Normal Reticulocytes Research Articles

Mature Sickle and Normal Red Cells Exhibit Regulatory Volume Decrease Activated by Urea and Mediated by KCl Cotransport.

KCl Cotransport (KCC) is active in normal (AA) reticulocytes and overly active in sickle (SS) reticulocytes. Cell swelling activates KCC and induces a powerful regulatory volume decrease (RVD) in reticulocytes, which increases cellular hemoglobin concentration (CHC) to new steady state values that are higher in SS than AA cells (Blood 2004; 104(9):2954–60). We recently showed that urea (300–900 mM), which strongly activates KCC, also induces an intense RVD with even higher final CHC values (SS>AA) (Blood 2004; 104 (11): 976a). Because KCC activity is high in reticulocyte-rich samples in both SS and AA blood, KCC activity has been assumed to be minimal in mature cells. We now report that mature RBC exhibit RVD stimulated by urea and mediated by KCC. AA and SS RBC were washed in HBS and treated with nystatin to increase cation content and decrease CHC to 22–24 gm/dl. During incubation at 37o in HBS (145 mM NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, pH 7.4) ± 600 mM urea, timed samples were taken into iced HBS, washed, and kept on ice until analyzed later that day on an Advia 120 automated cell counter, which reports frequency distributions for CHC of both mature RBC and reticulocytes. As previously reported, within 30 min reticulocytes achieved a new steady state CHC which was higher for SS than AA cells, though the speed of RVD was similar. Surprisingly, mean CHC of mature (non-reticulocyte) RBC in both AA and SS blood also increased upon incubation with urea. RVD in mature cells was slower than in reticulocytes and was apparently incomplete after 2 hours. RVD in mature RBC was completely abrogated (CHC was stable) in the absence of Cl- (sulfamate substitution) or in the presence of 100 uM DIOA (dihydro-indenyl-oxy-alkanoic acid), both of which inhibit KCC activity. Whereas reticulocyte CHC frequency distributions after urea-stimulated KCC-mediated RVD showed a single population, CHC distributions for mature RBC revealed two distinct sub-populations: One in which CHC changed little during incubation and a second which achieved a CHC similar to that achieved by reticulocytes after RVD. The relative size of the volume regulating (high CHC) sub-population increased steadily throughout the incubation, which was responsible for the progressive increase in mean CHC values. The high CHC sub-population was not apparent when cells were incubated in Cl- free media or with DIOA, indicating that RVD was mediated by KCC. After 2 hours incubation, 67 ± 8 % of SS RBC had shifted to higher CHC, compared to 37 ± 11 % of mature AA RBC (p<<0.001 by t-test). The progressive change in CHC histograms during incubation was consistent with cells achieving the same final CHC values at various rates. In preliminary studies with biotin-labeled AA cells ageing in vivo, urea-stimulated RVD in mature cells diminished with time, but persisted through most of RBC lifespan. These data indicate that the KCl cotransporter remains in the membrane of mature AA RBC, and is capable of producing RVD under the strong stimulation of urea. In SS RBC, which have shorter lifespan, a majority of non-reticulocytes retain urea-stimulated KCC activity.

Proficiency Testing of Reticulocyte Counting in Ontario

The reticulocyte count reflects the erythropoietic activity of the bone marrow and is thus useful in both the approach to the diagnosis of anemia and in monitoring bone marrow response to therapy. Traditionally, reticulocyte quantitation relied upon microscopic techniques; recently automated reticulocyte quantitation has become widely available. External quality assessment (EQA) of reticulocyte quantitation has not been widely reported; this paper presents data from reticulocyte EQA surveys conducted in Ontario between November 2000 and October 2004. Samples with normal and increased reticulocyte levels were obtained from adult donors and analyzed within 48 hours. We found that despite improved accuracy and precision, automated reticulocyte counting techniques do not translate into markedly improved inter-laboratory accuracy and precision. Further, we found that both microscopic and automated techniques can effectively differentiate between patients with normal and those with increased reticulocyte counts. Finally, we ascertained that reference intervals submitted with the initial surveys revealed that laboratories could have made erroneous clinical interpretations of their normal or elevated reticulocyte counts. The reestablishment of reference intervals by participants resulted in a marked improvement of the reference intervals submitted on subsequent surveys.

Activation of KCI Cotransport by Urea Induces Dehydration in Both Sickle and Normal Reticulocytes.

