Optimal Papanicolaou Smear Conditions for Manual Microdissection of Single Target Cells

Abstract

This study aimed to investigate the optimal conditions for Papanicolaou (Pap) smear to increase the success rate of target cell isolation through manual microdissection (MMD) and prevent cell spread. Pap smears were prepared using an HPV42-positive SurePath™ liquid-based cytology case, and 46 and 50 koilocytes were used in wet and dried Pap smears, respectively, to verify the success rate of target cell isolation using MMD based on the HPV detection rate. During MMD, the microscopic examination of both specimens revealed that cells in dried smears could be easily identified; however, cell debris remained in the surrounding area after MMD. Although it was difficult to observe cells in wet smears, there was no cell debris. When the needle tip was immersed in DNA lysate after cell isolation through MMD, a difference in cell solubility was found between dry and wet smears. HPV42 was detected in 94.7% and 97.4% of dried and wet Pap smears, respectively, via polymerase chain reaction genotyping using lysed cell solution; the detection rates were not significantly different. The isolation of target cells from wet Pap smears using MMD reduced the risk of contamination and increased the success rate of HPV detection. This study might facilitate the identification of new CIN-derived HPV-infected cells using MMD with wet Pap smears.

Keywords: PCR-based HPV testing; Papanicolaou smear; human papillomavirus; manual microdissection.

Figure 1

Before and after the isolation of single target cells from the Papanicolaou (Pap) smear. (a,c): Before the isolation of target cells using the manual microdissection (MMD) method. Arrows indicate koilocytes. (Pap stain, ×100); (b,d): after isolation of target cells using MMD (Pap stain, ×100). (a,b) Single target cells isolated via MMD from dried Pap smears. (c,d) Single target cells isolated via MMD from Pap smears wet with 100% ethanol.

Figure 2

Needle tip before and after dipping it in an alkaline lysis solution. Single target cell (arrow) isolated using MMD is visible at the needle tip. (a,c): Cell isolated from dried Pap smear was present at the needle tip. (e,g): Cell isolated from wet Pap smear adhered to the sides of the needle. (b,d,f,h): Needle tip images after immersing in it an alkaline lysis solution. The target cell was completely lysed and disappeared. After heat treatment, this lysed cell solution was used as a DNA template.