Acute pancreatitis during sickle cell vaso-occlusive painful crisis

Sickle cell disease is characterized by chronic hemolytic anemia and vaso-occlusive painful crisis. The vascular occlusion in sickle cell disease is a complex process and accounts for the majority of the clinical manifestations of the disease. Abdominal pain is an important component of vaso-occlusive painful crisis and may mimic diseases such as acute appendicitis and cholecystitis. Acute pancreatitis is rarely included as a cause of abdominal pain in patients with sickle cell disease. When it occurs it may result form biliary obstruction, but in other instances it might be a consequence of microvessel occlusion causing ischemia. In this series we describe four cases of acute pancreatitis in patients with sickle cell disease apparently due to microvascular occlusion and ischemic injury to the pancreas. All patients responded to conservative management. Acute pancreatitis should be considered in the differential diagnosis of abdominal pain in patients with sickle cell disease.     

Nucleosomes and neutrophil activation in sickle cell disease painful crisis

Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ⁰-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ^+-thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.     

Normal sublingual microcirculation during painful crisis in sickle cell disease

Obstruction of the microcirculation is the most important cause of painful crisis in sickle cell disease (SCD). Extensive microvascular obstruction has been observed in mouse models of SCD. A technique to determine the extent of the microcirculatory obstructions in humans may be helpful in the clinical setting and for research purposes. Therefore, we measured sublingual microcirculation longitudinally in patients with SCD admitted with painful crisis.Sublingual microcirculation was recorded with side-stream darkfield (SDF) imaging and semi-quantified with a microvascular flow index (MFI) on a range from 0 to 4 (arbitrary units; from 0 (no flow) to 4 (hyperdynamic flow)).Thirteen consecutive adult sickle cell patients admitted with painful crises were included and provided 47 measurements of MFI in 14 episodes of painful crisis. Seven patients provided baseline measurements and seven healthy controls were studied. The mean (± standard error of the mean) MFI during painful crisis was 2.6 ± 0.1 and did not change during the painful crisis. The mean MFI of patients with SCD during steady state (2.7 ± 0.1) and the mean MFI of the controls (2.7 ± 0.1) were not different from the mean MFI during painful crisis. During painful crisis irregular microvascular perfusion, expressed by the distribution width of the microvascular blood flow velocity, correlated negatively (r = − 0.484; P = 0.002) with hemoglobin concentration.We conclude that sublingual microcirculatory blood flow velocity is not disturbed in sickle cell patients during painful crisis.     

Red blood cell changes during the evolution of the sickle cell painful crisis.

A longitudinal study of the red blood cell (RBC) deformability, percent of dense erythrocytes, and hematologic parameters has been conducted during 117 painful crises affecting 36 patients with sickle cell anemia between January, 1985 and December, 1990. RBC deformability was determined by osmotic gradient ektacytometry and the percentage of dense cells was quantitated by centrifugation on a discontinuous Stractan density gradient. The data indicate that the painful crisis is a process that follows a bimodal form of evolution. The first phase of the painful crisis is characterized by increase in the severity of pain, increase in the number of dense cells, and a decrease in RBC deformability. In some patients the changes in dense cells and RBC deformability are evident 1 to 3 days before the onset of pain. In addition, the hemoglobin level decreases and the reticulocyte count increases during this initial phase. The second phase of the crisis is characterized by reduction in pain intensity, decrease in the number of dense cells, and increase in RBC deformability to values higher than those seen in the steady state. Moreover, the improvement in RBC deformability and the decrease in the number of dense cells at the end of a crisis seem to constitute new risk factors that may incite a recurrence of the crisis within 1 month in about 50% of painful episodes. The pathophysiologic events responsible for this bimodal behavior of RBCs during painful episodes may represent the appearance of factors that induce (1) preferential trapping of deformable cells in the microcirculation during the first phase of the crisis, followed by a decrease of dense cells and the appearance of new deformable RBCs released from the bone marrow during the second phase of the crisis; or (2) variable sickling of all circulating RBCs during the first phase followed by disappearance of dense RBCs and their replenishment by deformable cells during the second phase.     

