Wintrobe’s Clinical Hematology
11th Edition

Light Microscopy
Most lymphocytes in the blood are small (10 μm or smaller), although larger forms are common (Fig. 15.1A). Some of these large lymphocytes are known as large granular lymphocytes because they contain azurophilic granules in their cytoplasm (Fig. 15.1B) (17,18). Often, the distribution of lymphocyte diameters does not yield the commonly described three peaks of small, medium, and large lymphocytes because the dimensions of the cells vary according to the method of preparation (19). In blood smears stained with Romanovsky dyes, the nucleus is deep purplish blue, usually round or slightly indented and composed of dense aggregates of chromatin. Nucleoli are not visible with ordinary techniques, although a nucleolus may be seen in wet smears and histologic sections. The cytoplasm forms a narrow rim in small lymphocytes, but it may be abundant in larger cells. The cytoplasm is moderately basophilic and usually is devoid of granules, but larger cells may contain several bright reddish violet (azurophilic) granules that differ from the granules of myeloid cells in that they do not give the oxidase or peroxidase reaction.
Figure 15.1. Morphologic heterogeneity of human peripheral blood lymphocytes. A: Giemsa-stained blood smears: small and large lymphocytes. B: Large granular lymphocyte with azurophilic granules. C: Atypical lymphocyte. D: Lymphocytes resembling plasma cells (plasmacytoid) from the blood of a patient with viral pneumonia. E: One atypical lymphocyte and one plasmacytoid lymphocyte from peripheral blood. See Color Plate.
By phase contrast microscopic analysis, a well-defined centrosome can be observed. This area of the cytoplasm is adjacent to the nucleus, and because it is somewhat rigid, it may cause an indentation in the nucleus.
Often, transitional forms between lymphocytes and plasma cells are seen in the blood of patients with viral infections (Fig. 15.1C, Fig. 15.1D, Fig. 15.1E and Fig. 15.1E). These cells are variously known as lymphocytoid plasma cells or plasmacytoid lymphocytes. They obviously represent morphologic stages of differentiation of antigenically stimulated lymphocytes.
Transmission Electron Microscopy
The small lymphocyte (6 to 9 μm in size) reveals a smooth, bilaminar cytoplasmic membrane that contains only a few microvilli, except in the area of the uropod of motile cells (Fig. 15.2) (20,21,22,23 and 24). Occasional blebs, consisting of membrane-bound cytoplasm, are seen on the cell surface and may well be artifacts. The scanty cytoplasm of small lymphocytes shows a remarkable absence of organelles. The Golgi apparatus is small and usually is found near the nuclear notch. One or two centrioles are often seen in the appropriate plane of section. No organized endoplasmic reticulum is observed, although careful scrutiny may reveal one or two profiles. Many free ribosomes and occasional ribosome clusters are evident. Typical mitochondria are common, but lysosomes containing enzymes characteristic of these organelles are sparse. Dense bodies of unknown significance may also be seen occasionally. The cytoskeleton consists of occasional microtubules in the cytoplasm and microfilaments located adjacent to the cell membrane.
Figure 15.2. Ultrastructure of normal peripheral blood lymphocytes. A: A typical resting lymphocyte. The nucleus contains primarily heterochromatin in aggregates along the nuclear membrane. The nucleolus in lymphocytes is usually small. In the cytoplasm, the ribosomes are dispersed, but occasionally, a few short strands of rough endoplasmic reticulum are visible. The mitochondria are well developed and the centrioles (C), longitudinally sectioned in this illustration, show evidence that the cell is ready to enter mitosis on triggering. The Golgi apparatus (G) is small (×24,000). B: This lymphocyte contains granules (arrow) and is likely to correspond to the large granular lymphocyte variety. The cytoplasm is abundant and filled with ribosomes with a few strands of endoplasmic reticulum (×12,000). (From Zucker-Franklin D, Greaves MF, Grossi CF. Atlas of blood cells, 2nd ed. Philadelphia: Lea & Febiger, 1988, with permission.)
The nucleus is enveloped by a double membrane that fuses at the site of nuclear pores. Abundant dense heterochromatin forms aggregates close to the membrane and less often within the body of the nucleus. These aggregates are separated by interchromatinic spaces containing chromatin, small bits of aggregated chromatin, ribosome-like particles, and fibrils. A nucleolus usually is seen. The medium lymphocyte is larger (6 to 8 μm) because of an increase in the amount of cytoplasm. The nucleus contains chromatin that is looser, is distributed in small blocks, and adheres only in part to the nuclear membrane. The Golgi apparatus is more developed than in the small lymphocyte. The cytoplasm also contains numerous polyribosomes and a few strands of endoplasmic reticulum, mostly parallel with the nuclear membrane.
Lymphoblasts usually are larger cells (8 to 12 μm) with loose nuclear chromatin and a giant nucleolus that has a reticulated appearance. It occupies as much as one-third of the nuclear area. The cytoplasm contains many polyribosomes, but cisternae of endoplasmic reticulum are scarce. Lymphocytes carry the normal diploid number of 44 autosomes and two sex chromosomes.
Scanning Electron Microscopy
It was suggested originally that human peripheral blood lymphocytes could be separated into two broad categories on the basis of findings of scanning electron microscopy, depending on their cell-surface morphology (25,26). One population had a fairly smooth surface, whereas the other was described as hairy, being covered by numerous microvilli (Fig. 15.3). The former was considered to correspond to thymus-derived (or T) lymphocyte, whereas the latter was thought to represent bone marrow–derived (or B) lymphocyte. Results of subsequent studies demonstrated that the surface features of lymphocytes depend on the methods used for preparation of the cells as well as the functional state of the cells (27,28). Thus, lymphocytes stimulated by various mitogens have villi independent of their origin. In addition, both T and B lymphocytes have microvilli while in circulation and especially when they pass through the venules of LNs characterized by high endothelial cells (29). On the other

hand, all lymphocytes have a smooth surface when they reach their respective home microenvironment (30). Therefore, the smooth cell surface likely is associated with resting lymphocytes, whereas the appearance of microvilli is triggered by environmental stimuli that interact with cell-surface receptors. These newly formed microvilli may help lymphocytes to interact with target substrates.
Figure 15.3. Heterogeneity of human lymphocytes by scanning electron microscope. A: A smooth and a villous lymphocyte, thought to correspond to that of T and B lymphocytes, respectively. The appearance of the lymphocytes, however, depends on the method of preparation; both lymphocytes are smooth when they rest in their microenvironments (×14,500). B: Two villous cells in a preparation depleted of T cells (×14,500). (From Zucker-Franklin D, Greaves MF, Grossi CF. Atlas of blood cells, 2nd ed. Philadelphia: Lea & Febiger, 1988, with permission.)