Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

From cooperative to uncorrelated clogging in cross-flow microfluidic membranes.

The operational lifetime of filtration membranes is reduced by the clogging of pores and subsequent build-up of a fouling or cake layer. Designing membrane operations in which clogging is delayed or even mitigated completely, requires in-depth insight into its origins. Due to the complexity of the clogging process, simplified model membranes fabricated in microfluidic chips have emerged as a powerful tool to study how clogs emerge and deteriorate membrane efficiency. However, to date, these have focussed solely on dead-end filtration, while cross-flow filtration is of greater practical relevance at the industrial scale. As such, the microscopic mechanisms of clogging in crossflow geometries have remained relatively ill-explored. Here we use a microfluidic filtration model to probe the kinetics and mechanisms of clogging in crossflow. Our study exposes two findings: (i) the primary clogging rate of individual pores depends only on the trans-membrane flux, whose strong effects are explained quantitatively by extending existing models with a term for flux-controlled flow-enhanced barrier crossing, (ii) cross-membrane flow affects the pore-pore communication, leading to a transition from correlated to uncorrelated clogging of the membrane, which we explain qualitatively by deriving a dimensionless number which captures two essential regimes of clogging at the microscale.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

[Glucose-6-phosphate dehydrogenase deficiency: clinical presentation and eliciting factors]. / Glucose-6-fosfaatdehydrogenasedeficiëntie: klinische presentatie en uitlokkende factoren.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a hereditary X-linked disorder, is the most common enzymatic disorder of red blood cells in humans, affecting more than 200 million people worldwide. The prevalence is increasing in the Netherlands due to immigration of people from the Middle East and Africa. We present three different clinical manifestations of G6PD deficiency: neonatal jaundice, haemolysis provoked by infection and haemolysis caused by fava beans. The pathophysiology and treatment are discussed. Furthermore a recent update of chemicals which should be avoided in G6PD deficiency is provided.

[Acute anaemia in a Vietnamese patient with alpha-thalassaemia and a parvovirus infection]. / Acute anemie bij een Vietnamese patiënt met alpha-thalassemie en een parvovirus-infectie.

A 14-year-old girl of Vietnamese descent with an unremarkable medical history presented with haemodynamic shock due to severe anaemia. This was caused by an aplastic crisis resulting from the combined effects of a Parvovirus infection and HbH disease. The HbH disease was a result of compound heterozygosity for the South East Asia (SEA) deletion and the Constant Spring mutation in the genes coding for alpha-globin chains (HbH/Hb Bart's). The girl had multiple blood transfusions and recovered. Family investigation revealed that, in addition to these 2 mutations in the alpha-globin gene, some family members also carried the 3.7-kb deletion of the alpha-globin gene, a mutation in the beta-globin gene resulting in HbE, and a novel mutation of unknown clinical significance in the beta-globin gene. This case demonstrates that essentially asymptomatic carriership of thalassaemia can have serious consequences when coupled with a concurrent infection.

A family with complement factor D deficiency.

A complement factor D deficiency was found in a young woman who had experienced a serious Neisseria meningitidis infection, in a deceased family member with a history of meningitis, and in three relatives without a history of serious infections. The patient and these three relatives showed a normal activity of the classical complement pathway, but a very low activity of the alternative complement pathway and a very low capacity to opsonize Escherichia coli and N. meningitidis (isolated from the patient) for phagocytosis by normal human neutrophils. The alternative pathway-dependent hemolytic activity and the opsonizing capacity of these sera were restored by addition of purified factor D. The family had a high degree of consanguinity, and several other family members exhibited decreased levels of factor D. The gene encoding factor D was found to contain a point mutation that changed the TCG codon for serine 42 into a TAG stop codon. This mutation was found in both alleles of the five completely factor D-deficient family members and in one allele of 21 other members of the same family who had decreased or low-normal factor D levels in their serum. The gene sequence of the signal peptide of human factor D was also identified. Our report is the first, to our knowledge, to document a Factor D gene mutation. The mode of inheritance of factor D deficiency is autosomal recessive, in accordance with the localization of the Factor D gene on chromosome 19. Increased susceptibility for infections in individuals with a partial factor D deficiency is unlikely.

Normal pulse oximeter reading in a cyanotic infant.

A newborn infant who presented with central cyanosis was found to have hereditary methaemoglobinaemia. The pulse oximeter readings and physical findings were incompatible. Clinical assessment remains an important part in the management of such cases.

Seven new mutations in the nicotinamide adenine dinucleotide reduced-cytochrome b(5) reductase gene leading to methemoglobinemia type I.

Cytochrome b(5) reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.

A missense mutation in the heavy subunit of gamma-glutamylcysteine synthetase gene causes hemolytic anemia.

gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine. In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year-old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS- deficient grandson were identified as homozygous for a 473C-->T substitution, changing codon 158 from CCC for proline into CTC for leucine. Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation.

Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

A convenient diagnostic function test of peripheral blood neutrophils in glycogen storage disease type Ib.

Neutrophils from patients suffering from glycogen storage disease type Ib (GSD-Ib) show several defects. one of which is a decreased rate of glucose utilization. In this study, we established experimental conditions to show the stimulation of the neutrophil respiratory burst by extracellular glucose. With phorbol-myristate-acetate as stimulus of the burst, the activity of the NADPH oxidase in GSD-Ib neutrophils hardly increased on addition of glucose. In control and GSD-type Ia neutrophils, a clear increase was observed. The lack of response to extracellular glucose in GSD-Ib neutrophils is correlated with the inability to raise intracellular glucose-6-P levels on glucose addition, thereby limiting the activity of the generation of NADPH in the hexose-monophosphate shunt. Our study shows the usefulness of this test for the diagnosis of neutrophil function abnormality in GSD-Ib patients.

Hb Nijkerk: a new mutation at codons 138/139 of the beta-globin gene inducing severe hemolytic anemia in a Dutch girl.

We describe a new structural mutant of the beta-globin chain in a 17-year-old Dutch Caucasian girl. The mutant is associated with a severe pathology as a consequence of hyper-instability of the hemoglobin tetramer. The proband, whose parents had no history of hemolysis, was admitted to the hospital at 5 months of age with hemolytic anemia and splenomegaly. No indications for autoimmune defects or enzymopathies were found. Repeated hemoglobin electrophoresis on cellulose acetate revealed no abnormalities. At the age of 17 years, a minor abnormal band of less than 1% was detected on starch gel electrophoresis, migrating slightly faster than Hb A2. Sequencing of the beta-globin gene revealed heterozygosity for a 4 bp deletion (GCTA) in combination with a 1 bp insertion (T) at codons 138/139. This event eliminates two amino acids (Ala-Asn) and introduces a new residue (Tyr). We discuss the hematological and the pathophysiological consequences of this mutant, which is fully expressed as a gene product, and apparently assembled into unstable tetramers that precipitate shortly after.

[An opsonin-independent strain of Staphylococcus epidermidis]. / Opsoninnezavisimyi shtamm Staphylococcus epidermidis.

Human neutrophils, subjected to stimulation under different conditions (phorbol myristate acetate, opsonized zymosan, formylmethionyl-leucinephenylalanine, nonopsonized staphylococci), produced a factor (denoted as clumping factor, or CF) with a capacity for highly selective clumping and opsonization of staphylococci. Out of 68 strains of different species of staphylococci, only a single strains (S.epidermidis) was sensitive of CF. CF negative staphylococci were capable of inducing the release of CF by neutrophils, but were not bound by this factor. Extracts, obtained by the mechanical destruction of neutrophils (sonication, repeated freezing and thawing), had no clumping activity. CF had a mol. wt. exceeding 100 kD, was positively charged and disintegrated at 100 degrees C. The capacity of S.epidermidis 178 M for binding CF completely disappeared after the treatment of bacteria with pronase and partially disappeared after boiling and treatment with trypsin and periodate. Neuraminidase and heating at 80 degrees C produced no effect. These data are the first demonstration of highly selective (strain-specific) interaction between secretory products of neutrophils and bacteria.

A Dutch family with Hb Atlanta [beta 75(E19)Leu-->Pro].

We have investigated four members of a three-generation Dutch family for a suspected hemoglobinopathy. Chronic hemolysis and a moderate macrocytic normochromic anemia with slight morphological abnormalities of the red cells was observed in all four. Hemoglobin chain synthesis in vitro and separation of the globin chains by reversed phase high performance liquid chromatography revealed an abnormal beta-globin species in addition to the normal alpha and beta chains. The decreased amount of normal beta-globin and the low amount of unidentified protein suggested an unstable beta-globin variant. An abnormal band was detected by isoelectrofocusing. In one family member tested, the hemoglobin in an erythrocyte lysate had decreased heat stability. All carriers were positive in the isopropanol hemoglobin instability test. Treatment of erythrocytes with methylviolet gave rise to microgranular inclusions. Nucleotide sequencing of the polymerase chain reaction-amplified beta-globin gene revealed a heterozygous single base pair T-->C mutation at codon 75, which changes the normal CTG codon for leucine to a CCG codon for proline. This variant has previously been identified as Hb Atlanta or beta 75(E19)Leu-->Pro. The mutation creates a new Msp I restriction site, which was used to confirm the diagnosis in all four family members. A quantitative reverse transcriptase polymerase chain reaction procedure for determining the relative amounts of mRNA transcripts for the normal and abnormal globin chain showed a comparable stability for both transcripts.

Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst.

