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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.

If leukocyte esterase is detected, a color change occurs on the reagent pad after the strip is dipped in the urine sample. Be sure to follow the manufacturer's directions for read-time and test interpretation. A positive leukocyte esterase test indicates the presence of granulocytic white blood cells. Lymphocytes do not contain granules, and would not produce a positive leukocyte esterase test. Positive results should be confirmed by performing a microscopic examination on the sediment; being aware that white blood cells may be absent if they are lysed, yet releasing their esterases into the specimen. Positive results may occasionally be found in random specimens from females due to contamination of the specimen by vaginal discharge.

This slide is stained with Perls' Prussian blue. Three siderocytes containing blue granules are indicated by arrows. These inclusions may or may not have been visible on Wright's stain, as Pappenheimer bodies.

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

20 to 60% of red cell precursors seen in bone marrow slides normally contain siderotic iron granules visible with Prussian Blue stain. The presence of sideroblasts and siderocytes indicates that the red cell precursors have an ample supply of iron. When a red cell precursor contains too much iron, the siderotic granules form a ring around the nucleus and the resulting cells are referred to as ring sideroblasts. The ring sideroblast is an abnormal (pathological) form of sideroblast.

Coarse basophilic stippling is present in the cell in the center of this slide. The granules are larger and more prominent than those seen in fine stippling.

Fine basophilic stippling is also seen in the large red cell in the center of this field. The granules are not as prominent as those in the previous slide.

Kinetic Processes Specific to Platelets. Adhesion – When platelets adhere to exposed collagen, they take on a characteristic “spiny” shape. Their inherent stickiness, and the aforementioned spiny shape serve to compliment each other during this process. Von Willebrands Factor (vWF) is absorbed by surface receptors on both the platelet and exposed subendothelial tissue, thereby linking the platelets to the tissue. Release – This process occurs prior to aggregation. Platelets dump the contents of their granules (ADP, Serotonin, & Calcium), which aids the upcoming aggregation process by acting as a chemical signal. Aggregation – Platelets physically bind to each other, not just to the exposed subendothelial walls and collagen of the breached vessel. Platelet aggregation requires sufficient chemical signal stimulation. Stabilization (technically part of secondary hemostasis as fibrin is a product of secondary hemostasis)– This process strengthens the platelet plug with the addition of interwoven fibrin strands, ultimately producing a fibrin clot. The durable fibrin clot is the ultimate goal of hemostatic processes.

This slide shows a marrow aspiration smear with numerous ring sideroblasts. Normal red cell precursors have only one or at most two granules of iron in their cytoplasm. These abnormal red cell precursors have numerous iron containing granules in their cytoplasm indicating abnormal iron incorporation. This iron is actually incorporated into mitochondria. Ring sideroblasts can be seen in idiopathic sideroblastic anemia, and in sideroblastic anemia induced by drugs, lead poisoning, and alcohol abuse.

Lymphocytes "transform" in response to antigenic stimuli.Their nuclei becomes larger with more open chromatin and a greater degree of nuclear folding.The cytoplasm becomes abundant, the number of azurophilic granules may be increased and vacuoles may be present.The cytoplasmic membrane may be easily indented by surrounding red blood cells, resulting in a scalloped appearance of the cell's outer edge.These lymphocytes may also be referred to as reactive, activated or stimulated.

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.

Eosinophil is also known as eosinophilic granulocyte, or eo. Eosinophils are one of the easiest cells in the peripheral blood to recognize because of their large bright granules. The diameter of the eosinophil is 9-15 microns, and the N:C ratio is 1:3. Eosinophils are generally the largest granulocytes found in normal blood.

The chromatin pattern of the basophil nucleus is not quite as coarse as that of the neutrophil or eosinophil nuclei. Although the nucleus is usually segmented, the lobes are often difficult to discern because they tend to crowd together and are obscured by the cytoplasmic granules.

The abundant cytoplasm of a segmented neutrophil is of virtually the same appearance as that of the band. It stains faintly pink and contains numerous fine specific granules which are pinkish-lilac.

Basophil is also known as basophilic granulocyte and baso. Basophils are easily recognized because of their large, dark granules. The basophil's diameter is 9-15 microns, and its N:C ratio is approximately 1:3.

When examining a blood film you may find that some basophils have many dense granules while others appear washed out with only a few granules. This is because the granules are water soluble and tend to wash out during the rinse phase of the staining process.

Basophils serve as mediators of inflammatory responses, especially hypersensitivity reactions.IgE binds to the membrane receptors on basophils and degranulation is initiated.The enzymes released are vasoactive, bronchorestrictive and chemotactic (especially for eosinophils), so basophils seem to play a role in inducing and maintaining allergic reactions.The granules of basophils contain histamine, heparin and peroxidase.After degranulation occurs, basophils can synthesize more granules.The release of large numbers of these granules can cause anaphylactic shock and death.

Large lymphocytes have abundant pale blue transparent cytoplasm.If you imagine putting a printed page behind the cell, the cytoplasm looks as though you could see through it to read the words.Although there are usually no cytoplasmic granules present, a few large well-defined azurophilic granules (lysozomes) can occasionally be seen.

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.

Granular casts are composed of plasma protein aggregates and cellular remnants. Granular casts appear as cylinders of coarse, or fine, highly refractive particles. Coarsely granular casts (yellow arrow) contain large, coarse dark yellow or dark brown granules. They are very irregular in shape as shown in this high power magnification under the brightfield microscope. A hyaline cast can be seen just to the left of the coarse granular cast (blue arrow).

