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Clinical Chemistry 45: 1708-1717, 1999;
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(Clinical Chemistry. 1999;45:1708-1717.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Clinically Useful Information Provided by the Flow Cytometric Immunophenotyping of Hematological Malignancies: Current Status and Future Directions

Alberto Orfao1,a, Gerd Schmitz2, Bruno Brando3, Alejandro Ruiz-Arguelles4, Giuseppe Basso5, Raul Braylan6, Gregor Rothe2, Francis Lacombe7, Francesco Lanza8, Stefano Papa9, Paulo Lucio10, Jesus F. San Miguel1 and for the Standardization Committee on Clinical Flow Cytometry of the International Federation of Clinical Chemistry

1 Department of Medicine and Centro de Investigaciones del Cancer, Universidad de Salamanca, 37007 Salamanca, Spain.

2 Department of Laboratory Medicine, University of Regensburg, D-93053 Regensburg, Germany.

3 Renal Transplant Unit, Niguarda-Ca' Granda Hospital, 20162 Milan, Italy.

4 Laboratorios Clinicos de Puebla, Puebla 72530, Mexico.

5 Clinica Pediatrica, 10126 Torino, Italy.

6 Department of Pathology, University of Florida, Gainesville, FL 32610.

7 Laboratoire d'Hematologie, Hopital Haut-Leveque, 33608 Pessac, France.

8 Institute of Hematology, Ospedale S. Anna, 44100 Ferrara, Italy.

9 Istituto di Scienze Morfologiche, Universita degli Study di Urbino, 61029 Urbino, Italy.

10 Serviço de Hematologia, Instituto Portugues de Oncologia, 1093 Lisbon, Portugal.
a Address correspondence to this author at: Servicio General de Citometria, Laboratorio de Hematologia, Hospital Universitario, Paseo San Vicente s/n, 37007 Salamanca, Spain. Fax 34-23-294624; e-mail orfao{at}gugu.usal.es


   Abstract
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Background: At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications.

Methods: The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed.

Results: A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed.

Conclusions: The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.


   Clinical Utility of Immunophenotyping of Hematological Malignancies
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In the past two decades, it has been shown that immunophenotyping of abnormal hematological cells is very useful for the diagnosis, classification, prognostic evaluation, and detection of residual disease in patients with hematological malignancies (1)(2)(3)(4)(5). Although in the first immunophenotypical studies microscopic evaluation of antibody staining was used, at present flow cytometry is the preferred method for data analysis (3)(4)(5)(6)(7)(8).

Accordingly, there is general consensus that flow cytometry immunophenotyping is a primary diagnostic modality in chronic lymphoproliferative disorders, including non-Hodgkin lymphoma (1)(2)(4)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), as well as in both de novo acute leukemias and blast crises following either a chronic myeloproliferative disorder or a myelodysplastic syndrome (1)(2)(4)(7)(28)(29)(30)(31)(32)(33). Regarding the value of immunological classification, it is well established in acute lymphoblastic leukemia (ALL) (28)(34)(35)(36)(37) and in chronic lymphoproliferative disorders, including non-Hodgkin lymphoma (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), as well as in specific subtypes of acute myeloblastic leukemia (AML) (7)(28)(38)(39)(40)(41). From the prognostic point of view, several individual markers have been associated with disease outcome in both AML and ALL as well as in multiple myeloma, as summarized in Table 1 [reviewed in Refs. (1)(2)(3)(4)(5)(42)(43)(44)]; in contrast, no clear association has been demonstrated between the expression of surface antigens and prognosis of the disease in the different subgroups of chronic lymphoproliferative disorders.


