Nucleated Red Cells Research Articles

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.

Changes in alpha-globin gene expression in mice of two alpha-globin haplotypes during development

Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with the Hbag2 and Hbac haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins from Hbag2 and Hbac mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different in Hbag2 and Hbac mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers.

Nucleated red cell function: metabolism and pH regulation

The full extent and apportionment of aerobic and anaerobic contributions to energy transduction for membrane pumps associated with cellular pH regulation are very poorly understood. One way of approaching this problem at the cellular level is by using the nucleated erythrocyte as a model cell. Indeed, the aerobic and anaerobic capacity of salmonid erythrocytes and their β-adrenergic mediated pH regulation offers a model "pH regulating system" for examining cellular strategies of response to acute and (or) chronic changes in oxygen availability. Much of our work has focused on the balance between metabolic energy production and the maintenance of erythrocytic pH through primarily or secondarily active ionic exchange mechanisms at the cell membrane. Upon adrenergic stimulation, a rise in cyclic AMP activates the Na+–H+ exchanger, leading to cell alkalinization and an elevation of intracellular Na+. The increased Na+ evidently stimulates Na+,K+-ATPase activity and the increased ATP consumption is matched with aerobic energy production. The pHi that is subsequently established appears to be set by levels of poly anionic phosphates.

The Types of Hemoglobins and Globin Chains in Hydrops Fetalis

Details are presented of analyses of hemoglobins in blood samples from four newborn babies with hydrops fetalis using reversed phase and anion exchange high performance liquid chromatographic methodology. Three were homozygous for the alpha-thalassemia-1 (SEA) deletion, and one was a compound heterozygote for the same deletion and the larger alpha-thalassemia-1 (Fil) deletion. All four babies had beta, G gamma, A gamma, and zeta chains; these chains were present in Hb Bart's or gamma 4, Hb Portland-I (zeta 2 gamma 2), and Hb Portland-II (zeta 2 beta 2). Hb H (beta 4) could not be detected. The level of zeta was directly related to the level of beta and, thus, the fetal age. A lower level of zeta chain was present in the baby with the compound heterozygosity because the large deletion (Fil) on one chromosome included the zeta and psi zeta genes. Circulating red cells, i.e. reticulocytes and nucleated red cells, were unable to synthesize zeta chains, indicating that this capability must have ceased a few months prior to birth. Quantitative data obtained by chromatographic procedures were greatly influenced by the condition of the blood sample and the way it was stored. Hb Portland-II (zeta 2 beta 2) and Hb Bart's (gamma 4) are rather unstable when a red cell lysate is stored at 4 degrees C; this is in contrast to Hb Portland-I (zeta 2 gamma 2) which appears to be stable. Samples can be stored as washed red cells or red cell lysates at -70 degrees C.

Inclusion body beta-thalassemia trait in a Swiss family is caused by an abnormal hemoglobin (Geneva) with an altered and extended beta chain carboxy-terminus due to a modification in codon beta 114

We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

Inclusion Body β-Thalassemia Trait in a Swiss Family Is Caused by an Abnormal Hemoglobin (Geneva) With an Altered and Extended β Chain Carboxy-Terminus due to a Modification in Codon β114

Abstract We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors.

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.

Prenatal Asphyxia in Growth Retarded Fetuses

Members of the Department of Obstetrics at King’s College Hospital, London SE5 have measured the umbilical venous oxygen and carbon dioxide tensions, pH, lactate and glucose concentrations, nucleated red cell (erythroblast) content, and haemoglobin concentration in 38 fetuses with intra-uterine growth retardation in which blood sampling was performed by cordocentesis.

Prenatal asphyxia, hyperlacticaemia, hypoglycaemia, and erythroblastosis in growth retarded fetuses.

The umbilical venous oxygen and carbon dioxide tensions, pH, lactate and glucose concentrations, nucleated red cell (erythroblast) count, and haemoglobin concentration were measured in 38 cases of intrauterine growth retardation in which fetal blood sampling was performed by cordocentesis. The oxygen tension was below the normal mean for gestational age in 33 cases; in 14 it was below the lower limit of the 95% confidence interval for normal pregnancies. The severity of fetal hypoxia correlated significantly with fetal hypercapnia, acidosis, hyperlacticaemia, hypoglycaemia, and erythroblastosis. These findings indicate that "birth asphyxia" is not necessarily due to the process of birth.

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony- forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.

Changes of the phenotype of erythroid progenitor cells (BFU-E, CFU-E) and their progenies during early steps of differentiation.

