Decalcification options of bone material for consequential molecular biological and cytogenetic diagnosis – from the experimental model to trepanobiopsy

Molecular-biological and cytogenetic analysis of bone material has today a unique place in many areas of medicine. It is essential for the diagnosis of some lymphoproliferative disorders, and also for biomedical research. In biomedical research, the analysis concerns mainly the tissue engineering field, where mesenchymal stem cells differentiated into osteoblasts are used as a substitute in bioinplantology. Diagnostic in pathology includes especially the diagnosis of lymfoproliferative disease, mainly lymphomas, including both histological and immunohistochemical examination. But subtyping on the basis of cytogenetic and molecular-biological methods, detection of faults, translocations or amplification, is necessary.

For bioptical analysis and diagnostic ultra thin slices are required. Quality of these slices is dependent on superior material decalcification. However, most decalcifying techniques (e.g. formic acid) irreversibly damages the DNA and blocks subsequent molecular subtyping. Formic acid protons purine ring and thereby reduces the glycosidic bond to form covalent bonds. Furthermore, in the tissue degrades DNA to a string of less than 650 bp.

For decalcification of trepanobioptic rollers - diagnosis and experimentally for a mouse femur (Mus musculus) EDTA/ NaOH (10 mol/l - 40%) protocol was used (EDTA - ethylenediaminetetraacetic acid CH₂N (CH₂CO₂H) 2,2 - polyaminokarboxylic acid, which binds divalent cations). NaOH reacts with acid in form of ethylendiamine acid disodium salt (EDTA). This salt is more soluble in water than the acid itself. In reaction with metal ions (Mn+) such as Ca2+ complex ions of (MY) type are formed. We did the decalcification for three days on the shaker, changing EDTA every five hours and then rinse with water. Subsequently, the tissue was processed according to the protocol and paraffin blocks were prepared.

The trepanobioptic rollers were processed by FISH using different Visis FISH probes (Abbott), to determine the translocations. DNA purification using QIAamp DNA Mini Kit (Qiagen) and subsequent DNA amplification using the IdentiClone IGH Gene Clonality Assay kit (InVivoScribe) for the detection of the Ig heavy chains genes remodeling was performed.

A mouse femur was cut up and classical histological H/E staining was done to verify bone structure preservation. Afterwards Visis probe LSI BCR / ABL Dual Color Translocation EC Probe (Abbott) was used for cytogenetic examination.

All molecular methods were fully applicable and highly reproducible with very good visualization on specimens decalcified with this method. It can be concluded that this decalcification method is gentle, with tissue well preserved for routine histological staining and DNA remains in a condition suitable for further molecular biological and cytogenetic analysis.