The role of rib infarcts in the acute chest syndrome of sickle cell diseases.

The acute chest syndrome is a generic term for pulmonary complications of sickle cell diseases with heterogeneous etiologies that include pneumonia, vaso-occlusion of pulmonary arterioles, rib infarction, and fat embolism syndrome. My review summarizes these etiologies, the evidence, and pathophysiology supporting the hypothesis that infarction of segments of ribs by the same vaso-occlusive process responsible for the acute episodes of pain (characteristic of the sickle cell diseases) is often involved in the acute chest structure. Inflammation associated with the infarct then causes splinting, hypoventilation, and hypoxia and further vaso-occlusion. The relationship with adult respiratory distress syndrome and fat embolism is also discussed. Use of the incentive spirometer combined with effective analgesia when chest pain is present is advocated for prevention of the pulmonary infiltrates. Newer understanding of the role of nitric oxide in regulating oxygen transport and its relationship to blood transfusions used in therapy of the acute chest syndrome are discussed.

The survival characteristics of dense sickle cells.

Sickle red blood cells (RBCs) become depleted of potassium, leading to dehydration and abnormally elevated cellular density. The increased sickling that results is important for both hemolysis and vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells that were denser than normal to produce a high-density-enriched (HDE) population of sickle cells. Autologous HDE cells from 3 subjects were labeled with biotin and re-infused. The following determinations were performed: (1) the survival and density changes of HDE cells; (2) the amount of fetal hemoglobin (HbF) in labeled cells after magnetic isolation; (3) the percentage of labeled F cells; (4) the percentage of labeled cells displaying external phosphatidylserine (PS). For patients with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation half-time was 40, 80, and 180 hours, respectively. The percentage of HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion, indicating that HDE F cells have longer in vivo survival than HDE non-F cells. The percentage of PS(+), biotin-labeled HDE cells showed no consistent increase or decrease with time after re-infusion. These data provide evidence that HDE sickle cells, especially those that do not contain HbF, have a very short in vivo survival, and that the percentage of PS(+) cells in a re-infused HDE population does not change in a consistent manner as these cells age in the circulation.

Time-dependent changes in the density and hemoglobin F content of biotin-labeled sickle cells.

Sickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium.

The K+ efflux that mediates sickle-cell dehydration may occur through several pathways, including two with a high capacity for mediating rapid K+ loss, KCl cotransport and the Ca(2+)-dependent K+ channel [K(Ca2+)]. The rate and pathway of red blood cell (RBC) dehydration most likely depends on cell age and hemoglobin (Hb) composition, with the presence of HbF playing an important role. Oxygenated sickle RBCs have relatively stable cell volume during incubation in vitro, whereas deoxygenated cells become dehydrated, and therefore more dense, due to activation of one or more K+ efflux pathways. In this investigation, sickle RBCs were deoxygenated either continuously or in 15-minute cycles for 4 hours, and the density increases of very young, transferrin receptor-positive (TfR+) cells and the remaining TfR- cells were determined. The contribution of KCl cotransport was estimated by replacing Cl- with NO3-. K(Ca2+) was inhibited by removal of Ca2+ or addition of charybdotoxin (ChTX). For both continuous and cyclic deoxygenation, TfR+ cells had a greater density increase when compared with TfR- cells. The lower percentage of HbF found in the TfR+ population may contribute to this difference. With continuous deoxygenation, the density shift was decreased by inhibition of K(Ca2+), but not by inhibition of KCl cotransport. With cyclic deoxygenation, the density shift was decreased in an independent, additive manner by inhibition of both pathways. Thus, cyclic deoxygenation of sickle cells under these conditions appears to activate both K(Ca2+) and the KCl cotransporter.

Incentive spirometry to prevent acute pulmonary complications in sickle cell diseases.

