Flow Cytometry in Oncologic Diagnosis



Flow Cytometry in Oncologic Diagnosis


Michael J. Borowitz






Functional Components


A flow cytometer has three separate components: a fluidics system, an optical platform, and signal-processing electronics. The fluidic system aspirates the sample, mixes it with a sheath fluid to produce laminar flow, and conducts the cell suspension past the sensing zone where individual cells are examined. The optical platform consists of the laser light source(s), lenses to focus the light on the passing cell stream, filters that capture light of restricted wavelengths, and photomultiplier tubes that capture the emitted signals. The electronics convert photons to electrical signals in proportion to the total light contacting the photomultiplier tubes and amplify and scale the signals so that data can be readily analyzed. The value of each collected parameter is “quantitized” and assigned to a particular channel. Higher channels reflect brighter signals. Current software packages reprocess collected data electronically in a variety of formats; this flexibility of data analysis is a critical part of what makes this technology useful.


Electronically reprocessed data can be displayed by software programs in many forms, but the most commonly used forms are dot plots or histograms. The most useful displays usually correlate one parameter with another, with each dot representing a single event (i.e., cell) with the x- and y-channel values for the two chosen parameters. In addition to fluorescence, forward scatter and right-angle or side scatter can be displayed. Forward scatter is roughly proportional to the size of the cells, whereas side scatter is a measure of internal cellular complexity, which for hematopoietic cells usually means granularity. Typically, dot plots of either forward versus side scatter, or scatter versus fluorescence, are used to identify populations of interest in a process called gating, and additional displays show additional fluorescence or scatter characteristics of these populations. Because scatter measurements are made parallel to, and independent of, the fluorescence measurements, four-color flow cytometry, for example, is equivalent to six-parameter cytometry. The term multiparameter flow cytometry typically is used to define simultaneous analysis of five or more parameters on individual cells.



Fluorochromes and Fluorescence


Fluorochromes have spectral characteristics that allow them to absorb light of certain wavelengths and then to emit light at longer wavelengths. Emission, a specific characteristic of the compound, is not limited to a fixed wavelength but rather constitutes a spectrum, with variable numbers of photons emitted at different wavelengths. Different fluorochromes, including fluorescein isothiocyanate and phycoerythrin, as well as others, all have the capacity of absorbing light at 488 nm, but because they emit it at different wavelengths, it becomes possible to perform multicolor flow cytometry with a single laser emitting at 488 nm. Tandem conjugates, which covalently couple two fluorochrome molecules, allow light emitted by one molecule to be transferred to and excite the other; this process can greatly increase the number of colors that can be detected with a single laser. Additional dyes such as allophycocyanin, which emit longer wavelengths, cannot be excited by a 488 nm laser but require a second light source, most frequently one that emits light at 635 nm. The combination of new dyes, more tandem conjugates, and new instruments with even more lasers has made it possible to perform flow cytometry to detect more than a dozen colors; in the clinical laboratory today, instruments capable of detecting six to eight or even more colors are the norm, although many laboratories do not yet take advantage of these higher-order capabilities, in part because of the complexity associated with analyzing and quality controlling such complex data. However, 10-color flow cytometry has been adapted to routine clinical analysis.1



Applications of Flow Cytometry to Clinical Oncology


Flow cytometry plays a significant role in the diagnosis, classification, and management of patients with acute leukemia, chronic lymphoproliferative disorders, and non-Hodgkin lymphoma. The early promise of this technology in the management of patients with solid tumors has never been realized, but it still plays a role in certain areas.



Acute Leukemia


Flow-cytometric immunophenotyping has become standard in the evaluation of new patients with acute leukemia. The most obvious role of flow cytometry is in distinguishing lymphoid from myeloid leukemia, but flow cytometry can help in the diagnosis and management of these patients in many ways (Box 15-1).



Box 15-1   Applications of Flow Cytometry in Hematologic Neoplasia




Lymphoma and Lymphoproliferative Disorders




ALL, Acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia.



Lineage Assignment in Acute Leukemia


Phenotypic analysis of a bone marrow that is completely replaced by blasts is an almost elementary problem. Multiparameter flow cytometry, however, can dissect and categorize all populations in bone marrow, and most important, it can distinguish leukemic cells from normal cells, even when the leukemic cells are not the majority population. The antibody panel used to study patients with acute leukemia typically contains representative markers of all lineages, with some redundancy to allow recognition and classification because many antigens may be aberrantly lost or acquired in leukemic cells.2,3 No standard combination of antibodies is used by all laboratories, but certain combinations have proved particularly useful not only for the most economical classification of leukemia, but also to demonstrate characteristic aberrant patterns that may be extremely useful for the detection of residual disease in follow-up samples, as discussed later in this chapter.


Difficulties encountered in the interpretation of flow cytometry data derive largely from two problems. First, in cases in which leukemic cells are not an obviously dominant population, it is important to ensure that the cells of interest are analyzed. The most useful general approach for this difficulty takes advantage of the fact that the common leukocyte antigen CD45 is differentially expressed on different types of hematopoietic cells and, when combined with side scatter, produces a display in which blasts occupy a unique position not occupied by normal cells (Fig. 15-1).4 Thus combining CD45 in one color with multiple combinations of antibodies in additional colors allows detailed characterization of blast populations in marrow even when they are present only in low numbers.



