The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow-up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners. © 2007 Clinical Cytometry Society
Flow cytometric immunophenotyping (FCI) first appeared in clinical laboratories in the 1980s, in the wake of the AIDS epidemic. Initially utilized to assess CD4 T-cells, the technique was soon applied to lymphoid and eventually myeloid neoplasms. The intervening years have seen a marked rise in the utility of the technique, accompanied by improvements in technology that have enhanced the speed, specificity, and sensitivity. Despite its widespread use and in part, because the technique is so versatile and robust, it continues to suffer from a reputation for inconsistency due to a lack of strict standardization and variability in interpretative experience by its practitioners.
By 1995, practitioners recognized a clear need for consensus statements regarding the indications for FCI, and a group of U.S. and Canadian hematopathologists, hematologists, and laboratory scientists met in Bethesda, MD, on November 16–17, 1995. A series of consensus recommendations was generated by the group that addressed in detail a number of topics, including standardization and validation of laboratory procedures (1), selection of antibody combinations (2), data analysis and interpretation (3), data reporting (4), and medical indications (5). The last reference addressed medical indications largely from disease-specific perspective and did not necessarily identify those clinical signs and symptoms, which led to evaluation for those diseases in the first place. A subsequent consensus document generated by North American and European experts at ISAC 2000 summarized opinions regarding utility of various antibodies to diagnose various diseases (6), but once again neglected to consider the practical situation confronted by clinicians and laboratories: under what clinical circumstances is FCI appropriate? That is, what are the signs and symptoms most likely to prompt FCI and to yield clinically useful data? Conversely, what are the signs and symptoms that do not warrant FCI?
Partly in order to address this significant omission in the literature, a consensus conference was convened at the National Institutes of Health in Bethesda, MD, on July 14–15, 2006. Participating experts were primarily from North America and Europe (see Introduction). Participants were specifically asked to identify those signs and symptoms typically noted in patients with suspected hematolymphoid neoplasms and distinguish among those that do warrant FCI and those that do not warrant FCI. Note that a large and expanding literature documenting typical immunophenotypes of various diseases already exists (7, 8), and the present document makes no attempt to address this topic. Similarly, this document does not address the relative merits of other diagnostic modalities such as microscopic examination, immunohistochemistry, or cytogenetic and molecular studies.
CLINICAL SIGNS AND SYMPTOMS
Hematolymphoid malignancies are typically systemic diseases, with tumor involving more than one tissue site simultaneously. Bone marrow is often affected and resulting cytopenias may bring the patient to the attention of a physician. Some neoplasms elaborate cytokines and the resulting cytopenias, cytoses, and constitutional symptoms such as fever or weight loss may prompt investigation for a hematolymphoid neoplasm. Significant infiltration of tissues by neoplastic cells may present as mass lesions such as lymphadenopathy or organomegaly, lytic lesions of bone, or diffuse lesions such as skin rash or effusions. Infiltration may compromise the function of vital organs such as liver and brain. Clinical disease presentations are highly variable. For example, a patient with acute myeloid leukemia may present with bruising due to thrombocytopenia or with a skin rash due to leukemic infiltration. Furthermore, none of these findings is specific for acute myeloid leukemia, and in each instance the clinician and pathologist must consider a differential diagnosis that includes other hematolymphoid neoplasms, nonhematolymphoid neoplasms, and nonneoplastic etiologies.
Consensus was reached on a list of clinical indications which support FCI. In each instance it is assumed that a hematolymphoid neoplasm is suspected based on clinical or pathological findings and other causes for the abnormality (e.g., nutritional deficiency, infection, drug reaction, or autoimmunity) have been considered. Importantly, the association of the findings listed later with constitutional symptoms increases the likelihood of hematolymphoid neoplasia and strengthens the indication for FCI testing.
Any hematolymphoid neoplasia can present with anemia, leukopenia, and/or thrombocytopenia. In addition, monocytopenia may be a sign of specific hematolymphoid neoplasms. FCI may therefore be indicated in these situations (9–23). Since isolated cytopenias, particularly anemia, commonly occur in nonneoplastic diseases, these alone should not automatically trigger FCI. Pancytopenia is more likely to be associated with neoplasia and FCI is indicated. Because any hematolymphoid neoplasia can produce cytopenia, all lineages should be evaluated.
Elevated Leukocyte Count
Elevated leukocyte counts can be a sign of hematolymphoid neoplasia. The differential diagnosis of a lymphocytosis includes reactive conditions, chronic lymphocytic leukemia (CLL), and other neoplastic disorders of lymphocytes. A specific diagnosis of many chronic lymphoproliferative disorders can be made by flow cytometry (24–47). Monocytosis may be seen with chronic myelomonocytic leukemia or occasionally with other myeloproliferative disorders, and FCI may be helpful in distinguishing these from reactive monocytoses (11, 15, 16, 21, 23, 48). Finally, eosinophilia can be one of the first indications of acute myelogenous leukemia, mastocytosis, acute lymphoblastic leukemia, and T-cell lymphoproliferative disorders (7, 49–54). Neutrophilic leukocytosis is, in general, not an indication for flow cytometry in the absence of blasts, except for focused assays, such as CD64 expression, that provide objective evidence of a systemic acute inflammatory response to infection or sepsis (55, 56).
Isolated findings of polycythemia, thrombosis, or basophilia are not sufficient to warrant FCI, as they are more commonly associated with nonmalignant diseases or hematolymphoid neoplasms lacking characteristic FCI findings.
Observation of Atypical Cells or Blasts and Evaluation of Body Fluids
Identification of blasts in the marrow or peripheral blood is an absolute indication for flow cytometry. (21, 57–97). FCI confirms that atypical cells are blasts and plays an important role in the diagnosis and classification of acute leukemia. FCI is indicated in the evaluation of atypical mononuclear cells in body fluids to rule out reactive activated lymphoid cells and detect possible neoplasia (98–100) due to its greater sensitivity and specificity than morphology alone (101–103). Specifically, FCI is indicated in the evaluation of serous effusions and CSF, including aqueous or vitreous humor of patients with a history of hematolymphoid neoplasia (104–106).
Plasmacytosis or Monoclonal Gammopathy
These findings may be seen in plasma cell dyscrasias or multiple myeloma, characteristically associated with clonal proliferations of plasma cells, often with aberrant phenotypes. Monoclonal gammopathies can also be seen in chronic B lymphoproliferative disorders that can be diagnosed and often classified by FCI (5, 24, 107, 108). FCI assists in the differential diagnosis between plasma cell myeloma and monoclonal gammopathies of undetermined significance by determining the percentage of aberrant or clonal plasma cells of all bone marrow plasma cells (108–114) (Rawstron A, Orfao A, Mateo G. Report of the EMN (European Myeloma Network) workshop on flow cytometry in multiple myeloma and related disorders, to be submitted). FCI is useful in diagnostic evaluation of unexplained marrow plasmacytosis by assessing phenotypically aberrant or clonal plasma cells and its ability to detect other underlying monoclonal B-cell process (5, 108).
Organomegaly and Tissue Masses
Tissue-based lymphoid neoplasias commonly affect lymph nodes, spleen, mucosa-associated lymphoid tissue, skin, or nonlymphoid solid organs resulting in masses or organomegaly. With the exception of Hodgkin's lymphoma, FCI is extremely useful in the diagnosis and subclassification of tissue-based lymphoid neoplasias, and there is consensus that this testing is indicated in lymphadenopathy (46, 107, 115–126) (also see references24–47 for lymphocytosis), organomegaly (including but not limited to splenomegaly and hepatomegaly) (127), and tissue infiltrates (including but not limited to skin, mucosal sites, and bone) (34, 36, 39, 42, 124, 128–130). Recent work suggests that with careful assessment it is possible to make a diagnosis of classical Hodgkin's lymphoma by FCI (131), but the necessary procedures are not currently employed in most laboratories.
