Hematologic and hemorheological determinants of resting and exercise-induced hemoglobin oxygen desaturation in children with sickle cell disease

The aim of the study was to determine the factors associated with resting and exercise-induced hemoglobin oxygen desaturation. The well-established six-minute walk test was conducted in 107 sickle cell children (50 with sickle hemoglobin C disease and 57 with sickle cell anemia) at steady state. Hemoglobin oxygen saturation was measured before and immediately after the six-minute walk test. Blood samples were obtained on the same day to measure hematologic and hemorheological parameters. Exercise-induced hemoglobin oxygen desaturation was defined as a drop in hemoglobin oxygen saturation of 3% or more at the end of the six-minute walk test compared to resting levels. No children with sickle hemoglobin C disease, but approximately 50% of children with sickle cell anemia showed mild or moderate oxygen desaturation at rest, which was independently associated with the percentage of reticulocytes. Exercise-induced hemoglobin oxygen desaturation was observed in 18% of children with sickle hemoglobin C disease and 34% of children with sickle cell anemia, and was independently associated with the six-minute walk test, acute chest syndrome rate and the strength of red blood cell aggregates in children with sickle cell anemia. No association was found in children with sickle hemoglobin C disease between exercise-induced hemoglobin oxygen desaturation and the measured parameters. Hemoglobin oxygen desaturation at rest was common in children with sickle cell anemia but not in children with sickle hemoglobin C disease, and was mainly associated with greater hemolysis. Physiological strain during exercise and red blood cell aggregation properties may predict the occurrence of exercise-induced hemoglobin oxygen desaturation in children with sickle cell anemia.     

Determinants of red cell survival and erythropoietic activity in patients with sickle cell anemia in the steady state

We have studied 26 patients with sickle cell anemia to determine the factors that affect red blood cell (RBC) survival and other parameters of erythropoietic activity in the steady state. Determinants of erythropoietic activity included RBC survival by the 51Cr method, RBC production/destruction rate, alpha genotype, beta(s) haplotype, plasma 59Fe clearance, plasma iron turnover, erythron transferrin uptake), RBC Fe utilization, reticulocyte count, and erythropoietin levels. The alpha genotype was the most significant determinant of RBC survival followed, to a lesser extent, by the beta(s) haplotype. Hb F showed no correlation with RBC survival due to patient selection bias - the patients studied had comparable Hb F levels to start with. Other determinants of erythropoietic activity (hemoglobin level, mean corpuscular volume, reticulocyte count, RBC mass, RBC production/destruction rate, and erythropoietin level) were most likely secondary determinants associated with the alpha genotype, and not independent determinants in themselves. The data suggest that the alpha genotype and, and to a lesser extent, the beta(s) haplotype, might be determinants of the severity of the anemia of sickle cell disease, and should be considered in genetic counseling and patient selection for aggressive therapeutic interventions.     

Diagnostic value of anemia, red blood cell morphology, and reticulocyte count for sickle cell disease.

STUDY OBJECTIVE: To determine the diagnostic value of anemia, RBC morphology, and reticulocyte count for differentiating patients with sickle cell trait from those with sickle cell disease, who have acute medical or surgical conditions and a positive sickle cell screen. DESIGN: Retrospective chart review. SETTING: A midwest urban children's hospital with 220 beds and 36,000 emergency department visits per year. PARTICIPANTS: One hundred six patients with sickle cell trait and 152 patients with sickle cell hemoglobinopathies. RESULTS: Anemia was observed significantly more often in patients with sickle cell disease compared with sickle cell trait (P less than .001) at all ages 3 months and older. However, anemia alone as a diagnostic test lacked high sensitivity and specificity in children less than 4 years old. Sensitivity approached 100% with the presence of anemia, abnormal RBC morphology, or reticulocyte count of more than 2%. CONCLUSION: Absence of anemia alone does not exclude the diagnosis of sickle cell disease in children less than 4 years old. To differentiate trait from sickle cell disease, we recommend determination of not only hemoglobin adjusted for age but also of RBC morphology and reticulocyte count on all children presenting with acute medical and surgical conditions and a positive sickle cell screen.     