BACKGROUND: This study was designed to determine the incidence of thoracic bone infarction in patients with sickle cell diseases who were hospitalized with acute chest or back pain above the diaphragm and to test the hypothesis that incentive spirometry can decrease the incidence of atelectasis and pulmonary infiltrates. METHODS: We conducted a prospective, randomized trial in 29 patients between 8 and 21 years of age with sickle cell diseases who had 38 episodes of acute chest or back pain above the diaphragm and were hospitalized. Each episode of pain was considered to be an independent event. At each hospitalization, patients with normal or unchanged chest radiographs on admission were randomly assigned to treatment with spirometry or to a control nonspirometry group. Each patient in the spirometry group took 10 maximal inspirations using an incentive spirometer every two hours between 8 a.m. and 10 p.m. and while awake during the night until the chest pain subsided. A second radiograph was obtained three or more days after admission, or sooner if clinically necessary, to determine the incidence of pulmonary complications. Bone scanning was performed no sooner than two days after hospital admission to determine the incidence of thoracic bone infarction. RESULTS: The incidence of thoracic bone infarction was 39.5 percent (15 of 38 hospitalizations). Pulmonary complications (atelectasis or infiltrates) developed during only 1 of 19 hospitalizations of patients assigned to the spirometry group, as compared with 8 of 19 hospitalizations of patients in the nonspirometry group (P = 0.019). Among patients with thoracic bone infarction, no pulmonary complications developed in those assigned to the spirometry group during a total of seven hospitalizations, whereas they developed during five of eight hospitalizations in the nonspirometry group (P = 0.025). CONCLUSIONS: Thoracic bone infarction is common in patients with sickle cell diseases who are hospitalized with acute chest pain. Incentive spirometry can prevent the pulmonary complications (atelectasis and infiltrates) associated with the acute chest syndrome in patients with sickle cell diseases who are hospitalized with chest or back pain above the diaphragm.

Hemoglobin F-Cincinnati, alpha 2G gamma 2 41(C7) Phe-->Ser in a newborn with cyanosis.

A term infant presented with mild cyanosis without evidence of hypoxia. Cardiopulmonary disease, polycythemia, and methemoglobinemia were excluded. Standard hemoglobin electrophoresis, including isoelectric focusing, were normal. However, by reverse-phase C4 HPLC, an abnormal globin chain was detected. Analysis of tryptic peptides and amino acid sequence showed that the patient had an amino acid substitution Phe-->Ser at residue 41(C7) in the G gamma chain. This was confirmed by DNA sequencing that demonstrated a point mutation at the expected site in exon 2 of the G gamma gene, accounting for the appropriate change in the codon. This substitution, hemoglobin F-Cincinnati, alpha 2 gamma 2 41(C7) Phe-->Ser, not previously described, presumably decreased oxygen affinity of the hemoglobin. This substitution is very near the heme group and the alpha 1 beta 2 interface and, hence, in a crucial area of the globin chain. Abnormalities of gamma globin chains tend to be overlooked due to their transient presence and trivial clinical symptomatology, or due to "in utero" selection when physiologically abnormal. Mutant hemoglobins with altered oxygen affinity should be included in the differential diagnosis of newborns presenting with cyanosis, in whom all common causes have been excluded.

X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley.

In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations. Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway. To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced. A single transversion (A to C) was identified in exon 5. The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species. No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 5'-flanking and 350 nucleotides of 3'-flanking sequence. The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family. Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters. After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal. The cofactor, pyridoxal 5'-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.

Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes.

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Fat embolism syndrome associated with asthma and sickle cell-beta(+)-thalassemia.

A 25-year-old African-American man with sickle cell-beta(+)-thalassemia presented with acute asthma of 2 days' duration. The asthma was well controlled by 48 hr, and parenteral medications were changed to oral bronchodilators and steroids. Sixty hours after presentation, he developed pain similar to that of sickle cell vaso-occlusion, for which he received small amounts of analgesics. He died approximately 84 hours after presentation. Autopsy showed extensive marrow necrosis and massive fat embolism. This is the first reported case of fat embolism syndrome associated with this genotype, the mildest of the clinically significant sickle cell hemoglobinopathies. The relationship between these etiologic factors and indications for transfusions are discussed.

Deoxygenation-induced changes in sickle cell-sickle cell adhesion.

The tendency for sickle cells to adhere to each other is increased in oxygenated sickle blood in parallel with cell density. The increased adherence of these cells occurred despite their reduced deformability and diminished ability to form rouleaux. Using a method developed in our laboratory, we measured the yield stress: a sensitive index of cell-cell adhesion of deoxygenated suspensions of sickle cells. Deoxygenation of whole sickle blood to 30 to 50 mm Hg caused a significant increase in yield stress of all sickle blood samples. Deoxygenation caused a significant increase in yield stress of both dense and light sickle cells. Deoxygenation-induced increases in yield stress occurred at higher oxygen tensions for dense (> 55 mm Hg) than for light sickle cells (< 45 mm Hg). The increase in yield stress on deoxygenation was correlated with hemoglobin polymerization as assessed morphologically by sickling or by changes in relative viscosity. Thus, deoxygenation-induced cell sticking must involve small areas of strong membrane adhesion because the changes in yield stress occurred despite a reduction in rouleaux formation and surface area of membrane contact. Sickle trait red blood cells also exhibited increased yield stress on deoxygenation but only under hypertonic conditions where sickling occurred. Thus, deoxygenation-induced cell adhesion did not require prior membrane damage because it occurred in sickle trait cells. No change in yield stress was seen when deoxygenated sickle cells were suspended in buffer, but the addition of physiologic amounts of fibrinogen to buffer restored the deoxygenation-induced increase in cell adhesion. We speculate that the increase in sticking among sickle cells on deoxygenation results from spicule formation and may involve interaction of fibrinogen and possibly other plasma proteins with the cell membrane.

