EDUCATIONAL COMMENTARY – BLOOD CELL IDENTIFICATION

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Learning Outcomes Upon completion of this exercise, the participant will be able to: • Describe the morphologic features of normal peripheral blood leukocytes. • Identify toxic morphologic abnormalities in leukocytes.

Case History A 35 year old female presented to the ER with blurred vision, nausea, and vomiting. The patient also complained of recent severe headaches and fatigue. Laboratory findings included: WBC=25.3 x 109/L, RBC=4.85 x 1012/L, Hgb=13.8 g/dL, Hct=40%, MCV=92 fL, MCH=34.2 pg, MCHC=33.0%, RDW=12.8%, Platelets=265 x 109/L. WBC Differential: Neutrophils=68%, Bands=17%, Lymphocytes=6%, Monocytes=2%, Eosinophils=4%, Basophils=1%, Metamyelocytes=2%.

The patient presented in the case study for this testing event is being treated for breast cancer. The photographs for review represent several normal cells but also morphologic changes in leukocytes that may be seen in the peripheral blood in this situation.

Common cytoplasmic abnormalities seen in white blood cells include Döhle bodies, toxic granulation, and vacuoles. Such inclusions are generally part of the cell’s normal immune response to infection or inflammation. However, sometimes these inclusions indicate abnormal, inherited anomalies that affect leukocytes. Whenever inclusions are seen within cells, they are a significant finding and should be reported.

Photomicrograph BCI-15 shows a normal monocyte. Monocytes are the largest cells that can normally be present in the peripheral blood. Their cytoplasm is often abundant, is typically blue-gray in color, and often has vacuoles. The cytoplasm in monocytes may appear rough and uneven with cytoplasmic projections. Sometimes, fine pink or lilac (azurophilic) granules may be seen. The nuclei in monocytes vary in shape and may be round, oval, kidney-like, or lobulated. The chromatin is usually fine with minimal clumping and stains a light pink or purple.

Monocytes and reactive lymphocytes are sometimes difficult to distinguish. In general, the cytoplasm of monocytes is more vacuolated, extended, and uneven in appearance when compared to a reactive

American Proficiency Institute – 2005 3rd Test Event EDUCATIONAL COMMENTARY – BLOOD CELL IDENTIFICATION (cont.) lymphocyte. The nuclear chromatin in the monocyte tends to be folded, lacy, and loose versus the more condensed or coarsely clumped nucleus of a reactive lymphocyte.

Reactive lymphocytes represent one type of antigen-stimulated cell and their presence indicates a normal response to antigen. Therefore, a variety of morphologic features can be seen in reactive lymphocytes. In general, these cells tend to be large with abundant cytoplasm. Sometimes, the cytoplasm is intensely blue. The cytoplasmic membrane may be indented by surrounding red blood cells such that the cytoplasm "skirts" around the erythrocytes. The nuclei in reactive lymphocytes may be round, but are often elongated or irregularly shaped. The chromatin, though more dense than what is seen in a normal monocyte, is more dispersed than what would be seen in a normal lymphocyte.

BCI-16 illustrates a normal eosinophil. Eosinophils are distinguished by numerous, red-orange cytoplasmic granules. These granules are large and uniform in size. The nucleus of mature eosinophils is often bilobed. The chromatin is dense, clumped, and stains purple.

The cell shown in photomicrograph BCI-17 is a normal, small lymphocyte. In such small lymphocytes, the cytoplasm forms a scanty rim of blue around the nucleus. The nuclei are generally round, oval, or barely indented. The nuclear chromatin is dense, clumped, and stains a dark purple.

Photograph BCI-18 represents a normal segmented neutrophil. These cells typically have 2-5 nuclear lobes connected by thin strands of chromatin. The chromatin is clumped and condensed. The cytoplasm of neutrophils has numerous small, pink, tan, or violet-pink granules. It is difficult to distinguish individual granules in the cytoplasm of neutrophils, but their presence gives the cytoplasm an overall pink color and grainy appearance. Also, note that these granules are smaller and less intensely colored than the cytoplasmic granules of an eosinophil, as shown in BCI-16.

American Proficiency Institute – 2005 3rd Test Event EDUCATIONAL COMMENTARY – BLOOD CELL IDENTIFICATION (cont.)

The band cell is the first precursor of neutrophil maturation that can normally be seen in the peripheral blood. BCI-19 shows a classic band neutrophil. Band nuclei are characteristically shaped like a sausage, or the letters C or U. The chromatin is generally dense and clumped. The cytoplasm in bands contains numerous small, specific granules that resemble those seen in segmented neutrophils, staining pink, tan, or violet.

BCI-20 shows a segmented neutrophil with cytoplasmic toxic granulation. Toxic granules are primary or azurophilic granules that have retained their basophilia. Toxic granules are large and dark purple and may vary in number within any given neutrophil. Toxic granule formation is just one indicator of several disease states or conditions. They may be associated with bacterial infections, burns, cancer, and toxic or inflammatory states. They may also be seen in patients receiving cytokine therapy, such as granulocyte colony-stimulating factor (G-CSF) because these drugs accelerate production and packaging of enzymes, like lysozyme, that are normally synthesized by neutrophils in their primary granules.1

It is important not to confuse toxic granulation with basophils or artifacts from the staining process. Toxic granules are smaller than the granules seen in basophils, but larger than those in normal neutrophils. Furthermore, the specific granules in basophils often obscure the nucleus, whereas toxic granules are scattered only throughout the cytoplasm. Stain artifact may sometimes be confused with toxic granulation as well. Increased staining time, inadequate rinsing, or decreased pH of the buffer may cause toxic-like artifacts or inclusions. If artifact is suspected, look for similar structures outside of the cells to confirm your interpretation.

BCI-21 shows a Döhle body. These inclusions consist of denatured aggregates of rough endoplasmic reticulum or free ribosomes. Döhle bodies may appear as single or multiple blue or blue-gray masses within the cytoplasm of not only neutrophils, but more immature cells, such as bands and metamyelocytes. They are usually located near the cytoplasmic periphery of the cell and are variable in size and shape. The cytokine-induced appearance of Döhle bodies results from a shortened cell maturation time and persistent production activity in neutrophils.1 Döhle bodies are also seen in the conditions previously discussed for toxic granulation. In fact, a smear should also be evaluated for Döhle bodies whenever

American Proficiency Institute – 2005 3rd Test Event toxic granulation is observed because both reactive morphologic changes frequently occur together. There are additional morphologic and hematologic clues that indicate toxic or reactive changes are present in a patient. An elevated leukocyte count and increase in immature granulocytic cells, with the presence of toxic granulation and/or Döhle bodies, suggest a temporary activation of neutrophils.

Finally, note that the cell shown in BCI-19 also contains at least two Döhle bodies.

Reference 1. Glassy, EF. Color Atlas of Hematology. Northfield, Il: College of American Pathologists; 1998:45.

© ASCP 2005

American Proficiency Institute – 2005 3rd Test Event