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Cytoplasm Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Cytoplasm and links to relevant pages within the course.

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Alpha Thalassemia
Lactate Dehydrogenase

Lactate dehydrogenase (LD) is found in the cytoplasm of every cell. LD is present in the serum at a level of 100-190 U/L. The serum LD level will rise during increased cell damage.Persons with alpha thalassemia intermedia usually have an increased levels of lactate dehydrogenase (LD). This LD is of red blood cell origin, which leaks in to the plasma during hemolysis.

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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Cell Morphology

This slide is an illustration of a HEp-2 or HEp-2000® cell with several nuclear and cytoplasmic structures indicated. Antibodies to DNA, histones, centromere, nuclear RNP, cytoplasmic RNP, mitochondria, ribosomes, lysosomes, golgi apparatus, as well as a variety of cytoskeletal proteins such as microfilaments, intermediate filaments, and microtubules, can be detected using the HEp-2 or HEp-2000® cell lines.(Ref10) It is important to note that RNA constitutes the bulk of the nucleic acid in the cells, being 5-10 times more abundant than DNA.(Ref11) Generally we consider 2 compartments in the cell when detecting autoantibodies in systemic rheumatic disease: 1) nucleus, and 2) cytoplasm. Staining of the nucleus includes staining inside the nucleoli.

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Stages of Mitosis

The stages of mitosis include interphase, prophase, metaphase, anaphase and telophase. When we view mitotic cells we are generally interested in the metaphase mitotic cells. This is the only stage in the entire cell cycle in which the nuclear lamins and nuclear envelope are depolymerized and a major redistribution of nonchromosomal nuclear antigens occurs. Distinct differences in the staining characteristics of the metaphase mitotic cells can be observed with various autoimmmune sera.(Ref12)When reading ANA results it's important to be familiar with the different stages of mitosis. To be considered positive there must be a clearly discernible pattern staining the nucleus of the interphase cell. When a pattern is present in the nucleus of the interphase cell then the metaphase mitotic cells are examined to assist in identifying the pattern(s).If there is no staining in the nucleus of the cell but there is a discernible pattern in the cytoplasm of the cell, the sample is reported as ANA negative. However, the presence of a cytoplasmic pattern should be noted.

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Fluorescent ANA Testing

The most common method of ANA testing is indirect fluorescent assay (IFA) utilizing fluorescein isothiocyanate (FITC) as the marker on the secondary antibody.The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein. When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized with the aid of a fluorescent microscope. In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence. The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)

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Colorzyme®

A similar procedure that is also widely used is called Colorzyme®.(Ref7) This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope. The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANA's are present, a stable antigen-antibody complex is formed. After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody reagent conjugated to horseradish peroxidase. When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme specific substrate. The reaction between the enzyme labeled antibody and enzyme specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker staining. The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)

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The image on the right represents the result of a fluorescent antinuclear antibody (ANA) test. What pattern should be reported?Note: (a) points to the nuclei of several interphase cells, the primary consideration for discerning the ANA pattern and (b) indicates a metaphase mitotic cell. Observing the chromosomal area and cytoplasm of the metaphase cell may assist in identification of the ANA pattern.View Page
Overview

Autoantibodies to the nucleus of the cell are not the only antibodies with clinical significance. Autoantibodies to components of the cytoplasm can be very important pieces in solving the diagnostic puzzle for some patients and should be reported. When reading the ANA results always start by determining if there is a discernible pattern in the nucleus of the cells, if not then the result is ANA negative.Then examine the cytoplasm to see if there is a discernible pattern present there. If so, then the report should contain a comment on the cytoplasmic pattern present.The following photographs demonstrate the more common cytoplasmic patterns.

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Cytoskeletal

This sample is demonstrating an anti-cytoskeletal pattern in the cytoplasm of the cells.The cytoskeleton of eukaryotic cells is comprised of microfilaments such as actin, intermediate filaments such as vimentin and microtubules such as tubulin. Autoantibodies to these proteins are seen in a variety of autoimmune diseases.Whenever an ANA is read the first step is to see if there is a clearly discernible pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is considerable staining in the cytoplasm. This fibrous staining of the cytoskeleton is a discernible cytoplasmic pattern and should be reported (b).This sample is reported as ANA Negative, suspect cytoskeletal antibodies present. (The term "suspect" is used because the ANA substrate is not considered "confirmatory" for the identification of cytoskeletal antibodies. Additional follow-up testing is required).Follow-up testing would include testing for anti-smooth muscle antibodies. Anti-smooth muscle antibodies are seen in autoimmune liver disease.Reporting anti-Cytoskeletal Antibodies:Cytoplasmic pattern:• Fibrous strands in cytoplasmReport as:• Suspect cytoskeletal, suggest follow-up Clinical Significance:• Actin: Autoimmune hepatitis, PBC• Vimentin and others: SLE, RA, others

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Ribosomal P

This sample contains anti-ribosomal P antibodies.About 50% of the time when a sample contains anti-ribosomal antibodies, a positive nucleolar ANA is present along with fine granular staining in the cytoplasm. The other 50% of the time the nucleolar ANA is NOT present and only the fine granular cytoplasmic staining is seen. Therefore, depending on the status of the staining in the nucleus of the interphase cells, anti-ribosomal P antibodies can be either ANA positive or negative.The sample that is seen in this image is reported as ANA positive, Nucleolar (a), and (because of the strong cytoplasmic speckling (b)), suspect anti-ribosomal P antibodies present. Follow-up testing would include testing for anti-ribosomal P antibodies. Anti-ribosomal P antibodies are very specific for patients with SLE.Ribosomal Autoantibodies:Cytoplasmic pattern "Cloudy" fine speckling in cytoplasm Some samples may demonstrate nucleolar ANA

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Golgi

This sample is demonstrating an anti-golgi pattern.Whenever you read an ANA the first step is to see if there is a clearly discernable pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is a discernable pattern staining in the cytoplasm. This coarse granular staining cap like staining on one side of the cytoplasm is characteristic of anti-golgi antibodies and should be reported (b).This sample is reported as ANA Negative, suspect anti-golgi antibodies present. No follow-up testing is necessary. Anti-golgi antibodies are seen in various diseases.Golgi Autoantibodies 7 different antigens identifiedCytoplasmic pattern Coarse granular staining around one side of nucleusReport as Suspect anti-golgiClinical significance Sjögren's syndrome, SLE, RA Viral infections: HIV, EBV, CMV and rubella

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Mitochondrial

This sample is demonstrating an anti-mitochondrial pattern.Whenever you read an ANA the first step is to see if there is a clearly discernable pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is considerable staining in the cytoplasm. This granular "strands of beads" pattern is characteristic of anti-mitochondrial antibodies and should be reported (b).This sample is reported as ANA Negative, suspect mitochondrial antibodies present. Follow-up testing would include testing for anti-mitochondrial antibodies. Anti-mitochondrial antibodies are seen in patients with primary biliary cirrhosis.Mitochondrial antibodies:Cytoplasmic pattern Discrete speckling "strand of beads"

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Cytoplasmic patterns are interesting to look at but have no clinical significance and should NOT be reported.View Page

Body Fluid Differential Tutorial
Synovial Lining Cells

A joint space has a membranous lining similar to the mesothelium found in the pleural and peritoneal cavities. The synovial lining cells, which make up this membrane, produce synovial fluid which lubricates the joints.In a normal joint there is a minimal total volume of fluid present. With joint trauma, such as infection or inflammation, the volume will be increased and synovial lining cells may be noted on the cytospin preparation in addition to the cell types normally present with infection, inflammation or hemorrhage. Synovial lining cells (see arrows) resemble miniature mesothelial cells or small macrophages. They can be found singly or in clumps and can have "foamy"-looking cytoplasm.

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Synovial Lining Cells

In the image on the right, a clump of synovial lining cells (see arrow) is pictured. This slide originated from a patient with rheumatoid arthritis.Observe the size of these synovial lining cells relative to the lymphocytes and neutrophil present in the picture. They are smaller than mesothelial cells found in other fluids.Notice the fluffy, foamy appearance to the cytoplasm. While these cells are found in a cluster, it is still possible to make out indistinct cytoplasmic boundaries. Just like mesothelial cells, these may occasionally be bi-nucleate.

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Adenocarcinoma in Peritoneal Fluid

This is a cytospin of an ascites fluid from a patient with widely metastatic adenocarcinoma.Notice the size of these tumor clumps (see arrows) when compared to the size of the background neutrophils, lymphocytes and macrophages.Also, note how close together the nuclei appear in the tumor clump. Think about the separation you would see in a mesothelial clump. These tumor cells are larger than mesothelial cells would normally be. They have a considerably larger and more dysplastic-looking nucleus and have much less cytoplasm than a mesothelial would normally have. These are key differentiating features in the identification of adenocarcinoma tumor clumps in fluids.

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Neuroblastoma in Pleural Fluid (NBL)

Neuroblastoma is a tumor that arises from embryonic neural crest tissue. It is the most common tumor diagnosed in children under the age of five. Since it arises from nerve tissue, it can be found in many locations throughout the body and therefor can be found in several body fluids.This image shows a tumor clump in the pleural fluid of a patient with stage IV neuroblastoma. Like many of the other metastatic tumors shown in this section, the cells are large in size with a large nucleus and a soft, fine chromatin pattern with prominent nucleoli. The cytoplasm is basophilic with little distinction between individual tumor cells. Since the nuclei are so close to one another, the cytoplasm is much more scant; indicating that this is tumor is not a mesothelial clump.

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Neuroblastoma Tumor Clump vs. Mesothelial Clump in Pleural Fluid

This photo shows a neuroblastoma tumor clump (blue arrow) in the same field as several mesothelial cells (red arrows).While the individual cells are the same overall size, the tumor cells have larger nuclei and a smaller amount of cytoplasm than the mesothelial cells. The chromatin is finer in these tumor cell nuclei. Also, note the differences in the mesothelial cell chromatin pattern, which is much more coarse in texture with darker nucleoli present. The mesothelial cells have a distinct demarcation between adjacent cells, while this line of demarcation is not as apparent in the sheet of tumor cells.

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Agranular Metastatic Melanoma in Cerebrospinal Fluid

Most malignant cells in this patient's cerebrospinal fluid appear similar to the tumor cells shown in the prior case of metastatic melanoma, with the exception of the presence of melanotic granules. The characteristic shaggy look to the cytoplasm and the very prominent nucleoli in a cell that resembles a mesothelial cell is highly suggestive of metastatic melanoma.

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Alveolar Rhabdomyosarcoma (ARMS) in Plerual Fluid

Metastatic alveolar rhabdomyosacroma tumor cells are not as large as adenocarcinoma , but will also present in clumps.Notice the extremely fine chromatin texture and the very large and prominent nucleoli. Several of these tumor cells are bi-nucleate and a few have very prominent glycogen storage vacuoles in the cytoplasm.

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Metastatic Tumors in Fluid Cytospins.

There are a wide variety of solid tumors that can metastasize and spread into body fluids. As with cytospins positive for leukemia or lymphoma, any smear with tumor or suspected tumor should be sent for pathology or hematologist review.Body fluids tend to be a good growth medium for metastatic tumors. These tumor cells tend to be present in sheets and clumps. Frequently there will be reactive changes with increased mesothelial cells and macrophages associated with metastatic tumors as well.Tumor cells, in general, typically appear large with fine/open chromatin patterns, dismorphic or dysplastic nuclei and prominent nucleoli. They will have varying amounts of basophilic cytoplasm depending on the tissue of origin.

