Principles of Testing and Publications

Flow cytometry involves staining live cells with fluorescently labeled antibodies that bind to proteins expressed on the cell surface. Different types of lymphocytes express different proteins (for example, T-cells express the protein CD3, and B cells express the protein CD21). The cells are analyzed on a flow cytometer, which tells us how many cells of each type are present. This information allows us to determine the lineage of the cells present, and whether they are homogeneous (more consistent with neoplasia) or heterogeneous (more consistent with a reactive process).

The image below demonstrates how T zone lymphoma is diagnosed by flow cytometry. In these graphs, each dot represents a cell. The cells are plotted based on their level of expression of the T cell antigen CD5 on the X axis, and the level of their expression of the pan-leukocyte antigen CD45 on the Y axis. The graph on the left shows that a reactive lymph node is a mixture of T cells (in green) and non-T cells (in gray). All the cells in this lymph node express CD45. The graph on the right is from a dog with T zone lymphoma. In this lymph node, most of the cells express CD5, but a significant subpopulation of these cells have lost expression of CD45. This observation is considered diagnostic for T zone lymphoma.

The PARR assay is a PCR assay in which the DNA of immunoglobulin (antibody) and T cell receptor genes is amplified. The results tell us if the majority of cells in the sample are derived from a single clone (most consistent with neoplasia), or from multiple clones (most consistent with a reactive process).

We estimate we detect 93% of confirmed canine lymphomas and 91% of confirmed feline lymphoma cases with the PARR assay. Therefore, a negative result does not rule out a diagnosis of lymphoma in either species.

The figure below shows PARR assay results for a normal lymph node (top row), B-cell lymphoma (middle row) and T-cell lymphoma (bottom row). The B-cell lymphoma and T-cell lymphoma have single sized PCR products in the immunoglobulin gene reaction (left column) and T-cell receptor reaction (right column), respectively.

Ki67 is a protein that is expressed in the nucleus of a cell during proliferation but is absent when cells are quiescent. It is a commonly used marker of proliferation in immunohistochemistry. In some cancers, high Ki67 expression predicts a more aggressive disease.

We have found that Ki67 can help to distinguish cases of B-cell chronic lymphocytic leukemia with a short survival from those with a more indolent clinical course. Testing for Ki67 is not intended to guide therapy, but to provide some additional prognostic information about this disease, which has a very heterogeneous clinical course. Ki67 is detected using a flow cytometry assay. We have not established the value of Ki67 detection in other forms of lymphoid neoplasia.

The figure below shows flow cytometry of Ki67 expression in B-cells from control dogs and from dogs with B-cell chronic lymphocytic leukemia (BCLL). Representative plots are shown from 3 control dogs without lymphoproliferative disease (2 lymph node samples [top left, top center] and 1 blood sample [top right]), 3 BCLL cases with low Ki67 expression (middle), and 3 BCLL cases with high Ki67 expression (bottom). Samples were stained with anti-Ki67-FITC (red histogram) or isotype-FITC control (grey histogram) and CD21+ B-cells were gated for flow cytometry analysis. The isotype control was strictly used to determine the percent positive for Ki67 expression. Control B-cells and low Ki67 BCLL cases had <20% Ki67-expressing B-cells. High Ki67 BCLL cases had >40% Ki67-expressing B-cells.

Cell block techniques can be used to fix fluid samples (i.e. blood, effusions, or aspirates) into a paraffin block that can be used for immunohistochemical staining. This technique can increase diagnostic accuracy by applying additional antibodies that are not readily available for flow cytometry and allow us to investigate expression of intracellular antigens. Immunohistochemistry typically provides clearer and more consistent results than immunocytochemistry.

Utility of cell block

Occasionally, we will not be able to characterize a hematopoietic neoplasm based on the readily available antibodies used for flow cytometry. Cell blocks are useful for investigating the expression of additional antigens in lineage ambiguous hematopoietic neoplasms, particularly those found in the cytoplasm or nucleus of cells. For example, we currently do not have an antibody that can recognize surface antigens that are specific for plasma cells. Therefore, we can use cell block techniques to determine if the cells express intracellular IRF4/MUM1, an antigen associated with plasma cell differentiation. Cell blocks are not limited to plasma cell neoplasms and we can apply additional antibodies that are available for immunohistochemistry. Other antigens detected include, but aren’t limited to: CD204, CD18, IBA1, CD20, PAX5 and CD3.

Schematic overview of cell block technique

• (A) A capillary tube is filled with liquid or a sediment (in low cellular samples).
• (B) Capillary tubes are sealed with clay; in the absence of significant blood contamination, a high-density medium (eg, percoll) may be aspirated to facilitate separation of the cell pellet from the clay (arrow).
• (C) After centrifugation, the capillary tube is broken at the liquid–solid interface using a glass-writing diamond pen.
• (D) After fixation in formalin for at least 24 hours, the CTB is pushed out of the glass capillary using a paper clip and placed in a cassette for routine paraffin embedding.
• (E) Paraffin-embedded CTB sections are stained with H&E or other stains

Case study example

The blood from a 7-year-old English setter was analyzed by flow cytometry and expressed CD34 but no other lineage-specific antigens, leading to a diagnosis of acute leukemia. The lineage (myeloid vs lymphoid) was not clear from flow cytometry. The buffy coat was embedded in a paraffin block and stained with standard immunohistochemical procedures. The cells express the T-cell antigen CD3 (brown staining) but not B-cell antigens CD20 and PAX5, nor the plasma cell antigen MUM1. The expression of CD3 indicates T-cell lineage (acute T-cell leukemia). CD3 was not detected by flow cytometry because acute T-cell leukemias typically do not express surface CD3, but will express cytoplasmic CD3 which is detected by immunohistochemistry.