Failure to select the leukemic cell population for analysis, or including a mixture of leukemic and normal cells in a gate, may cause confusion in the reporting of flow cytometry results. It is best to identify the leukemic population visually and to provide a detailed description of the antigens expressed on the leukemic population, especially in myeloid leukemias, in which the dynamic patterns of maturation associated with morphologic variation in acute myeloid leukemia (AML) are reflected in changes in both light scatter and antigen expression as the leukemic cells mature. Tabular arrays of “percentage positive” are not recommended as part of a flow cytometry report, because they cannot reflect this complexity and may cause confusion.5


The second problem in interpreting flow cytometry results in leukemia derives from the fact that most of the reagents used in classifying leukemia are only relatively rather than absolutely specific. Thus correct classification requires not only a panel with some redundancy but also an understanding of patterns of reactivity of the antibodies. Markers such as CD13 and CD33, which are considered myeloid antigens because they originally were produced against myeloid leukemia cells, are found in up to 50% of cases of lymphoid leukemia,6 and interpretation of leukemias positive for these markers continues to be a cause of confusion. Generally speaking, the most specific markers of a given lineage are not highly sensitive, and the most sensitive ones are not specific. This finding is true more of myeloid markers than of lymphoid markers, and thus lymphoid leukemias usually can be recognized precisely, whereas poorly differentiated myeloid leukemias usually are defined by the presence of myeloid markers in the absence of specific lymphoid antigens.



Acute Leukemias of Indeterminate or Ambiguous Lineage


Although almost all cases of acute leukemia can be categorized easily regarding lineage, the lack of absolute specificity of most markers, and the promiscuity of their expression, implies that some cases cannot be resolved easily. Unfortunately, considerable controversy exists about the use of the terms mixed lineage, bilineal, and biphenotypic leukemia, and the new World Health Organization 2008 classification has recommended a more descriptive term: “mixed phenotype acute leukemia” (MPAL).7 These leukemias, which express myeloid- and lymphoid-associated markers in various combinations, represent a heterogeneous group of diseases. Previously, these leukemias were identified using a scoring system that assigned various point values to different antigens, with a diagnosis of “biphenotypic leukemia” rendered if the score were high for more than one lineage.8 The new World Health Organization classification recommends a different approach, recognizing the limitations of defining lineage with arbitrary scores. In this system, MPAL is defined using fewer, more specific markers, and, most significantly, cases of MPAL associated with specific genetic lesions are considered specific entities.



Association of Immunophenotype and Molecular Abnormalities


Many phenotypes in both acute lymphoblastic leukemia (ALL) and AML are highly associated with characteristic cytogenetic abnormalities.9 In ALL, these include cases associated with mixed lineage leukemia rearrangements, TEL-AML1 or E2A-PBX1. In AML, the most important link is in promyelocytic leukemia with the t(15;17) translocation. Whereas lack of human leukocyte antigen-DR subregion (HLA-DR) is the best-known abnormality, only about half of cases of DR-negative AML turn out to be acute promyelocytic leukemia, and other combinations of marker expression are much more sensitive and specific for acute promyelocytic leukemia.10 AML associated with the t(8;21) translocation also shows a characteristic phenotype.11



Minimal Residual Disease Detection in Acute Leukemia


Several studies have demonstrated that the presence of residual leukemic cells in the marrow of patients who are in clinical and morphologic remission is a very strong adverse prognostic factor.1220 Although the most extensive data exist in cases of childhood ALL,1317,19 the principle has also been shown to apply to persons with adult ALL21,22 and to persons with AML.18,20


Both molecular and flow-based methods have been used to detect minimal residual disease (MRD). Flow-based assays of MRD are based on the principle that nearly all leukemias show a pattern of expression of antigens that is aberrant compared with the pattern seen in normal differentiation.15,2329 This aberrancy can take several forms. Some leukemic cells can abnormally express antigens of a different lineage or show loss of expression of a normal lineage marker. A more common finding is expression of normal differentiation antigens, but at an intensity that is different from that expected for a particular stage of differentiation. This latter attribute makes flow MRD analysis applicable to most cases of leukemia. Nonetheless, recognizing these deviations requires a clear understanding of patterns of maturation in normal differentiation, including marrow regeneration, as viewed in multiparameter space.


The pattern of antigen acquisition and loss during B-cell maturation in the bone marrow has been very well characterized, and certain markers are particularly useful for distinguishing normal and leukemic maturation. Consideration of markers including intensity of CD45, CD34, CD10, CD58, and CD38 or aberrant coexpression of myeloid or other unexpected antigens can allow detection of as few as 1 in 104 leukemic cells, even when normal B-cell precursors are present in significant numbers (Fig. 15-2). Marrow T-cell acute lymphoblastic leukemia (T-ALL) also can be distinguished from normal T cells, in part because T-ALL will typically coexpress cytoplasmic CD3 and TdT, which is never seen on any normal cell.15 However, after treatment with steroid-based therapies, T-cell leukemias may undergo therapy-induced maturation and lose expression of TdT and other markers useful for distinguishing immature and mature T cells.30 Thus T-ALL MRD detection often relies on detecting the same type of phenotypic abnormalities as seen in B-cell acute lymphoblastic leukemia (B-ALL). Detection of myeloid MRD usually is a more elaborate process because of the greater phenotypic heterogeneity in AML. Certain aberrant combinations, including coexpression of CD34 and CD56, CD117 and CD15, or CD7 and myeloid antigens occur with sufficient frequency to be useful in a large number of cases.24,28,31 Achieving 10−4 sensitivity on a consistent basis is difficult, and sensitivities are more typically closer to 10−3. Because abnormal populations at diagnosis may not persist at recurrence, monitoring patients with acute leukemia is best done by looking for phenotypic differences from normal, rather than for a specific abnormal phenotype present at diagnosis.



Jun 13, 2016 | Posted by in ONCOLOGY | Comments Off on Flow Cytometry in Oncologic Diagnosis

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