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Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks
Published on: May 20, 2018
Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and
H Merz1, O Rickers, S Schrimel
1Institute of Pathology, Medical University of Lübeck, Germany.
Insights
Microwave detection systems improve immunohistochemistry sensitivity in formalin-fixed tissues. This method enhances immunoglobulin detection, offering results comparable to fresh frozen material with better morphology.
Area of Science:
- Histopathology
- Immunology
- Biochemistry
Background:
- Immunohistochemistry (IHC) sensitivity is crucial for diagnosing various diseases.
- Formalin-fixed, paraffin-embedded (FFPE) tissues offer excellent morphology but present antigen masking challenges for IHC.
- Fresh frozen tissues provide high sensitivity but poor morphology preservation.
Purpose of the Study:
- To evaluate a novel microwave-based detection system for enhancing immunoglobulin (Ig) detection in FFPE tissues.
- To compare the sensitivity and morphological preservation of the microwave method with traditional techniques.
- To assess the utility of this method for diagnosing lymphoid malignancies.
Main Methods:
- Combination of protease digestion and microwave treatment with heavy metal salts or urea.
- Application of the protocol to formalin-fixed, paraffin-embedded Burkitt's lymphoma and B-chronic lymphocytic lymphoma (B-CLL) specimens.
- Comparison of Ig detection results with those from fresh frozen sections.
Main Results:
- The microwave-based method achieved high sensitivity for Ig heavy and light chain detection in FFPE tissues, comparable to fresh frozen material.
- Urea treatment provided sensitivity similar to heavy metal salts but without toxicity.
- Formalin-masked cytoplasmic and surface immunoglobulins became accessible, with Burkitt's lymphoma consistently showing surface Ig expression.
- Ig detection in B-CLL and immunocytoma suggests quantitative differences rather than absolute absence, impacting differential diagnosis.
Conclusions:
- Microwave-assisted IHC significantly enhances Ig detection sensitivity in FFPE tissues, preserving morphology.
- The use of urea offers a safer, effective alternative to heavy metal salts.
- This technique improves antigen accessibility and has implications for the differential diagnosis of lymphoid neoplasms.
Abstract:
The introduction of microwave detection systems has significantly enhanced the sensitivity of immunohistochemistry in formalin-fixed, paraffin-embedded tissue. The combination of protease digestion and microwave treatment including the use of heavy metal salts or urea gives an Ig heavy and light chain detection system in formalin-fixed and paraffin-embedded material as good as that achieved using fresh frozen material, with the advantage of a well-preserved morphology. The sensitivity of this method depends on the heavy metal salt used and its concentration. The use of urea yields equally sensitive results, while avoiding the toxicity of heavy metal salts. Heating times in the microwave oven also have an important influence on the results. The microwave-based technique may allow for the improved accessibility of formalin-masked antigens, as was shown here for cytoplasmic and surface immunoglobulins. It was demonstrated that formalin-fixed specimens of Burkitt's lymphoma when subjected to the new protocol almost always showed surface Ig expression, giving results comparable to those obtained in frozen sections. Moreover, it could be shown that in cases of B-chronic lymphocytic lymphoma (B-CLL) or immunocytoma, surface and cytoplasmic Ig could be detected, so that the difference may be just a quantitative one and thus may no longer be safely used as a criterion in differential diagnosis. In future, the wide use of this technique will allow for the study of Ig expression at the single cell level in morphologically well-preserved material.
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