Number Of Nucleated Red Cells Research Articles

Homozygosity for the Mediterranean α-thalassemic deletion (hemoglobin Barts hydrops fetalis)

Hemoglobin Barts hydrops fetalis syndrome is the most severe and generally fatal clinical phenotype of α-thalassemia. We diagnosed a fetus at 23-weeks gestation with having hydrops fetalis, by ultrasound. At 32 weeks, intrauterine death was detected. Molecular studies revealed that the fetus had the hemoglobin Barts hydrops fetalis syndrome due to homozygosity for the Mediterranean α-thalassemia deletion. This clinical phenotype is generally rare in the Eastern Mediterranean, and this is the first report of this syndrome from Iraq. Techniques for molecular characterization became available only very recently in this country, in a diagnostic setting. Thus, the detection of further cases might be expected in future.

Sox6 Directly Silences Epsilon Globin Expression in Definitive Erythropoiesis

Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG) DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p100H, ɛy globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic) cells define a 36–base pair region of the ɛy proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that Sox6 acts as a repressor by directly binding to the ɛy promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of ɛy in p100H homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of ɛy globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of ɛy globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia.

Haematological abnormalities in the growth-restricted infant

The sequelae of intra-uterine growth restriction (IUGR) and placental insufficiency are frequently encountered in the neonatal intensive care unit, particularly in the preterm baby. These babies have a number of distinctive haematological abnormalities that include the triad of (i) neutropenia, (ii) thrombocytopenia, and (iii) increased red cell production (large numbers of nucleated red cells and/or polycythaemia), together with signs of hyposplenism (red cell targets, spherocytes and Howell-Jolly bodies). These changes are present at birth and therefore can be assumed to begin in fetal life. The haematological abnormalities involve all blood cell lineages and affect mature cells and progenitors, suggesting that a common factor(s) causes fundamental alterations in the process of haemopoiesis in the fetus. This review discusses the pathogenesis, clinical consequences and management of these abnormalities in the newborn infant with IUGR.

Enrichment of fetal nucleated cells from maternal blood: model test system using cord blood.

The presence of small numbers of fetal nucleated red cells in the maternal circulation has been a stimulus for the development of technologies for non-invasive prenatal genetic analysis. Our laboratory has been assessing the feasibility of density gradient centrifugation followed by magnetic activated cell sorting (MACS) of cells expressing CD32 and CD45, to deplete maternal nucleated blood cells. We have examined the efficiency of each of the steps of this procedure using cord blood from term pregnancies as a source of nucleated red blood cells. Cord blood was shown to contain highly variable numbers of nucleated red cells. Three different density gradients were examined. There was no major difference in the performances of the double and triple gradients. Density gradient centrifugation resulted in enrichments of nucleated red blood cells of about 1000-fold relative to the total cell count. However, it was apparent that the selection of the cell layers which were most enriched for these cells would result in significant losses of nucleated red cells in other layers. MACS sorting of cells using CD45 resulted in white cell depletions ranging from 7 to 34-fold. These data provide a foundation for comparison with other methods and for optimization of the MACS technique.

Developmental rescue of an embryonic-lethal mutation in the retinoblastoma gene in chimeric mice.

The requirement for a functional retinoblastoma gene, Rb-1, in murine development around days 12-15 of gestation precludes monitoring the effect of loss of Rb-1 function on later stages of development and on tumorigenesis in adult mice. Here we describe the developmental rescue of embryonic stem cells carrying two inactive Rb-1 alleles in chimeric mice. Rb-1- cells contributed substantially to most tissues in adult chimeras, including blood, liver and central nervous system, which were severely affected in pure Rb-1- embryos. The adult chimeric erythroid compartment appeared completely normal, but an increased number of nucleated red cells was observed during fetal liver erythropoiesis in highly chimeric embryos. No ostensive abnormalities were seen in the developing and adult CNS. However, the developing retina of chimeric Rb-1- embryos showed ectopic mitoses and substantial cell degeneration, while the contribution of Rb-1- cells to the adult retina was much reduced. Moreover, the formation of lens fibre cells was severely disturbed. No retinoblastomas developed in any of these mice. Instead, nearly all animals died of pituitary gland tumours which were exclusively derived from Rb-1- cells.

