Air drying as a preparatory factor in cytology: Investigation of its influence on dye uptake and dye binding

Schulte, Erik

doi:10.1002/dc.2840020213

“…The IOD of nuclei fixed in alcohol fixatives is higher than IOD of air-dried nuclei in all tissue types, which is in agreement with previous reports [19,20,24]. The possible explanation for higher values of alcohol fixed nuclei was offered in previous reports, which stated that alcohol precipitates the nuclear proteins and more binding sites are recovered on the DNA for the stain.…”

Section: Discussionsupporting

confidence: 91%

The Effect of Primary Fixation with Standard Postfixation and the Duration of Hydrolysis on Nuclear Features in Image Cytometry

Fležar

¹

,

Doudkine²,

Us‐Krašovec

³

1998

Analytical Cellular Pathology

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The effect of three primary fixation procedures, used in the preparation of routine cytological samples: air-drying, Delaunay, and Saccomanno fixation, with postfixation in modified Böhm–Sprenger fixative, on nuclear features as a function of hydrolysis time is reported. Three different cell types: lymphatic cells (tonsil), epithelial cells (buccal mucosa) and mesenchymal cells (uterine myometrium) were used for the study. Our findings show, that generally not all features have the same plateau times as the IOD (integrated optical density), and that many features show different values depending on cell type and fixation method. It is therefore recommended that for any primary fixative used in routine clinical work and for each cell type, the hydrolysis curve for all nuclear features to be used in sample analysis should be established.

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“…Differences in the segmentation procedure between programs and glare (having a larger effect on smaller dense particles) may explain the generally higher IOD values in lymphocytes than in granulocytes. In general, critical remarks on DNA image cytometry should address not only instrumentation but also sampling (10) and specimen preparation (2,3,6,9,15).…”

Section: Discussionmentioning

confidence: 99%

Quality assurance in DNA image analysis on diploid cells

Thunnissen

¹

,

Ellis

²

,

Jütting

³

1997

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DNA image analysis is presently performed in many laboratories. Before general extrapolation of results between different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis. The aim of this study was to establish the performance of different DNA image analysis instruments when measuring different diploid cells. On three separate parts of the same object slide, human liver cells, white blood cells, and imprints of a breast fibroadenoma were sampled. In this quality assurance interlaboratory study, 13 laboratories participated voluntarily. Two slides were sent to each participating laboratory: one Feulgen and one unstained to be stained according the participating laboratory in‐house procedure. The features integrated optical density (IOD) and object AREA were recorded for each nucleus. For calculation of the results, the average IOD value of liver diploid cells was set at 2c. A striking difference was observed between the different presumed diploid cell types, from almost 1c to 3c. This variation was not dependent on central or in‐house staining. Although in‐house calibration was performed for each image analysis system, a surprisingly large variation existed in the reported object AREA, irrespective of the cell type. These results clearly demonstrate that measurements performed in one laboratory cannot be extrapolated to the outcome of others and support the need for standardization. The use of external control cells works well for comparison of instruments. In conclusion, in DNA image analysis quality control is necessary, standardization is obligatory, and the use of an internal control for determination of the diploid peak in a histogram of patient samples is recommended. Cytometry 27:21–25, 1997. © 1997 Wiley‐Liss, Inc.

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“…Because different objectives are used, the distribution error varies (13). In general, critical remarks on DNA image cytometry should address not only instrumentation but also sampling (10) and specimen preparation (2,3,6,9,15). Differences in the segmentation procedure between programs and glare (having a larger effect on smaller dense particles) may explain the generally higher IOD values in lymphocytes than in granulocytes.…”

Section: Discussionmentioning

confidence: 99%

Quality assurance in DNA image analysis on diploid cells

Thunnissen

¹

,

Ellis

²

,

Jütting

³

1997

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DNA image analysis is presently performed in many laboratories. Before general extrapolation of results between different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis. The aim of this study was to establish the performance of different DNA image analysis instruments when measuring different diploid cells. On three separate parts of the same object slide, human liver cells, white blood cells, and imprints of a breast fibroadenoma were sampled. In this quality assurance interlaboratory study, 13 laboratories participated voluntarily. Two slides were sent to each participating laboratory: one Feulgen and one unstained to be stained according the participating laboratory in-house procedure. The features integrated optical density (IOD) and object AREA were recorded for each nucleus. For calculation of the results, the average IOD value of liver diploid cells was set at 2c. A striking difference was observed between the different presumed diploid cell types, from almost 1c to 3c. This variation was not dependent on central or in-house staining. Although inhouse calibration was performed for each image analysis system, a surprisingly large variation existed in the reported object AREA, irrespective of the cell type. These results clearly demonstrate that measurements performed in one laboratory cannot be extrapolated to the outcome of others and support the need for standardization. The use of external control cells works well for comparison of instruments. In conclusion, in DNA image analysis quality control is necessary, standardization is obligatory, and the use of an internal control for determination of the diploid peak in a histogram of patient samples is recommended. Cytometry 27:21-25, 1997. r 1997 Wiley-Liss, Inc.Key terms: DNA image analysis; interlaboratory study; quality control; reproducibility; staining DNA image analysis is persently performed in many laboratories. Before general extrapolation of results among different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis.The feasibility of interlaboratory studies on DNA image analysis in Europe had been examined as part of a voluntary quality assurance program within an EECfunded concerted action in analytical cytometry. The results showed that (a) this type of study may facilitate improvement in quality standards, (b) the precision of the image analysis systems was usually of high standard, and (c) the accuracy was 2-17% (17). Because one organ, the liver, was used and in the imprints the diploid values were used for interlaboratory calibration for this study, the question remained as to how well different image analysis systems performed when different diploid cells were compared.For an adequate interpretation of the DNA histogram, the precise location of the diploid peak can be determined on external or internal controls. In image analysis, several cell types have been used as external control including rat liver cells, lymphocytes and granulocytes (7,14,18,1...

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Air drying as a preparatory factor in cytology: Investigation of its influence on dye uptake and dye binding

Cited by 18 publications

References 16 publications

The Effect of Primary Fixation with Standard Postfixation and the Duration of Hydrolysis on Nuclear Features in Image Cytometry

The Effect of Primary Fixation with Standard Postfixation and the Duration of Hydrolysis on Nuclear Features in Image Cytometry

Quality assurance in DNA image analysis on diploid cells

Quality assurance in DNA image analysis on diploid cells

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