Overall, though, double staining failed to show any significant improvement. technical aspects of double staining, especially the effects of antigen retrieval, give hope that this technique could be applied to other immunocytochemical staining that would possess a greater ability to improve ploidy analysis. strong class=”kwd-title” Keywords: Ploidy, Early malignancy detection, Cervical malignancy, Quantitative image cytometry, Proliferation, Immunocytochemistry, Heat-mediated antigen retrieval Intro Cervical malignancy is the third most commonly diagnosed malignancy in females globally.1 While testing programs based on the Papanicolaou (Pap) smear have significantly reduced mortality due to cervical malignancy in industrialized nations,1C3 more than 85% of instances today arise in low-resource settings, making cervical malignancy the second-leading cause of cancer death among women in developing countries.1,2 This presents a distinct 7-Amino-4-methylcoumarin challenge to establishing cervical testing programs where they may be needed most, as testing programs 7-Amino-4-methylcoumarin based on the Pap smear require an extensive and costly infrastructure, in addition to significant teaching and skill to interpret the patient slides. Potential alternatives include visual inspection with acetic acid4 and human being papillomavirus (HPV) screening.5C9 Unfortunately, visual inspection methods continue to rely on adequate training of practitioners while rollout of HPV testing programs in low-resource settings has been hindered by cost and logistics.10 Moreover, high-risk HPV testing has a higher sensitivity but lower specificity for detecting high-grade cervical precancers than conventional cytology11 and low-resource settings are particularly sensitive to the follow up costs of false positive cases. We have previously demonstrated that ploidy analysis using Feulgen-thionin staining performs comparably with standard cytology and HPV screening for detecting cervical high-grade lesions.12 Further study suggests that ploidy and HPV mRNA may be indie predictors of cervical dysplasia.13 However, the amount of DNA present within the nucleus of a normal cycling cell changes as it progresses through the cell cycle. A normal cycling cell can be diploid, tetraploid, or somewhere in between. Frankly irregular cells ( 2.5 times the normal complement of DNA) are rare and occur in widely disparate and very low frequencies, even in high grade squamous intraepithelial lesions (HGSIL).14C16 Hence, ploidy might be an improved biomarker for cervical cancer screening if normal dividing cells could be distinguished from abnormal non-cycling cells by using an immunostain for Ki-67 like Rabbit polyclonal to KIAA0317 a marker of cell proliferation. Ki-67 is an antigen indicated in the nuclei or on chromosome surfaces during all active phases of the cell cycle (ie. all except G0).17 As such, it has been used for many years like a proliferation marker and in the assessment of many cancers,17C21 including cervical malignancy.22C25 Previous attempts to simultaneously determine DNA content and assess proliferation status in the same cell have relied heavily on fluorescent labeling recognized by flow cytometry, in which abnormal cell identification is hindered from the uncertainty of only individual cell passage through the flow 7-Amino-4-methylcoumarin cytometer, which can mistakenly detect signals from non-cellular material, especially when seeking to detect a relatively rare event.26,27 We instead propose to use 7-Amino-4-methylcoumarin absorbance stains on slide-mounted samples. Absorbance stains are permanent and less costly to image, as a simple light microscope will suffice. A slide-based assay would enable the study of a wider range of sample types without picking up signals from non-cellular material. By double staining cervical cytological specimens, normal cycling cells can be removed from the analysis, focussing on those cells whose abnormal DNA content might be indicative.