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Formalin fixed paraffin embedded (FFPE) tissue samples - are they good enough for routine molecular clonality testing?

BACKGROUND-AIM: The diagnostic material for hematopathology, where the special tool is molecular clonality testing by polymerase chain reaction (PCR), almost exclusively consists of formalin fixed paraffin embedded (FFPE) tissue samples. Although there is better DNA quality Unauthenticated Download Date | 4/30/19 2:29 PM 5th EFLM Conference on Preanalytical Phase Zagreb (HR), 22-23 March 2019 eA67 in fresh tissues, getting DNA from FFPE tissues is still a challenge. The aim was the quality assessment of DNAs extracted from FFPE tissue samples for molecular clonality testing. METHODS: DNAs were extracted from 66 FFPE tissue samples by proteinase K digestion followed by alcohol precipitation. The quality assessment of extracted DNAs was performed by the BIOMED-2 multiplex PCR method resulting in a ladder of PCR fragments (100, 200, 300, 400 and 600) in base pairs (bp). The extracted undiluted and diluted DNAs (1:5) were amplified with Control Size Ladder (CSL) master mix and analyzed by agarose gel electrophoresis. The size of PCR fragment was an ultimate parameter for the correct interpretation of the results and for final conclusion. DNA of adequate quality was considered when PCR fragment size ≥ 200 bp was obtained and heavily degradated DNA when PCR fragment size was below 200 bp. RESULTS: During two years 66 DNAs were extracted from FFPE tissue samples. Most of them were of adequate quality with PCR fragment size ≥ 200 bp (61/66, 92, 4%). Heavily degradated DNAs were only few (5/66, 7, 6%). Amplified PCR fragments of max 400 bp was the upper limit for DNAs from FFPE tissue samples (21/61, 34, 4%), those of 300 bp were considered as very good quality DNAs (26/61, 42, 6%) and amplified PCR fragments of 200 bp as good quality DNAs (12/61, 19, 7%) that could be used for further molecular clonality testing. CONCLUSIONS: The protocol for DNA extraction from FFPE tissue samples was used appropriately and DNAs extracted from FFPE tissue samples were succesfully amplified. BIOMED-2 control gene PCR method showed as an objective and reliable tool for the quality assessment, but also essential for routine molecular clonality testing

FFPE tissue samples, DNA extraction, molecular clonality testing

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