatophilic erythrocytes with micronuclei versus the previous stage. Mexidole, when used at 50 mg/kg prior to blood reinfusion, decreased the quantity of polychromatophilic erythrocytes with micronuclei to the basic level, which is indicative of reversibility and instability of cytogenetics impairments in the marrow cells of animals observed in the early post-resuscitation period.     

红细胞内疟原虫影响网织红细胞检测的研究

目的探讨疟疾患者红细胞内疟原虫对德国西门子ADVIA 2120全自动血细胞分析仪测定网织红细胞的影响。方法用ADVIA2120全自动血细胞分析仪检测疟疾患者的网织红细胞并与健康人群进行比较。结果疟疾患者的网织红细胞计数的绝对值(RET#)、百分比(RET%)、低吸光度网织红细胞百分率(%L Retic)、中吸光度网织红细胞百分率(%M Retic)与对照组比较,差异无统计学意义(P>0.05);而高吸光度网织红细胞百分率(%H Retic)与对照组比较,差异有统计学意义(P<0.05)。结论疟原虫对ADVIA2120全自动血细胞分析仪测定网织红细胞无明显的影响,但如果高吸光度网织红细胞百分率(%H Retic)增高且临床出现发烧的患者,应复检血片仔细查找疟原虫。     

Erythropoietic protoporphyria: two populations of reticulocytes, with and without protoporphyrin

Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin. The photosensitivity experienced by these patients is assumed to be due to a leakage of protoporphyrin from the erythrocytes and transfer to the skin, where protoporphyrin acts as a photosensitizer. The leakage of protoporphyrin from the erythrocytes has been offered as an explanation for the great variety in protoporphyrin content observed among erythrocytes in this disease. Based on density gradient separation of red cells, it has been concluded that all reticulocytes and young erythrocytes contain large amounts of protoporphyrin. From our results, density gradient centrifugation is not suitable for age separation of red cells from patients with erythropoietic protoporphyria. By developing a new method for isolation of reticulocytes and applying flow cytometry to determine protoporphyrin content in individual cells, it was observed that two populations of reticulocytes were present in patients with erythropoietic protoporphyria, one with and the other without protoporphyrin. The half-life of protoporphyrin in red cells was found to be 12–14 days, in contrast to 1–2 days described previously, suggesting a slower release of protoporphyrin from the red cells than previously anticipated.     

Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.

Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TOP and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure.     

Rh antibodies against the pretransplant red cells following Rh- incompatible bone marrow transplantation

A 22-year-old, blood group O, Rh-positive (R2r) man received bone marrow from his blood group A, Rh-negative (rr), HLA-identical sister for treatment of acute lymphocytic leukemia. The patient's pretransplantation serum contained anti-A in a low concentration; therefore, plasmapheresis was not done prior to transfusion of bone marrow. To prevent graft-versus-host disease, bone marrow was incubated with absorbed rabbit antithymocyte globulin prior to infusion, and the patient was treated with methotrexate in the posttransplantation period. After transplantation, the patient received 6 units of group O, Rh-negative (rr) packed red cells from random donors and 6 units of platelets from the marrow donor. Three months after transplantation, 0.5 percent of his red cells were still of the host's type (group O, Rh-positive), as detected by immunofluorescence technique in blood smears. Four months after transplantation, three different Rh antibodies--anti-D, -E, and -G--were detected. Since the patient received only Rh-negative red cell transfusions, it is concluded that he was immunized to his original red cells.     

