We examined inter-and intraobserver reproducibility and concordance between histological diagnosis and independently collected clinical findings in a large series of patients with the major subtypes of myeloproliferative neoplasms (MPNs) and controls. Seven hematopathologists reviewed 272 bone marrow biopsies including 43 controls. Diagnoses were determined according to the 2008 criteria of the World Health Organization (WHO). The participants were blinded to all clinical data except patient age. After initial evaluation all hematopathologists participated in a 3-day meeting with a leading clinician chaired by an expert hematopathologists. In cases with lack of consensus on fiber grading (n 5 57), a new evaluation was performed. In cases with discordance on morphological diagnosis (n 5 129), an additional nonblinded evaluation taking clinical data into consideration was carried out. For remaining cases with a lack of concordance between morphological diagnosis and clinical diagnosis (n 5 33), a similar nonblinded evaluation was performed. Consensus on final histological diagnosis and concordance with clinical diagnosis were determined. Blinded histological evaluation resulted in a 53% consensus rate. After re-evaluation of fiber content, consensus was reached in 60% of cases. Adding clinical data increased the histological consensus to 83%. For cases with a histological consensus, we found a concordance of 71% with the clinician's diagnoses. This is the first study to present a larger cohort of MPN patients mimicking the diagnostic challenges that hematopathologists face in their daily practice. The results support the postulates of the WHO that both morphological and clinical findings are essential for a valid diagnosis Am.
Early prefibrotic myelofibrosis (early PMF) is a diagnosis that clinically and histologically mimic essential thrombocythemia (ET), but is important to distinguish from ET, polycythemia vera (PV) and primary myelofibrosis (PMF) due to its different prognosis and clinical evolution. In this study, we assessed the allele burden of JAK2V617F in bone marrow biopsies from patients with these chronic myeloproliferative neoplasms. We correlated our findings with the amount of phosphorylated STAT3 (P-STAT3) and STAT5 (P-STAT5) in megakaryocyte nuclei in the bone marrow. The JAK2V617F allele burden was significantly higher in patients with PV (median: 50.99, range: 23.08-97.29, p < 0.01 and p < 0.01) and PMF (median: 44.13, range: 33.61-92.17, p < 0.05 and p < 0.01) compared with a low allele burden in ET (median: 23.465, range: 8.67-47.92) and early PMF (median: 25.68, range: 0.61-49.13) respectively. In addition, we found a significantly higher phosphorylation of STAT5 and STAT3 in the JAK2V617F positive group than in the negative group. There was no positive correlation between increasing JAK2V617F allele burden and the amount of P-STAT3 and P-STAT5. However, we found low values of P-STAT5 in bone marrow biopsies from patients with ETJAK2V617F+ as compared with patients with early PMFJAK2V617F+. Although this difference was statistically significant, larger studies are needed to firmly support this conclusion.
We examined the learning effect of a workshop for Danish hematopathologists led by an international expert regarding histological subtyping of myeloproliferative neoplasms (MPN). Six hematopathologists evaluated 43 bone marrow (BM) biopsies according to the WHO description (2008), blinded to clinical data. All panelists then participated in the workshop. The case biopsies - mixed with 251 other MPN BM biopsies - were reviewed again. Consensus regarding the histological subtype was significantly improved; from 49% to 72% (Fleiss kappa value 0.302 pre-workshop, 0.474 post-workshop; p = 0.004). There was no significant effect on the isolated morphological characteristics. Agreement between cases with histological consensus and clinical diagnosis was 86% without significant change during workshop sessions. Our study demonstrates that experienced hematopathologists can significantly improve their diagnostic ability by a workshop led by an international expert; not by improving the evaluation of individual histological parameters but by weighting these in their conclusive diagnosis.
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.
4108 Background: In Philadelphia-negative chronic myeloproliferative neoplasms (MPN) increased microvascular density, bizarre vessel architecture and increased number of pericytes are distinct histopathological features; apart from the characteristic proliferation of myeloid cell lines and the progressive accumulation of connective tissue in the bone marrow. Pericytes express several markers such as CD146, CD271, Smooth Muscle Actin (SMA), Desmin, Platelet-derived growth factor receptor beta and Neuron-glial 2. However, these markers are also expressed in other cell types of which some are related to vascular structures. Immunofluorescence labelling is the golden standard for detection of co-expressed cellular antigens, but due to the crowded cellular environment in bone marrow and excessive autofluorescence, identification of cell types by light microscopy is preferred. Aim: This is a methodological study aiming to identify pericyte marker profiles by light microscopy in bone marrow biopsies, contributing to our understanding of the pathogenesis of MPN. Method and results: Formalin fixed, decalcified and paraffin-embedded blocks of bone marrow trephine specimens from normal donor (n=1) and patients with primary myelofibrosis (PMF) (n=3) were included. Specimens were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique), inspired by the work of Glass et al. 2009 (J Histochem Cytochem). Briefly, antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualised with Amino-Ethyl-Carbazole (AEC). After imaging, erasing of AEC with 96% alcohol and blocking of immunoreagents, the slides were stained with a traditional double immuno-labelling procedure. We successfully applied up to four layers of antibodies using CD146, SMA, CD34, CD271, and Ki67 in different combinations; either displayed as single or single followed by traditional double sequential staining runs (figure 1). In addition to the conventional light microscopy analysis we applied a Photoshop color palette, where the different immunohistochemical reactions in the staining sequence were assigned to the different color channels creating a single composite image. The SE-technique significantly improves morphological studies especially in bone marrow trephines with the cells of interest intermingled with other cells. Additionally, the SE-technique makes it possible to detect more than two antigens regardless of immunoglobulin type or animal host. Conclusion: To our knowledge, the SE-technique described in this study, is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow trephines at light microscopic level. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms. The SE-technique displayed as single staining images and Photoshop color palette combined image. Panel A-D shows an identical area in the different steps of the method. A) CD146. Positive pericytes (arrow). B) SMA. Positive pericytes (arrow). C) CD34. Negative pericytes (arrow). D) Combined image with CD146 (green channel), CD34 (red channel), and SMA (blue channel). Coexpression of CD146/CD34 is seen as yellow reaction deposit, and coexpression of CD146/SMA as cyan reaction deposit (arrow). Note, as a negative control, the CD146 positive fat cell in A (arrowhead) - negative in B and C, and SMA positive pericyte in B (arrow) – negative in C. Disclosures: No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.