Comparative proteomics of erythrocyte aging in vivo and in vitro.

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.

The red cell revisited--matters of life and death.

An erythrocyte-fractionating method combining volume and subsequent density separation is described. Iron isotope (59Fe)-validation proved this combination of methods to be complementary. By deploying HbA1c as cell age marker, obtained fractions demonstrated that circulating erythrocytes lose 20% of hemoglobin and membrane by shedding vesicles. Vesiculation from older cells proved to be facilitated by the spleen. Animal studies revealed that such vesicles are rapidly removed from the circulation by scavenger receptors on Kupffer cells with phosphatidylserine acting as the principal ligand. These studies reveal the existence of an alternative pathway of erythrocyte breakdown. This means that the premortal substrate of 20% of any erythrocyte is at our disposal. As this kind of vesiculation takes place during the entire erythrocyte lifespan, loss and sometimes reutilisation of marker substances limits the usefulness of isotope studies to the first half of the erythrocyte lifespan, thereby putting the dogmatic lifespan of 120 days into question. Furthermore, these studies add to the understanding of hemoglobin A1c (HbA1c) metabolism and the origin of the wide variation of erythrocyte parameters in peripheral blood. Removal of old erythrocytes from the circulation and from donor blood may open new ways into the treatment of both bilirubin and secondary iron overload.

Feasibility of a new in vitro approach to evaluate cellular damage following co-infusion of red blood cell concentrates and intravenous drug solutions.

INTRODUCTION: Transfusion guidelines may result in unwanted delay in infusion schemes, as simultaneous infusion of blood components and drug solutions is universally prohibited. The aim of this study was to measure possible damage to red cells by drug solutions, as manifested by haemolysis, using a dynamic model that resembles the clinical setting. METHODS: Stored filtered and irradiated RBC concentrates and drug solutions were co-infused in an in vitro dynamic model. Also, incubation in a static model was performed. The haemolytic potency of the drug solutions was measured by determining free haemoglobin (fHb) levels. RESULTS AND DISCUSSION: Neither in the dynamic tests nor in the static tests did fHb levels exceed the maximally acceptable standard for filtered RBC concentrates according to Dutch specification guidelines. In the static test model, fHb levels were slightly elevated compared with those of control samples, as well as those in the dynamic test model. CONCLUSION: A novel in vitro dynamic infusion system appears to represent a useful technique to calculate possible damage to RBCs resulting from co-infused drug solutions. Co-infusion of the drug solutions tested with filtered and irradiated RBC concentrates did not produce fHb levels above the levels accepted by the Dutch national guidelines. Apart from haemolysis, other parameters reflecting RBC damage should be investigated in future studies.

High prevalence of celiac disease in apparently healthy blood donors suggests a high prevalence of undiagnosed celiac disease in the Dutch population.

BACKGROUND: In the last few years the prevalence of celiac disease (CD) seems to have increased. It is clear that subclinical and silent CD exist in a large subgroup of the celiac population. METHODS: The aim of this study was to evaluate the prevalence of CD in an apparently healthy population. Blood samples were obtained from 1000 apparently healthy blood donors at Arnhem and Nijmegen Blood Donation Centers from January 1997 through April 1998. Sera from 660 blood donors were assayed for total IgA. By means of immunofluorescence, antibodies, including those to endomysium (EMA), were determined. Serum immunoglobulin levels (IgA) were assayed by means of nephelometry. All donors who had positive serology for EMA underwent small-intestinal biopsy. RESULTS: Of the 1000 healthy blood donors 3 had positive EMA. Small-intestinal biopsy of two of these showed subtotal villous atrophy (Marsh IIIb), and the third had intraepithelial lymphocytosis and crypt hyperplasia (Marsh II). The prevalence of gluten sensitivity was 1 of 330. Low IgA (0.60-0.23 g/l) in our study group was found in 9 of 660 (1%), but no one showed an IgA < or = 0.02 g/l. CONCLUSION: Our study shows that the prevalence of gluten-sensitivity in apparently healthy blood donors is 3 of 1000, which suggests a high prevalence of CD in the Dutch population, in contrast to the results of the last published Dutch epidemiologic studies. The recorded prevalence will increase further with greater recognition of subclinical and asymptomatic forms detected by screening tests.