Non relationship of climatologic factors and painful sickle cell anemia crisis

Conflicting results have arisen from studies concerning the correlation (if any) between climatological changes and the frequency of painful episodes in the sickle cell population. During a 13 month period records of 71 patients with hemoglobin genotypes SS or SC were reviewed. Data analysis failed to reveal an association between the frequency of painful sickle cell crisis and a number of weather and environmental variables. We were unable to demonstrate relationships between the climatologic factors of temperature, humidity, carbon monoxide level and precipitation in the frequency of 362 pain crises in 71 sickle cell patients during a 13 month period.     

The painful crisis of homozygous sickle cell disease: clinical features

The details of onset, perceived precipitating factors, associated symptoms, and pain distribution in the painful crisis of homozygous sickle cell (SS) disease have been prospectively recorded in 183 painful crises in 118 patients admitted to a day-care centre in Kingston, Jamaica. Painful crises developed most frequently between 3 p.m. and midnight, most commonly affected patients aged 15-29 years, affected the sexes equally, and were not obviously influenced by menstrual cycle. Of the perceived precipitating factors, skin cooling occurred in 34%, emotional stress in 10%, physical exertion in 7%, and pregnancy in 5% of women of child-bearing age. Cold as a precipitant was not less common in patients with more subcutaneous fat. Pain affected the lumbar spine in 49%, abdomen in 32%, femoral shaft in 30%, and knees in 21%. There was a highly significant excess of bilateral involvement in limb and rib pain. Recurrent painful crises occurred in 40 patients but showed no evidence of involving similar sites on successive occasions. Abdominal painful crises were associated with abdominal distention in 18 (31%) and with referred rib pain in a further 15 (26%) of crises. Fever was common even in apparently uncomplicated painful crises, suggesting that fever is characteristic of the painful crisis itself and not necessarily indicative of infection. Following investigation and treatment in a day-care centre, over 90% of patients returned home.     

Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis

We developed a microfluidics-based model to quantify cell-level processes modulating the pathophysiology of sickle cell disease (SCD). This in vitro model enabled quantitative investigations of the kinetics of cell sickling, unsickling, and cell rheology. We created short-term and long-term hypoxic conditions to simulate normal and retarded transit scenarios in microvasculature. Using blood samples from 25 SCD patients with sickle hemoglobin (HbS) levels varying from 64 to 90.1%, we investigated how cell biophysical alterations during blood flow correlated with hematological parameters, HbS level, and hydroxyurea (HU) therapy. From these measurements, we identified two severe cases of SCD that were also independently validated as severe from a genotype-based disease severity classification. These results point to the potential of this method as a diagnostic indicator of disease severity. In addition, we investigated the role of cell density in the kinetics of cell sickling. We observed an effect of HU therapy mainly in relatively dense cell populations, and that the sickled fraction increased with cell density. These results lend support to the possibility that the microfluidic platform developed here offers a unique and quantitative approach to assess the kinetic, rheological, and hematological factors involved in vasoocclusive events associated with SCD and to develop alternative diagnostic tools for disease severity to supplement other methods. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in SCD.Sickle cell disease (SCD) is characterized by acute and chronic vasoocclusion that can cause pain (1), acute chest syndrome (2), organ damage (3), stroke, and even death (4, 5). The pathogenic basis of “painful crisis” arising from vasoocclusion in SCD is extremely complex (6–8). It is triggered by many factors, including poor deformability of red blood cells (RBCs), adhesion among multiple cell types and blood components (e.g., sickle RBCs, endothelial cells, adherent leukocytes, and possibly platelets), as well as the local microenvironment (e.g., low oxygen concentration and acidosis). Under conditions of low oxygen partial pressure (pO₂), sickle RBCs experience intracellular sickle hemoglobin (HbS) polymerization, thereby reducing cell deformability (9). Such reductions in deformability can severely impact blood flow in narrow vessels, ultimately causing a transient or persistent blockage (10). Competition between the delay time for HbS polymerization and the RBC transit time in microcirculation is likely a key determinant of disease severity (11). Both in vitro (12) and ex vivo (13) models reveal that HbS polymerization and its effect on cellular rigidity play important roles in causing vascular obstruction. For example, HbS polymerization alone could be sufficient to cause complete RBC blockage in vasculature (12). Increases in microvascular transit time, arising from higher rigidity, of sickle RBCs cause peripheral vascular resistance to blood flow (13).The search for better means to predict painful vasoocclusion crises has focused on a range of hematological and rheological abnormalities. Significant correlations have been shown between pain rates and early death in patients with sickle cell anemia (14) and between early death and several risk factors such as fetal hemoglobin (HbF), hematocrit, and white cell count (15). However, factors such as patient age, sex, HbF (16), intracellular HbS polymerization (17), or fraction of dense RBCs (18) do not appear to show a sufficiently direct correlation with the frequency and/or severity of pain crises. Although HbF level is generally considered important, its direct connection to disease severity is not fully established (19, 20). Some possible links between the incidence of painful crises and steady-state cell hydration (21) and/or deformability at isotonic osmolarity have been identified (22). Such connections, however, do not account for the observation that cell deformability and the proportion of dense cells vary longitudinally in the same patient during crisis (23). Changes have also been reported in the biorheological characteristics of sickle RBC suspension following deoxygenation (DeOxy) in an in vitro vascular model (24).An in vitro model with a well-defined vascular structure and a well-controlled hypoxia condition would serve as an ideal tool to investigate many complex pathophysiological processes in vasoocclusion. Recent advances in microfluidics technology have enabled us to design unique in vitro capabilities with biophysically appropriate microenvironments that mimic the geometric features of vascular systems, thereby facilitating quantitative characterization of DeOxy blood flow (12, 24), detection of HbS polymerization in DeOxy liquid drops (25), and investigation of pathologic adhesion in blood rheology (26). Several methods have been developed to mimic oxygen depletion whereby HbS polymerization and subsequent cell sickling can be triggered; they include long-term gas perfusion at low pO₂ level (13, 27), DeOxy medium exchange (25, 28), reducing agents (29–31), and laser photodissociation of carbon monoxide (22, 32). Along with complex in vivo models that reflect the dynamic response of cells, an in vitro model would have the potential to predict the conditions that would lead to vasoocclusion and to improve the assessment of disease severity by quantifying the individual parameters that modulate vasoocclusion.We designed a microfluidic platform (Fig. 1) that mimics the rheology of microcirculation in vivo. It also characterizes the isolated effects of cell morphologic sickling, unsickling, and altered cell rheology. With this design, we explored in a systematic and controlled manner possible correlations of these effects with hematological parameters (e.g., %HbS), cell density, and hydroxyurea (HU) therapy.Open in a separate windowFig. 1.Microfluidic platform for investigation of biophysical alterations in sickle RBCs under transient hypoxia conditions. (A) Schematic of microfluidic device with O₂ control for studying kinetics of cell sickling and unsickling. (B) Identification of cell sickling events from a microscopic image (arrows indicate sickled RBCs). (C) Schematic of microfluidic device with capillary-inspired structures for single-cell rheology study. Note: Schematics are not drawn to scale, and the dimensions are in microns.Cell sickling was measured using a double-layer device with a cell channel (5 µm high), a gas channel (100 µm high), and an in-between polydimethylsiloxane (PDMS) film 150 µm in thickness (Fig. 1A). The O₂ concentration was controlled by exchanging gas flow in the channel through the PDMS membrane, which is gas-permeable (33). Although it is known (34) that the morphology of sickled cells depends on the DeOxy rate, we observed heterogeneity in cell morphology at the same DeOxy rate. Sickle RBCs typically form spiky edges and dark coarse texture due to intracellular HbS polymerization, the visual identification of which was enhanced by a band-pass filter (Fig. 1B and Movie S1). We thus define sickled cells as those obviously distorted from their original shape and/or texture under the Oxy state [O₂ concentration ∼20% (vol/vol)] to the DeOxy state (O₂ concentration <5%). This visual determination of cell sickling was further confirmed with an independent single-cell rheology test, where similar trends were observed in cell sickling and single-cell capillary obstruction (Results). The kinetics of cell sickling was quantified by two parameters: the sickled fraction (the fraction of all RBCs in the sample that are sickled) and the delay time of cell sickling (the time elapsed between the initiation of DeOxy and the point when a cell shows optically visible features of morphologic sickling). The delay time of cell unsickling was defined as the time elapsed between the initiation of reoxygenation (ReOxy) and the point when the RBC fully recovered its presickle morphology in a visibly identifiable manner.Individual-cell rheology was measured using a microfluidic channel that consisted of periodic obstacles forming microgates 4 µm wide, 5 µm deep, and 15 µm long (Fig. 1C). The channel dimensions were chosen to mimic the size of the smallest capillaries in the human body (4–10 µm in diameter) (35). Cell velocity was measured as the average velocity of individual RBCs flowing through periodic gates under a constant differential pressure. The obstruction fraction was determined as the ratio of obstructed RBCs to all RBCs entering into the microgate arrays during the DeOxy period.     

