Splenic sequestration associated with sickle cell trait and hereditary spherocytosis.

Coexistence of sickle cell trait and hereditary spherocytosis (HS) is unusual, and only 16 cases have been reported in the literature. These patients have the same clinical and hematological features as individuals having HS alone. We report a serious complication, acute splenic sequestration crisis (ASSC), occurring in two patients with sickle cell trait and HS. One patient experienced four episodes of ASSC during an 11-year span, while the other had two episodes of this complication during a 4-year period. Red blood cell studies and membrane protein analysis confirmed the diagnosis of HS as a consequence of spectrin deficiency. Splenectomy resulted in marked clinical and hematological improvement in both patients. Histological examination of spleens following splenectomy confirmed that significant erythrostasis and sickling had indeed occurred. ASSC can occur in patients with coexistence of sickle cell trait and HS, and this potentially life-threatening complication should be considered in this condition.     

Interaction of sickle cell trait with hereditary spherocytosis: splenic infarcts and sequestration

The association of sickle cell trait (SCT) and hereditary spherocytosis (HS) has been reported in only 18 patients. Three of these 18 patients experienced splenic infarct or acute splenic sequestration. We report here a 46-year-old African-American male, the oldest reported case to date, who experienced episodes of hemolysis and severe left upper quadrant pain for the past 26 years. The patient had compensated hemolysis with splenomegaly. A CT scan of the abdomen revealed a large infarct in the spleen. The diagnosis of SCT was confirmed with isoelectric focusing, cation exchange and reverse-phase HPLC. The presence of a silent, interacting globin variant as the cause of hemolysis and sickling in the spleen was ruled out by sequencing of the alpha1-, alpha2- and beta-globin genes. The diagnosis of HS was established by an osmotic fragility test. The interaction of HS and SCT leads to RBC dehydration with increased MCHC and intracellular Hb S concentration presumably favoring intrasplenic sickling and resultant splenic infarcts and sequestration as seen in this case.     

The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.     

Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen

Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.     

A fatal case of acute splenic sequestration in a 53-year-old woman with sickle-hemoglobin C disease.

Acute splenic sequestration crisis (ASSC) is a common complication in infants and young children with homozygous sickle cell disease, but it is infrequent in patients with sickle-hemoglobin C (SC) disease. When it does occur in such patients, it is often associated with exposure to high altitude, either by air travel or mountainous environment. Since 1970, only 15 cases of ASSC have been reported in patients with SC disease who were not exposed to a high altitude. Nine of these were children and adolescents aged 11 to 18 years, while six were adults aged 21 to 44 years. A review of these cases shows only two fatalities from ASSC: a 12-year-old West Indian girl living in England and a 13-year-old black girl in the United States. In this report, we describe the sudden death from ASSC of a 53-year-old black woman with SC disease at low altitude. To our knowledge, death from ASSC has not been previously reported in an adult patient with SC disease.     

Atypical Hereditary Spherocytosis: Biochemical Studies and Sites of Erythrocyte Destruction

A family with hereditary spherocytosis has been investigated and found to have the following unusual features: atypical pre- and post-incubation red cell osmotic fragility and absence of splenic sequestration of red cells. Ouabain, an inhibitor of active cation transport, was found not to inhibit the corrective effect of glucose on the rate of autohaemolysis of whole blood, a finding present in some cases of known hereditary spherocytosis, but not in others. The red cells of the propositi showed evidence of increased A.T.P.-ase activity as well as increased utilization of A.T.P. for purposes other than for active cation transport, and are metabolically similar to hereditary spherocytic cells in this respect. The reason for the absence of splenic sequestration of red cells was not established.     

Co-existence of hereditary spherocytosis and a new red cell pyruvate kinase variant: PK mallorca

BACKGROUND AND OBJECTIVE: A partial red blood cell (RBC) pyruvate-kinase (PK-R) deficiency was found in a patient with concomitant hereditary spherocytosis (HS) and chronic hemolytic anemia. Clinical, biological and molecular studies were performed in the patient, his parents and a brother, in order to characterize the specific PK-R gene mutation and the inheritance mechanism of the transmission of both red cell defects in this particular family. DESIGN AND METHODS: Conventional biological studies were used to identify the PK-LR gene mutation responsible for hereditary transmission of PK-R deficiency and HS. The family study was completed with genotypic and RBC membrane protein analyses in the patient and his family. RESULTS: Molecular study of the PK deficiency was performed in all the family members and demonstrated a heterozygous condition for the 1516 G->A (506Val->Ile) mutation at the PK-LR gene in both the patient and his mother. Since this mutation has not been reported previously, it is provisionally named PK "Mallorca". The study of RBC membrane proteins demonstrated the existence of partial band 3 and protein 4.2 deficiencies in the propositus and his father but not in the mother and brother, who were also studied. These results support the dominant mode of inheritance of HS and PK-LR gene in this family. INTERPRETATION AND CONCLUSIONS: HS and PK deficiency are not exceptional in Spain. The co-existence of both RBC defects in the same patient, however, is very rare; only a few cases have been described to date. Our findings suggest that performing an elementary RBC enzyme survey in all patients with HS would help to determine the real frequency of this apparently rare association.     

