Evaluation of the iron status of a population

The iron status of a population of 1564 subjects living in the northwestern United States was evaluated by measurements of transferrin saturation, red cell protoporphyrin, and serum ferritin. The frequency distribution of these parameters showed no distinct separation between normal and iron-deficient subjects. When only one of these three parameters was abnormal (transferrin saturation below 15%, red cell protoporphyrin above 100 mug/ml packed red blood cells, serum ferritin below 12 ng/ml), the prevalence of anemia was only slightly greater (10.9%) than in the entire sample (8.3%). The prevalence of anemia was increased to 28% in individuals with two or more abnormal parameters, and to 63% when all three parameters were abnormal. As defined by the presence of at least two abnormal parameters, the prevalence of iron deficiency in various populations separated on the basis of age and sex ranged from 3% in adolescent and adult males to 20% in menstruating women. It is concluded that the accuracy of detecting iron deficiency in population surveys can be substantially improved by employing a battery of laboratory measurements of the iron status.     

Role of transferrin in determining internal iron distribution

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.     

Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long- term repopulating stem cells

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca- 1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.     

The p53 gene in pediatric therapy-related leukemia and myelodysplasia

We investigated the frequency of p53 mutations in 19 pediatric cases of therapy-related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2- basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting.     

Enhanced production of B lymphocytes after castration

Castration has long been recognized to stimulate thymic growth and augment cellular immunity. We sought to determine whether castration affects B lymphopoiesis by analyzing the phenotype of bone marrow and spleen cells from animals postcastration. In this report, we show that the bone marrow cells from castrated male mice show a sustained, twofold to threefold increase in numbers of B220+/IgM- cells and of newly formed B220+/IgM+ B cells. Most of the expanded B220+/IgM- cell population consisted of small, HSAhi, CD43- cells characteristic of pre- B cells. The castrated animals also showed increased numbers of splenic B cells, primarily consisting of small IgM+, IgDlo, B220lo, HSAhi cells. Taken together, these results show that castration causes dramatic, long-lived enhancement of B lymphopoiesis in bone marrow and increased numbers of mature B cells in the periphery.     

Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4- dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on hemoglobin

Erythrocyte glutathione (GSH) can be rapidly depleted by incubating the cells with 1-chloro-2,4-dinitrobenzene (CDNB), which forms 2,4- dinitrophenyl-S-glutathione with GSH through the reaction catalyzed by glutathione S-transferase. GSH-CDNB conjugate thus formed stays undegraded within the erythrocytes. This indicates that in the erythrocytes, mercapturic acid pathway is inoperative. Depletion of GSH in the intact erythrocytes by CDNB results in rapid oxidation of large amounts of hemoglobin to methemoglobin. When glutathione S-transferase- free hemolysate of erythrocytes is incubated with CDNB, the depletion of GSH as well as methemoglobin formation are minimal. Glutathione peroxidase and glutathione reductase activities of the erythrocytes are not affected by CDNB. These studies provide a specific enzymatic method for rapid removal of erythrocyte GSH and also indicate that GSH is vital in maintaining a reduced environment within the erythrocytes.     

Alloimmunization following platelet transfusion: the absence of a dose- response relationship

A major concern about the use of prophylactic platelet transfusions is the development of alloimmunization. To determine whether the rate of alloimmunization is related to the number of platelet transfusions, we measured the development of lymphocytotoxic antibody in the first 2 mo of induction therapy in patients with acute nonlymphocytic leukemia. All patients received prophylactic random donor platelets and packed red blood cells during induction. No patient had lymphocytotoxic antibody present at admission. One hundred and six patients received an average of 9.3 platelet transfusions (range 2-34) containing an average of 61 U (range 9-236). The rate of alloimmunization was 38% overall and correlated with refractoriness to platelet transfusions. Ten of 19 patients receiving less than or equal to 4 transfusions became immunized, compared with 30/87 patients receiving less than 4 transfusions. There was no relationship between the number of platelet transfusions given and the rate of severity of alloimmunization, suggesting prophylactic platelets need not be withheld expressly to prevent alloimmunization.     

Monoclonal and oligoclonal gammopathy after bone marrow transplantation

Serial serum protein electrophoreses were performed on 60 patients undergoing allogeneic and syngeneic bone marrow transplantation (BMT). More than 50% of patients (31 of 60) developed transient oligoclonal and monoclonal gammopathies that appeared an average of 84 days posttransplantation (range 27 to 336 days) and persisted an average of 175 days (range 14 to 652 days). Immunofixation analysis revealed 82% of the M components to be of the immunoglobulin G (IgG) type and 18% to be IgM; 56% were kappa and 44% were lambda. A strong correlation between development of graft versus host disease (GVHD) and appearance of M components was observed (73% incidence in GVHD patients v 27% in non-GVHD patients, P = .0003). Two of the three syngeneic graft recipients also developed monoclonal gammopathies. Evidence of oligoclonal circulating B-cell populations was found in 68% of patients posttransplantation by flow cytometric B-cell clonal excess assay. No correlation of recovery of particular B- or T-lymphocyte subsets and development of M components was seen. The development of transient oligoclonal and monoclonal gammopathies after transplantation may be a ubiquitous finding reflecting recapitulation of early B-cell ontogeny.     

A colony assay for blast cell progenitors in non-B non-T (common) acute lymphoblastic leukemia

A clonal method in cell culture is described that permits the quantitation of blast precursors in common (non-T, non-B) ALL; the method also yields information about progenitor properties, based on analysis of cells in colonies. The technique is identical to that used successfully for normal and malignant B-cell progenitors except that it requires culture at below O2 tension (5%-7%); mononuclear cells from blood or marrow are depleted of T cells and cultured with media conditioned by T cells in the presence of phytohemagglutinin and irradiated T cells. Cultures are incubated for 5-7 days in a moist atmosphere at 5% CO2 and 5%-7% O2. Colonies were obtained from marrow or blood of 16 of 18 ALL patients. Cells in colonies had the same characteristics (E-, slg-, cALL+ and clgM+ or clgM-) as the cells in the patient. By replating pooled colonies, self-renewal of progenitors was shown. The findings are considered in light of a model of leukemic blasts that depicts such populations as lineages maintained by progenitors that either renew themselves or give rise to blast cells with little or no proliferative capacity.     

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.