Fixation of Brain Tumor Biopsy Specimens with RCL2 Results in Well‐Preserved Histomorphology, Immunohistochemistry and Nucleic Acids

Abstract

RCL2 is an alcohol-based fixative reported to preserve histomorphology and nucleic acids in non-CNS neoplasms. We compared histomorphology, immunohistochemistry, DNA and RNA in brain tumor specimens preserved frozen at -80°C, and after formalin or RCL2 fixation. RCL2-fixed and paraffin-embedded (RCLPE) samples showed well-preserved histomorphology and specific immunoreactivity comparable to formalin-fixed and paraffin-embedded (FFPE) specimens testing a broad panel of antibodies. In all the analyzed cases, high-molecular weight DNA (up to a fragment length of 600 bp) was amplifyable from RCLPE samples, even after prolonged fixation times. Beta-actin (ACTB) and O6-methylguanine-methyltransferase (MGMT) gene concentrations were significantly higher in DNA isolated from RCLPE specimens as compared with FFPE specimens. Testing of MGMT promoter methylation status using methylation-specific polymerase-chain reaction (MSP) yielded conclusive results in 8/8 analyses in RCLPE and 6/8 analyses in FFPE material, respectively. Amplification of three reference genes (ABL, RAR-alpha, BCR-1) from cDNA showed good RNA preservation in frozen and RCLPE tissue specimens and significant RNA degradation in all FFPE samples. In conclusion, RCL2 fixation of brain tumor biopsies does not seem to significantly compromise histological tumor typing or immunohistochemistry and preserves nucleic acids (DNA and RNA) at a better quality than formalin fixation.