Maxwell P. Westerman

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy.

Levels of circulating red blood cell (RBC)‐derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6‐fold greater in SCA and 4‐fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40–50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)‐treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

Age-Related Somatic Structural Changes in the Nuclear Genome of Human Blood Cells

Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.

Alteration of red cell membrane organization in sickle cell anaemia

Bee venom phospholipase A2 and the fluorescent probe merocyanine 540 were used to examine plasma membrane phospholipid organization in the spicules released by deoxygenation and reoxygenation of sickle red cells, as well as in reversibly and irreversibly sickled erythrocytes. Digestion of phosphatidyl ethanolamine in spicules was comparable to that of phosphatidyl choline, and these structures were stained by the fluorescent probe. Both assays suggest that membrane lipid asymmetry is disrupted in spicules. The residual cells, from which the spicules were derived, retain the normal asymmetry in phospholipid distribution between the outer and inner leaflets of the plasma membrane bilayer. Comparable experiments with cell fractions enriched in irreversibly sickled cells revealed a partial enhancement of phosphatidyl ethanolamine digestion, confirming the similar experiments of Lubin et al (1981). Staining of these cells with merocyanine 540, however, did not reveal a subfraction of stainable cells, indicating that this increase in phosphatidyl ethanolamine digestion is not due to the presence of a small fraction of cells which have completely lost their membrane asymmetry.

Microvesicles from Sickle Erythrocytes and their Relation to Irreversible Sickling

Summary. Incubation of sickle (HbS) erythrocytes for periods up to 96 h leads to the formation of irreversibly sickled cells (ISCs) and to the release of spectrin‐free microvesicles similar to those derived from aged or Ca2+‐ionophore‐treated normal erythrocytes. The sickle microvesicles were somewhat larger than those from normal cells and showed minor differences in their membrane polypeptide composition. Sickle microvesicles were no different from their parent cells in their content of fetal haemoglobin. Neither microvesiculation nor formation of irreversibly sickled cells required the presence of Ca2+ in the medium but Ca2+ did accelerate both processes. Although in these prolonged incubations microvesiculation appeared to occur concomitantly with the formation of ISCs, it is not clear whether or not microvesiculation is a necessary prelude to irreversible sickling.

Gaucher's disease: A morphologic study and measurements of iron metabolism

Abstract A study of the intracellular location of Gaucher cerebroside, the deposition of iron and iron metabolism was made in a group of patients with Gauchers disease. Purified cerebroside obtained from normal tissue or from tissue of patients with Gauchers disease had an electron microscopic appearance identical with the cytoplasmic tubular structures observed within Gaucher cells, hence the tubular structures most likely are the morphologic representation of the cerebroside. Iron was present in all Gaucher cells, frequently in an increased amount, and body iron stores were moderately increased. No avidity between iron and cerebroside, however, was demonstrated. Iron metabolic studies were most consistent with mild ineffective erythropoiesis. The combined findings suggest that the iron in Gaucher cells comes from phagocytized red cells and support the concept that the erythrocyte is the source of the cerebroside in the Gaucher cell.

Acupuncture: An evaluation in the painful crises of sickle cell anaemia

&NA; An evaluation of acupuncture for pain relief was made in 10 patients with sickle cell anaemia during 16 pain crises. A model was developed in which the patient served as his own control and in which both patient and examiner were unaware of whether an acupuncture point or a sham site was treated. The results show (1) that pain relief was obtained in 15 of the 16 painful episodes regardless of whether an acupuncture point or a sham site was treated, demonstrating considerable overlap between the effects of needling acupuncture points and sham sites; (2) that needling at acupuncture points for pain relief is not significantly superior to treatment at sham sites; (3) that needling, per se, whether at acupuncture points or at sham sites can be useful for alleviating pain in sickle cell crises. The model could be useful for evaluation of pain relief by needling in other diseases.

Measurement of Iron Stores Using Deferoxamine

Abstract Deferoxamine (DFO)-induced urinary iron excretion was evaluated as a test for the measurement of iron stores. Iron stores were assessed by both phlebotomy and histologic techniques or by h...

The effect of spicules obtained from sickle red cells on clotting activity.

Summary. Spicules from sickle red cells were examined for their effects on the clotting activity of blood. The spicules were obtained from the sickle red cells after deoxygenation and oxygenation and were tested for clotting activity with Russells viper venom assay. A marked increase in clotting activity was observed when spicules were added to the system. The increase was distinctly greater than that observed after the addition of sickle red cells while normal red cells had little effect. Vesicles prepared from sickle or normal red cells by incubation with the ionophore A‐23187 + Ca2+ also markedly increased clotting activity. The effect of spicules or vesicles on the clotting system may be related to reorganization of phospholipid in the spectrin‐poor membrane of the spicules or vesicles. Because of these effects, the spicules from the sickle red cells may contribute to the hypercoaguable state in these patients and possibly to their vaso‐occlusive crises since free spicules are present in their plasma. Vesicles from red cells from other types of anaemia with hypercoaguability may have a similar effect on coagulation.

Assessment of painful episode frequency in sickle‐cell disease

Frequency of painful episodes in sickle‐cell disease is considered to be related to clinical severity and possibly to other aspects of the disease. Measurements of frequency often include only hospital‐related or more severe, longer‐lasting episodes. Since painful episodes, however, may regularly occur in nonhospital settings or be shorter‐lasting with possible different pathologic effects, we measured all painful episodes in 10 adults with sickle‐cell disease for 1.0–3.8 years, using a daily questionnaire. The results were related to other indices of disease severity and to possible precipitating factors, such as cold weather and menses. Sixty‐one percent (on average) of the total number of episodes (243) were nonhospital‐related, and 33% (on average) were shorter‐lasting. Episode frequencies, whether determined as total, hospital‐related, nonhospital‐related, or shorter‐lasting, were not related to each other or to other indicators of disease severity. The highest incidence of episode frequency occurred in the winter. The association of episodes with menses was moderately close in individual patients. The findings suggest that nonhospital‐related painful episodes and shorter‐lasting episodes may contribute significantly to episode frequency. Measurement of frequency of all painful episodes would require consideration when evaluating episode frequency and its relationship to disease severity, to possible precipitating factors of episodes, and to treatment of the disease, and for study of the natural course of the disease. Am. J. Hematol. 54:183–188, 1997

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. Am. J. Hematol. 65:174–175, 2000.