Bone marrow transplantation corrects the splenic reticuloendothelial dysfunction in sickle cell anemia

In sickle cell anemia (SCA), the loss of reticuloendothelial function is the result of vasoocclusive events occurring in the spleen. Such asplenia occurs early in the course of the disease and is considered to be permanent in late childhood. In this report, three patients 10, 11, and 14 years of age suffering from severe SCA and found to be asplenic were treated by bone marrow transplantation (BMT). Before transplantation, all three patients had loss of reticuloendothelial splenic function, as assessed by the presence of abundant Howell-Jolly bodies on blood smears and absence of technetium 99m (99mTc) splenic uptake. After BMT, Howell-Jolly bodies disappeared from blood smear, whereas 99mTc isotopic scan found normal isotope uptake. Our data indicate that BMT can correct "permanent asplenia" in SCA patients. However, it remains to be determined if such treatment can also correct other SCA-related organ dysfunctions.     

Splenic function in persons with sickle cell trait at moderately high altitude

We investigated the possibility that persons with sickle cell trait who reside chronically at moderately high altitude might develop impaired splenic reticuloendothelial function. Seventeen healthy young black men with sickle trait who had lived at greater than or equal to 1,609 m for greater than or equal to 10 years participated in the study along with 25 matched control subjects with normal hemoglobin. Splenic function was assessed by radionuclide liver-spleen scanning and by red cell pit counts. No evidence of impaired splenic function was found in the sickle trait group. The data suggest that long-term residence at moderately high altitude does not place persons with sickle cell trait at risk for splenic dysfunction.     

The Splenic Platelet Reservoir in Sickle Cell Anemia

The presence of hypersplenism andfunctional asplenia occurring concomitantly in a child with sickle cell anemiaprompted a study of the splenic platelet reservoir in this hemoglobinopathy.The young child with sickle cell anemiaand a large spleen, who is unable toremove Howell-Jolly bodies, concentrate ^99mTc sulfur colloid in his spleen,or respond to intravenous particulateantigen, retains the splenic reservoirfunction to pool platelets. This reservoir function is lost in the older patientin whom the spleen has become autoinfarcted. Thus, an independence ofcertain splenic functions is present inyoung children with sickle cell anemiawho have splenomegaly.

Submitted on February 29, 1972 Revised on June 2, 1972 Accepted on June 11, 1972     

Hyposplenism: comparison of different methods for determining splenic function

Asplenic patients are at risk for pneumococcal sepsis. Patients with hyposplenic function, such as associated with sickle cell disease (SCD), are also at risk. However, tests to assess splenic function are either unavailable or lacking standardization. The aim of this study was to compare different methods for determining splenic function. Eighteen patients with SCD (i.e., 10 heterozygous (SC) and 8 homozygous (SS) SCD patients), and eight splenectomized patients were compared to 10 controls. All subjects underwent spleen scintigraphy, after which functional splenic volumes (FSV) were calculated. FSV was compared to immunological function and B cell-subsets, as well as phagocytic function represented by the presence of Howell Jolly bodies (HJB) and percentages of pitted red cells (PIT). Heterozygous SCD (SC) patients had increased splenic volumes, but diminished FSV, homozygous SCD (SS) patients were asplenic. Splenectomized and SS patients had a strongly reduced phagocytic and immunological function. SC patients had reduced anti-polysaccharide responses without an increase in PIT. FSV correlated significantly with phagocytic and immunological function. HJB were indicative of splenic dysfunction, HJB absence was not indicative of normal functioning splenic tissue. Although visualizing HJB is methodologically advantageous to PIT, both are valid biomarkers of splenic dysfunction. The amount of non-switched memory B cells is strongly correlated to FSV.     

Endotoxinaemia in sickle cell disease

Fifty-nine children with sickle cell anaemia (HbSS) or associated haemoglobinopathies were studied prospectively using a chromogenic Limulus amoebocyte lysate assay to detect circulating endotoxin. The 41 children with HbSS (mean age 8 years 9 months) had more serious disease than the 18 with HbSC disease (n = 14) or HbS-beta-thalassaemia (n = 4) (mean age 7 years 2 months), with a greater degree of splenomegaly, lower haemoglobin, and higher white cell counts, platelet counts and bilirubin values (P less than 0.05 for all). Twenty-nine children with HbSS had evidence of poor reticuloendothelial function, with red cell pitting of greater than or equal to 2%. Three of these 29 had low levels of endotoxin in plasma (0.12-0.24 endotoxin units (EU)/ml); two were clinically well, one had a painful crisis. Eight of 18 children with other sickle haemoglobinopathies had greater than or equal to 2% pitted red cells; none was endotoxinaemic. Therefore, in 37 patients with reticuloendothelial dysfunction, three were endotoxinaemic; all had sickle cell anaemia. Although not statistically significant, this suggests that endotoxinaemia may occur predominantly in patients with reticuloendothelial dysfunction, and is compatible with the hypothesis that systemic endotoxinaemia can derive from the intestine especially when reticuloendothelial function is depressed.     

