Educational Commentary – Blood Cell Id: Leukocyte and Abnormal Erythrocyte Identification

EDUCATIONAL COMMENTARY – BLOOD CELL ID: LEUKOCYTE AND ABNORMAL ERYTHROCYTE IDENTIFICATION

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Learning Outcomes

On completion of this exercise, the participant should be able to: • describe morphologic characteristics of normal peripheral blood leukocytes; • discuss morphologic features specific to target cells and nucleated red blood cells; • compare the appearance of a normal lymphocyte with that of a nucleated red blood cell; and • describe characteristic morphologic features of immature leukocytes.

Case Study An infant was delivered prematurely. The CBC results from this one day old infant are as follows: WBC=71.1 x 109/L, RBC=2.18 x 1012/L, Hgb=7.3 g/dL, Hct=21.7%, MCV=99.6 fL, MCHC=33.7 g/dL, RDW=15 %, Platelet=56 x 109/L.

Overview The images provided in this test event represent both normal and abnormal cells seen in the peripheral blood of an infant delivered prematurely with a diagnosis of Down syndrome and transient myeloproliferative syndrome.

Image BCI-01 shows a target cell (codocyte). These red blood cells have a central core area of hemoglobin surrounded by a white circle and then a final rim of more hemoglobin. Codocytes actually circulate shaped as bells or Mexican hats. They acquire the target appearance when flattened and dried on a glass slide during the preparation of the blood smear. Several mechanisms can cause target cells to form. These include conditions such as liver disease, associated with excess cholesterol and phospholipid. Lipids accumulate on the

American Proficiency Institute – 2015 1st Test Event

EDUCATIONAL COMMENTARY – BLOOD CELL ID: LEUKOCYTE AND ABNORMAL ERYTHROCYTE IDENTIFICATION (cont.)

erythrocyte membrane, resulting in increased red blood cell membrane surface area relative to the hemoglobin content and codocyte formation. Likewise, some anemias (e.g. iron deficiency, thalassemia, and hemoglobinopathies) cause a decreased hemoglobin level within the red blood cell while the same membrane surface area is maintained and target cells can then appear. Finally, codocytes may be found as artifacts when a wet blood slide is blown dry instead of air dried during smear preparation or if the slide is made in an especially humid environment.

Image BCI-02 is a nucleated red blood cell. It is not unexpected to see such a cell in the peripheral blood of an infant. The nuclear chromatin is clumped and dense, although areas of hyper-clumping may be seen. The moderate amount of cytoplasm in this example is blue-gray or a dull blue. Sometimes, depending on how much hemoglobin has been synthesized in any individual cell, the cytoplasm may have a slight pink or pink-gray hue. Nucleated red blood cells are immature erythrocytes that retain their nucleus. Even in infants and in abnormal conditions it is typical to see only the later maturation stages of these cells, as in this example.

Image BCI-03 is an eosinophil. Eosinophils are readily distinguished by their abundant orange-red cytoplasmic granules. The granules are typically large and uniform in size and shape. The nuclear chromatin is dense, clumped, stains purple, and is characteristically bi-lobed. Eosinophils are not identified according to maturation stage when reported in a differential cell count.

American Proficiency Institute – 2015 1st Test Event

EDUCATIONAL COMMENTARY – BLOOD CELL ID: LEUKOCYTE AND ABNORMAL ERYTHROCYTE IDENTIFICATION (cont.)

The cell depicted in Image BCI-04 is a normal lymphocyte. Lymphocytes vary in size, but this is a nice example of a small cell. Small lymphocytes usually have a high nuclear to cytoplasmic ratio and blue cytoplasm. The clearer area in this particular cell is unusual. However, the dense, clumped chromatin is characteristic. Nuclei in small lymphocytes may be oval, round, or slightly indented.

Occasionally, small lymphocytes and nucleated red blood cells, as shown in Image BCI-02, may be confused. Although both of these cells in this test event are similar in size, slight differences in nuclear and cytoplasmic features can help differentiate them. The nuclear chromatin in the lymphocyte is generally uniformly dense, whereas the chromatin in the nucleated erythrocyte appears patchier. The cytoplasm in the small lymphocyte is blue. The clear areas within the cytoplasm, although not typical, are not surprising to see. The cytoplasm in nucleated red blood cells is a more dull blue or blue-gray, more abundant, and less likely to show areas of clearing.