Evidence for the disruption of the bone marrow microenvironment by combined exposures to inhaled benzene and ingested ethanol

Studies have been performed to investigate the effects of combined in vivo exposures to inhaled benzene and ingested ethanol on the earliest known murine erythropoietic precursor cells, the Burst Forming Unit--Erythroid (BFU-E) and the Colony Forming Unit--Erythroid (CFU-E). Previously we had determined that murine erythropoietic cell populations were particularly susceptible to combined benzene + ethanol treatments. The most striking example of erythropoietic disruption was the transient appearance of large numbers of nucleated red cells (normoblasts) in the circulating blood. In the present studies, male C57Bl/6 mice were exposed to 300 ppm benzene via inhalation for 6 h/d x 5 d/wk x 9 wks. Groups of mice were also exposed to 5% ethanol in the drinking water 4 d/wk x 9 wks. Appropriate controls were also maintained. The hematological assays were performed after 1, 4, and 9 weeks of exposure. After 4 weeks of exposure large numbers of normoblasts appeared in the circulating blood of mice exposed to benzene + ethanol. However, there were no corresponding increases in the numbers of the earliest erythroid progenitor cells in the bone marrow. There were, however, marked increases in the numbers of these cells in the spleen. Previous work in this laboratory had confirmed that the marrow was the source of circulating normoblasts among animals exposed to benzene + ethanol. We conclude, therefore, that circulating normoblasts appear in the peripheral blood because of changes in the bone marrow microenvironment rather than as a consequence of increased erythropoietic proliferation in the marrow.

Marrow erythroid and neutrophil cellularity in the dog.

This paper describes a method for determining the number of marrow erythroid and neutrophil cells in which the cellularity of marrow sections was related to that of the total marrow by radioiron dilution. Tissue sections were prepared from methacrylate-embedded dog marrow biopsies, and neutrophils were identified by staining of their primary granules. After correction of direct section counts for multiple counting error, accurate neutrophil-erythroid ratios were established with a coefficient of variation of less than 10 percent when 10-4 cells were examined. An average neutrophil-erythroid ratio of 1.2 was found in six normal dogs. The total number of nucleated red cells in the dog was 5.48 plus or minus 0.78 times 10-9/kg (plus or minus 1 SD), and the corresponding erythron iron turnover was 0.90 plus or minus 0.11 mg Fe/100 ml whole blood/day. The total number of marrow neutrophils, derived from the neutrophil-erythroid ratio, was 6.6 plus or minus 0.59 times 10-9 cells/kg, of which 1.4 were promyelocytes and myelocytes, 2.3 were metamyelocytes and bands, and 3.0 were segmented neutrophils. Leukopheresis studies were carried out in six dogs to confirm the accuracy of these cellular measurements. Marrow counts showed a mean decrease of 22.7 times 10-9 cells or 35 percent of the postmitotic neutrophil pool, and it was calculated that 10.2 times 10-9 additional cells had been taken from already circulating blood. This estimated deficit of 32.9 times 10-9 was almost identical to the 33 times 10-9 cells actually counted in the removed blood.

Defectivity of Rauscher murine erythroblastosis virus.

THE Rauscher leukaemia virus (RLV) induces a rapidly developing fatal erythroblastosis in mice, characterized by splenomegaly and a highly increased number of nucleated red cells in the peripheral blood1. The first sign of action of the virus is the production of erythroblast foci in the spleen within a week2. A linear dose-response relationship for virus and number of foci has been observed in several mouse strains, suggesting that a colony can be initiated by a single infective unit2.