Biomarkers of splenic function in infants with sickle cell anemia: baseline data from the BABY HUG Trial

We evaluated spleen function in 193 children with sickle cell anemia 8 to 18 months of age by (99m)Tc sulfur-colloid liver-spleen scan and correlated results with clinical and laboratory parameters, including 2 splenic biomarkers: pitted cell counts (PIT) and quantitative Howell-Jolly bodies (HJB) enumerated by flow cytometry. Loss of splenic function began before 12 months of age in 86% of infants in association with lower total or fetal hemoglobin and higher white blood cell or reticulocyte counts, reinforcing the need for early diagnosis and diligent preventive care. PIT and HJB correlated well with each other and liver-spleen scan results. Previously described biomarker threshold values did define patients with abnormal splenic function, but our data suggest that normal spleen function is better predicted by PIT of ≤1.2% or HJB ≤55/10(6) red blood cells and absent function by PIT ≥4.5% or HJB ≥665/10(6). HJB is methodologically advantageous compared with PIT, but both are valid biomarkers of splenic function. This trial was registered at www.clinicaltrials.gov as #NCT00006400.     

Quantitative analysis of Howell-Jolly bodies in children with sickle cell disease

OBJECTIVES: Although functional asplenia in sickle cell disease (SCD) begins early in life and has important clinical consequences, quantitative measurement of splenic function is not readily available. A novel high-throughput flow cytometric method for quantitating Howell-Jolly bodies (HJB) has been developed which isolates HJB-containing CD71(+) and CD71(-) erythrocytes. Analysis of these cell populations allows quantitative measurement of splenic filtrative function and possible chromosomal damage. METHODS: Blood specimens from 147 children with SCD were analyzed using a high-throughput flow cytometric method. Enumeration of the following populations was accomplished: 1) CD71(+) reticulocytes among total erythrocytes, identifying the youngest erythroid cell population; 2) HJB-containing CD71(+) reticulocytes, which isolate young erythrocytes containing micronuclei as an index of cytogenetic damage; and 3) HJB-containing CD71(-) erythrocytes, identifying older erythrocytes containing micronuclei, indirectly measuring splenic function. RESULTS: Children with HbSC (n = 24) had slightly elevated HJB frequencies, while children with HbSS (n = 125) had highly elevated frequencies within CD71(+) cells (0.44% +/- 0.40%, normal 0.12% +/- 0.06%, p < 0.001) and CD71(-) cells (2493 +/- 2303 per million RBC, normal 20 +/- 11, p < 0.001). Using a multiple regression model, the frequency of HbSS CD71(+) reticulocytes containing HJB was significantly influenced by hydroxyurea use (p < 0.0001), age (p = 0.0288), and splenectomy (p = 0.0498). Similarly, mature CD71(-) erythrocytes containing HJB were positively correlated with hydroxyurea (p = 0.0001), age (p < 0.0001), and splenectomy (p = 0.0104). CONCLUSIONS: HJB quantitation by flow cytometry is a novel assay for measuring splenic function and may be valuable for investigating the efficacy and safety of therapeutic options for children with SCD.     

Transient functional asplenism in sickle cell-C disease

During a painful crisis, the spleen of a patient with sickle cell-C disease failed to visualize with 99^m Tc-sulfur colloid although it was significantly enlarged by palpation. At that time, the peripheral blood smear showed numerous sickle cells, normoblasts and erythrocytes containing Howell-Jolly bodies. After therapy with hypotonic saline solution and with clinical improvement, the enlarged spleen was completely visualized on repeat scanning. The normoblasts and erythrocytes containing Howell-Jolly bodies also decreased in number. This case of transient asplenism is similar to the cases previously reported only in children with homozygous sickle cell disease. The significance of this phenomenon with respect to increased susceptibility to infection is discussed.     

Splenic sepsis in sickle cell disease

We describe two patients with sickle cell disease (SCD) who developed infections situated in the spleen. One patient had a splenic abscess and there was strong clinical evidence for an infected splenic infarct in the second patient. SCD predisposes to splenic infection because of functional hyposplenism, defective phagocyte function and splenic infarction. Splenic infections can occur in patients who might be considered to have an absent spleen and the diagnosis of splenic abscess should be considered in individuals with SCD who present with fever and abdominal pain.     

