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IMMUNOPHENOTYPING OF
LEUKEMIAS & LYMPHOMAS
INTRODUCTION
Immunophenotyping is the process used to identify and quantify cells
of the blood, bone marrow and lymph tissues according to their biological
lineage and stage of differentiation as defined by glycoproteins and associated
structures of the cell membrane. These lineage-specific cell surface
markers are produced by the normal genetic program of the cells or by aberrant
expression patterns that are pathologic. The cell markers are designated
according to a standard nomenclature that defines Clusters of Differentiation
(CD) by scientific consensus. They are detected by a process that combines
fluorescently-labeled, monospecific immunological reagents and a flow cytometer
to count and analyze the cell populations. The cells are then classified
by size, marker reactivity, clonality, and proportion. The procedure is
used clinically in diagnosis, prognosis, residual disease assessment, therapeutic
monitoring, and case management of leukemias, lymphomas, and related conditions.
A variety of tissues and body fluids may be analyzed. To ensure the quality
and clinical utility of the interpretations, all cytometric data are interpreted
in the context of a microscopic review of the specimen, and all interpretations
are written by a pathologist or hematologist.
COMMON INDICATIONS:
Classification of undifferentiated leukemia as lymphoid or myeloid
Subclassification of leukemias within lineage type
Identification of acute biphenotypic leukemias
Monitor therapy, residual disease, or recurrence
Evaluate leukocytosis
Evaluate lymph nodes for non-Hodgkin's lymphoma
Perform staging work-up in non-Hodgkin's lymphoma
SPECIMEN REQUIREMENTS:
Blood: 5-7 mL of blood in Na Heparin (green top) tube and 5-7 mL of blood in EDTA (purple top) tube. Mix well by inverting. To ensure optimal results, samples should be received within 24 hours. If the sample cannot be received within 24 hours, the sample may stand at room temperature, however, a blood smear must be made from the EDTA blood and sent along with the specimen. Alternatively, a 7 or 10 mL ACD tube (yellow top) may be submitted accompanied by a white blood count and differential obtained at the same time the ACD tube was drawn (counts from a separate tube must be provided to avoid dilutional effects of ACD).
Bone Marrow: 1-2 mL of bone marrow drawn in a sodium heparinized syringe (approximately 500 USP sodium heparin per mL of specimen). Mix well. Transfer specimen to a sodium heparinized tube. More specimen may be required if marrow is hypocellular. Samples should be received within 24 hours. If samples cannot be shipped to arrive within 24 hours, the specimen should be put into transport media (a heparinized syringe is still necessary for the initial draw).
Tissue: Place tissue biopsy in sterile container with tissue culture media. Samples should be received within 24 hours to ensure optimum viability. A viability check is performed prior to analysis of cells isolated from tissue.
MARKER GROUPS & ADD-ON STUDIES:
Leukemia Profile, Acute: Putative or known leukemic cell populations are analyzed for expression of T cell markers (CD2, CD3, CD4, CD5, CD7, and CD8), B cell markers (CD19 and CD20) and myeloid/monocytic markers (CD13, CD14, CD15, and CD33). Maturity status is assessed with CD34, HLA-DR, and CD10 (CALLA). Terminal transferase (TdT) may also be requested as a separate test or added by the pathologist when warranted. Other markers may be added after review by a staff pathologist or hematologist.
CLL & Lymphoma Profile: The total T cell population is enumerated by testing for the presence of three pan-T cell markers: CD2, CD3, CD5; CD4 (helper) and CD8 (suppressor) subpopulations are routinely included. The total B cell population is quantitated by determining the number of cells that express the pan-B markers CD19 and CD20. Clonality of B cell population is determined via the ratios of the kappa and lambda immunoglobulin light chain isotypes or their co-expression with pan-B markers or both. Co-expression of CD5 and CD20, a finding frequently associated with neoplastic proliferations, is also measured. CD10 (CALLA), CD23, FMC-1 and HLA-DR are included with the standard profile. Other markers may be added in assessing T-cell disorders (CD1, CD30), Hodgkin's disease (CD15, CD30), or anaplastic (Ki-1) lymphoma (CD30).
Hairy Cell Leukemia (HCL), Prolymphocytic Leukemia (PLL), or Mantle Cell Lymphoma / Leukemia: These B-cell diseases are assessed by performing a CLL & Lymphoma profile plus the hairy cell markers CD11c (complement receptor), CD 25 (IL-2 receptor), and CD103, and the prolymphocytic / hairy cell marker FMC-7. The B-lymphnoid marker CD23 is evaluated in relationship to CD5 expression for the different diagnosis of CLL vs. MCL. CD23 is part of the fundamental lymphoma profile.
Anaplastic (Ki-1) Lymphoma and Hodgkin's Disease: The markers CD1, CD15, and CD30 (Ki-1) are used in evaluation of these disease disorders. There are currently no specific markers for either inclusion or exclusion of HD, yet cytometric and microscopic evaluation provide supplementary data to the histopathologic data.
RESULTS
Verbal results are typically available within 24 hours of sample receipt; written reports within 48 to 72 hours. (Providing the background diagnostic information, relevant histopathologic information and clinical status in the space provided on the requisition can help to avoid delays.)
The final written report contains the cytometric marker percentages, morphologic correlation with a Wright-stained smear and full immunophenotypic interpretation by a clinical pathologist.
Relative values are reported for each marker tested; absolute values are also reported when whole blood is analyzed for CLL or immunodeficiency. Marker expression levels (intensity) are also evaluated and described when relevant.
Results are telephoned, faxed, or both, per your specifications.