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© Borgis - New Medicine 1/2008, s. 13-15
*Jolanta Kłopocka
Flow cytometric analysis of eosin-5-maleimide binding to protein of band 3 and Rh-related proteins in diagnosis of hereditary spherocytosis
Department of Biophysics Medical Centre of Postgraduate Education Warsaw, Poland
Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS).
Interaction of EMA with the ĺ-NH2 group of lysine in band 3 protein was previously reported.
The aim of this work was to introduce modified flow cytometric analysis of eosin-5-maleimide binding to protein of band 3 and Rh-related proteins as the basis of a screening test in diagnosis of hereditary spherocytosis.
Materials and methods
Red cells from 12 patients with membrane deficiencies in HS (spectrin in 4 patients, band 3 in 3 patients, protein 4.2 in 2 patients and combined spectrin/ankyrin in 3 patients) and from 26 healthy volunteers.
Flow cytometric analysis of chemically modified EMA-labelled red cells.
Fluorescence intensity of EMA-labelled HS red cells is sensitive to changes
in the relative amounts of band 3, Rh AG, CD 47 and Rh proteins.
Binding of EMA to the integral proteins (band 3, RhAG, CD 47 and Rh proteins) implicated in red cell cytoskeleton underpins the high specificity of the flow cytometric test for HS

The biochemical basis of red cell membrane cytoskeletal defects associated with hereditary spherocytosis (HS) is heterogeneous and connected with deficiency of proteins such as spectrin, ankyrin, protein 4.2 and band 3 [1, 2, 3, 4, 5]. These proteins can be tested by the sodium dodecyl sulfate polyacrylamide gel electrophoresis method (SDS–PAGE) [6], which is currently the reference laboratory test for the identification of membrane protein deficiencies in HS.
The reaction between eosin-5-maleimide (EMA) and the ĺ-NH 2 group of Lys-430 on erythrocyte band 3 protein was first used as the basis for the flow cytometric analysis of band 3 in HS patients [7, 8, 9]. Eosin-5-maleimide binds stoichiometrically to band 3 on intact red cells. Thus, a reduction of fluorescence intensity indicates a quantitative reduction of erythrocyte band 3.
A decrease of fluorescence intensity was also detected with spectrin and protein 4.2 deficient cells. The inability to detect ankyrin-deficient HS red cells may suggest that either location of ankyrin within the cytoskeleton is distal to the EMA binding site of band 3 or that ankyrin is ´immobilized´ within the cytoskeleton.
Densitometric analysis of red cell membrane components after SDS-PAGE is also known to be insensitive to estimating the ankyrin content [6] because of the compensation effect produced by ankyrin content in reticulocytes and young red cells that are present in greater numbers in non-splenectomized HS patients. The gel method is strictly a quantitative technique. It lacks sensitivity in detecting a slight protein deficiency in mild HS samples containing a mixture of normal and abnormal erythrocyte proteins.
The dye method is more sensitive in detecting a mixed population of red cells in a blood sample because flow cytometry analyses fluorescence intensity of single cells (King 2000) [9]. The dye method is a reliable, rapid diagnostic test (2 h from sample collection to result) and can serve well as a screening test for the diagnosis of HS. In patients with typical features of HS, diagnosis of HS can be made if the fluorescence intensity of EMA-labelled red cells falls within the reference range for HS red cells [9].
Further investigations showed that EMA also reacts with the exofacial sulfhydryl group on intact red cells (heat labile reaction) [10, 11, 12, 13]. Chemical modification of EMA binding in the flow cytometric method, i.e. selectively blocking amine and sulfhydryl groups on red cells, was used for further investigation of EMA binding [11].
Materials and methods
Red cells from 12 patients with HS: 4 patients with spectrin deficiencies, 3 with band 3 deficiencies, 2 with protein 4.2 deficiencies and 3 with combined spectrin/ankyrin deficiencies. Red cells from 26 healthy volunteers as normal controls, 4 samples of Rh null red cells used as negative controls in EMA binding.
Flow cytometric analysis of EMA-labelled intact red cells from normal, Rh null and hereditary spherocytosis was performed.
Chemical modifications: N-ethylmaleimide (NEM) and acetic anhydride were used to block reactive sulfhydryl and amine groups, respectively, on intact red cells. Rh null red cells lack the two thiol-containing Rh proteins (Mr 30-32 kD); therefore they were used as a control for the absence of fluorescence in the 32 kD region.
The average mean channel fluorescence (MCF) reading for normal red cell samples was 20% greater than that of Rh null red cell samples, whereas the fluorescence of HS red cells was 30% lower than that of normal red cells, confirming an earlier observation [9].
NEM treatment of normal red cells resulted in 25% decrease in EMA fluorescence, whereas this treatment only produced a small difference in fluorescence intensity between untreated and NEM-treated Rh null red cells.

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Adres do korespondencji:
*Jolanta Kłopocka
Department of Biophysics
Medical Centre of Postgraduate Education 01-813 Warsaw, 99 Marymoncka Str. tel.: 022 569 38 21 e-mail: biofmat@cmkp.edu.pl

New Medicine 1/2008
Strona internetowa czasopisma New Medicine