First report of reversal of organ dysfunction in sickle cell anemia by the use of hydroxyurea: splenic regeneration

Much of the morbidity associated with sickle cell anemia (SCA) is due to ongoing infarction resulting in organ dysfunction. Because the spleen is often the first organ damaged in this illness, there is a significant impairment of the immune system in these patients. Hydroxyurea (HU) has been shown to increase fetal hemoglobin (HbF) and decrease painful episodes in patients with this disease. It is unclear whether HU can prevent organ damage. We treated two SCA patients with HU for several years and found evidence of reversal of previously documented splenic dysfunction. Patient no. 1 was treated for 30 months with an increase in HbF to 30%. HU was stopped because of cytopenia. She developed left upper quadrant pain. A splenectomy was performed due to the possibility of splenic abscesses. A pathologic review found no evidence of infection and an enlarged spleen that showed active germinal centers. Patient no. 2 was treated for 24 months with HU before developing splenomegaly. His HbF levels were 25% to 30%, his pit counts averaged 2%, and his liver spleen scans showed uptake. These two cases show that chronic HU therapy may reverse splenic dysfunction in certain patients and suggest that this drug may have efficacy beyond the elimination of pain in SCA.     

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.     

Functional asplenia in hemoglobin SC disease

The incidence of functional asplenia in sickle-hemoglobin C (SC) disease has not been defined, and the use of prophylactic penicillin to prevent life-threatening septicemia in this disorder is controversial. The percentage of red blood cells with pits (pit count) is a reliable assay of splenic function in other disorders but has not been validated in hemoglobin SC disease. To address these issues, we conducted a prospective, multicenter study of splenic function in persons with hemoglobin SC disease. Baseline clinical data were recorded, and red blood cell pit counts were performed on 201 subjects, aged 6 months to 90 years, with hemoglobin SC; 43 subjects underwent radionuclide liver- spleen scanning. Pit counts greater than 20% were associated with functional asplenia as assessed by liver-spleen scan, whereas pit counts less than 20% were found in subjects with preserved splenic function. Pit counts greater than 20% were present in 0 of 59 subjects (0%) less than 4 years of age, in 19 of 86 subjects (22%) 4 to 12 years of age, and in 25 of 56 subjects (45%) greater than 12 years of age. Other subjects with hemoglobin SC, who had previously undergone surgical splenectomy, had higher pit counts (59.7% +/- 9.5%) than splenectomized subjects without hemoglobinopathy (38.5% +/- 8.8%) or with sickle cell anemia (20.5% +/- 1.9%; P < .001). Two subjects with hemoglobin SC disease (not splenectomized), ages 14 and 15 years, with pit counts of 40.3% and 41.7% died from pneumococcal septicemia. These data indicate that functional asplenia occurs in many patients with hemoglobin SC disease, but its development is usually delayed until after 4 years of age. The pit count is a reliable measure of splenic function in hemoglobin SC disease, but values indicative of functional asplenia (> 20% in our laboratory) are higher than in other disorders. The routine administration of prophylactic penicillin to infants and young children with hemoglobin SC disease may not be necessary.     

Howell-Jolly body counting as a measure of splenic function. A reassessment

Non-surgical and surgical asplenia predisposes to fatal infections; therefore, simple, non-invasive and repeatable tests for assessing splenic function are required, even in non-specialized medical institutions. Howell-Jolly bodies are the most characteristic peripheral blood abnormality after splenectomy, but their counting is not considered a reliable measure of splenic function. In this study, in a group of splenectomized subjects and of patients with non-surgical hyposplenism, we have compared counting of Howell-Jolly bodies, stained by both the May-Grünwald/Giemsa method and the Feulgen reaction, with pitted cell counting which is considered a reliable technique for the assessment of splenic hypofunction. A significant correlation has been found between Howell-Jolly body counts, stained by either technique, and pitted cell counts (P less than 0.0001). Through Howell-Jolly bodies were never detectable when pitted cell counts fell between 4 and 8%, values consistent with a very mild splenic hypofunction, for pitted cell counts above 8% their increase was always associated with increasing Howell-Jolly body counts. These data suggest that, although pitted cell counting represents a more sensitive method for evaluating splenic function, Howell-Jolly body counting may still be regarded as a simple and reliable technique for identifying and monitoring those cases associated with a real risk of overwhelming infections.     

