Deficient Red Blood Cells Research Articles

Detection of paroxysmal nocturnal hemoglobinuria-phenotype in patients with chronic lymphocytic leukemia and multiple myeloma

Paroxysmal nocturnal hemoglobinuria (PNH) results due to decrease or absence of glycosylphosphatidylinositol-anchored (GPI) molecules, such as CD55 and CD59, from the surface of the affected cells. PNH-phenotype has been described in various hematological disorders, mainly aplastic anemia and myelodysplastic syndromes; recently it has been reported in patients with lymphoproliferative syndromes and multiple myeloma (MM). We evaluated the presence of CD55 negative and/or CD59 negative red blood cell (RBC) populations in newly diagnosed treatment naive-54 chronic lymphocytic leukemia (CLL) and 29 MM patients by flow cytometry. PNH-phenotype was not reported in any patient; however, RBC populations deficient in CD55 were detected in 16.66% (9/54) CLL and 6.89% (2/29) MM patients. Clinical presentation or the hematological parameters did not show any relationship with the presence of CD55 deficient RBC population. Our study showed absence of PNH-phenotype in patients with CLL and MM; however, isolated CD55 deficient RBC were identified in both CLL and MM. Larger prospective studies by other centers, including simultaneous analysis of granulocytes for the presence of PNH-phenotype, are needed to corroborate these findings and to work out the mechanisms and the significance of the existence of this phenotype in these patients.

A novel FUT1 allele was identified in a Chinese individual with para‐Bombay phenotype

The para-Bombay phenotype is characterised by H-deficient or H partially deficient red blood cells (RBCs) in individuals who secrete ABH antigens in their saliva. Samples from an individual whose RBCs had an apparent para-Bombay phenotype and his family members were investigated and a novel FUT1 allele was identified. RBCs' phenotype was characterised by standard serologic technique. Genomic DNA was sequenced with primers that amplified the coding sequence of FUT1 and FUT2, respectively. Routine ABO genotyping analysis was performed. Haplotypes of FUT1 were identified by TOPO cloning sequencing. Recombination expression vectors of FUT1 mutation alleles were constructed and transfected into COS-7 cells. The pα-(1,2)-fucosyltransferase activity of expression protein was determined. B101/O02 genotype of the proband was correlated with ABH substances in saliva. The proband carried a new FUT1 allele which showed 35C/T, 235G/C and 682A/G heterozygote by directly DNA sequencing. Two haplotypes, 235C and 35T+682G, were identified by TOPO cloning sequencing and COS-7 cells transfected with five recombination vectors including wild-type, 35T, 235C, 682G and 35T+682G alleles were established respectively. The α-(1,2)-fucosyltransferase activities of cell lysates which had transfected with 35T, 235C, 682G and 35T+682G recombination vectors showed 79·45, 16·23, 80·32 and 24·59%, respectively, compared with that of the wild-type FUT1-transfected cell lysates. A novel FUT1 allele 235C was identified, which greatly diminished the activity of α-(1,2)-fucosyltransferase.

Nutrition and depression: the role of folate.

A relationship between folate and neuropsychiatric disorders has been inferred from clinical observation and from the enhanced understanding of the role of folate in critical brain metabolic pathways. Depressive symptoms are the most common neuropsychiatric manifestation of folate deficiency. Conversely, borderline low or deficient serum or red blood cell folate levels have been detected in 15-38% of adults diagnosed with depressive disorders. Recently, low folate levels have been linked to poorer antidepressant response to selective serotonin reuptake inhibitors. Factors contributing to low serum folate levels among depressed patients as well as the circumstances under which folate and its derivatives may have a role in antidepressant pharmacotherapy must be further clarified.

AE1 and carbonic anhydrase II: are they colocalized and do they exhibit direct functional interaction in cell membranes?

