Evaluation of the optimal provision of formalin-fixed, paraffin-embedded material for reverse transcription-PCR in soft-tissue tumour diagnosis.
Author(s): Thway K, Wren D, Lee J, Thompson L, Fisher C, Gonzalez D
Publication: J Clin Pathol, 2017, Vol. 70, Page 20-24
PubMed ID: 27207012 PubMed Review Paper? No
Purpose of Paper
The aim of this paper was to determine if RNA extraction of formalin-fixed paraffin-embedded (FFPE) scrolls or slide-mounted sections affects qRT-PCR success rates.
Conclusion of Paper
The beta-2 microglobulin (B2M) qRT-PCR amplification failure (> 30 Ct value) rate in soft tissue malignant and benign tumor specimens was 13.4% when scrolls were used for extraction (collected in 2011, five 10 µm thick scrolls), 4.4% when five 5 µm thick slide mounted sections were used (collected in 2012), and 7.9% when one 10 µm thick slide-mounted section was used (collected in 2013). However, the sample sets differed in the proportion of biopsy to excision specimens (2011= 3:15; 2012= 2:5; 2013= 13:3) and storage duration and processing regimes differed among the groups. Further, some of the observed effects the authors postulate may be due to too much or too little material being used for the extraction method. The authors noted that all specimens that failed to produce qRT-PCR results had a 100% success rate by FISH analysis. Potential effects of FFPE processing regime, and storage durations and conditions of FFPE blocks, scrolls, and slide-mounted sections were not investigated. The authors concluded that RNA extraction is most effective when thin slide-mounted FFPE sections from surgical excisions were used, and that the type of specimen (biopsy versus surgical excision), the percent tumor cellularity, and formalin fixation and paraffin processing regime employed should be taken into consideration when choosing specimen format.
The aim of this paper was to determine if RNA extraction of formalin-fixed paraffin-embedded (FFPE) scrolls or slide-mounted sections affect qRT-PCR success. Samples used for RNA extraction included several different types of benign and malignant soft tissue tumors (alveolar rhabdomyosarcoma, embryonal rhabdomyosarcome, spindle cell sarcoma, synovial sarcoma, intramuscular myxoma, fibrosarcoma, fibromyxoid sarcoma, fibromatosis, melanoma, liposarcoma, Ewing sarcoma, sclerosing epithelioid fibrosarcoma, chondrosarcoma, myxoid adipocytic tumor, small cell sarcoma) that were preserved by formalin fixation and paraffin embedding, and included both core needle biopsies and surgical excisions. FFPE specimens that were processed as five 10 µm thick scrolls were collected and preserved in 2011 (209 specimens), those processed as five 5 µm thick slide-mounted sections were from 2012 (158 specimens), and those processed as one 10 µm thick slide-mounted section was from 2013 (203 specimens). Although the authors noted that formalin fixation and paraffin embedded processes improved over the course of the study, details on formalin fixation, paraffin processing, storage duration and conditions of FFPE blocks, scrolls, or slides were not provided. The method of RNA extraction and purification were not provided but remained constant for all FFPE specimens analyzed. RNA quality was determined by technical success or failure of the beta-2 microglobulin (B2M) transcript by qRT-PCR. Technical failure was defined by a cycle threshold (Ct) of > 30.
Summary of Findings:
The rate of qRT-PCR amplification of B2M failure (> 30 Ct value)in soft tissue malignant and benign tumor specimens was 13.4% when scrolls were used for extraction (collected in 2011, five 10 µm thick scrolls), 4.4% when five 5 µm thick slide mounted sections were used (collected in 2012), and 7.9% when one 10 µm thick slide-mounted section was used (collected in 2013). Each sample set differed in the proportion of biopsy to excision specimens (2011= 3:15; 2012= 2:5; 2013= 13:3), storage duration and potentially processing regime all of which may have been a confounding factor in the qRT-PCR technical failure rate. The authors postulate that RNA extraction columns may have been saturated when 50 µm of FFPE tissue collected by excisions were used for RNA extraction (for specimens collected in 2011). Conversely, extraction of RNA from a single 10 µm thick slide-mounted section may have resulted in technical failure due to an insufficient amount of tumor from biopsy specimens. The authors noted that B2M was detected by FISH analysis in all of the specimens with B2M qRT-PCR CT values >30. Potential effects of FFPE processing regime, and storage durations and conditions of FFPE blocks, scrolls, and slide-mounted sections were not investigated.
- Neoplastic - Carcinoma
- Neoplastic - Benign
- Neoplastic - Sarcoma
Analyte Technology Platform RNA In situ hybridization RNA Real-time qRT-PCR
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume Five 10 µm thick scrolls
Five 5 µm thick scrolls
One 10 µm thick slide-mounted section
Real-time qRT-PCR Specific Targeted nucleic acid B2M
Biospecimen Acquisition Method of tissue acquisition Core needle biopsy