Intracellular oxidation of dihydrorhodamine 123 (DHR) to the fluorescent compound rhodamine 123 (Rho123) was used to detect the production of oxygen metabolites in activated neutrophils. Total leukocyte preparations can be used in this assay, which is a great advantage when priming of the respiratory burst is studied. We have defined the conditions that should be taken into account when priming is studied with this assay. We found that neither the extent nor the kinetics of DHR oxidation match those of NADPH oxidase activity. In addition, DHR oxidation is influenced by the absolute and relative number of neutrophils in the leukocyte suspension, by the DHR concentration and by myeloperoxidase availability. The results presented in this study emphasize the need for carefully designed experiments when DHR is used to study the respiratory burst in neutrophils.

[Determination of spectrin in erythrocytes: an important aid in the diagnosis of hereditary spherocytosis]. / Bepaling van spectrine in erytrocyten: een belangrijk hulpmiddel bij de diagnostiek van hereditaire sferocytose.

OBJECTIVE: Assay of spectrin in erythrocytes as a diagnostic test in hereditary spherocytosis (HS). DESIGN: Validation of a diagnostic test. SETTING: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands. METHOD: A radiolabelled rabbit antiserum against human spectrin was used to determine the amount of spectrin in erythrocytes from 64 patients with proven or supposed HS, suffering from inborn, sometimes familial anaemia and a decreased osmotic resistance of the erythrocytes. These amounts of spectrin were compared with those of 12 patients with decreased osmotic resistance suffering from haemolytic anaemia of unknown cause, 16 patients with various other erythrocyte disorders and 30 healthy blood donors. RESULTS: The intradonor and interdonor variations in the amount of spectrin in erythrocytes from healthy blood donors were found to be less than 7%. In 56 of the 64 patients with HS (88%), the erythrocytes contained less than 86% of the normal amount of spectrin. A similar result was found in 4 of the 12 patients suffering from non-characterised haemolytic anaemia (33%). In contrast, a normal amount of spectrin was found in the erythrocytes of patients with other erythrocytic disorders. CONCLUSION: The radio-immunoassay of spectrin in erythrocytes is more specific for the diagnosis of HS than the osmotic fragility test of the erythrocytes. The normal amount of spectrin found in 8 of the 64 patients possibly suffering from HS may be due to a rare molecular origin of HS not leading to a decreased spectrin level or may be related to other causes of anaemia than HS.

Neutrophil Fc gamma RIIIb deficiency, nature, and clinical consequences: a study of 21 individuals from 14 families.

Several individuals have been described whose neutrophils lack the normally abundantly expressed IgG Fc gamma receptor IIIb (Fc gamma RIIIb). We now studied the responsible genomic defect and analyzed the medical history in detail of 21 Fc gamma RIIIb-negative donors identified in 14 unrelated families. We developed a polymerase chain reaction allele-specific-primer annealing assay to genotype for the NA polymorphism of the Fc gamma RIIIB gene. All Fc gamma RIIIb-deficient individuals were negative for both the NA1 and the NA2 allele. In all cases the complete absence of the Fc gamma RIIIB alleles was confirmed using a Southern blot-based restriction fragment length polymorphism assay. Furthermore, an additional deletion of the next more telomeric located Fc gamma RIIC gene was found. Family studies showed that at least one Fc gamma RIIIB allele was absent in both parents in 6 families, whereas in 2 families the father had a normal phenotype. Two individuals suffered from an autoimmune thyroiditis. Four individuals had had multiple episodes of infection, 3 had only incidental infections, and 14 never had any serious infection. Genotyping showed a normal Fc gamma RIIa phenotype distribution among the Fc gamma RIIIb-negative individuals, thus excluding the possibility that the presence of the favorable IgG2-binding low-responder isoform of Fc gamma RIIa (131-H) contributed to the overall absence of recurrent bacterial infections.

[Molecular-genetic studies of carrier stage of glucose-6-phosphate dehydrogenase deficiency]. / Moleculair-genetisch onderzoek naar draagsterschap van glucose-6-fosfaat-dehydrogenase-deficiëntie.

OBJECTIVE: Determining the reliability of a new DNA analysis in the detection of carriers of 6 mutations that cause glucose-6-phosphate dehydrogenase (G6PD) deficiency. DESIGN: Validation of a diagnostic test. SETTING: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands. METHOD: With polymerase chain reactions (PCR) and restriction enzyme analyses, the DNA of 78 proven patients or carriers was compared with the DNA of 51 patients suffering from haemolytic anaemia (possibly due to G6PD deficiency) and of 50 healthy blood donors. RESULTS: In 60 of the 78 patients, 1 or 2 of the 6 mutations were found that lead--according to the literature--to G6PD deficiency. In 2 of the 51 anaemic patients a clinically relevant mutation was found, while such a mutation was revealed in 3 of the 50 blood donors. All 3 had been born in Curaçao or Surinam, areas with a higher incidence of G6PD deficiency than the Netherlands. CONCLUSION: In comparison with G6PD activity tests, which leave 50% of carriers undetected, the described PCR method is a reliable test. Because G6PD activity measurement is independent of mutation analysis, we conclude that a combination of these tests will detect carriers of G6PD deficiency with a higher sensitivity than either of these tests separately.