A cold agglutinin disease,difficult to distinguish from paroxysmal cold hemoglobinuria

A 58-year-old male started passing dark brown colored urine in January 2002. An annual medical examination in the same month revealed a mild anemia and an increased serum LDH level. Because of the instability of the data of his peripheral red cell count and hemoglobin concentration as measuring with a blood cell auto-analyzer, and also the hemolytic findings in a test tube at room temperature (25 degrees C), he was referred to our hospital. Laboratory data revealed Hb 11.2 g/dl, reticulocytes 73.1% (233,000/microliter), indirect-bilirubin 2.8 mg/dl, LDH 757 U/l, and hemosiderinuria, suggesting some intravascular hemolysis. The cold agglutinin titer was > 1,024, direct and indirect Coombs tests were both positive, and the Donath-Landsteiner antibody (D-L antibody) was initially assessed as false positive because of a high titer of cold agglutinin. He was finally diagnosed as having a cold agglutinin disease (CAD) with anti-I autoantibody. Serologic tests for syphilis and mycoplasma, and various virus tests were all negatives. After avoiding cold exposure, his symptoms, including hemoglobinuria, disappeared. However, during 9 months follow-up, he still showed a high titer of cold agglutinin. This case suggests that CAD should be considered when peripheral blood cell count data are unstable as assessed by a blood cell auto-analyzer.     

Sickle red cell microrheology and sickle blood rheology

The hallmark of the phenotypic expression of sickle cell disease is the remarkable degree of heterogeneity in the clinical manifestations. They vary latitudinally among patients and longitudinally in the same patient. The pathogenesis of sickle cell anemia centers on the sequence of events that occur between polymerization of deoxy hemoglobin S and increased red cell destruction, vasoocclusion, and end organ damage. Cellular dehydration, changes in sickle red blood cell rheology, adhesion of sickle red cells to vascular endothelium, inflammatory response, and tissue injury are some of the factors that contribute to hemolytic anemia, vasoocclusion, and eventual multiorgan damage. The focus of this review is on the rheology of sickle blood and microrheology of sickle RBC. Determinants of sickle RBC rheology and the factors that modulate its severity are discussed.     

Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease

Background

Recent evidence suggests that red blood cell aggregation and the ratio of hematocrit to blood viscosity (HVR), an index of the oxygen transport potential of blood, might considerably modulate blood flow dynamics in the microcirculation. It thus seems likely that these factors could play a role in sickle cell disease.

Design and Methods

We compared red blood cell aggregation characteristics, blood viscosity and HVR at different shear rates between sickle cell anemia and sickle cell hemoglobin C disease (SCC) patients, sickle cell trait carriers (AS) and control individuals (AA).

Results

Blood viscosity determined at high shear rate was lower in sickle cell anemia (n=21) than in AA (n=52), AS (n=33) or SCC (n=21), and was markedly increased in both SCC and AS. Despite differences in blood viscosity, both sickle cell anemia and SCC had similar low HVR values compared to both AA and AS. Sickle cell anemia (n=21) and SCC (n=19) subjects had a lower red blood cell aggregation index and longer time for red blood cell aggregates formation than AA (n=16) and AS (n=15), and a 2 to 3 fold greater shear rate required to disperse red blood cell aggregates.

Conclusions

The low HVR levels found in sickle cell anemia and SCC indicates a comparable low oxygen transport potential of blood in both genotypes. Red blood cell aggregation properties are likely to be involved in the pathophysiology of sickle cell disease: the increased shear forces needed to disperse red blood cell aggregates may disturb blood flow, especially at the microcirculatory level, since red blood cell are only able to pass through narrow capillaries as single cells rather than as aggregates.     