Note that the cell in image BCI-04 has several features that distinguish it from a reactive (atypical, variant) lymphocyte. Generally, reactive lymphocytes are large cells with abundant cytoplasm. The cytoplasm color is inconsistent in reactive lymphocytes. Cells may appear a deep blue while others are a more intense blue only at the cell’s periphery and even others may look gray or gray-blue. Reactive lymphocytes also often have an irregular shape and are not as uniformly round or oval as the normal lymphocyte shown. The cytoplasm may even appear to skirt around adjacent blood cells. The nuclear shape may be slightly indented, oval, or round. The chromatin pattern in reactive lymphocytes varies. It may be more open or dense and clumped. The chromatin pattern in small, normal lymphocytes is condensed. Evaluating several morphologic features is important when attempting to identify peripheral blood cells.

Image BCI-05 shows a monocyte. These cells are large and often have abundant cytoplasm. The cytoplasm is usually blue-gray, may contain faint pink or red-purple granules and, as in this example, vacuoles. The cytoplasm also often appears grainy or uneven. Monocyte nuclei may be round, oval, lobulated, or indented. The chromatin is generally fine, shows minimal clumping, and stains a lighter purple.

American Proficiency Institute – 2015 1st Test Event

EDUCATIONAL COMMENTARY – BLOOD CELL ID: LEUKOCYTE AND ABNORMAL ERYTHROCYTE IDENTIFICATION (cont.)

Image BCI-06 illustrates a blast. It is not normal to see blasts in the peripheral blood, even in an infant. Notice the large size of this cell and the high nuclear to cytoplasmic ratio. Note the scanty, agranular, deep blue cytoplasm characteristic of blast cells. Nuclei in blasts are generally oval or round. The chromatin pattern is loose and open; the nucleus stains a lighter purple color. In some blasts, prominent and numerous nucleoli may be seen. The blast in this image has nucleoli, but they are small and indistinct. The differential cell count indicated an increase in blast cells in this patient. But, because blasts of various cell types share similar features, it is often difficult to classify these cells based on morphology alone. Therefore, additional techniques are needed to determine the specific cell line of blasts (myeloblast, lymphoblast, monoblast, and so forth).

The cell in Image BCI-07 is a myelocyte. Myelocytes are immature granulocytes and, as with blasts, are not normally seen in the peripheral blood. The cytoplasm in myelocytes contains specific granules that stain light purple or pink. Sometimes, darker nonspecific granules are also visible. A faint blue background is often still present. Likewise, a clear area adjacent to the nucleus may be seen. The nucleus in myelocytes is usually round or oval and often eccentrically located in the cell. The chromatin is beginning to clump.

While the cell identified in this image is defined as a myelocyte, there are a few morphologic characteristics that are suggestive of a megakaryocyte, especially given the patient’s diagnosis. Megakaryocytes are not normally seen in the peripheral blood, but small cells can be circulating associated with myeloproliferative neoplasms and myelodysplastic syndromes. Although the granules in myelocytes may be both specific (primary) and nonspecific (secondary), the granules in this cell appear too small and more purple-blue in color than would be expected for either primary (red-purple) or secondary (lilac or tan) granules. Though some blue cytoplasm may be visible in myelocytes, it is unusual to see areas free of any granules. The morphologic discrepancies seen in this cell emphasize the difficulties sometimes associated with classifying peripheral blood cells. It is important to always evaluate cellular size, nuclear features, and cytoplasmic characteristics to inform the best possible identification for a cell.

American Proficiency Institute – 2015 1st Test Event

EDUCATIONAL COMMENTARY – BLOOD CELL ID: LEUKOCYTE AND ABNORMAL ERYTHROCYTE IDENTIFICATION (cont.)

Summary The patient whose peripheral blood was evaluated in this testing event was diagnosed as having Down syndrome and a transient myeloproliferative disorder. This condition, which often spontaneously resolves, is identified in approximately 10% of infants with Down syndrome.1 The disorder is often indistinguishable from an acute leukemia. 10-20% of children with Down syndrome have an increased chance for a fully manifested acute leukemia, especially acute megakaryoblastic leukemia.1 The genetic and environmental factors associated with the progression to an acute leukemia, however, have not been established. At this time, there are no prognostic indicators to identify which children with Down syndrome and transient myeloproliferative disorder will develop acute leukemia. The neonate with transient myeloproliferative disorder often presents with thrombocytopenia and an increase in blasts, as in this case. The blasts are usually megakaryoblasts. Marked elevation in the white blood cell count may also be seen.2

The images presented represent both normal and abnormal cells that were seen in the peripheral blood. This exercise underscores the importance of carefully reviewing a peripheral blood smear to identify morphologic variations that can support a diagnosis.

References 1. Kjeldsberg C, Perkins S. Practical Diagnosis of Hematologic Disorders. Volume 2: Malignant Disorders. 5th ed. Chicago, IL: ASCP Press; 2010.

2. McKenzie S, Williams L. Clinical Laboratory Hematology. 3rd ed. Upper Saddle River, NJ: Prentice Hall; 2015.

American Proficiency Institute – 2015 1st Test Event