KCl Cotransport (KCC) is highly expressed in sickle red blood cells (SS RBC) and recent data have demonstrated its abnormal response to cell swelling and acid pH. We showed that the final MCHC achieved by SS reticulocytes upon activation of KCC by these stimuli was higher than that of normal (AA) reticulocytes (Joiner et al, Blood, in Press). Here we report studies examining the sensitivity of KCC to activation by urea at concentrations present in the kidney and the effect of urea stimulation of KCC on reticulocyte MCHC. KCC fluxes were assayed as Rb uptake over 20 min in isotonic saline solutions buffered with HEPES to pH 7.4 (37°C) containing 0.1 mM ouabain, and 0.01 mM bumetanide, with 27 mM RbCl replacing equimolar NaCl. Under these conditions > 95 % of Rb uptake was Cl-dependent (assessed by sulfamate replacement of Cl). The maximal volume-stimulated KCC flux (VSmax KCC) was measured for each sample in cells swollen isotonically to MCHC < 27 gm/dl (nystatin method). Urea (100 to 1000 mM) increased osmolality of buffers, but did not alter initial MCHC. MCHC of reticulocytes (detected by flow cytometry) was tracked by measuring density changes on calibrated OPTIprep® gradients. Cl-dependent, ouabain- and bumetanide-insensitive Rb influx in both AA and SS RBC was increased by urea, reaching a plateau at 1000 mM urea that was similar to VSmax KCC. SS RBC were more sensitive to urea stimulation than AA RBC: 50% VSSmax KCC was achieved at 330 mM urea in SS RBC vs 550 mM in AA RBC. This effect was sulfhydryl dependent: exposure to the reducing agent dithiothreitol (preincubation for 30 min at 37°C with 10 mM DTT, then 1 mM in flux media) normalized the response to urea in SS RBC, with no effect in AA RBC. When swollen to MCHC 30 gm/dl, SS and AA retics exhibited Regulatory Volume Decrease (RVD) which increased MCHC. RVD in both AA and SS retics was abolished by incubation in sulfamate media, indicating mediation by KCC. As previously reported, final MCHC achieved after two hours incubation by SS retics was greater than AA retics (see Table, Control).Final Reticulocyte MCHC, gm/dl [mean (SD), n = 3]AASSp (AA vs SS)Control31.9 (0.7)34.7 (1.20.03Urea35.3 (0.5)37.8 (0.3)0.002p (Control vs Urea)0.0050.03Urea (600 mM) enhanced RVD in both AA retics and SS retics. Sulfhydryl reduction with DTT had no effect on urea-stimulated RVD in SS reticulocytes. RVD stimulated by urea was complete within 60 minutes, and was irreversible: additional incubation without urea did not lower MCHC. The partially dehydrating effect of brief (10 min) exposure to urea was also irreversible, and was cumulative: cells exposed to two 10 min exposures to high urea (600 mM), separated by 10 min at a low, non-stimulating concentration of urea (100 mM), yielded the same MCHC as a continuous 20 min exposure. These data demonstrate that urea, at concentrations found in the renal medulla, is a powerful stimulant of KCC and intiates a striking RVD in reticulocytes. To the extent that intermittent stimulation of KCC by urea in the kidney occurs in vivo, this could contribute an exaggerated RVD resulting in dehydration of SS reticulocytes.

A Second Promoter for the Human KCl Cotransporter 1 (KCC1) Gene Drives Differential Expression of a Variant Isoform in Sickle Versus Normal Reticulocytes.