Biochemical Lesion in Dilantin-Induced Erythroid Aplasia

Abstract Clinical and biochemical studies are reported in a patient who developed pure red cell aplasia after 2 years therapy with Dilantin but in whom aplasia could subsequently be induced with 4-5 Gm. of the drug. Recovery occurred regularly when the drug was discontinued. The administration of 1500 ml. of patient’s plasma obtained during the aplastic phase to a normal volunteer with Dilantin had no effect on the recipient’s reticulocyte count, number of nucleated red cells in his bone marrow, or Fe59 clearance. Attempts to demonstrate binding of gamma globulin to the patient’s nucleated red cells in the presence of Dilantin by immunofluorescence technic were unsuccessful. Dilantin, in a concentration of 20 µg./ml. in vitro, caused significant inhibition of the uptake of C14 formate, glycine, adenine, orotic acid, and uridine into DNA but not into RNA of patient’s bone marrow studied when fully recovered. There was no effect on the uptake of deoxyuridine or thymidine. The Dilantin effect was specific to the erythroid cells as shown by radioautographic studies and by the absence of inhibition when these cells were absent from the bone marrow. Vitamin B12 and folinic aci dgiven to the patient in large doses did not reverse the hematologic effects of Dilantin. The daily intravenous administration of all four deoxyribonucleosides, deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine had a suggestive, but not clear-cut, effect. In an effort to examine a possible relationship of the pure red cell aplasia in this patient to riboflavin metabolism, the patient was started simultaneously on Dilantin and riboflavin. Dilantin was now without effect. However, the patient remains refractory to Dilantin a year after riboflavin was discontinued. It is concluded that Dilantin exerted its toxic effect in this patient by specifically inhibiting DNA synthesis in erythroid cells probably at the step of deoxyribotide formation. The role of riboflavin in the development of resistance to the toxic effect of Dilantin in this patient remains uncertain.

The placenta in cases of hemolytic disease of the newborn

The changes in the placenta associated with hemolytic disease of the newborn were studied. Twenty-seven placentas from rhesus-isoimmunized mothers and one from a mother with anti-Le were collected and examined histologically and by the large section technique of Gough and Wentworth. The more severely the baby was affected the larger the placenta in relation to the baby. In the severe cases, the placentas were extremely pale and more friable than normal. The diagnostic histologic features in the placenta were a marked decrease in the number of fetal vessels and an increase in the number of nucleated red cells in these vessels. Langhans’ layer persisted in all cases but in itself was not considered a specific change. A direct effect of the rhesus antibody-antigen reaction was postulated as the possible cause of these changes.