Simultaneous occurrence of rib infarction and pulmonary infiltrates in sickle cell disease patients with acute chest syndrome.

In order to determine if a relationship exists between rib infarction and the acute chest syndrome (ACS) in sickle cell disease patients, bone scans were reviewed in 55 episodes in 38 patients with pain of suspected osseous origin. A bone scan was positive for thoracic bone infarction if abnormally increased or decreased uptake was present in ribs, sternum or thoracic spine. Radiographs were considered to be positive for ACS if there was pulmonary infiltrate or pleural effusion in the absence of laboratory or clinical evidence of bacterial pneumonia. ACS by chest x-ray was present in 22 episodes, 21 of which showed evidence of infarction of the bony thorax on bone scan. Thoracic bone infarction occurred in the absence of chest x-ray changes in only 11 episodes. This association between bone infarction and radiographic ACS was statistically significant (p < 0.001, Fisher's exact test). A strong association exists between ACS and infarction of the bony thorax. It is possible that bone infarction leads to pain, hypoventilation and the clinical picture of ACS.

Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

Influence of plasma and red cell factors on the rheologic properties of oxygenated sickle blood during clinical steady state.

Yield stress is a sensitive index of blood fluidity at low shear stress. Using a method that measured the stress required to cause motion of a thin sedimenting layer of red cells, we found significant elevations of yield stress in patients with homozygous sickle cell anemia during clinical steady state. Mixing studies of sickle cells in normal plasma and buffered saline and of normal red cells in sickle plasma showed (1) that the increased yield stress of sickle blood was not due to differences between sickle and normal plasma factors and (2) that yield stress of sickle cells was not increased in the absence of plasma proteins. Multivariate regression analysis was performed to determine the dependence of sickle blood yield stress on several red cell and plasma factors. The yield stress measurements were normalized for differences in plasma fibrinogen concentration. Other factors studied included cell density, fetal hemoglobin concentration, alpha globin genotype, cell deformability as measured by high shear viscosity, and fibronectin and von Willebrand factor concentrations. Cell density was the primary determinant of yield stress. Measurements of yield stress on density fractionated sickle cells confirmed that the increased yield stress of sickle blood was due to the dense sickle erythrocyte. We conclude that the increased yield stress of sickle blood during clinical steady state was due to an abnormal interaction between the dense sickle cell membrane and plasma protein(s).

Rib infarcts and acute chest syndrome in sickle cell diseases.

In the absence of evidence for pneumonia or pulmonary embolus, primary pulmonary infarction has been assumed to be the cause of the syndrome of chest pain, fever, and pulmonary infiltrate on chest X-ray that commonly complicates sickle cell anaemia. To find out whether the syndrome might be due to rib infarction, 99mTc-diphosphonate bone scans were done. In the eleven episodes thus investigated (10 patients) the scans showed segmental areas of increased radionuclide uptake in ribs, indicative of bone infarction. A possible sequence of events is that the rib infarcts are primary and cause bone pain, followed by soft tissue reaction, pleuritis, and splinting. The resultant hypoventilation leads to atelectasis and subsequent development of the radiographic changes of the acute chest syndrome. Prevention of hypoventilation and treatment of bone pain are important therapeutic goals.

Hb Port Huron [alpha 56 (E5)Lys----ARG]: a new alpha chain variant.

We have determined the structural abnormality of a putative Hb E detected in an African-American family with no apparent Asian ancestry. The tryptic peptide map performed by high performance liquid chromatography showed that the electrophoretic variant was indeed Hb E [beta 26 (B8)Glu----Lys]. In addition, the tryptic map showed an abnormal peptide adjacent to the alpha T-6 peptide. The amino acid analysis and confirmatory restriction analysis of the DNA showed that a second mutation was also present, characterized by a substitution of arginine for lysine at residue 56 of the alpha chain. The variant is clinically silent and has been named Hb Port Huron for the city in Michigan where the family resides.

