Identification of Megakaryocytes by Flowcytometry; Immunophenotypic Expression in Reactive States and Hematopoietic Malignancies
JN Punia, T Fennell, V Douglas-Nikitin, JZ Huang, MD Smith. William Beaumont Hospital, Royal Oak, MI
Background: The use of flow cytometry in the diagnosis of hematopoietic neoplasms has focused primarily on granulocytic and erythroid cell lines but not megakaryocytes (MKCs). The reasons for this are likely related to 1) relatively fewer numbers of MKCs in the marrow and 2) fragility of MKCs to specimen processing. We have observed an increased yield of MKCs using a modified fix and permeabilization (perm) procedure. Thus, the aim of our study was to 1) verify a method for isolation of MKCs in bone marrow samples, and 2) determine if expression of von Willebrand factor (vWF), which is normally expressed in MKCs, varies by diagnosis.
Design: We collected 57 cases that we divided into 6 groups: normal (13), secondary thrombocytosis (4), benign thrombocytopenia-BT (6), myelodysplasia-MDS (10), chronic myelogenous leukemia, CML (7) and myeloproliferative neoplasm, not CML or BCR/ABL(-)MPN (17). Slides were reviewed for confirmation of the diagnosis and to assign a dysplasia score in MKCs (1-3). We used a modified fix and perm procedure and isolated suspected MKCs using a CD45 positive, high side scatter gate.
Results: Cells in the MKC gate showed strong expression of vWF, above isotype control and granulocytes. These cells also stained positive for CD36 and negative for CD14. Flow sorting on two cases was able to show MKCs morphologically. Univariate statistical analysis showed that there was no significant difference in vWF expression for age, gender, platelet count, WBC count and dysplasia score. However, cases with MDS showed lower vWF expression than BT (25.7 vs. 66.7; p=0.04) and there was a positive correlation between vWF and hemoglobin (hgb, r=0.49, p=0.01). There was a trend indicating that MDS cases show less vWF expression than other benign categories and BCR/ABL(-)MPN, but there were likely too few cases to reach statistical significance.
Conclusions: Our results show that MKCs can be consistently analyzed by flow cytometry using a modified fix and perm procedure. We also show that there may be differences in MKC vWF expression between diagnostic categories, with decreased vWF expression in cases of MDS vs. benign thrombocytopenia, and possibly vs. other benign cases and BCR/ABL(-)MPN. This may be diagnostically useful in distinguishing MDS from benign cases. In addition, there was a positive correlation between hgb and MKC vWF expression that appeared to be independent of diagnosis.
Monday, March 22, 2010 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 187, Monday Morning