The K+Cl- cotransporter plays a significant role in the maintenance of red cell volume. During cellular maturation, this cotransporter actively moves K+ and Cl- out of the cell. The accompanying movement of water results in dehydration and shrinking of the red cell. Because KCl cotransporter activity is higher in sickle compared to normal reticulocytes, it has been considered a potential modifier gene for sickle cell disease. We have evidence for expression of three KCC genes in human reticulocytes and have investigated the promoter for KCC1. While the expression of the principal KCC1 transcript did not differ in SS compared to normal reticulocytes, we now describe an alternative transcript of the KCC1 gene emanating from a second promoter and exhibiting a restricted tissue distribution. Investigation of the EST databases revealed spliced ESTs corresponding to the use of four distinct N-terminal exons in the KCC1 gene, each reported multiple times in the dataset. Primers were developed for these 5′ regions (exon1, 1a, 1b, and 1c) and used in an RT-PCR reaction with human reticulocyte RNA. The exon1 form and the exon1b variant were expressed. When the relative levels of these forms were compared, expression of the exon1 transcript was unchanged, while significantly higher levels of the exon1b variant was evident in the AA reticulocyte RNA compared to numerous SS samples. In an analysis of seven other human tissue samples, the exon1b isoform was highly expressed in kidney, lung, and heart, while the KCC1ex1 transcript was expressed at a constant level in all tissues. Although the transcript for this variant could arise from the KCC1 promoter we have previously characterized, the pattern of expression suggested control from a second promoter. A 915bp region corresponding to −787 to +128 was isolated and cloned into a reporter construct to test for promoter activity. This clone was compared with KCC1 promoter constructs in transient transfection assays. The exon1b construct not only exhibited promoter activity by directing high levels of luciferase expression in K562 cells, it also demonstrated tissue-specificity with a low level of activity in Jurkat cells. This recapitulates the endogenous levels detected by RT-PCR analysis of these cell lines. The −787/+128 exon1b construct is also 3-fold more active than the ubiquitously expressed exon1 promoter. To identify the control elements for this promoter, we produced a series of deletion constructs; the smallest construct contained 181bp. No reduction in reporter gene activity was evident, indicating the major regulatory elements lie very close to this promoter. Since exon1 encodes 39aa and exon1b only 7aa, the use of this smaller first exon effectively produces an N-terminal truncation in the protein. Studies with the mouse KCC1 cDNA have demonstrated that proteins produced by an N-terminal truncation are not only inactive for K+Cl- cotransport, but also function as dominant negative regulators of a full-length KCC1 protein. High level expression of this variant in AA cells compared to SS cells would therefore be consistent with the low reported activity in the AA reticulocytes. Induction or modulation of the expression of the KCC1ex1b variant may be an key factor in the control of red cell hydration.

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

Pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase deficiency.

Anti-CD20 monoclonal antibody for the treatment of severe, immune-mediated, pure red cell aplasia and hemolytic anemia

Immune-mediated, acquired pure red cell aplasia (PRCA) is a rare disorder frequently associated with other autoimmune phenomena. Conventional immunosuppressive treatment is often unsatisfactory. Rituximab is a monoclonal antibody against the CD20 antigen, highly effective for in vivo B-cell depletion. An 18-month-old girl with both severe PRCA and autoimmune hemolytic anemia, refractory to immunosuppressive treatment, received 2 doses of rituximab, 375 mg/m(2) per week. The drug was well tolerated. After anti-CD20 therapy, substitutive treatment with intravenous immunoglobulin was started. The treatment resulted in marked depletion of B cells; a striking rise in reticulocyte count ensued, with increasing hemoglobin levels, finally leading to transfusion independence. The child is now 5 months off-therapy, with normal hemoglobin and reticulocyte levels. This case suggests a role of anti-CD20 monoclonal antibody for treatment of patients with antibody-mediated hematologic disorders. (Blood. 2001;97:3995-3997)

CDNA cloning of mutant catalase in acatalasemic beagle dog: single nucleotide substitution leading to thermal-instability and enhanced proteolysis of mutant enzyme

The mutant catalase purified previously from acatalasemic dog liver was heat-labile but possessed normal activity, suggesting a mutation within the coding region distal from the catalytic site. The nucleotide and deduced amino acid sequences of acatalasemic beagle dog catalase were determined by analysis of cDNA obtained by 5′- and 3′-RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Comparative analysis of cDNA sequences of normal and acatalasemic dog catalases indicated a single nucleotide difference where alanine 327 (G̱CT) was substituted with threonine (A̱CT). The mutant catalase, which was overexpressed in COS-1 cells, was heat-labile as previously observed with the purified enzyme from acatalasemic dog liver, indicating that this amino acid substitution can lead to structural instability. No catalase protein and activity were detected by immunoblotting and spectrophotomeric assay in acatalasemic dog reticulocytes although almost the same level of mRNA expression as that in the normal reticulocytes was observed. Pulse-labeling and immunoprecipitation examination indicated that the level of catalase synthesis in the acatalasemic dog reticulocytes was almost the same (approximately 80%) as that in the normal reticulocytes. On the other hand, the synthesized mutant catalase in reticulocytes was rapidly degraded ( t 1/2: 1.8 h) compared with the normal catalase ( t 1/2: 14.0 h) and this degradation was almost completely inhibited by lactacystin (LC). These results suggested that the proteolytic degradation mediated most likely by proteasome might be involved in disposing of the mutant catalase in acatalasemic erythroid cells.

