Deformation measurement of individual cells in large populations using a single-cell microchamber array chip.

We analyze the deformability of individual red blood cells (RBCs) using SiCMA technology. Our approach is adequate to quickly measure large numbers of individual cells in heterogeneous populations. Individual cells are trapped in a large-scale array of micro-wells, and dielectrophoretic (DEP) force is applied to deform the cells. The simple structures of micro-wells and DEP electrodes facilitate the analysis of thousands of RBCs in parallel. This unique method allows the correlation of red cell deformation with cell surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous populations.

Hemoglobin Hakkari: an autosomal dominant form of beta thalassemia with inclusion bodies arising from de novo mutation in exon 2 of beta globin gene.

Certain beta globin gene mutations produce a thalassemia major phenotype in the heterozygous state. While most such patients have thalassemia intermedia, we describe a young Guatemalan child with a de novo mutation in the beta globin gene, codon 31 T --> G (Hemoglobin Hakkari), who developed severe anemia at the age of 10 months and remains transfusion-dependent. The substitution of B13 leucine with arginine in the beta globin results in alteration of a critical heme contact point resulting in an extremely unstable variant hemoglobin and a clinical picture that is characterized by ineffective erythropoiesis and numerous intracytoplasmic inclusions within the erythrocyte precursors of the bone marrow. .

Modelling and rapid simulation of multiple red blood cell light scattering.

The goal of this work is to develop a computational framework to rapidly simulate the light scattering response of multiple red blood cells. Because the wavelength of visible light (3.8 x 10(-7) m < or = lambda < or = 7.2 x 10(-7) m) is approximately an order of magnitude smaller than the diameter of a typical red blood cell scatterer (d approximately 8 x 10(-6) m), geometric ray-tracing theory is applicable, and can be used to quickly ascertain the amount of optical energy, characterized by the Poynting vector, that is reflected and absorbed by multiple red blood cells. The overall objective is to provide a straightforward approach that can be easily implemented by researchers in the field, using standard desktop computers. Three-dimensional examples are given to illustrate the approach and the results compare quite closely to experiments on blood samples conducted at the Children's Hospital Oakland Research Institute (CHORI).

The role of phosphatidylserine in recognition and removal of erythrocytes.

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.

The role of secretory phospholipase A2 in acute chest syndrome.

Acute chest syndrome (ACS) is the leading cause of death in sickle cell disease. Severe ACS often develops in the course of a vasoocclusive crisis (VOC), and frequently involves pulmonary fat embolism. Secretory phospholipase A2 (sPLA2), a potent inflammatory mediator, is elevated in ACS, and sPLA2 levels in serum or plasma predict impending ACS. In addition sPLA2 may play a major role in the actual damage to the lung resulting in a new pulmonary infiltrate on chest radiography, respiratory symptoms, and ultimately alveolar collapse and the impairment of gas exchange. The data indicate that measurement of sPLA2 can be useful in alerting the clinician to patients with impending ACS, and suggest that instituting early therapies based on sPLA2 levels, including inhibition of sPLA2 activity, may be useful to prevent or reduce the clinical morbidity of ACS in sickle cell disease.

Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia.

Several transgenic murine models for sickle cell anemia have been developed that closely reproduce the biochemical and physiological disorders in the human disease. A comprehensive characterization is described of hematologic parameters of mature red blood cells, reticulocytes, and red cell precursors in the bone marrow and spleen of a murine sickle cell model in which erythroid cells expressed exclusively human alpha, gamma, and betaS globin. Red cell survival was dramatically decreased in these anemic animals, partially compensated by considerable enhancement in erythropoietic activity. As in humans, these murine sickle cells contain a subpopulation of phosphatidylserine-exposing cells that may play a role in their premature removal. Continuous in vivo generation of this phosphatidylserine-exposing subset may have a significant impact on the pathophysiology of sickle cell disease.

Characterization of the phosphatidylserine-exposing subpopulation of sickle cells.

Phosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor-expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na(+) and low K(+) content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca(++) levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca(++). (Blood. 2001;98:860-867)

Secretory phospholipase A2 activity correlates with postinjury multiple organ failure.

UNLABELLED: Postinjury multiple organ failure (MOF) may result from overwhelming systemic hyperinflammation. Secretory phospholipase A2 (sPLA2) produces many inflammatory lipid mediators, and levels have been correlated with both the severity of patient injury and postinjury mortality. The objective of this study was to characterize the plasma activity of sPLA2 type IIa in severely injured patients and to determine whether the activity of this enzyme correlates with the subsequent development of MOF. PATIENTS: Seventeen severely injured patients at known risk for MOF had blood sampled on postinjury days 0, 1, 2, 3, and 5. DESIGN: sPLA2 activity was sequentially measured and correlated with MOF scores. RESULTS: Six patients (35%) developed MOF. In comparison with non-MOF patients, MOF patients had elevated sPLA2 activity beginning 36 hrs postinjury (MOF sPLA2, 2.4 +/- 0.97, vs. non-MOF sPLA2, 0.86 +/- 0.16 active units (AU); p < .05) and continuing over the ensuing 5 days. To rule out the possibility that stored blood components required for patient resuscitation was the source of sPLA2, the sPLA2 was measured in packed red blood cells, platelet concentrates, and fresh frozen plasma over the routine storage time. None of the products tested had elevated levels of sPLA2 compared with fresh plasma from healthy adult volunteers. CONCLUSIONS: We conclude that increased sPLA2 activity is associated with the development of postinjury MOF.

