Hereditary spherocytosis is the most common red blood cell (RBC) membrane disorder, that occurs in all ethnic groups, in Caucasians with the prevalence ranging from 1:2000 to 1:5000 (1). This disorder is highly heterogeneous in clinical presentation, inheritance, molecular basis and biochemical phenotype. HS is clinically characterized by anemia, jaundice, splenomegaly and gallstones. However the clinical severity of HS varies from symptom-free carrier to severe anemia. During microscopic examination of peripheral blood smear, RBC reveal spheroidal shape and the number of reticulocytes is significantly increased. The abnormal red cell morphology results in shortened cell survival due to RBC protein deficiency. Subjects with HS are characterized by inherited deficiency of one or several of membrane/cytoskeleton proteins: α-spectrin, β-spectrin, ankyrin, anion exchanger 1 (AE1, protein band 3) or/and protein 4.2. The molecular defect in one of the protein underlying HS is a cause of so-called primary deficiency. In some cases of HS more than one RBC membrane protein is deficient. This is because the primary protein defect “triggers” secondary protein deficiencies (2). Either a deficiency or dysfunction of one or more of RBC membrane proteins leads consequently to the detachment of the lipid bilayer from the spectrin-based cytoskeleton resulting in weakening of the vertical protein interaction (3, 4).

Single or combined protein deficiency in RBCs can be determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (5).

In this study we report red cell membrane proteins alterations in previously HS diagnosed patients of Polish population by densitometric analysis of SDS-PAGE separated proteins.

In our study we aimed: 1) to identify RBC membrane protein defects in Polish HS patients, 2) to analyze the distribution of membrane protein defects among members of analyzed HS families, and 3) to correlate the defined by SDS-PAGE protein deficiencies with the values of EMA test.

In this study 41 consecutive HS patients and 60 healthy blood donors as controls were investigated. Peripheral blood was collected from patients and controls during diagnostic procedures after obtaining informed consent and approval from the Bioethics Committee of the Centre of Postgraduate Medical Education. The procedures were followed in accordance with the Helsinki international ethical standards on human experimentation.

Patients were from 31 unrelated families: 19 males and 22 females, range of age 1.5-74 years; 31 patients were aged < 18 and 10 were adults. Only four (three adults and one < 18 y.a) patients were splenectomized before the time of the study. In all but two cases, HS was diagnosed on the basis of the clinical history, physical examination ant the results of the laboratory tests: positive EMA (eosin-5-maleimide) binding assay, complete blood count, blood smear examination, reticulocyte count. None of the patients had had transfusions within the 3 months preceding the study.

Blood samples of healthy blood donors were used as a controls. For each RBC ghost preparation blood of three blood donors was taken and after separation from plasma and WBC, RBC were pooled and treated as a control sample.

Peripheral venous blood samples (4-10 ml) drawn from the patients and controls were collected on EDTA as anticoagulant and if needed stored in 4°C until processed, but no longer than 24 h. Erythrocyte ghost were prepared according to the Dodge et al. (6) with minor modifications. Briefly, erythrocyte suspension was washed with phosphate buffered saline (pH 7.2), and then cells were lysed with 5 mM phosphate buffer (pH 8.0) supplemented with 0.2M PMSF and spun at 15 000 x g for 15 min, 4°C. The supernatant was removed and cells were washed by using lysis buffer until haemoglobin-free ghosts were obtained. Purified RBC ghost were frozen in small aliquots in -70°C. Sample protein concentration was determined using Roti-Quant (Roth). Isolated membranes were subjected to SDS-PAGE in the Laemli buffer system (7). Gels were loaded with 20 μg of total membrane proteins. Before loading samples were denatured for 45 min in 37°C and reduced with sample buffer containing 5% β-mercaptoethanol. Polyacrylamide gels (8%, Rothiphorese, Roth), were stained with 0.012% Coomassie blue (R-250, Roth) in 10% ethanol, 5% acetic acid and destained in 10% ethanol and 5% acetic acid. The electrophoretic analysis for each RBC membrane protein sample from HS patient was performed in duplicate gels. Each gel was loaded as follows: controls, n = 3 and HS n = 6. Quantitative analyses were performed using ImageJ 1.48v Windows Application (8). Each band was quantified as an area under densitometry curve and its quantity was expressed as percent of total, when for total the sum of bands: α-spectrin, β-spectrin, ankyrin, anion exchanger1 (AE1), protein 4.1, protein 4.2 and actin was accounted. The level of each analyzed HS protein was compared to controls and presented as percent of control, assuming the level of control as 1.

In this study 41 membrane protein samples of HS diagnosed patients have been analyzed. Quantitative analyses were performed using ImageJ 1.48v Windows Application (8). Each band was quantified as an area under densitometry curve and its quantity was expressed as percent of total, when for total the sum of bands: α-spectrin, β-spectrin, ankyrin, anion exchanger 1 (AE1), protein 4.1, protein 4.2 and actin was accounted. The level of analyzed RBC membrane protein was compared to controls and presented as proportional value, assuming the level of control as 1. As a relevant value of protein deficiency the deficit ≥ 5% was adopted.