Quantification of loss of haemoglobin components from the circulating red blood cell in vivo.

Previous studies have shown that a considerable amount of haemoglobin is lost from the intact red cell during its lifespan. The aim of this study was to determine the relative contribution of all the haemoglobin components to this process. Therefore, the relative amount of haemoglobins A0, A2, F and the glycated haemoglobins were determined in 24 fractions of different cell age. These fractions were obtained by the combination of counterflow and density centrifugation. When the absolute amount of all haemoglobin components were calculated using the MCH-values of each fraction, it appeared that the mean red cell loss of haemoglobins A0, A2, F, an unknown X and "rest" comprised, respectively, 440, 23, 1, 4 and 1 amol per cell, while the mean gain of the glycated haemoglobins was 84 amol per cell. This resulted in a net loss of 385 amol of haemoglobin per cell. One of the glycated haemoglobins (HbA1e2) turned out to be the product of further carbamylation. It was concluded that in the first half of the red cell lifespan HbA0 and HbA2 decreased by glycation and carbamylation and that in the second half some of the HbA0 and HbA2 but also some of the glycated and carbamylated haemoglobin components leave the red cell. The total loss amounted to about 20%.

Breast-cancer mortality in a non-randomized trial on mammographic screening in women over age 65.

Recent case-referent studies in the Nijmegen breast-screening programme have shown a reduction in breast-cancer mortality of approximately 50% due to screening of women aged 65 years and older. In this type of study, however, the results may be biased because of self-selection. The purpose of our present study was to compare the breast-cancer mortality rate in a population invited for screening with that of a reference population from an area without a screening programme. In 1977-1978, 6773 women aged 68-83 years were enrolled in the mammographic screening programme in Nijmegen, The Netherlands. The women were followed up until 31 December, 1990. The reference population consisted of women from the same birth cohort from Arnhem, a neighbouring city without mass screening, for whom the entry date was 1 January, 1978. The ratios of the Nijmegen and Arnhem breast-cancer mortality rates with 95% confidence intervals (CI) were calculated. In the study period, 173 patients were diagnosed with primary breast cancer in Nijmegen vs. 183 in Arnhem; 40 Nijmegen patients had died of breast cancer vs. 51 Arnhem patients. The cumulative mortality-rate ratio was 0.80 (95% CI = 0.53-1.22). In the periods 1978-1981, 1982-1985 and 1986-1990, the mortality rate ratios were 1.44 (95% CI = 0.67-3.10), 081 (95% CI = 0.37-1.79) and 0.53 (95% CI = 0.27-1.04), respectively. After adjustment for the difference in incidence rate that existed between the Nijmegen and Arnhem populations, mammographic screening of women older than 65 can be expected to yield a 40% reduction in breast-cancer mortality after 10 years.

Mammographic screening after the age of 65 years: evidence for a reduction in breast cancer mortality.

We evaluated whether regular mammographic screening of women aged 65 years or older affected breast cancer mortality. In Nijmegen, a population-based screening program for breast cancer was started in 1975, with biennial mammography for women aged 35-64 years. Since 1977, elderly women have also been participating. For the present case-control study, women were selected who were over 64 years of age at the most recent invitation. Eighty-two of them had died from breast cancer. For these cases, 410 age-matched population controls were selected. The ratio of breast cancer mortality rates of the women who had participated regularly (ie., in the 2 most recent screening rounds prior to diagnosis) vs. the women who had not participated in the screening was 0.56 (95% CI = 0.28-1.13). The rate ratio was 0.45 in the women aged 65-74 years at the most recent invitation (95% CI = 0.20-1.02), whereas it was 1.05 in the women aged 75 years and older (95% CI = 0.27- 4.14). While the breast cancer survival rate of the non-participant patients was fairly equal to that of patients from a control population, the underlying incidence rate of breast cancer was higher in the participants than in the non-participants. Therefore, we conclude that bias was present, but that it had decreased our effect estimate. The real reduction in breast cancer mortality due to regular screening will be even larger. Regular mammographic screening of women over age 65 (at least up to 75 years) can reduce breast cancer mortality by approximately 45%.

Effect on breast cancer mortality of biennial mammographic screening of women under age 50.

