Implementation of a real‐time reference and calibration grid platform for improved screening – mapping in Pap test slides

Abstract

Cervical cancer screening based on the Papanicolaou (Pap) test is a widely applied but not always efficient practice for detecting Human Papillomavirus (HPV) mediated lesions, partially due to a non-systematic and inadequate screening process. Our aim was to introduce an inexpensive easy-to-use direct screening platform for improved detection of abnormal cells indicative of underlying cervical neoplasia as well as persisting HPV infection. By employing a novel, efficient technique of laser-based micromachining, we achieved the fabrication of spatial grids on commercially available coverslips allowing visual segmentation of the slide for efficient screening. Abnormal and formerly diagnosed as negative for intraepithelial lesion or malignancy (NILM) Pap test slides (n = 200) were analyzed by conventional and grid-based screening. Grid-based microscopy led to a more reliable diagnosis compared to the conventional by identifying an increased number of abnormal cells (P = 0.001). It decreased borderline ASCUS, AGC diagnosis, increasing LSIL, HSIL and in situ AdenoCa detection rates closely related with biopsy (P = 0.015; kappa = 0.978). Concerning the set of NILM diagnoses in rapid re-screening, the method upgraded six cases (n = 6) to LSIL (P = 0.001). The proposed technical solution offers a calibration and orientation visual aid during the on-site screening process providing significant advantages compared to expensive digital imaging techniques.

Comparative p16INK4A Expression in Laryngeal Carcinoma and Cervical Cancer Precursors: A Real-time Grid-based Immunocytochemistry Analysis

“…Finally, the current experimental study is characterized by the pilot implementation of an innovative reference and calibration grid on conventional coverslips in the screening process of p16 ICC stained slides. Recently this technique has been successfully performed in cervical screening based on Pap test slides (8). Our study group strongly supports its introduction in routine cytology as a significant improvement in eye-based slide scanning, in detecting abnormal cells that modify diagnoses especially in cytological borderline cases.…”

“…Laser techniques can allow direct writing and transfer of predetermined patterns by means of surface or sub-surface micromachining in a variety of materials ranging from glasses to soft polymeric materials. The technique of femtosecond laser micromachining (FLM) inscription was applied towards the fabrication of a customized visible rectangular grid, on the coverslip of microscope slide (8). The applied FLM inscription set-up is composed by an infrared laser source, generating laser pulses of ~350 fs pulse duration at ~1,064 nm wavelength.…”

“…The laser beam passes through a power control system in order to adjust its power at the required level, and then it is directed by an optical mirror onto the coverslip flat surface, which is mounted on a three-dimensional XYZ-axis computercontrollable translation stage, for the inscription. The optical delivery system of the FLM set-up provides accurate control of laser writing characteristics, such as exposure time, laser beam spot size, laser power density and total delivered laser energy, allowing consequently the accurate control of the laser-based defined grid structures, in terms of geometrical, optical, and surface quality properties (8). That enables the full customization of thickness, and depth, of grid lines in the glass coverslips providing optimum visibility in microscope screening of the corresponding slides, without covering and hiding any vital cytological or biological information.…”

Impact of Chromosome 9 Numerical Imbalances in Oral Squamous Cell Carcinoma: A Pilot Grid-Based Centromere Analysis

“…The grid’s size and observation windows’ density could be modified according to the researcher’s needs. Based on our previous experience in Pap test slide screening-mapping, we selected a prototype grid consisting of seventy-two ( n = 72) rectangular squares of a typical surface area of 4 × 4 mm, equal to 16 mm 2 , arranged in 12 columns and 6 rows [ 27 ]. Each square segment can have also appropriate spatial-indexing marks (printed with various techniques like FLM) in a suitable way to assure minimum visual interference under microscope inspection ( Figure 1 b).…”

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis

“…Concerning the CISH slides, screening was based on a set of cover slips integrated with spatial rectangular grid (now GCS) in order to perform a systematic way of slide eye-scanning. We used in this study a novel technique of micromachining with the use of ultra short Laser pulses at the femtosecond pulse duration regime (Femtosecond Laser Micromachining-FLM) in combination with high precision translation systems (Tsiambas and Riziotis, 2017). Laser techniques can allow direct writing and transfer of predetermined patterns by means of surface or sub-surface micromachining in a variety of materials ranging from glasses to soft polymeric materials.…”