Comparative response to splenectomy in Coombs-positive autoimmune hemolytic anemia with or without associated disease.

We reviewed our experience in 30 patients with direct Coombs-positive (DAT+) autoimmune hemolytic anemia (AHA) who underwent splenectomy. Twelve patients had idiopathic "warm" AHA (group I) and 18 had AHA associated with systemic diseases (group II). Complete response to splenectomy was defined as having normal hemoglobin and reticulocyte count lasting for at least 6 months without subsequent medical therapy. Subnormal but greater than 50% improvement in these parameters with or without medical therapy was considered to be a partial response. Median age was 64 (23-81) in group I and 68 (23-76) in group II. Median follow-up duration was 18 and 10.9 months, respectively. Nine of 11 (82%) evaluable patients with idiopathic AHA and 3 of 16 (19%) patients with associated disease achieved a complete response. Partial response was obtained in 2 (18%) and 6 (37%) patients in groups I and II, respectively. Both complete-response and overall-response rates were statistically different between two groups (P = 0.001 and 0.02). Postoperative courses of group I patients were uneventful except for one who developed a subphrenic abscess. Five patients in group II developed bacterial infections, which were mostly pneumonias. Our findings indicate that splenectomy is an effective treatment approach with low morbidity and mortality in patients with refractory idiopathic AHA. It should, however, be considered cautiously in AHA patients with underlying systemic diseases because of its decreased efficacy and increased surgical morbidity in this subgroup.

Serum transferrin receptor as a marker of erythropoiesis suppression in patients on chronic transfusion.

In the management of patients requiring chronic transfusion, various parameters may be used to evaluate the degree of erythroid marrow suppression. The aim of our study was to assess which of these parameters provide the most useful assessment of erythropoiesis. We studied 27 chronically transfused patients, 19 with sickle cell disease (SS patients) and 8 with thalassemia. Thirty-one nonchronically transfused SS patients and 74 healthy children served as controls. We measured serum transferrin receptor levels, reticulocyte counts, hemoglobin (Hb) concentrations and erythropoietin levels. The serum transferrin receptor levels were very elevated in control SS patients and remained significantly elevated in those on transfusion therapy, but were normal in thalassemia patients, indicating a more complete suppression of erythropoiesis. The reticulocyte counts were elevated in all SS patients, even when on chronic transfusion, but were in the normal range in patients with thalassemia. Erythropoietin levels were elevated in patients with thalassemia and in all the SS patients. Hb levels negatively correlated with serum transferrin receptor and erythropoietin in all SS patients. In the transfused SS patients, a higher HbS level correlated with higher reticulocyte counts, transferrin receptor, and erythropoietin levels. In thalassemia patients, erythropoiesis was more completely suppressed, as reflected both by normal reticulocyte counts and near-normal transferrin receptor levels. Though the reticulocyte counts were not significantly different in the transfused SS patients, the serum transferrin receptor levels were less elevated than in SS patients not on transfusion. The serum transferrin receptor level appears to be the most useful marker of marrow erythropoietic activity in chronically transfused SS patients. We recommend that reticulocyte counts be integrated with periodic measurements of serum transferrin receptor levels.

An In Vitro Model of Human Red Blood Cell Production From Hematopoietic Progenitor Cells

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

Decay-Accelerating Factor (CD55) and Membrane Inhibitor of Reactive Lysis (CD59) Are Released Within Exosomes During In Vitro Maturation of Reticulocytes

Exosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.

Decay-Accelerating Factor (CD55) and Membrane Inhibitor of Reactive Lysis (CD59) Are Released Within Exosomes During In Vitro Maturation of Reticulocytes

AbstractExosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.

Cytometric analysis and maturation characteristics of reticulocytes from myelodysplastic patients.

Myelodysplasia is characterized by a hypoproliferative anaemia with ineffective intramedullary erythropoiesis. We have used the novel technology of the Bayer H3 analyser to characterize reticulocytes (RNA containing red cells) from 32 MDS patients and 10 elderly normal subjects. In comparison with reticulocytes from normal subjects, those from MDS patients were larger with a lower haemoglobin concentration. Reticulocytes from sideroblastic patients had a lower haemoglobin content and concentration than for refractory anaemia patients but no other differences between FAB subtypes were found. H3 reticulocyte RNA content parameters correlated poorly with those derived by the Sysmex R-1000, particularly in the MDS group. On reticulocyte maturation to red cells MDS patients concentrated haemoglobin more than normal subjects and this was most evident in the sideroblastic group. Platelet depletion of whole blood suggested that large platelets in the sideroblastic group may have partly contributed to this observation. Prospective evaluation of changes towards normal reticulocyte cytometric parameters may assist in assessment of early erythroid response to therapy in MDS patients.