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  		Table 1. Prognostic value of the expression of individual antigens on leukemic cells according to different disease groups.a

In addition, it has been demonstrated in recent years that flow cytometry is a sensitive method for detecting residual disease in several hematological malignancies, including B-cell chronic lymphoproliferative disorders (45)(46)(47) and both ALL (48)(49)(50)(51)(52)(53)(54) and AML (32)(33)(55)(56)(57)(58)(59)(60). Although the clinical impact of immunological detection of residual disease in these patients has not yet been definitively demonstrated, preliminary reports (32)(48)(49)(50)(51)(52)(53)(54)(60) indicate that it may contribute to a more accurate monitoring of patients and management of chemotherapy and transplantation on an individual basis; in addition, it will also contribute to a more precise evaluation of possible tumor contamination of the cell products that will be used for autologous stem cell transplantation (48)(58)(59).


   Standardization of Flow Cytometry Immunophenotyping of Hematologic Malignancies: State of the Art
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Because of the potential clinical utility of the information provided by the flow cytometric immunophenotyping of hematological malignancies, this technology has expanded rapidly in the diagnosis and monitoring of patients. The intra- and interlaboratory reproducibility of a test must be achieved and optimized before it can become a diagnostic laboratory test. This applies to both the methods used for the assessment of antigen expression in hemopoietic cells and the criteria used for the clinical interpretation of the results obtained. A careful analysis of the literature shows the existence of disturbing degrees of variability, a clear example of which is the reported incidence for cross-lineage antigen expression in both ALL (50)(61)(62)(63)(64)(65)(66)(67) and AML (57)(68)(69)(70)(71)(72)(73) and its potential prognostic value, especially among AML cases (1)(57)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80). A critical analysis of these reports shows that there are a wide range of different reagents and methods used for both sample preparation and data acquisition and analysis. In addition, a lack of standardized criteria for data interpretation is a common finding that introduces disturbing variability. This has been confirmed in several different reports on external quality assessment of immunophenotyping procedures (81)(82)(83)(84). Such situations have led in recent years to an enormous effort by several groups to obtain specific consensus protocols for leukemia immunophenotyping and common criteria for data interpretation and reporting (1)(2)(3)(4)(5)(6)(7)(28)(34)(81)(84)(85)(86)(87). Although these efforts have led to a common language and improved methodologies, they have been only partially successful. In addition, morphology continues to be the standard of reference for the immunophenotypic characterization of leukemia, which according to Paietta et al. (39) should be considered as a relic of the past.

The factors that may affect the results of the immunophenotypic analysis of neoplastic hemopoietic cells have already been identified, at least to a large extent (1)(2)(3)(4)(5)(6)(7)(28)(34)(85)(86)(87). Among them, technical aspects such as the type and quality of the sample, the reagents and the sample preparation protocols, instrument set-up and calibration, and the potential component of subjectivity introduced during data analysis or with the interpretation of the results represent the most common sources of variability. Based on this knowledge, several scientific groups (1)(2)(3)(4)(5)(6)(7)(28)(34)(85)(86)(87) have discussed these aspects in detail and have reported their consensus opinions and recommendations. However, in many of these reports, the type of information provided by the flow cytometry immunophenotyping of hematological malignancies is not analyzed in depth. Such a detailed analysis would certainly contribute to understanding the answers provided by flow cytometry to the specific questions posed in current practice that are related to the immunophenotype of hematologic malignancies: Does this lymphocytosis correspond to a chronic lymphoproliferative disorder? Is this monoclonal component associated with a malignant disease condition? Are the blast cells present in a certain sample of myeloid or lymphoid hemopoietic cell lineages? Are there residual leukemic cells in a sample that is in morphologically complete remission? In general it may be considered that, at least to a certain extent, the type of information requested influences the analytical procedure to be used. In other words, one of the most relevant aspects when performing or requesting flow cytometric immunophenotyping of hematologic malignancies is to have a clear idea of the type of information provided by this test that could help in answering a specific clinical question. Accordingly, the major goal of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact. In addition, where appropriate, we will comment briefly on the technical approaches that should be selected to obtain this information with the highest sensitivity, specificity, objectivity, and simplicity as well as the quickest way to get it.


