Flow cytometry (immunophenotyping)
Synonym(s): Flow cytometric analysis for immunophenotyping of lympho and myeloproliferative disorders
- Lymphoma Lymphoma : neoplastic malignant proliferation of lymphoid cells arising in peripheral lymphoid tissues (eg lymph node, spleen, thymus, lymphoid tissue associated with the mucosa).
- Leukemia Leukemia : neoplastic malignant proliferation of either lymphoid or myeloid cells arising in the bone marrow and usually also observed in the peripheral blood. It may be either myeloid or lymphoid in origin, acute or chronic.
- Leukemic lymphoma: also called stage V lymphoma. It is a lymphoma with secondary involvement of bone marrow and/or blood.
- Cluster of differentiation (CD): surface or intracytoplasmic molecules expressed by lymphoid and myeloid cells and useful for their recognition.
- The main applications of flow cytometry in lymphoproliferative and myeloproliferative disorders are:
- Distinction between reactive and neoplastic lymphocytosis:
- Lymphocytosis is a common finding in both dogs and cats. Marked lymphocytosis is usually neoplastic (leukemia) although mild/moderate lymphocytosis may be difficult to classify and may reflect either a reactive or a neoplastic process (eg reactive lymphocytosis due to chronic inflammation). Flow cytometric evaluation of the different lymphoid sub populations in the peripheral blood may help to clarify this.
- Distinction between reactive lymphoid hyperplasia and lymphoma:
- Lymphadenomegaly Abdominal organomegaly is a common clinical finding in both dogs and cats. It may be due to lymphoid hyperplasia, a reactive condition secondary to non specific antigenic stimulation characterized by a proliferation of a mixed population of lymphoid cells (mostly B-lymphocytes). Lymphoma may also present in form of generalized lymphadenomegaly and is characterized by the expansion of a monomorphic population of either T or B lymphoid cells. Flow cytometry is helpful in the characterization and differentiation of these disorders which may have a similar clinical presentation.
- Immunophenotyping of feline and canine lymphomas:
- T-cell phenotype has been reported to be a negative prognostic factor in canine multicentric lymphoma. The prognostic significance of the immunophenotype in feline lymphoma is still unclear.
- Classification of acute and chronic leukemias:
- Acute leukemias are characterized by the presence of large blastic cells in the peripheral blood which express CD34 (stem cell marker). Acute leukemias have a poorer prognosis compared with the chronic forms in both dogs and cats with short survival times (weeks-months). In chronic leukemias, high numbers of small or intermediate sized lymphocytes CD34 are usually seen. These forms have a slow clinical progression with survival times frequently > 1 year.
- Distinction between myeloid and lymphoid leukemias:
- Myeloid leukemias are rare in both dogs and cats and may be either acute or chronic in origin. They are the result of the clonal proliferation of immature myeloid cells and express specific myeloid markers (eg CD14, MPO). Lymphoid leukemias are more common and may also be acute or chronic. They express specific T or B lymphoid markers. Most of the chronic lymphoid leukemias in dogs are T-cytotoxic in origin (CD3+, CD4-, CD8+) while in the cat they are usually T-helper (CD3+, CD4+, CD8-). B-cell phenotype has been reported to be a negative prognostic factor in canine chronic lymphoid leukemia Chronic lymphoid leukemia.
Source of test material
- The samples required for flow cytometric analysis are either blood or multiple fine needle aspirates (FNA Fine-needle aspirate) of tissues depending on the disease suspected (lymphoma or leukemia).
Sample collection technique
- Leukemia: 2.5 ml of whole blood in EDTA (if the test will be performed within 24 hours), 2.5 ml of blood into fixative solution (if the analysis will be performed in more than 24 hours, up to 7 days). Special tubes containing fixative solution are usually provided by the laboratory that performs the test.
- 2-3 fresh blood smears (air dried and unstained) should always accompany the sample since evaluation of the cell morphology on blood film examination is essential for a correct interpretation of the flow cytometric results.
- Lymphoma: multiple lymph node FNAs into fixative solution. Special tubes containing fixative solution are usually provided by the laboratory that performs the test. At least one cytological preparation from a lymph node aspirate should be provided since evaluation of the cell morphology on smear examination is essential for a correct interpretation of the flow cytometric results .
- Samples can be shipped by post and should reach the laboratory as soon as possible (ideally in less than 24 hours) unless specific fixative solutions are used. Samples for flow cytometric analysis are stable at room temperature.
- If a lymphoproliferative disorder is suspected, collection of the sample for flow cytometric analysis should be performed before any treatment is given to the animal since the administration of steroids and chemotherapeutics may affect the expression of the CDs on the surface of the lymphoid/myeloid cells.
- Specific isotype control antibodies are run every time. They have no specificity for a target cell yet retain all the non-specific characteristics of the antibodies used in an analysis. The purpose of such a control is to confirm the specificity of primary antibody binding and rule out non-specific Fc receptor binding or other cellular protein interactions.