Previous studies have reported that intracellular carbonic anhydrase (CA) II binds to the C‐terminal domain of AE1 and it has been suggested that this interaction directly increases the transport rate of AE1. The goal of our study was a) to detect the intracellular distribution and possible colocalization of CAII and AE 1 in living cells and b) to measure bicarbonate transport rate of AE1 in the absence and presence of CAII in HEK 293 cells und human red blood cells. We expressed fusion proteins of AE1 and yellow fluorescent protein and of CAII and cyan fluorescent protein in HEK 293 cells and observed the distribution of CAII and AE1 with a confocal laser scanning microscope. AE1 was exclusively associated with the plasma membrane, while CAII was homogeneously distributed in the cytoplasm. There was no apparent enrichment of CAII in the plasma membrane. In mass spectrometric measurements of bicarbonate permeability (PHCO3−) we observed a marked increase of PHCO3− over controls upon expression of AE1. Coexpression of AE1 and CAII did not lead to a further increase in PHCO3−. In another study, we measured the bicarbonate permeability of normal and CAII‐ deficient human red blood cells and found no difference in PHCO3−. Both results suggest that there is no direct functional interaction between AE1 and CAII in HEK 293 and human red blood cells.

Suivi biologique des patients atteints d’hémoglobinurie paroxystique nocturne et traités par eculizumab

Summary Paroxysmal nocturnal haemoglobinuria is a rare but serious disease. It is treated today with eculizumab, a humanized monoclonal antibody, that blocks the complement activation of C5. Six patients were treated, with blood collection at each eculizumab infusion; intravascular haemolysis remained with a sharp drop in the haptoglobin level in all patients and a fairly high level of total biliburin in at least 50% of patients despite a significant decrease in the lactate dehydrogenase level. In two patients, the direct antiglobulin test was positive for the complement only (C3d) after a treatment period of two weeks and eight months without associated blood transfusion. The percentage of deficient red blood cells (CD59 negative) increases either rapidly or slowly or may remain steady as seen through flow cytometry. In the six patients of our study, the size of deficient granulocytes and monocytes clones remained unchanged.

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Impaired red cell deformability in iron deficient subjects

Iron deficiency is a systemic disorder, which affects a variety of different cell types and is one of the most frequent diseases throughout the world. The influence of iron deficiency upon erythrocyte deformability is controversial and could be a consequence of membrane peroxidation damage or cross linking of membrane proteins. The aim of this study was to determine the overall elasticity (the deformability of the entire cell is evaluated) of iron deficient red blood cells (RBC) using laser optical tweezers. In this study, the laser trapped the cell and the elasticity was then analyzed measuring cell deformation at six different drag velocities. Twenty-five RBCs from 11 healthy blood donors (controls) and 7 patients with iron deficiency anemia were analyzed. Iron deficiency subjects were classified into 3 groups based on Hb concentration for statistical analysis (group I: Hb = 7.0-7.9; group II: 8.0-10.2 and group III: 7.0-10.2 g/dl). The results showed an increased rigidity in the iron deficiency of deficient red blood cells when compared to normal control blood cells, and, this impaired deformability seems to be correlated to the hemoglobin concentration. In conclusion, the results obtained by optical tweezers showed that iron deficiency affects the elasticity of whole RBC.

Direct Antiglobulin Test, Ferritin and GPI-Negative Cell Populations in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) during Eculizumab Therapy: Extravascular Hemolysis Unmasked by Efficient Eculizumab-Mediated Inhibition of Intravascular Hemolysis.

Introduction: Eculizumab is a humanized monoclonal antibody inhibiting the cleavage of complement factor C5 and preventing terminal complement activation. Therapy with eculizumab leads to a highly significant reduction of complement-mediated intravascular hemolysis and related morbidities including thromboembolic events in Paroxysmal Nocturnal Hemoglobinuria (PNH). It is a new targeted, disease-modifying treatment of PNH. In a placebo-controlled, double-blind randomized study of eculizumab more than 50% of patients (pts) achieved transfusion independence with the remaining pts showing a 44% reduction in the number of red blood cell (RBC) units transfused. In this study we analyzed the proportion of cells with deficiency of GPI-anchored proteins, i.e. PNH cell populations, during eculizumab therapy. Furthermore, we examined a potential mechanism to explain residual hemolysis in some pts despite complete blockade of the terminal complement cascade.Methods: We performed flow cytometric analysis of GPI-anchored proteins, measured levels of hemolysis (LDH), direct bilirubin, hemoglobin and ferritin, and carried out a direct antiglobulin test in samples from 15 (7 female, 8 male) PNH pts during eculizumab therapy. To identify PNH cells, flow cytometric analyses of CD58/CD59 on reticulocytes and erythrocytes, CD66b/CD24/CD16 on granulocytes as well as CD14/CD48 on monocytes were performed. For comparison, the direct antiglobulin test was also performed in 22 (11 female, 11 male) PNH pts without eculizumab therapy.Results: Flow cytometric results of the 15 PNH pts before eculizumab (baseline) and at the time of latest follow-up during eculizumab were compared: GPI-deficient erythrocyte population doubled (29.3% +/−19.3% vs. 56.9% +/−24.0%). The proportion of GPI-deficient reticulocytes (79.1% +/−6.8% vs. 84.6% +/−8.7%), granulocytes (87.5% +/−9.7% vs. 85.5% +/−12.4%) and monocytes (79.0% +/−20.6% versus 73.6%+/−16.2%) did not change significantly. We observed a mean decrease in LDH from 2802 U/l +/− 1015 U/l to 240 U/l +/− 62 U/l; while total bilirubin was stable (47.2 +/− 33.3 μmol/l versus 45.0 +/− 27.8 μmol/l), direct bilirubin showed a threefold increase (3.2 +/− 3.2 μmol/l versus 9.6 +/− 2.4 μmol/l). An increase of ferritin levels was also observed (270 +/− 609 ng/ml versus 651 +/− 652 ng/ml). We performed a direct antiglobulin test in the control group of 22 pts not receiving eculizumab therapy and in 12 pts that received eculizumab. 10 of the 12 pts were tested before and during eculizumab therapy. Only three of all 34 pt who had not received eculizumab at time of antiglobulin test showed a positive antiglobulin test with polyspecific antiglobulin serum, two of them with monospecific anti-IgG and one of them was positive with monospecific anti-C3d. In contrast, all but one of the 12 pts during eculizumab therapy reacted positive with polyspecific antiglobulin serum and monospecific anti-IgG and anti-C3d; in addition, one pt was positive with anti-C3c. The only patient with a negative antiglobulin test during eculizumab therapy was treated for PNH-related thromboembolic events without having transfusion dependent hemolytic anemia. Two of the 12 pts had anti-erythrocytic allo-antibodies (anti-D, -C, -E, -Lua, and -Kpa) before initiation of eculizumab therapy and one of them had additional warm auto-antibodies.Conclusions: 1) A positive direct antiglobulin test (due to C3d loading of PNH RBC) seems to be a consistent finding during eculizumab therapy, which has therefore to be taken into account as a new differential diagnosis of a positive direct antiglobulin test. 2) Eculizumab does not block the early stages of complement activation and therefore C3 cleavage products may occur and bind to CD55/CD59-deficient RBC. The inhibition of terminal membrane attack complex formation by eculizumab allows the survival of these cells which otherwise would undergo a rapid intravascular hemolysis due to C5b-9 formation. 3) The endogenous population of GPI–anchored protein deficient RBC increases during eculizumab therapy. 4) Our data are preliminary and need to be confirmed in larger patient groups: For this purpose, we recommend the monitoring of signs of extravascular hemolysis and ferritin during eculizumab therapy.