Encapsulation of ribonucleic acid in human red blood cells for use as a reticulocyte quality control material for flow cytometric analysis.

The osmotic lysis procedure was employed to encapsulate ribonucleic acid (RNA) in human red blood cells in order to prepare a reticulocyte reference control. The procedure required the hypotonic dialysis of erythrocytes in the presence of RNA and cytosolic components of red blood cells followed by a short hypertonic dialysis to restore isotonicity and reseal the pores formed on the cell membrane during the hypotonic swelling. The procedure was monitored by a dedicated flow cytometer for reticulocyte counting and required 120 min. Approximately 20% of the erythrocytes undergoing the reversible osmotic lysis were encapsulated with various amounts of RNA. The morphology of the RNA-loaded erythrocytes were similar to those of normal erythrocytes and reticulocytes, however, their mean cell volume (MCV) was slightly smaller than normal cells. RNA-loaded erythrocytes prepared by this method were stable for several months as a reference control for identification and enumeration of reticulocytes using flow cytometric as well as manual analysis methods and resulted in a high correlation coefficient between these counting techniques.

Increased Cation Permeability in Mutant Mouse Red Blood Cells With Defective Membrane Skeletons

Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

ANEMIA AND ERYTHROPOIETIN LEVELS IN LUNG TRANSPLANT RECIPIENTS

An evaluation of 26 surviving outpatient lung transplant recipients at one center showed that 65% (17/26) had significant anemia (hemoglobin < 11 g/L for women, < 14 g/dl for men) at a median follow-up of 13.5 months after transplantation (range, 1-41 months). There were 14 men and 12 women with a mean age of 45.1 years (range, 23.1-66.7 years). Fifteen had a double allograft and 11 had a single allograft. Anemia was normochromic and normocytic/macrocytic with a tendency to anisocytosis, with normal reticulocyte counts. Iron deficiency (transferrin saturation < 20%) was found in 35% (6/17) of anemic patients, and two of them also had ferritin levels < 15 micrograms/L. In addition, vitamin B12 was decreased in 1 patient. Folate levels were all normal. Erythropoietin levels were significantly decreased in anemic lung transplant recipients as compared with nontransplanted iron-deficient anemic patients (median, 1 mU/ml, range 1-41 mU/ml, vs. 53 mU/ml, 15-88 mU/ml; P < 0.05). In nonanemic lung transplant recipients, erythropoietin levels were decreased too, as compared with normal controls (median, 2 mU/ml, range 1-21 mU/ml, vs. 5 mU/ml, 3-32 mU/ml; P < 0.05). Investigation of peripheral stem cells in 9 patients showed normal stimulation of erythroids (burst-forming unit, erythroid; median, 573 cells/ml; range, 128-1898 cells/ml) independent of erythropoietin concentrations. Analysis of putative prognostic factors, such as age, surgical procedure (double vs. single lung allograft), indication for transplantation, time after transplantation, infection status, presence of bronchiolitis obliterans, immunosuppression (+/- azathioprine), serum creatinine, creatinine clearance, hypertension, and arterial partial pressure of oxygen, did not demonstrate any difference in erythropoietin concentrations. Only the sex variable revealed a trend to higher levels in women than in men (median, 4 mU/ml, range 1-41 mU/ml, vs. 1 mU/ml, 1-16 mU/ml; P > 0.05). The causes for low erythropoietin levels are not quite understood yet; however, they offer a rationale for the treatment of chronic anemia with recombinant human erythropoietin.

Normal Reticulocyte Count and Subtype HFR in Antenatal Blood

Normal Reticulocyte Count and Subtype HFR in Antenatal Blood

Complete Nucleotide Sequence of the Murine Erythroid β-Spectrin cDNA and Tissue-Specific Expression in Normal and Jaundiced Mice

AbstractSpectrin, a heterodimer of α and β subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid β-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2. chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the α/β subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid β-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.

Complete nucleotide sequence of the murine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice

Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.