A lifelong, wide-range radiation biodosimeter: erythrocytes with transferrin receptors.

Rats exposed to 0.1-600 cGy x-rays developed significantly prolonged increases (1.9-11%) of normoblasts (red cell precursors) in the bone marrow as well as a subpopulation of transferrin receptor marked erythrocytes (E-Tr, 1.9-20%) in the blood. Several characteristics are shared in common by these two progenies of the hematopoietic stem cell (HSC): a heretofore unrecognized dose-response curve extending from 0.1-600 cGy composed of logarithmic and quadratic dose response segments, the joining of the segments at approximately 150 cGy, and an identical 29-wk period for one-half repair of the quantified cell damage. This suggests that both of these cells could be surrogates for assessing radiation injury. Preliminary findings suggest that this phenomenon is identifiable in humans.

The phylogenetic odyssey of the erythrocyte. IV. The amphibians.

Amphibians manifest permanently nucleated, oval, flattened, biconvex erythrocytes. These cells demonstrate a cytoskeleton which is responsible for their morphogenetic conversion from a sphere to an ellipse and imparts to their cellular mass reversibility of traumatic deformation. The class Amphibia has the largest of all erythrocytes attaining volumes greater than 10,000 femtoliters in the Amphiuma. The large dimensions reflect evolutionary processes, genomic size, ploidy and the relative size of other somatic cells. Conversely, the erythrocyte count and hemoglobin concentration of these species are low. Occasional denucleated red cells can be seen in the peripheral blood but may attain levels of 90-95% of the total circulating population in certain members of the tribe Bolitoglossini (e.g. Batrachoseps attenuatus). These erythroplastids retain the marginal band thus remaining different from mammalian erythrocytes. Embryologically, erythropoiesis initiates in the yolk sac and then progresses to the kidney, liver, and possibly spleen. The yolk sac cohort is transitory and is successively replaced by the larval and definitive populations of erythrocytes. Red cell production (along with thrombocytopoiesis) in adult urodeles is conducted intravascularly in the spleen. In anurans this organ is usually the major site although the liver also serves as a secondary locus for this activity. Medullary (bone marrow) erythropoiesis makes its phylogenetic debut in anurans and typically occurs during heightened hemopoiesis following metamorphosis or hibernation. Maturation of the erythrocyte in the circulation is commonplace (especially in urodeles) while proliferation at this site is inducible by splenectomy and/or hemolysins. Erythrocyte-related values demonstrate variable differences associated with age, weight, season, gender, and environment.

The phylogenetic odyssey of the erythrocyte. III. Fish, the lower vertebrate experience.

The piscine erythrocyte can be considered the prototype of the red cells that are distributed among inframmalian vertebrates. It is a permanently nucleated, hemoglobin-ladened, oval, flattened, biconvex disc. Ultrastructurally it demonstrates a cytoskeleton comprised of a marginal band and a membrane skeleton which are responsible for the erythrocyte's conversion to an ellipsoid during morphogenesis and endow it with resilience to physical trauma. Erythropoiesis initiates in the yolk sac, followed in many fishes, by the intermediate cell mass. These sites are the sources of the transitory, primitive generation red cells which apparently make their first phylogenetic appearance in fishes and which are subsequently represented in all classes of vertebrates including mammals. Production of definitive generation erythrocytes is centered in evolutionary "pre-splenic" tissue of the gastrointestinal tract or in the spleen in cyclostomes, dipnoi, and chondrichthyes while in teleosts it is typically located in the kidneys with or without splenic participation. The blood is a major site of erythrocyte maturation in the lower fishes and exhibits significant numbers of immature erythroid cells plus occasional mitotic figures. Some teleosts also circulate developing erythroid cells. Certain fishes have occasional circulating erythroplastids, conceptually a portent of phylogenetic changes in higher vertebrates. Remarkably, some bristlemouths have denucleated erythrocytes exclusively in the circulation. The largest piscine erythrocytes are found in the dipnoi, myxines, and chondrichthyes. Primitive fish with the exception of the endothermic sharks tend to have lower hemoglobin concentrations than the modern teleosteans. The very highest hemoglobin concentrations are attained by the endothermic scombrids. Erythrocyte-based data have a broad extent and are variably affected by age, sex, season and environment. This report includes a substantial selection of illustrations (fish species and rbc micrographs).

