April 2012
Feature Story

A practical, case-based guide to flow cytometric analysis in the workup of hematopoietic neoplasms. That’s what the latest book is from CAP Press. Authors Sindhu Cherian, MD, and Brent Wood, MD, PhD, start with an introduction detailing immunophenotypic changes seen in normal hematopoiesis. Four chapters follow on B-cell neoplasms, T-cell and NK-cell neoplasms, chronic myeloid stem-cell neoplasms, and acute leukemia. They provide 36 cases, each dedicated to a specific disease entity. Here, as a sampling of the new book, is their chapter on acute leukemia and one of several related cases.

To order (Pub 221, $95 for CAP members, $110 for others ), call 800-323-4040
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Dr. Cherian is assistant professor of laboratory medicine at the University of Washington, Seattle. Dr. Wood, also of the University of Washington, is professor of laboratory medicine, director of the hematopathology laboratory and Seattle Cancer Care Alliance Pathology, and medical director of SCCA Laboratory. The authors write in the preface to their book that there are many ways to display flow cytometric data that are roughly equivalent and that the displays in their book reflect the standard approach used in their laboratory.

Acute leukemia is a neoplasm of immature myeloid or lymphoid cells characterized by a block in maturation, usually at the stage of an early progenitor (blast), with an associated increase in proliferation leading to an expansion of immature cells.¹ Immature lymphoid cells or lymphoblasts are expanded in acute lymphoblastic leukemia (ALL), while immature myeloid cells, including myeloid progenitors, abnormal promyelocytes, monoblasts, or promonocytes, are expanded in acute myeloid leukemia (AML). In most cases, a diagnosis of AML requires a morphologic blast percentage of 20% or greater in the blood or marrow. For ALL, a similar cutoff is preferred but not required for diagnosis. ALL, more commonly T-cell ALL, may primarily involve tissues, and when this is the primary manifestation, this process is called lymphoblastic lymphoma. AML may involve the tissues as well and when present is designated myeloid sarcoma.

By flow cytometry, acute leukemia is typically first recognized on a CD45 versus side scatter (SSC) plot as an expansion of cells with low CD45 and intermediate SSC.² Lymphoid blasts may have a slightly lower SSC in comparison with the slightly higher SSC commonly seen on myeloid blasts or the increased SSC characteristic of abnormal promyelocytes, which may approximate that of granulocytes (Fig. 1 (PDF, 1.7 MB)). Other cell types, including basophils, plasmacytoid dendritic cells, and hypogranular neutrophils, may also fall into the “blast gate” as defined by CD45 versus SSC; therefore, more specific markers are required for definitive blast identification. Such markers should enable one to confirm immaturity of the population of interest, define lineage, and define the population of interest as immunophenotypically aberrant and different from normal progenitor cells, including normal myeloid progenitors or B-cell precursors (hematogones) present in the marrow or T-cell precursors (thymocytes) present in the thymus. For myeloid blast equivalents, a combination of CD34 and CD117 is often employed to define immaturity, while antigens including CD5, CD7, CD11b, CD11c, CD13, CD14, CD15, CD16, CD33, CD38, CD56, CD64, CD65, HLA-DR, and MPO may be used to define lineage and demonstrate aberrancies in antigen expression. Markers including CD10, CD19, CD20, CD34, CD38, immunoglobulin light chains, and TdT achieve these goals for B cells, and markers including CD1a, CD2, CD3 (surface and cytoplasmic), CD4, CD5, CD7, CD8, CD10, CD34, and TdT may be employed in evaluating T cells. The assignment of lineage is a principal component of the diagnosis of acute leukemia. A relatively small number of antigens are sufficiently lineage associated to allow definitive lineage assignment when present and are discussed in detail in conjunction with Case 35.

Flow cytometry is also useful in enumerating blast populations, particularly in the blood. However, blast enumeration in bone marrow is complicated by two principal factors: hemodilution and under-representation of nucleated red blood cells. The former is invariably present in marrow specimens and increases in amount with increasing volume of marrow aspirate and decreased aspirability of the marrow, eg, fibrosis. The latter is dependent on the method of processing and the instrument detection threshold used for specimen acquisition. In addition, it is important to recognize that blasts are defined by morphology and that the immunophenotypic equivalent is the sum of populations as diverse as stem cells, CD34+ progenitors, B-cell progenitors, promyelocytes, monoblasts, and promonocytes. For these reasons, morphology, rather than flow cytometry, should be used for blast enumeration in the marrow when historic or World Health Organization criteria are applied.

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