Methodologies

Lymphomas are considered clonal proliferations of lymphocytes arrested at different stages of differentiation, thereby recapitulating stages of normal lymphocyte differentiation. Immunohistochemistry (IHC) with various antibodies enables identification of the specific lineage and developmental stage of the lymphoma.1 IHC utilizes antibodies tagged with a visible label to detect the presence of specific proteins in cells or tissue. The current armamentarium includes more than 50 antibodies used in diagnosing various lymphomas. The beginning IHC panel of markers is decided based on morphologic differential diagnosis.2 No single marker is specific. Most IHC panels contain antibodies specific for B-cell lesions, which comprise the majority of lymphomas. These include CD20, CD3, bcl-2 and CD43. CD30 may be added if HL or other CD30-expressing lymphomas are suspected.3

Tissue biopsies routinely undergo fixation (chemical cross-linking of proteins to preserve proteins, cellular morphology and tissue architecture) in formalin buffer, are embedded in a paraffin block and are then thinly sectioned and mounted on glass slides for IHC. The paraffin from the tissue sections must be completely removed, and an antigen retrieval procedure is used to unmask the epitope sites of proteins before starting the immunostaining process. Tissue sections undergo blocking of nonspecific sites before being treated with the primary antibody targeting the protein of interest. A secondary antibody tagged with the visible label binds to the primary antigen-antibody complex. If needed, signal amplification techniques can be used to enhance the sensitivity of IHC.

Flow cytometry (FC) is a technique used to characterize and separate individual cells. Cells are passed through a laser beam in single file, and their light-scattering properties are recorded to analyze their microscopic properties, such as the size of the cells, cytoplasmic granules, etc. Cells can also be immunostained with fluorescent antibodies to identify proteins of interest and then passed through a flow cytometer to characterize them into distinct populations by their light-scattering and fluorescent qualities. FC is a sensitive and specific method for diagnosis and classification of NHL.4

IHC vs FC

IHC has the advantage that the cells of interest are identified morphologically and can be used retrospectively on fixed tissues. However, only a single marker is routinely used on tissue sections, and some investigators have reported difficulty using IHC to characterize immunoglobulin light chains.4

FC provides a more precise definition of individual cell types through a combination of physical characteristics and the use of multiple antibodies directly conjugated with different fluorochromes. Applying multiple markers permits detection of multiple antigens on the same cell in a single procedure. FC can also assess monoclonality by detecting immunoglobulin light chain expression. FC is rapid since it requires fresh specimens to maintain viability and avoid loss of antigenicity through the process of fixation; processing of fresh tissue within minutes or a few hours is therefore mandatory. It is clearly an advantage that results are available within hours.4 However, fresh tissue is often not available in a clinical situation, leaving IHC as the most practical option.

References

  1. Rao IS. Role of immunohistochemistry in lymphoma. Indian J Med Paediatr Oncol. 2010;31(4):145-147.
  2. Higgins RA, Blankenship JE, Kinney MC. Application of immunohistochemistry in the diagnosis of non-Hodgkin and Hodgkin lymphoma. Arch Pathol Lab Med. 2008;132(3):441-461.
  3. Lu J, Chang KL. Practical immunohistochemistry in hematopathology: a review of useful antibodies for diagnosis. Adv Anat Pathol. 2011;18(2):133-151.
  4. El-Sayed AM, El-Borai MH, Bahnassy AA, El-Gerzawi SMS. Flow cytometric immunophenotyping (FCI) of lymphoma: correlation with histopathology and immunohistochemistry. Diagn Pathol. 2008;3:43-55.