Abnormal white blood cells: Leukemia or not? (Proceedings)

Leukemia = the presence of neoplastic cells in circulation. Leukemia can arise independently of, or concurrently with tissue neoplasia. The opposite is also true. A hematopoietic cell neoplasm can be localized to the BM or tissue without being seen in circulation.

Lymphoproliferative disease is the general term to describe an abnormal expansion (often neoplastic, but occasionally reactive) of the lymphoid line. Lymphoma or lymphosarcoma describes a solid, sarcomatous lymphoid tumor which may involve a variety of organs, such as lymph nodes, liver, spleen, kidney, and intestines. Bone marrow is involved in a small percentage of cases.

Myeloproliferative disease (MPD) is the proliferation of granulocytic, megakaryocytic, erythrocytic, and/or stromal connective tissue (fibrous and osseous) cells in the bone marrow and includes the following:

• Undifferentiated MPD or blastic form

• Granulocytic

• Eosinophilic

• Basophilic

• Monocytic

• Myelomonocytic (neutrophils and monocytes)

• Megakaryocytic

• Erythemic myelosis (RBC)

• Erythroleukemia (RBC and WBC)

Myelofibrosis is a poorly understood entity characterized by proliferation of hemic cells, as well as diffuse fibrosis in the bone marrow. Extramedullary hematopoiesis accompanies these changes. Myelofibrosis is a form of MPD and may also be a terminal stage of other forms of MPD.

Diagnosis

Leukemia is suspected when the white cell count is high with increased numbers of blasts cells and/or atypical cells. The bone marrow should be sampled when the blood is not definitive or when trying to identify the specific type of leukemia. A bone marrow aspirate provides more information on cell morphology. A bone marrow core biopsy provides more information regarding bone marrow architecture. This is best used to identify focal lesions, fibrosis, and cellularity.

If the bone marrow contains greater than 30% blast cells and lack orderly maturation, it is consistent with myeloproliferative disease. If less than 30% blast, the process is classified as dysmyelopoiesis. Cytochemistry and electron microscopy may be necessary to identify the cell type. Focal aggregates of neoplastic cells can be seen with lymphoma, myeloma, and mast cell tumors.

Leukemia can be classified by several processes. The terms acute, subacute, and chronic have been used to describe the degree of cellular differentiation.

1. Acute very little differentiation of neoplastic cells (mostly blasts).

2. Subacute more differentiated cells (intermediate).

3. Chronic welldifferentiated neoplastic cells.

Acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) are relatively easy to diagnose due to the presence of significant numbers of blasts or other atypical cells. In the dog, acute leukemia appears slightly more likely to be of myeloid than lymphoid origin. However, it may be difficult to differentiate AML from ALL through examination of hematologic or cytologic features. In addition, AML in dogs and cats is difficult to diagnose through flow cytometry immunophenotyping as the available antibody panels do not provide lineage specific myeloid or myelomonocytic markers. While combinations of antibodies, coupled with flow scatter patterns can be used to help characterize myeloid precursors, no currently available panel of antibodies can consistently identify myelomonocytic leukemia or AML. CD34 is detected on both AML and ALL. There is also no PCR test for clonality for AML. One of the best markers for AML is myeloperoxidase. Currently, this is detected by most labs through cytochemistry on cytology or histology slides.

Subtle signs that are often used to differentiate AML from ALL on the blood film include the presence of granules or vacuoles in the cytoplasm, a finer nuclear chromatin pattern and as slightly more centralized nuclear placement. If there is even a slight continuum to more differentiated cells, this can be helpful but many blastic or intermediate leukemias are poorly differentiated.

Acute lymphoid leukemia may be comprised of cells of B, T, or NK origin. CD34 is often detected on AML and ALL, but is rarely (or not) detected on lymphoma and therefore serves as a useful marker to differentiate ALL with tissue involvement from lymphoma with marked leukemia. Flow phenotyping and PCR for antigen receptor rearrangement (PARR) are very useful for the characterization of lymphoproliferative processes. However a negative result does not exclude the possibility of a lymphoid (versus myeloid) neoplasia.

Well-differentiated neoplastic cells are easily identified. The problem is differentiating between a reactive versus a neoplastic process. For example:

Neutrophilic left shifts can (rarely) go back to blasts. Frequently they extend to myelocytes and sometimes progranulocytes with many inflammatory and infectious diseases. Thus, the presence of immature cells in circulation does not necessarily equal leukemia.

A chronic lymphocytosis can be seen as a reactive process against Ehrlichia and other agents. The range of lymphocyte counts reported overlaps that seen for leukemias, thus counts alone may not be useful for differentiation.

Eosinophilia can be seen as a reactive process against parasites or other neoplastic processes, can be part of the syndrome of hypereosinophilic disorders, or can represent an eosinophilic leukemia.

Significant monocytosis can be seen with some immune mediated diseases (e.g. IMHA) and chronic inflammatory diseases as well as part of monocytic and myelomonocytic leukemias.