Patient Monitoring Indications
Once an initial diagnosis of hematolymphoid neoplasm is rendered, additional FCI may be performed. Indications for testing in this setting are as follows:
Staging disease to document the extent of involvement (111, 132, 133). Although flow cytometry is more sensitive than conventional morphology for detecting disease in bone marrow and blood, clinical decisions based on upstaging due to detection of submicroscopic disease should be carefully considered because in many diseases the significance of staging using more sensitive technology has not been well established. The value of performing flow cytometric staging on both bilateral marrow aspirates, compared to pooled specimens from both sides, has not been established, though in the case of minimal residual disease analysis studying two independent marrow samples does not provide significant additional information (134).
Detecting potential therapeutic targets, e.g., CD20, CD52 (135, 136).
Assessment of response to therapy (including “minimal residual disease” testing) and persistence of FCI-detectable MRD following therapy, which is often an adverse prognostic factor. However, the optimal frequency of such monitoring has not been established (112, 137–163).
Documentation of progression or relapse.
Diagnosis of additional intercurrent hematolymphoid neoplasm, either treatment- related (such as MDS/AML or PTLD) or coincidental (164–170).
Evaluation of disease acceleration (e.g., CML blast crisis) or transformation (e.g., diffuse large B cell lymphoma in low grade lymphoma or CLL) (66, 74, 96, 171, 172).
Prognostication, especially in CLL, with detection of ZAP70 or CD38 (135, 136, 173–179), acute leukemia (59, 77, 78, 180–185), or myeloma, whereby phenotype, presence of circulating plasma cells, or assessment of proliferation rate can predict progression (111, 114, 186–188).
Samples submitted for FCI may not also not show hematolymphoid neoplasia. Some patients, despite highly suspicious signs and symptoms of hematolymphoid neoplasm, will have another cancer type, or a nonneoplastic condition such as an occult infection or autoimmune process. Samples from patients with a hematolymphoid neoplasm may not be involved and/or therapy may reduce the disease burden to undetectable levels. In such cases, a negative evaluation for hematolymphoid neoplasm by FCI may still be informative (25, 35, 44, 48, 122). Of course, the extent to which a negative FCI study is valuable is related in part to the quality of the evaluation, which is dependent on the antibody panel; specifically which antibodies, how many, and in what combinations (reference Technical consensus document, Wood et al., 2007).
Flow cytometry is generally not indicated in the following conditions because they are usually not associated with hematolymphoid malignancy or associated with hematolymphoid neoplasms that are not detectable by FCI.
Flow cytometry is diagnostically productive and cost-effective. Therefore, flow cytometric studies are an integral component of contemporary hematopathology practice. Knowledge of the flow cytometric findings of the hematopoietic cells involved in hematolymphoid neoplasms is critical in patient management, providing data that aids in diagnosis, staging, prognosis, therapeutic targets, and disease monitoring. FCI, however, is not indicated in all clinical situations and these consensus guidelines are meant to aid decisions on the use of flow cytometry. We present guidelines for appropriate clinical settings for flow cytometric testing for hematolymphoid neoplasia developed by an international panel of experts. Previous consensus recommendations (5, 6) utilized a disease-oriented approach which, while informative, tended to obscure the practical aspects of best practice in the setting of a flow cytometry laboratory. The current consensus document was developed as a practical guide to FCI based on clinical settings that confront clinicians and laboratories. Although this is the first consensus document to explicitly summarize the clinical signs and symptoms that warrant the use of FCI, it in fact reflects clinical practice exercised since the inception of the technique.
The biologic analyses of neoplasms outpace personalized treatment options. Indeed, FCI provides an important tool for the development or selection of personalized treatment and stimulates the development of new strategies. However, the clinical application of the biologic information provided by flow cytometric analyses must be evaluated in large, well designed, clinical trials. Furthermore, future clinical trials to evaluate novel therapeutic regimens must consider the usefulness of flow cytometric data and other biologic parameters. Consequently, the contribution of flow cytometry to patient care is a dynamic and evolving process with important clinical applications. Thus periodic review and assessment of medical indications for flow cytometric evaluation is necessary to keep pace with scientific and clinical advancements.