Collection of mobilized peripheral blood stem cells from a donor with severe iron deficient anemia

We report a 45-year-old woman with iron deficient anemia (IDA) who underwent a collection of allogeneic peripheral blood stem cells (PBSCs) induced by granulocyte-colony stimulating factor (G-CSF) after a rapid improvement of IDA by iron replacement. Her peripheral red blood cells (RBCs) after iron therapy were composed of two different-sized subpopulations; one consisted of microcytes, which were iron deficient RBCs, and another of normocytes, which were produced after iron replacement. On the first day of PBSC collection, the interface setting was maintained aiming at 2% hematocrit as usual; however, PBSCs could not be collected adequately. Sedimentation of iron deficient, lighter RBCs under centrifugation within a blood cell separator could be similar to that of mononuclear cells, and the lighter RBCs could contaminate the mononuclear cell layer, resulting in the collection of the lighter layers of mononuclear cells than desired. On the second day, we succeeded in obtaining enough PBSCs by collecting heavier layers than those collected on the first day by using a 4% hematocrit and monitoring white blood cell counts of the collection line serially. It should be noted that the lighter RBCs from a donor with a history of IDA could complicate collection of PBSCs.

Isolated Granulocytopenia in Patients with Unclassifiable MDS May Be Related to a Mutation of PIG-A Gene Leading to GPI-APs Deficiency Expressed Predominantly on Granulocytes.

An acquired somatic mutation of PIG-A gene leads to a deficiency in glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) in affected cells. A typical clinical manifestation of the GPI-APs deficiency is paroxysmal nocturnal hemoglobinuria (PNH) with intravascular hemolysis, various degree of cytopenia reflecting marrow failure and trombophilia. Besides so-called „classic“ PNH, clinical and laboratory features of PNH may also be present in some patients with aplastic anemia (AA) or myelodysplasia (MDS). Moreover, a small subset of AA or MDS patients may exhibit in peripheral blood small populations of GPI-APs deficient red blood cells (RBC) and granulocytes, not accompanied by clinical and laboratory signs of hemolysis. This subset of patients with a small PNH clone may respond to immunosuppressive therapy. Herein, we report on three patients who were referred to our hospital with the diagnosis of unclassified MDS (RA unclassifiable according to the WHO classification). In all patients, bone marrow aspiration and biopsy revealed normocellular marrow with only minimal to mild dysplasia with no excess of blasts and no karyotype abnormalities. The main laboratory feature was moderate to severe granulocytopenia (absolute neutrophil count: 0.23x109/l, 1.39x109/l and 0.81x109/l, respectively). All patients had normal Hb level, RBC and platelet counts and none of them exhibited any clinical and laboratory signs of hemolysis (reticulocyte count, serum bilirubin, LDH and haptoglobin were within normal limits). Flow cytometric analysis of peripheral blood revealed a significant number of GPI-AP-deficient granulocytes in all patients, but no CD59 or CD55 deficient RBC except for a small PNH RBC clone in patient Nr.3. In patient Nr.1 (50 years old male) who exhibited 30–35% of GPI-AP-deficient granulocytes, sequencing analysis of PIG-A gene discovered transition C/T in exon 3, causing a missense substitution at codon 248 (P248L). Patient Nr.2 was a 28 years female with 20% of GPI-AP-deficient granulocytes, with sequentially identified transition G/A in exon 6 of PIG-A gene resulting in replacement of glutamic acid by lysine at codon 482 (E482K). In patient Nr.3 (34 years old male), flow cytometric analysis revealed 40–45% of PNH granulocytes and 8–12% of CD59 and CD55 deficient RBC. In this patient, sequencing analysis of PIG-A gene revealed transversion A/T in exon 5, leading to a non-sense mutation at codon 367, forming a preterminal STOP codon. This mutation resulted in the loss of 117 aminoacids from the C-terminus of the protein. Our results suggest that unexplained profound granulocytopenia may be in some patients related to mutation of PIG-A gene leading to GPI-APs deficiency expressed predominantly on granulocytes. MDS patients with isolated granulocytopenia who are mostly diagnosed as unclassifiable RA according to the WHO criteria should be screened by sensitive flow cytometry methods for presence of GPI-APs deficient granulocytes. Evidence of a PNH clone may have a prognostic value and may be helpful for decision of an optimal treatment strategy, including immunosuppression.