A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer‐wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 μm drove flow into mechanical filtering units where RBCs flew either slowly through 5‐ to 2‐μm‐wide slits or rapidly along 10‐μm‐wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage‐dependent retention of Plasmodium falciparum‐infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.Am. J. Hematol. 90:339–345, 2015. © 2015 Wiley Periodicals, Inc.     

Initial assessment of the beneficial effect of partial splenectomy in hereditary spherocytosis

Clinical manifestations of hereditary spherocytosis (HS), the most common red blood cell (RBC) membrane disorder, can be abrogated or markedly reduced by splenectomy. However, concerns regarding risks from overwhelming infections after splenectomy have restricted its use, especially in children. This study was designed to determine if partial splenectomy can decrease the hemolytic rate while maintaining phagocytic function of the spleen. Partial splenectomy was performed in 11 children (age 2 to 13) with HS. The effect on hemolytic rate was assessed by comparing the presurgical and postsurgical values for hemoglobin, reticulocyte number, and RBC life span. The residual splenic phagocytic function was assessed using technetium 99m scans and by enumerating the percentage of pitted RBCs in circulation. There were no complications from the surgical procedure in any of the 11 individuals. Following partial splenectomy, hemoglobin increased on the average by 3 g/dL, reticulocyte count decreased by 300 x 10(6)/L, and RBC life span was substantially prolonged. Normal technetium uptake was noted in the splenic remnant and the percentage of pitted RBCs was in the normal range. Partial splenectomy is effective in decreasing the hemolytic rate while maintaining residual splenic phagocytic function of the spleen in HS. We conclude that the use of this procedure as treatment for RBC membrane disorders warrants consideration, especially in infants under 5 years of age who need frequent transfusions.     

Targeted disruption of the β adducin gene (Add2) causes red blood cell spherocytosis in mice

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.     

Acute splenic sequestration crisis in an adult with sickle β-thalassemia

Acute splenic sequestration crisis (ASSC) is a major cause of morbidity and mortality in children with sickle cell disease. Reports of ASSC in adults with sickle β-thalassemia (S-β^thal) are rare and consist of isolated case reports comprising a total of seven patients, three of whom died during the crisis. We report a 22-year-old man with S-β^thal who developed ASSC 1 day after suffering multiple blunt trauma. Systemic inflammatory response to severe blunt trauma may have precipitated ASSC in our patient. ASSC in adults with S-β^thal is a potentially life-threatening complication with a high risk of recurrence. Splenectomy is recommended after the first attack of ASSC in adults with S-β^thal.     

Growth of Plasmodium falciparum in human erythrocytes containing abnormal membrane proteins.

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, we have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp+)], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1(0))], HE due to a spectrin alpha I domain structural variant that results in increased content of spectrin dimers [HE(Sp alpha I/65)], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [3H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp+) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. Preincubation of HS(Sp+) RBCs in culture for 3 days did not alter these results. Normal parasite growth was observed in RBCs from one HS subject with normal membrane spectrin content. The extent of decreased parasite growth in HS(Sp+) RBCs closely correlated with the extent of RBC spectrin deficiency (r = 0.90). Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth inhibition, the extent of which correlated with the content of spectrin dimers (r = 0.94). RBCs from two unrelated subjects with structural variants of band 3 sustained normal parasite growth. Decreased growth in the pathologic RBCs was not the result of decreased ATP or glutathione levels or of increased RBC hemolysis. We conclude that abnormal parasite growth in these RBCs is not the consequence of metabolic or secondary defects. Instead, we suggest that a functionally and structurally normal host membrane is indispensable for parasite growth and development.     