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Lymphocytes vs. Monocytes

Lymphocytes, for the most part, have a more regular cytoplasmic border without the cytoplasmic blebbing and pseudopods that are present in monocytes.Observe the differences in character of the cytoplasm between the two cell types. Both cells have blue cytoplasm, however the monocyte (red arrow) has a grainy, gritty texture that is absent in the lymphocytes. In addition, there are fine red cytoplasmic granules present in the monocyte that are not apparent in the lymphocytes.Notice the relatively regular nuclear shape of the lymphocytes (blue arrow) versus the more complex nuclear shape of the larger monocyte. There is also a difference in the texture of the chromatin between the two cell types. The lymphocyte chromatin is more dense and clumped.

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Lymphocytes

The image in this slide depicts slightly activated normal lymphocytes (see arrows).Observing the cytoplasm, it is apparent that the cell it is pastel blue and non-granular; identical to what one would expect to see in a slightly viral peripheral lymphocyte.It is also important to note the chromatin texture and staining. While the chromatin is a bit more loose than a normal lymphocyte observed on a peripheral blood smear, it still has course, smudge-like, clumped chromatin of a mature lymphocyte.

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Viral Lymphocytes vs. Monocytes

The smear shown on the right was taken from a patient with partially treated bacterial meningitis. It is obvious to see that neutrophils are the predominant cell type, however there are also monocytes and a few atypical lymphocytes as well.The atypical lymphocytes (blue arrows) have a more generous and basophilic cytoplasm than the normal, smaller lymphocytes. They also have a more regular nuclear shape compared to the monocytes (red arrows). The lymphocytes also have a more regular cytoplasmic border without the blebs and pseudo-pods present in the monocytes. Another key feature in the differentiation of these two cell types is the vacuolation present in the cytoplasm of the monocytes which is not present in the lymphocytes.

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Lymphocytes and Atypical Lymphocytes

Similar to peripheral blood, lymphocytes in fluids come in all sizes. A larger lymphocyte is not necessarily an abnormal or atypical lymphocyte, however. In the image shown here there is one atypical lymphocyte (blue arrow) adjacent to the monocyte at the top of the smear. The atypical lymphocyte is almost as big as the monocyte, however the nucleus is more regular. The amount of cytoplasm is similar between the two cells, but the atypical lymphocyte has a deeper blue shading at the edge of the cytoplasm. Though there are nucleoli present in this atypical lymphocyte, it does not necessarily mean that this is a malignant cell. Cyto-spinning can expose nucleoli in cells that would not normally appear to have nucleoli in the peripheral blood. The balanced amount of cytoplasm in comparison to the nucleus and the overall size of the cell are consistent with the range of variation found with atypical lymphocytes.In this smear, there is also a plasmatoid lymph in the lower left of the cluster (green arrow) and a basophil in the lower right (orange arrow).

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Atypical Lymphocytes

The image shown to the right depicts a cluster of cells containing both normal and atypical (reactive) lymphocytes. The variations in size, depth of color, and cytoplasmic volume similar to what one would expect to observe on a peripheral blood smear. There is a difference in the density of the chromatin in the lymphocytes as well as the uniformly regular nuclear shape. The atypical lymphocytes on the left (green arrow) are larger than the other lymphocytes while exhibiting a larger amount of generally non-granular cytoplasm. One of the atypical lymphocytes has a few azurophilic granules in the Golgi area.The single cell at the bottom right with the finer, less dense chromatin and irregular nucleus is a monocyte (blue arrow).

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Viral Lymphocytes

The smear in this slide came from a patient suffering from viral meningitis. Notice the absence of neutrophils and the large numbers of lymphocytes, most of which are normal. There is much greater amount of cytoplasm in the three atypical lymphocytes grouped in the center of the image (see arrows). These atypical lymphocytes have a chromatin pattern similar to a mature lymphocyte, even though the cells have increased size.The more activated a lymphocyte becomes in response to a viral infection, the more likely it is to see nucleoli on a cytospin, especially in the pediatric population.Nucleoli alone does not make an atypical lymph malignant or leukemic. Chromatin textures and cytoplasmic volumes will be altered as well in leukemia and lymphoma.

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Plasma Cells

Plasma cells and plasmacytoid lymphocytes can be found in any viral-reactive effusion. This image is a pleural fluid from a patient with viral pneumonia. Notice the plasma cells (see arrows) which contain a large amount of cytoplasm compared to the smaller lymphocytes. The color is more basophilic and there is a noticeable clearing adjacent to the nucleus. Notice the dense and clumped chromatin pattern. Some of these cells have a hint of cytoplasmic vacuolation which can be prominent storage vacuoles.

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A patient with an infectious mononucleosis infection presents in the emergency room. Physicians order a spinal tap which is immediately sent to the laboratory for review. Please identify the cell in the image below from this patient's cerebrospinal fluid sample.View Page
Monocytes and Macrophages

Monocytes and macrophages are frequently found together in body fluids. In fact, macrophages are actually monocytes that have arrived in the fluid earlier, and become more active than their more recently arrived brethren. The function of a monocyte or macrophage is to remove and recycle dead or dying cells so they do not become an irritant to the body. Macrophages (see arrows) are larger with much more cytoplasm which is frequently heavily vacuolated. The nucleus tends to be pushed to the edge of the cell and the cytoplasm may have ingested materials present. You may see ingested RBC's or WBC's, lipid droplets, hemosiderin and even ingested crystals.

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Monocytes vs Lymphocytes

While the cytoplasm of the two monocytes in this image (red arrows) is not as grainy as some; the larger size, complex nuclear shape, fine chromatin pattern and cytoplasmic vacuoles help to identify them as monocytes.Lymphocytes (blue arrows) keep the shape of their nuclei much more simple, maybe displaying a bit of an indent, which is different compared to monocyte clefting.

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Monocytes

Monocytes (red arrows) are larger than lymphocytes (blue arrows). They tend to have a more complex nuclear shape, and cytoplasm that is more grainy than lymphocytes. Their chromatin is softer, finer, and more "lacy" when compared to lymphocyte chromatin.Notice the greater irregularity of the cytoplasmic membranes in the monocytes compared to the lymphocytes.

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Histiocytes

The large multinucleate macrophage shown in this bronchialalveolar lavage image (see arrow) is a histiocyte. Notice the similar cytoplasm characteristics and chromatin texture and staining between the histiocyte and macrophages. If you were to fuse together 5 of the macrophages in this photo, it would not be easily distinguished from the histiocyte in the center.Note: The terms histiocyte and macrophage are sometimes used interchangeably. The choice of which term to use can be institution and pathologist dependent. Some institutions designate large multinuclear macrophages as histiocytes.

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Acute Myeloid Leukemia (AML)

This cytospin is from a patient diagnosed with Acute Myeloid Leukemia (AML) who had central nervous system involvement at the time of diagnosis.Notice the large size of these blasts. They have very fine, soft chromatin with very prominent multiple nucleoli. The cytoplasm has a hint of the background granularity that myeloid blasts have on a peripheral smear. These characteristics help to identify immature myeloid blast cells in fluid differential analysis.

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Acute Myeloid Leukemia (AML) continued

This cytospin shows three myeloid blasts (blue arrows) and a cell that is in mitosis (red arrow). These three blasts have varying amounts of cytoplasm and nuclear complexity, but all have similar chromatin /cytoplasmic textures and staining characteristics.Mitotic figures are not usually seen in benign fluids and should be commented upon in the differential report according to your hospital's protocols.

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Prominent vacuolation involving the cytoplasm of abnormal lymphoblast-like cells seen in a body fluid preparation is a distinctive feature of Burkitt Lymphoma.View Page
Central Nervous System (CNS) Relapse: L2 Acute Lymphoblastic Leukemia (ALL)

This image shows a cerebrospinal fluid cytospin prepared from a known leukemia patient who presented during therapy with new onset of severe headache and mental status changes. The cell count revealed 350 white blood cells (WBCs) and 5 red blood cells (RBCs)/ mL. Notice the large mononuclear cells that are the predominant population in this sample. They are quite large compared to the few normal lymphocytes and occasional RBC that are present. Notice the very fine chromatin and markedly irregular nuclear shape of the blasts. These cells should not be confused with monocytes; the cytoplasm is lymphoid; without the fine, "ground glass" cytoplasm that is typical for a monocyte. Another differentiating feature is the scant cytoplasm present, which discourages identification as a monocyte. Although monocytes can have irregular nuclear shapes, these cells have nuclear irregularities which exceed those seen in normal monocytes.

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L3 Burkitt Lymphoma

This photo is a peritoneal fluid from a patient with stage IV Burkitt lymphoma. While this smear is more cellular than is ideal for optimum evaluation of morphology, it is still possible to recognize the characteristic morphology of the lymphoma cells present.The Burkitt cells are as large or larger than the few neutrophils present and somewhat resemble other types of lymphoblasts. However, they have course dense chromatin with very basophilic and markedly vacuolated cytoplasm (see arrows).This cytospin demonstrates the typical cytoplasmic vacuolation of Burkitt lymphoma in which the vacuoles break through the background of dense chromatin and intensely basophilic cytoplasm.

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Non-Hodgkin Lymphoma

This cytospin was prepared from a pleural fluid obtained from a patient with multiply recurrent non-Hodgkin lymphoma. The patient had repeated pleurocentesis to remove excess and to improve his quality of life while on palliative therapy.The three large mononuclear cells in the center are the lymphoma cells (blue arrows). Notice their large total and nuclear size compared to the background lymphocytes and the scant amount of basophilic cytoplasm present with the few fine cytoplasmic vacuoles. Notice also the wide range of normal and reactive cells in the background. In patients with recurrent malignant effusions, it is not uncommon to see such mixed cell populations.It is important to look for low numbers of lymphoma cells in known lymphoma patients, as they may be present in low numbers during and after therapy, rather than in the large numbers that are usually present upon initial diagnosis.

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Anaplastic Large Cell Lymphoma (ALCL)

This cytospin is from a patient who presented in respiratory distress and was found to have a large mediastinal mass and large bilateral pleural effusions.The lymphoid cells in this image are large and immature in appearance. These lymphocytes were initially believed to be consistent with lymphoma cells but, after immunophenotyping, were found to be reactive T-cells instead of lymphoma cells.The three larger cells in the image look similar. The two larger cells on the left are just macrophages. The one larger cell on the right is actually the malignant cell (see arrow). The malignant cell has a larger nucleus with softer more open chromatin and a slightly more prominent nucleoli. The cytoplasm is also more basophilic, and the vacuoles are atypical. They are not the typical round vacuoles seen in macrophages/histiocytes; these vacuoles are more elongated.The diagnosis of ALCL was confirmed when the cytogenetics proved positive for the specific translocation, t(2;5), that defines this lymphoma.

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Acute Lymphoblastic Leukemia (ALL): L1 Morphology

This is a cytospin from the CSF of a patient with L1 acute lymphoblastic leukemia (ALL) obtained at the time of diagnosis. Notice the monotonous look to the cells present. They are of moderate size with soft fine chromatin and have a scant amount of basophilic cytoplasm. There is some irregularity and slight cleavage to the nuclear shape. Some of these blasts have cytoplasmic vacuoles. Though these blasts have a hint of a nucleolus, it not necessary for them to be present in order for these cells to be considered blasts. The relative size, chromatin texture and scant amount of cytoplasm define these cells as L1 lymphoblasts. Notice the three small densely staining normal lymphocytes indicated by the arrows, that allow for a contrast of the relative sizes and chromatin textures of the blasts with those of the normal small lymphocytes.