Molecular basis for para‐Bombay phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele

The para-Bombay phenotype is characterized by H-deficient or H-partially deficient red blood cells (RBCs) in persons who secrete ABH antigens in their saliva. The studies that determined the genotypes for two Chinese individuals with the para-Bombay phenotype are described. RBC phenotypes were characterized by conventional serologic methods. Exons 6 and 7 of the ABO gene were amplified, as well as the entire coding region for FUT1 and FUT2, with four independence polymerase chain reactions (PCRs) from genomic DNA. PCR products were excised, purified from agarose gels, and sequenced directly. Mutations of FUT1 were identified by TOPO cloning sequencing. For both individuals, RBC ABO genotypes correlated with ABH substances in their saliva. One individual (a patient) had two heterozygous mutations of FUT1 by direct DNA sequencing, namely, a C-->T heterozygous mutation at position 293(C293T) and AG heterozygous deletion (CAGAGAG-->CAGAG) at position 547 to 552. These two mutations were confirmed to be compound heterozygotes; that is, each mutation was determined to be on a separate homologous chromosome by TOPO cloning sequencing. The FUT2 genotype was Se(357)Se(357). The other individual (a blood donor) had an AG deletion at position 547 to 552 homozygous allele in FUT1. The FUT2 genotype was Se(357)Se(357,385). C293T mutation can cause Thr/Met at amino acid position 98. AG deletion at position 547 to 552 caused a reading frameshift and a premature stop codon. A novel nonfunctional FUT1 allele C293T was identified in a person with the para-Bombay phenotype. This rare H-deficient phenotype may result from different nonfunctional alleles.

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.

Pathogenesis and Therapy of a New Model of SOD2 Deficiency-Induced Sideroblastic Anemia.

Manganese superoxide dismutase (SOD2) is a major antioxidant protein which protects cells against the toxicity of superoxide anion, a by-product of mitochondrial respiration. While knockout of SOD2 results in late embryonic or early neonatal death, SOD2 deficient hematopoeitic stem cells (HSC) are capable of rescuing lethally irradiated normal hosts. The resulting hematopoeitic chimeric mice show a persistent hemolytic anemia similar to human sideroblastic anemia. Loss of SOD2 in erythroid progenitor cells results in mitochondrial proliferation and excess iron deposition within mitochondria of reticulocytes. Mature red blood cell (RBC) show abundant iron inclusions with evidence of a defect in iron incorporation into heme and altered survival. SOD2 deficient cells show both enhanced reactive oxygen species production and protein oxidative damage. We recently published a comparative RBC proteome analysis. Our results show that the expression of several proteins involved in folding/chaperone function, redox regulation, ATP synthesis and RBC metabolism is altered in SOD2 deficient cells. To define early events in the pathogenesis, we are currently performing a gene expression array strategy. The overall goal of this procedure is to characterize differential gene expression at the level of TER-119+ CD71+ erythroid precursor cells from the bone marrow of SOD2 deficient HSC mice versus wild-type and antioxidant-treated SOD2 deficient recipients. Preliminary analysis generated a list of 718 genes as significantly discriminating between wild-type and SOD2 deficient RBC progenitors. Taken together, our results and subsequent validation of candidate genes in in vivo experiments will identify genes involved in response of erythroid progenitors to oxidative stress as well as determinants of RBC lifespan in other hemolytic disorders.

A band 3–based macrocomplex of integral and peripheral proteins in the RBC membrane

AbstractWe have studied the membrane proteins of band 3 anion exchanger (AE1)–deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3(−/−) RBCs differed from the human band 3–deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3–deficient RBCs. Proteins of the glycophorin C complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO2/O2 gas exchange unit (metabolon) in the erythrocyte.