Absence of mitochondrial superoxide dismutase results in a murine hemolytic anemia responsive to therapy with a catalytic antioxidant.

Manganese superoxide dismutase 2 (SOD2) is a critical component of the mitochondrial pathway for detoxification of O2(-), and targeted disruption of this locus leads to embryonic or neonatal lethality in mice. To follow the effects of SOD2 deficiency in cells over a longer time course, we created hematopoietic chimeras in which all blood cells are derived from fetal liver stem cells of Sod2 knockout, heterozygous, or wild-type littermates. Stem cells of each genotype efficiently rescued hematopoiesis and allowed long-term survival of lethally irradiated host animals. Peripheral blood analysis of leukocyte populations revealed no differences in reconstitution kinetics of T cells, B cells, or myeloid cells when comparing Sod2(+/+), Sod2(-/-), and Sod2(+/-) fetal liver recipients. However, animals receiving Sod2(-/-) cells were persistently anemic, with findings suggestive of a hemolytic process. Loss of SOD2 in erythroid progenitor cells results in enhanced protein oxidative damage, altered membrane deformation, and reduced survival of red cells. Treatment of anemic animals with Euk-8, a catalytic antioxidant with both SOD and catalase activities, significantly corrected this oxidative stress-induced condition. Such therapy may prove useful in treatment of human disorders such as sideroblastic anemia, which SOD2 deficiency most closely resembles.

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

p45NF-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. Surviving p45nf-e2(-/-) adults have mild anemia characterized by hypochromic red blood cells (RBCs), reticulocytosis, and splenomegaly. Erythroid abnormalities in p45nf-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 in p45nf-e2(-/-) mice. (Blood. 2001;97:2151-2158)

Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease.

To determine the effects of L-arginine (L-Arg) supplementation on nitric oxide metabolite (NOx) production, oral L-Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso-occlusive crisis (VOC). L-Arg (0.1 g/kg) increased NOx formation by 18.8 +/- 68% in normal controls, whereas steady-state SCD patients demonstrated a paradoxical decrease in NOx of -16.7 +/- 4% (P = 0.004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77.7 +/- 103%, a response that was dose dependent. L-Arg appears to be the rate-limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

Patterns of arginine and nitric oxide in patients with sickle cell disease with vaso-occlusive crisis and acute chest syndrome.

PURPOSE: Our objective was to evaluate L-arginine and nitric oxide metabolite (NOx) levels in children with sickle cell disease (SCD) at steady-state and during vaso-occlusive crisis (VOC). Because alterations in nitric oxide production may have an important role in the pathophysiology of SCD, our second aim was to determine if a relationship exists between these levels and vaso-occlusive crisis (VOC). PATIENTS AND METHODS: Plasma L-arginine and serum NOx levels were examined in 36 patients with SCD with 39 episodes of VOC and 10 children with SCD at steady-state. Daily levels were obtained in children requiring hospitalization. RESULTS: Steady-state L-arginine levels were normal in children with SCD. L-arginine levels were low, however, in children with VOC (37.4 +/- 2.7 vs. 53.6 +/- 4.6 micromol/L; P = 0.008) but returned to baseline during hospitalization. In contrast, NOx levels were normal at presentation but decreased during hospitalization for both patients with VOC and patients with acute chest syndrome (ACS) (21.1 +/- 2.0, 17.4 +/- 2.4, and 12.3 +/- 1.6 micromol/L, respectively; P < 0.05). In the patients with VOC who had ACS develop, L-arginine decreased to the lowest levels at the time of the ACS diagnosis, correlating with decreasing NOx levels. CONCLUSION: These data suggest that there may be a relationship between the L-arginine-nitric oxide pathway and vaso-occlusion in SCD. Low arginine levels during VOC could reflect a state of acute substrate depletion that results in a decrease in nitric oxide production.

Alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent.

