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Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone
Published on: April 8, 2015
Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I
S A Loyson1, L H Rademakers, P Joling
1Department of Pathology, University Hospital Utrecht, The Netherlands.
Insights
This study shows that immunohistochemistry on decalcified bone marrow trephine biopsies can identify all hematopoietic and stromal cells. Optimized antigen retrieval methods allow for accurate cell lineage identification in paraffin-embedded samples.
Area of Science:
- Hematology
- Immunohistochemistry
- Bone Marrow Histology
Background:
- Accurate identification of bone marrow cellular components is crucial for diagnosing various hematological disorders.
- Traditional methods for bone marrow analysis often involve frozen sections, which can limit antigen preservation and detailed morphological assessment.
- Decalcification of bone marrow biopsies can compromise the integrity of cellular antigens, posing challenges for immunohistochemical analysis.
Purpose of the Study:
- To establish optimal immunohistochemical protocols for identifying stromal structures and hematopoietic cell lineages in decalcified bone marrow trephine biopsies.
- To evaluate the efficacy of different antigen retrieval methods on paraffin-embedded, decalcified bone marrow samples.
- To enable reliable characterization of the bone marrow microenvironment using routine histological techniques.
Main Methods:
- Two antigen retrieval methods (pepsin and acid citrate buffer) were compared for their effectiveness on decalcified bone marrow paraffin sections.
- Various antibodies targeting specific cell surface markers and intracellular proteins were tested for optimal reactivity.
- Staining patterns were analyzed after both prolonged (48h EDTA) and short electrolytic decalcification methods.
Main Results:
- Heating in acid citrate buffer was generally preferred for antigen demonstration and background reduction, with specific exceptions for elastase, von Willebrand Factor, Ulex europaeus agglutinin (UEA-1), and alpha-smooth muscle actin (alpha-SMA).
- Both prolonged EDTA and short electrolytic decalcification yielded comparable staining results.
- Successful identification of myeloid, erythroid, megakaryocytic, B-cell, and T-cell populations was achieved, alongside visualization of stromal cells and the bone marrow capillary network.
Conclusions:
- Immunohistochemistry on decalcified paraffin-embedded bone marrow trephine biopsies is a viable method for identifying all hematopoietic and stromal components.
- Optimized antigen retrieval techniques significantly enhance the ability to detect cellular markers in processed bone marrow samples.
- The decalcification time can be reduced to 1-1.5 hours without compromising the quality of immunohistochemical analysis.
Aims:
To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.
Methods And Results:
Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and alpha-smooth muscle actin (alpha-SMA) required no pretreatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.
Conclusions:
This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1-1.5 h.