The effects on breast cancer mortality seen after 16 years of biennial screening of younger women are assessed in this prospective cohort study. Since 1975 some 13,500 women, aged 35-49 in 1975, were invited to participate in the Nijmegen screening programme comprising a mammographic examination every 2 years. By the end of 1990, 75 women had died of breast cancer out of the 332 cases diagnosed after the start of the screening project. Women from the same birth cohort, living in Arnhem, a neighbouring city with a comparable population and without a screening project, were used as controls. In this city, 74 breast cancer deaths out of 284 cases occurred during the same period. In Nijmegen, after 16 years of follow-up, breast cancer mortality showed a non-significant reduction of 6% (95% confidence interval: 32% reduction, 29% excess). In the relevant period, after a time lag of 10 years from the start of the programme, this reduction rose to 20% (95% confidence interval: 48% reduction, 23% excess). No reduction in breast cancer mortality was observed in the first decade of screening. For a later period, a shift towards a reduction emerges, but the data are as yet inconclusive.

Determinants of red blood cell deformability in relation to cell age.

Red blood cell (RBC) deformability was determined with an ektacytometer in fractions separated on the basis of differences in cell volume or density. Deformability was measured with ektacytometry (rpm-scan and osmo-scan). We studied three groups of RBC fractions:1. By counterflow centrifugation we obtained fractions of different cell age which showed a slight decrease in mean corpuscular haemoglobin concentration (MCHC) and an increase in surface-to-volume (S/V) ratio in fractions with older cells. 2. By Percoll fractionation fractions were obtained which showed a pronounced increase in (MCHC) but no change in S/V ratio. 3. By a combination of both fractionation techniques, fractions were obtained which showed an increased MCHC and an increase in S/V ratio. Deformability in group 1,2 and 3 showed respectively no change, a moderate decrease and a pronounced decrease in fractions of older cells. A decline in deformability occurs during the aging process of the red blood cell. This decline in deformability in old red cells is greater than originally thought. This decline is the result of an increase in haemoglobin concentration and a second factor, probably a decrease in membrane elasticity.

Characteristics of red blood cell populations fractionated with a combination of counterflow centrifugation and Percoll separation.

Red blood cell (RBC) fractions were studied after separation of whole blood by means of counterflow centrifugation, Percoll column (Pharmacia, Uppsala, Sweden), and a combination of both separation techniques. Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hemoglobin A1c (HbA1c) were measured in each fraction. From the results it was obvious that the combination of both techniques was the best separation technique of these three. MCV had a good correlation with cell age as measured with HbA1c concentration gradient; MCH and MCHC less so. MCV and MCH decreased in parallel to an increase in HbA1c. MCHC increased with increasing HbA1c. From these data it is concluded that there is a steadily ongoing loss of cellular hemoglobin and proportionally more cellular water during the life of the RBC.

[A survey of the epidemiology of breast cancer in The Netherlands]. / Een overzicht van de epidemiologie van borstkanker in Nederland.

Breast cancer is responsible for the main part of the total cancer incidence (+/- 30%) and cancer mortality (+/- 20%) among Dutch women. Due to aging of the Dutch population alone, the absolute numbers of breast cancer cases and deaths will increase considerably. Risk factors for breast cancer give no clues for primary prevention. By means of periodic screening patients are diagnosed at an earlier stage of disease, with a better prognosis. Even without a screening programme symptomatic women are diagnosed at earlier stages of disease. This paper gives a review of trends in breast cancer incidence and mortality in The Netherlands, as well as a risk profile of breast cancer patients and the relation between disease stage and survival. Its purpose is to obtain a better insight into the intended effects of the national breast cancer screening programme.

Evaluation of overdiagnosis of breast cancer in screening with mammography: results of the Nijmegen programme.

After 12 years of screening for breast cancer in Nijmegen (1975-86), during which period six mammographic examination rounds were carried out, the extent of overdiagnosis was evaluated. Overdiagnosis is defined as a histologically established diagnosis of invasive or intraductal breast cancer that would never have developed into a clinically manifest tumour during the patient's normal life expectancy if no screening examination had been carried out. The whole 12-year period shows an excess of 11% of breast cancer cases in Nijmegen, compared with the neighbouring city of Arnhem, where no mass screening was performed. The incidence of breast cancers in Nijmegen in the period 1975-78 is higher, compared with the incidence rates in Arnhem; the rate ratio is 1.30. For the time-intervals 1979-82 and 1983-86 the rate ratios are 1.03 and 1.01 respectively with (0.89; 1.18) and (0.86; 1.16) as 95% confidence intervals. This leads to the conclusion that there is no evidence that screening programmes using modern mammography constitute a significant risk for overdiagnosis of breast cancers.