The phylogenetic odyssey of the erythrocyte. II. The early or invertebrate prototypes.

Freely existing hemoglobin-bearing cells suspended in a plasmic milieu (erythrocytes) are found in a relatively small number of taxanomically scattered invertebrates. These species include some annelids, echiurids, molluscs, phoronids, nemerteans and echinoderms, e.g. Pista pacifica, Urechis caupo, Noetia ponderosa, Phoronis australis, Lineus fuscoviridis and Cucumaria miniata respectively. The typical invertebrate erythrocyte (hemocyte, coelomocyte) can be described as permanently nucleated, considerably larger than the human red cell, oval or circular in configuration and spherical, biconvex or flattened in profile. The marginal band of the erythrocyte, a bundle of subplasmalemmal microtubules that circumscribes the periphery of the cell and lies in the plane parallel to its flat surface makes its first appearance in certain invertebrates. This structure in association with the cell surface-associated cytoskeleton is responsible for the flattened elliptical shape seen in some invertebrate erythrocytes and endows them with flexibility and resilience to mechanical forces. This in an evolutionarily persistent characteristic that is retained throughout the submammalian vertebrates. The erythrocytes of invertebrates are more morphologically and functionally diversified than the mammalian model. In addition to respiratory activities (oxygen storage and transport) they can sometimes function as vendors of nutrients and participate in other less obvious processes. These cells therefore frequently not only retain organelles that are usually discarded by vertebrate erythrocytes (ribosomes, golgi apparatus, etc.) but may also depending upon the species, manifest in their cytoplasm organelles and inclusions that are not a normal component of developing or mature submammalian vertebrate and mammalian erythroid cells. Examples of the latter are pigment granules, lipid droplets, extensive glycogen stores and prominent Prussian blue positive inclusions. Erythrocytes in the invertebrates, though presenting certain cytologic and functional features in common, are a heterogenous collection of cells, each tailored for a specific species or group of organisms.

The phylogenetic odyssey of the erythrocyte. I. Hemoglobin: the universal respiratory pigment.

Hemoglobin is a molecular entity that is capable of reversibly binding and releasing oxygen in either extra- or intracellular milieus. It is present in scattered invertebrates in physical solution or in cellular sites while in vertebrates it is universally located in circulating erythrocytes. These cells serve as the vehicle for and otherwise foster the optimum utilization hemoglobin. Hemoglobin's variable sphere of respiratory activities can be viewed as reflecting the specific requirements for each organism in which it is observed. Once these concepts have been established and the advantages and limitations of its cytologic packaging recognized, the study of the erythrocyte as expressed in its dimensions, colligative aspects, geometry, internal morphology and pathologic variations can be approached in a purposeful manner.

Formation of intracellular vesicles in neonatal and adult erythrocytes: evidence against the concept of neonatal hyposplenism.

Intraerythrocytic vesicles accumulate in the peripheral blood as a result of impaired clearance of these intracellular inclusions by the spleen. The observation that neonates demonstrate an increased percentage of erythrocytes containing these vesicles constitutes the primary evidence supporting the concept that the newborn is functionally hyposplenic. Neonatal erythrocytes also demonstrate an increased propensity to undergo a variety of endocytic processes. We therefore questioned whether the increase in red cell vesicles in the neonate might be the result of increased vesicle formation as opposed to impaired splenic clearance. Newborn and adult erythrocytes were incubated in vitro in synthetic medium at 37 degrees C. Several parameters confirmed the maintenance of physiologic conditions, including levels of erythrocyte phosphate metabolites monitored by nuclear magnetic resonance. The acquisition of intraerythrocytic vesicles during the course of these incubations was compared. Over a period of 144 h, 19.2% of neonatal erythrocytes acquired vesicles compared to 3.7% of the adult cells (p less than 0.001). The increase in vesicles was greater in younger density-separated erythrocytes in both the neonate (37.6%, p less than 0.0005) and the adult (10.3%, p less than 0.002), but persisted even in the oldest erythrocytes (12.2% and 2.4%, respectively). We conclude that the increase in erythrocytic vesicles in the neonate may not simply be an indication of hyposplenism, but a reflection of increased vesicle formation which overwhelms the clearance capability of the spleen.

