Expression of Myeloma-Specific Markers in Bone Marrow Spicules Using a Novel Immunohistochemical Technique

In addition to its role in diagnosis and assessing the prognosis of numerous malignancies, immunohistochemistry (IHC) is an important tool for the analysis of protein biomarker expression in tissue sections. The combination of bone marrow (BM) biopsy and IHC is frequently used to study histopathological alterations in diseases such as multiple myeloma (MM). However, due to the harsh decalcification process generally used for processing of bone marrow biopsies, protein epitopes are occasionally rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin-clot matrix that allows for IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Briefly, bone marrow aspirates drawn from MM patients were subjected to a brief processing step to isolate the marrow spicules. Once isolated, the spicules were admixed with thromboplastin and pooled normal human plasma to create a clot. The clot was allowed to harden at 37°C after which it was placed into an embedding cassette for fixation and histological processing. The result was a formalin-fixed, paraffin-embedded clot suitable for IHC studies, including the construction of TMAs. Using patient paired spicule-clot and core biopsies from patients diagnosed with myeloma, we studied several antibodies including, but not limited to, kappa and lambda immunoglobulin light chains, CD138 and Cyr61, a member of the CCN family of extracellular matrix-associated signaling proteins. Results demonstrated that IHC staining for all antibodies was comparable in both the spicules and in the cores. While BM core biopsies had areas of non specific staining for several antibodies, this was not generally observed in the BM spicule samples with the exception of immunoglobulin light chains which displayed an element of non-specific staining with both samples. Moreover, we observed a consistently superior morphology in the BM spicule samples than in matched bone marrow core biopsies. In conclusion, our study demonstrates that BM spicule samples processed from MM patients using this novel fibrin-clot matrix technique were suitable for IHC and had generally lower background and non-specific staining coupled with better morphology when compared to matched core biopsies.