The development of hemolytic alloantibodies and erythrocyte autoantibodies complicates transfusion therapy in thalassemia patients. The frequency, causes, and prevention of this phenomena among 64 transfused thalassemia patients (75% Asian) were evaluated. The effect of red blood cell (RBC) phenotypic differences between donors (mostly white) and Asian recipients on the frequency of alloimmunization was determined. Additional transfusion and patient immune factors were examined. 14 (22%) of 64 patients (75% Asian) became alloimmunized. A mismatched RBC phenotype between the white population, comprising the majority of the donor pool, and that of the Asian recipients, was found for K, c, S, and Fyb antigens, which accounts for 38% of the alloantibodies among Asian patients. Patients who had a splenectomy had a higher rate of alloimmunization than patients who did not have a splenectomy (36% vs 12.8%; P =.06). Erythrocyte autoantibodies, as determined by a positive Coombs test, developed in 25% or 16 of the 64 patients, thereby causing severe hemolytic anemia in 3 of 16 patients. Of these 16, 11 antibodies were typed immunoglobulin G [IgG], and 5 were typed IgM. Autoimmunization was associated with alloimmunization and with the absence of spleen (44% and 56%, respectively). Transfused RBCs had abnormal deformability profiles, more prominent in the patients without a spleen, which possibly stimulated antibody production. Transfusion of phenotypically matched blood for the Rh and Kell (leukodepleted in 92%) systems compared to blood phenotypically matched for the standard ABO-D system (leukodepleted in 60%) proved to be effective in preventing alloimmunization (2.8% vs 33%; P =.0005). Alloimmunization and autoimmunization are common, serious complications in Asian thalassemia patients, who are affected by donor-recipient RBC antigen mismatch and immunological factors.

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2).

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.

Secretory phospholipase A(2) predicts impending acute chest syndrome in sickle cell disease.

Acute chest syndrome (ACS) is the leading cause of death in sickle cell disease. Severe ACS often develops in the course of a vaso-occlusive crisis (VOC), but currently there are no predictors for its development. Secretory phospholipase A(2) (sPLA(2)), a potent inflammatory mediator, is elevated in ACS, and previous work suggests that sPLA(2) predicts impending ACS. We prospectively evaluated sPLA(2) concentration during 21 admissions for VOC; 6 of these patients went on to develop ACS. Elevation of sPLA(2) was detected all 6 patients 24 to 48 hours before ACS was clinically diagnosed. Adding the requirement for fever raised the specificity of sPLA(2) to 87% while retaining 100% sensitivity. These data indicate that sPLA(2) can be useful in alerting the clinician to patients with impending ACS. In addition, sPLA(2) may be useful for instituting early therapies to prevent or reduce the clinical morbidity of ACS.

Adherence of phosphatidylserine-exposing erythrocytes to endothelial matrix thrombospondin.

Phospholipid asymmetry is well maintained in erythrocyte (RBC) membranes with phosphatidylserine (PS) exclusively present in the inner leaflet. The appearance of PS on the surface of the cell can have major physiologic consequences, including increased cell-cell interactions. Because increased adherence of PS-exposing RBCs to endothelial cells (ECs) may be pathologically important in hemoglobinopathies such as sickle cell disease and thalassemia, we studied the role of PS exposure in calcium ionophore-treated normal RBC adherence to human umbilical vein endothelial cell (HUVEC) monolayers. When HUVEC monolayers were incubated with these PS-exposing RBCs, the ECs retracted and the RBCs adhered primarily in the gaps opened between the ECs. A linear correlation was found between the number of PS-exposing RBCs in the population and the number of adhering RBCs to the monolayer. Pretreatment of RBCs with annexin V significantly decreased adherence by shielding PS on the RBCs. Similarly, PS-containing lipid vesicles decreased RBC binding by competing for the PS binding sites in the monolayer. PS-exposing RBCs and PS-containing lipid vesicles adhered to immobilized thrombospondin (TSP) and matrix TSP, respectively, and adherence of PS-exposing RBCs to EC monolayers was reduced by antibodies to TSP and to its EC receptor, alpha(v)beta(3). Together, these results indicate a role for PS and matrix TSP in the adherence of PS-exposing RBCs to EC monolayers, and suggest an important contribution of PS-exposing RBCs in pathologies with reported vascular damage, such as sickle cell anemia. (Blood. 2000;95:1293-1300)

Identification and molecular characterization of acyl-CoA synthetase in human erythrocytes and erythroid precursors.

Full-length cDNA species encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukaemic cell line were cloned, sequenced and expressed. The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase with high similarity to rat brain ACS2 (91% identical). The second form (66% identical with LACS5) was 97% identical with human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kb mRNA transcript in human haemopoietic stem cells from cord blood, bone marrow, reticulocytes and fetal blood cells derived from fetal liver. An additional 6.3 kb transcript is also found in these erythrocyte precursors; 2.9 and 9.6 kb transcripts of LACS5 are found in human brain, but transcripts are virtually absent from human heart, kidney, liver, lung, pancreas, spleen and skeletal muscle. The 78 kDa expressed LACS5 protein used the long-chain fatty acids palmitic acid, oleic acid and arachidonic acid as substrates. Antibodies directed against LACS5 cross-reacted with erythrocyte membranes. We conclude that early erythrocyte precursors express at least two different forms of acyl-CoA synthetase and that LACS5 is present in mature erythrocyte plasma membranes.

Hereditary spherocytosis and elliptocytosis erythrocytes show a normal transbilayer phospholipid distribution.

Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment with N-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells.

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid.

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase.

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.