Physiologic formation of intracellular vesicles in mature erythrocytes.

The ability of mature erythrocytes to spontaneously form intracellular vesicles has been implied from clinical studies but has not been examined experimentally. An in vitro model was developed to demonstrate whether mature erythrocytes are capable of spontaneously forming intracellular vesicles. Normal human erythrocytes were incubated in vitro at 37 degrees C for 144 hr in a synthetic medium. During the course of these incubations, approximately 4% of erythrocytes developed intracellular vesicles which were quantitated by using interference contrast microscopy. Electron microscopic studies confirmed the intracellular nature of these vesicles. Incubation of erythrocytes in autologous plasma produced similar results. The rate of vesicle acquisition in vivo was measured by quantitating erythrocyte vesicles immediately prior to and following splenectomy. The rates of vesicle acquisition in vivo and in vitro were comparable. This in vitro model confirms the ability of mature erythrocytes to spontaneously form intracellular vesicles and strongly supports the concept that this is a physiologic process.

Iron kinetics effects of 88 millirads. Partial-versus-total body X-irradiation.

Rats were irradiated with one tibia shielded (95% marrow exposure), total body exposed (TBI, 100%), and only one tibia exposed (5%), or they were sham irradiated (SI, 0%). Plasma Fe-59 clearance time (T1/2) and Fe-59 content ratio in the right and left tibia (RT/LT) were assayed to determine the erythroid activity of the overall marrow of the animals and the relative marrow activity in the exposed and shielded tibias, respectively. When a major fraction of the overall marrow was shielded or irradiated, the overall erythroid activity levels were identical to those of the SI and TBI animals, respectively. Interestingly, enhanced normoblastosis was observed in the marrow of the exposed tibia of individual animals exhibiting normal erythroid activity in 95% of the marrow. Conversely, localized marrow with normal erythroid activity was found in a shielded tibia of individual rats, demonstrating an enhanced erythroid activity in a major fraction of the total body. It was concluded that 88 mrad can alter marrow functions in a small isolated skeletal region as effectively as in the whole body, and tandem assays of the Fe-59 T1/2 and Fe-59 RT/LT can facilitate ultra-low-dose X-ray studies involved with partial body exposures.

Leucocyte alkaline phosphatase in the Mongolian gerbil and other species: a comparative view.

Cytochemical analyses performed on peripheral blood smears of the Mongolian gerbil reveal the presence of variable amounts of leucocyte alkaline phosphatase (LAP) activity in the neutrophil of this species. Levels of activity in individual cells ranged from 1 to 4+. Cells completely devoid of activity were rarely encountered. Immature neutrophils residing in the bone marrow also displayed moderate amounts of enzymatic activity. The results of a semiquantitative assessment of overall LAP activity in this rodent, expressed as a mean LAP score, were compared with similar analyses carried out on a diverse sample of vertebrates. Intermediate levels of activity were recorded for the gerbil (means = 241). This score was comparable to that obtained for the rat (means = 199) and significantly higher than that seen in man (means = 79). A wide range of LAP scores were noted for closely related species, as well as phylogenetically distant species.

An effective surrogate for impact studies.

Nineteen unembalmed (fresh) cadavers were used in 21 experiments to test restraint systems in automobile impact studies. Some were mounted in a rearward firing sled; others were placed in standard cars during collisions. Prior to and after testing, each cadaver was evaluated. After each experiment a postmortem examination was performed. High-speed registering cameras (1,000 to 2,000 frames per second) were used and multiple telemetric recordings were made. The radiographic and pathologic changes were similar to those seen in patients following high-velocity accidents. Results were compared with those of car accident victims admitted dead on arrival to one of our hospitals. This study is a progress report. The results are being used to improve existing restraint systems and to evaluate experimental models.

The effects of low dose (less than 1 rad) X-rays on the erythropoietic marrow.

