Lesson 4-3. Leukocytes
4-6. GRANULOCYTIC SERIESThe stages in the normal maturation of the granulocytes are: myeloblast, promyelocyte, myelocyte (neutrophilic, eosinophilic, and basophilic), metamyelocyte (neutrophilic, eosinophilic, and basophilic), band cell (neutrophilic, eosinophilic, and basophilic), and segmented cell (neutrophilic, eosinophilic, and basophilic). As the granulocytes mature, the granules increase in number. These granules later become specific and differ in the affinity for various dyes. Neutrophilic granules do not stain intensely with either dye. Basophilic granules have an affinity for the basic or blue dye. Eosinophilic stain red with an affinity for the acid dye. The criteria for identification of the various stages of the granulocytic series are: size of cell, nucleus-cytoplasm ratio, nuclear shape, number of nucleoli, and the type and size of cytoplasmic granulation.a. Myeloblast. See figure 4-18.
Figure 4-18. Granulocytic series: Myeloblast
(1) Size. 15 to 20 microns in diameter.
(2) Nucleus. The nucleus is round or ovoid and stains predominantly reddish-purple. The interlaced chromatin strands are delicate, well defined, and evenly stained. Two to five pale blue nucleoli are demonstrable. The nucleus occupies most of the cell with a nucleus-cytoplasm ratio of 4:1. It is separated from the cytoplasm by a definite nuclear membrane.
(3) Cytoplasm. The cytoplasm is a narrow, moderate blue and smooth, no granules, and a rim around the nucleus.
b. Promyelocyte. See figure 4-19.
Figure 4-19. Granulocytic series:
a. Promyelocyte. b. Promyelocyte with auer body.
(1) Size. 15 to 21 microns in diameter.
(2) Nucleus. The nucleus is round or ovoid with coarse-clumping, purple chromatin material. Two to three oval, light-blue nucleoli are usually present. The nucleoli are less distinct than in the myeloblast. This cell has a nucleus-cytoplasm ratio of 3:1.
(3) Cytoplasm. The cytoplasm is light purple and contains varying numbers and sizes of dark nonspecific granules that stain red to purplish-blue. The granules usually overlie the nucleus.c. Myelocyte. See figure 4-20. In the myelocytic stage, the granules are definite and so numerous that frequently they obscure nuclear detail. While promyelocytes are sometimes distinguished as neutrophilic, eosinophilic, or basophilic, the differentiation is generally considered as first occurring in the myelocytic stage.
Figure 4-20. Granulocytic series: Myelocyte.
d. Neutrophilic Myelocyte.
(1) Size. 12 to 18 microns in diameter.
(2) Nucleus. The nucleus is round, oval, or flattened on one side. The chromatin strands are light purple, unevenly stained, and thickened. Nucleoli are usually absent. The nucleus is smaller than the earlier cells of this series with a nucleus-cytoplasm ratio of 2:1 to 1:1.
(3) Cytoplasm. The cytoplasm is pink with blue patches and contains a small relatively light area of ill-defined, pink granules, which develop among the dark, nonspecific, azurophilic granules of the promyelocyte. As the myelocyte ages, the dark granules become less prominent and the light-pink-colored neutrophilic granules predominate.e. Metamyelocyte. See figure 4-21.
Figure 4-21. Granulocytic series:
a. Metamyelocyte, b. Band neutrophil.
(1) Size. 10 to 15 microns in diameter.
(2) Nucleus. The nucleus is indented or kidney-shaped. The indention is less than half the width of an arbitrary round nucleus. The nucleus is eccentric or centrally located in the cell. The nuclear chromatin pattern is coarse and clumped. Nucleoli are absent. The nucleus-cytoplasm ratio is approximately 2:1 to 1:1.
(3) Cytoplasm. The cytoplasm is pinkish-blue and has moderate to abundant specific granules.
f. Neutrophilic Band.
(1) Size. 9 to 15 microns in diameter.
(2) Nucleus. The nucleus is shaped like a horseshoe with a dark pyknotic mass at each poIe of the nucleus where the lobes develop. The nucleus is deeply indented from the metamyelocyte stage. The nucleus-cytoplasm ratio is approximately 1:2.
(3) Cytoplasm. The cytoplasm moderate to abundant pink and contains many small evenly distributed violet-pink granules.
g. Neutrophilic Segmented Cell.
(1) Size. 9 to 15 microns in diameter.
(2) Nucleus. The nucleus has two to five definite lobes separated by a very narrow filament or strand. The nucleus has dense, coarse, clumped, irregular chromatin pattern. The granules are small pinpointed pink to rose-violet and specific. The nucleus-cytoplasm ratio is approximately 1:3.