Evaluation of the yield stress of normal blood as a function of fibrinogen concentration and hematocrit.

The yield stress is a sensitive index of blood fluidity at low shear. Seven healthy adults were studied at hematocrits varying between 40 and 80% and fibrinogen concentrations from 0.0 to 0.935 g/dl. Multivariable analysis was used to determine the functional dependence of yield stress on hematocrit and fibrinogen level. The major findings from this analysis include a decreasing effect of fibrinogen at high concentrations (saturation effect), a relative insensitivity of yield stress to fibrinogen at low concentration (threshold effect), and a strong interaction between the effects of hematocrit and fibrinogen concentration on yield stress. Our results give the normal range of yield stress for a given value of fibrinogen and hematocrit and can be used to predict the effect of reductions in hematocrit or fibrinogen on the yield stress of normal blood.

The relationship between fetal hemoglobin and disease severity in children with sickle cell anemia.

A study was conducted in a sample of 140 children with sickle cell anemia to evaluate the relationship between hematological variables (%HbF, %HbA2, %Hb, and mean cell volume) and disease severity. A patient's severity status was determined by whether he/she was hospitalized, had a transfusion, and/or had a pain crisis at 2 evaluation periods; the first was based on a patient's history taken at the initial assessment visit to the Wayne State Comprehensive Sickle Cell Center, and the second was based on a 1-3 year follow-up at the center. Fetal hemoglobin was a strong predictor of a patient's hospitalization and transfusion status. A decrease in %HbF of 4.76% (one SD of %HbF) was associated with a 3.58 fold (95% confidence interval, 1.18-7.28) greater odds of being hospitalized both prior to initial assessment and on follow-up, compared to not being hospitalized at either evaluation. Similarly, a decrease in %HbF of 4.76% was associated with a 5.56 fold (95% confidence interval, 1.67-18.96) greater odds of having a transfusion both prior to initial assessment and on follow-up compared to not having a transfusion at either evaluation. Patients who were both hospitalized and transfused at initial assessment and on follow-up (n = 12) had a mean %HbF of 7.59%, while patients who were not hospitalized or transfused at either evaluation (n = 19) had a mean %HbF of 13.61%. Fetal hemoglobin was not a significant predictor of pain crises in this sample of patients. None of the other hematological variables were significant predictors of disease severity in this study. The strong relationship between %HbF and disease severity identified in this study suggests that a single %HbF measurement may be useful in predicting important aspects of the clinical course of children with sickle cell anemia.

Painful crisis and dense echinocytes: effects of hydration and vasodilators.

We have conducted a series of studies using discontinuous arabinogalactan density gradient ultracentrifugation of erythrocytes from the peripheral blood of: patients with sickle cell anemia (SCA), in and out of pain crisis; hydrated SCA, hemoglobin SC, and normal individuals all of whom were pain-free; and patients with SCA given short courses of oral vasodilator compounds. Our results indicate that in pain crisis patients develop an echinocytic change that is most prominent in the denser layers (specific gravity greater than 1.128 g/ml) of the discontinuous gradient and effects both irreversibly sickled cells (ISC) and non-ISC. This echinocytic change, we have previously shown, correlates with a decrease in erythrocyte reduced glutathione, a major intraerythrocyte anti-oxidant compound. We could find no consistent change in the percentage of dense cells in pain crisis versus out of crisis. However, out of crisis, hydration led to a marked increase in the percentage of dense erythrocytes in sickle cell anemia and in a HbSC patient, compared to the same individual out of crisis and on ad lib fluids. There was no consistent change in echinocytic forms. Because hydration may be expected to have produced an increase in intravascular volume and vasodilation, we determined whether short courses of three different vasodilators would increase the dense fraction in patients with SCA who were pain-free. There was no change in percentage of dense erythrocytes or in the percentage of echinocytes. We concluded that painful crisis occurs in association with an echinocytic change that may be induced by oxidant injury and that in the pain-free state, hydration, but not short courses of vasodilator drugs, increased the percentage of dense erythrocytes but not the degree of echinocytosis they displayed. The differential effect of hydration, with respect to painful crisis, may indicate that these dense cells are bound to vascular endothelium or trapped in blood vessels at the time of crisis but mobilized by hydration in the out-of-crisis state.