Canis ISSN: 2398-2942

Flow cytometry (immunophenotyping)

Synonym(s): Flow cytometric analysis for immunophenotyping of lympho and myeloproliferative disorders

Contributor(s): Joy Archer, Francesco Cian

Overview

  • 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.

Sampling

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Tests

Methodologies

Control

  • 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.

Methodologies

  • 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

Availability

  • Only specialized laboratories offer the service of immunophenotyping for lympho/myeloproliferative disorders by flow cytometry.

Validity

Sensitivity

  • 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 null 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).

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Cian F, Guzera et al (2013) Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis. Vet Clin Path,in press.
  • Campbell M W, Hess P R et al (2012) Chronic lymphocytic leukemia in the cat: 18 cases. Vet Comp Oncol early view 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, 100-106PubMed.
  • 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, 145-164PubMed.

Other sources of information

  • Withrow S J, Vail D M et al (2012) Small animal clinical oncology. (5th edition). Saunders, Missouri, USA.
  • Sun Tsieh (2008) Flow cytometry and immunohistochemistry for hematologic neoplasms. Lippincott Williams and Wilkins, Philadelphia, USA.


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