(3) Cytoplasm. The cytoplasm is light pink and the small, numerous, and evenly distributed neutrophilic granules have a light pink color.Development of the Eosinophilic Group. Cells of the eosinophilic group are characterized by relatively large, spherical, cytoplasmic granules that have a particular affinity for the eosin stain. The earliest eosinophil (myelocyte) has a few dark spherical granules with reddish tints that develop among the dark, nonspecific granules. As the eosinophilic cells pass through their various developmental stages, these granules become less purplish-red and more reddish-orange. The dark blue, nonspecific granules, characteristic of the promyelocyte and the early myelocyte stages, disappear. Because the percentage of eosinophils is usually low in bone marrow peripheral blood smears, no useful clinical purpose is served by routinely separating the eosinophils into their various myelocyte, metamyelocyte, band, and segmented categories. On the other hand, in situations such as eosinophilic leukemia in which the eosinophils are greatly increased, an analysis of the incidence of the various stages would be useful in diagnosis.
i. Eosinophil. See figure 4-22.
Figure 4-22. Granulocytic series: Eosinophil
(1) Size. 9 to 15 microns in diameter.
(2) Nucleus. The nucleus usually has two definite lobes separated by a very narrow filament or strand. Has clumped, coarse chromatin pattern. Seldom does an eosinophil have more than two lobes.
(3) Cytoplasm. The cytoplasm contains bright reddish-orange, distinct granules. The granules are spherical, uniform in size, and evenly distributed throughout the cytoplasm, but rarely overlie the nucleus.
j. Development of the Basophilic Group. These cells have round, indented, band, or lobulated nuclei and are classified according to the shape of the nuclei, as basophilic rnyelocytes, metamyelocytes, bands, and segmented forms. These cells are so few in peripheral blood and bone marrow that there is little clinical value in differentiation of the various maturation stages. See figure 4-23.
Figure 4-23. Granulocytic series:
a. Basophil, b. Neutrophil: segmented.
k. Mature Basophils.
(1) Size. 10 to 16 microns in diameter.
(2) Nucleus. The nucleus has definite lobes and are separated by a very narrow filament or strand. The nuclear details are obscured by the large coarse cytoplasmic granulation.
(3) Cytoplasm. The cytoplasm is covered by many blue to black granules. These granules are unevenly distributed and vary in number, size, shape, and color usually blue-black.
Granulocytes are leukocytes devoid of specific granulation. These cells generally originate in the lymphatic system, but can be found in normal bone marrow. Granulocytes include the lymphocytic series, monocytic series, and plasmocytic series.
The stages in the development of the lymphocytic series are: lymphoblast, prolymphocyte, and lymphocyte. These cells are fragile and can show shape variants. Lymphocytes usually have round contours, blue cytoplasm, and eccentrically located round nuclei. Cells of this series are differentiated on the basis of the nuclear chromatin.a. Lymphoblast. See figure 4-24.
(1) Size. 10 to 18 microns in diameter.
(2) Nucleus. The nucleus has an oval or round shape and stains reddish- purple. The nuclear chromatin is fine, well distributed, and coarser than in the myeloblast. Chromatin is condensed at the edges of the nucleus to form a definite nuclear membrane. One to two nucleoli are present. The nucleus is prominent with a nucleus-cytoplasm ratio of 6:1.
(3) Cytoplasm. The cytoplasm is moderate to dark blue and smooth with a frequent perinuclear clear zone.
Figure 4-24. Lymphocytic series:
a. Lymphoblast. b. Lymphocyte. c. Smudge cel.l
b. Prolymphocyte. See figure 4-25.
Figure 4-25. Lymphocytic series: .Prolymphocyte.
(1) Size. 10 to 18 microns in diameter.
(2) Nucleus. The nucleus is oval and slightly indented. The nuclear chromatin is coarse, slightly clumped, am dark purple. One light blue nucleolus is usually present. The nucleus- cytoplasm ratio is 5:1.
(3) Cytoplasm. The cytoplasm varies from moderate to dark blue and it can show a few red-purple ( azurophilic) granules.c. Lymphocyte. See figure 4-26.
(1) Size. The mature cell of this series varies greatly in size. Small lymphocytes are 7 to 9 microns in diameter. The large lymphocytes are 6-16 microns in diameter.
(2) Nucleus. The nucleus is round or oval and can be slightly indented.The nuclear chromatin is markedly condensed, dark purple-blue, and clumped. Nucleoli are absent and a definite nuclear membrane ids present. The nucleus-cytoplasm ratio is approximately 1.5:1.0.
(3) Cytoplasm. The cytoplasm is light blue to blue with a perinuclear clear zone around the nucleus. A few azurophilic granules can be seen in the cytoplasm of larger lymphocytes.