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     

Gel-filtration of sickle erythrocytes: separation based on cell deformability

Filtration of red blood cells (RBC) through columns of pre-swollen agarose-based beads has been evaluated using cells from subjects with sickle cell disease. Elution profiles from these gels showed elution times close to normal controls for a large fraction of sickle erythrocytes and a prolonged elution time for a sub-population of these cells. Analysis of red blood cell deformability using a computerized micropore filtration system (CTA) indicated that the deformability of sickle red blood cells in the first fraction was similar to controls but that the last fraction contained a sub-population of rigid RBC. We thus conclude that sickle red blood cell separation in columns of agarose-based beads is based upon cell deformability. Gel filtration therefore appears to be an interesting tool for the study of red blood cells in a variety of disorders with sub-populations of rigid, abnormal cells, and seems especially suited for studies in various sickle cell diseases. Keywords: Deformability, gel filtration, sickle cell disease, erythrocyte     

Ascorbate levels in red blood cells and urine in patients with sickle cell anemia

Ascorbic acid can be important in sickle cell anemia (SCA) because significant oxidative stress occurs in the disease. Ascorbate could contribute to reduction of the increased oxygen free radicals generated in sickle red blood cells (SRBC) and to the recycling of vitamin E in the cells, while renal loss could contribute to the low plasma levels. Evaluation of red blood cell (RBC) and urine ascorbate in SCA has not been reported. Results showed (1) ascorbate levels in SRBC were similar to those in normals; (2) urine ascorbate excretion was increased in 36% of patients; (3) plasma levels of ascorbate were decreased. Conclusions: (1) Ascorbate is present in SRBC, most likely due to ascorbate recycling, despite increased free-radical generation. (2) The increase in renal excretion may contribute to the low plasma levels of ascorbate. (3) The presence of ample ascorbate in SRBC and decreased plasma ascorbate suggests that ascorbate movement across the SRBC membrane may differ from normal RBC.     

A Cold Agglutinin: Om

An IgM kappa cold agglutinin reacted with cord blood as well as with adult red blood cells (RBC). The agglutinin reacted strongly with papainized and neuraminidase (RDE)-treated RBC. It resembled anti-Me cold agglutinin in these serological findings. However, the activity of this agglutinin against RBC was markedly decreased in the presence of human milk. The data suggest that the agglutinin is directed against a new glycolipid antigen common to human RBC, termed Om antigen.     

L-Glutamine therapy reduces endothelial adhesion of sickle red blood cells to human umbilical vein endothelial cells

Background

We have previously demonstrated that therapy with orally administered L-glutamine improves nicotinamide adenosine dinucleotide (NAD) redox potential of sickle red blood cells (RBC). On further analysis of L-glutamine therapy for sickle cell anemia patients, the effect of L-glutamine on adhesion of sickle RBC to human umbilical vein endothelial cells (HUVEC) was examined.

Methods

The first part of the experiment was conducted with the blood samples of the 5 adult sickle cell anemia patients who had been on L-glutamine therapy for at least 4 weeks on a dosage of 30 grams per day compared to those of patient control group. In the second part of the experiment 6 patients with sickle cell anemia were studied longitudinally. Five of these patients were treated with oral L-glutamine 30 grams daily and one was observed without treatment as the control. t-test and paired t-test were used for determination of statistical significance in cross-sectional and longitudinal studies respectively.

Results

In the first study, the mean adhesion to endothelial cells with the autologous plasma incubated cells were 0.97 ± 0.45 for the treated group and 1.91 ± 0.53 for the nontreated group (p < 0.02). Similarly with lipopolysaccharide (LPS) incubated cells the mean adhesion to endothelial cells were 1.39 ± 0.33 for the treated group and 2.80 ± 0.47 for the untreated group (p < 0.001). With the longitudinal experiment, mean decrease in the adhesion to endothelial cells was 1.13 ± 0.21 (p < 0.001) for the 5 treated patients whereas the control patient had slight increase in the adhesion to endothelial cells.

Conclusion

In these studies, oral L-glutamine administration consistently resulted in improvement of sickle RBC adhesion to HUVEC. These data suggest positive physiological effects of L-glutamine in sickle cell disease.     

