A total of nine MM patients were studied. Eight of the patients were male and one female, with a median age of 68 years. All patients were treated with high doses of chemotherapy followed by autologous stem cell transplant.
Bone marrow (BM) biopsy specimens were obtained from MM patients enrolled in trials at University of Arkansas for Medical Sciences (UAMS). The UAMS institutional review board approved the clinical trials and all patients provided informed written consent.
BM trephine biopsy specimens were fixed in 10% neutral buffered formalin and briefly decalcified by acid chelation and embedded in paraffin. Sequential 4 µm thick sections were cut and slides were prepared for BM biopsy immunostaining. One slide from each patient was stained using hematoxylin and eosin (H&E) to check cell morphology.
BM spicule samples were prepared by ficoll gradient separation of BM aspirates with BM spicules being collected from the top lipid layer. The BM spicule samples were then transferred into a 50 ml ClearSpin Filter (Novagen, Gibbstown, NJ) and centrifuged at 250 rpm for 2 minutes. If fluid still remained on the top of the filter, the sample was centrifuged for an additional 2 minutes or until filter clears from fluids. After centrifugation, BM spicules from the filter were transferred very carefully into a 1.5ml eppendorf tube appropriately labeled. Then 200 µl each of previously warmed (37°C) normal pooled human plasma with EDTA and Thromboplastin-DS (Pacific Hemostasis, Cape Town, South Africa) was added to the tube and mixed by hand. The tube was then placed at 37°C for 5 to 10 minutes to allow for clot formation. The tube was carefully removed and the clot and transferred into a HistoScreen™ mesh cassette (Perk Scientific Inc, Lansdowne, PA). The cassette was then placed into formalin (1:10 dilution, Fisher Scientific, Pittsburgh, PA) and fixed overnight followed by embedding in paraffin. For IHC staining, 4 µm sections of paraffin-embedded bone marrow biopsy cores or BM spicule fibrin-clot matrixes were cut and placed onto positively charged glass microscope slides and allowed to air-dry overnight prior to de-waxing.
Tissues were deparaffinized in xylene, dehydrated with ethanol, and rinsed in phosphate buffered saline (PBS). When necessary, heat-induced epitope retrieval was carried out on deparaffinized tissue sections using a modified pressure cooker (Decloaker™, Biocare Medical, Concord, CA) and a pH 6.0 citrate buffer (Bull’s Eye Retrieval solution, Biocare Medical). Slides were stained separately for expression of five antibodies: kappa (DakoCytomation, Carpenteria, CA), lambda (DakoCytomation), CD138 (clone B-B4, Biocare Medical), CYR61 (kindly provided by Dr. L.F. Lau, University of Illinois at Chicago) and DKK1 (Novus Biologicals, Littleton, CO). All reactions were performed using an automated immunostainer (DakoCytomation, Carpenteria, CA) in conjunction with the Envision+-HRP detection system (Dako). Appropriate negative controls were included for each case. Endogenous peroxidase was inhibited with methyl alcohol containing 0.01% H2O2 with additional blocking performed utilizing Background Sniper™ (Biocare Medical, Concord, CA) and/or Background Terminator™ (Biocare Medical). Nuclei were counter-stained with hematoxylin and sections evaluated by light microscopy using an Olympus BH-2 microscope (Olympus, Melville, NY) fitted with a SPOT2 digital camera (Diagnostic Instruments, Sterling Heights, MI). Scoring was done based on staining intensity of antibodies on a scale of 0 to +3 (0 scant, +1 mild, +2 moderate, +3 strong).