1. Systemic Mastocytosis Associated with Chronic Idiopathic Myelofibrosis
Karl Sotlar, Anja Bache, Florian Stellmacher, Burkhard Bültmann, Peter Valent, Hans-Peter Horny 
The Journal of Molecular Diagnostics 
Volume 10, Issue 1, Pages (January 2008)
DOI: /jmoldx
Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2. Figure 1
SM with associated CIMF = SM-AHNMD/CIMF (case no. 302). A: Lower magnification clearly shows CIMF with extremely hypercellular BM, increased neutrophilic granulocytopoiesis (depicted in red), clustering of pleomorphic megakaryocytes, and a large (pale-appearing) infiltrate in the left central portion of the picture. A diagnosis of SM cannot be made without additional stainings. B: Higher magnification of this infiltrate exhibits an abundance of spindle-shaped cells with round nuclei and inconspicuous nucleoli. The overwhelming majority of spindle-shaped cells is not stained with CAE. C: Immunostaining clearly shows a strong cytoplasmic reactivity of these cells with an antibody against tryptase. Spindle-shaped tryptase-positive cells in the BM are atypical MCs allowing a diagnosis of SM to be established. D: To further emphasize the neoplastic character of the MCs, immunostaining with anti-CD25 should be performed, here leading to strong reactivity of MCs. The cytoplasmic staining of perifocal megakaryocytes can be used as an internal control. A and B: Naphthol AS-D chloroacetate esterase (CAE); C: ABC method; AA1 (anti-tryptase); D: ABC method; anti-CD25 (Novocastra, New Castle, UK).
The Journal of Molecular Diagnostics  , 58-66DOI: ( /jmoldx )
Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3. Figure 2
Effects of LNA-mediated PCR clamping. A: In melting curve analyses with Light Cycler hybridization probes wild-type (wt) JAK2 and mutation V617F can be discriminated through different melting peaks at ∼60°C (wt) and 65°C (V617F) (black continuous line). The method allows detection of the mutation in a 10:1 excess of normal versus mutated cells (dark gray broken line). B: LNA-mediated PCR-clamping results in suppression of the amplification of the JAK2 codon 617 wt allele (black continuous line and dark gray broken line) and in a 10-fold increase of sensitivity, yet allowing detection of the mutated allele in a 100-fold excess of wt background DNA (light gray broken line).
The Journal of Molecular Diagnostics  , 58-66DOI: ( /jmoldx )
Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4. Figure 3
Microdissection of bone marrow cells in SM-CIMF. A: Typical compact MC infiltrate with numerous spindle-shaped MCs before microdissection. B: The same detail as depicted in A after microdissection. Replaced single cells are indicated by white dots. C and D: Loosely scattered MC infiltrates before (C) and after (D) microdissection. Position of cells is indicated with circles. E and F: CD15+ myeloid cells before (E) and (F) after microdissection. Replaced single cells are indicated by white dots. (A–-F, ABC method/AEC; A-–D, anti chymase; E and F, anti-CD15).
The Journal of Molecular Diagnostics  , 58-66DOI: ( /jmoldx )
Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5. Figure 4
KIT and JAK2 mutation screening by melting point analysis of microdissected cells. A: Representative results obtained after melting point analysis of nested PCR-amplified, microdissected MCs (red continuous line) and CD15+ cells (blue broken line), both carrying the KIT mutation D816V (case 302). HMC-1 DNA served as control. The KIT codon 816 wt-specific melting peak is located at ∼60°C whereas the D816V-specific melting peak is located at ∼65°C. B: Detection of the JAK2 mutation V617F in microdissected CD15+ cells (blue broken line) and in MCs (red continuous line). The wt-specific melting peak is located at ∼60°C whereas the V617F-specific peak is located at ∼64°C (case 661). HMC-1 DNA served as the wt control (gray dotted line) and HEL cells as the V617F-specific control (gray continuous line).
The Journal of Molecular Diagnostics  , 58-66DOI: ( /jmoldx )
Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions