Uticaj formalina i parafinskog bloka na fragmentaciju molekula DNK u humanim tkivima izuzetih prilikom izvođenja sudskomedicinskih obdukcija
The influence of formalin fixation and paraffin embedding on DNA fragmentation in human tissues excluded during medico-legal autopsies
Doktorand
Vitošević, KatarinaMentor
Todorović, DanijelaČlanovi komisije
Milošević-Đorđević, OliveraAlempijević, Đorđe
Sazdanović, Maja
Metapodaci
Prikaz svih podataka o disertacijiSažetak
Cilj ove studije je bio da se ispita efekat formalina i parafinskog bloka na
kvalitet DNK izolovane iz različitih zdravih tkiva izuzetih tokom
sudskomedicinskih obdukcija. Korišćena su zdrava tkiva srca, jetre, mozga, pluća i
bubrega koja su fiksirana u 10% puferizovanom ili 4% nepuferizovanom formalinu
od 6h do 2 meseca, kao i parafinski blokovi zdravih tkiva srca, jetre i mozga
arhivirani od 1988-2017. godine. Morfološke odlike tkiva su proučavane nakon
H&E bojenja preparata. DNK je izolovana pomoću fenol-hloroform-izoamil alkohola
(PCI) i komercijanog kita. Čistoća i prinos DNK su određeni spektrofotometrijski.
Integritet izolovane DNK je određen PCR amplifikacijom GPD1 (150bp), ACTB
(262bp) i RPL4 (407bp) gena. Na histomorfološke karakteristike tkiva ne utiču ni
fiksiranje tkiva u formalinu ni kalupljenje u parafinski blok. Ekstrakcija DNK iz
uzoraka tkiva PCI metodom omogućuje dobijanje DNK boljeg kvaliteta od DNK dobijene
korišćenjem kita. Bolji kvalitet DNK ...je u uzorcima tkiva fiksiranih u 10%
puferizovanom formalinu. Ne postoji razlika u čistoći i integritetu DNK između
različitih fiksiranih tkiva, već samo u prinosu: najveći prinos DNK je dobijen u
uzorcima iz tkiva jetre, a najmanji iz srca i mozga. Dužina fiksiranja utiče na
prinos i integritet DNK: do 72h fiksiranja je moguće amplifikovati fragmente
dužine do 407bp, a do 14 dana fragmente do 262bp. Najveći prinos DNK je dobijen iz
ukalupljenog tkiva jetre, a iz tkiva srca i mozga je izlovana DNK očuvanijeg
integriteta. Dužina arhiviranja parafinskih blokova tkiva utiče na kvalitet DNK:
RPL4 je amplifikovan do 6 godina, ACTB do 26 godina i GPD1 do 30 godina
arhiviranja parafinskih blokova tkiva. DNK izolovana PCI metodom iz tkiva srca i
mozga koja su fiksirana u 10% puferizovanom formalinu ima najbolji kvalitet, pa su
ova tkiva pogodana za molekularno biološke analize i nakon 30 godina arhiviranja.
The aim of this study was to examine the effect of formalin and paraffin block on the quality
of DNA isolated from various healthy tissues excluded during medico-legal autopsies.
Healthy heart, liver, brain, lung and kidney tissues fixed in 10% buffered or 4% unbuffered
formalin from 6h to 2 months were used, as well as paraffin blocks of healthy heart, liver and
brain tissues archived from 1988-2017. Morphology of tissues was studied using H&E
staining. DNA was isolated using phenol-chloroform-isoamyl alcohol (PCI) and commercial
kit. The purity and yield of DNA were determined spectrophotometrically. The integrity of
the isolated DNA was determined by PCR amplification of GPD1 (150bp), ACTB (262bp)
and RPL4 (407bp) genes. The histomorphological characteristics of the tissue are not affected
by tissue fixation in formalin or embedding into a paraffin block. Extraction of DNA from
tissue samples by PCI method enables obtaining better quality of DNA than DNA obtained ...
using commercial kit. Better DNA quality is obtained from samples isolated from tissues
fixed in 10% buffered formalin. There is no difference in the purity and integrity of DNA
between different tissues, but only in yield: the highest DNA yield was obtained in samples
from fixed liver tissues, and the lowest from the heart and brain. The duration of fixation
аffects on yield and integrity of DNA: fragments up to 407bp in length can be amplified up to
72h of fixation, and fragments up to 262bp in length up to 14 days of fixation. The highest
yield of DNA was obtained from embedded liver tissue and DNA of more preserved integrity
was extracted from heart and brain tissue. The duration of archiving of paraffin tissue blocks
аffects the quality of DNA: RPL4 is amplified up to 6 years, ACTB up to 26 years and GPD1
up to 30 years of archiving paraffin tissue blocks. DNA isolated by PCI method from heart
and brain tissues fixed in 10% buffered formalin has the best quality, so these tissues are suitable for molecular biological analysis even after 30 years of archiving.