Educational Commentary – Morphologic Abnormalities in Leukocytes

EDUCATIONAL COMMENTARY – MORPHOLOGIC ABNORMALITIES IN LEUKOCYTES

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Learning Outcomes Upon completion of this exercise, participants will be able to: • identify the morphologic characteristics of normal peripheral blood leukocytes. • describe two types of cytoplasmic inclusions that represent morphologic changes in leukocytes.

The images provided in this testing event represent several normal cells as well as immature granulocytes and morphologic changes in leukocytes that suggest the patient has an infection.

Image BCI-08 shows a normal monocyte. Monocytes are large cells. A monocyte is the largest normal cell seen in the peripheral blood. While size is important when identifying any cell, cytoplasmic characteristics and nuclear features should be considered as well. The cytoplasm in monocytes is typically blue-gray in color and frequently contains vacuoles. The cytoplasm may also appear rough or uneven. Cytoplasmic projections are often seen. Sometimes, fine pink or lilac (azurophilic) granules are present. Nuclear features are important to evaluate in monocytes. Monocyte nuclei vary in shape and may be round, oval, lobulated, or kidney-like. The chromatin stains a light pink or purple and is usually fine with minimal clumping.

Image BCI-09 shows a band (stab) neutrophil. This cell is the earliest precursor of neutrophil maturation that can normally be seen in the peripheral blood. The band in this image with a nucleus shaped like the letter C is a classic example. The chromatin is characteristically dense and clumped. The cytoplasm contains numerous small, specific granules that stain pink, tan, or violet.

American Proficiency Institute – 2009 2nd Test Event EDUCATIONAL COMMENTARY – MORPHOLOGIC ABNORMALITIES IN LEUKOCYTES (cont.)

The arrow in Image BCI-10 points to a small lymphocyte. Note the scanty rim of blue cytoplasm surrounding the nucleus. Lymphocytes vary in size, and larger cells are sometimes seen normally in the peripheral blood. Nuclei in lymphocytes are usually oval, round, or only slightly indented. The nuclear chromatin is dense, clumped, and stains a dark purple.

Distinguishing between monocytes and reactive lymphocytes can sometimes be difficult. Remember that reactive lymphocytes are a normal cellular response to an antigen. Therefore, a variety of morphologic changes can be associated with these cells. In general, reactive lymphocytes also tend to be large, with abundant cytoplasm. The cytoplasm sometimes may be intensely blue. The cytoplasmic membrane may be indented by surrounding erythrocytes such that the cytoplasm “skirts” around the RBCs. The nuclei in reactive lymphocytes may be round, but it is frequently irregularly shaped or elongated. The chromatin is more dispersed and fine than what would be seen in a normal small lymphocyte. In comparison, the cytoplasm of monocytes is more vacuolated, extended, and uneven in appearance. The nuclear chromatin tends to be lacy, folded, and loose. The nuclear shape in monocytes is variable as well. Table 1 summarizes morphologic characteristics of small lymphocytes, reactive lymphocytes, and monocytes.

TABLE 1. Characteristics of Lymphocytes and Monocytes.

Characteristic Small Lymphocytes Reactive Lymphocytes Monocytes

Cell size 8-12 µm Variable, can be large Large (15-18 µm) (9-30 µm)

Nuclear shape Round Irregular (lobulated, oval, Horseshoe, round, notched) folded

Nuclear chromatin Clumped; parachromatin More fine (but not like a Lacy, loose strands, not evident and more blast); parachromatin brainlike lavender more evident and white

Nucleoli Usually absent May be prominent Absent

Cytoplasm Amount Scant Abundant Abundant

Cytoplasm Color Blue Often deep, intense blue Light bluish gray with darker edges at contact points with other cells

American Proficiency Institute – 2009 2nd Test Event EDUCATIONAL COMMENTARY – MORPHOLOGIC ABNORMALITIES IN LEUKOCYTES (cont.)