   Information Provided by the Flow Cytometry Immunophenotyping of Hematological Malignancies
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Flow cytometry has been used for more than two decades for the identification, enumeration, and/or characterization of both healthy and leukemic hemopoietic cells on the basis of their immunophenotypic features. One well-accepted advantage of flow cytometry for the immunological analysis of hemopoietic cells compared with microscopy is that it provides an objective and sensitive multivariate analysis of high numbers of cells, this information being obtained in a single cell basis. In spite of this, for a long time immunophenotyping of hematological malignancies has been considered as a second-line test after morphological and cytochemical diagnosis has been established. However, because of the objectivity, the high statistical accuracy, and the unique type of information on neoplastic cells provided by this analytical approach, immunophenotyping is increasingly being used in routine practice. In any case, a prerequisite for the precise flow cytometric analysis of neoplastic hemopoietic cells is the ability to identify them in a sensitive and specific way. Only under these conditions will we be in a position to accurately enumerate and characterize these pathologic cells.

identification of leukemic cells
A precise definition of the presence of pathological hemopoietic cells is essentially based on the ability to clearly distinguish them from the healthy cells present in the specimen. Accordingly, phenotypic patterns of healthy cells present in all types of samples used for the diagnosis of hematological malignancies must be well established not just on the basis of their relative/absolute distribution but by considering their phenotypic characteristics, which define a precise place for these cells in a multidimensional space created by the different light scatter and fluorescence-associated markers analyzed.

Classically, it has been considered that leukemic/lymphomatous cells reflect the immunophenotypic characteristics of healthy cells blocked at a certain differentiation stage. In recent years, accumulating evidence has shown that pathological cells display aberrant phenotypes as defined by cross-lineage antigen expression, asynchronous antigen expression, ectopic phenotypes, and abnormal differentiation pathways among others (32)(50)(58)(59)(88)(89). These aberrant phenotypes are believed to reflect, at least to a certain extent, the genetic abnormalities present in the pathologic cells. Recent data have shown that the incidence of these aberrant phenotypes increases with the use of appropriate multiple-staining techniques (72), and at least in acute leukemias (54)(58)(59)(60)(72)(88)(89)(90), chronic lymphoproliferative disorders (46), and plasma cell dyscrasias (91)(92), they may be present in almost all patients. Such a high incidence of aberrances allows discrimination between healthy and pathological cells within a sample, thus avoiding the need for enrichment steps such as density gradient centrifugation procedures (4)(5)(85).

From a practical point of view, identification of pathologic cells cannot be achieved by selecting them exclusively on the basis of their light scatter properties, as was initially proposed for the analysis of peripheral blood lymphoid subsets (93). Accordingly, apart from the light scatter properties, which certainly are of great help by providing useful information, other markers are necessary for sensitive and specific identification of pathological hemopoietic cells. The use of orthogonal light scatter (SSC)/CD45 gating has been proposed (4)(5)(34)(94)(95)(96) as a helpful tool for the identification of pathological cells in acute leukemias because blasts usually appear in a position where few healthy cells are located in the SSC/CD45 dot plot, especially in peripheral blood samples. Other criteria, such as the use of low SSC/CD19+, low SSC/CD7, and intermediate SSC/strong CD38, may be more specific and useful than CD45 gating for B-cell disorders (46)(51)(97)(98)(99), T-cell ALL (51), and plasma cell dyscrasias (91)(92)(100), respectively. Although none of these latter three markers allows the simultaneous study of all types of malignant hemopoietic cells because they are restricted to a specific cell lineage or maturation-associated stage, these criteria produce a highly sensitive approach for the identification of leukemic cells in lymphoid neoplasias, especially when leukemic cells are present at low frequencies (46)(91). Once the main cell population (e.g., CD19+ B cells) has been selected, additional stainings performed simultaneously within that particular cell subset will contribute to a precise discrimination between the healthy and pathological counterparts, as was clearly shown in previous reports (46)(50)(51)(91). Therefore, multiple stainings must be used and indirect immunofluorescence methods, in principle, avoided because of inherent technical problems (4)(85).