Early differentiation processes of human erythroid progenitor cells (BFU-e, CFU-e) have been studied during in vitro proliferation using a panel of monoclonal antibodies with known reactivity on different levels of the erythroid cell line. Two antibodies recognizing structures on BFU-e (VIP-2b, BMA 021), two antibodies reactive with CFU-e and nucleated red cells (5F1, CLB-Ery-3) and one antibody directed against glycophorin A (VIE-G4) were used for this study. Normal human bone marrow cells were induced to proliferation in an erythroid progenitor cell assay and, after different periods of incubation, agar cultures were treated with these antibodies and complement. Thereafter, the remaining erythroid cells were incubated again to continue their proliferation with the same stimulators as before. The changes of the phenotype of BFU-e and CFU-e progenies during in vitro proliferation were determined by the reduction of colony formation in comparison with untreated control cultures. Our results indicate that the loss of HLA-DR antigens and the p45 structure is accompanied by the acquisition of structures recognized by the antibodies 5F1 and CLB-Ery-3. After 5-7 d of incubation BFU-e derived progenies exhibit the same antigenic structure as has been found for CFU-e. Glycophorin A expression could only be demonstrated at a late differentiation stage of the erythroid cell lineage.

Expression of Two Natural Killer Cell Antigens, H-25 and H-366, by Human Immature Myeloid Cells and by Erythroid and Granulocytic/Monocytic Colony-Forming Units

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony-forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.

Elimination of murine erythroleukemic stem cells with a novel anti-erythroid antibody conjugated to ricin A-chain: a model for studies of bone-marrow transplantation therapy.

We have produced a rat monoclonal antibody (MAb) MAE15 (IgG), specific for murine erythroid cells, using a murine erythroid cell line as immunogen. This MAb specifically binds to the surface of normal and neoplastic murine erythroid cells. Murine mature erythrocytes and non-erythroid cells as well as rat and human erythroid and non-erythroid cells are not recognized by MAb MAE15. Immunoblotting analysis and mixed precipitation in agar gel showed MAb MAE15 to be specific for murine epitope of 69 kDa antigen of erythroblasts (Ag-Eb), an interspecies antigenic marker of nucleated red cells and reticulocytes. A conjugate (immunotoxin) was prepared, comprising ricin A-chain and MAb MAE15. The immunotoxin inhibited protein synthesis of murine erythroleukemic Ag-Eb-positive K-2 cells and completely inhibited (at the concentration of 2 X 10(-7) M) spleen colony formation by erythroleukemic stem cells of the Ag-Eb-positive RAL cell line. Approximately 35% of the murine normal stem-cell (CFU-S) population was not affected by the immunotoxin at the concentration of 2 X 10(-7) M. This experimental system may be a convenient model for studies of bone marrow transplantation therapy of erythroleukemias.

Adenine phosphoribosyl transferase: assay using high performance liquid chromatography

Adenine phosphoribosyl transferase (APRT) (EC 2.4.2.7) is the enzyme which catalyses the reaction of the purine base adenine with S-phosphoribosyl-l-pyrophosphate (PP-ribose-P) to form the mononu~Ieotide adenosine monophosphate. Human APRT is a cytoplasmic enzyme present in all tissues with the highest specific activities being found in nucleated red cells. Severe homozygous deficiency of APRT is associated with nephrolithiasis and passage of urinary calculi composed of 2,8-dihydroxyadenine [1,2], suggesting that the enzyme has an important salvage function in man. Partial APRT deficiency [3,4] with 25-40% residual enzyme activity in red cell lysates is a relatively common heterozygous trait occurring in about 1% of the population [5] and is not associated with disease. Elevated levels of red cell APRT activity are found in the neonatal period [6] and in some patients with megalobtastic anaemia [7], as a direct consequence of reticulocytosis. They are also a characteristic finding in patients with Lesch-Nyhan syndrome [8] and the X-linked form of severe gout and hyperuricaemia associated with hypoxanthine-guanine phosphoribosyl transferase deficiency 191, where substrate stabilisation of PP-ribose-P may be important. Elevated erythrocyte APRT levels have also recently been reported in patients with hereditary erotic aciduria [lo], an inborn error of pyrimidine metabolism. Current methods for the quantitative assay of the enzyme are mainly radiochemical [ll-131. In this paper we describe an assay for red cell APRT activity utilising high performance liquid chromatography which is suitable for use in clinical laboratories,

Discrimination of Na+-independent transport systems L, T, and asc in erythrocytes. Na+ independence of the latter a consequence of cell maturation?

