Vpliv različnih dejavnikov na čas dekalcinacije kostnih tkivnih vzorcev

Ličen, Anja (author), Locatelli, Igor (mentor), Pižem, Jože (comentor)

Kosti dajejo našemu telesu oporo, varujejo notranje organe ter so zaloga kalcijevih in fosforjevih ionov. V histopatološkem laboratoriju so vzorci kostnega tkiva podvrženi dekalcinaciji, saj ta omogoči njihovo zmehčanje. Zmehčanje je potrebno, da lahko kostne vzorce režemo na mikrotomu. Poznamo dva najpogostejša načina, dekalcinacijo s kislinami in dekalcinacijo s pomočjo kelatorjev. Kisline so lahko močne ali šibke, kot kelator pa se uporablja EDTA (etildendiamintetraocetna kislina). Po končani dekalcinaciji gredo vzorci skozi običajen postopek obdelave histoloških vzorcev. Diagnozo poda zdravnik po pregledu preparatov in rezultatov molekularno genetskih preiskav. Prvi namen magistrske naloge je bil s pomočjo statistične analize ugotoviti, kako različni dejavniki vplivajo na čas dekalcinacije. Kot drugo nas je zanimalo, če se čas dekalcinacije z uporabo mikrovalovne pečice in segrevanjem na 50°C z reagentom MoL-DECALCIFIER® (10% raztopina EDTA proizvajalca Milestone) skrajša v primerjavi s segrevanjem na samo 37°C v istem reagentu oziroma brez segrevanja z reagentom TBD (komercialno pripravljena raztopina mravljične kisline proizvajalca Thermo Fischer Scientific). Pregledali smo 587 izvidov vzorcev prejetih na Inštitut za patologijo Medicinske fakultete v Ljubljani med leti 2017 in 2020 ter opravili statistično analizo s programom SPSS. Ugotovili smo, da na čas dekalcinacije statistično značilno vplivajo način določanja končne točke dekalcinacije, vrsta dekalcinacije, razlike pa lahko vidimo tudi med posameznimi vrstami vzorcev. Čas dekalcinacije je najkrajši pri dekalcinaciji z EDTA (MoL-DECALCIFIER®) v mikrovalovni pečici, vendar moramo pri tem vedeti, da z EDTA dekalciniramo predvsem vzorce, ki so že v osnovi mehkejši kot tisti, ki jih tretiramo s TBD. Čas merimo v dnevih, saj le redko kateri vzorec potrebuje samo nekaj ur za dekalcinacijo. Tudi pri neposredni primerjavi časa dekalcinacije, ko smo isti vzorec tretirali z EDTA v mikrovalovni pečici na 50°C ter v sušilniku na 37°C oziroma s TBD na sobni temperaturi, smo ugotovili, da so bili časi dekalcinacije z EDTA v mikrovalovni pečici krajši. Ta način smo tudi vpeljali v rutinsko uporabo histopatološkega laboratorija Inštituta za patologijo, Medicinske fakultete v Ljubljani.

• Language: Slovenian
• Keywords: dekalcinacija, EDTA, TBD, skrajšanje časa, vnaprejšnje določanje, segrevanje
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-148535
• Publication date in RUL: 26.08.2023

Secondary language

• Language: English
• Title: Influence of various factors on decalcification time of bone tissue samples

Abstract

Bones give our body support, protect internal organs, and are a storehouse of calcium and phosphorus ions. In the histopathology laboratory, bone samples are decalcified allowing them to soften. Softening is needed so the tissue samples can be cut on the microtome. The two most common methods of decalcification are decalcification with acids and decalcification with chelators. Acids can be strong or weak and the chelator EDTA (ethylenediaminetetraacetic acid) is being used. After decalcification is complete, the samples go through the usual process as any other histological samples. The diagnosis is made by pathologists after they review all the slides and get the results of the molecular tests. In the master's thesis, we first used statistical analysis to determine what influence different factors have on the time of decalcification. Secondly, we wanted to find out if the time of decalcification can be short-ed - using a microwave oven and heating to 50°C with EDTA reagent compared to heating to 37°C in a drying oven with the same reagent or with TBD (commercially prepared solution of formic acid from Thermo Fischer Scientific) on room temperature. We reviewed 587 results of the samples which were brought to the Institute of Pathology Faculty of Medicine, Ljubljana, and did the statistical analysis in the SPSS program. The type of decalcification and how we determine the end point of it have a statistically significant influence on the time of decalcification. When we compared individual types of samples we also found that differences can be also seen. The time of decalcification was the shortest when samples were treated with EDTA in the microwave oven, but here we need to emphasize that samples that undergo decalcification with EDTA are usually softer than those treated with TBD. Time is usually measured in hours as samples rarely need only a few hours to decalcinate. In the second part, we treated the same sample with EDTA in the microwave at 50°C and in the drying oven at 37°C or with TBD at room temperature. The time of decalcification was shortened when the sample was treated in the microwave oven. This method was also introduced into the routine work of the histopathology laboratory of the Institute of Pathology, Faculty of Medicine in Ljubljana.

• Keywords: decalcification, optimization, reduction of time, in advance determination, heating