enumeration of leukemic cells
The number of pathological cells identified morphologically in bone marrow and/or peripheral blood samples is currently used for the diagnosis of several disease conditions. As an example, diagnostic criteria based on the proportion of blasts in patients with myelodysplastic syndrome (101) or acute leukemia (102), the percentage of plasma cells in plasma cell dyscrasias (103), and the number of mature-appearing lymphocytes in chronic lymphocytic disorders (104) are used even if the observer is not able to distinguish between healthy and malignant cells within a particular cell population on the basis of their morphology and cytochemical characteristics.

For a long time flow cytometry has been used in diagnostic laboratories for the enumeration of peripheral blood CD4+ T lymphocytes in HIV infection, which has been shown to be the most useful index for patient monitoring (104). More recently, flow cytometry has also become the preferred method to assess the number of hemopoietic progenitor cells (CD34+ cells) in cell products that will be used for bone marrow or peripheral blood stem-cell transplantation (105). For these purposes, flow cytometry has been shown to be a robust and reproducible technology (106)(107)(108).

If all pathological cells present in a sample can be identified as such, their number may be easily monitored. The only prerequisite is that single cells can be distinguished from those events that do not correspond to a cell and from cell multiplets that may be present at variable proportions among all the acquired events; moreover, dead cells must be excluded because of nonspecific staining (4)(85)(109)(110)(111). To monitor how many of the events acquired actually correspond to single nucleated cells, several methods, such as the use of nucleic acid stains for both viable and nonviable cells and/or the light scatter properties of the events acquired, may be used reliably (4)(85)(107)(109)(110)(111). Caution should be taken with other approaches, such as gating on CD45+ cells, because mature nucleated bone marrow erythroid cells (112) as well as some leukemic cells may be negative for this antigen (113)(114)(115). In addition, appropriate analysis of the light scatter properties of these events must be made to take into account the possible existence of selective cell doublets such as those that usually occur in B-cell chronic lymphoproliferative disorders (116). In general, cell multiplets show increased scatter and fluorescence values compared with single cell.

According to what has been mentioned above, flow cytometric immunophenotyping of hematological malignancies will certainly become a useful and powerful method for the enumeration of pathological cells present in a given sample. As a matter of fact, recent reports have demonstrated that the immunophenotypic enumeration of leukemic cells is of both diagnostic and prognostic value. Two clear examples of this are the value that has been associated with the assessment of the percentage of bone marrow plasma cells corresponding to healthy residual polyclonal plasma cells in the differential diagnosis between monoclonal gammopathies of undetermined significance and multiple myeloma (91) and the prognostic impact of the number of residual bone marrow cells displaying leukemia-associated phenotypes in patients with either AML (32)(55)(56)(57)(58)(59)(60) or ALL (48)(49)(50)(51)(52)(53)(54)(59)(88) who are in morphologically complete remission. Nevertheless, it should be mentioned that in some specimens, such as bone marrow or spinal fluid samples, the enumeration of pathologic cells should be taken with caution because contamination with peripheral blood cells may occur and in such a case it may affect the final count of pathological cells present in the sample.

A special comment should be made on the analysis of solid tissues such as lymph node and spleen. In these tissue types, the disaggregation procedures usually affect cell viability, producing relatively high numbers of dead cells. This may largely limit the accuracy of the enumeration of neoplastic cells present in these samples.

characterization of leukemic cells
The aim of the immunological characterization of pathological cells present in a sample once they have been identified and enumerated should include three consecutive steps: (a) lineage assignment; (b) analysis of the degree of heterogeneity of the abnormal cell population attributable to either the existence of different pathological clones or the presence of cells in different maturational stages; and (c) further phenotypic characterization of each of the pathologic cell subsets identified.

