Comparison of formalin and FineFIX in preserving DNA material in small biopsies

To the Editor: Formalin is a routine fixative in surgical pathology wards. Although morphological assessment is very desirable in formalin-fixed materials, DNA and RNA are not preserved well and molecular studies cannot be performed with reliable results. Biopsies and surgical specimens, as well as postmortem tissue samples, represent an important resource, especially for studies of molecular epidemiology, rare disease, neuropathology, and case studies with very long follow-up periods. A major advantage of using paraffin-embedded material is that it is easier to collect tissues from already existing archives in comparison to frozen fresh tissues that need a specific sample collection, with dedicated spaces and specific equipment. Because formalin fixation results in poor preservation of DNA and RNA, there are efforts to introduce new molecular friendly fixatives by some investigators. Recently a new fixative named FineFIX, which is ethanol based, has been introduced (Milestone, Sorisole, Italy) but few published articles exist about its advantages in improving molecular analysis. Nowadays many small samples are taken during endoscopy or needle biopsies and sent to pathology laboratories, and these may be the only available specimen for future molecular studies. We found no published data on DNA studies in small samples, so we decided to examine this fixative at a molecular level and compare it with formalin in small biopsies. Ninety small gastric biopsies, similar in size, taken during endoscopy, were put in formalin and in FineFIX in equal numbers, in our laboratory (Pathology Department, Children Medical Center affiliated with Tehran University of Medical Sciences). Due to the small size of the biopsies we could not divide each sample into two halves. Duration of fixation was equal for the two groups. Standard processing was performed, and HEand Giemsa-stained slides were prepared for each case, and those of unsuitable quality and quantity were excluded from the study. The morphology of two groups was compared among the two methods. Seventy biopsies were selected for the study (20 samples were excluded because they were superficial or very small). DNA was extracted from 5 mm sections of both FineFIX and formalin fixed-paraffin wax-embedded tissues, until no tissue remained in the blocks (Genomic DNA extraction kit, Cat. No: k3032, Lot No: 0602; Bioneer Inc., Alameda, CA, USA). Each sample was amplified using specific primers for human b2-microglobulin. Polymerase chain reaction conditions (PCR) and amplicon lengths are listed in Table 1. Polymerase chain reaction (PCR) was performed in a 50 mL final volume using standard conditions (Dynamic BioSciences (PouyaZistech), Tehran, Iran; kit Cat. No. KP175). Every reaction included 50–100 ng of DNA, 2 mL of each primer, which were added to lyophilized mastermix composed of KCl, MgCl2, Tris HCl, dNTP and Taq DNA polymerase. The following amplification program was used for every PCR reaction analysis: initial denaturation at 94°C for 3 min; 40 cycles composed of denaturation, at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min. Final extension was performed for one cycle for 3 min at 72°C. Statistical analysis was performed using c test for comparison of the two groups. Morphological studies of the two groups indicated shrinkage of tissue in the FineFIX group, which is undesirable but does not interfere with final diagnosis (Fig. 1). Mast cell count in gastric biopsies, which is performed routinely in our lab for Giemsa-stained slides, could not be done due to degranulation of mast cells or staining quality in the FineFIX group, which has not been previously reported. In the formalin-fixed group 20 of 35 specimens had a b2-microglobulin band (248 bps), but 30 out of 35 had this band in the FineFIX group (Fig. 2). The difference between these two groups in preserving DNA for this gene was statistically significant (P = 0.008). The PCR reaction for 10 specimens with no amplification product was repeated but the results were the same. Although formalin is a good preservative for morphological purposes, molecular study, especially in small biopsies, is not