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Central Nervous System (CNS) Toxoplasmosis

This image represents a cerebrospinal fluid (CSF) cytospin preparation from a patient who recently received a bone marrow transplant for recurrent central nervous system (CNS) Burkitt's lymphoma. The patient was admitted to the hospital two weeks after transplant due to rapidly altering mental status. When the CSF cell count demonstrated a high white blood count (WBC) count, the first concern was a possible CNS relapse of the Burkitt's lymphoma. However, the cytospin showed many neutrophils in spite of the patients peripheral blood neutropenia. No malignant cells were identified.On closer examination of the neutrophil clusters, ovoid inclusions were noted (see arrow) as well as free banana shaped organisms (see circled area). The ovoid inclusions in the neutrophils and the free forms have lavender cytoplasm with a centrally placed cluster of reddish granules.

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Central Nervous System (CNS) Toxoplasmosis continued

This is a higher power view of this same smear demonstrating a neutrophil that is filled with Toxopasma gondii tachyzoites (blue arrow).There are a few free organisms in this image well, indicated by the red arrows. Again, the typical morphology for toxoplasmsa organisms is lavender cytoplasm with a red granular cluster in the center of each parasite.This patient was negative for Toxoplasma gondii prior to a transplant but had received 15 units of blood products due to cytopenias.It is believed that a donor for one of the transfused units had been exposed to Toxoplasma gondii either through cats or contaminated food and had transient circulating Toxoplasma gondii in his or her blood when the donation was made. In this case, the recipient was profoundly immunocompromised, which lead to rapidly developing systemic disease.

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Bronchial Lining Cells continued

Bronchial lining cells may appear on cytospin preparations in sheets and clumps.In the image on the right, notice the grouping of bronchial lining cells. A few bronchial lining cells are intact and have identifiable cilia (see arrows).Many of these cells appear as rectangular smears with a smudged nucleus at the base. This is not uncommon in cytospins of BALs. The location of the nucleus at the base of the elongated cytoplasm shape identifies these cells as bronchial lining cells.

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Mesothelial Cells

The mesothelium is the name given to the membrane that lines most body cavities and surrounds the internal organs. Cells that shed from these membranes are commonly found in pleural, peritoneal and pericardial fluids. Mesothelial cells are large cells that may be found as single cells or in clusters and clumps. They tend to have a large round centrally placed nucleus with a generous amount of basophilic cytoplasm which can appear frayed at the edges. They will have one ore two small, well-defined, deeply staining nucleoli. While they may have small pinpoint vacuoles, they will not have the larger "foamy" vacuoles seen in macrophages or histiocytes.There are two mesothelial cells in the image below (see arrows). While they are different in size, they are definitely larger than the background lymphocytes and plasmacytoid lymphocytes. Notice the irregular frayed edge to the cytoplasmic membrane.

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Reactive Mesothelial Cells

Reactive mesothelial cells can be found when there is an infection or an inflammatory response present in a body cavity. This condition can be due to the presence of a bacterial, viral or fungal infection. It can also be the result of trauma or the presence of metastatic tumor.Reactive mesothelial cells tend to come in clusters and clumps and have a more washed out cytoplasm in body fluids. Notice in the image on the right, how indistinct the cytoplasmic borders are in this clump compared to normal mesothelial cells. The wide separation of the nuclei and the well defined nucleoli help to identify these as reactive mesothelial cells. However if there is any doubt, the smear should be sent for hematology or pathology review.Note: It is not uncommon for macrophages to be mixed into a reactive mesothelial clump.

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Mesothelial Cells continued

Mesothelial cells are frequently found in clusters and clumps. In Image 1 below, the individual cells in this clump still maintain the round nucleus and generous basophilic cytoplasm that can be seen in individual mesothelial cells. Also, the boundaries between cells are clear and distinct, unlike a multinucleate histiocyte in which there are no clear boundaries between the previously discrete cells. In this particular image, there are two binucleate mesothelial cells in this mesothelial clump.Binucleate mesothelial cells are a normal variant found in any fluid with mesothelial cells. Rarely trinucleate mesothelial cells can be seen. However, any fluid that has mesothelial cells with more than 3 nuclei is abnormal and should be sent for hematology or pathology review. Image 2 depicts binucleate mesothelial cells.

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Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Iron Staining

Iron staining on bone marrow aspirate smears is commonly part of the standard order protocol for bone marrows aspirates. The iron staining procedure utilizes the Prussian Blue stain for ferric iron to assess bone marrow iron stores. This procedure is particularly helpful when evaluating patients with anemia, iron overload, myelodysplasia, etc. In the adult setting, it is commonly performed on the bone marrow biopsy, but can be requested on the aspirates as well.In the pediatric setting, it is less likely to be part of the standard order set since young children rarely have stainable iron stores. However, iron staining may be requested on patients with congenital anemia and possible mitochondrial defects to look for sideroblastic anemia.In this technique, iron will stain blue and will normally be found in bone marrow stromal/ macrophages, which are found in the spicules. On aspirate smears, without fragments/spicules, it is not possible to evaluate for iron stores. However, if there are nucleated red blood cells (NRBCs) present, it is still possible to look for the ringed sideroblasts, common in sideroblastic anemias.The image on the right is a field from a bone marrow slide from a patient with congenital sideroblastic anemia. The NRBC indicated by the red arrow is a normal siderocyte with few granules of hemosiderin scattered through the cytoplasm. The NRBC that is indicated by the blue arrow has a large number of hemosiderin granules concentrated in the mitochondria that surround the nucleus. This is a pathologic ringed sideroblast.

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Myeloblast

Under normal circumstances, the segmented neutrophil is the most common nucleated cell in the peripheral blood. These bacterial-infection-fighting cells are produced in the bone marrow and arise from their precursor cell, the myeloblast. The myeloblast is the youngest cell in the myeloid lineage. It is approximately 12-20 microns in size with very basophilic cytoplasm. The nucleus takes up around 2/3 of the total cell volume with a soft, finely stranded chromatin with very little clumping. The nucleus is eccentrically placed and ovoid, but can also be slightly flattened. Myeloblasts will typically have two or more nucleoli with well defined nucleolar membranes. In a well-stained preparation, you should be able to observe the outline and blue color of the nucleoli.The myeloblast's cytoplasm is basophilic and can have a hint of background "ground glass" graininess. This graininess is separate from any primary granules that develop as the cell progresses toward the progranulocyte stage. The cytoplasmic membrane tends to be regular without much denting, bumps, pseudopods, or shredding.The cell in the first image on the right shows the relative size, nucleus, and gritty basophilic cytoplasm of a classic myeloblast. Note that there is a small cluster of red primary granules present which, in addition to its other features, help to identify this cell as a myeloblast.The second image shows a myeloblast (blue arrow) at a later stage that is not quite a promyelocyte but is very close. The nucleoli are still prominent, the size has not changed much, and the cytoplasm is still only about 1/3 of the cell. There are a few more primary granules but they are not prominent enough to consider this cell a progranulocyte.While the myeloid sequence tends to be the predominant cell type found in normal bone marrows, myeloblasts should make up less than 5% of the bone marrow's nucleated cells.

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Promyelocyte

Promyelocytes are generally larger than myeloblasts, measuring approximately 12 to 20 microns. The nucleus is similar in size to the myeloblast but the cytoplasm is more abundant at this stage. The nucleoli will begin to close and become less prominent than in the blast stage. The chromatin strand texture in promyelocytes tends to become slightly more coarse and clumped than the chromatin pattern present in a myeloblast. Promyelocyte cytoplasm will have a gritty basophilic color and texture; however, there will also be prominent primary granules. These granules will look like red/purple grains of sand. With careful observation, one can note the cuboid nature of the granules. In the top image to the right notice the size of the promyelocyte on the right hand edge (red arrow),versus the other myeloid cells in the frame. Notice how basophilic the cytoplasm is compared to the more mature myelocytes that are present. Observe the prominent, red, primary granules, which stand out against the basophilic background.In the bottom image on the right, the promyelocyte (blue arrow) has matured a bit more, giving it an appearance closer to an early myelocyte. Though the overall size of the cell has not decreased noticeably (as what happens as cells mature), the depth of the basophilia is not as prominent, nor are the primary granules as obvious as they were in the cell shown in the top image. While the nucleoli are obvious in both cells, the chromatin texture in the cell indicated by the arrow in the bottom image is a bit more clumped and coarse. Also notice the clearing/ lighter color in the Golgi (perinuclear) zone of the bottom cell (indicated by the green arrow). This is where the first development of neutrophil secondary granules will become evident as the cell progresses to the next stage of maturation.

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Myelocyte

The next stage of the myeloid maturation sequence is the myelocyte. The cytoplasm of this cell begins to produce specific, secondary granules. If the cell is destined to be a neutrophil these secondary granules will be pink/tan and will cause the basophilic color to lighten and breakup. At the "dawn" of neutrophilia, these secondary granules are most obvious in the golgi area. As the cell matures closer to a metamyelocyte, they fill the entire cytoplasm.While the cytoplasm shifts to producing secondary granules it also looses the prominence of its primary granules. In situations where the bone marrow is stressed or forced to make neutrophils quickly, as in sepsis or during certain therapeutic injections, some of these primary granules may persist as "toxic granules".At the same time the secondary granule production begins, the nucleus is shrinking and condensing. The nucleoli close and disappear, the chromatin gets coarser/denser and more clumped, and the chromatin gets tighter darker and more compact.The very early myelocyte (red arrow) in the top image to the right still displays its immature features. While the chromatin is not as condensed as in the intermediate and late stage myelocytes in the bottom image, notice how the cytoplasm no longer has the darker basophilic color of a promyelocyte. There are clusters of neutrophil secondary granules that are changing and breaking up the solid basophilic color. Notice too, that you can no longer see any red/purple primary granules. In this cell the cytoplasm is leading the maturational dance and the nucleus is lagging.The bottom image to the right shows two myelocytes (blue arrows): one intermediate in maturity, one a bit more mature, as well as a metamyelocyte (green arrow). Notice how the size of the cell continues to shrink as the cell matures. It is apparent that both the nucleus and the cytoplasm of the metamyelocyte adjacent has decreased in size and the chromatin has condensed/clumped as the cell matured toward a metamyelocyte.

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Metamyelocyte

In the metamyelocyte stage, the cytoplasm and nucleus continue to decrease in size. The cytoplasm achieves full secondary granule content. The chromatin becomes more dense, knotted, and compact, while the nucleus begins to indent and acquire the familiar "kidney bean" shape. By the end of the stage, the cell will be similar in size to a mature neutrophil with similarly cytoplasmic granularity.The top image to the right shows a fairly classic metamyelocyte. Observe the indented kidney bean-shaped nucleus and neutrophil-colored cytoplasm. Notice the clumped aggregates of chromatin in each pole of the nucleus. The vacuolated cytoplasm in this cell is an indication of toxic stress.In the bottom image to the right, notice the metamyelocytes (see red arrows) and their chromatin patterns. The patterns becomes more dense and clumped as the metamyelocytes continue to mature to the band neutrophil stages (see blue arrows).

