Administrative Office
St. Joseph's Hospital Site, L301-10
50 Charlton Avenue East
HAMILTON, Ontario, CANADA L8N 4A6
PHONE: (905) 521-6141
FAX: (905) 521-6142 http://www.fhs.mcmaster.ca/hrlmp/
The Flow Cytometry Laboratory at the Henderson Site is responsible for the analysis of blood and/or marrow samples where there is a suspicion of a malignant monoclonal population of cells. In some cases the abnormal population of cells present in the blood or marrow are the majority of all nucleated cells (i.e. leukemia). In other cases, a suspicious population of cells is subtle and requires a comprehensive analysis to establish monoclonality. This is often the case when a suspicious population of lymphoid or lymphoid-appearing cells are noted following examination of a peripheral blood smear. Assessment of lymphocytosis incorporates the skills of an experienced morphologist to recognize features associated with malignancy as compared to lymphocytosis in response to infection (e.g. EBV). More often these cases will be referred to the flow cytometry laboratory for further more definitive analysis. Standard Immunophenotypic profiling can readily determine the lineage of the target population. In some cases where the proportion of cells are minimal, this does require careful gating and exploration of CD marker combinations.
Analysis of an abnormal lymphocytic population will readily define these cells as being of B- or T-cell lineage.
On some occasions, cells will also express NK (natural killer cell) markers. In the case of B cells, monoclonality can be readily noted by comparing the relative proportions of immunoglobulin light chains present on the cell surface. In this regard, if the ratio of kappa to lambda light chains is greater than 10:1 or less than 1:3 for population of B-lymphocytes, it is highly suggestive of a monoclonal population (light chain restriction). This will be further assayed and confirmed by molecular analysis (immunoglobulin heavy chain PCR).
Screening assessment of monoclonality in T cell disorders, including peripheral T cell lymphocytosis at present does not have a convenient flow-based screening assay (such as light chain restriction). T cells do have a unique T cell receptor, which undergoes mutation to create diversity in the same way B cells produce unique Ig rearrangements. The majority of peripheral blood T cells express the T cell receptor (TCR), which is comprised of

units. Each T cell will, in theory, express a unique combination of V

, J

, V

, D

, J

TCR gene segments which results in an extensive TCR

repertoire. If a malignant/monoclonal T cell phenotype is present, analysis of the TCR repertoire should be skewed to a particular V

subtype. Recently, the flow cytometry laboratory has implemented a monoclonal antibody based V

analysis into the clinical service laboratory. This process has greatly enhanced the rapid ability to recognize potential malignant states prior to the use of molecular confirmation, which in the case of the T cells is sometimes inconclusive.
Types of T cells Disorders in Peripheral Blood and Marrow
1. T-cell large granular lymphocyte leukemia – In T-cell large granular lymphocyte (LGL) leukemia. Patients demonstrate invasion of bone marrow, spleen and liver by clonally expanded T-cell LGLs. The large granular lymphocyte is a morphologically distinct lymphoid subset comprising 10 to 15% of normal peripheral blood mononuclear cells. LGLs arise from two major lineages: 1) CD3+, CD57+), CD56- T cells, representing activated cytotoxic effector T cells, and 2) CD3-, CD56+ natural killer cells. Secondary benign (nonclonal) LGL expansions have been reported following viral infections (EBV, HBV, HCV, HIV, CMV), connective tissue diseases, ITP, non-Hodgkin's lymphoma, some skin disorders, and hemophagocytic syndromes. Patients with
LGL lymphoproliferative disorder typically have an associated neutropenia.
2. Mycosis fungoides — Mycosis fungoides typically presents as an indolent cutaneous eruption with erythematous scaly patches or plaques, often resembling common skin disorders such as eczema or psoriasis.
Patients with Sezary syndrome present with generalized erythroderma, lymphadenopathy, and atypical T-cells
(Sezary cells) in the peripheral blood.
3. T-Cell Prolymphocytic Leukemia (T-PLL) — T-cell prolymphocytic leukemia is a distinct subtype of PLL with a clinical course that is significantly different from that of B-PLL, which is a much more common phenotype.
4. NK cell neoplasms-Natural killer cell large granular lymphocyte leukemia The clinical presentation of natural killer (NK) cell LGL leukemia is considered more aggressive than that of T-cell LGL leukemia.

5. Adult T cell lymphoma/leukemia: ATLL, according to the REAL and WHO classifications, is defined as a peripheral T-cell neoplasm associated with infection by the human T-lymphotropic virus, type I (HTLV-I). The morphology of these entities includes cells with bizarre hyperlobated nuclei (clover leaf or "flower cells"), often resembling Sezary cells. Tumor cells express T-cell associated antigens (CD2, CD3, CD5), but usually lack
CD7. CD25 is expressed in a majority of the cases.
Identification of Vβ Restriction in the Analysis of
Complicated T cell Lymphoproliferative Disorders
We are now utilizing a panel of 24 mAbs capable of detecting T cell TCR restriction using Vβ restriction analysis.
Analysis is limited to CD3+ cases where determination of monoclonality is problematic. Once monoclonality is established further panels of CD markers are employed to further characterize and make a formal diagnosis.
Analysis of Suspicious T cell Population by Flow Cytometry
Detailed immunophenotypic analysis of T cell lymphoproliferative abnormalities include a panel of immunophenotypic assays. These include initial characterization of CD4+ T cells, which include ATLL and
Sezary/Mycosis vs. CD8+ T cell disorders comprised of LGL-T cell and LGL NK type disorders. T-CLL/T-PLL may express either CD4 or CD8. A panel of markers can be employed to delineate each condition. In addition morphological features are also useful.
The use of V

analysis is a useful tool with rapid turnaround time and will increase the diagnostic accuracy for specific cases T cell lymphoproliferative disorders.
References
1.
Beemd, R, Boor PC, van Lochem EG et al. Flow Cytometric Analysis of the VB Repertorie in Healthy
Controls. Cytometry 40:336-345 (2000).
Praniti Soamboonsrup, Cathy Ross and Ronan Foley
Discipline of Special Hematology
Hamilton Regional Laboratory Medicine Program
Henderson General Hospital Site