Lineage assignment of pathological cells does not represent a major difficulty in the study of mature (i.e., chronic) hematologic malignancies because in these patients neoplastic cells already express highly specific markers such as the CD3/TCR complex in T-cell disorders (1)(2)(3)(4)(5)(6)(8). In contrast, lineage assignment is a major challenge in the diagnosis of acute leukemias because of the immaturity of the cells. The production of the first monoclonal antibodies against epitopes present in healthy leukocytes has led reports on the existence of lineage specific markers. However, preliminary studies on leukemic samples showed that in some cases malignant cells display cross-lineage antigen expression. Accordingly, reactivity for the lymphoid markers Tdt, CD2, CD7, and CD19 was demonstrated in MPO+ myeloid leukemic cells (68)(69)(70)(71)(72)(73)(75)(76)(77)(78)(79)(80), whereas positivity for the myeloid antigens CD13, CD33, and CD15 can be found in a variable proportion of ALL cases (50)(54)(74). This has led to enormous confusion in the literature and efforts to clarify the pathogenetic and clinical implications of these findings (2)(28)(117)(118). In addition, a search for more specific markers that could be of help in distinguishing between the different hematopoietic cell lineages has started. At present it is well established that there are several ways to obtain lineage assignments in acute leukemias and that most of the methods are effective, the incidence of real biphenotypic/bilineage cases being very low (1)(2)(4)(28)(85). The most specific markers for cells of the B-lymphoid lineage are cytoplasmic CD79a and either surface or cytoplasmic immunoglobulins, whereas for T-cell lineage, the most specific markers are cytoplasmic or surface CD3 and T-cell receptor (2)(3)(4)(5)(7)(34)(119). For myeloid cells, myeloperoxidase and lysozyme are the most highly specific markers (3)(4)(5)(7)(28)(34)(119). Subclassification of myeloblasts according to the different myeloid cell lineages largely remains a challenge for the future because, with the exception of the megakaryocyte-associated markers CD61, CD41, and CD42 (7)(28)(34)(40) and the erythroid related antigen glycophorin A (7)(28)(34)(41), no good markers have been identified for discriminating granulocytic from monocytic cell lineages as well as for the identification of a high proportion of erythroid cases (3)(4)(5)(7)(28)(34)(41). Finally basophilic, eosinophilic, mast cell, and dendritic cell disorders probably remain largely undiagnosed because of the lack of information on the immunological characteristics of the immature counterparts of these cell subsets (120)(121)(122)(123). Investigation of the normal differentiation pathways of CD34+ precursors as regards the acquisition of MPO, lysozyme, CD64, CD15, or CD123 expression will certainly contribute to a better discrimination among the different myeloid cell lineages (120)(121)(122)(124)(125).

Pathological cells from patients suffering from different hematological malignancies usually have been thought to represent cells that display a blockade in their differentiation ability, giving rise to the accumulation of a relatively homogeneous population of hemopoietic cells. In spite of this, in vitro studies have shown that leukemic cells derive from a more immature leukemic progenitor (126). Accordingly, most CD34+ progenitor cells in chronic myeloid leukemia patients belong to the pathological clone, although they retain their ability to differentiate into mature appearing granulocytes (127). On the other hand, an oligoclonal origin has been postulated for several hematologic malignancies, and it is feasible to detect different cell clones, as has been demonstrated by cytogenetic and molecular studies. Thus, one would expect that from the immunophenotypic point of view, pathological cells from an individual patient may be heterogeneous. As a matter of fact, recent reports on immunophenotyping have shown the existence of more than one population of pathological cells in patients with acute leukemias, their frequency being as high as 80% in AML (33)(127). However, common differentiation pathways frequently are found between different subsets of pathological cells, suggesting that these subpopulations might belong to the same pathological clone (33)(58)(128). In contrast, in chronic lymphoproliferative disorders the existence of more than one pathological cell population, although being much less frequent, is usually associated with the presence of more than one cell clone as demonstrated by molecular studies (129).