Figure 4-26. Lymphocytic series:
Lymphomcyte, azurophilic granulation.
4-9. MONOCYTIC SERIES
The stages in the development of the monocytic series are monoblast, promonocyte, and monocyte. Cells of the series are slightly larger than granulocytes. They are round with smooth margins and seldom show shape variants. The mature monocyte is differentiated from the lymphocyte and metamyelocyte by the very fine, light staining nucleus.a. Monoblast. See figure 4-27.
Figure 4-27. Monocytic series:
a. Monoblast. b. Stem (ferrata cell).
(1) Size. 12 to 20 microns in diameter.
(2) Nucleus. The nucleus is round or oval fine lacy light blue- purple in color. The nuclear chromatin is moderately basophilic to blue-gray. One to two distinct nucleoli are present. The nucleus-cytoplasm ratio is 4:1 to3:1.
(3) Cytoplasm. The cytoplasm is a clear, deep blue and follow a thin rim around the nucleus.
b. Promonocyte. See figure 4-28.
Figure 4-28. Monocytic series: Promonocyte.
(1) Size. 14 to 18 microns in diameter.
(2) Nucleus. The nucleus is oval or single fold. The nuclear chromatin is fine and spongy1 to 5 nucleoli may represent. The nucleus-cytoplasm ratio is 3:1 to 2:1.
(3) Cytoplasm. The cytoplasm is blue-gray with ground glass appearance with fine dust like azurophilic (red-purple) granules.c. Monocyte. See figure 4-29.
Figure 4-29. Monocytic series:
a. Neutrophil (late band). b. Monocyte.
(1) Size. 14 to 20 microns in diameter.
(2) Nucleus. The nucleus is round or kidney-shaped, but can be deeply indented or slightly lobed. One of the most distinctive features of the monocyte is the presence of superimposed lobes, giving the nucleus the appearance of brain-like convolutions. Heavy lines marking the edges of the folds and grooves are features that are not seen in other cells. Another feature of the nucleus, which is of value in diagnosis, is the tendency for the nuclear chromatin to be loose with light spaces in between the chromatin strands, giving a coarse linear pattern in contrast to the Iymphocyte that has clumped chromatin. Nucleoli are absent. The nucleus- cytoplasm ratio is approximately 2:1 to 1:1.
(3) Cytoplasm. The cytoplasm of the monocyte is dull gray-blue while the cytoplasm of the neutrophils in the adjacent fields is definitely lighter and is pink rather than gray-blue. The nonspecific granules of the monocyte are usually fine and evenly distributed, giving to the cell a dull, opaque or ground-glass appearance. In addition to the background of evenly distributed nonspecific granules, there may be a few unevenly distributed larger azurophilic granules. Vacuoles are often demonstrable in the cytoplasm.
Plasmocytes constitute approximately one percent of the white cells in the normal bone marrow. These cells can represent in the peripheral blood in chronic infections, granulomatous and allergic diseases, and multiple myeloma. The stages of development are: plasmoblast, proplasmocyte, and plasmocyte.
Plasmoblast. See figure 4-30.
Figure 4-30. Plasmocytic series: Plasmoblast.
(1) Size. 18 to 25 microns in diameter.
(2) Nucleus. The nucleus is large, oval or round, and located off center in the cell. Nuclear chromatin is more clumped than in reactive lymphocyte. There may be lighter staining area near the nucleus (perinuclear halo). There are multiple nuclei that may or may not be visible. The cytoplasm is basophilic, abundant and non-granular. The nuclear cytoplasm ratio is 4:1.
Proplasmocyte. See figure 4-31.
Figure 4-31. Plasmocytic series: Proplasmocyte.
(1) Size. 15 to 25 microns in diameter.
(2) Nucleus. The nucleus is ovoid and located eccentrically. The chromatin is purple, coarser, and more clumped. One to two nucleoli are present. The nucleus- cytoplasm ratio is 3:1.
(3) Cytoplasm. The cytoplasm is intensely basophilic, usually bluer than a blast and is nongranular. A lighter-staining area in the middle of the cell (in the cytoplasm, next to the nucleus) may become visible. This is termed a hof or perinuclear halo. May have occasional nucleoli.
Plasmocyte. See figure 4-32.
Figure 4-32. Plasmocytic series: Plasmocyte
(1) Size. 8 to 20 microns in diameter.
(2) Nucleus. The nucleus is round to ovoid, and eccentrically located (located near the edge of the cell). The chromatin is coarse, lumpy, and purple. Nucleoli are not usually present. The nucleus-cytoplasm ratio is 2:1 to 1:1.
