Title
Evaluation of various methods to quantify endothelial cells attached to vascular prostheses: Comparison with a new "gold standard" FACS method
Author
Visser, M.J.T.
van Lennep, A.C.D.
van Bockel, J.H.
van Hinsbergh, V.W.M.
van der Keur, M.
Hermans, J.
Gaubius Instituut TNO
Publication year
1996
Abstract
For in vitro evaluation of functional properties of endothelial cells seeded on synthetic vascular prostheses accurate and reproducible quantification of cells is mandatory. Comparison of these properties with those resulting from other studies requires correlation of the functional parameters to reliably counted cell numbers. The accuracy of methods of quantification currently being used is unknown due to the lack of a "gold standard" method to which these methods can be compared. To determine the accuracy and reproducibility of four widely used methods, we have developed a "gold standard" model, using a flow cytometer (FACS). Endothelial cells, attached to collagen-coated Dacron vascular prostheses, were counted by four conventional methods and a new method of quantification after the attached number of cells had been determined with 99% accuracy by FACS. Subsequently, ratios were computed by dividing the cell numbers determined by the methods under investigation by those determined by FACS (×100%). The four conventional methods investigated were (1) removal and subsequent counting of cells from substrata by trypsin (T), (2) digestion of cells by citric acid and counting of crystal violet-stained cell nuclei (CV), (3) light microscopy after hematoxylin staining (LM), and (4) scanning electron microscopy (SEM). The new method consists of the measurement of cell fluorescence after labeling with fluorescein-diacetate (FDA). T and CV had average accuracy ratios of 127 ± 58% and 96 ± 48%, respectively (± standard deviation). The ratios for LM and SEM were 116 ± 101% and 44 ± 10% (respectively). FDA had a ratio of 99 ± 7%. Reproducibility of cell quantification by T and CV was significantly less than that of quantification by LM, SEM, and FDA, as expressed by data on inter- and intraobserver agreement. Our results indicate that the investigated conventional methods of quantification failed to meet criteria of both high accuracy and reproducibility. Light microscopy and scanning microscopy methods were inaccurate but yielded reproducible countings. We conclude that the FACS method can serve as a "gold standard" to compare the accuracy and reproducibility of cell quantification methods. Moreover, the FDA method results in both accurate and reproducible quantification of endothelial cells attached to vascular prosthetic material. © 1998 Academic Press, Inc. Chemicals/CAS: 3',6'-diacetylfluorescein, 596-09-8; Fluoresceins
Subject
Biology
citric acid
collagen
crystal violet
fluorescein diacetate
hematoxylin
trypsin
article
cell adhesion
cell count
dacron vascular prosthesis
endothelium cell
fluorescence activated cell sorter
human
human cell
microscopy
priority journal
reproducibility
scanning electron microscopy
umbilical vein
Blood Vessel Prosthesis
Cell Adhesion
Cell Separation
Cells, Cultured
Cytological Techniques
Endothelium, Vascular
Flow Cytometry
Fluoresceins
Humans
Microscopy, Electron, Scanning
Observer Variation
Reproducibility of Results
To reference this document use:
http://resolver.tudelft.nl/uuid:d335de6f-ea74-461f-8fd1-75dfec9f235b
DOI
https://doi.org/10.1006/jsre.1996.0110
TNO identifier
233223
ISSN
0022-4804
Source
Journal of Surgical Research, 61 (1), 237-243
Document type
article