Ethylene Diamine Tetra Acetic Acid (EDTA) Decalcification of Paediatric Bone Marrow Trephines In a Diagnostic Laboratory

In paediatric patients with haematological disorders such as acute lymphoblastic leukaemia (ALL), bone marrow aspiration is sometimes difficult to obtain and bone marrow trephine biopsy (BMTB) is a valuable source of material. In a diagnostic laboratory, the turnaround time is critical for a bone marrow trephine to be decalcified, processed and embedded. In our laboratory, 48 hours was routinely required from the time the bone marrow was performed until the sections were ready for reporting. A hydrochloric acid-EDTA (Ethylene-Diamine-Tetra-Acetic acid) decalcifying solution was used for 4 hours but rendered the trephines unsuitable for special studies such as Fluorescence in Situ Hybridisation (FISH).

To evaluate an alternative decalcification method which preserved the ability to perform FISH on formalin-fixed paraffin-embedded (FFPE) tissue without compromising the turnaround time as a laboratory quality improvement measure.

Following overnight formalin fixation, the BMTB was decalcified in a solution containing 20% EDTA with continuous stirring for 7.5 hours. The 20% EDTA, pH 7.1 stock solution was prepared by adding Ammonium Hydroxide (25%, concentrated ammonia) (Merck) to distilled water. EDTA disodium salt (372.24; Ajax Finechem) was added and the pH adjusted to pH 7.1 using concentrated ammonia. BM trephines were then processed routinely, embedded in paraffin and 4μm sections were mounted on Super Frost Plus slides. Haematoxylin-Eosin (H&E) staining, Silver Nitrate staining for Reticulin was performed on all slides and Immunocytochemistry, Immunofluorescence and FISH on selected slides.

20 trephines from 15 patients underwent 7.5 hour EDTA decalcification. The diagnosis in 9 patients was Precursor-B ALL while one each had T-Cell ALL, Acute Myeloid Leukaemia, Hodgkin's disease, Refractory Anaemia, Drug Induced Anaemia and Idiopathic Thrombocytopenic Purpura. The mean age was 9.3 years (range 1.9–16.7years) and the mean trephine length was 12.8mm (range 6–21mm).

100% decalcification was achieved in 18 trephines while in 2 trephines 95% decalcification was achieved on morphological examination. The turnaround time was 48 hours. The quality of H&E, reticulin stain, immunohistochemistry and immunofluorescence was maintained and FISH was successful on these FFPE BMTB tissues. This has lead to incorporation of this method for routine use in our laboratory.

20% EDTA decalcification of paediatric BMTB specimens is feasible without affecting the quality of histological preparations or turnaround time. The main advantage of the EDTA decalcification process is that the tissue is amenable to FISH analysis should it be required.

No relevant conflicts of interest to declare.