To be sure that we are correctly characterizing the pathological cells present in the sample, they must be identified through several multiple-staining combinations. Accordingly, some markers, usually conjugated with the same fluorochrome, may be used in each of the multiple-staining combinations analyzed. As an example in B-cell disorders such as B-precursor ALL and in B-cell chronic lymphoid leukemias, the use of CD19 in all tube combinations tested highly improves and facilitates the sensitive and specific characterization of the pathological cells by reducing the number of healthy cells present in the SSC/CD19+ selected population (46)(48)(129).

Further immunological characterization of pathological cells in the clinical laboratory should be performed whenever a specific feature of these cells has been shown to be of clinical importance for the diagnosis (immunological features that are specific of a certain cell lineage and maturation stage), prognosis (subclassification of leukemias affecting the same hemopoietic cell lineage according to differentiation features or phenotypic characteristics associated with certain genetic abnormalities), or monitoring of the disease (investigation of phenotypic aberrations and abnormal differentiation pathways). In general, information on the characteristics of leukemic cells should be obtained when comparing leukemic with healthy phenotypes to explore how similar and how different leukemic cells and their healthy counterparts are. To date, the clinical utility of the flow cytometric immunophenotypic characterization of hematological malignancies has been based mainly on the investigation of the similarities between leukemic and healthy cells (1)(2)(3)(4)(5)(6)(7)(28)(34). In fact, the assessment of the lineage and differentiation stage of the pathologic cells represents the basis of the current immunophenotypic diagnosis and classification of hematological malignancies. Representative examples of what is mentioned above are the assessment of myeloid or B- or T-lymphoid involvement in the differential diagnosis between AML and ALL of either the B- or T-cell lineages (1)(2)(4)(7)(28)(34) and the subclassification of precursor B-ALL cases into pro-B, common, pre-B, and B-ALL according to the expression of B-cell differentiation markers (CD10, cytoplasmic immunoglobulin µ, surface immunoglobulins) in leukemic cells (28)(35)(36)(37). Nevertheless, as mentioned above, at present it is well established that leukemic cells display immunophenotypic features that are different from those present in healthy cells of the same cell lineage and maturation stage (32)(33)(46)(51)(58)(59)(72)(91)(92)(108). Most probably these phenotypic aberrations are related to the existence in leukemic cells of underlying genetic alterations. To support this hypothesis, it would then be expected that specific genetic abnormalities are associated with characteristic phenotypic aberrations. In fact, recent reports have shown the existence of an important association between specific genetic alterations and the immunophenotypic characteristics of leukemic cells in both AML (29)(57)(130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140) and ALL (60)(90)(141)(142)(143)(144)(145)(146)(147)(148)(149)(150) patients. However, in most of these cases the sensitivity and specificity of the aberrant immunophenotype for the identification of those cases carrying a specific genetic abnormality is relatively poor, which limits the use of immunophenotyping as a screening method to select those cases in which confirmatory molecular studies will be performed. This relatively low sensitivity and/or specificity is probably related to technical questions such as the use of individual antigen expression instead of multivariate phenotypic patterns and the use of information on just the presence/absence of individual antigens without considering the extent of antigen expression and its pattern of reactivity (homogeneous vs heterogeneous, unimodal vs multimodal). As a matter of fact, in a more recent study (140) on a series of 111 AML patients in which multivariate information provided by the use of multiple stainings analyzed at flow cytometry was obtained, it has been shown that the combination of three phenotypic variables (the number of major blast cell populations, the pattern of CD34/CD15 expression, and the reactivity for CD13) was highly sensitive (100%) and specific (99%) for the selection of AML cases carrying PML/RAR-{alpha} gene rearrangements as demonstrated by the combined use of the reverse transcription-PCR and fluorescence in situ hybridization.

Based on these findings, it can be concluded that because of the clinical utility of comparing the results obtained from the immunophenotypic characterization of healthy and leukemic cells, a major challenge for the near future is the possibility of performing stable, calibrated, and standardized measurements in such a way that identical cells provide identical phenotypic patterns whenever they are analyzed at different times and in different laboratories.


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