Investigation of the survival of paroxysmal nocturnal hemoglobinuria red cells through the immunophenotyping of reticulocytes

BACKGROUND: Complement-mediated lysis of red cells (RBCs) is a classic feature of paroxysmal nocturnal hemoglobinuria (PNH) that is traditionally studied with a combination of radiolabeling of RBCs and in vitro hemolysis tests. Phenotyping of reticulocytes was used as an alternative method for the evaluation of the relative life span of normal RBCs (PNH I) and RBCs that were partially (PNH II) or completely (PNH III) deficient in CD59. STUDY DESIGN AND METHODS: Murine monoclonal antibodies CD55, CD58, and CD59 and thiazole orange were used to phenotype reticulocytes by two-color flow cytometry in nine PNH patients. RBC survival could be calculated from the ratio of CD59- or CD59low mature RBCs to CD59- or CD59low reticulocytes obtained from these patients who had not received a transfusion. RESULTS: The life span of PNH III RBCs varied from about 17 to 60 days. PNH II reticulocytes were found in the four patients with PNH II RBCs. The life span of PNH II RBCs varied with their residual expression of CD59, and cells with 15 to 20 percent of the normal amount of CD59 were protected against in vivo hemolysis. CONCLUSION: Phenotyping of reticulocytes is a convenient and reliable tool for evaluating the relative survival of normal and PNH RBCs. PNH II and PNH III reticulocytes are phenotypically distinct, and some PNH II RBCs may be sensitive to complement-mediated lysis in vitro, but normally they are complement-resistant in vivo.     

苯肼对兔网织红细胞微观流变学特性影响的研究

目的 利用苯肼注入造成的动物贫血模型,对兔网织红细胞微观流变学特性进行研究.总结兔网织红细胞微观流变学特性的变化规律.方法 采用苯肼注入动物体内,能使动物造成贫血,从而诱发动物幼红细胞增多症,造成一种贫血的动物模型.在几天内连续研究苯肼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[1],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.结果 注入苯肼后,新生的网织红细胞流变学特性与成熟红细胞流变学特性存在明显异常,网织红细胞的变形指数、取向指数、和小变形指数与成熟红细胞的相应指数存在明显异常.结论 网织红细胞流变学特性与成熟红细胞流变学特性存在明显异常,为研究苯肼对网织红细胞及红细胞的血液流变学特性的影响提供了理论与实验基础.     

Studies on Red Cell Aplasia. V. PRESENCE OF ERYTHROBLAST CYTOTOXICITY IN γG-GLOBULIN FRACTION OF PLASMA

The marrow cells of a patient with pure red cell aplasia markedly increased their rate of heme synthesis when they were freed from the host environment and were incubated in vitro. When the red cell aplasia was treated with cyclophosphamide and prednisone, marrow cell incorporation of (59)Fe into heme in vitro increased several weeks before a reticulocytosis was apparent, and was the earliest effect noted. The plasma gammaG-globulins of this patient inhibited heme synthesis by normal marrow cells or the patient's own marrow cells obtained after remission of the disease.Since the inhibition of heme synthesis could be the result of damage to erythroblasts, the patient's posttreatment marrow cells or normal marrow cells were labeled with (59)Fe and were then incubated with the patient's pretreatment, treatment, and posttreatment gammaG-globulins as well as normal gammaG-globulins. At the end of this incubation the supernatant and cells were separated and counted. Heme was extracted and also was counted. Treatment of the cells with the patient's pretreatment gammaG-globulins resulted in a release of 40% of the radioactive heme from the cells. This represented the loss of radioactive hemoglobin and was an index of erythroblast cytotoxicity. A progressive disappearance of the cytotoxic factor in the gammaG-globulins occurred in the 3 wk period preceding the onset of reticulocytes in the patient's blood. Posttreatment and normal gammaG-globulins did not produce this effect and increased injury of red cells and lymphocytes was not produced by the patient's pretreatment gammaG-globulins. These studies demonstrate a method for measuring erythroblast cytoxicity and show that red cell aplasia is associated with gammaG-globulins that specifically damage erythroblasts. Whether interference with new erythroblast development also occurs and contributes to the inhibition of heme synthesis has not yet been ascertained.     

A mathematical model of the volume, pH, and ion content regulation in reticulocytes. Application to the pathophysiology of sickle cell dehydration.

