Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Methodology for processing mastectomy and cryopreservation of breast cancer tissue in a resource- poor setting: A pilot study.

Author(s): Okoli UA, Okafor MT, Agu KA, Ndubuisi AC, Nwigwe IJ, Nna EO, Okafor OC, Ukekwe FI, Nwagha TU, Menkiti VC, Eze CO, Onyekwelu KC, Ikekpeazu JE, Anusiem CA, Mbah AU, Chijioke CP, Udeniya IJ

Publication: Cryobiology, 2020, Vol. 97, Page 179-184

PubMed ID: 32562613 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of cold ischemia  (20-45 min) and frozen storage duration (0, 7, or 14 days) on the morphology, cell viability, and RNA yield and purity of a breast tumor tissue specimen. Specimens were collected from a woman diagnosed with invasive, ductal, triple negative breast cancer undergoing a mastectomy. Resected tumor tissue was cut into 2-3 mm3 pieces and fixed immediately in 10% buffered formaldehyde for histopathological review (details not provided) or placed in 4°C Hanks Balanced Salt Solution within 1 min to investigate effects of cold ischemia. To investigate effects of cold ischemia, 3 specimen pieces were snap-frozen after 3 minute intervals of cold ischemia (range: 20-45 min; 20, 23, 26, 29, 32, 35, 38, 42, and 45 min) in cryovials containing a freezing medium (10% Me2SO4 and 90% heat inactivated fetal bovine serum) in liquid nitrogen vapor, and then stored at -80°C for 0, 7, or 14 days. Tissue stored frozen for 7 and 14 days were fixed in 10% buffered formaldehyde, paraffin embedded, and H&E stained for histopathological analysis and results were compared to specimens fixed immediately after collection. Specimens stored frozen for 0, 7, and 14 days were assessed for tissue viability using a modified MTT Proliferation Assay Kit protocol.  RNA was extracted using the RNeasy Mini Kit, quantified by Qubit, and purity assessed by spectrophotometry (OD 260 nm/280 nm).

   Summary of Findings:

   Tissue morphology and viability of snap-frozen tumor specimens from a single patient subjected to up to 45 min cold ischemia and stored frozen for 7 or 14 days before analysis were comparable to tumor specimens processed immediately after collection. Overall, mean RNA yield per tissue was 8.47 ng/mg ± 2.28 (mean ± SD) and the A260/A280 ratios were between 1.7 and 1.9. RNA yield and purity of both the fresh and frozen breast tumor tissues from the patient did not differ significantly, regardless of cold ischemia time or frozen storage duration, (P>0.05, all).

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Colorimetric assay
   RNA	Spectrophotometry
   RNA	Fluorometry
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	20 min 23 min 26 min 29 min 32 min 35 min 38 min 42 min 45 min
   Storage	Storage duration	0 days 7 days 14 days
   Biospecimen Preservation	Type of fixation/preservation	Formaldehyde Snap frozen

   View more