Changes that raise the index of suspicion for myeloid leukemia

• Persistent and/or increasing numbers despite treatment

• Irregular nuclear membrane (particularly for monocytic precursors)

• Giant or bizarrely segmented neutrophils

• Lack of differentiation to mature neutrophils

• A more blue grey cytoplasm as opposed to the deeply basophilic cytoplasm of lymphoblasts

• Vacuoles

• A dusting of azurophilic granules

• In cats, dark basophilic (versus lilac) granules within basophils and basophilic precursors

• Marked thrombocytosis

• Hemoglobinization of cells

• Asynchrony of nuclear and cytoplasmic maturation

• Cytoplasmic blebs in circulation

• Mitotic figures in circulation

Lymphocyte morphology is often used to help characterize lymphoproliferative processes

Small lymphocytes are smaller in size than a neutrophil and have a round nuclei that takes up the majority of the cell. The nuclei contain densely aggregated chromatin forming large chromocenters (condensed chromatin). Nucleoli are not seen. The cytoplasm is scant (sometimes only a very thin rim is visible) and lightly basophilic in color. These are typically called 'mature lymphocytes'. However, early lymphoid progenitor cells, hematopoietic stem cells, certain stages and types of dendritic cells, and other immature precursor cells may have a very similar morphology to 'mature, well-differentiated, small, resting lymphocytes'.

Intermediate to large lymphocytes range in size from slightly larger than small lymphocytes to the size of neutrophils. The nuclei still takes up the majority of the cell, however more abundant cytoplasm is visible in these cells. Often, the nuclei is placed eccentrically within the cytoplasm. The nuclear chromatin is finely clumped to granular. Typically, nucleoli are not seen although strands of loosely clumped nuclear chromatin may be mistaken for nucleoli. The cytoplasm is lightly basophilic in color. Occasionally these cells contain azurophilic granules suggestive of a natural killer (NK) phenotype.

Lymphoblasts are as large as a neutrophil or larger. Size alone does not indicate neoplasia. Very large lymphoblasts (2-4x the size of neutrophils) may be seen in reactive and hyperplastic processes although the presence of significant numbers of blasts in blood almost always indicates a neoplastic process. Lymphoblasts contain round to oval nuclei with fine or stippled chromatin (loosely aggregated chromatin). One or more nucleoli may be visible. The cytoplasm is moderately to deeply basophilic. Occasionally (seen more in cats than dogs) the cytoplasm may contain punctate vacuoles.

Reactive lymphocytes are similar in morphology to small lymphocytes but are slightly larger and have more abundant, more basophilic cytoplasm.

Plasma cells are intermediate sized cells that contain small, round, eccentrically placed nuclei with condensed chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent, eccentric, perinuclear, clear zone that corresponds to the Golgi. These cells are rarely seen in blood, but commonly seen in tissue aspirates and bone marrow.

Morphologic features and typical findings used to characterize lymphoproliferative processes

Lymphoglandular bodies are round, homogeneous, basophilic structures comprised of cytoplasmic fragments. The presence of lymphoglandular bodies is seen in cytologic preparations of lymphoid tissue that contains increased numbers of lymphoblasts. This can be due to neoplasia (lymphoma) or hyperplasia.

The presence of an eccentric, perinuclear clearing Golgi clearing zone is often suggested as a feature of B-cells and plasma cells. The clearing zone is the Golgi and it is a prominent feature in plasma cells. However, the Golgi apparatus is an organelle found in most cells, including T-cells and myeloid cells.

Sezary cells are described as medium to large lymphocytes with ceribriform nuclei. These neoplastic T-cells are characteristic features of Sezary syndrome in people which encompasses mycosis fungoides and other epitheliotropic lymphomas. A similar syndrome and cell morphology occurs in dogs but has been rarely reported in cats. In dogs, epitheliotropic T-cell lymphoma is also seen in the gastrointestinal tract.

In humans, the presence of flower cells or cloverleaf cells is most often associated with T-cell disease (especially infection with human T-lymphotrophic virus-1 (HTLV-1)). In dogs and cats, similar morphology has been seen in both B-cell and T-cell lymphoproliferative disease as well as myeloproliferative disease (especially myelomonocytic leukemia).

Chronic lymphocytic leukemia

Canine CLL. In dogs, CLL appears to be primarily a T-cell disease although B-cell CLL has also been reported (3:1 ratio of T:B). The cytologic morphology of T-cell CLL in dogs is typically of granular lymphocytes. The granular cells primarily express CD3 and CD8. About a third of the reported cases express the gamma/delta TCR rather than the alpha/beta TCR. Non-granular T-cell CLL more commonly express the alpha/beta TCR but may be either CD4 (helper T cells) or CD8 (cytotoxic) positive. B-cell CLL in dogs has also been reported. B-cell CLL and T-cell CLL appear to have somewhat different patterns of disease progression. B-cell CLL affects the bone marrow early in disease and may be considered a primary bone marrow disease. T-cell CLL typically does not affect the bone marrow until late in the disease and may spread from the marrow after splenic involvement.

In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis. These cells are also CD8+ and must be differentiated from CLL.

Feline CLL. Similar to that seen in dogs, feline CLL is primarily a T-cell disease. However, unlike that seen in dogs, feline CLL is primarily a result of CD4 or helper T-cell proliferation although occasional cases of CD8+, CD4CD8 double positive, and CD4CD8 double negative CLL have also been reported. Often, but not always, these cells may contain granules however they are more often small than the large granules seen with large granular lymphocyte neoplasia.