Potential Diagnostic Application of the Quantitative Assessment of Red Blood Cell Membrane Protein Expression

Special Article - Red Blood Cells

Thromb Haemost Res. 2019; 3(2): 1024.

Potential Diagnostic Application of the Quantitative Assessment of Red Blood Cell Membrane Protein Expression

Várady Gy1, Szabó E1,2, Kulin A1, Zámbó B1, Mózner O1, Pálinkás M3, Poór Gy3 and Sarkadi B1,4*

1Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok krt. 2, Budapest, Hungary, 1117

2Biospirál-2006 Kft., Temesvári krt 62. Szeged, Hungary, 6726

3National Institute of Rheumatology and Physiotherapy, Frankel Leó u. 25-29. Budapest, Hungary,1023

4Department of Biophysics and Radiation Biology, Semmelweis University, Tuzoltó u. 37-47, Budapest, Hungary, 1094

*Corresponding author: Sarkadi B, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudósok krt. 2, Budapest, Hungary; Email: sarkadi@biomembrane.hu

Received: May 06, 2019; Accepted: May 31, 2019; Published: June 07, 2019

Abstract

We have recently developed an efficient flow cytometry-based method which allows to quantitatively determine the expression levels of selected Red Blood Cell (RBC) membrane proteins. Interestingly, RBCs express several hundred membrane proteins, the expression of which is determined by the individual genetic background and complex regulatory effects. Many of these membrane proteins are closely linked to the development of human diseases or diseasesusceptibilities, thus the determination of specific RBC protein expression levels may help to perform a patient-specific, stratified diagnosis. In this technology we generate RBC ghosts with a simple fixation procedure, then use monoclonal antibodies optimized to recognize specific RBC membrane proteins. The data obtained in selected groups of patients and control subjects are compared, and the genetic basis of the alterations in membrane protein expression is further examined by performing specific SNP and mutation analysis from genomic DNA samples. In this mini-review we summarize our recent studies related to changes in RBC protein expression in gout patients, and describe an interesting discovery based on the RBC expression studies of a plasma membrane calcium pump.

Keywords: Red blood cells; Flow cytometry; ABCG2; Gout; PMCA; Malaria; Biomarkers

Abbreviations

FACS: Fluorescence-Activated Cell Sorter; GWAS: Genome- Wide Association Studies; RBC: Red Blood Cell; SNP: Single Nucleotide Polymorphism; WGA: Wheat Germ Agglutinin

Introduction

Methodology for assessing RBC membrane protein expression and its medical role

Relevant RBC membrane proteins are selected based on the literature (red blood cell CD antibodies, GWA and medical diagnostic studies), and these data are compared to Mass Spectrometry (MS) measurements and the data in the erythrocyte database [1-3]. For Flow Cytometry (FACS) measurements, RBC ghosts (hemoglobin depleted erythrocyte membranes) are prepared from a small drop of blood and these ghosts are labeled by a fluorescent lectin derivative (e.g. wheat germ agglutinin-AlexaFluor647, WGA-A647) to allow selective gating. Thereafter, the ghosts are labeled with specific monoclonal antibodies, then with suitable fluorescent secondary antibodies (for details see [3,4]). The measurements are carried out in a flow cytometer, preferably equipped with automatic high throughput plate sampler.

In order to obtain quantitative results in the FACS assay, each membrane protein and each selective first antibody has to be carefully titrated to give maximum level interactions. Still, the results of the FACS measurements do not provide an actual number of the given membrane protein per RBC, and data have to be evaluated based on the protein levels obtained in a certain population or under specific disease conditions.

The advantage of the FACS assay lies in the quick and cost-efficient method that requires only a small amount of blood. However, a major issue in this work is the selection of proper antibodies for trustworthy, quantitative membrane protein measurements. In most cases no information is available about the use of commercially available antibodies in flow cytometry, and the quantities of most of the red blood cell membrane proteins are also unknown. In this latter regard, recently improving quantitative mass spectrometry measurements may provide proper estimates [1,2,4]. Still, for the quantitative detection of a particular RBC membrane protein, the use of at least two different antibodies, favorably recognizing different epitopes of the same protein, are preferred. In our research projects we have examined numerous commercially available monoclonal antibodies, potentially suitable for the characterization of RBC membrane proteins that is showing specific and sensitive detection of membrane protein levels even at low protein concentrations (Table 1).