Oral analgesia for treatment of painful crisis in sickle cell anemia

A therapeutic plan that emphasized oral narcotic analgesia was instituted for the treatment of painful crisis of sickle cell anemia. Of the 100 adult sickle cell syndrome patients registered at North Central Bronx Hospital, 15 were identified as using the emergency department facilities three or more times per year. This "frequent user" patient population was tracked in their hospital and drug usage patterns during the first full year of the oral protocol and compared to their own patterns during the year prior to the protocol. The patients used the ED at the same rate but their frequency of admissions to the hospital dropped by 75%. The oral program produced a significant fall in the amount of narcotics dispensed in the ED (P less than .01).     

Oxygen-induced marrow red cell hypoplasia leading to transfusion in sickle painful crisis

The benefit of oxygen (O2) therapy in non-hypoxic sickle cell patients in painful crisis is uncertain. We report a case of a non-hypoxic sickle cell patient in painful crisis who developed marrow red cell hypoplasia requiring transfusion support after O2 therapy. The uncertain benefits of O2 use in such cases must be weighed against the serious and underrecognized risks of transfusion. In patients who develop O2-induced marrow red cell hypoplasia, cessation of O2 therapy may reverse the anemia and obviate the need for transfusion.     

The painful crisis of homozygous sickle cell disease. A study of the risk factors

Some epidemiologic features of the painful crisis in homozygous sickle cell disease were examined in a retrospective study of 995 painful crises. Previously reported associations with cold weather and pregnancy were confirmed. There was a striking increase in painful crises in male patients between the ages of 15 and 25 years, whereas female patients showed little age-related change. The frequency of painful crises correlated positively with hemoglobin levels and reticulocyte counts in both sexes and negatively with mean corpuscular volume in female patients. There was a striking increase in painful crises in male patients with hemoglobin levels above 8.5 g/dL (greater than 85 g/L). High hemoglobin levels appear to be an important risk factor for painful crises.     

