A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Purpose: Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. Methods: We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. Results: The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. Conclusion: We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

Paratonsilar Approach to Styloid Process in Eagle's Syndrome-A Retrospective Analysis.

Eagle's syndrome, defined by Eagle in 1949, is characterised by morphological abnormality/ossification of the styloid process. Traditionally styloid process is approached after tonsillectomy or through an invasive external cervical approach. In the present study we propose a novel Paratonsillar approach for elongated styloid process in eagle's syndrome. The study was retrospective analysis of the medical records of patients who underwent styloid process excision through paratonsillar approach over 2 years period. The present study included 26 patients who underwent paratonsillar styloid process excision. Elongated styloid process was mostly prevalent in the middle age group 30-50 years constituting 73% of patients included in our study. 65% patients had symptomatic elongated on the right side and 35% had on left side. In 62% patients the procedure was over in 15-30 min. There was no documented post operative complication. Paratonsillar approach for styloid process excision is a safe, minimally invasive, day care procedure with almost no postoperative complications.

"A Systematic Review on Guidelines and Recommendations for Tracheostomy During COVID-19 Pandemic".

World is under threat of COVID-19 pandemic, associated with many numbers of critically ill patients. To manage these intubated patients there are need of more ventilators but world is not prepared for this type of situation and there are lacunae of such arrangements in most of the countries. As we know patients cannot be intubated for long time and they should be given preference to alternative airway in the form of tracheostomy. COVID-19 is aerosol transmitted disease which lead to indeed challenge to health care providers to safely perform tracheostomy and provide post tracheostomy care to these patients with minimising risks of nosocomial transmission to themselves and accompanying nursing staff. There are so many guidelines and recommendations for the timing, desired place of tracheostomy, change in tracheostomy steps related to conventional method and the subsequent management of patients. So, the aim of this systematic review is to give a brief review of available data on COVID-19 related to the timing, personal protections, operative steps modifications, and subsequent post tracheostomy care during this pandemic.

Oral Malignant Melanoma.

To present a case of malignant melanoma. A 55-year old female presented with swelling over left upper gingiva which bled on brushing. She was diagnosed with left oral malignant melanoma on histopathology of punch biopsy specimen, which was then followed by C.T. P.N.S and Neck to know the lesion's extent and Ultrasonography of Abdomen and Pelvis to rule out any metastatic deposits. Patient was managed by left lower partial maxillectomy with left radical neck dissection. No intra-operative or post-operative complications seen. Patient was then sent for chemoradiotherapy. No any complications seen at fifteen months follow-up. Oral malignant melanoma is amenable to surgery and chemoradiotherapy with excellent results if detected at an early stage. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-021-02735-1.