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Band Neutrophil

When a neutrophil reaches the band stage, the cytoplasm is normally fully granulated and should be identical to its fully mature segmented neutrophil counterpart. The chromatin has continued to condense and should appear as knotted and clumped as a mature neutrophil. When a cell matures from a metamyelocyte to a band, the condensation and indentation of the nucleus reaches the point where all parts of the nucleus are of uniform width. While a U-shaped nucleus is the most classic shape, the band nucleus can be curled or coiled as well.The cell in the top image to the right is an early band. It has a U-shaped nucleus, but still has a bit of openness to the chromatin texture. As the band moves on to the mature segmented neutrophil stage, the entire nucleus will become quite condensed.In the second image, there is a late band neutrophil just above the promyelocyte (see red arrow). Observe the increased amount of clumped and condensed chromatin present here. The cytoplasm appears similar to the segmented neutrophils in the same frame. It is also important to note how much more condensed the chromatin becomes in the mature segmented neutrophils, also in this frame.When it becomes difficult to see if the band has pinched enough to be considered a mature segmented neutrophil, the degree of chromatin condensation and clumping can be used as an additional deciding factor.

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Segmented Neutrophil

The segmented neutrophil is the end stage of maturation in the myeloid lineage. The cell is similar in size to the band neutrophil and has a well granulated cytoplasm with a deeply condensed, knotted and clumped chromatin pattern. The chromatin pinches into several segments, usually separated by visible filaments. In some segmented neutrophils, this filament is inferred by the folding and shape of the nucleus.The top image on the right shows the classic morphology of a segmented neutrophil. The nucleus of a normal segmented neutrophil has two to five lobes, connected by thin filaments. Six or more lobes is an indication of abnormal development, usually related to B12 or folate deficiency.The bottom image shows the progression from band neutrophil (red arrows) to early segmented neutrophil (blue arrow) and finally to fully-mature segmented neutrophil (green arrow). Take a close look at the cell closest to the promyelocyte. You can see a drumstick-like projection arising from the end terminal segment. This can be seen in smears on female patients and is a Barr body or inactivated X-chromosome.

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Myelocytes: Eosinophils and Basophils

As stated previously, the myelocyte stage is the first stage where cell-specific maturation appears in the granulocyte lineage. When you look at a promyelocyte, you cannot yet identify if it will mature into a neutrophil, eosinophil, or basophil. It is only when secondary granules are produced that the endpoint of maturation can be identified. The size and shape of these initial secondary granules help with this identification. While the cytoplasm and granules may look different depending on the lineage of the cell, the progression of nuclear maturation is the same for all granulocytes.When eosinophil secondary granules are first produced, they may not show the same bright orange color found in the mature eosinophil. It is the larger, spherical shape of the granules that identifies the early eosinophil myelocyte, not necessarily the eosinophilic color. In fact, early eosinophil granules may appear somewhat basophilic in color. The larger, three-dimensional, spherical shapes of the granules help to identify cells as eosinophil precursors (see red arrows in top image).Immature basophils (see blue arrow in lower image) can be hard to distinguish from promyelocytes as well. It is important to note the differences in color and shape between primary neutrophil granules and basophil secondary granules. The primary granules in promyelocytes appear as red/purple grains of sand - think of red/purple cubes with defined edges. Basophil granules have a purple /black color and look more like splinters - think of a purple/black sheet of glass that is shattered into the cytoplasm of a cell. The cytoplasmic color in an early basophil myelocyte may be similar to a promyelocyte since there are no fine granules in the basophilic cytoplasm. Remember that mature basophils are not as fully granulated as neutrophils and have a clear/uncolored cytoplasmic background rather than the pink/tan background of neutrophils.

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Pronormoblast (Proerythroblast)

The pronormoblast, or erythroblast, is the earliest stage in erythroid maturation. It is a very round cell that is about the same size as a myeloblast. It has a distinctive deeply basophilic, velvety cytoplasm that does not have the fine background grittiness found in the myeloblast. A pronormoblast typically has a round, centrally-located nucleus , unlike a myeloblast that typically has an eccentric nucleus.The chromatin texture is coarser than myeloid chromatin and is more reticular and bumpy, almost like beads on a string. The pronormoblast will have multiple prominent nucleoli. The nuclear membrane appears highlighted compared to other cell types and there will be small breaks in the membrane that are known as nuclear pores. The erythroid lineage is the only cell line that has nuclear pores, which can help to distinguish intermediate erythroid precursors from lymphocytes.The upper image on the right shows a pronormoblast (red arrow) adjacent to a few monocytes (blue arrows). Notice that the pronormoblast is round and regular and the cytoplasm is intensely basophilic. Observe the central placement of the round nucleus and the nucleoli. Notice the coarse and grainy chromatin texture as well.The lower image on the right shows a late pronormoblast (red arrow) with a few later stage erythrocyte precursors (blue arrows). While the overall size of the late pronormoblast shown in this image is similar to the cell in the upper image, notice the less prominent nucleoli with the classic reticular grainy pattern of the chromatin. The cytoplasm still has the midnight-blue, velvety-look of a pronormoblast.

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Basophilic Normoblast

The basophilic normoblast is slightly smaller in size than the pronormoblast. The chromatin is a bit more condensed, while just beginning to clump. At this stage, the nucleoli will have closed completely. The absence of nucleoli is the major feature that distinguishes a basophilic normoblast from a pronormoblast. The midnight-blue, velvety-look of the cytoplasm is still very prominent, which makes this cytoplasm morphology indistinguishable from that found in a pronormoblast. As a basophilic normoblast continues to mature, the overall cell size will decrease and the chromatin will condense. The cytoplasm will gradually begin to lighten as globin chain synthesis begins.The first image to the right shows three early basophilic normoblasts (red arrows), including one that is binucleate. Notice the grainy, reticular texture of the chromatin. The chromatin has clumped where the nucleoli have closed. The nuclear pores are more prominent. The deep basophilia is starting to lighten in the golgi area, which is normal as globin synthesis progresses. Binucleated red blood cells are normal so long as the two nuclei are of even size. They can be observed most commonly in bone marrows with increased erythroid production.The second image shows a group of basophilic normoblasts (red arrow) maturing toward the polychromatophilic normoblast stage. Notice that the size of the cell continues to shrink. The chromatin is becoming more condensed. Also notice that the cytoplasm remains quite basophilic.

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Polychromatophilic Normoblast

In the polychromatophilic normoblast stage, the cytoplasm has begun to produce hemoglobin and, as a result, the color starts to shift from deep basophilic to a slate blue/gray shade. The cell continues to slowly shrink in size while the chromatin becomes much more knotted and clumped. The spoke-like pattern of the chromatin accentuates the nuclear membrane and the nuclear pores.The top image on the right shows a clump of polychromatophilic normoblasts. Notice they are all very similar in size, shape and stage of maturation. This is a classic pattern in erythroid development and these clusters are frequently associated with macrophages or histocytes in the marrow as they are the RBC precursors' source of iron. Note that the cytoplasm color is now a blue/gray rather than the deep midnight blue of the basophilic pronormoblast stage. The lower image on the right shows a range of RBC precursors. At the bottom is a cluster of basophilic normoblasts (see red arrow), one of which is binucleate. There are also two cells above the cluster: the top cell is an early polychromatophilic normoblast (blue arrow) while the lower is a late basophilic normoblast (green arrow). Note the difference in cytoplasm color. The polychromatophilic normoblast is slate blue/gray while the basophilic normoblast still maintains the midnight blue hue. Observe the nuclei and the chromatin pattern: the chromatin is much more condensed in the polychromatophic normoblast.

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Orthochromic Normoblast

Orthochromic normoblasts are the last nucleated stage of erythroid maturation. In this stage, the nuclei of the cells completely shrink to a pyknotic remnant. The cytoplasm color approaches the color of a peripheral RBC as it becomes fully hemoglobinized. This is the stage that is most commonly seen when NRBCs are found in the peripheral blood. In the top image on the right there are many orthochromic normoblasts scattered across this section of bone marrow. Note the pyknotic-appearing nuclei which make them easy to spot, even at lower magnification. It is also evident that the cytoplasm is well hemoglobinized and the color is just slightly more blue than the non-nucleated red bloods cells present.In the higher magnification (second image), notice the orthochromic normoblast (blue arrow) to the right of the basophilic normoblasts. The color of the cytoplasm of the orthochromic normoblast is almost identical to the background RBCs. Notice how condensed the nucleus has become as well. You can actually observe the nucleus in the early stages of extrusion/elimination from the cell. Once the nucleus has been extruded, the slight blue color, also known as polychromasia, will begin to fade and the now non-nucleated RBC will be indistinguishable from any other circulating RBC.

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Monoblast

Monocytes progress through maturational stages in a similar fashion to the myeloid series before entering the peripheral blood circulation. The final stage of monocyte maturation into macrophages occurs after they have migrated out of the peripheral blood and into the surrounding tissues via diapedesis. Mature macrophages are also found in the bone marrow. The monocyte lineage does not maintain a maturational pool in the bone marrow as large as the myeloid pool. As a result, the monoblast stage is infrequently noted in most normal bone marrows.Monoblasts are the largest blasts of all the hematopoeitic cell lines present in the bone marrow. They have a large, round, centrally-placed nucleus with soft, fine-stranded chromatin. They normally have a single, large, prominent nucleolus. The cytoplasm is very generous and has a fine, grainy texture. In the monoblast stage, the cytoplasm will be basophilic, similar to other blasts, but will possess a slightly lighter shade of blue. In the monoblast, the color will shift to blue-gray as the cell matures into a monocyte.The top image on the right shows a single monoblast. Notice the large, round nucleus, the single large nucleolus and the generous blue, grainy cytoplasm. The second image shows a group of monocyte precursors. The large cell at the top is a monoblast (see red arrow). Notice the round and flat look of the nucleus in the blast compared to the other stages. Observe the nuclear shape becoming more folded and three-dimensional as the cell matures.

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Promonocyte

In the promonocyte stage of development, the nucleolus is still visible while the nucleus begins to indent and fold. This may be observed as pleated or creased-looking chromatin or as a definite flattening or indenting of the nucleus. The chromatin will begin to condense but will still be finer and more "lacy" than what is found in a mature monocyte. The cytoplasm of the promonocyte will begin to mature as well as the color begins to shift toward the blue-gray, grainy texture found in mature monocytes. The fine pink granules found in mature monocytes will also begin to appear. The image on the right is from a patient with monoblastic leukemia (M5b according to the French-American-British system) . This slide permits the observation of several promonocytes in one image. These cells would only rarely be seen in a normal bone marrow . Notice the folded and indented nuclei of the promonocytes (see red arrows). Note that as the promonocyte matures, the cell size decreases and the complexity of the nucleus increases . Notice the fine pink granules, which increase in number as the cell size decreases.

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Monocyte

The monocyte is the final stage of monocyte maturation found in the peripheral blood before it migrates into tissues and further develops into a macrophage (histiocyte).When seen in the bone marrow, a mature monocyte will look identical to its peripheral counterpart. It will have fine, lacy chromatin pattern with varying degrees of nuclear folding and condensation. The cytoplasm will be blue-gray in color with a slightly grainy texture. The cytoplasm may have a light sprinkling of fine pink cytoplasmic granules. The mature monocyte will be larger than mature segmented neutrophils, but not quite as large as promyelocytes or early myelocytes. The top image to the right shows several monocytes with varying degrees of nuclear folding (see red arrows). Notice that the chromatin clumping is not as dense as that found in neutrophils. Notice also that the cytoplasm is blue-gray and grainy, not the pink/tan of a neutrophil. Observe that the mature monocytes are slightly smaller than the promyelocytes in the image.The lower image to the right shows a monocyte (red arrow) adjacent to a segmented neutrophil (blue arrow). The monocyte is clearly larger. Notice the increase in size of the two monocytes below (green arrows) as they begin to transform into macrophages (histiocytes). The vacuolation is an indication of this transformation occurring.