Erythroblastic Anemia

Erythrobiastic anemia, Mediterranean anemia, thalassemia, or Cooley's anemia was separated by Cooley in 1927 from the group of anemias showing the von Jaksch syndrome, on the basis of its racial incidence. The disease is familial, occurring only in children of the races derived from the Mediterranean region, especially Greece and Italy. It is characterized by a slowly progressing anemia and by the presence of a large number of nucleated red cells. The extraordinary proliferation of erythroblastic tissue leads to changes in the bones, especially in the skull. Extreme pallor of the skin with an icteric tint may be the first sign, then a prominent abdomen from splenomegaly. These signs and symptoms may increase, and evidences of cardiac insufficiency may become manifest. Periods of unexplained fever may occur. Physical development may be retarded. A mongoloid appearance is the next condition that goes to make up the picture. There may be epicanthus and mild exophthalmos. The skull shows prominent frontal and parietal bosses; by contrast the sutures may appear depressed. The liver and lymph nodes are generally enlarged, great enlargement of the spleen is constant, frequently the heart is hypertrophied, and there may be a systolic murmur. In severe cases of long standing, myocardial damage may be demonstrated. Roentgen examination of the skeleton shows osteoporosis of the long bones with trabeculae standing out clearly. The cortex is thin. The medullary cavity is widened and transverse lines are conspicuous. In the skull the space between the tables is widened, and the outer table is reduced to such thinness as not to be visible on the roentgenogram. The trabeculae connecting the inner and outer tables give the appearance of “hair standing on end.” Blood studies usually show a low color index, marked anisocytosis and poikilocytosis, immaturity of red cells, increase in platelets and a polymorphonuclear leukocytosis with numerous immature cells. Reticulocytes are somewhat increased, constituting 5 to 20 per cent of the red cells. Fragility tests have shown a prolongation of the span of hemolysis, beginning at 0.50 to 0.54 per cent, complete at 0.30 to 0.20. According to Cooley and Lee, the red cells have certain specific characteristics. They tend to be large and to have an uneven distribution of hemoglobin. An abnormality of the stroma has also been described, which, however, may also be encountered in sickle-cell anemia. Increased blood destruction occurs but is not so marked as in sickle-cell anemia. The bilirubin content of the blood is increased, the Van den Bergh reaction is indirect. Whether a “latent” form of erythroblastic anemia exists remains a question. The course of the disease is progressive, but some patients may survive into adult life. At times there are periods of hyperpyrexia, without other evidence of infection. Death, however, is usually due to infection.

ERYTHROBLASTIC ANEMIA OF COOLEY (FAMILIAL ERYTHROBLASTIC ANEMIA) IN AN INDIAN BOY

From the ill defined group of conditions known as von Jaksch's anemia, Cooley¹in 1927 separated a type of anemia which occurred in children which he regarded as a clinical entity and which presented characteristic clinical, roentgenologic and microscopic features. He reported that clinically children with this disease exhibited splenomegaly, muddy, yellowish discoloration of the skin and mongoloid facies, due to abnormal prominence of the malar bones. The patients were invariably of Greek, Italian or Syrian stock. Roentgenologically, osseous dystrophy, with thickening and sponginess of the bones, was a characteristic feature; microscopically, there was evidence of hemolytic (hypochromic) anemia, with an unusually large number of nucleated red cells in the blood. Cooley stressed the ancestry of these children, and it came to be believed that this type of anemia was almost exclusively confined to children of Mediterranean stock, so much so that one of the synonyms for familial erythroblastic

ERYTHROBLASTIC (COOLEY'S) ANEMIA AND NEUROLOGIC COMPLICATIONS (STATUS DYSMYELINATUS)

Erythroblastic anemia, a disturbance of the hemopoietic system, is a well established disease characterized by racial and familial incidence, mongoloid appearance, progressive anemia, deficiency of hemoglobin with decrease in the number of red cells but with large numbers of nucleated red cells in the circulation, leukopenia of high degree, enlargement of the spleen and slight enlargement of the liver and destructive changes in the bones with lesions in the bone marrow. About 60 cases so far have been reported in the literature. As far as could be ascertained, in none were there neurologic symptoms. The case to be described is of interest clinically because of the signs and symptoms of an extrapyramidal disorder and histologically for the changes in the striatum and pallidum. REPORT OF A CASE J. V., a 10 year old boy of Italian extraction born in the United States, was admitted to the Montefiore Hospital on Feb.

A Case of Cooley's Anæmia showing Characteristic Skull Changes

This condition, one of the “congenital anaemias,” was described by T. B. Cooley in the American Journal of Diseases of Children, 1927, 34, 347, and occurs in children of Mediterranean parentage; it is associated with severe anaemia, enlarged spleen and liver, and large numbers of nucleated red cells in the circulating blood. In addition there is an unusual appearance of the bones of the skull. As the disease is rarely seen in this country, a fairly typical case is worth putting on record. A child, a female of 17 months, was brought to Out-patients Dept., Bristol General Hospital, in August 1936, with debility and anaemia. On examination it was found to be very anaemic, to have a large liver and spleen and very enlarged glands in the neck.