(3) Cytoplasm. The cytoplasm adjacent to the nucleus is lightly stained in contrast to the periphery of the cell that has a high saturation of red and blue dyes. In same cells, the dark cytoplasm has a greenish or larkspur-blue color. The cytoplasm contains multiple small and relatively unstained globules embedded in a bluish-red filamentous matrix. It is the presence of these tapioca-like globules in the dark surrounding medium that gives the plasmocyte its characteristic mottled and foamy appearance and its brilliant translucency. In occasional cells, the globules can be quite prominent and take a red or bluish-red stain. Such globules are called Russell or fuchsin bodies, or eosinophilic globules. Vacuoles of various sizes are frequently demonstrable.
VARIATIONS OF LEUKOCYTES
Variations of leukocytes occur as a result of abnormal maturation of the nucleus and/or cytoplasm. These variations are induced by leukemic states, infectious diseases, and toxicity. Described below are the most frequently occurring variations.
Dohle Bodies. Dohle bodies are light blue or blue-gray, small, round inclusions found in the cytoplasm of neutrophilic leukocytes. Contains RNA and may represent localized failure of the cytoplasm of neutrophils. The variation may occur in toxic conditions such as severe infections, burns, poisoning, and following chemotherapy.
Auer Rods. Auer rods are rods or spindle-shaped, cytoplasmic inclusions. They stain reddish-purple and are 1 to 6 microns long and less than 1.5 microns thick. They are frequently found in myelogenous leukemia. Also, found in the cytoplasm of myeloblast, and monoblasts.
Toxic Granullation. Toxic granulation (figure 4-33) occurs in the neutrophilic metamyelocyte, band, and segmented cells. These granules are distinguished from the normal granulation because they are large, coarser and stain a dark purple. The variations occur in toxic states, severe infections, and burns. Believed to be primary granules which show increased alkaline phosphate activity.
Figure 4-33. Variations in leukocytes: Band Neutrophil: Toxic Granulation
Basket Cell. A basket cell is a ruptured leukocyte that has a network appearance. These cells result from a partial breakdown of the immature and fragile leukocytes. Basket cells are found predominantly in diseases with an acute shift toward immature forms, for example, leukemias.
Vacuolated Cell. A vacuolated cell is a degenerated cell with holes or vacuoles in the cytoplasm. Vacuolated cells can be seen in severe infections, poisoning, and leukemias, and in cells that have been in Heller & Paul oxalate too long.
Hypersegmentation. A normal neutrophilic segmented cell has a nucleus with an average of three lobes or segments. In a hypersegmented cell the nucleus is broken up into six or more lobes (see figure 4-34). This cell usually has a larger diameter than a normal neutrophilic segmented cell. Hypersegmentation is often seen in pernicious anemia and folic acid deficiency. They may also be found in chronic infections.
Figure 4-34. Variations in leukocytes: Neutrophili: hypersegmented.
Atypical Lymphocytes. See figure 4-35. These lymphocytes are characteristic of infectious mononucleosis but they may also be seen in apparently healthy individuals and those with certain other diseases. Atypical lymphocytes are larger than normal and vary in appearance. Downey and McKinley described three types of atypical lymphocytes, but this classification has no real clinical purpose.
Figure 4-35. Variations in leukocytes: Atypical lymphocytes
(1) Size. Large, up to 20 microns in diameter.
(2) Nucleus. The nucleus is oval or kidney shaped with very coarse chromatin strands not as lumpy as a normal lymphocyte.
(3) Cytoplasm. The cytoplasm is blue to dark blue. Often it is vacuolated which gives rise to a foamy appearance.
L.E. Cells. See figure 4-36.
Figure 4-36. Variations in leukocytes: L.E. cells.
Rosettes. Rosette formation is the intermediate stage in the formation of an L.E. cell. A rosette formation consists of neutrophilic leukocytes surrounding free masses of lysed nuclear material.
(1) Persons having lupus erythematosus, one of the “collagen” diseases, have an abnormal; plasma protein that causes swelling and breakdown of certain blood cell nuclei in vitro. This degenerated nuclear material attracts phagocytic cells, particularly segmented neutrophils, which engulf this nuclear mass. The resulting phagocyte and inclusion material is termed an "L.E.” cell.
(2) The nucleus of an L.E. cell is adjacent to the peripheral outline of the inclusion material. The inclusion is smooth and silky or light purple and has no visible chromatin network.
Tart cells. A tart cell, which may be confused with the L.E. cell, contains lysed nuclear material within its cytoplasm. It differs from an L.E. cell because the inclusion retains characteristic nuclear structure. This inclusion is not smooth and has a darker staining periphery. The significance of tart cells is not known but their presence in an L.E. preparation does not signify a positive test for systemic lupus erythematosus.