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Two segmented neutrophils and a lymphocyte (indicated by an arrow) are in the center of this picture. Notice the mature chromatin structure in the nucleus of the lymphocyte. Three mature red cells are present around the lymphocyte. Two macrophages are also present in this picture.

Blast cells may be seen in the spinal fluid when cell proliferation in acute leukemia or lymphoma spreads to the central nervous system. The arrows indicate the two blasts in this field. Notice the smooth chromatin pattern in the nucleus and prominent nucleoli in both cells. Notice that an Auer rod is present in the cytoplasm in the blast to the right. The Auer rod indicates that these blasts are myeloblasts rather than lymphoblasts. A segmented neutrophil and several red cells can also be seen.

Malignant cells that have broken away from tumors located in other areas of the body may be seen in spinal fluid. All of the cells in this field are tumor cells. The cells in this slide are characterized by an open, loose chromatin pattern, nucleoli and vacuoles. Notice that the vacuoles are present in both the nucleus and the cytoplasm. Vacuoles in the nucleus are an unusual finding even in tumor cells. Tumor cells are often found in clumps and may have more than one nucleus due to their erratic mitotic patterns. Malignant cells sometimes have an irregular nuclear shape. Bizarre granules may be found in malignant cells but are absent in mesothelial cells.

The Howell-Jolly body in this nucleated red cell is right next to the nucleus and not as clearly visible as the one in the preceding slide.

A sideroblast, shown at the arrow, is a nucleated red cell containing siderotic granules. However, these granules are generally not clustered around the nucleus, but are found in the periphery of the cell. Siderocytes are present normal bone marrow.

Another example of a ringed sideroblast. The granules, although clustered around the nucleus, are not as prominent as the ones in the previous slide.

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells. They appear blue/gray on the Wright's stained smear. The residual RNA in the cytoplasm causes the blue/gray color. The terms, polychromasia or polychromatophilic, are used to describe these cells on a Wright's stained preparation. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count.

Another example of a cell rarely seen in the bone marrow is an osteoblast. Osteoblasts are cells which are similar in appearance but somewhat larger than plasma cells or tumor cells. The nucleus is eccentric and the "hoff" area is sometimes located away from the nucleus. The cytoplasm appears rather foamy when compared to a plasma cell. The size of an osteoblast is 20-25 microns. Osteoblast produce bone.

It is also important to examine the morphology of platelets. One megakaryocyte shows a single nucleus surrounded by cytoplasm which will eventually break off to form platelets. The other one at the arrow shows a lobated nucleus which has divided several times; the large amount of cytoplasm surrounding this nucleus means that this cell will be able to produce more platelets. In general, as the megakaryocyte gets older, it forms more nuclear lobes, more cytoplasm and therefore is able to produce more platelets.

High power (40x objective) examination can be used to estimate the myeloid-to-erythroid ratio. The erythrocytes are nucleated, immature erythrocytes. Under high power, nucleated red cells appear to have a dark purple nucleus as opposed to the lighter staining nucleus of the myeloid or granulocyte series. Lymphocytes also have a dark staining nucleus and some may be erroneously included in the erythroid estimate. In the normal marrow these numbers are insignificant.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

When viewing a confusing cell, it is helpful to focus through several planes, taking special note of overall chromatin structure, appearance or presence of any filaments, and general cytoplasmic characteristics.For example, here we see a neutrophilic cell that is folded over itself making exact identification difficult.However, due to the thickness of the nucleus and the absence of a filament, we would classify it as a band.

Any cell containing specific granules in which the lobes of the nucleus are connected by a filament is considered a segmented cell.A filament is defined as a thread-like structure.Since at times, in viewing a three dimensional object from one direction it is impossible to be certain whether two parts of the nucleus are connected by a filament or band, it is suggested that such cells always be placed in the segmented category, since this is the more differentiated and more common cell.

As time progresses, the band nucleus gradually develops constrictions, resulting in the formation of nuclear lobes. In this way, the band neutrophil matures into a segmented neutrophil.

The nucleus of the large lymphocyte is larger than that of the small lymphocyte, and is more irregular in shape. Sometimes it is rounded, oval or indented.

At other times the nucleus appears to be stretched across the cell, attached to the cell membrane at each end. A nucleus like this seems to have visible "stretch lines" through it.

Small lymphocytes have only a thin rim of clear, homogenous, moderate blue cytoplasm around the nucleus.

Overall, the nucleus has a soft, spongy, three-dimensional appearance, in contrast to the hard, flat nucleus of the large lymphocyte and the densely clumped nucleus of the band.

At times it can be very difficult to differentiate monocytes from large lymphocytes.Monocytes may be mistaken for large lymphs when their cytoplasm stains too lightly, when the characteristic granules are indistinct, or when the nucleus is rounded or only slightly indented.Sometimes a cell will have the nucleus of a lymphocyte and the cytoplasm of a monocyte, or some other confusing combination of characteristics.In order to properly identify the cell, it is necessary to weigh all of the characteristics together to determine which cell type it most resembles.Even then it is occasionally necessary to judge the cell on the basis of the company it keeps.For instance, if there are many monocytes, but few large lymphocytes around, the confusing cell is probably a mono.

