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34 Part I: Clinical Evaluation of the Patient Chapter 3: Examination of the Marrow 35

and erythroblasts (best evaluated in the direct film or concentrate) are PRINCIPLES OF FLOW CYTOMETRY
examined for the presence of iron granules in the cytoplasm (siderob-
lasts). Late erythroblasts are readily identified by their small size and INTERPRETATION
the size, shape, and chromatin pattern of the nucleus. The proportion
of normal late erythroblasts that contain one to four small Prussian Immunophenotyping is complementary to morphology in the contem-
70
blue granules is extremely variable (3–69 percent) in normal subjects. porary practice of marrow cell identification. Flow cytometers use simi-
Pathologic ring sideroblasts are characterized by an increased number lar principles to the automated hematology analyzers discussed in Chap.
of iron granules arranged in a ring encircling at least 1/3 of the nucleus, 2, with the notable difference that fluorescence-labeled monoclonal
reflecting accumulation of iron in mitochondria (Chap. 87). antibodies directed toward cluster of differentiation (CD) antigens are
the primary diagnostic tool. As described in the World Health Organi-
zation classification of hematologic malignancies, immunophenotypic
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MEGAKARYOCYTES data (expression of cell surface, intracytoplasmic, and nuclear antigens)
Chapter 111 discusses the megakaryocyte in detail. Megakaryocytes are are key determinants of diagnosis and classification of hematopoietic
large cells (30–150 μm) with darkly stained, irregularly lobed nuclei. malignancies. The principle of immunophenotyping is to diagnose and
The cytoplasm is blue “cotton candy” textured, and the more mature follow neoplastic cell populations by virtue of differential patterns of
cells contain many azurophilic granules. protein expression. Only the basic principles of flow cytometry analysis
are described in this chapter, so that the reader has the basis for under-
LYMPHOCYTES standing the phenotypic characteristics associated with the hematopoi-
etic disorders described in greater detail in other chapters of this book.
In normal marrow, lymphocytes similar to those found in the blood
occur in variable numbers, depending on the degree of blood contami-
nation of the marrow (Chap. 73). Immature lymphoid cells with a high METHODOLOGY
nuclear-to-cytoplasmic ratio and moderately dense, but finely distrib- Flow cytometers are automated hematology analyzers that use princi-
uted, chromatin (“hematogones”) often are seen in marrow aspirates of ples of light scatter and fluorescence to define cellular populations in
children, and mostly represent B-cell precursors. These cells may cause which to analyze expression of proteins typically identified by fluores-
71
diagnostic difficulty in some clinical settings, such as the “rebound” cent tagged antibodies. A single-cell suspension is aspirated into a lam-
lymphocytosis that occurs after cessation of maintenance chemotherapy inar flow of isotonic diluent that passes in front of one or more laser
for acute lymphoblastic leukemia. beams. Light scatter and fluorescence data are collected using specific
photomultiplier tubes with appropriate filtration to collect scattered
PLASMA CELLS light (same wavelength as the incident laser light) or fluorescence emit-
Normal plasma cells vary in size but usually are 12 to 16 μm in diameter ted light (at a longer wavelength determined by the dye used). Multiple
when spread on a slide. They are round or oval. The nucleus is small, detectors with different filtration coupled with single or multiple lasers
round, eccentrically placed, and stained densely purple. The chromatin are used to collect highly multiplexed data. As with automated hema-
is coarse and clumped. Nucleoli are not visible. The cytoplasm is deep tology analyzers, light scatter information is collected at a low angle
blue, often with a paranuclear clear zone (Chap. 73). Binucleate forms (correlates with cell size) and 90-degree angle (correlates with cellular
may be found in normal marrow. granularity and nuclear complexity; Chap. 2, Fig. 2–1). The latter mea-
surement is especially useful in separating developing myeloid progen-
itors, monocytes, and mature granulocytes from lymphoid cells and
OTHER CELL TYPES blasts.
Mast cells are readily recognized by their content of dark-blue gran- Immunophenotyping can be achieved by using monoclonal anti-
ules, which usually completely fill the cytoplasm and may obscure the bodies specific to certain cell surface proteins, most of which have CD
nucleus (Chap. 63). The cells are round or spindle-shaped and often are designations as defined by international workshops. A primary require-
located deep in the particles, frequently lying along blood vessels. The ment for flow cytometry analysis is that cells must be viable and in
nucleus often is not visible but when seen is round or oval with a vesic- single-cell suspension prior to staining, which is why this method is used
ular chromatin pattern. largely for hematopoietic malignancies and immunologic disorders,
Osteoclasts and osteoblasts are uncommon, and are more likely seen and not for analysis of solid tumors. This consideration also explains
in hypocellular marrow or marrow obtained from children and from differences in results between flow cytometry and morphologic or
adults with hyperparathyroidism or osteoblastic reactions to tumors. immunohistochemical observations when samples with highly adher-
Osteoclasts are large cells and may be larger than 100 μm in diameter ent neoplastic cells are analyzed. For instance, in multiple myeloma
(see Fig. 3–3). They superficially resemble megakaryocytes but contain or large cell lymphoma the proportion of malignant cells is typically
multiple separated nuclei that have a moderately fine chromatin pattern lower (or absent) by flow cytometry compared with marrow biopsy. In a
with nucleoli. The cytoplasm varies from slightly basophilic to intensely well-equipped and appropriately staffed clinical laboratory, preliminary
acidophilic because of the content of acidophilic granules. Osteoclasts information often can be provided within 3 to 4 hours after the initial
may contain coarse basophilic debris. Osteoblasts usually are oval cells sample collection, thereby facilitating institution of appropriate therapy
up to 30 μm in the longest diameter (see Fig. 3–3). They often occur (e.g., in the case of newly diagnosed acute leukemias).
in groups. The nucleus usually is quite eccentric and may seem to be Clinical laboratories typically use four- to six-color analysis, plus
spilling out of the cell. The chromatin pattern is uniform, and one to side and forward light scatter, for routine diagnostic panels. For research
three nucleoli are present. The cytoplasm is light blue and may contain studies, simultaneous analysis of up to 20 simultaneous fluorochromes
a few red granules. Osteoblasts may be mistaken for plasma cells. In is possible, by excitation with up to 5 lasers and separate collection of
osteoblasts, the pale centrosomal region of the cytoplasm is separated the emitted light produced by interaction with each laser. At present,
from the nucleus, in contrast to that of the plasma cell, in which the routine use of that many simultaneously measured markers is not nec-
centrosomal region directly abuts the nucleus. essary for clinical diagnosis. An important consideration is that analysis

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