Effects of oxidative stress on red blood cell rheology in sickle cell patients

Sickle cell anaemia (SS) and sickle cell‐haemoglobin C disease (SC) patients exhibit severe red blood cell (RBC) rheological alterations involved in the development of several complications. The contribution of oxidative stress in these haemorheological abnormalities is still unknown. We compared RBC reactive oxygen species (ROS) and glutathione (GSH) content, and the haemorheological profile of SS (n = 11), SC (n = 11) and healthy subjects (n = 12) at baseline and after in‐vitro treatment with t‐butyl hydroperoxide (TBHP). We showed: (i) higher RBC ROS content in SS and SC patients, with the highest level observed in SS patients; (ii) lower RBC GSH content in sickle syndrome patients, especially in SS patients; (iii) TBHP increased RBC ROS production and decreased RBC GSH content in all groups; (iv) TBHP decreased RBC aggregation and increased the strength of RBC aggregates in all groups but the increase in RBC aggregates strength was greater in sickle cell patients; (v) TBHP decreased RBC deformability in the three groups but with a higher magnitude in sickle cell patients. These data suggest that RBCs from sickle cell patients have an exaggerated response to oxidative stress, which is accompanied by a profound abnormal haemorheological profile, with greater alterations in SS than in SC patients.     

Erythropoietic activity in patients with sickle cell anaemia before and after treatment with hydroxyurea

In this project we have prospectively studied the erythropoietic activity in patients with sickle cell anaemia (SS) before and after treatment with hydroxyurea (HU). Some of the patients were enrolled in a double-blind placebo controlled trial of HU in patients with SS and others were enrolled in an open label study. Determinants of erythropoietic activity included the reticulocyte count, red blood cell (RBC) survival by the 51Cr method, plasma 59Fe clearance, plasma iron turnover (PIT), erythron transferrin uptake (ETU), RBC production/destruction rate, and RBC Fe utilization. Therapy with HU increased the mean corpuscular volume (MCV), haemoglobin (Hb)F, RBC survival and t1/2 59Fe clearance; it decreased the reticulocyte count, the white blood cell (WBC) count, ETU, and the PIT. Most of the changes in parameters of erythropoiesis could be explained by the increase in 51Cr RBC survival after therapy with HU. Together the data showed that in selected patients the net effect of HU on Hb level was a function of the difference between the suppressive effect of HU (decreased RBC production) and the increase in RBC survival. In the majority of patients who responded to HU, there was a preferential effect on RBC survival.     

Assessment of Red Cell Autoantibodies in Autoimmune Hemolytic Anemia of Warm Type by a Radioactive Anti-IgG Test1

To evaluate the applicability of a radioactive ¹²⁵I-anti-IgG test (RIAT) for the detection of small amounts of IgG antibodies on red blood cells (RBC) of patients with autoimmune hemolytic anemia of warm type (AIHA), RBC of 125 patients were studied (AIHA, n=53; Coombs'-negative AIHA, n=6; chronic cold agglutinin disease, n=7; non-immune anemias, n=59). It was found that the RBC of all cases (33/33) with a positive direct IgG antiglobulin test (DAT-IgG), but also 13 out of 20 patients with a negative DAT-IgG, but detectable complement (C3/C4), and 4 out of 6 cases with Coombs'-negative AIHA gave positive results in the direct RIAT. RBC-associated IgG was higher in the DAT-IgG positive group (n=33; x?=8.1%) than in the DAT-IgG negative group (n=26; x?3.4%). There was no correlation between hypergammaglobulinemia and RBC-associated IgG. The sensitivity of the indirect RIAT was not remarkably better as compared to the indirect antiglobulin test. The RIAT is valuable in the serology of borderline cases of AIHA.     

A Combination of IgG and IgM Autoantibodies in Chronic Cold Agglutinin Disease: Immunologic Studies and Response to Splenectomy

A patient with chronic cold agglutinin disease and anti-I antibody was studied. The titer of the patient's serum at 4 degrees C was 700 with adult I RBC, 256 with cord RBC, 256 with adult i RBC. At room temperature the titers were decreased and the serum also reacted at a titer of 4,000 with enzyme treated adult I RBC. Dithiothreitol treatment of the serum and purified antibody decreased reactivity only slightly, indicating that the antibody was IgG. The heat eluate contained 1.2 mg/ml IgG kappa, but only 80 micrograms/ml IgM kappa. A hybridoma made by fusing the patient's peripheral blood lymphocytes with a human myeloma cell line contained only IgM kappa (30 micrograms/ml) anti-I activity. The IgM fraction of the heat eluate reacted similarly to the hybridoma supernatant. The IgG fraction resembled the serum and heat eluate. In this case study, IgG kappa and IgM kappa immunoglobulins, both possessing cold agglutinin activity, were present in the patient's serum. This finding is unusual in idiopathic cold agglutinin disease and in view of the predominance of an IgG cold agglutinin, splenectomy was recommended for treatment.     

