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Updated: Sep 8, 2026
Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
Published on: January 8, 2010
Bone marrow one step fixation-decalcification in Lowy FMA solution: an immunohistological and in situ hybridization
A Gaulier1, C Fourcade, G Szekeres
1Service d'Anatomie Cytologie Pathologiques, C. H. Victor Dupouy, Argenteuil, France.
Insights
This study demonstrates that Lowy formalin mercuric chloride acid (FMA) fixation is effective for bone marrow biopsies (BMB), preserving immunoreactivity for various cell types and enabling RNA visualization. Optimal antibody dilutions and microwave pre-treatment are key for accurate results.
Area of Science:
- Histopathology
- Immunohistochemistry
- Molecular Pathology
Background:
- Bone marrow biopsies (BMB) require specific fixation for accurate analysis.
- Evaluating antibody performance on fixed BMB is crucial for diagnostic accuracy.
- Assessing RNA preservation for in situ hybridization in BMB is important.
Purpose of the Study:
- To evaluate the efficacy of a one-step fixation-decalcification method using Lowy formalin mercuric chloride acid (FMA) for bone marrow biopsies (BMB).
- To assess the immunoreactivity of various antibodies on FMA-fixed BMB.
- To determine the suitability of FMA fixation for in situ hybridization of RNA.
Main Methods:
- Bone marrow biopsies (BMB) underwent a 20-hour one-step fixation-decalcification in Lowy FMA solution.
- Immunoreactivity of antibodies against myeloid, megakaryocytic, erythroid cells, lymphocytes, mastocytes, and metastatic cells was tested.
- Optimal antibody dilutions and microwave pre-treatment protocols were established.
- In situ hybridization was performed to visualize mRNA Kappa/Lambda and EBV RNAs.
Main Results:
- Lowy FMA fixation allowed excellent histological staining and preserved immunoreactivity for nearly all tested antibodies on paraffin-embedded BMB.
- Optimal working dilutions were defined, and trypsinization was found to be unnecessary.
- Microwave pre-treatment was essential for certain antibodies (e.g., CD20 L26, CD8, CD3, CD34, MB1 Kappa/Lambda) but suitable for others (UCHL1, LN2, CD30).
- FMA fixation enabled successful visualization of mRNA Kappa/Lambda in myeloma and EBER 1 EBV RNAs in HIV lymphoma via in situ hybridization.
Conclusions:
- Lowy FMA fixation is a versatile and effective method for BMB, supporting both immunohistochemistry and in situ hybridization.
- The study provides essential guidelines for optimizing antibody staining and RNA detection in FMA-fixed BMB.
- Consideration of safety handling for mercuric chloride is necessary.
Abstract:
The immunoreactivity of paraffin embedded bone marrow biopsies (BMB) was studied following a one step 20-hour-fixation-decalcification in Lowy formalin mercuric chlorid acid solution which permits excellent histological stainings. Antibodies reactive with myeloid, megakaryocytic, erythroid cells, T and B lymphocytes, mastocytes and metastatic cells were compared. Nearly all antibodies working on paraffin sections were demonstrated on Lowy FMA fixed BMB. Special care was taken to define an optimal working dilution. Trypsinization was not necessary. A slide microwave pre-treatment appeared essential before testing CD20 L26, CD8, CD3, CD34, MB1 Kappa and Lambda antibodies. It was suitable for UCHL1, LN2, CD30 antibodies. The same fixative allowed an m RNA Kappa or Lambda in myeloma and EBER 1 EBV RNAs in HIV lymphoma visualization by in situ hybridization. The safety handling of the toxic mercuric chloride component is discussed.
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