Algorithm(s)
PDF algorithm(s) available at www.arupconsult.com.
Leukemia and Lymphoma Phenotyping AlgorithmClinical Background
During evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL), the possibility of a malignant disorder requires evaluation.
Refer to Leukemia-Lymphoma Phenotyping topic at www.arupconsult.com for table of Common Hematologic Antigens
See Also
Algorithm(s)
PDF algorithm(s) available at www.arupconsult.com.
Leukemia and Lymphoma Phenotyping AlgorithmDiagnosis
Diagnosis
- Indications for testing - evaluation of peripheral lymphocytosis (absolute lymphocytosis >4,000/µL)
- Laboratory testing
- Must rule out other possible disorders associated with lymphocytosis
- If lymphoproliferative disorder remains a significant possibility after clinical evaluation, cell surface phenotyping of lymphocytes should be performed
- Phenotyping of cell surface antigens on peripheral blood lymphocytes usually performed using flow cytometry
- Technique provides percentage of lymphocytes positive for a particular antigen and density of antigens
- Normal peripheral blood lymphocytes consist of approximately 10% B-cells, 80% T-cells and 10% NK cells
- Most commonly used markers (CD = cluster designation)
- B-cell - CD10, CD19, CD20, CD22, CD23, CD24, CD79b, CD103, Kappa, Lambda, FMC7, Cytoplasmic Kappa, Cytoplasmic Lambda
- T-cell - CD1, CD2, CD3, CD4, CD5, CD7, CD8, T-cell receptor (TCR) alpha- beta, TCR gamma-delta, Cytoplasmic CD3
- Myeloid/Monocyte - CD11b, CD13, CD14 (Mo2), CD14 (MY4), CD15, CD33, CD64, CD117, myeloperoxidase
- Miscellaneous - CD11c, CD16, CD25, CD30, CD34, CD38, CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin, TdT, bcl-2
- Phenotyping of cell surface antigens on peripheral blood lymphocytes usually performed using flow cytometry
Lymphoproliferative disorders, malignancies of the lymph system, can be divided into B-cell and T-cell types
- B-cell lymphoproliferative disorder is probable if immunoglobulin light chain restriction is demonstrated by surface typing of kappa or lambda
- B-cell chronic lymphocytic leukemia (CLL) or mantle cell lymphomas are suspected if CD5 is positive and CD10 is negative
- Circulating mantle cell lymphoma can be mistaken morphologically for B-cell CLL
- Mantle cell lymphoma considered when
- CD20, CD19 - strong intensity
- Surface immunoglobulin - strongly expressed
- CD23 - absent
- Diagnosis
- Molecular and FISH testing
- Requires t(11;14) translocation demonstration
- CLL is more likely when:
- CD20 - strong intensity
- Surface immunoglobulins - weakly expressed
- CD 23 - present
- Mantle cell lymphoma considered when
- Circulating germinal center cell derived lymphoma is probable if CD10 is positive and CD5 is negative
- Germinal center lymphomas - follicular, Burkitt’s, diffuse large B-cell
- Diagnosis
- Some cases can be confirmed by the demonstration of the t(14;18) breakpoint by PCR or FISH testing
- PCR detects approximately 80% of t(14;18) translocations found in follicular lymphoma
- FISH is more sensitive for this translocation in fixed tissue
- FISH can also detect a c-MYC or BCL-6 rearrangement for Burkitt’s or diffuse large B-cell lymphoma
- Hairy cell leukemia has a characteristic phenotype that is CD5-, CD10-, CD11c+, CD25+ and CD103+
- CD103 antigen (also known as B-ly7) is present in virtually all cases
- CD11c and CD25 are less specific, but present in virtually all cases of hairy cell leukemia
- T-Cell lymphoproliferative disorders
- Most show abnormalities of pan T-cell antigens CD2, 3, 5, or CD7
- T-cell disorders
- Proliferating lymphocytes are usually positive for CD3
- Most common form is large granular lymphocytosis
- Usually show rearrangement of T-cell antigen receptor locus
- Clonality assessed by flow cytometry, PCR or Southern blot
- Large granular lymphocytosis is suspected if percentage of CD16, CD56 or CD57 positive cells >50%, or if absolute count of these cells >2000/µL
- Peripheral T-cell lymphoma (NOS)
- Phenotypic abnormalities
- NK cells
- CD3 is absent
- Occurs in a minority of cases
- Angioimmunoblastic lymphoma has characteristic CD10 and CD4 positive and CD52, 56 and 16 negative
- Anaplastic large cell lymphoma - positive CD30 and ALK(+)
- Some pan T-cell antigens are frequently deleted
- Sézary syndrome should be considered if CD7 and CD26 are negative
- Clinical spectrum and prognosis are variable in both T-cell and NK-cell types
- Most behave in indolent fashion
Differential Diagnosis
- Epstein-Barr virus (EBV)
- Chronic infections
- Granulomatous disease
Algorithm(s)
PDF algorithm(s) available at www.arupconsult.com.