Pure red cell aplasia of long duration complicating major ABO-incompatible bone marrow transplantation

In 3 of 15 consecutive patients receiving a human leukocyte antigen (HLA)-identical but major ABO incompatible bone marrow transplant (BMT), pure red cell aplasia (PRA) lasting 5 to 8 months was observed. Titers of the incompatible anti-A agglutinin before infusion of the red blood cell (RBC)-depleted BMT was very high in one, and in the usual range in two patients. Decrease of agglutinin titers during the first 4 weeks after BMT were comparable between PRA patients and those of ABO-incompatible BMT recipients with timely RBC recovery. However, in PRA patients, agglutinin titers rose again and remained elevated for 19 to 28 weeks. RBC engraftment and reticulocyte recovery ultimately occurred spontaneously and coincided with the decrease of agglutinin titers below 16. These observations indicate that PRA is antibody-dependent in this setting. Furthermore, it is conceivable that cyclosporine facilitates recipient-derived antibody synthesis after major ABO-incompatible BMT.     

Impaired vasodilation by red blood cells in sickle cell disease

Red blood cells (RBCs) have been ascribed a unique role in dilating blood vessels, which requires O2-regulated binding and bioactivation of NO by Hb and transfer of NO equivalents to the RBC membrane. Vasoocclusion in hypoxic tissues is the hallmark of sickle cell anemia. Here we show that sickle cell Hb variant S (HbS) is deficient both in the intramolecular transfer of NO from heme iron (iron nitrosyl, FeNO) to cysteine thiol (S-nitrosothiol, SNO) that subserves bioactivation, and in transfer of the NO moiety from S-nitrosohemoglobin (SNO-HbS) to the RBC membrane. As a result, sickle RBCs are deficient in membrane SNO and impaired in their ability to mediate hypoxic vasodilation. Further, the magnitudes of these impairments correlate with the clinical severity of disease. Thus, our results suggest that abnormal RBC vasoactivity contributes to the vasoocclusive pathophysiology of sickle cell anemia, and that the phenotypic variation in expression of the sickle genotype may be explained, in part, by variable deficiency in RBC processing of NO. More generally, our findings raise the idea that defective NO processing may characterize a new class of hemoglobinopathy.     

Complement-Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.     

Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol‐12‐myristate‐13‐acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA‐activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA‐dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.     

Nitric oxide attenuates normal and sickle red blood cell adherence to pulmonary endothelium

Increased adherence of sickle red blood cells (RBC) to endothelium is implicated as an initiating event of vaso-occlusion in sickle cell disease. Although much is known about the humoral influences of this interaction, there has been little investigation regarding endothelial contributions. Endothelial derived nitric oxide (NO) inhibits adhesion of platelets and leukocytes to endothelium and decreases expression of VCAM-1, an endothelial adhesion site implicated in sickle RBC/endothelial adherence. However, whether NO inhibits RBC adherence to endothelium is unexplored. We tested this hypothesis with endothelial monolayers exposed to RBC from normal (Hb AA) and sickle cell (Hb SS) volunteers in a parallel plate flow chamber. To decrease NO production, endothelial monolayers were exposed to 100 microM nitro-L-arginine (NLA), an inhibitor of nitric oxide synthase, resulting in an 87% increase in normal RBC adherence (P = 0.002). Because adherence of normal RBC to endothelium was low, the effect of DETA-NO, an NO donor, was tested after activation of endothelium with TNF-alpha increased adherence by 130% (P < 0.001). Subsequent addition of 2 mM DETA-NO produced a 75% decrease in adherence of normal RBC to endothelium (P = 0.03). At baseline, sickle RBC were significantly more adherent than normal RBC (P < 0.001) and DETA-NO decreased sickle RBC adherence by 54% (P = 0.04). Thus, NO inhibits both normal and sickle RBC adherence to endothelium. Strategies that enhance NO activity may be therapeutic in sickle cell disease.