Adult female Sprague-Dawley rats were exposed (200 kvp X-rays) to whole body doses of 22-1320 mrad and examined for changes in the level of red blood cell precursors (RBCp) in the marrow at 5-30 weeks post-irradiation, under nonbled and phlebotomy-induced anemic stress conditions. Increases in the RBCp %, RBCp/mg marrow, and RBCp/skeleton under nonbled conditions, and a suppressed erythroid response to an induced anemia, were found after acute doses in the range of at least 70 mrad. Dosages of 22 or 44 mrad that induced no measurable changes when applied only once were found to be effective when they were employed 4 or 2 times/week, respectively. The results suggested the presence of a linear-quadratic dose-response relationship in which the quadratic function exists between 88 and 981 mrad, and the linear dependency, below 88 mrad.

Haemolytic anaemia in rhesus monkeys induced by methylcellulose.

The rhesus monkey was evaluated in its haemopoietic and histological response to intraperitoneal injections of methylcellulose. The haematologic alterations included a mild haemolytic anaemia, lymphopaenia, monocytosis, a shortened survival of Cr51 -labelled autologous erythrocytes (17 . 1 vs 13 . 3 days, P less than 0.025) and normoblastic hyperplasia of the bone marrow. There was a diffuse sequestration of the polymer in the phagocytic cells of the spleen, liver, bone marrow, lymph nodes and adrenal glands. The renal glomerular endothelium also consistently stored this material. Overt splenomegaly was not induced. The monkey appears to present, along with other animals, a rather species-specific response to methylcellulose that is accompanied by fundamental responses observable in all subjects.

"Hemogenic endothelium" of the embryonic aorta: Does it exist?

Aggregates of intravascular, presumably endothelial-derived, undifferentiated cell were found in the aorta of 14-15 day Mongolian gerbil embryos, in several 10 mm pig embryos, in one 10 day mouse embryo and in one 9.5 mm human embryo. As in other species in which they have been identified, these "aortic cell clusters" generally occurred in groups of 25-100 cells and were typically observed adherent to the ventral luminal wall of the abdominal aorta during a brief period in gestation. The present electron microscopic study shows that these cells possess many characteristics in common with undifferentiated primitive blood cells of the yolk sac blood island. Their in situ origin is supported by ultrastructural similarity to underlying endothelium as well as the presence of numerous intercellular junctions among themselves and with subjacent endothelium. It is suggested that they may be morphologically undifferentiated hemopoietic precursor cells. Hypotheses are proposed for the mechanism of their origin from aortic endothelium.

The Mongolian gerbil (Meriones unguiculatus): its histological and haematological response to methylcellulose.

After intraperitoneal injections of 2.5% aqueous methylcellulose twice a week for 15 weeks gerbils showed a mild haemolytic anaemia, heterophilia, lymphopenia and monocytosis. Many monocytes and a few lymphocytes had a foamy vacuolated cytoplasm. There was a sequestration of methylcellulose in the phagocytic cells of the spleen, liver, bone marrow and in other scattered foci, and consistent storage in the glomerular endothelium. Myeloid metaplasia was noted in the spleen, and splenic weights were markedly increased (P Less than 0.05); hepatomegaly was also present. The survival at the end of 15 weeks was 60%. From this experiment, the gerbil is considered to be a potential model for a stimulated monocyte-macrophage system.

Cytology of the bone marrow in the Mongolian gerbil.

Heterophils were the most numerous cells (39%), followed by the normoblasts (27%) and lymphocytes (8%). Significant differences between males and females were observed in the total number of heterophils (42 and 35% respectively, P less than 0.002), heterophil myelocytes (12 and 10%, P less than 0.01) and the myeloid:erythroid ratio (1.9:1 and 1.4:1, P less than 0.03). Heterophils presented both annular and polymorphous nuclei, which appeared to develop along separate lines. The ring series began with a small nuclear 'vacuole' which increased in size with maturation of the cell. Ring-forms comprised 1/3 of the total heterophil population in the marrow. Eosinophils and basophils also demonstrated annular nucleated forms. Mature heterophils tended to have a scarcity of stainable specific granulation. Eosinophil granules were larger than those of the heterophil, slightly refractile and did not react avidly with eosin. Basophil granules, conversely, reacted strongly with the methylene-blue component of Wright-Giemsa stain and were metachromatic. Normoblasts retained their cytoplasmic basophilia to a greater degree than comparable cells in man.