- Perform a sample cell count with the hematology analyser and manual film examination to verify the sample has adequate cellularity and preservation and is suitable for the test.
- Aliquot sufficient amount of sample in multiple tubes in order to have approximately 1x10*6 nucleated cells per tube.
- Add specific directly conjugated antibodies (antibody+fluorochrome) to appropriate tubes and incubate for 30 minutes at 4°C in the dark. For intracellular markers (eg CD79a) before adding the antibodies permeabilize the cells with specific solutions and incubate for 15 minutes.
- Wash all cells with PBA solution (PBS + Na azide + bovine serum albumin), centrifuge for 5 minutes at low speed, decant supernatant and re-suspend pellet. Repeat the procedure.
- Lyse the red blood cells by adding ammonium chloride lysing buffer and incubate for 15 minutes. Centrifuge tubes at low power speed, decant supernatant and re-suspend the pellet.
- Wash all cells with PBA solution, centrifuge for 5 minutes at low speed, decant supernatant and re-suspend pellet. Repeat the procedure.
- Perform flow cytometric analysis
- Only specialized laboratories offer the service of immunophenotyping for lympho/myeloproliferative disorders by flow cytometry.
- Flow cytometry is one of the main techniques used for immunophenotyping solid lymphoid masses (eg lymphoma) together with immunocytochemistry (ICC) and immunohistochemistry (IHC). It has a sensitivity and specificity superior to ICC and IHC. It is the only technique available for immunophenotyping leukemias.
Technique intrinsic limitations
- Flow cytometry cannot be performed if the cell count of the sample is too low. This happens more frequently in samples from lymphoma and reflects poor FNA sampling. Prior examination of a slide made from one of these aspirates may be useful to assess their cellularity.
- When flow cytometry shows the presence of a mixed population of cells with distinct phenotypes the interpretation of the results may be difficult. This may indicate the presence of a reactive process (eg lymphoid hyperplasia) although an early lymphoma/leukemia cannot be ruled out. In those cases re evaluation of the lesion in a short period of time may be recommended. Histopathological examination of the lesion may be another consideration (ideal for evaluation of the tissue architecture).
- Rarely the sample tested may be negative to all the markers used. This may be due to poor sample preservation (old sample) with lost of CDs from the surface of the lymphoid/myeloid cells. Other possible explanations would be: previous treatment with steroids/chemotherapeutics which may affect the expression of the surface markers or the presence of a cell lymphoma (eg NK lymphoma, undifferentiated lymphoma/leukemia).
- Only a limited panel of antibodies is available for cats therefore immunophenotyping feline disorders may be more difficult especially in poorly differentiated forms (eg CD34 for the identification of acute leukemias does not cross react with feline leukocytes).
Normal (reference) values
- Flow cytometric results will be reported as percentage of cells that express each CDs tested (see list of CDs in dogs in table 1). At the end of the report, a final recapitulatory interpretation related to the flow and CBC/cytology is usually provided.
- Samples from patients with either lymphoma or leukemia will show a monomorphic population of either lymphoid of myeloid cells with a specific phenotype (T or B in the case of a lymphoma/lymphoid leukemia).
- Samples from patient with either a reactive lymphocytosis or lymphoid hyperplasia will show a mixed population of T and B lymphoid cells.
Clusters of differentiation (CDs) Targeted cells Table 1. Most common CDs tested for immunophenotyping lymphoma/leukemia in dogs CD3 T lymphoid cell CD5 T lymphoid cells CD4 T-helper lymphocytes CD8 T-cytotoxic lymphocytes CD14 Myeloid cells (monocytic) MPO Myeloid cells CD45 Pan leukocyte marker CD34 Immature lymphoid/myeloid cells CD79a ß lymphoid cells CD21 Mature ß lymphoid cells
- Recent references from VetMedResource and PubMed.
- Cian F, Guzera et al (2014) Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis. Vet Clin Path 43 (1), 101-108 PubMed.
- Campbell M W, Hess P R et al ( 2012) Chronic lymphocytic leukemia in the cat: 18 cases. Vet Comp Oncol 11 (4),256-64 PubMed.
- Comazzi S & Gelain M E (2011) Use of flow cytometric immunophenotyping to refine the cytological diagnosis of canine lymphoma. Vet J 188, 149-155 PubMed.
- Comazzi S, Gelain M E et al (2011) Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia. J Vet Intern Med 25, 100-106 PubMed.
- Vernau W, Moore P F (1999) An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol 69, 145-164 PubMed.
Other sources of information
- Withrow S J, Vail D M et al (2012) Small animal clinical oncology. 5th edn. Saunders, USA.
- Sun Tsieh (2008) Flow cytometry and immunohistochemistry for hematologic neoplasms. Lippincott Williams and Wilkins, USA.