Vacuoles in red cells from splenectomized subjects originate during cell life: association with glycosylated haemoglobin?

Whole blood from splenectomized subjects (n = 8) contained a substantial percentage of vacuolated ('pitted') red cells (34.7 +/- 8.3%), while blood from controls revealed none. The percentage of haemoglobin A1 (HbA1) had increased significantly compared with controls (p less than 0.01). Fractionation on cell density revealed that the number of 'pitted' cells and the HbA1 percentage were associated with increased cell density. Fractionation on cell volume showed that 'pitted' cells are equally distributed in fractions with varying mean cell volume (MCV) and that decreasing MCV was associated with a linear rise of HbA1. It appeared that, shortly after splenectomy (n = 4), 'pits' develop early in cell life and that also older cells, after previous splenic contact, are capable of pit formation. A positive correlation found between the number of 'pitted' cells and the percentage of HbA1 might point to an impaired release of HbA1, manifest in the presence of 'pits'. The increased percentage of HbA1 in whole blood from splenectomized subjects may thus be explained.

Reticulocyte crisis after splenectomy: evidence for delayed red cell maturation?

In whole blood from splenectomized subjects (n = 8) in a steady state an increased number of reticulocytes was observed (14.0 +/- 7.8% versus 3.6 +/- 2.4% controls: p less than 0.05). Cell fractionation on density showed that reticulocytes, as in normals, are more or less confined to the least dense fraction; cell fractionation on volume revealed that reticulocytes form a heterogeneous cell population with diverging volume, but sharing a low density. Immediately after splenectomy (n = 4), a reticulocytosis is observed, mainly manifest in the top fractions (262.0 +/- 49.0% versus 40.0 +/- 32.4% preoperatively; p less than 0.01). This reticulocytosis is not due to pre- or postoperative complications, but is associated with the splenectomy itself and probably is the consequence of a delayed, but ultimately occurring red cell maturation. The increase of reticulocytes is not accompanied by significant alterations in cell morphology, as far as cell volume is concerned.

Decreased iron absorption in patients with active rheumatoid arthritis, with and without iron deficiency.

Mucosal uptake, mucosal transfer, and retention of a physiological dose of ferrous iron were studied in women with active rheumatoid arthritis (RA): 19 with normal and 17 with depleted bone marrow iron stores. Control subjects were 26 normal women and 20 women with uncomplicated iron deficiency. Iron absorption was measured with 59Fe as a tracer and by whole body counting. Compared with controls, final iron retention was considerably decreased in both groups of patients with RA. Analysis of the two sequential steps of iron absorption showed that mucosal uptake was normal in iron replete patients with RA but was significantly lower in patients with RA with depleted iron stores compared with iron deficient controls. Mucosal transfer of iron was considerably decreased in patients with RA with normal iron stores. The impaired absorption of iron in patients with active RA may delay the correction of the haemoglobin concentration when anaemia of chronic disease is complicated by iron deficiency.

Alterations in size, shape and osmotic behaviour of red cells after splenectomy: a study of their age dependence.

In whole blood from splenectomized subjects (n = 20), red cells showed a significant increase of mean surface area (MSA), mean cell volume (MCV), MSA/MCV-ratio and osmotic resistance, with the mean cell haemoglobin concentration (MCHC) being decreased. Studies on red cell populations of different cell age revealed that the increase of MSA affects younger and older cells, whereas the increase of MCV can mainly be ascribed to young cells with low density. The increased osmotic resistance is mainly determined by older cells due to a more favourable MSA/MCV-ratio. Shortly after splenectomy (n = 5) the MSA of younger and older cells increased, whereas the increase of MCV affected only young cells with a lowered density; moreover, the MSA/MCV-ratio increased in older cells in particular, resulting in a relatively greater increase of osmotic resistance. An impaired maturation of the reticulocyte may underlie the initial increase of MSA and MCV of young cells, but the present results contradict the current view that delayed maturation explains the changes in morphology and osmotic resistance of asplenic red cells.