A Teaching Database for Diagnosis of Hematologic Neoplasms Using Immunophenotyping by Flow Cytometry

Context.—In the diagnosis of lymphomas and leukemias, flow cytometry has been considered an essential addition to morphology and immunohistochemistry. The interpretation of immunophenotyping results by flow cytometry involves pattern recognition of different hematologic neoplasms that may have similar immunologic marker profiles. An important factor that creates difficulty in the interpretation process is the lack of consistency in marker expression for a particular neoplasm. For this reason, a definitive diagnostic pattern is usually not available for each specific neoplasm. Consequently, there is a need for decision support tools to assist pathology trainees in learning flow cytometric diagnosis of leukemia and lymphoma. Objective.—Development of a Web-enabled relational database integrated with decision-making tools for teaching flow cytometric diagnosis of hematologic neoplasms. Design.—This database has a knowledge base containing patterns of 44 markers for 37 hematologic neoplasms. We have obtained immunophenotyping data published in the scientific literature and incorporated them into a mathematical algorithm that is integrated to the database for differential diagnostic purposes. The algorithm takes into account the incidence of positive and negative expression of each marker for each disorder. Results.—Validation of this algorithm was performed using 92 clinical cases accumulated from 2 different medical centers. The database also incorporates the latest World Health Organization classification for hematologic neoplasms. Conclusions.—The algorithm developed in this database shows significant improvement in diagnostic accuracy over our previous database prototype. This Web-based database is proposed to be a useful public resource for teaching pathology trainees flow cytometric diagnosis.

Identification of Rat Targets of Anti-Soluble Liver Antigen Autoantibodies by Serologic Proteome Analysis

Background: Anti-soluble liver antigen (SLA) autoantibodies are specific for autoimmune hepatitis type 1 and are the only immunologic marker found in 15–20% of hepatitis cases previously considered cryptogenic. Anti-SLA antibodies react with the 100 000g supernatant from rat liver homogenate, but the molecular targets remain controversial. Methods: We characterized anti-SLA targets by one- and two-dimensional immunoblotting analysis. The recognized proteins were identified by peptide mass fingerprint analysis after matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Results: Three proteins of 35 kDa and pI 6.0, 50 kDa and pI between 6.0 and 6.5, and 58 kDa and pI between 6.5 and 7.0 were stained more intensely by anti-SLA positive-sera than by control sera. After in-gel tryptic digestion, MALDI-TOF analysis of the generated peptides enabled the clear identification of N-hydroxyarylamine sulfotransferase, isoforms of α-enolase, and isoforms of catalase. Conclusions: Possible antigens for anti-SLA antibodies include a sulfotransferase, α-enolase(s), and catalase(s). Two-dimensional electrophoresis combined with mass spectrometry offers a versatile tool to identify molecular targets of autoantibodies and thus to improve diagnostic tools and the understanding of the immune process.

Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia

Immunologic marker studies of the lymphoid leukemias have greatly improved the precision of diagnosis of these disorders by providing specific information regarding the lineage and stage of maturation of the malignant cells. Such studies have also enhanced our understanding of normal lymphocyte development, permitting reproducible identification of lymphoid cells in discrete developmental stages. By elucidating the functions of lymphoid cell differentiation antigens, it has been possible to gain insight into the signal transduction mechanisms by which these cells interact among themselves and with other cell types. Similar studies have shown that ALL is an immunophenotypically heterogeneous disease with clinically important subtypes representing clonal expansions of lymphoblasts at different stages of maturation. Furthermore, successful correlation of immunophenotype with certain karyotypic and molecular abnormalities, which appear to underlie most or all leukemias, were made possible by the inclusion of immunologic marker assessment. Interestingly, many of these phenotype-related abnormalities have involved either the Ig or TCR genes, thus providing additional clues to the mechanisms of leukemogenesis. Knowledge of the immunologic features of leukemic cells has been essential for the generation of phenotype-specific response data in the context of modern therapy for ALL. With wider use of intensive treatment, the traditional prognostic distinctions among immunophenotypes have begun to disappear; however, certain classes of agents have more favorable toxicity/efficacy ratios against some immunophenotypes than others, justifying continued efforts to target therapy by immunologic species of ALL. Antibody-toxin conjugates, or immunotoxins, have induced complete responses in preliminary trials and may prove clinically useful, perhaps in combination with chemotherapy, if their toxic side effects can be controlled. Finally, immunologic markers may serve as sensitive targets for the detection of minimal residual disease; the clinical usefulness of this approach will depend on prospective comparisons with molecular methods.