Splenic state in surviving patients with visceral heterotaxy

AIM: To identify patients with visceral heterotaxy who are at risk from fulminant sepsis. METHODS: We studied 38 patients, 37 having undergone abdominal ultrasound, all 38 having examination of blood films to establish presence of Howell-Jolly bodies, and all 38 documented to have had pneumococcal vaccination and prophylaxis with penicillin. We checked whether the parents were aware of the splenic state of their child, and when possible, we compared current results of blood films with those obtained postnatally. RESULTS: Two of the 17 patients with multiple spleens, all 11 without a detectable spleen, and 1 of 9 patients with a normal spleen, showed Howell-Jolly bodies in their blood films. In 5 of 23 patients with serial blood films, Howell-Jolly bodies had not been seen postnatally, but could now be detected in current blood films. Of these patients, 2 had multiple spleens, 1 did not have a spleen, and 1 had a solitary spleen of normal size. In the other patient, ultrasound could not be performed. Only one of these patients was receiving penicillin prophylactically, and had received pneumococcal vaccination. Of the 15 patients in whom Howell-Jolly bodies were present in the blood, only 8 parents knew about the potential risk for infection. Another 7 parents were sure that their child was taking penicillin regularly, and had received pneumococcal vaccination. CONCLUSIONS: Howell-Jolly bodies can be found in the blood of patients with visceral heterotaxy independent of the anatomical state of the spleen. As Howell-Jolly bodies can be encountered in the blood of such patients with increasing age, those with multiple and solitary spleens should be monitored regularly to identify those at risk. Parental knowledge of the splenic state, and compliance for prophylaxis using penicillin, and pneumococcal vaccination, were unsatisfactory in our cohort.     

Splenic Red Cell Pooling in Hairy Cell Leukaemia

The splenic red cell volume has been measured directly by an isotope method with quantitative scanning in 10 patients with leukaemic reticuloendotheliosis (hairy cell leukaemia). The volume ranged between 211 and 726 ml (mean 410 ml, SD 158) and this constituted 15–48% (mean 28.1%, SD 9.5) of the total circulating red cell volume. This is an exceptionally large pool when compared with that found in myeloproliferative and lymphoproliferative disorders with the same degree of splenomegaly. It is consistent with the histological features which show marked red cell accumulation in the splenic cord areas. The red cell pooling in the spleen thus appears to be a significant factor in the anaemia and there was fairly good correlation between the percentage of improvement in the anaemia and the percentage of red cell volume contained in the spleen. By direct measurement of the splenic red cell pool, it is possible to predict the extent to which splenectomy will benefit the anaemia and this may also provide an indirect measure of the extent of bone marrow dysfunction in the causation of the anaemia.     

Splenic function in children with sickle cell disease: two different patterns in Saudi Arabia

Splenic function in 35 Saudi children homozygous for sickle cell disease (age range 3-9 years) was studied using radioactive colloid scans. Two different patterns emerged. Splenic dysfunction was demonstrated in more than 80% of children who were originally from the south-western part of the country. They were found to have low HbF levels. In contrast normal or nearly normal splenic function was found in all patients from the Eastern Province in whom HbF levels were high. These different patterns of splenic function may contribute to the severe and mild forms of sickle cell disease seen in Saudi Arabia.     

Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for α-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.     

Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease

Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT.     

Thrombosis and sickle cell disease

Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin, which has the unique property of polymerizing when deoxygenated. The pathophysiology of acute and chronic clinical manifestations of SCD have shown the central role of dense, dehydrated red cells in acute and chronic clinical manifestations of this pathology. Recent studies have indicated that SCD is characterized by a hypercoagulable state that contributes to the vaso-occlusive events in microcirculation, leading to acute and chronic sickle cell-related organ damage. This review discusses, in the context of SCD, (1) abnormalities in the coagulation system, (2) perturbation of platelet activation and aggregation, (3) vascular endothelial dysfunction, (4) the contribution of cell inflammatory responses, and (5) the connection with nitric oxide metabolism. We also review the available studies on the therapeutic approaches in clinical management of hypercoagulability in SCD.     

Abnormalities of serum transcobalamins in sickle cell disease (HbSS) in Black Africa

The unsaturated vitamin B12 binding capacity (UBBC) of serum, the three transcobalamins (TC I, TC II, TC III), and the total leucocyte and neutrophil counts have been studied in paediatric patients with sickle cell disease (SCD). Increase in the level of the binding capacities of TC I and TC II with concomitant increase of UBBC was observed in these children who also had increased total white blood and neutrophil counts. There was a significant reduction in the level of endogenous B12. These abnormalities are discussed in relation to the deficiency of the splenic reticuloendothelial function, immunologic defect, hepatic degenerative changes and aplastic crisis observed in SCD. These results lend support to the view that transcobalamins are involved in the defence mechanism of the body. The significant reduction in serum cobalamin in SCD suggests a higher demand on this vitamin for metabolic functions, especially for the sparing of folate.