We developed a mathematical model of the reticulocyte, seeking to explain how a cell with similar volume but much higher ionic traffic than the mature red cell (RBC) regulates its volume, pH, and ion content in physiological and abnormal conditions. Analysis of the fluxbalance required by reticulocytes to conserve volume and composition predicted the existence of previously unsuspected Na(+)-dependent Cl- entry mechanisms. Unlike mature RBCs, reticulocytes did not tend to return to their original state after brief perturbations. The model predicted hysteresis and drift in cell pH, volume, and ion contents after transient alterations in membrane permeability or medium composition; irreversible cell dehydration could thus occur by brief K+ permeabilization, transient medium acidification, or the replacement of external Na+ with an impermeant cation. Both the hysteresis and drift after perturbations were shown to depend on the pHi dependence of the K:Cl cotransport, a major reticulocyte transporter. This behavior suggested a novel mechanism for the generation of irreversibly sickled cells directly from reticulocytes, rather than in a stepwise, progressive manner from discocytes. Experimental tests of the model's predictions and the hypothesis are described in the following paper.     

New capabilities for diagnosis of iron-deficiency anemia by computer morphometry of reticulocytes

With the advent of new cytological techniques, such as computer morphometry, the problems in the study of the structure of reticulocytes that characterize insignificant changes in the parameters of erythroid population functioning in the bone marrow in response to altered hemoglobin synthesis in patients with iron-deficiency anemia (IDA) become topical. The changes in peripheral blood reticulocytes in IDA determine those in red blood cells and may be a reliable differential diagnostic tool to identify IDA.     

Reticulocyte cellular indices: a new approach in the diagnosis of anemias and monitoring of erythropoietic function

Reticulocyte analysis has been extended from the simple enumeration of reticulocytes to precise measurements of mRNA content and of cellular indices such as volume, hemoglobin (Hb) concentration, and content. Assessment of reticulocyte maturity is based on the fluorescence intensity of reticulocytes, which depends on RNA content. The appearance of high fluorescence reticulocytes has been shown to be associated with engraftment in the setting of bone marrow or peripheral stem cells transplantation, although it is still not clear how this parameter can improve quality or cost of care compared with the traditional use of absolute neutrophil counts. Reticulocyte indices have been studied especially in the setting of iron deficiency and functional iron deficiency during recombinant human erythropoietin (r-HuEPO) therapy. Reticulocyte hemoglobin content (CHr) may allow prompt identification of an imbalance between r-HuEPO therapy and iron availability by detecting the presence in reticulocytes of iron-restricted erythropoiesis. Diagnosis of simple iron deficiency can also be achieved in a more cost-effective fashion by using CHr in conjunction with the regular complete blood count (CBC), rather than relying on the traditional biochemical parameters of iron metabolism. Response to therapy of megaloblastic anemia can also be monitored with CHr. These new reticulocyte parameters provide a real-time assessment of the functional state of erythropoiesis.     

Hemolysis of “stress” reticulocytes: a source of erythropoietic bilirubin formation

The formation of bilirubin-(14)C was measured in rats given transfusions of red blood cells containing (14)C-labeled hemoglobin heme. Per cent conversion of hemoglobin-(14)C to bilirubin was 4 times greater with transfusion of "stress" reticulocytes from rats responding to hemorrhage than with normal reticulocytes from unstimulated donors. When the increased number of labeled reticulocytes produced by hemorrhaged donors was also considered, the total magnitude of labeled bilirubin formation was almost 20 times higher with stress as compared to normal reticulocytes. The findings were not influenced by splenectomy of either donor or recipient rats, iron loading of donors, or bleeding of recipients. However, bilirubin-(14)C formation fell off progressively as studies were performed at longer intervals after erythroid stimulation.Total bilirubin-(14)C formation in rats transfused with stress reticulocytes was compared to the production of early-labeled bilirubin from all potential sources in intact rats bled according to the same schedule used in the transfusion experiments. It is estimated that degradation of hemoglobin from sress reticulocytes accounts for virtually the entire rise in erythropoietic bilirubin formation from 24 to 96 hr after glycine-2-(14)C administration, but that additional sources make a major contribution before that time. These findings are consistent with the concept that destruction of immature erythroid cells in the peripheral blood, and probably in the bone marrow, accompanies the physiologic response to erythroid stimulation.     

Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation

Chimerism after bone marrow transplantation (BMT) was investigated by flow cytometry analysis of red blood cells (RBCs) and of reticulocytes using a series of selected monoclonal or polyclonal antibodies directed against ABO, Rhesus, Kell, Duffy or MNSs antigens. The method allows the routine detection of less than 0.1% of positive cells in artificial mixed field populations. Blood samples from 135 patients undergoing BMT were investigated around days 15, 30, 45, 60, 90, 180, and then every 6 months after transplantation. Characteristic patterns showing expression of donor red blood cell antigens (expansion markers) and concomitant decrease of recipient specific antigens (depletion markers) within days 16-20 were observed for 125 successfully engrafted patients. Distinct patterns were obtained in 10 patients. A delay in engraftment was evidenced in four patients by the absence of chimerism during the first 6 months without any sign of relapse. Re-appearance of recipient RBCs and reticulocytes was observed in five patients; it was consistent with relapse that was later confirmed by clinical, haematological and cytogenetic studies. Finally, a stable and partial chimerism with 20% of RBCs expressing a marker from the recipient was observed in one patient without any sign of relapse. The reported investigation demonstrated that flow cytometry of RBCs and reticulocytes represents a powerful method to efficiently monitor bone marrow transplanted patients on a long-term basis.     

Characteristics of marrow production and reticulocyte maturation in normal man in response to anemia

Erythropoiesis in normal man was studied during periods of phlebotomy-induced anemia of varying severity. This study permitted a comparison of marrow production measurements over a wide range of marrow production levels. As long as the serum iron remained above 50 mug/100 ml, measurements of plasma iron turnover provided an excellent index of marrow production at all levels of red cell production. In contrast, the absolute reticulocyte count demonstrated a poor correlation with the other measurements. This was shown to be the result of a prolongation of the time required for circulating reticulocytes to lose their reticulum, which correlated with the severity of the anemia. For the clinical application of the reticulocyte count as a measurement of marrow production, an adjustment must be made for this alteration in the circulating reticulocyte maturation time.     

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.     

Variations in the Agglutinability of Red Blood Cells from Patients with Hematologic Disorders

The agglutinability of red blood cells from patients with various hematologic disorders was compared with that of erythrocytes from normal persons by quantitative hemagglutination in the ABH blood group system. Red blood cells from six patients with hypoplastic anemia or metastatic marrow disease as well as normal reticulocytes (five experiments) were less agglutinable than normal mature cells. Erythrocytes from one patient with lymphoma and an acquired hemolytic anemia showed enhanced agglutinability. Our data suggest that changes in erythrocyte reactivity may be more common than suspected, although usually undetected by less sensitive methods.     

绿色荧光蛋白转基因大鼠骨髓间充质干细胞的分离鉴定

背景：转基因动物提取的间充质干细胞自身携带绿色荧光蛋白,与传统病毒、质粒转染相比,能在活细胞中稳定地表达,可较快筛选其修饰的细胞。目的：观察绿色荧光蛋白转基因大鼠骨髓间充质干细胞的生物学特性。方法：取2周龄的绿色荧光蛋白转基因大鼠双侧长骨骨髓,采用全骨髓贴壁法分离培养骨髓间充质干细胞。流式细胞术分析第5代绿色荧光蛋白阳性骨髓间充质干细胞的细胞表型,并分别加入成骨及成脂条件培养液进行体外多向诱导分化,采用茜素红钙盐染色和油红O染色进行鉴定。结果与结论：成功获得了稳定表达绿色荧光蛋白的骨髓间充质干细胞,流式细胞仪检测细胞表达 CD90和CD105,不表达或弱表达CD14和CD45。经成骨诱导3周后茜素红染色可见有橘红色钙盐沉积,经成脂诱导3周后油红O染色见红色的脂滴。结果表明稳定表达的绿色荧光蛋白未影响到骨髓间充质干细胞的多向分化潜能,可作为良好的示踪因子。