Epithelial cells are larger than white blood cells and red blood cells, and contain a single nucleus. White blood cells (pus cells) usually show a segmented nucleus. Red blood cells are 1/2 to 2/3 as large as white blood cells, contain no nucleus, and are gram negative.Hyphae are gram positive tubular filamentous fungal elements which may show branching or intertwining. Yeast cells are round to oval, often budding, gram positive fungal elements, about the same size as RBCs. They are generally much larger than bacteria.

As mentioned earlier, the nucleus of a small lymphocyte is approximately the same size as normal erythrocytes and can be used as a guideline for determining the size of the red cells in the field. Notice that most of the red cells in this field are about the same size as the lymphocyte in the center.

Another form of renal epithelial cells originates in the collecting ducts and measures 12-20 microns. These are identified by their characteristic cuboidal shape and large usually slightly eccentric nucleus. Cuboidal cells are sometimes confused with neutrophils. Neutrophils, however, are slightly larger to nearly twice as large (20-35 microns) as cuboidal cells and have a segmented or band-shaped nucleus while cuboidal cells have a single round nucleus. This view under high power phase contrast shows two cuboidal cells lying between squamous cells.

The most common type of cell seen in the urine sediment is the epithelial cell. This slide shows squamous epithelial cells under low power brightfield microscopy. They appear as large flattened cells with abundant cytoplasm and small round central nucleus. Although squamous epithelial cells have little clinical significance they must be differentiated from other cellular elements.

Using phase contrast and high power the cytoplasm and nucleus appear much darker. Note the folded edge of the cytoplasm which is characteristic of squamous cells. The long, thread-like structures are mucous strands.

A normal mature neutrophil is 9-13 microns in diameter and contains 3-5 lobes or segments.When the number of segments is increased to six or more the cell is hypersegmented. Some hypersegmented cells will be larger than in 15 microns. Hypersegmentation is seen most frequently in neutrophils but can also occur in eosinophils and basophils. The nuclear segments are composed of deoxyribonucleic acid (DNA). A defect in the production of DNA causes the maturation process to be slower than normal which in turn causes the nucleus to hypersegment. The cytoplasm will be normal in appearance and function, indicating that these cells are capable of phagocytosis.These cells are considered pathological

When examining a slide made from an EDTA tube of normal blood, an occasional cell containing a round pyknotic nucleus and neutrophilic-appearing cytoplasm may be seen. Rare cells such as these do not indicate the presence of Pelger-Huet anomaly.

Hyposegmented cells are neutrophils with fewer than three nuclear lobes. The nucleus may be round, peanut-shaped, band-shaped or bilobed. Since nuclear lobe development is abnormal, the chromatin structure often appears more mature than normal. It is sometimes very smooth, almost pyknotic.

Large inclusions in leukocyte cytoplasm appear with Alder-Reilly syndrome. Inheritance patterns are not completely clear. The condition is characterized by larger than usual azurophilic and deeply violet staining granules clustered throughout the cytoplasm (even covering the nucleus)in all granulocytes. There are variations in which some lymphocytes and monocytes may be affected. These inclusions represent partially degraded mucopolysaccharides within lysosomes.Alder-Reilly bodies may be found independently of genetic mucopolysaccharidoses as an inherited anomaly (Jordan's anomaly). Cytoplasmic vacuoles of toxic origin are not present in Alder-Reilly cells. The background condition in Alder-Reilly syndrome is mucopolysaccharidosis with various types of bone and cartilage disorders, reported first in gargoylism, then in Hunter and Hurler syndromes. Accompanying conditions are hepatosplenomegaly, corneal opacities, and mental retardation. Reference: Brunning, Richard D. Morphologic Alterations in Nucleated Blood and Marrow Cells in Genetic Disorders. Human Pathol: 99-124, March, 1970

A basophil and a small lymphocyte are compared in the same field of the upper photograph, A single basophil is shown in the lower photograph.The cytoplasmic granules of the basophil are larger than the granules of toxic granulation.They contain chemical mediators of immediate hypersensitivity, and are found in the cytoplasm and overlying the nucleus (better seen in the lower photograph). Basophilic granules stain metachromatically with toluidine blue indicating the presence of acid mucopolysaccharide or proteoglycans, both thought to be heparin or heparin-like substances.Basophils are related to tissue mast cells, each involved in hypersensitivity responses and following anaphylactic episodes.Under the stimulation of complement components C3a and C5a, many mediators are released from the basophil granules, including histamine, heparin, and eosinophil chemotactic factors of anaphylaxis, or ECF-A.Basophils are the least common neutrophils in the peripheral blood, comprising 2% or less of the differential count.The presence of large granules of irregular size in basophils and the admixture of eosinophilic granules may indicate dysplastic changes associated with myelodysplastic disorders and leukemia.

Plasma cells are uncommonly observed in the peripheral blood smear.They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities.In the upper and lower photographs are plasma cells with features mindful of myeloma cellsThe large myeloma cell in the upper photograph has an eccentric immature nucleus with a muddy chromatin pattern.Note also clumping and stacking of the erythrocytes, bordering on rouleaux formation ,implicating an increase in plasma gamma globulin.The plasma cell with the double nucleus in the lower photograph is particularly suggestive of myeloma.Further studies are in order including a bone marrow examination where at least 30% of bone marrow cells should be variations of mature and immature plasma cells.Serum electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine.The presence of lytic bone lesions is a convincing clinical clue.With these findings in combination, a diagnosis of myeloma can be made with assurance.