Human thiopurine methyltransferase activity varies with red blood cell age.

Inherited differences in thiopurine methyltransferase (TPMT) activity are an important factor in the wide interindividual variations observed in the clinical response to thiopurine chemotherapy. The aim of this study was to establish a population range for red blood cell (RBC) TPMT activity in children with acute lymphoblastic leukaemia (ALL) at disease diagnosis. An additional aim was to investigate factors that can influence TPMT activity within the RBC. Blood samples were collected from children with ALL at disease diagnosis, prior to any blood transfusions, as part of the nationwide UK MRC ALL97 therapeutic trial. RBC TPMT activity was measured by h.p.l.c. RBCs were age-fractionated on Percoll density gradients. Pretreatment blood samples were received from 570 children within 3 days of venepuncture. TPMT activities at disease diagnosis ranged from 1.6 to 23.6 units/ml RBCs (median 7.9) compared with 0.654-18.8 units (median 12.9), in 111 healthy control children (median difference 4.5 units, 95% CI 3.9, 5.1 units, P < 0.001). A TPMT quality control sample, aliquots of which were assayed in 60 analytical runs over a 12 month period, contained a median of 11.98 units with a CV of 11.6%. Seven children had their RBCs age-fractionated on density gradients. TPMT activities in the top gradient (young cells) ranged from 4.2 to 14.1 units (median 7.5) and in the bottom gradient (old cells) 1.5-12.6 units (median 4.7 units), median difference 2.3 units, 95% CI 0.7, 4.1, P = 0.035. Circulating RBCs do not constitute a homogeneous population. They have a life span of around 120 days and during that time undergo a progressive ageing process. The anaemia of ALL is due to deficient RBC production. The results of this study indicate that RBC TPMT activities are significantly lower in children with ALL at disease diagnosis. This may be due, at least in part, to a relative excess of older RBCs.

Reduced oxidative-stress response in red blood cells from p45NFE2-deficient mice

Abstractp45NF-E2 is a member of the cap ‘n' collar (CNC)-basic leucine zipper family of transcriptional activators that is expressed at high levels in various types of blood cells. Mice deficient in p45NF-E2 that were generated by gene targeting have high mortality from bleeding resulting from severe thrombocytopenia. Survivingp45nf-e2−/− adults have mild anemia characterized by hypochromic red blood cells (RBCs), reticulocytosis, and splenomegaly. Erythroid abnormalities inp45nf-e2−/− animals were previously attributed to stress erythropoiesis caused by chronic bleeding and, possibly, ineffective erythropoiesis. Previous studies suggested that CNC factors might play essential roles in regulating expression of genes that protect cells against oxidative stress. In this study, we found that p45NF-E2–deficient RBCs have increased levels of reactive oxygen species and an increased susceptibility to oxidative-stress–induced damage. Deformability of p45NF-E2–deficient RBCs was markedly reduced with oxidative stress, and mutant cells had a reduced life span. One possible reason for increased sensitivity to oxidative stress is that catalase levels were reduced in mutant RBCs. These findings suggest a role for p45NF-E2 in the oxidative-stress response in RBCs and indicate that p45NF-E2 deficiency contributes to the anemia inp45nf-e2−/− mice.

Inhibition of p. falciparum invasion in PK- and PGI deficient red blood cells by inducing band 3 protein degradation

Inhibition of p. falciparum invasion in PK- and PGI deficient red blood cells by inducing band 3 protein degradation

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5′ nucleotidase deficiency: 25 years later

In 1972, Valentine et al described, under the name of ‘non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency’, an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden–Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of ‘haemolytic disorders associated with increased ATP’. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5′ nucleotidase (P5N). In conclusion, all these cases were formerly referred to as ‘high ATP syndromes’ because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

Correction of the PNH Defect by GPI-Anchored Protein Transfer

Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.

Correction of the PNH Defect by GPI-Anchored Protein Transfer

AbstractHemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.