The cellular blood picture of the Mongolian gerbil throughout the first year of life: a longitudinal study.

The Mongolian gerbil was subjected to a longitudinal study of its cellular blood picture throughout its first year of life. This laboratory rodent initiates its neonatal period with an erythrocytic macrocytosis, a subadult red cell count and panleukocytosis, followed by a transient leukopenia. The adult blood picture is attained by 8 weeks with an erythrocyte count of 8.5--9.0 X 10(6) rbc/mm3 and a leukocyte level of 10--12 X 10(3) cells/mm3. The erythrocytes are microcytic (approximately 53 mu3) and frequently demonstrate polychromasia as well as basophilic stippling. There is a sexual dimorphism favoring the male in the erythrocyte corpuscular volume, mean corpuscular hemoglobin, hematocrit and hemoglobin values.

Embryonic and fetal hemopoiesis in the Mongolian gerbil (Meriones unguiculatus).

A study of the development of hemopoiesis in the Mongolian gerbil (Meriones unguiculatus) was conducted in order to determine the temporal sequence, the organs involved and the cytology of blood cell formation in this species. Hemopoiesis in the intrauterine life of the gerbil can be divided into four phases based on the site of blood cell formation: (1) the vitelline phase, (2) the hepatic phase, including thymic histogenesis, (3) the splenic phase and (4) the medullary phase, with the development of secondary lymphoid tissues. At the onset of each of these phases a blast-like cell was identifiable in each hemopoietic organ which, because of its morphology and its presumed multipotentiality was classified as a "lymphoid cell". In the yolk sac phase (gestational day 12) two generations of erythrocytes, a primitive and a definitive, are formed. The liver is by day 15 erythropoietic and megakaryopoietic, but later, a few granulocytes are also found in its extravascular compartment. The thymus is exclusively lymphopoietic from the appearance of its earliest cells on day 15. Splenic hemopoiesis is initiated with the presence of lymphoid cells (day 20) followed later by the appearance of morphologically identifiable blood cell lines. Early normoblastic and granulocytic activity begins in the marrow cavities on day 23, though the marrow is not considered to be a source of circulating blood cells during fetal life. Lymph node histogenesis occurs during the last four days of gestation, first in the cervical region and then in other parts of the body. The finding of undifferentiated lymphoid cells in all organs at the initiation of hemopoiesis and in the peripheral blood throughout gestation is discussed in light of the migratory theory of hemopoiesis.

Erythrocyte survival in children as studied by labeling with stable 50Cr.

The survival of 50Cr- and 51Cr-labeled autologous and/or homologous erythrocytes was compared simultaneously in eight pediatric patients and one adult. 50Cr, a stable, nonradioactive nuclide, had values comparable to those of standard radioactive 51Cr labeling. The data also demonstrated the capability of 50Cr-51Cr labeling to reveal differences in survival between two populations of erythrocytes monitored simultaneously in the same individual. The technique permitted the use of the nonradioactive isotope in volumes of blood that are appropriate for the pediatric age group.

Erythrocyte basophilic stippling in the Mongolian gerbil.

Basophilic stippling of the circulating erythrocytes is characteristic of the Mongolian gerbil. Its enzymatic digestion along with the concomitant removal of diffuse erythrocytic polychromasia by the action of ribonuclease demonstrates that it represents microscopically visible aggregates of cytoplasmic ribonucleoprotein, presumably of ribosomal origin. Up to 40% of the total circulating erythrocytes may be stippled in foetal and newborn animals. There is then a progressive decline in incidence until adult levels are attained at least by 20 weeks of age. The bone marrow of the adult gerbil contains a higher proportion of stippled red cells than the circulating blood. The stippling can be either coarse or fine and observable in both polychromatophilic and orthochromic cells. It is suggested that erythrocytes with basophilic stippling are relatively immature red cells still demonstrating remnants of cytoplasmic ribonucleoprotein.