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Macrophage (Histiocyte)

The macrophage is the final stage of development in the monocyte lineage. It is a phagocyte whose roles include the removal of dead and dying tissue and the destruction and ingestion of invading organisms. Macrophages (histiocytes) act as immune modulators as they will present antigens from ingested pathogens to helper T-cells.Their primary role in the bone marrow is the removal of cellular debris, including old red blood cells (RBCs). As a result, they become a source of iron for maturing RBC precursors. A histiocyte is a less phagocytic form of a macrophage with fewer lysosomal granules. Histiocytes may form clusters, or even fuse together into mulitnucleated giant cells. These giant cells are particularly evident on bone marrow biopsy from a patient with a marrow granuloma.The top image on the right shows the early transformation of a monocyte into a macrophage (see red arrow). Notice the increase in the amount of cytoplasm present as the cell begins to ingest debris in the bone marrow. This is demonstrated by the increasing vacuolization present in the cytoplasm. The larger the debris ingested, the larger the vacuoles will be.The lower image on the right shows a macrophage with large vacuoles (red arrow) adjacent to an RBC cluster (blue arrow). This is a common placement, since the macrophage is the iron source for these developing RBCs in the bone marrow.

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Lymphocyte

Lymphocytes mature in the lymph nodes rather than in the bone marrow and therefore are not routinely assessed when deciding if a marrow has "trilinear" (myeloid, erythroid, megkaryocytic) maturation. However, they are normally present in the bone marrow and, when clustered in a lymphoid follicle, can be very prominent. Since lymphocytes mature in the lymph nodes, they will appear identical to peripheral blood lymphocytes when viewed in the bone marrow. They will have the same range of variation in size and cytoplasm and will demonstrate the same types of viral transformations noted in the peripheral blood. Viral/atypical lymphocytes are combined together with normal lymphocytes in a bone marrow differential count and not placed into their own category, as they are in a peripheral blood differential. However, the hematopathologist may include this information in the interpretation, if these changes are noted.Lymphocytes can be found scattered throughout the bone marrow and must be distinguished from early erythroid precursors, which they can closely resemble. Lymphocytes are frequently found in and around early NRBC clusters. In the top image on the right, notice the medium-sized lymphocyte (red arrow) next to the two basophilic normoblasts (blue arrow). The color and texture of the scant lymphoid cytoplasm is almost identical to the NRBC, which can be a bit confusing. However, observe the differences in the nuclei between the two cell types. The lymphocyte has a less distinct chromatin clumping pattern than the basophilic normoblasts and the lymphocyte does not have any "nuclear pores." Also, the lymphocyte has an irregularly-shaped nucleus that is hugging the cytoplasmic border, while the NRBC has a round and regular, centrally-placed nucleus. Identify the three lymphocytes circling the NRBCs in the second image (see red arrows). Notice the chromatin of the lymphocytes; the lymphoid smudgy/clumpy pattern is certainly not as dense and clumped as what is noted in the NRBCs. This nuclear difference becomes more pronounced as the erythroids mature. The cytoplasmic differences should be more apparent as well, since lymphocytes will never produce hemoglobin.

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Hematogone

Hematogone is a term applied to a subset of early B-lymphocytes, found in normal bone marrow, whose morphology greatly resembles that of leukemic lymphoblasts. These cells are larger than the average mature lymphocyte, have scant cytoplasm, and a fine, soft chromatin texture; however, they are not quite as immature in appearance as a true leukemic lymphoblast. Hematogones are more common in younger children but can be found in bone marrow samples of patients at any age. They tend to be found in increased numbers within the bone marrows of patients recovering from bone marrow suppression. Common causes of increased concentrations of hematogones include: viral illness, chemotherapy recovery, and immune mediated cytopenias, such as idiopathic thrombocytpenic purpura (ITP). Hematogones are also common in patients with neuroblastoma.With experience, knowledge of the patients underlying clinical condition, and the ability to review a patient's bone marrow, it is possible to distinguish hematogones from blasts. When necessary, a hematogone flow cytometry panel can be obtained to distinguish these benign cells from lymphoblasts.Notice the size of these blast-like hematogones (see red arrows). They are larger than the few background lymphocytes present in these images. Notice the fine chromatin and scant cytoplasm. They are usually found mixed in with the full range of bone marrow cellular lineages, but can cluster with other lymphocytes within the spicules.

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Plasma Cell

Plasma cells are terminally differentiated B-lymphocytes that have developed a characteristic morphology while actively producing and releasing immunoglobulins. While plasma cells have their origins in the bone marrow as B-cells, they usually leave the bone marrow to develop and mature in the lymph nodes or spleen. Plasma cells begin to produce immunoglobulins after being stimulated by T-cells and exposed to processed antigens.Under normal circumstances, plasma cells are not a large percentage of the lymphoid cells found in a marrow. They are usually placed in a separate category in the differential, unlike viral/atypical lymphs. There can be a relative increase in plasma cells in reactive marrows, and both plasma cells and their early precursors will be markedly increased in plasma cell disorders.While mature plasma cells somewhat resemble lymphocytes, there are a few important differences. The size of the cell is usually larger with more abundant cytoplasm. The nucleus is eccentrically placed and the overall shape of the cell generally resembles a wedge or comet with the nucleus leading the cytoplasm. The chromatin is just as thick and clumpy as a lymphocyte's but is aligned in a more "spokey" or "clockwork" pattern. The cytoplasm is usually more basophilic than the cytoplasm of a normal lymphocyte and will have a well-defined perinuclear halo or noticeable clearing in the golgi area. Vacuoles may or may not be present.Notice the size of the single plasma cell in the top image (see red arrow). It is larger than the neutrophil precursors surrounding it and is almost rectangular in shape. Observe that the nucleus leads the cytoplasm, causing the wedge or comet shape. Notice the prominent perinuclear halo. Find the two plasma cells in the upper left corner of the second image. There is much more cytoplasm in these plasma cells compared to the occasional lymphocyte present in the field. Notice the eccentric nuclear placement as well as the characteristic clearing in the golgi area.

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What are the cells that are indicated by the red arrows in the image on the right?View Page
Megakaryocyte: Immature

The megakaryocyte lineage is the cell line responsible for the production of platelets found in the peripheral blood. Unlike the other bone marrow lineages that decrease in size as they mature, the megakaryocyte starts smaller and increases in size as it matures. The megakaryocyte begins as a mononuclear cell that has the same physical size and nuclear/cytoplasmic proportions as a lymphoblast. Eventually the megakaryocyte matures into a multinucleated giant cell with vast amounts of cytoplasm. As this cell matures, it can actually increase to more than ten times the size of other nucleated cells found in the bone marrow.In the early stages of development, the cytoplasm of a megkaryocyte is basophilic without any obvious platelet granules. The cytoplasm will be darker near the edges of the cell and may have a "foamy" look in the golgi area adjacent to the nucleus. Cytoplasmic granule development is not usually noticeable until the cell's cytoplasm color begins to lighten.Notice the sizes of the early-intermediate stage megakaryocytes (red arrows) in comparison to the background bone marrow cells present in the two images to the right. The megakaryocyte nucleus makes up the largest part of the cell at this early stage. Notice the increasing lobulation as the cell increases in size and how the cytoplasm becomes more foamy and slightly more granular as well.

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Megakaryocyte: Mature

The mature megakaryocyte is the largest cell found in the bone marrow. It can easily reach more than ten times the size of the other cells and usually has as much, if not more, cytoplasmic volume than it has nucleus.Once nuclear/DNA replication found in the early megakaryocyte has halted, the amount of cytoplasm will begin to increase until the megakaryocyte reaches its maximum mature size. As platelet granule production increases, the cytoplasm color will shift from basophilic to the grainy pink texture and light lavender color we are familiar with in peripheral platelets. When the cytoplasm has matured enough to produce platelet granules, the process of shedding platelets can begin. This can occur either as an ongoing continuous process or as a single complete release leaving a naked megakaryocyte nucleus.Notice the extremely large sizes of the megakaryocytes in the images to the right. The megakaryocytes tend to be found in the heart of the bone marrow fragments rather than loosely scattered throughout the smear. Observe the proportion of nucleus to cytoplasm. Notice the foamy, shaggy, irregular cytoplasmic border of the single megakaryocyte that is circled in the bottom image. This megakaryocyte is in the process of releasing platelets and small clusters of platelets can be seen in its vicinity.

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Osteoclast

Osteoclasts are the cells responsible for bone resorption. They work in conjunction with osteoblasts. Both cells under normal circumstances are constantly in the process of rebuilding/reshaping/repairing bone to ensure strength and function. Osteoclasts are only infrequently seen in bone marrow aspirates. They become more obvious when the cellularity is depressed.Osteoclasts are large multinucleate cells somewhat similar in appearance to megakaryocytes, which can cause confusion. Notice in the images to the right that the nuclei of the osteoclasts are flat, even in number, oval or round, uniform in size, and well separated in the cytoplasm. In contrast, megakaryocyte nuclei are segmented and clump in three dimensional clusters. The cytoplasm of an osteoclast is grainy and paler in color than a megakaryocyte. Observe how fluid and irregular the cytoplasmic borders are in the osteoclast.

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Osteoblast

Osteoblasts are the cells responsible for the production and deposition of bone. They may not be apparent in normal cellular bone marrow, since they appear in low frequency. In situations where the total bone marrow cellularity is decreased, they become more visible.Osteoblasts are individual cells but tend to travel in small groups or clusters. They are quite large compared to the normal background blood cells and resemble giant plasma cells. They are oval-shaped cells and tend to have quite basophilic cytoplasm. An osteoblast has a single round nucleus with a fairly open chromatin texture. Notice in the images to the right how the nucleus of the osteoblast is eccentrically placed. On some smears it will almost appear as if the nuclei are in the process of being extruded from the cells. This effect is more commonly seen on extremely hypocellular bone marrows and is less pronounced in bone marrows with a higher cellularity. Notice the large size of the osteoblasts in comparison to the background bone marrow elements.

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Case Studies in Clinical Microbiology
Review 3

Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).

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Cerebrospinal Fluid (retired 7/17/2012)
Pia Arachnoid Mesothelial Cells (continued)

Seven mesothelial cells are seen in this slide. Notice that all of the nuclei have a distinct shape with no evidence of irregular division. Chromatin pattern is typical of cells that originate in the tissues. Cytoplasm is irregular and some pseudopods are evident, especially in the lower portion of the field.

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Pia Arachnoid Mesothelial Cells (continued)

A reactive pia arachnoid mesothelial cell as noted by the darker cytoplasm is present in this field. Reactive cells are a common finding in cytospin smears from spinal fluid samples and are sometimes difficult to distinguish from tumor cells. Mesothelial cells are usually interspersed among the other cells, rather than appearing in clumps. They have a single distinct nuclei that may be eccentric. The macrophages (histiocytes) are seen next to the mesothelial cell. Macrophages are distinguished from circulating monocytes by the irregular appearing cytoplasm. Bacteria, red cells or other debris can often be seen in the cytoplasm of macrophages.