Leukemia and Lymphoma Phenotyping Algorithm| Tests |
Tests generally appear in the order most useful for common clinical situations
| Test name: Leukemia/Lymphoma Phenotyping (Comprehensive-Whole Blood) |
| ARUP #: 0096299 |
| Methodology: Flow Cytometry |
| Use: Use for whole blood specimens to aid in selection of appropriate therapy and assessment of clinical behavior and prognosis Available antigens included: |
| Test name: Leukemia/Lymphoma Phenotyping (Comprehensive - Bone Marrow) |
| ARUP #: 0095244 |
| Methodology: Flow Cytometry |
| Use: Use for bone marrow specimens to aid in selection of appropriate therapy and assessment of clinical behavior and prognosis Available antigens included: |
| Test name: Leukemia/Lymphoma Phenotyping (Comprehensive - Miscellaneous) |
| ARUP #: 0095243 |
| Methodology: Flow Cytometry |
| Use: Use for tissue specimens or fluids to aid in selection of appropriate therapy and assessment of clinical behavior and prognosis Available antigens included: |
| Test name: T-Cell Clonality by Flow Cytometry Analysis of TCR V-Beta |
| ARUP #: 0093199 |
| Methodology: Flow Cytometry |
| Use: Further characterize phenotypically abnormal T-cell populations identified by flow cytometry Determine the existence of monoclonality based on expression of T-cell antigen receptor beta chain variable regions (TCR V-Beta) |
| Limitations: Tests only for T-cell alpha and beta antigen receptors; if T-cell delta or gamma antigen receptors testing is desired, PCR testing is recommended |
| Test name: T-Cell Clonality Screening Assay by PCR, Fluid |
| ARUP #: 0055567 |
| Methodology: Polymerase Chain Reaction/Fragment Analysis |
| Use: Determine presence of a monoclonal T-cell population in whole blood or bone marrow |
| Limitations: A negative result does not entirely exclude the presence of a T-cell receptor gamma rearrangement (or monoclonal T-cell population) in the sample |
| Follow-up: Confirm results with T-cell clonality assessment by restriction fragment souther blot hybridization, fluid |
| Test name: T-Cell Clonality Screening Assay by PCR, Tissue |
| ARUP #: 0055568 |
| Methodology: Polymerase Chain Reaction/Fragment Analysis |
| Use: Determine presence of a monoclonal T-cell population in tissue |
| Limitations: A negative result does not entirely exclude the presence of a T-cell receptor gamma gene rearrangement (or monoclonal T-cell population) in the sample |
| Follow-up: Confirm results with T-cell clonality assessment by restriction fragment souther blot hybridization, fluid |
| Test name: T-Cell Clonality Assessment by Restriction Fragment-Southern Blot Hybridization, Tissue |
| ARUP #: 0055594 |
| Methodology: Restriction Fragment Southern Blot Hybridization |
| Use: Confirm presence of T-cell receptor beta gene arrangement presence in tissue |
| Limitations: A negative result does not entirely exclude the presence of a T-cell receptor gene rearrangement (or monoclonal T-cell population) in the sample |
| Test name: T-Cell Clonality Assessment by Restriction Fragment-Southern Blot Hybridization, Fluid |
| ARUP #: 0055596 |
| Methodology: Restriction Fragment Southern Blot Hybridization |
| Use: Confirm presence of T-cell receptor beta gene arrangement presence in whole blood or bone marrow |
| Limitations: A negative result does not entirely exclude the presence of a T-cell receptor gene rearrangement (or monoclonal T-cell population) in the sample |
| Test name: bcl-2/JH, t(14;18) Translocation by PCR, Fluid |
| ARUP #: 0055616 |
| Methodology: Polymerase Chain Reaction |
| Use: Detect chromosomal translocation t(14:18) (BCL2/IGH gene rearrangements) [bcl-2/JH] Components include BCL2/IGH, t(14;18) [bcl-2/JH] translocation major breakpoint and minor cluster regions |
| Limitations: A negative result does not entirely exclude the presence of a BCL2/IGH [bcl-2/JH] chromosomal t(14;18) translocation |
| Test name: bcl-2/JH, t(14;18) Translocation by PCR, Tissue |
| ARUP #: 0055619 |
| Methodology: Polymerase Chain Reaction |
| Use: Use to determine presence of BCL2/IGH, t(14;18) [bcl-2/JH] chromosomal translocation (non-Hodgkin lymphoma) Components include BCL2/IGH, t(14;18) [bcl-2/JH] translocation major breakpoint and minor cluster region |
| Limitations: A negative result does not entirely exclude the presence of a BCL2/IGH [bcl-2/JH] chromosomal t(14;18) translocation |
