Identification of intracellular markers in induced sputum and bronchoalveolar lavage samples in patients with respiratory disorders and healthy persons.

Abstract:

:Induced sputum and bronchoalveolar lavage (BAL) are widely used for retrieving cells and soluble materials for studies of airway inflammation. Centrifuged cell samples are suitable for immunochemical identification of cellular products. The aim was to determine the optimal fixation procedure to detect intracellular antigens in situ. In immunocytochemistry, an appropriate choice of fixation method is a prerequisite for identification of cells and, consequently, for reliability results. We compared eight fixation and permeabilization methods to detect intracellular antigens in cytocentrifuged cell samples. Four granular proteins specific to eosinophils (eosinophil cationic protein, ECP; eosinophil peroxidase, EPO) and neutrophils (human neutrophil lipocalin, HNL; myeloperoxidase, MPO) were the antigens studied. We found that the organic solvents often used in immunocytochemistry are unsuitable fixatives for detection of these intracellular low-molecular-weight proteins. Treatment with crosslinking fixatives alone resulted in incomplete penetration of antibodies into the cell interiors. Best results were obtained using a commercial reagent Ortho PermeaFix (OPF) for flow cytometry. With this, fixation and permeabilization take place simultaneously OPF-treated cells retained their structural characteristics, and the antibodies studied penetrated both cellular and granule membranes. With OPF treatment, ECP EPO, HNL, and MPO were fixed on their places in granules, and their antigenicity was retained. Correct identification of intracellular proteins is important in characterization of the respiratory inflammatory response in clinical work and research.

journal_name

Respir Med

journal_title

Respiratory medicine

authors

Metso T,Haahtela T,Sevéus L

doi

10.1053/rmed.2002.1373

subject

Has Abstract

pub_date

2002-11-01 00:00:00

pages

918-26

issue

11

eissn

0954-6111

issn

1532-3064

pii

S0954-6111(02)91373-9

journal_volume

96

pub_type

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