Quantitative analysis of Howell-Jolly bodies in children with sickle cell disease.

OBJECTIVES: Although functional asplenia in sickle cell disease (SCD) begins early in life and has important clinical consequences, quantitative measurement of splenic function is not readily available. A novel high-throughput flow cytometric method for quantitating Howell-Jolly bodies (HJB) has been developed which isolates HJB-containing CD71(+) and CD71(-) erythrocytes. Analysis of these cell populations allows quantitative measurement of splenic filtrative function and possible chromosomal damage. METHODS: Blood specimens from 147 children with SCD were analyzed using a high-throughput flow cytometric method. Enumeration of the following populations was accomplished: 1) CD71(+) reticulocytes among total erythrocytes, identifying the youngest erythroid cell population; 2) HJB-containing CD71(+) reticulocytes, which isolate young erythrocytes containing micronuclei as an index of cytogenetic damage; and 3) HJB-containing CD71(-) erythrocytes, identifying older erythrocytes containing micronuclei, indirectly measuring splenic function. RESULTS: Children with HbSC (n = 24) had slightly elevated HJB frequencies, while children with HbSS (n = 125) had highly elevated frequencies within CD71(+) cells (0.44% +/- 0.40%, normal 0.12% +/- 0.06%, p < 0.001) and CD71(-) cells (2493 +/- 2303 per million RBC, normal 20 +/- 11, p < 0.001). Using a multiple regression model, the frequency of HbSS CD71(+) reticulocytes containing HJB was significantly influenced by hydroxyurea use (p < 0.0001), age (p = 0.0288), and splenectomy (p = 0.0498). Similarly, mature CD71(-) erythrocytes containing HJB were positively correlated with hydroxyurea (p = 0.0001), age (p < 0.0001), and splenectomy (p = 0.0104). CONCLUSIONS: HJB quantitation by flow cytometry is a novel assay for measuring splenic function and may be valuable for investigating the efficacy and safety of therapeutic options for children with SCD.

Persistent reticulocytosis in a case of poodle macrocytosis.

A healthy 14-year-old, male neutered, Miniature Poodle was found to have a persistent erythrocyte macrocytosis and reticulocytosis with a normal and stable HCT. The hematologic features of macrocytosis, increased Howell-Jolly bodies, and metarubricytosis, in the absence of anemia or other cytopenias, combined with the cytologic evidence of bone marrow erythroid dysplasia, including megaloblastosis, binuclearity, increased mitotic activity, and nuclear fragmentation, are consistent with previous reports of congenital dyserythropoiesis termed poodle macrocytosis. We speculate that the additional presence of persistent reticulocytosis in the absence of an identifiable stimulus for accelerated erythropoiesis may represent a phenotypic variation of this inherited condition, and the morphologic abnormalities of the dyserythropoiesis are described.

Which common test should be used to assess spleen autotransplant effect?

BACKGROUND: Historically, total splenectomy was the only choice of treatment for traumatic splenic injuries. However, nonoperative management and spleen-preserving surgical techniques are preferred in modern medicine. In some situations in which the surgeon has to perform splenectomy, spleen autotransplant may preserve the splenic function. Selecting the best method for evaluating the splenic autotransplant effect has been debated for several years. In this study, we compared three common tests in evaluating the implanted spleen function. METHODS: Participants included 10 patients who were candidates for laparotomy and splenectomy. After performing splenectomy, we implanted five pieces of the spleen in the greater omentum of each patient. After 3 months, the implanted spleen function was evaluated by nuclear red blood cell (RBC) scan, serum immunoglobulin (Ig) M level, and presence of Howell-Jolly (HJ) bodies in the peripheral blood smear. RESULTS: All patients had normal peripheral blood smear. The IgM level was lower than normal in one patient, and scintigraphy did not demonstrate the transplanted spleen in another patient. CONCLUSION: All these tests may have comparable results, but because of availability and low cost of peripheral blood smear, which is also easily performed, it can be considered as the first option to evaluate the implanted spleen function.

DNA degradation during maturation of erythrocytes - molecular cytogenetic characterization of Howell-Jolly bodies.

Howell-Jolly bodies (HJBs) are small DNA-containing inclusions of erythrocytes and are often present after splenectomy. The genetic composition of HJBs is unknown at present. We isolated individual erythrocytes that had inclusion bodies from five splenectomized patients and performed DNA amplification using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) with subsequent reverse painting on normal male metaphase spreads. We also measured the sizes of HJBs in erythrocytes from a splenectomized patient using an inverted microscope. Two-dimensional positions of HJBs were projected onto a virtual erythrocyte. The average size of HJBs was 0.73 +/- 0.17 microm (range 0.4-1.1 microm). Inside the erythrocyte the HJBs were found to be equally distributed. Small HJBs contained DNA from one or two centromeres and larger HJBs contained DNA from up to eight different centromeres. Centromeric DNA from chromosomes 1/5, 7, 8, and 18 was most frequently observed. Signals from the centromeric regions of chromosomes 3, 4, 9, and 10 were not observed. Signals from euchromatic regions were detected in a few cases. We hypothesize that in addition to enucleation and nucleus fragmentation DNA degradation during maturation of erythrocytes preferentially eliminates euchromatic DNA. Similarities between these processes and those described for embryonic stem cells suggest that most stem cells are able to degrade DNA in a genetically controlled manner.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.

Pocked erythrocyte counts in patients with hereditary spherocytosis before and after splenectomy.

The pocked (pitted or vacuolated) erythrocyte count has become increasingly utilized as a simple inexpensive test of splenic reticuloendothelial function. Values are less than 2.0% in normal subjects and 20 to 70% following splenectomy. Because scant and conflicting data are available about pocked erythrocyte measurements in hemolytic anemias other than the hemoglobinopathies, we performed pocked erythrocyte counts in 27 patients with hereditary spherocytosis. Prior to splenectomy patients often had elevated values (mean 4.9%). This unexpected observation suggests that hemolytic anemia may result in congestion of the red pulp and/or induced mild splenic reticuloendothelial blockade. As expected, but contrary to a previous report, pocked erythrocyte values following splenectomy were markedly increased (mean 54.9%).

Formation and disappearance of pocked erythrocytes: studies in human subjects and laboratory animals.

The pocked or "pitted" RBC count is being increasingly utilized as a test of splenic function. Since little is known about patterns of formation and removal of the characteristic organelles in the pocked RBC, we performed serial pocked RBC counts following splenectomy in six patients and in three animal species (dogs, rats, and rabbits). In the patients, pocked RBC counts began to rise within 1 week following splenectomy and reached a plateau (40-60%) by 60-100 days. Similar results were obtained following splenectomy of dogs, except that the plateau value was less. Pocked RBCs in splenectomized rats rose initially, but after the sixth week there was a progressive decline in their numbers; splenosis or accessory spleens were not visualized at autopsy. Rabbits had only a slight and inconsistent rise in pocked RBCs after splenectomy. When the rate of removal of pocked RBCs from the circulation was determined by transfusion of blood from splenectomized dogs in eusplenic animals, the pocked RBC count rapidly decreased within 3 to 6 hours. Pocked RBCs did not disappear when crosstransfused into a splenectomized recipient animal. Prior treatment of the recipient dog with either corticosteroids or vincristine did not affect the pattern of removal of pocked RBCs. We conclude that pocked RBCs rise slowly following splenectomy, disappear rapidly from the circulation in the presence of a normal spleen, and vary in pattern of rise and peak levels following splenectomy of different laboratory animals.