The events in the hemolytic crisis of hereditary spherocytosis,with particular reference to the reticulocytopenia,pancytopenia and an abnormal splenic mechanism

The course of hereditary spherocytosis (congenital or familial hemolytic anemia) is subject to major or minor exacerbations or crises. Pancytopenia, reticulocytopenia, and extreme spherocytosis characterize the major crises, during whichhypersplenic effects appear to play a major role. These are characterized by thecombination of (1) an unusual degree of hemolysis with (2) inhibitory effects uponmaturation and delivery of bone marrow cells. At the height of the crisis, an extreme degree of maturation arrest in erythropoiesis is present. Splenectomy, whichis often urgently necessary, results in a very rapid increase in all the cellular elements of the blood, confirming the phagocytic and inhibitory effects of the abnormal spleen. Following splenectomy, no further crises occur. The presence of successive cases in the same family suggest the possible role of infection as aprecipitating or trigger agent in initiating an abnormal splenic mechanism leadingto crisis. The cause of the hereditary spherocytosis is commented upon and evidencebearing upon an autopsecific mechanism is discussed.

     

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.     

A family with sickle cell trait and hereditary spherocytosis

The rare combination of sickle cell trait with hereditary spherocytosis in a Sudanese family is reported. The splenic hypofunction and fibrosis as a result of sickling has ameliorated the clinical course of the 35 year old father. The symptoms of a six years old son seem to be getting milder with age too. The family is managed conservatively and splenectomy was not required.     

Acute Splenic Sequestration Crisis in Adult Sickle Cell Disease: A Report of 16 Cases

Abstract

Acute splenic sequestration crisis (ASSC), characterized by rapidly progressive anemia and circulatory compromise in the setting of sudden splenic enlargement, is an uncommon entity among adult sickle cell patients. We reviewed cases of adult ASSC encountered at our institution to generate insight into the recognition, diagnosis, and treatment of the condition. Cases of adult ASSC during a 10-year period were identified retrospectively. Patient charts were reviewed for laboratory and imaging results; demographic data and clinical course were collected and reviewed. Sixteen cases of adult ASSC were identified. Most patients presented with pain crisis; only four of 16 patients presented with abdominal pain. The maximum decreases in hemoglobin (Hb) (42.0%) and platelets (62.1%) occurred at day 2.9, delaying identification and treatment. Hemodynamic instability played a large role in dictating risk stratification. Therapy consisted of transfusion (14/16) and splenectomy (5/16). No recurrences were noted in a mean follow-up time of 5.3 years but review of patients’ charts demonstrated that at least one of the patients had two prior episodes. Adult ASSC may present with non specific findings and patients may not deteriorate until several days into a previously uneventful hospital course. Changes in platelet counts may be more reliable markers than changes in Hb level since red cell transfusions may interfere with assessments of the sequestration process. This case series of adult ASSC, the largest reported in the literature to date, highlights common clinical, laboratory, radiological, and pathological features of this uncommon entity and helps to guide recognition, diagnosis, and treatment.     

Iron overload in hereditary spherocytosis: Association with hla-linked hemochromatosis

Heavy iron loading is a rare clinical finding in patients with hereditary spherocytosis. A pedigree was studied in which the proband, a 38-year-old man, had both hereditary spherocytosis and overt hemochromatosis. He had never received blood transfusions. The 8-year-old son of the proband also had hereditary spherocytosis and mildly increased iron stores. The 39-year-old brother of the proband did not have spherocytosis but did have overt hemochromatosis. Since hemochromatosis is transmitted as an HLA-linked autosomal recessive disorder, HLA haplotypes serve as markers of hemochromatosis alleles. In this pedigree only individuals with two hemochromatosis alleles (homozygosity) had heavy iron loads, whether hereditary spherocytosis was present or not. The presence of hereditary spherocytosis may have contributed to the magnitude of the iron loading but was not a major factor. Our findings suggest that nontransfusional hemochromatosis found in association with hereditary spherocytosis is due primarily to homozygosity for hemochromatosis.     