| Test name: Chromosome Analysis, FISH-Interphase |
| ARUP #: 0092615 |
| Methodology: Fluorescence in situ Hybridization |
| Use: Detect chromosome abnormalities associated with mantle cell lymphoma FISH probes must be specified and include c-MYC (c-Myc) rearrangements, t(11;14) (IGH/CCND1), t(14;18) (IGH/BCL2), IGH rearrangement with unknown partner, ALK rearrangements, and BCL6 rearrangements Fresh tissue sample required |
| Test name: IgVH Mutation Analysis by Sequencing |
| ARUP #: 0040227 |
| Methodology: Polymerase Chain Reaction/Sequencing |
| Use: Assists in the clinical management of patients with established diagnosis of chronic lymphocytic leukemia |
| Test name: ZAP-70 Analysis by Flow Cytometry |
| ARUP #: 0092392 |
| Methodology: Flow Cytometry |
| Use: Assist in the clinical management of patients with established diagnosis of chronic lymphocytic leukemia |
| Limitations: ZAP-70 results should not be used for diagnostic purposes Results of this test should always be correlated with morphologic and clinical information |
| Test name: Chromosome Analysis, Chronic Lymphocytic Leukemia (CLL) Panel by FISH |
| ARUP #: 0092616 |
| Methodology: Fluorescence in situ Hybridization |
| Use: Fluorescence in situ hybridization panel is performed for CLL prognosis Specific genomic abnormalities tested for are: ATM, D13S25, p53, and trisomy 12 |
| Limitations: Limit of detection is probe dependent and around 1-5% in interphase nuclei |
| Follow-up: Repeat testing as clinically indicated to monitor disease progression |
| Test name: Chromosome Analysis, Bone Marrow |
| ARUP #: 0097605 |
| Methodology: Giemsa-Band Analysis |
| Use: Detect chromosome abnormalities associated with lymphoproliferative disorders in bone marrow |
| Follow-up: Repeat testing as clinically indicated to monitor disease progression |
| Test name: t(11;14) IgH-CCND1 Translocation by FISH |
| ARUP #: 0049381 |
| Methodology: Fluorescence in situ Hybridization |
| Use: Assist in the clinical management of patients with established diagnoses of mantle cell lymphoma Fixed tissue sample required |
| Test name: bcl-1/JH, t(11;14) Translocation by PCR, Fluid |
| ARUP #: 0055557 |
| Methodology: Polymerase Chain Reaction |
| Use: Assist in the clinical management of patients with established diagnoses of mantle cell lymphoma Detect CCND1/IGH, t(11,14) [bcl-1/JH] translocation |
| Limitations: A negative result does not entirely exclude the presence of a CCND1/IGH [bcl-1/JH] chromosomal t(11;14) translocation |
| Test name: Immunohistochemistry Stain Offering |
| ARUP #: arup005 |
| Methodology: Immunohistochemistry |
| Use: For fixed tissue samples, diagnostic consultative services as well as immunohistochemical staining for ALK-1, BCL-2, BCL-6, Beta F-1, BOB.1, BAX, CD!a, CD2 (LFA-2), CD3, CD4 (4B12), CD5, CD7 CD8, CD10, CD15 (Leu M1), CD20 (L26), CD21, CD22 (BL-CAM), CD23, CD25, CD30, CD34, CD42b, CD43 (L60), CD44 (HCAM), CD45 (LCA), CD45R (4KB5), CD45RO (UCHL-1), CD56, CD57, CD61 (gpIIIA), CD74 (LN1), CD79a, CD95, CD99 (013), CD117, CD138 (sydican-1), Caspase3, c-Myc, Cyclin D1 (BCL-1), D2-40, DBA-44, Fascin, Fli-1, glycophorin A, kappa, lambda, Muc-1, myeloperoxidase, Oct-2, p80, PAX-5, Rb1, TIA-1, TRAP and TdT are available |
References
General References
Bahler DW. Flow Cytometric Analysis. In Kjeldsberg C, ed. Practical Diagnosis of Hematologic Disorders, 4th ed. Chicago: ASCP Press, 2006. pp. 827-843.References from the ARUP Institute for Clinical and Experimental Pathology Research®
Medical Reviewers
Bahler, David W., M.D., Ph.D. Medical Director, Hematologic Flow Cytometry, and Acting Medical Director, Molecular Hematopathology at ARUP Laboratories; Associate Professor, Pathology, University of Utah
Lamb, Allen N., Ph.D. Medical Director, Cytogenetics, ARUP Laboratories; Associate Professor, Department of Pathology, University of Utah
Perkins, Sherrie L. , M.D., Ph.D. Medical Director, Hematopathology at ARUP Laboratories; Professor, Anatomic Pathology, University of Utah
South, Sarah T. , Ph.D. Medical Director, Cytogenetics at ARUP Laboratories; Assistant Medical Director, CGH Microarray Laboratory, and Assistant Professor, Department of Pediatrics, University of Utah
Comprehensive Review: January 2008
Last Update: September 2008