On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Repeated exposure of C57B1 mice to inhaled benzene at 10 ppm markedly depressed erythropoietic colony formation

Exposure of C57B1 mice to 10 ppm benzene (the current occupational exposure limit) for 6 h/day, 5 days/week causes a progressive depression in the in vitro colony forming ability of one of the erythroid progenitor cells, the colony-forming unit-erythroid (CFU-E). Colony growth of cells from exposed mice was only 5% of control colony growth after 178 days of exposure. Burst-forming-cell growth was depressed to 55% of control growth after 66 days but returned to control growth values at 178 days. In addition, benzene-exposed mice exhibited depressions in the numbers of splenic nucleated red cells and in the numbers of circulating red cells and lymphocytes. These results suggest that low-level exposure to benzene may be hematotoxic.

The characterisation of monoclonal antibodies against haemopoietic cells: Comparison of an immunoperoxidase method with fluorescence activated cell sorting

Nucleated cells from normal human peripheral blood and bone marrow have benn analysed with 2 rat monoclonal antibodies of known specificity, one (YAML 555.6.6) directed against the P28,33 complex (anti-HLA-DR), and the other (YAML 501.4.4) directed against leucocyte common antigen (anti-LCA). The patterns of reactivity with an indirect immunoperoxidase method on fixed smeared cells were in close agreement with those obtained with a fluorescence activated cell sorter. A further new monoclonal antibody of unknown antigen specificity (YAML 537.2) reacts with an intracellular antigen present in neutrophils and their precursors from the promyelocyte stage onwards, megakaryocytes, and a proportion of monocytes, but not with eosinophils, nucleated red cells or lymphocytes. This reactivity could not be demonstrated using the fluorescence activated cell sorter. The immunoperoxidase method allows the identification of individual positive and negative cells and therefore provides a method of identifying minor reactive and non-reactive cell populations in a heterogeneous cell sample such as normal bone marrow. Cytoplasmic binding sites can be differentiated from membrane binding.

Conversion of sodium channels to a form sensitive to cyclic AMP by component(s) from red cells.

Sodium transport has been measured in the isolated epithelium from colons of male Sprague-Dawley rats. Sodium transport in colons was induced by pretreating the animals with dexamethasone (6 mg kg-1) which caused the appearance of an amiloride-sensitive short circuit current within a few hours. Forskolin, a diterpene, which activates adenylate cyclase, was found to increase the cyclic adenosine monophosphate (cyclic AMP) content of rat colons and also to increase short circuit current at the same time. However, measurements of chloride and sodium fluxes across the epithelium indicated that forskolin activates chloride secretion but has no effect on sodium transport. In confirmation of (3) it was found that the amiloride-sensitive short circuit current was unchanged after the short circuit current had been increased by forskolin under a variety of conditions. The behaviour of the mammalian colon as indicated in (3) and (4) is unlike that of amphibian sodium transporting epithelia. It is shown that in toad urinary bladder forskolin increases amiloride-sensitive short circuit current. Procedures were investigated which might make sodium transport in the mammalian colon sensitive to cyclic AMP. Exposing the apical surface to sonicated suspensions of nucleated red cells (frog, toad and duck), followed by washing, gave preparations with amiloride-sensitive short circuit currents which were increased by forskolin or dibutyryl cyclic AMP. It would appear that the sodium channel in the mammalian colon, unlike that of amphibian tissues, has lost the ability to have its properties modified by cyclic AMP. Incubation of colons with sonicated suspensions of nucleated red cells apparently modifies the tissues such that sodium transport across the tissue becomes sensitive to the nucleotide.

Assessment of bone marrow and splenic erythropoiesis in myelofibrosis.

The iron uptake in bone marrow and spleen was measured in 29 patients with myelofibrosis using 52Fe and quantitative scanning. In 10 patients, no iron uptake in the marrow could be observed and active erythropoiesis was extramedullary only. In the bone marrow of patients with myelofibrosis, the iron uptake per nucleated red cell was less than that observed in conditions without myelofibrosis or extramedullary erythropoiesis. Increasing splenic iron uptake was likely to be associated with a decreasing bone marrow iron uptake and was related to the size of the spleen. The data suggest that in myelofibrosis, the spleen dominates iron uptake through intense erythropoiesis and a high splenic blood flow, thus restraining iron supply to the bone marrow.