EDUCATIONAL COMMENTARY DISTINGUISHING MORPHOLOGIC LOOK-ALIKES

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1 EDUCATIONAL COMMENTARY DISTINGUISHING MORPHOLOGIC LOOK-ALIKES Educational commentary is provided through our affiliation with the American Society for Clinical Pathology (ASCP). To obtain FREE CME/CMLE credits look under Continuing Education on the left side of the screen. Learning Outcomes Upon completion of this exercise, participants will be able to: discuss morphologic characteristics of normal peripheral blood leukocytes. describe morphologic features of reactive lymphocytes. differentiate cells, cellular inclusions, and artifacts that may mimic true morphologic abnormalities. A CBC with differential was requested for a 25 year old male with an unexplained fever. He had recently returned from a trip to Brazil. Laboratory findings included: WBC= 5.8 x 10 9 /L, RBC= 5.42 x /L, Hgb= 15.6 g/dl, Hct= 45%, MCV= 83 fl, MCH= 29 pg, MCHC= 34.9 g/dl, and Platelets= 90 x 10 9 /L. The images for review represent both normal and abnormal blood cells as well as organisms that may be seen in the peripheral blood in a malarial infection. All images are Wright-stained, Image BCI-01 depicts an eosinophil. Eosinophils are easily identified because of their numerous, redorange granules. These characteristic granules are large and uniform in size and have been compared to new copper pennies. The nucleus of an eosinophil is frequently bilobed and has dense, clumped chromatin. Image BCI-02 represents a monocyte. Monocytes are large cells. In fact, they are the largest cells that can normally be present in the peripheral blood. They typically have abundant, blue-gray cytoplasm; vacuoles are often present. Note the numerous vacuoles in this monocyte. The cytoplasm of monocytes characteristically has a rough or uneven

2 appearance. Sometimes cytoplasmic projections may be seen, as well as a fine dusting of pink or lilac (azurophilic) granules. This monocyte does have numerous azurophilic granules. Monocytic nuclei vary in shape and may be indented, lobulated, kidney-like, oval, or round. Nuclear chromatin is generally fine with minimal clumping and stains a light purple or pink. Image BCI-03 is a normal segmented neutrophil. The characteristic morphologic feature and origin for the cell s name is the segmentations seen in the nucleus. These cells typically have 2 to 5 nuclear lobes separated by only thin strands of chromatin. The chromatin in the nuclear lobes is dense and coarsely clumped. The cytoplasm of segmented neutrophils contains numerous small granules that normally stain violet, tan, or pink. Image BCI-04 shows a band neutrophil. Similar to the segmented neutrophil in Image BCI-03, band cells have many small cytoplasmic granules that usually stain violet, tan, or pink. Note this cell is about the same size as the eosinophil shown in Image BCI-01; however, the cytoplasmic granules are smaller and not as intensely colored as those in the eosinophil. The characteristic morphologic feature of the band cell is the nucleus that is shaped like a sausage or the letter C or U. The nuclear chromatin is clumped and dense. Image BCI-05 is a reactive lymphocyte. Other terms used to describe this cell are variant or atypical. Reactive lymphocytes can display a variety of morphologic features, reflecting the heterogeneity of the immune response that results in their formation. However, some general characteristics can be described. Their cytoplasm is often abundant and areas of clearing are frequently seen. The cytoplasm may also appear a darker blue where the cell is

