The hemolysis kinetics of psoriatic red blood cells
Introduction
Psoriasis is linked to plasma membrane alterations in different types of cells and that pathological changes are not limited only to the cell membrane of keratinocytes [1], [2], [3], [4]. Psoriasis is associated with several red blood cell (RBC) membrane alterations such as: membrane fluidity decrease [2], [3], [4], significant elevation of Na+-K+pump activity [5] and quantitative changes of erythrocyte membrane proteins, especially spectrin deficiency and increase in band 4 protein [6]. These changes may indicate red cell cytoskeleton impairment. Our previous results revealed that the decrease of membrane fluidity corresponded with exacerbation of skin lesions [4]. The biochemical composition and molecular organization of erythrocyte membranes are strictly related to their biophysical properties. Modifications in composition or molecular organization can affect rheological properties, such as cell deformability [7]. In fact, we found that in psoriatic erythrocytes the main erythrocyte rheology parameter-erythrocyte deformability was affected [8]. Among different factors involved in RBC deformability, membrane fluidity has been assigned a major role [7], [9].
The aim of the present study is to analyse the hemolitic behaviour of psoriatic RBCs. Using a variety of methods based on hemolysis one may obtain some information concerning cell membrane stability and flexibility that reflects erythrocyte deformability. Several hemolysis tests, e.g., estimation of the osmotic fragility and the autohemolysis tests have been used as the diagnostic standard for many years [10]. The osmotic fragility test is a commonly used technique to detect changes in the erythrocyte shape and membrane flexibility as well as being a rough index of red cell surface area-to-volume ratio [11]. Membrane rigidity (flexibility) is therefore considered to be one of the determinants of red cell osmotic fragility. On the other hand, flexibility of the cell membrane is also one of the major factors determining erythrocyte deformability [12]. Therefore, analysis of the osmotic behaviour of RBCs can provide information concerning red cell deformability.
In the present experiment RBC deformability has been measured by osmotic gradient ektacytometry [13], [14]. This technique allows cell deformability to be measured in response to applied shear stress as a function of medium osmolality. As was shown by Clark et al. [13] the measurement of the whole cell deformability in isotonic and various hypotonic media by the use of laser diffractometry is a sensitive method capable of detecting alterations in surface area-to-volume ratio.
The osmotic behaviour of RBCs was examined by analysing the red cell kinetics of hemolysis. The method is based on the measurement of the transmitted light (λ = 700 nm) scattered by a suspension of red blood cells, while the cells are exposed to osmotic stress in the stopped-flow regime [15].
Section snippets
Preparation of blood samples
Venous blood samples were obtained from a group of 23 patients with moderate psoriasis aged 46 ± 9 years and from a control group of 45 healthy donors from a Blood Centre aged 40 ± 11 years. The samples were drawn in sterile vacuum tubes containing anticoagulant (K3EDTA). RBCs were separated from the blood by centrifugation at 1500 ×g for 15 min. and then washed three times with PBS buffered saline (pH 7.4). Each sample of washed erythrocytes was prepared for further experiments.
Kinetics of hemolysis
Washed RBCs were
Statistical analysis
The data are expressed as means ± S.D. The statistical analysis used the unpaired Student's t-test. The value P < 0.05 was considered as significant.
Results
Selected examples of the experimentally-derived dependence of transmittance on time for control and psoriatic samples are presented in Fig. 1. The value of parameter A for psoriatic erythrocytes was lower than for control cells (Fig. 2). The fraction of hemolyzed erythrocytes for control samples was about 20% and for psoriatic ones 12.6%. The difference was statistically significant (P < 0.001). The fast kinetic component described by the exponential B⁎exp(-t/Tfast) of psoriatic erythrocytes was
Discussion
A comparison of the erythrocyte hemolytic kinetics of blood samples from psoriatic patients and healthy subjects showed distinct differences which can be quantitatively evaluated. The fitting of experimental data to a mathematical model [15], [16] allowed us to estimate the fraction of cells that undergo lysis (parameters A, B and C) and/or the haemolytic properties of the cell plasma membrane (parameters Tfast and Tslow).
The value of parameter A can be correlated with overall blood sample
Conclusions
The results of this study showed that the kinetics of hemolysis may be a promising method for detecting erythrocytes with defective plasma membrane components and/or cytoskeleton. The measurement of this phenomenon can not only be informative with regard to the extent of membrane disturbance but also with regard to cell functioning, since changes of the membrane structure and properties result in the alteration of cell metabolism [18], [22]. Furthermore, the biochemical composition and
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