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Monocytes

The arrow in this slide is pointing to a monocyte. The nucleus has an open chromatin pattern which gives it a spongy appearance. The other two nucleated cells could be classified as macrophages (histiocytes) because the nucleus in each cell is oval or kidney bean-shaped and the cytoplasm is very irregular. After circulating in the blood for one to three days, monocytes enter the tissues. The tissue form of the monocyte is called a macrophage or histiocyte.

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Lymphocytes

Many lymphocytes are present in this field. Two larger, reactive lymphocytes with intact cytoplasm and slightly indented nuclei are indicated by the blue arrows. Two other large cells with irregular, trailing cytoplasm are macrophages (histiocytes). These are indicated by the red arrows. Increased lymphocytes may be seen in viral meningoencephalitis, partially treated bacterial meningitis, multiple sclerosis, Guillian-Barre's syndrome, or polyneuritis.

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Blast Cells

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.

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Malignant Cells

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.

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Another Malignant Cell

Another example of a malignant cell. This cell has a smooth chromatin pattern similar to the chromatin pattern commonly seen in blast cells. This cell has a high nuclear to cytoplasm (NC) ratio which is typical for malignant cells. No nucleoli are visible in this cell although malignant cells often have large nucleoli.

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Pia Arachnoid Mesothelial Cells

Pia arachnoid mesothelial (PAM) cells are often found in spinal fluid because they line the arachnoid space between the skull and the cerebrum. Ependymal cells which line the ventricles may also be present. Since ependymal and mesothelial cells are normal findings, the term mesothelial is sometimes used as a general term for both. It is essential to differentiate these tissue cells from blast cells or tumor cells which have diagnostic significance. An example of a pia arachnoid mesothelial cell, as indicated by the arrow, is present in this slide. Differentiating characteristics of this type of cell include an off-center, single, round nucleus and irregular cytoplasm that exhibits pseudopods.

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Erythrocyte Inclusions
Reticulocytes

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. 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. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. Reticulocytes appear blue-gray on the Wright or Wright-Giemsa-stained smear and are referred to as polychromatophilic red cells (indicated by the arrow in Image A). The residual RNA in the cytoplasm causes the blue-gray color. A supravital stain, such as new methylene blue N or brilliant cresyl blue, is used to stain reticulocytes (indicated by the arrows in Image B) for an actual count.Automated methods are available for performing reticulocyte counts. An automated method provides a more accurate absolute retiulocyte count than the manual method that is reported as a percentage of total RBCs.

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Basophilic Stippling

The presence on a Wright-stained peripheral blood smear of relatively evenly-distributed dark-blue particles or granules of varying size in the cytoplasm of erythrocytes is referred to as basophilic stippling. These dark-blue or blue-purple granules are predominantly precipitates of ribosomes (RNA) and may indicate impaired hemoglobin synthesis, probably due to the instability of RNA in the young cell. The erythrocyte containing these inclusions may stain normally in other respects or it may be polychromatophilic.The basophilic stippling that is clinically significant is coarse stippling. Fine stippling is often noted in polychromatophilic red cells, and sometimes in other red cells, and is generally not significant. Coarse basophilic stippling may be seen in sideroblastic anemias, lead poisoning, myelodysplasias, and thalassemias.

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Howell-Jolly Body in Nucleated Red Cell

Since Howell-Jolly bodies are nuclear remnants, they can also be seen in the cytoplasm of the young nucleated red cells.

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Pappenheimer Bodies

Pappenheimer bodies may be seen in the cytoplasm of mature and immature erythrocytes on a Wright-stained peripheral blood smear. They appear as small dark purple granular bodies of varying size, frequently clustered in groups of two, three, or more near the edge of the cell. They are composed of degenerating cellular remnants that contain iron. Pappenheimer bodies are most likely caused by accelerated red cell division or impaired hemoglobin synthesis.

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Erythrocyte Inclusions (retired 7/10/2012)
Multiple small, dark blue particles scattered throughout the cytoplasm of erythrocytes is/are called:View Page
Howell-Jolly Bodies in Cytoplasm

Since Howell-Jolly bodies are nuclear remnants, they can also be seen in the cytoplasm of the young nucleated red cells.

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What are Pappenheimer bodies?

Pappenheimer bodies are seen in the cytoplasm of mature and immature erythrocytes on a Wright's stained smear. They are composed of degenerating cellular remnants, which contain iron. Pappenheimer bodies are most likely caused by accelerated red cell division, or impaired hemoglobin synthesis. Pappenheimer bodies appear as small dark purple granular bodies of varying size frequently clustered in groups of two, three or more near the edge of the cell.

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In a Wright's-stained smear, Pappenheimer bodies must be differentiated from basophilic stippling and Howell-Jolly bodies. Pick the statement which best describes each of the following.View Page
Which of the following statements best describes Pappenheimer bodies?View Page
Reticulocytes

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. 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. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. Reticulocytes appear blue-gray on the Wright-Giemsa-stained smear and are referred to as polychromatophic red cells (image on the left). The residual RNA in the cytoplasm causes the blue-gray color. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count as seen in the image on the right.

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What is Basophilic Stippling?

On a Wright's-stained smear, the presence of multiple dark blue particles or granules of varying size, scattered throughout the cytoplasm of erythrocytes in the reticulocyte stage is called basophilic stippling. There are two types of stippling, fine or diffuse, and coarse or punctate. The erythrocyte containing them may stain normally in other respects or it may be polychromatophilic.

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Fundamentals of Molecular Diagnostics (retired 2/12/2013)
Types of Nucleic Acid Synthesis

A gene is a hereditary unit or sequence of the nucleotide bases ACGT, occupying a fixed location or locus on the chromosome. It is these genes that carry all the information for life processes.DNA is rewritten into 3 types of RNA, each with a specific task:Messenger RNA (mRNA) carries the protein message to the cytoplasm.Ribosomal RNA (rRNA) is the location of protein synthesis.Transfer RNA (tRNA) is responsible for amino acid transport.Each 3-base nucleotide sequence (codon) codes for a specific amino acid. Some amino acids have more than one codon to direct their placement; this is termed degeneracy.

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Which of the following types of RNA is responsible for amino acid transport?View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
What is the cell indicated by the arrow in this illustration:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the leukocyte seen in this illustration:View Page
The cell indicated by the arrow in the illustration is occasionally seen in the bone marrow and can be mistaken for a plasma cell - what is this cell called:View Page
Pelger-Huet anomaly is characterized by:View Page
The precursor of the platelet which is commonly only found in the bone marrow is:View Page

Histology Special Stains: Carbohydrates
Basophilic and Acidophilic Staining

Synthetic dyes/stains are generally created so that they are one of the following stain types:Basic - contains basic groups that have an affinity for acidic tissue elements. Acidic - contains acidic groups that have an affinity for basic tissue elements. Basic stains are used to stain nuclei and other basophilic (basic loving) cellular structures in tissues. Acidic stains are used to stain cytoplasm and other acidophilic (acid loving) cellular structures in tissues.Many biological staining procedures rely on acid-base chemistry.

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Colloidal Iron: Staining Protocol

Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides. Fixative:10% Neutral Buffered Formalin Step Reagent Time Technical Notes 1 12% Acetic Acid Solution Brief Rinse 2 Working Colloidal Iron Solution (10mL Stock Colloidal Iron, 18mL Distilled Water and 12mL Glacial Acetic Acid) 1 Hour 3 12% Acetic Acid Solution 3 Changes x 3 Minutes Each 4 Ferrocyanide-Hydrochloric Acid Solution (25mL Potassium Ferrocyanide, 2% and 25mL Hydrochloric Acid, 2%) 20 Minutes 5 Running Tap Water 5 Minutes 6 Nuclear Fast Red Solution 5 Minutes 7 Running Tap Water 1 Minute Clouding will result when slides are placed in the alcohols if slides are not washed well after Nuclear Fast Red staining. Expected Results Acid Mucins and Sialomucins = Deep Blue Nuclei = Pink to Red Cytoplasm = PinkPost Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped.

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Histology Special Stains: Connective Tissue
Basophilic and Acidophilic Staining

Synthetic dyes/stains are generally created so that they are one of the following stain types:Basic - Contains basic groups that have an affinity for acidic tissue elements.Acidic - Contains acidic groups that have an affinity for basic tissue elements.Basic stains are used to stain nuclei and other basophilic (basic loving) cellular structures in tissues. Acidic stains are used to stain cytoplasm and other acidophilic (acid loving) cellular structures in tissues. Many biological staining procedures rely on acid-base chemistry.

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Masson's Trichrome Staining - Chemistry

Using acid-base chemistry, three dyes are employed to selectively stain muscle, collagen fibers, fibrin, and erythrocytes. Bouin’s solution is used first as a mordant to link the dye to the targeted tissue components. Nuclei are stained with Weigert’s hematoxylin, an iron hematoxylin, which is resistant to decolorization by the subsequent acidic staining solutions. Biebrich scarlet acid fuchsin stains all acidophilic tissue elements such as cytoplasm, muscle, and collagen. Subsequent application of phosphomolybdic/phosphotungstic acid is used as a decolorizer causing the Biebrich scarlet acid fuchsin to diffuse out of the collagen fibers while leaving the muscle cells red. Application of aniline blue will stain the collagen after which, 1% acetic acid is applied to differentiate the tissue section.

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HIV: Structure and Replication (retired 2/20/2013)
Penetration and Infection

After penetration of the cell membrane by the gp41, the HIV capsid enters the cell's cytoplasm. Next, cellular enzymes strip away the capsid so that the HIV genome is released. Also stripped away are proteins p24 and p17. Protein 24 coats the HIV genome and protein 17 lines the inside of the capsid.

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Introduction to Bone Marrow
Choose the marrow cell from the list below which has highly granular basophilic cytoplasm but does not show nuclear lobation.View Page
These cells tend to occur in tight clusters. They may have prominent nucleoli, immature chromatin, and scant cytoplasm.View Page
Supporting Cells

Reticular cells (adventitial cells) provide structural support for the endothelial cells that line the venous sinus and the developing blood cells within the hematopoietic cord. The cytoplasm of the reticular cells is capable of extending itself in fiberlike strands deep into the hematopoietic cords. These strands provide a meshwork for the blood cells. Other types of cells which furnish support in the cord include macrophages and fat cells.

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Location of Cells within Cord

Within the hematopoietic cords each cell line has a specific location for development. Erythroid precursors are located near a venous sinusoid and cluster around a macrophage. This is referred to as an erythroblastic island. Developing red cells obtain iron needed for hemoglobin production from macrophages. Megakaryocytes are also located close to a venous sinus. They extend their cytoplasm in fingerlike projections through the sinus wall in order to release their platelets directly into the blood in the sinus. Immature granulocytes lie within the hematopoietic cords. The metamyelocyte stage is the first stage of the granulocyte series that is motile and able to move toward the sinus area. Mature neutrophils, eosinophils and basophils enter the sinusoidal blood through the basement membrane. As maturing erythrocytes also move toward the sinus wall any remaining nuclei are lost as the red cells move through small openings in the cells lining the sinus wall.

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Ring Sideroblasts

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.

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Promegakaryocyte

The next stage after the megakaryoblast is the promegakaryocyte. It is intermediate in maturity between a megakaryoblasts and mature megakaryocytes. It may have multiple nuclei, coarse chromatin, and more cytoplasm than a megakaryoblast.