3 indented by surrounding red blood cells (RBCs). Overall, the cytoplasm may appear gray, pale blue, or deep blue. Nuclear shape variations are also possible and the cell may have a round, oval, indented, or folded nucleus. The nuclear chromatin may be fine or moderately to coarsely clumped. Sometimes reactive lymphocytes resemble monocytes. When identifying any cell, it is important to consider cell size, cytoplasmic characteristics, and nuclear features. Compare the reactive lymphocyte in Image BCI-05 with the monocyte in Image BCI-02. Note the more open nuclear chromatin in the monocyte versus the dense chromatin in the reactive lymphocyte. The cytoplasm of the monocyte is also more vacuolated, with uniform cytoplasmic margins when compared with the peripheral blueing and cytoplasmic indentations of the reactive lymphocyte. Image BCI-06 uses arrows to demonstrate malarial parasites. These organisms were identified as Plasmodium falciparum. Three other species also infect humans: Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. The 4 species can be differentiated by specific morphologic features. Malaria organisms complete a complex life cycle with several stages infecting and maturing in erythrocytes. The RBCs shown in Image BCI-06 are infected with ring trophozoites, a common stage seen in peripheral blood smears. Notice the prominent, red chromatin dots and the faint blue or purple circles. Multiple trophozoites within a single erythrocyte are a common finding in P. falciparum infections. Image BCI-07 shows a platelet superimposed on an RBC. Platelets represent cytoplasmic fragments of a bone marrow precursor cell called a megakaryocyte. They are usually small, granular, and variably round or oval in shape. Platelets stain light purple or blue-gray. Platelets overlying RBCs can often be mistaken for the ring form of malarial parasites. Therefore, the stained blood smear should always be examined carefully. Several clues help to determine if abnormal RBC inclusions are really present on a peripheral

4 blood smear. Organisms that are intracellular will focus with the cell, whereas platelets focus separately. Platelets are also granular and tend to have a halo or clearing surrounding them. This is in contrast to the well-defined ring of malaria. Likewise, using other images or fields of view under the microscope can help in identifying platelets versus malarial organisms. Look for extracellular platelets that are similar in size, shape, and color to the object that is superimposed. Although not as common and therefore not as easily mistaken for malaria, another infectious agent, Babesia, may be confused with the ring forms of malaria. The organisms in babesiosis may also appear in the peripheral blood as rings. As in malaria, individual erythrocytes may have single or multiple rings. However, as many as 12 parasites may be seen in Babesia whereas no more than 3 are seen with Plasmodium. The rings in babesiosis are generally smaller than those in malaria, and sometimes 4 rings arranged as a tetrad are visible. These rings stain blue and may be oval or elongated as well as round. Multiple chromatin dots may be seen in babesiosis. In fact, >3 chromatin dots is a feature unique to Babesia. Infections with the various species of Plasmodium may also be evident in forms other than ring trophozoites. Schizonts and gametocytes (banana-shaped organisms) may also be found in the peripheral blood. Sometimes, the trophozoite in malarial infections will appear ameboid or sprawling within the RBC rather than the classic ring shape. The ring form of Babesia is the only stage that will be seen in the peripheral blood. Likewise, Babesia does not appear ameboid within the erythrocyte cytoplasm. Finally, in patients with severe babesiosis, organisms may appear outside of RBCs. Malarial parasites are never observed extracellularly. The Table below compares selected morphologic features of malaria, platelets, and babesiosis. Table. Morphologic features of malaria, platelets, and babesiosis. Cell Feature Malaria Platelets Babesiosis Organisms per RBC 1-3 N/A Often >3 Shape Circular, sometimes ameboid Round, oval Round, oval, elongated Color Blue, purple Blue, purple Blue Chromatin dots per organism Other 1 or 2 N/A Different stages may be seen Granular; do not focus with cell if superimposed 1-3; tetrads possible May be extracellular

5 Summary The patient was diagnosed with malaria. The images provided for evaluation represent both normal and abnormal peripheral blood findings that may be associated with this condition. A careful review of the stained peripheral blood smear is critical to an accurate diagnosis of many disorders but especially those caused by certain infectious organisms. In the case of infection with malaria (and others such as Babesia), visually identifying the organisms in the peripheral blood is the only way to diagnose the infection. It is also important to differentiate cells, cellular inclusions and artifacts that may be confused with true morphologic abnormalities. Therefore, the laboratory professional must always be vigilant to the possibility of infectious organisms as well as morphologic look-alikes and complete a careful examination of the peripheral blood smear. ASCP 2010