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.

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.

Counterflow centrifugation of red cell populations: a cell age related separation technique.

Red cell populations were fractionated on the basis of differences in density by a centrifugation method described by Murphy and on the basis of differences in mean cell volume (MCV) by counterflow centrifugation. By 59Fe-incorporation and determination of the HbA1c content, both methods were studied for their ability to separate red cell populations into fractions of different mean cell age. It can be concluded that separation on the basis of differences in cell volume results in a linear separation according to age whereas separation on the basis of density only results in an accumulation of very young cells in the top fractions. A gradual decrease in cell volume with age, combined with a constant haemoglobin concentration in the cells, indicates release of haemoglobin from the red cells during their lifespan.

Membrane characteristics and osmotic fragility of red cells, fractionated with anglehead centrifugation and counterflow centrifugation.

Red cell populations were separated on the basis of differences in density using anglehead centrifugation and on the basis of differences in mean cell volume using counterflow centrifugation. In the different fractions, mean surface area was calculated, phospholipid and cholesterol content determined as well as the osmotic behaviour in hypotonic salt solutions. Older red cells appeared to be more resistant to hypotonic salt solutions, due to favourable surface area to volume ratio.