Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic staining methods with respect to colour segmentation.
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 The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate.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.
Mesker
W E
WE
Department of Cytochemistry and Cytometry, Sylvius Laboratories, University of Leiden, The Netherlands.
Knepflé
C F
CF
Ploem-Zaaijer
J J
JJ
Schipper
N W
NW
Boland
G J
GJ
Tanke
H J
HJ
D003587:Cytomegalovirus / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis*; D006098:Granulocytes / Q000821:virology*; D006801:Humans; D007089:Image Enhancement; D007124:Immunoenzyme Techniques; D013194:Staining and Labeling / Q000379:methods