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Immunophenotyping is a routine method to evaluate B-cell non-Hodgkin lymphomas. Flow cytometry plays a complementary role in diagnosis and classification of these types of lymphomas, since combination of morphologic, immunophenotypic and genotypic features is needed to correctly classifying each disease entity. Multiparameter flow cytometry, which is now carried out with routine combinations of six to eight monoclonal antibodies, allows identifying even small lymphomatous cell populations on the basis of aberrant B-cell marker expression and clonality. The immunophenotypic patterns obtained by multiparameter flow cytometry are useful to correctly diagnose most of cases of specific subtypes of B-cell non-Hodgkin lymphomas and to discover peculiar clinical presentations, such as discordant and composite lymphomas. Immunophenotypic variability, however, characterizes B-cell lymphomas. Therefore, flow cytometry should always be used in combination with other techniques to correctly classify each disease entity. Finally, multiparameter flow cytometry is characterized by high sensitivity in detecting residual disease.

Ly-1/CD5 is a 68-kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells. Although its function remains the subject of speculation, CD5 expression serves as a useful marker for a functionally distinct population of B cells that has attracted a considerable amount of interest from investigators of both murine and human immune systems. Of critical importance in much of this work is the isolation of pure populations of CD5+ B cells along with appropriate control populations. A flow cytometry technique is presented in this unit, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules.

The introduction of monoclonal antibodies (mAbs) for cell immunophenotyping and use of flow cytometry with the progressively improving software for multivariate analyses have revolutionized the diagnosis and influenced the classification of hematologic neoplasms. In this review we focus on the practical application of flow cytometry in the diagnosis and classification of malignant lymphomas and related lymphoproliferative disorders with special emphasis on differential diagnosis. A general approach to the utilization of flow cytometry (FC) in hematopathology with an algorithm to diagnose the most common neoplasms is presented. We discuss precursor B-cell neoplasms, mature B-cell neoplasms (SLL/CLL, mantle cell lymphoma, marginal zone lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, plasma cell dyscrasias and lymphomas with plasmacytic differentiation), precursor T-lymphoblastic leukemia and mature (peripheral) T-cell neoplasms, including T-SLL/PLL, anaplastic cell lymphomas and large granular cell leukemia/lymphoma. The text is accompanied by characteristic FC scatterplots of the discussed entities.

The present review summarizes our efforts in developing a novel immunologic approach ("Comparative Phenotype Mapping") targeted at assessing minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients. The method relies on quantitatively aberrant, leukemia-associated antigen expression patterns which allow to discriminate leukemic from normal BCP using a limited panel of antibody combinations and multidimensional flow cytometry. In an analysis of 63 follow up bone marrow samples of patients with BCP-ALL we show that this approach enables to efficiently detect MRD. Further clinical observation revealed that the patients which were MRD-positive by flow cytometry (although in morphological remission) had a very high probability of early disease recurrence compared to the good chances of a relapse-free survival (RFS) in the MRD-negative cohort (RFS 0.0 vs. 0.76 at 3 years). Comparative Phenotype Mapping thus proves to be a reliable method for MRD detection in BCP-ALL. Concluding remarks relate to the optional applications of the method as well as to future perspectives. An ongoing large prospective study which we are now conducting on the basis of Comparative Phenotype Mapping will clarify the clinical significance of MRD detection in ALL patients by this method, and will determine its value compared to related as well as molecular-genetic techniques.

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.