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Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic
W E Mesker1, C F Knepflé, J J Ploem-Zaaijer
1Department of Cytochemistry and Cytometry, Sylvius Laboratories, University of Leiden, The Netherlands.
Abstract:
The detection of human cytomegalovirus (HCMV) infected poly-morphonuclear leukocytes (PMNLs) for early finding of the pp65 antigen using automated image analysis has been improved. The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate. The number of automatically detected false positive objects due to incomplete inactivation of endogenous peroxidase and strong variations in counterstain intensity could be reduced by 81% using this AP staining method. The number of detected truly positive cells with both staining methods was not significantly different. Furthermore, a new image analysis system providing processing of colour images was evaluated. Since plain differences in absorption wavelength are required for colour segmentation, the red immuno-staining was combined with a green counterstain using methyl green. Screening of AP- instead of PO-stained slides in combination with colour segmentation resulted in a further reduction of the number of falsely detected alarms from 61% to 11%. Consequently, the sensitivity of the automated detection was improved. For AP staining detection of cells in frequencies of approximately one to one million was demonstrated. Screening for CMV-positive, alkaline phosphatase labelled cells using an image analysis system with colour segmentation resulted in a reduced false alarm rate, a better visual interpretation of the images and subsequently an increase in the sensitivity of the automated screening process.
Insights
Automated detection of human cytomegalovirus (HCMV) infected cells is improved by using alkaline phosphatase (AP) staining and colour image analysis. This new method significantly reduces false alarms, enhancing early HCMV diagnosis.
Area of Science:
- Virology
- Immunology
- Medical diagnostics
Background:
- Early detection of human cytomegalovirus (HCMV) is crucial for patient management.
- Automated image analysis for detecting HCMV-infected cells using pp65 antigen shows promise but faces challenges with false positives.
- Traditional immunoenzyme peroxidase (PO) staining methods can be affected by endogenous enzyme activity and counterstain variations.
Purpose of the Study:
- To improve the automated detection of HCMV-infected poly-morphonuclear leukocytes (PMNLs) by optimizing staining and image analysis techniques.
- To reduce false positive signals in automated screening for HCMV.
- To enhance the sensitivity and reliability of early HCMV diagnosis.
Main Methods:
- Replaced traditional PO staining with alkaline phosphatase (AP) staining using new fuchsin substrate.
- Implemented a new image analysis system capable of processing colour images.
- Combined AP staining with a green counterstain (methyl green) for colour segmentation.
Main Results:
- AP staining reduced false positive objects by 81% compared to PO staining.
- The number of truly positive cells detected remained comparable between AP and PO staining methods.
- Utilizing colour segmentation with AP staining further decreased false alarms from 61% to 11%, significantly improving automated detection sensitivity.
- Detection of cells at frequencies as low as one in a million was demonstrated with AP staining.
Conclusions:
- Alkaline phosphatase (AP) staining combined with colour image analysis offers a superior method for automated HCMV detection in PMNLs.
- This approach significantly reduces false alarm rates, leading to improved sensitivity and reliability in early HCMV diagnosis.
- The enhanced method allows for more accurate and efficient screening of CMV-positive cells, even at low frequencies.