Erythrocyte sedimentation rate during steady state and painful crisis in sickle cell anemia

To define its diagnostic utility in sickle crisis, the erythrocyte sedimentation rates of oxygenated blood were studied in patients with sickle cell anemia and healthy normal subjects using the Guest-Westergren method. A normal range for sedimentation rate as a function of hematocrit was established in 22 normal subjects. Twenty-seven asymptomatic patients with sickle cell anemia had abnormally low sedimentation rates in relation to their hematocrits. Those low sedimentation rates were not increased by substituting plasma from healthy control subjects, which suggests that the low sedimentation rate was a cell-related phenomenon. Sedimentation rates measured in 28 patients with sickle cell anemia at the end of uncomplicated painful crisis increased to levels appropriate for their degree of anemia. In patients with sickle crisis and medical complications, the sedimentation rates were even higher. At the end of an uncomplicated painful crisis, the mean plasma fibrinogen level was significantly higher than at the onset (p less than 0.005). When red cells from patients with sickle cell anemia at the end of crisis were suspended in normal plasma from control subjects, the sedimentation rates remained high. It is concluded that the erythrocyte sedimentation rate of asymptomatic patients with sickle cell anemia is abnormally low due to cellular factors, and the increase during painful crisis is due primarily to red cell changes, modified by plasma factors.     

The incidence of painful crisis in homozygous sickle cell disease: correlation with red cell deformability

To determine whether the vasoocclusive severity of homozygous sickle cell (SS) disease is influenced by cellular dehydration, we correlated the incidence of painful crisis with steady-state measurements of red cell hydration. Sixteen children with SS disease were followed for 3.3 to 8 years (mean, 6.8 years), and a single crisis rate was calculated for each patient. At the time of well visits, cellular hydration was assessed by measuring cell deformability, the percentage of red cells with a density greater than or equal to 1.1056 g/mL, and the percentage of irreversibly sickled cells (ISC). The incidence of painful crisis showed a strong positive correlation with Omax, a deformability measurement reflecting cellular hydration (r = .84, P less than .002), and with hemoglobin concentration (r = .59, P = .04). That is, higher crisis rates were observed in patients with less dehydrated, more deformable red cells and also in patients with higher hemoglobin concentrations. Furthermore, cell deformability and hemoglobin concentration were independent predictors of the incidence of painful crisis, which is consistent with separate effects of these two red cells parameters on vasoocclusive severity.     

The percentage of dense red cells does not predict incidence of sickle cell painful crisis

To test the hypothesis that the tendency of hemoglobin S (HbS) to polymerize within cells is the major determinant of the incidence of vaso-occlusive episodes, we have examined the effect of the percentage of dense cells (as measured by Percoll-Stractan continuous density gradient centrifugation) on the frequency of painful crises in a group of 36 patients with sickle cell disease. No correlation was found between the percentage of dense cells and admissions for crisis. Among the patients with known alpha-gene status (n = 25), the strong correlation between decreased dense cells and alpha-thalassemia (- alpha/alpha alpha) reported previously was confirmed (P less than .001). In addition, in this small subset, patients with alpha- thalassemia (-alpha/alpha alpha) appeared to have a marginally increased number of admissions for sickle cell crisis (t = 2.1910, P less than .05), which was independent of the percentage of dense cells. We conclude that the percentage of dense sickle cells cannot predict the incidence of painful crisis, suggesting that other factors (microcirculatory regulation or other humoral and cellular factors) are more important in the generation maintenance of painful crises than the necessary, but not sufficient, tendency of HbS-containing red cells to sickle.     

Hemoglobin oxygen saturation discrepancy using various methods in patients with sickle cell vaso-occlusive painful crisis

