The diagnostic Laboratory for Immunocytology
Leukemic and lymphoma cells can be recognized in their benign counterparts by means of immunophenotyping. The differentiation scheme in which the phenotype of the cells in different maturation-stages are shown, can be used to determine the lineage of the malignant cell and his maturation state. In general acute leukemia's are the counterparts of cells in immature stages of differentiation whilst chronic diseases are usually more mature. The expression of these differentiation antigens are measured using monoclonal antibodies. The design of the antibody-panels is based on the guidelines from the Dutch Society for Cytometry (NVC). The NVC is part of the section Immunological Cell diagnostics of the Dutch Foundation for Quality Assessment in Clinical Laboratories. This foundation organizes quality control rounds in which the laboratory for Immunocytology takes part.
The laboratory for immunocytology is represented in the working party flowcytometry of the Dutch Society for Cytometry. This working party keeps track of new methods and protocols that can be used for diagnostic purposes. Subgroups with specific fields of interest are formed. At present there are two subgroups in which the laboratory plays a role: ‘the role of immunophenotyping in the diagnosis of MDS’ and ‘Measurement of ZAP70-expressie in B-CLL’. The MDS-subgroup has contacts with other investigators in Europe and the United States.
B and T cell differentiation scheme
Another possibility for the diagnosis of leukemia and lymphomas is the use of RNA/DNA analysis. From cytogenetic research it has become clear that certain chromosomal abnormalities are correlated to specific haematological malignancies. These abnormalities play a role in the arising of the disease. The genes concerned in these abnormalities are so-called ‘proto-oncogenes’; genes that are conserved during evolution that can lead to cancer when the expression of the genproduct is qualitative or quantitative changed. An example is the Philadelphia-chromosome: a part of chromosome 9 is translocated to chromosome 22 leading to the fusion-gene bcr-abl. This protein is not expressed in normal cells but is detected in almost all CML-patients. The expression of these fusion-genes can be used for diagnosis but also for therapy-evaluation. Real-time PCR (Taqman-technology) makes it possible to quantify 1 malignant cell in 100.000 normal cells.
The laboratory for immunocytology played a role in the European study to standardize the measurement of fusion transcripts and control genes by means of real-time PCR and is still board member of the Modhem (MOlecular Diagnostics for HEmatological Malignancies). The Modhem organizes quality control rounds for the quantification of fusion transcripts.
MRD-measurement in childhood ALL
Finally the laboratory for immunocytology works together with the department of immunology of the Erasmus MC and the Dutch Childhood Oncology Group. Children diagnosed with Acute Lymphoblast Leukemia (ALL) are all treated in conformity with the protocol described by the DCOG. Part of this protocol is the measurement of the tumorload during the first three months of therapy. Based on this tumorload, therapy becomes heavier or is lowered. For each individual patient is determined how the immunoglobulin- and T-cell receptor-genes are rearranged. These rearrangements follow a strict pattern that is correlated to different differentiation stages and can be used to design patient-specific real-time PCR reactions using patient-specific forward primers, consensus reverse primers, and consensus probes. The laboratory is part of the European Study Group on Acute Lymphoblast Leukemia – Minimal Residual Disease (ESG-ALL-MRD) and participates twice a year in a quality round organized by this study group.
Gene rearrangement immunoglobuline heavy chain