Waxy casts appear as cylinders of smooth, highly refractive material. They are yellow, homogeneous and their ends may be square or broken off. Cracks may occur within the cast, giving it a segmented appearance. Waxy casts are believed by some to be the final stage of degeneration of the fine granules of granular casts. Since the granules need time to degrade, this finding implies localized nephron obstruction. Waxy casts are seen in chronic renal failure, and acute and chronic renal allograft rejection. Unusually broad waxy casts are known as renal failure casts. These very broad casts are created in the dilated tubules seen in end-stage renal disease.

Amorphous urates appear as dark or yellow red granules while phosphates are white or colorless. The pH of the urine determines the type of amorphous crystals present. They may be urates in acid urine or phosphates in alkaline urine.

In the center of this slide is a neutrophil from a patient with Chediak-Higashi syndrome. Notice that some of the granules are very large and purple.

This slide is also from a patient who has Alder-Reilly anomaly. Notice that neutrophil seen in this slide has granulation which is much heavier than in the previous slide. The amount of granulation may vary from cell to cell with some cells being unaffected. A lymphocyte showing abnormal granules is also present in this slide.

This slide is an example of a neutrophil showing normal granulation. Notice that on close examination the granules are present but not prominent. Some normal neutrophils may have granules slightly more prominent than this example.

Dohle bodies are sometimes faintly stained, irregular in shape and are therefore easy to miss when examining a slide. The Dohle body in this cell can be seen near the edge of the cytoplasm. Notice that the cytoplasmic granules are somewhat heavier and darker than normal.

The neutrophils found in Chediak-Higashi can be differentiated from toxic granulation. In conditions causing toxic granulation, the granules are smaller and more numerous and only the neutrophils are affected. In Chediak-Higashi, eosinophils, basophils, lymphocytes and monocytes are affected. In eosinophils larger than normal eosinophilic granules may be seen, basophils may exhibit larger than normal basophilic granules, lymphocytes, large azurophilic granules. Larger pale granules/bodies may appear in monocytes.

A lymphocyte from a patient with Chediak-Higashi. The azurophilic granules appear much larger than those seen in normal lymphs.

Another example of toxic granulation. Notice that the granules are larger and redder than those in the previous slide.

Alder Reilly Anomaly is a rare autosomal recessive hereditary disorder in which the basic defect involves protein-carbohydrate complexes called mucopolysaccharides. The accumulation of partially degraded (broken down) protein-carbohydrate complexes within the lysosomes account for the larger than normal purple-staining granules seen in the granulocytes, monocytes and/or lymphocytes. The granules may occur in clusters, rather than diffusely, throughout the cytoplasm as in toxic granulation. These inclusions may be seen in the bone marrow more frequently than in peripheral blood. The physical characteristics associated with this disorder include gargoylism and dwarfism. The function of the cells involved is not affected. This morpholical change would be classified as pathological since the body is responding abnormally even though the function is not affected.

The following pages in this presentation includes a series of white blood cell abnormalities that may be identified in a peripheral blood smear. Many of the cases will simulate the practice of a peripheral smear review by a hematology morphologist. He/she must asses what responses in patient care may be triggered by the clinician attempting to interpret the reported findings on a peripheral smearObservations of white blood cell abnormalities in the peripheral blood smear should be reported so as to direct the physician to an immediate specific diagnosis, such as: (1) atypical lymphocytes suggesting infectious mononucleosis rather than leukemia, (2) toxic granules in neutrophils as in acute infections, or atypical granules suggesting a genetic disorder, (3) an unusual mix of cells, such as too many or too few neutrophils, monocytes, or other myeloid cells, and (4) the presence of giant platelets, myelocytes, or other cells suggesting a myelodysplastic syndrome.In summary, laboratory data should be presented to clinicians in a user friendly way to promote effective decision making. The design of the data base of information must be directed toward providing clinically helpful information clearly and quickly in order to facilitate appropriate action in terms of optimizing patient care outcomes.d

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome in itself is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome. The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

In 1952 Chediak (a Cuban physician) reported a childhood disorder in which abnormal cytoplasmic inclusions appeared in the neutrophils of four family members. In 1954 Higashi reported a similar abnormality in an 11-month old Japanese infant. These inclusions were identified as lysosomal in origin and found in this rare autosomal recessive disorder Death was usually related to recurrent infections or hemmorhage though now some of the affected patients live to reproduce. Ocular and cutaneous albinism, increased susceptibility to pyogenic infections, abnormal granules in neutrophils, and a bleeding tendency are prominent findings. The striking neutrophilic inclusions appear as coarse intra-cytoplasmic azurophilic granules (see photograph).These granules arise from dilated portions of the Golgi-endoplasmic reticulum lysosomal apparatus. Aleutian mink and other animals are known to have Chediak-Higashi syndrome. Azurine pelts from infected mink were once prized by coat makers.

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

Illustrated in the photograph is a immature granulocyte with a distinct rod-shaped intracytoplasmic inclusion. This inclusion is known as an Auer rod, which is seen in up to 10% of blast cells in patients with acute myelogenous leukemia. An Auer red is the fusion of primary granules into rod-like inclusions.

The cytoplasm of eosinophils is evenly filled by numerous orange-red granules of uniform size. They do not overlie the nucleus.The eosinophil granules contain numerous enzymes including peroxidase, phospholipase D, catalase, acid phosphatase, and vitamin B12-binding proteins.Their ability to kill bacteria is less than that of neutrophils. Their main purpose is to counteract parasitic infections and to participate in immune allergic reactions.They may also be increased in a variety of nonimmunologic inflammatory responses from bacteria and fungi causing chronic infections. Malignancies, collagen vascular diseases, and myeloproliferative disorders may also may be settings for prominent eosinophils.