OBJECTIVE: To evaluate agreement among various methods for measuring oxyhemoglobin (O2Hb) saturation in adult hypoxic patients with sickle cell disease (SCD) during painful vaso-occlusive crisis and to compare those results with a control group. PATIENTS AND METHODS: The hemoglobin oxygen saturation was determined simultaneously by pulse oximetry (SpO2), co-oximetry [SO2 (functional oxyhemoglobin saturation) and FO2Hb (oxyhemoglobin fraction)] and by calculation (SaO2) using a normal O2Hb dissociation curve in 18 adult patients with SCD during vaso-occlusive crisis and 12 non-SCD patients with various cardiopulmonary diagnoses. The method proposed by Bland and Altman was used to evaluate agreement of various methods in each of the two groups. RESULTS: Mean differences between various methods in patients with SCD were significantly larger than the control group. Limits of agreement (LOA) were also wider in the SCD group than in the control group. Mean bias between SpO2 and SO2, and SpO2 and FO2Hb in patients with SCD were -3.1 +/- 4.4 (LOA: -11.9 to 5.7) and 2 +/- 4.1 (LOA: -6.2 to 10.2) respectively, compared with -1.4 +/- 1.4 (LOA: -4.2 to 1.4) and 1.2 +/- 1.5 (LOA: -1.9 to 4.3) in the control group. A mean bias of -4.5 +/- 4 (LOA: -12.5 to 3.5) between SpO2 and SaO2 was noted in patients with SCD compared with -0.1 +/- 2.1 (LOA: -4.3 to 4.1) in the control group. The width of LOA for various methods in patients with SCD ranged from 9.8 to 17.6 compared with 1.3 to 8.4 in the control group. CONCLUSION: Patients with SCD during vaso-occlusive crisis have discrepancies in O2Hb saturation measurements by various methods. Abnormal pulse oximetry values in these patients should be interpreted cautiously and supplemented by arterial blood gas analysis and co-oximetry.     

Evolution of laboratory parameters during sickle cell painful crisis: evidence compatible with dense red cell sequestration without thrombosis

We find that during 51 episodes of sickle cell painful crisis indirect bilirubin fell 52% from its steady state value of 2.3 +/- 1.9 mg% to a value of 1.1 +/- 0.37 mg% at the end of crisis (p less than .00000085). The indirect bilirubin decline correlates with a decrease in the dense sickle cells during crisis (r = .31, p less than .0009). During steady state, both indirect bilirubin and lactic acid dehydrogenase correlate significantly with number of dense red cells (r = .62, p less than .000002 and r = .32, p less than .02 respectively). Platelet counts, beta-thromboglobulin, Platelet Factor 4, and Fibrinopeptide A levels all were elevated during steady state and did not change during the evolution of crisis. These data demonstrate that elevated indices usually associated with platelet activation are a feature of the steady state of sickle cell disease but argue against thrombosis as a factor in the progression of a sickle cell painful crisis episode. The parallel decline of both dense cells and bilirubin during painful crises indicates that the disappearance of dense cells during crisis is not caused by hemolysis and supports the hypothesis that dense red cell sequestration, in the absence of evidence of thrombosis, is an intrinsic component of the evolution of sickle cell painful crisis.     

Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease

Background

Little is known about the effects of blood rheology on the occurrence of acute chest syndrome and painful vaso-occlusive crises in children with sickle cell anemia and hemoglobin SC disease.

Design and Methods

To address this issue, steady-state hemorheological profiles (blood viscosity, red blood cell deformability, aggregation properties) and hematologic parameters were assessed in 44 children with sickle cell anemia and 49 children with hemoglobin SC disease (8-16 years old) followed since birth. Clinical charts were retrospectively reviewed to determine prior acute chest syndrome or vaso-occlusive episodes, and rates of these complications were calculated.

Results

Multivariate analysis revealed that: 1) a higher steady-state blood viscosity was associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, but not in children with hemoglobin SC disease; 2) a higher steady-state red blood cell disaggregation threshold was associated with previous history of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia.

Conclusions

Our results indicate for the first time that the red blood cell aggregation properties may play a role in the pathophysiology of acute chest syndrome in children with hemoglobin SC disease and boys with sickle cell anemia. In addition, whereas greater blood viscosity is associated with a higher rate of vaso-occlusive crises in children with sickle cell anemia, no association was found in children with hemoglobin SC disease, underscoring differences in the etiology of vaso-occlusive crises between sickle cell anemia and hemoglobin SC disease.Key words: sickle cell anemia, hemoglobin SC disease, red blood aggregation, blood viscosity, red blood cell deformability