3.1 Introduction
Production o f paraffîn-section material from tissue samples that contain bone requires décalcification. Techniques such as acidic décalcification or EDTA chelation are suitable methods. Acid décalcification is generally quicker than EDTA chelation, but studies have suggested that it results in hydrolysis of DNA (Alers et a l
1999), impairing the ability to perform molecular studies. Samples in this study were processed into paraffin-embedded sections at the Department o f Histopathology, University College London Hospitals NHS Trust. In this department, décalcification is routinely undertaken using formic acid solutions o f varying strengths. Such décalcification is suitable for certain routine m olecular studies such as immunohistochemistry, but fluorescence in situ hybridization (FISH) is not routinely performed within the department.
3.2 Methods
Acid Décalcification of Trephine Bone Samples
All bone marrow trephine samples were routinely decalcified for 12-18 hours using a 5% formic acid solution (Becton Dickinson Laboratory Supplies) in distilled water. For larger, autopsy bone marrow samples, a 10% formic acid solution was used for a 7-10 day period with replenishment of the solution on one to two occasions during the décalcification period. All samples were then cut into 5-micron thickness sections and mounted on silane-coated slides in preparation for FISH. Adjacent sections were mounted on uncoated slides for staining with haematoxylin and eosin.
3.3 Results
Five percent formic acid décalcification resulted in successful FISH in all trephine samples analysed for AR gene copy number in bone métastasés from HRPC (12/12 cases). In contrast, for the five larger autopsy bone marrow specimens, décalcification in 10% formic acid solution resulted in no successful FISH studies (see Chapter 4 for the results of the FISH investigations). Where there were soft tissue métastasés from the same autopsy cases available for study (three out of five cases), FISH was successful in two out of three samples (the remaining two samples coming from patients with only metastatic bone disease at post-mortem).
3.4 Discussion
The literature on the best methods for décalcification of bone marrow samples for molecular studies is small. Routine acid décalcification of bone marrow trephine and autopsy bone samples has been reported to result in total failure to obtain DNA for in situ hybridization (ISH), comparative genomic hybridization (CGH) and flow cytometry studies in ante-mortem trephine and post-mortem bone marrow samples from prostate cancer, in a small study by Alers et al, 1999. In contrast, within this study, décalcification using a 10 % EDTA solution for three autopsy bone marrow specimens alone, resulted in successful ISH, CGH and flow cytometry and was also reported to give better preservation of architecture for routine haematoxylin and eosin staining and immunohistochemistry. The authors concluded on this basis, that EDTA was highly preferable to their routinely used acid décalcifier where studies on DNA were required.
Acid décalcification was also used for the specimens in the current study, but a different preparation was used (5-10% formic acid rather than RDO, a proprietary agent produced by Apex Engineering Products, Plainfield, IL, USA), as used by Alers et ah, 1999. The finding here suggest that brief décalcification of small samples in a 5% formic acid solution does not result in significant DNA acid hydrolysis.
There is little published information available on the effects o f formic acid décalcification on DNA degradation. A Medline literature search for the years 1966- 2002 was unable to find other reports of successful studies using FISH, from bone marrow trephine biopsies decalcified in formic acid. Sarsfield et al. reported that formic acid décalcification of bone marrow trephines degraded DNA for polymerase chain reaction (PCR) studies using specific PCR primers (Sarsfield et al. 2000). In this small comparative study o f formic acid and EDTA décalcification from eleven bone marrow trephine specimens, similar quantitative amounts o f DNA could be retrieved using both décalcification methods, but formic acid pre-treatment resulted in a degraded DNA smear and failure to obtain specific PCR products in the majority of samples tested.
Provan et al. have also investigated formic acid décalcification and were able to generate a 294 base pair (bp) DNA PCR product in only six out of ten samples following formic acid décalcification o f paraffin-embedded bone marrow biopsies (Provan er ai/. 1992).
In contrast, EDTA décalcification resulted in successful amplification of specific DNA PCR products up to 643 bp in the study by Sarsfield et a l and the author’s previous experience o f EDTA for décalcification o f bone marrow trephines alone strongly supports this finding (Wickham et a l 2000). None o f the samples in the current study were decalcified using EDTA.
In conclusion, it has been shown that routine acid décalcification with 5% formic acid can satisfactorily preserve DNA for certain types o f molecular biological studies (such as FISH) on prostate cancer bone métastasés. Longer and stronger acid décalcification resulted in unsuccessful FISH in all autopsy specimens whilst non calcified material from the same post-mortem samples was shown to work satisfactorily, strongly suggesting it is the décalcification process that is the cause for the failure (as the remaining methodology for the FISH is identical for all types of samples).
Décalcification using EDTA solution has been reported to offer the best chance of successful DNA retrieval from bone tissue in two studies (Alers et a l 1999; Sarsfield et a l 2000) and appears to be the method o f choice for décalcification in a prospective study of bone marrow trephine and autopsy samples. However, the current study has shown for the first time that limited 5% formic acid décalcification can be used for FISH studies in bone métastasés from carcinoma o f the prostate.