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Comparison of cell fixation methods of induced sputum
specimens: an immunocytochemical analysis.J
Immunol Methods. 2006 Jan 20;308(1-2):36-42. Epub 2005 Nov
21.
Induced sputum
(IS) is a non-invasive method to evaluate airway inflammation.
Various techniques are used to fix IS cells but their respective
value has never been compared. We aimed to determine the best IS
cell fixation technique for cellular markers staining. Cells were
fixed using four methods: 1) periodate-paraformaldehyde-lysine (PLP)-sucrose,
2) paraformaldehyde 4% on slide and 3) in solution and 4)
acetone-methanol. Analysis was based on percentage of positive cells
compared to total cell counts stained by hematoxylin and quality of
staining. Using PLP-sucrose resulted in a higher percentage of
positive cells for CD3 and a better quality of staining.
Acetone-methanol showed a lower percentage of positive cells for
CD68 and a poor quality. PLP-sucrose gives the best results for the
preservation of the studied cell markers and acetone-methanol the
worst.
Identification of
intracellular markers in induced sputum and bronchoalveolar lavage
samples in patients with respiratory disorders and healthy persons.Respir
Med. 2002 Nov;96(11):918-26
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.
Cell specific markers for eosinophils and neutrophils in sputum and
bronchoalveolar lavage fluid of patients with respiratory conditions
and healthy subjects.Thorax.
2002 May;57(5):449-51.
BACKGROUND:
Highly specific protein markers for eosinophils and neutrophils
could be a valuable diagnostic aid in various respiratory disorders.
The cell specificity of monoclonal antibodies against eosinophil
peroxidase (EPO), eosinophil cationic protein (ECP), human
neutrophil lipocalin (HNL), and myeloperoxidase (MPO) was
investigated using immunocytochemical techniques. METHODS: Induced
sputum and bronchoalveolar lavage fluid samples from 14 patients
with respiratory conditions and four healthy individuals were
studied. Antigens were detected at their intracellular sites in
cells with well preserved structures using optimal techniques for
fixation, permeabilisation, and immunolabelling. RESULTS: Anti-EPO
antibodies reacted only with eosinophils, and anti-HNL antibodies
only with neutrophils. Anti-ECP antibodies reacted with both
eosinophils and neutrophils and anti-MPO antibodies with neutrophils
and monocytes. Cells not stained by monoclonal anti-EPO and anti-HNL
antibodies included lymphocytes, monocytes, macrophages, squamous
epithelial cells, and ciliated epithelial cells. CONCLUSIONS: EPO, a
unique component of eosinophils, and HNL, a unique component of
neutrophils, are useful markers for the identification of
eosinophils and neutrophils, respectively, in sputum and
bronchoalveolar lavage fluid. |