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Megakaryoblast

This slide shows an example of the youngest cell in the megakaryocyte series, the megakaryoblast. Megakaryoblasts have a fine chromatin structure with multiple nucleoli, and scant basophilic cytoplasm. Cytoplasmic tags are frequently seen. It may vary 20-50 microns in diameter.

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Megakaryocyte

The next stage is the fully developed megakaryocyte. It typically shows nuclear divisions and abundant very granular cytoplasm. Megakaryocytes are the largest cell found in normal bone marrow and can range in size from 30-100 microns. The nuclear chromatin pattern is coarse. Nucleoli are absent.

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Meta Megakaryocyte Stage

The next stage is the meta megakaryocyte. By this point, platelets are actually breaking away from the meta megakaryocyte cytoplasm. The example shown in this slide illustrates the platelets forming and breaking away near the arrow.

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Other Large Cells

It is also important to scan the slide for the presence of other large cells which are not usually seen in normal marrow. An osteoclast is an example of this type of cell. Osteoclasts are large multiinucleated cells (up to 100 microns) which may be confused with megakaryocytes. One striking difference is that an osteoclast has multiple nuclei which are separate from each other. The multiple nuclei in the megakaryocyte are joined together. The cytoplasm, although somewhat finer in texture, could be mistaken for platelets.

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Osteoblast

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.

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Plasma Cells

An occasional plasma cell is a normal finding in the bone marrow. The nuclear chromatin pattern is coarse, the cytoplasm is varying shades of blue with a "hoff" or light staining area adjacent to the nucleus.

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Mott Cell

Another rare but abnormal type of plasma cell is the Mott cell (morula cell). The compartments visible in the cytoplasm are immunoglobulins which have not been released. Mott cells may be seen in parasitic infections and malignant tumors.

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Gaucher Cell

A Gaucher cell is a histiocyte (macrophage) whose cytoplasm is filled with linear or fibrillar material (kerasin). This cell is characteristic of the congenital glycolipid disorder, Gaucher's disease. Gaucher cells may also be seen in the marrow of patients with chronic granulocytic leukemia. When seen in this condition, they are referred to as pseudo-Gaucher cells.

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Megakaryocyte Morphology

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.

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Tumor Cells

This slide shows a cluster of tumor cells. Notice that the cytoplasm scant in contrast to the abundant cytoplasm of osteoblasts.

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Another Example of Tumor Cells

Another example of tumor cells under higher magnification, showing large cells with loose immature chromatin structure and ill-defined cytoplasm.

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Macrophage

The large cell in the center of this slide is a macrophage, which is normally present in low numbers in the marrow. Macrophages have a loose chromatin pattern and on some smears the nucleoli appear blue/green. The cytoplasm is irregular in shape and contains granules.

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Nuclear Division in Megakaryocytes

Megakaryocytes differ from other cell lines because the nucleus divides during mitosis but the cytoplasm does not.

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Microbiology / Serology Question Bank - Review Mode (no CE)
This parasite is found in blood.View Page
This suspicious form was recovered in blood.View Page
India Ink is used to:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
A hematology technologist observed the intracellular forms seen in the field of view of a Wright-Giemsa-stained peripheral blood smear shown in this photomicrograph. In consultation, the microbiology technologist advised that the form seen most likely represents:View Page

Normal Peripheral Blood Cells
Platelet Kinetics

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are found within the circulation while one third sequestered within the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

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Platelet Appearance

Platelets are anucleate cells, measuring only 1-4 microns in diameter. They are the smallest of the formed elements found in normal peripheral blood. The arrows point to platelets.Their shape varies greatly, but they are usually round, oval or rod-shaped. In addition, platelets stain light blue to purple in color, and are very granular. The cytoplasm of platelets can be divided into two areas: the chromomere and the hyalomere. The chromomere is located centrally where the granules tend to aggregate. The hyalomere surrounds the chromomere and is a clear, blue, non-granular zone. The diagram on the right illustrates the central granular chromomere, and the peripheral clear hyalomere of a platelet.

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Appearance of the Erythrocyte

Erythrocytes are non-nucleated, round, biconcave, disc-shaped cells They are 6.7 to 7.7μ in diameter, 2μ thick, and have an average volume (Mean Corpuscular Volume, MCV) of 80-100μ3. In stained blood films, only the flattened surfaces of the RBC's are seen. Therefore, they appear circular with an area of central pallor corresponding to the indented area. The central pallor occupies about 1/3 of the diameter of the cell. The overall red blood cell diameter is slightly less than that of the nucleus of the small lymphocyte. The cytoplasm stains pink to brick-red, and no nucleus is present.

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Glossary of Terms A through M.

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.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Appearance of a Band Neutrophil

A band neutrophil has moderate to abundant cytoplasm staining pale blue to pink. Present within the cytoplasm are fine lilac to pink granules. These specific granules, called neutrophilic granules, are small and somewhat resemble pencil stippling. Notice that in the image to the right, the band neutrophil has a non-segmented nucleus. Instead, the nucleus shows an indented shape which appears similar to a horseshoe. The nucleus of a band neutrophil is typically U-shaped. Frequently, the nucleus of a band appears folded or twisted, thus making identification a bit more difficult. The nucleus stains a deep purplish-blue color, and the nuclear chromatin appears condensed, coarse, and clumped. Band neutrophils are also referred to as stabs or simply as bands. The diameter of a band is approximately 9-16 microns, and its nuclear to cytoplasmic (N:C) ratio is approximately 1:2.

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Segmented Neutrophils

Segmented neutrophils may also be referred to as segs, polymorphonuclear leukocytes, polys and PMNs. Segmented neutrophils are the most mature neutrophilic granulocytes present in circulating blood. Their diameter is approximately 9-15 microns, and their N:C ratio is approximately 1:3. 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.In order to identify a segmented neutrophil, the cell must have the following characteristics:The nucleus is a deep reddish-purple color, and the chromatin has a coarse, clumped texture.The seg nucleus normally has from 2-5 lobes, with an average of 3.The lobes are connected to each other by a fine filament or strand of nuclear membrane. A filament is a thread-like strip which is so narrow that there is no visible nuclear material between the two sides.

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Which of the following phrases best describes a segmented neutrophil?View Page
Eosinophils

Eosinophils are also known as eosinophilic granulocytes, or eos. Eosinophils are easy to recognize in the peripheral blood because of their large, bright reddish-orange granules. The diameter of the eosinophil is 9-15 microns, and the nuclear to cytoplasmic (N:C) ratio is 1:3. Eosinophils are generally the largest granulocytes found in normal blood.Their cytoplasm is usually colorless or light blue. However, the color is usually masked by the large granules that are present. These granules take up the acid components of Wright's stain, and are therefore reddish-orange.

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All of the following statements describe an eosinophil EXCEPT:View Page
Basophils

A basophil is also known as basophilic granulocyte or a 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. The color of the basophil cytoplasm is light pink to light blue or colorless, but as with eosinophils, the specific basophilic granules are prominent making the cytoplasm difficult to see. The granules are quite large and, because they take up the basic components of Wright's stain, are purplish-black in color.

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Lymphocytes

Lymphocytes are a heterogeneous group of cells that have different origins, lifespans and functions, and vary markedly in size. Some have a diameter of approximately 7µ, while others are as large as 18µ. The variations in size are mainly due to different amounts of cytoplasm. Therefore, the N:C ratio may range from 5:1 in some lymphocytes to 1:2 in others. The nucleus is slightly larger than a normal RBC. It is usually round or oval in shape, but may be slightly indented. The chromatin is very dense and clumped. Small lymphocytes have only a thin rim of clear, homogenous, moderate blue cytoplasm around the nucleus.

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Large Lymphocytes and Reactive Lymphocytes

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. In this case, the cells would be called large granular lymphocytes. A large lymphocyte can be found in the upper image to the right.Reactive, or atypical, lymphocytes are relatively fragile cells, and as a result can be squeezed out of shape by surrounding cells, giving them a scalloped appearance instead of a smooth cytoplasmic edge. The nucleus of the reactive lymphocyte is larger than that of the small lymphocyte, and is more irregular in shape. Sometimes it is rounded, oval or indented with a typical "stretched" appearance. A reactive lymphocyte can be found in the lower image to the right.

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Which of the following cells is characterized by a thin rim of cytoplasm around the nucleus?View Page
When Lymphocytes Transform

Lymphocytes "transform" in response to antigenic stimuli. As discussed earlier, 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. Again, these lymphocytes may also be referred to as reactive or atypical lymphocytes.

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Monocyte Appearance

Monocytes are the largest of the normal peripheral blood cells, ranging from 14-20µm in diameter with an N:C ratio of approximately 3:1. Monocytes have abundant blue-gray cytoplasm containing many fine lilac granules. These give the cytoplasm a "ground glass" appearance. However, these granules may be difficult to see if the blood film is poorly stained. Frequently, cytoplasmic vacuoles are present. These vacuoles appear as unstained areas or "holes" in the cytoplasm; an example of which can be found in the lower image to the right.Because monocytes are extremely motile cells, blunt pseudopods may be seen. These should not be confused with the apparent cytoplasmic projections produced when large lymphocytes are indented by surrounding cells. Monocytes have generally lighter staining nuclei than do other leukocytes. The nucleus stains a pale bluish-violet, and the chromatin is fine. 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. The nucleus may be round, kidney-bean shaped, folded, indented, or horseshoe, and may show "brain-like" convolutions.

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Differentiating Monocytes from Large Lymphocytes

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 monocyte.

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Differentiating Monocytes from Large Lymphocytes: Table.

The table below lists some characteristics that help to distinguish large lymphocytes from monocytes. CellImageNucleusCytoplasmLarge lymphocyteOval, round, indented, "stretched Deep purplish-blue Dense Sky-blue Clear, transparent No granules or azurophilic (reddish) granules Infrequent vacuoles Cytoplasm may be indented by surrounding cells MonocyteRound, oval, indented, convoluted Pale purplish- blue Fine, lacy, spongy Blue-grayCloudy, opaque, "ground-glass" appearanceFine granulesFrequent vacuolesCytoplasm may have pseudopods

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All of the following descriptions are characteristic of monocytes EXCEPT:View Page
Match the characteristics with the cell type.View Page

Normal Peripheral Blood Cells (retired 6/20/2012)
Match the descriptions with the cells.View Page
A ________ has colorless cytoplasm with large purplish-black granules.View Page
Monocytes have a blue-gray cytoplasm with a "ground glass" appearance. Vacuoles may be present in the monocyte cytoplasm.View Page
When Lymphocytes Transform

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.

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Platelet Cytoplasm

The cytoplasm of platelets can be divided into two areas: the chromomere and the hyalomere. The chromomere is located centrally where the granules tend to aggregate. The hyalomere surrounds the chromomere and is a clear, blue, non-granular zone.

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Platelet Kinetics

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are in the circulation and one third are present in the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

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Platelet Cytoplasm

This diagram of platelets illustrate the central granular chromomere, and the peripheral clear hyalomere.

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Cell Diameter

The cell diameter is slightly less than that of the nucleus of the small lymphocyte. The cytoplasm stains pink to brick-red, and no nucleus is present.

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Glossary of Terms A through M.

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.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Appearance of Cytoplasm

Their cytoplasm is usually colorless or light blue. However, the color is usually masked by the large granules that are present. These granules take up the acid components of Wright-Giemsa stain, and are therefore reddish-orange.