The xerocytosis of Hb SC disease

Patients with Hb SC disease were found to have microcytic and hyperchromic red cell indices despite mild reticulocytosis. Iron deficiency anemia was ruled out by the finding of normal serum ferritin levels. In order to determine whether the microcytosis was due to coexistent alpha-thalassemia, restriction endonuclease mapping was performed on genomic DNA extracted from peripheral blood leukocytes. Patients with Hb SC disease had microcytic indices despite the presence of a full complement of four alpha-genes (alpha alpha/alpha alpha), suggesting that the microcytosis may be due to cellular dehydration (or xerocytosis), since the mean corpuscular hemoglobin concentration in Hb SC disease patients was significantly higher than in controls. This possibility was investigated further by the determination of RBC cation content. RBC Na levels were similar in SC and normal red cells. Hb SC RBCs, however, had significantly reduced K levels. These findings show that RBC cation content, and thus cell water, is decreased in Hb SC disease. The decreased RBC K level in the presence of normal cellular Na concentration suggests selective K loss that is not due to inhibition of the Na K pump. Ouabain-insensitive K+ efflux was increased to four times normal in SC cells. Cell dehydration was confirmed by the demonstration of increased high-density RBCs on discontinuous Stractan density gradients and by osmotic gradient ektacytometry. Cellular dehydration and its sequelae were worse in CC erythrocytes and milder in AC cells than in Hb SC red cells. Taken together, these data indicate that in Hb SC disease the RBCs are severely dehydrated and typically microcytic and hyperchromic. Hb SC RBCs seem to be dehydrated due to selective K loss. These findings suggest a functional interrelationship between Hb SC, the red cell membrane, and cation regulation.     

Shear Rate Dependency of Red Cell Sequestration in Skin Capillaries in Sickle Cell Disease and Its Variation with Vasoocclusive Crisis

Objective: To develop techniques for assessing the sequestration of red blood cells (RBCs) in skin capillaries of sickle cell disease (SCD) patients due to RBC adhesion to endothelium (EC) and RBC aggregation, and to determine the extent to which these processes correlate with onset of painful vasoocclusive crisis. Methods: Video recordings of nailfold capillaries in the skin of patients with SCD were made during steady-state periods and episodes of painful crisis. A transient low-flow state was induced with a pressure cuff, and reductions in RBC velocity were measured by spatial cross-correlation of light intensity along arterial and venous capillary limbs. An RBC accumulation index (AI) was calculated from RBC flow to represent the percentage of arterial in-flow sequestered in a capillary. An index of hematocrit (HI) was derived from axial distributions of light intensity, and a sequestration index (SI) was calculated to represent the relative increase of venous limb HI relative to that in the arterial limb with onset of stasis. Results: Both AI and SI increased dramatically from zero at steady flow to a maximum as shear rates within the capillary were reduced to zero. The increase was small until shear rates (γ) fell below a transition value (γ_T), following which both AI and SI increased sharply with onset of stasis. For 20 ≤ γ < γ_T the transient increase in AI was significantly elevated in the order AI_CRISIS > AI_{STEADY STATE} > AI_CONTROL, thus reflecting increasing RBC sequestration in the venous limb due to either adhesion or aggregation. Compaction of RBCs in the venous limb was evidenced by increased SI that was greater than control for both steady-state and crisis subjects, but insignificantly elevated during crisis compared to steady state, thus supporting a lesser role of RBC aggregation. Conclusions: Transient sequestration of sickle RBCs in the low-flow state appears to be dominated by RBC-EC adhesion, which becomes enhanced during crisis. Although aggregation may enhance adhesive contact of RBCs with EC, it does not increase to the same extent as the rate of sequestration, thus reflecting a greater role of RBC-EC adhesion.     

The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology

Knowledge concerning SS (homozygous for the beta s gene) red blood cell (RBC) heterogeneity has been useful for understanding the pathophysiology of sickle cell anemia. No equivalent information exists for RBCs of the compound heterozygote for the beta s and beta c genes (SC) RBCs. These RBCs are known to be denser than most cells in normal blood and even most cells in SS blood (Fabry et al, J Clin Invest 70:1284, 1981). We have analyzed the characteristics of SC RBC heterogeneity and find that: (1) SC cells exhibit unusual morphologic features, particularly the tendency for membrane "folding" (multifolded, unifolded, and triangular shapes are all common); (2) SC RBCs containing crystals and some containing round hemoglobin (Hb) aggregates (billiard-ball cells) are detectable in circulating SC blood; (3) in contrast to normal reticulocytes, which are found mainly in a low-density RBC fraction, SC reticulocytes are found in the densest SC RBC fraction; and (4) both deoxygenation and replacement of extracellular Cl- by NO3- (both inhibitors of K:Cl cotransport) led to moderate depopulation of the dense fraction and a dramatic shift of the reticulocytes to lower density fractions. We conclude that the RBC heterogeneity of SC disease is very different from that of SS disease. The major contributions of properties introduced by HbC are "folded" RBCs, intracellular crystal formation in circulating SC cells, and apparently a very active K:Cl cotransporter that leads to unusually dense reticulocytes.