Image BCI-11 is a normal segmented neutrophil. These cells characteristically have 2 to 5 nuclear lobes. Notice that the lobes are connected by just a narrow strand of chromatin. Compare this segmented neutrophil with its filaments to the band cell shown in Image BCI-09, with its wide, bridging chromatin. Also note that the segmented neutrophil has clumped and condensed nuclear chromatin. The cytoplasm in neutrophils has numerous small, pink, tan, or violet granules. It is sometimes difficult to distinguish individual granules, especially in photomicrographs such as this, but their presence gives the cytoplasm an overall grainy and pink appearance.

The arrow in Image BCI-12 points to a metamyelocyte. It is not normal to see this cell in the peripheral blood. However, the patient in this case study had an elevated WBC count and a possible infection, so the presence of an immature cell such as a metamyelocyte in the peripheral blood is not surprising. Metamyelocytes feature a nucleus that is slightly indented and characteristically described as kidney-shaped. Sometimes it is difficult to distinguish a metamyelocyte from a band, such as the cell identified in Image BCI-09. Note that in a metamyelocyte the indentation of the nucleus is less than half of the diameter of the hypothetical round nucleus. In comparison, the nuclear indentation in the band is more than half of a possibly round nucleus. Also notice that a segmented neutrophil, such as that seen in Image BCI-11, has nuclear lobes connected by thin filaments and not bridges of chromatin. The segmented neutrophil, band, and metamyelocyte are also post-mitotic cells. So, the nuclear chromatin in all of them is dense and clumped. They are all similar in size as well, though this metamyelocyte appears slightly larger. Numerous pink, tan, or violet specific granules can be seen in the cytoplasm of the metamyelocyte and also in the band and segmented neutrophil. Table 2 compares the morphologic features of a segmented neutrophil, band, and metamyelocyte.

American Proficiency Institute – 2009 2nd Test Event EDUCATIONAL COMMENTARY – MORPHOLOGIC ABNORMALITIES IN LEUKOCYTES (cont.)

TABLE 2. Morphologic Features of Segmented Neutrophils, Bands, and Metamyelocytes.

Feature Segmented Neutrophil Band Metamyelocyte

Cell Size Medium Medium Medium

Nuclear shape 2-5 lobes connected by C or U; indentation >½ Kidney-bean; indentation filaments of “round” nucleus <½ of “round” nucleus

Nuclear chromatin Dense, clumped Dense, clumped Dense, clumped

Cytoplasm Numerous pink, tan, or Numerous pink, tan, or Numerous pink, tan, or violet granules violet granules violet granules

The cell in Image BCI-13 is a segmented neutrophil. However, it is not normal. This cell has cytoplasmic toxic granulation. Toxic granules are primary or azurophilic granules that have retained their basophilia. Compared with the normal granules in the segmented neutrophil shown in Image BCI-11, the toxic granules are slightly larger and darker purple. Toxic granules may be seen not only in bacterial infections, but also in several other conditions, including burns, trauma, and the therapeutic use of cytokines.

Image BCI-14 depicts a Döhle body. Döhle bodies may appear as single or multiple blue inclusions within the cytoplasm of neutrophils and bands. They represent denatured aggregates of rough endoplasmic reticulum. Döhle bodies are usually located near the inner periphery of the cell wall and vary in size and shape. They may be seen in the same conditions as discussed for toxic granulation. In fact, evaluating a peripheral blood smear for Döhle bodies whenever toxic granulation is seen is good laboratory practice because both morphologic changes often occur together.

Editor's note: A few laboratories reported May-Hegglin inclusion for Image BCI-14. Döhle bodies must be differentiated from May-Hegglin bodies which are morphologically identical. Signs of toxicity, such as

American Proficiency Institute – 2009 2nd Test Event EDUCATIONAL COMMENTARY – MORPHOLOGIC ABNORMALITIES IN LEUKOCYTES (cont.) a shift to the left in the myeloid series, elevated total leukocyte count, or toxic granulation suggest Döhle bodies, whereas a normal leukocyte count, no shift to the left, the presence of giant abnormal platelets, or lack of toxic granulation of neutrophils suggests May-Hegglin anomaly.

Case Summary The patient had an elevated WBC count, immature granulocytes in the peripheral blood, and morphologic changes to leukocytes that suggested a possible infection. Although more laboratory evaluation and other testing were needed to make a diagnosis of the patient’s condition, the initial findings reported by the laboratory provided the clinician with important information that guided additional clinical assessments.