title: A novel immunohistochemical sequential multi-labelling and erasing technique enables epitope characterization of bone marrow pericytes in primary myelofibrosis.
abstract: OBJECTIVE
  In Philadelphia (Ph)-negative chronic myeloproliferative neoplasms, increased microvascular density, bizarre vessel architecture and increased number of pericytes are among the distinct histopathological features. The aim of this study was to characterize bone marrow pericytes in primary myelofibrosis (PMF) using a novel multi-labelling immunohistochemical technique.
  
  
  RESULTS
  Bone marrow biopsies from a normal donor (n = 1) and patients with PMF (n = 3) were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique). Antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualized with aminoethylcarbazole. After imaging, erasing and blocking of immunoreagents, the slides were stained with a traditional double immunolabelling procedure. In addition, we applied a Photoshop® colour palette, creating a single composite image of the sequential staining procedures. We successfully applied four layers of antibodies on one slide using CD146, smooth muscle actin, CD34, CD271 and Ki67 in different combinations. The SE-technique significantly improves morphological and phenotypical studies in bone marrow specimens.
  
  
  CONCLUSIONS
  To our knowledge, the SE-technique is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow by light microscopy. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms.
authors.affiliation: Department of Clinical Pathology, Hospital South Naestved, Ringstedgade 61, Naestved, Denmark. abmu@regionsjaelland.dk
authors.forename: Ann
authors.initials: A
authors.lastname: Madelung
authors.forename: Michael
authors.initials: M
authors.lastname: Bzorek
authors.forename: Henrik
authors.initials: H
authors.lastname: Bondo
authors.forename: Eva
authors.initials: E
authors.lastname: Zetterberg
authors.forename: Ole Weis
authors.initials: OW
authors.lastname: Bjerrum
authors.forename: Hans Carl
authors.initials: HC
authors.lastname: Hasselbalch
authors.forename: Stefan
authors.initials: S
authors.lastname: Scheding
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Ralfkiaer
mesh_terms: D001854:Bone Marrow Cells / Q000276:immunology* / Q000473:pathology; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D020286:Pericytes / Q000276:immunology* / Q000473:pathology; D055728:Primary Myelofibrosis / Q000276:immunology* / Q000473:pathology