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spring 2008: volume 13, issue 1 newsletter of the myelodysplastic syndromes foundation From the Guest Editor’s Desk Flow Cytometry: Providing Additional Information in Diagnosis, Prognosis and Monitoring Treatment of MDS simultaneously along with light scattered by the cells as they traverse the laser light at rates of 500–1000 per second. The data generated by the flow cytometer can be analyzed by correlating the intensity of each dye along with cell size and cell granularity on each individual cell, Denise A. Wells, MD resulting in an identifying phenotype for that Contents Michael R. Loken, PhD cell. The cell surface proteins or antigens detected by monoclonal antibodies are the From the Operating Director’s Desk 5 Hematologics, Inc. Seattle, Washington final translated products of genes which are Young Investigator Grants Program 6 highly regulated during maturation from Flow cytometry is only beginning to be Foundation Initiatives 7 hematopoietic stem cells found in the bone used to study patients with MDS. We would marrow to fully functional mature cells Meeting Highlights/Announcements 8 like to provide background and an overview observed in peripheral blood. With proper of technology, and its basis for assessing Patient Services 11 selection of antibodies, and a careful multi- patients with MDS which will have dimensional analysis, it is possible to identify Patient Forums and Support Groups 12 increasing importance in the near future in every cell in a marrow aspirate specimen, Living With MDS 13 being able to separate MDS from other classifying it to a lineage and a maturational causes of decreased blood counts. stage within that lineage, and assessing Drug News 15 whether or not it displays a normal or an Patient Registries and Referrals 17 Flow Cytometry: abnormal phenotype.1,2 A 30 Second Primer MDS Centers of Excellence 18 Hematopoiesis (the production of blood The technology of flow cytometry cells) can be described as a series of Information on Clinical Trials 21 discriminates between different types of steps with progression from one stage to Educational Resources 25 blood cells based on the quantitative the next characterized by multiple “intensity” amounts of unique proteins (and some changes in antigenic expression identified Foundation Publications 29 sugars) found primarily on the cell surface. by different antibody/dye combinations. In Contributions to the Foundation 30 These proteins are recognized by specific normal developing bone marrow cells, the antibodies, each labeled with a different constellation of antigens and their relative In Memorium 31 colored fluorescent dye. By incubating the intensities are highly regulated. The changes General Information 36 cells with the specific monoclonal observed in antigen intensities represent antibodies, only the cells which exhibit the stepwise changes in gene product target protein become tagged with the expression that mark developing cells fluorescent dyes. The amount of dye is progress to the next stage. These stages of directly related to the amount of the specific normal hematopoietic cells are established protein on that cell which can be detected in fetal life, are invariant with age and by the flow cytometer as the cells pass remain intact in stressed or regenerating single file through a laser beam. Multiple bone marrow, following chemotherapy and dyes of different colors can be detected even after stem cell transplantation. 1 Phenotypic Changes in which antigens are present on each lineage, Flow cytometric analysis (focusing on MDS Bone Marrows at each maturational stage, and how their normal/abnormal blasts, myeloid and expression is related to other cellular monocyte development) can be a compli- Once the patterns of gene product antigenic markers. Multiple studies have mentary addition to a morphologic analysis expression during the development of shown that the multiple antigenic profiles, or of bone marrow in facilitating an accurate normal blood cells were elucidated, a “phenotypes,” of bone marrow cells in comparison to the patterns on leukemia diagnosis. Dyspoietic features in the patients with MDS exhibit differences from cells showed that these neoplastic cells did erythroid and megakaryocytic lineages are the patterns identified in normal bone not match the normal relationships. Every often prominent by morphology, whereas the marrow, not only among the blast cells, but leukemia analyzed by flow cytometry was assessment of abnormal myeloblasts, also on the maturing myeloid cells and found to be different from normal and maturing myeloid cells and especially 3-12 17 different from other similar leukemias, i.e., monocytes. The variability in published monocytes, is more difficult by morphology. the intensities and relationships between reports on flow cytometry in studying MDS is Flow cytometry, however, focuses on these antigens (the leukemic phenotype) were due, in part, to the uniqueness of each maturing leukocytes. Together the two unique to individual patients. The differences patient and which antibodies are used to technologies can provide a better total picture in intensities of antigens on the leukemia as distinguish between the normal presentation of the bone marrow in MDS patients. At compared to normal cells provided a “tumor of antigens and those observed on each present, technical considerations limit the specific marker” that can be used to monitor unique patient. analysis of erythroid and megakaryocytic response to chemotherapy and to detect Because of the extensive variability from cells by flow cytometry. residual disease following treatment. patient to patient as well as the changes that occur over time for an individual patient, Identifying, Enumerating and The flow cytometric assessment of acute Classifying Blasts (and chronic) leukemias consists of single specific antigenic changes are not Flow cytometry has a distinct advantage evaluating the major, usually homogenous, sufficient to fully capture the abnormalities 13,14 over morphology for blast cell enumeration neoplastic cell population. When used in a present. The abnormalities observed are diagnostic role for unexplained cytopenias in not just in the expression of single antigens because of the ability to count many more suspected MDS, the manner in which the but affect the relationships between multiple cells (10,000 –100,000 or more as technology is applied changes. Instead of antigens. Hence, a multidimensional approach opposed to 300–500 cell analyzed by detecting a major abnormal immature cell is required to relate all cell proteins to microscope) and a definable set of cellular population (found in leukemia), the approach each other. This analysis must distinguish characteristics that can be used to identify becomes a search for a more subtle between a stressed normal marrow (with an the cells, and is potentially less subjective abnormality causing, or related to, the increase in more immature forms called a than morphologic criteria of defining what inadequate production of hematopoietic shift-to-the-left) from a true uncoupling of constitutes a blast. Since several different cells resulting in the cytopenia. This requires the co-regulation of gene products as combinations of antibodies can be used to evaluation of subsets of abnormal cells such evidenced by abnormal antigens present in discriminate the myeloblasts, it has been as monocytes, maturing myeloid cells, and neoplastic processes that may occur at any recommended that a phenotypic blast count myeloblasts present in the bone marrow as of the developmental steps.5,15 should be obtained using multiple the abnormalities in protein expression are In addition to abnormal increase or combinations of antigenic markers, found not only on the immature cells (blasts) decrease in intensity of antigens, the types comparing the results for consistency and but also on the more mature cells. In this of abnormalities observed in MDS include as an internal quality control check of the manner, flow cytometric data for MDS must inappropriate lineage expression (lymphoid data.5 When applied to MDS, the be used in the same way as a morphologic antigens on myeloid cells), asynchronous identification and enumeration of blasts review of an aspirate smear, comparing the expression where antigens normally must incorporate the multiple antibody features of normal (non-neoplastic) bone identified on immature cells appear on combination approach since the process of marrow elements to their neoplastic mature cells, and complete absence of neoplastic transformation may involve the counterparts. In a morphologic analysis, antigen expression (possibly due to loss of one or more of the antigens used to each lineage is examined to determine the complete loss of the gene, regulators, classify the cells as immature. The use of frequency of cells, the degree of maturation, or over-inhibition).16 Other abnormalities 3 or more combinations of antibodies to and suspected abnormalities observed detected by flow cytometry can be observed. define a flow cytometric blast provides a relative to previous knowledge of cytologic Physical changes of cell size and decreased redundant system with internal controls for appearance. Likewise, a flow cytometric cellular granularity are often observed in the loss or inappropriate expression of any approach requires an intimate knowledge of MDS marrows. one of the markers. Flow cytometric blast 2 counts are expressed as per non-erythroid Scoring System (IPSS)19 score indicating the These quite