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Which of the following phrases best describes a segmented neutrophil?View Page
All of the following statements describe an eosinophil EXCEPT:View Page
Appearance of a Band Neutrophil

A band has moderate to abundant cytoplasm staining pale blue to pink. Present within the cytoplasm are fine lilac to pink granules. These specific granules, called neutrophilic granules, are small and somewhat resemble pencil stippling.

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Appearance of the Segmented Neutrophil

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.

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Appearance of Cytoplasm

The color of the cytoplasm is light pink to colorless, but as with eosinophils, the specific basophilic granules are prominent.These granules are large and, because they take up the basic components of Wright-Giemsa stain, are purplish-black.

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Cytoplasmic Vacuoles

Frequently, cytoplasmic vacuoles are present. These vacuoles appear as unstained areas or "holes" in the cytoplasm.

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Large Lymphocytes

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.

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Identify the nucleated blood cell:View Page
Lymphocyte or Lymph

Lymphocytes are a heterogeneous group of cells that have different origins, lifespans and functions, and vary markedly in size. Some have a diameter of approximately 7µ, while others are as large as 18µ. The variations in size are mainly due to different amounts of cytoplasm. Therefore, the N:C ratio may range from 5:1 in some lymphocytes to 1:2 in others.

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Small Lymphocytes

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

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Which of the following cells is characterized by a thin rim of cytoplasm around the nucleus?View Page
Opaque vs. Transparent

The monocyte cytoplasm is opaque rather than transparent. One would be unable to read imaginary print placed behind the cell.

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Apprearance of Cytoplasm

Monos have abundant blue-gray cytoplasm containing many fine lilac granules. These give the cytoplasm a "ground glass" appearance. However, these granules may be difficult to see if the blood film is poorly stained.

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Differentiating Monocytes from Large Lymphocytes

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.

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Monocytes often posses blunt pseudopods, have soft spongy nucleus, frequently has vacuoles in the cytoplasm.View Page
Match the characteristics with the cell type.View Page
All of the following descriptions are characteristic of monocytes EXCEPT:View Page

Parasitology Question Bank - Review Mode (no CE)
Arrange the following Plasmodium morphologic forms in order from immature to mature:View Page
This suspicious form measures 12 µm by 7 µm and was found in a stool sample.View Page
Recovered in a stool sample, this suspicious form measures 7 µm.View Page
This parasite was found on a blood smear.View Page
These suspicious forms were seen on a blood smear.View Page
This suspicious form measures 15 µm and was recovered in stool. Which of the following conditions is/are associated with the presence of this form?View Page
The protozoa are classified and placed in groups based on which of the following criteria? View Page
Match each parasite pictured with its respective classification:View Page
Match the parasite pictured with its corresponding name & morphologic form(s):View Page
Match each picture with its respective morphologic form:View Page
Label the morphologic structures on this parasite form:View Page
A 27 year old female graduate student recently returned from South America, where she completed a nature study of the rain forest. She spent months "living off the land." The woman went to her physician seeking treatment for a sinus infection, which she thought was responsible for several recent bouts of diarrhea. Upon questioning the patient, the doctor decided to collect stool for culture and parasitic examination. The stool culture was reported as "no enteric pathogens isolated." This suspicious form was seen on both wet preparations and on permanent stain. It measures 17 µm. The identify of this form is most likely:View Page
A stool was received in the laboratory for parasitic examination on a 49 year old female who just returned from missionary work in numerous third world countries around the world. The patient had been suffering from mild diarrhea over the past two weeks. These two suspicious forms were seen. Form 1 measures a mere 6 µm whereas form 2 measures 35 µm. Label these two forms:View Page
Match each amebic cyst with its respective name:View Page
This suspicious form measures 18 µm and was seen in stool. Name that parasite!View Page

Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Conditions suggested by the macrocytes and the neutrophil in the photograph to the right include which of the following?View Page
Splenectomy Morphologies

Several erythrocyte abnormalities are present in both the upper and lower images. Many of these atypical cells are most likely present as a result of the patient's splenectomy.Considerable anisocytosis and poikilocytosis with many tear-drop cells, bite cells, fragmented forms, and a few target cells are apparent. Some of the erythrocytes in the upper image contain Howell-Jolly bodies (DNA fragments) that may be single or multiple, especially in myeloproliferative disorders. These inclusions stain negatively for iron and are eccentrically placed in the red cell cytoplasm.

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By utilizing a Prussian Blue stain, which of the following red blood cell inclusions would be identifiable if present? Note: Upper image = Wright-Giemsa stainLower image = Prussian Blue stainView Page

Semen Analysis
Abnormal Forms

There are a number of abnormalities of sperm morphology. Abnormal heads can include enlarged head, double head, round head, constricted head, amorphous head, pinhead, and acute tapering forms. There are also heads with abnormal numbers of vacuoles (>2 in the acrosomal region and/or vacuoles in the post-acrosomal region are abnormal). Midpiece abnormalities include distended and thin midpiece regions. Abnormal tails include short tails, double, triple or multiple tails, coiled tails, broken tails, or absent tail. Cytoplasmic droplets are also seen in some specimens. These are large regions of cytoplasm just below the head assumed to represent failure of complete sperm maturation or a sign of either toxicity or oxidation. There have also been reports that cytoplasmic droplets may be artifacts from the fixation and staining for morphology analysis.WHO 5th edition contains multiple examples of normal sperm and borderline/abnormal variations that cause a sperm to be classified as abnormal. It is an excellent resource.

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Evaluating Sperm Morphology

Smear evaluation technique Two slides should be prepared and at least 200 sperm should be counted per slide to minimize sampling error. A 100X oil immersion objective should be used (1000X magnification) to more clearly identify sperm detail, although it is NOT necessary to report all variations in head size and shape or various midpiece and principal piece defects.Use a systematic approach to prevent biased selection of sperm. Assess every intact sperm that is viewed within a field (having both a head and a tail) but is not overlapping other sperm. Systematically select areas of the slide to view.General morphology guidelinesNormal sperm morphology requires that the head (including the neck) and tail (including the midpiece and principal piece) of the sperm are normal.Head staining characteristics and morphologyThe heads of normal mature sperm are slightly oval in shape and measure approximately 4.0 to 5.5µm in length and 2.5-3.5µm at the widest part. The acrosomal region should comprise 40-70% of the head area. The acrosomal region stains pale blue and the post-acrosomal region stains dark blue with Papanicolaou, Shorr, and Diff-Quik stains. The acrosomal region of the head should contain no large vacuoles and not more than two small vacuoles. Tail staining characteristics and morphology Sperm have a long tail which is slightly thicker near the head than at the end. Sperm tails measure about 50µm. A normal midpiece is slender, regular, and about the same length as the sperm head; it does not bulge out or contain excess cytoplasm. The midpiece may show some red staining.The principal piece has a uniform thickness, which is thinner than the midpiece. It can be looped so that it is bent back on itself, although a sharp angle would be abnormal as would coiled tails (>360°). An example of a normal sperm is represented on the right.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Squamous Epithelial 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 (upper image) and a squamous epithelial cell under high power. The sediment in the lower image was stained with a supravital stain. The cells 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.

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Squamous Cells Under High Power

Under high power magnification the cytoplasm appears granular and a small round nucleus may be apparent.

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Squamous Cells Under Phase Contrast

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.

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Sternheimer-Malbin Stain

The Sternheimer-Malbin (SM) stain is a commonly used supravital stain containing crystal-violet and safranin. WBC's, epithelial cells, and casts stain well with SM stain. Sedi-Strain (Clay Adams, Sparks, MD) and Kova stain (ICL Scientific) are among those commercially available. Nuclei and cytoplasm of various cells can be stained with a 0.5% solution of toluidine blue.

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Theoretical and Practical Aspects of Routine H&E Staining
Eosin as a Counterstain

Eosin is a rose-colored fluorescent dye that is created through the action of bromine on fluorescein. Although it is not the only dye used to stain the cytoplasm of cells, it is the most common. It can be used in either an aqueous or alcoholic solution. The strength of the solution varies, but generally a 0.5% to 2% solution is used. Some advantages of using alcohol-based eosin formulations over aqueous are that they: React in a more stable manner chemically. Minimize, if not eliminate entirely, the unpredictable effects of various impurities such as water-soluble salts that, in water, may interfere with dye uptake. Tend to stain more slowly than water-based formulations, resulting in a wider range of shades.

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Eosin Differentiation

Differentiation of the eosin staining occurs in the subsequent water wash and continues as the tissue goes through the dehydration alcohols. The desired result should yield the following three shades of pink: Orange-pink: Red blood cells Light pink: Connective tissue Dark pink: Muscle fibersThe cytoplasm of the cells will contain varying shades of pink.

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Other than the cytoplasm of cells, what other tissue constituents are stained with eosin? (Choose all that apply.)View Page
Breast

These two images show breast tissue.Numerous nuclei line the ductal formations formed by this cancer.The interlacing fibrous tissue between the ducts is staining varying shades of pink with the eosin. The large white empty spaces are adipose tissue. There are a few clusters of red blood cells on the periphery.The second image shows the ducts at a higher magnification; the nuclear detail is well demonstrated as is the pale cytoplasm of the neoplasm.

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Three Broad Categories of Biological Stains

There are three broad categories of biological stains: General or Routine Stains: Used to differentiate between the nucleus and the cytoplasm. This allows for the different tissue types to be distinguished from one another. These stains use one, two, or sometimes three dyes. The H&E stain falls into this category. Special Stains: These are used to demonstrate specific elements within the tissue, such as connective tissue, muscle, carbohydrates, lipids, pigments, and nerve tissue. Heavy Metal Impregnation: Involves the deposit of metallic substances onto targeted cells. This is often used to demonstrate nerve cells and certain microorganisms.

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Which one of the following categories of stains is used to differentiate between a cell's nucleus and cytoplasm?View Page
Which category of stain would you employ if asked to demonstrate muscle in a tissue sample ?View Page
Chemistry of Dyes

The chemical structure determines the color of dyes. More specifically, it is the atomic groupings, or chromophores that are the determining factors. The more chromophores present, the deeper the color. The chromophores can be acidic or basic. Generally speaking, stain is taken up by tissues due to dye-tissue or reagent-tissue affinities. Affinity is used to describe the attractive forces binding the dye to the tissue components. This is called a chemical bond which is formed through an electrostatic attraction between two oppositely charged ions.AcidsAn acidic dye is the salt of a color acid and has the affinity for basic tissue components such as the cytoplasm of all cells.BasesA basic dye is the salt of a color base and has the affinity for acidic tissue components such as the nucleic acids in the nucleus of all cells.

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Variations in White Cell Morphology -- Granulocytes
A patient has a WBC count of 4.0 x 109/L. Giant platelets, such as the one indicated by the red arrow in the image, are observed on the Wright stained peripheral smear. Blue-staining inclusions are also observed in several of the neutrophils. What is the identity of this blue-staining inclusion indicated by the black arrow in the image?View Page
Auer rods are seen in the cytoplasm of blasts from which cell line?View Page
Abnormal granulation can be seen in the cytoplasm of leukocytes in which of the following conditions?View Page
Hypersegmentation

A normal mature neutrophil is 10-15 µm 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.

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Degenerate Neutrophils in EDTA blood

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.

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Döhle Bodies

Döhle bodies are small, blue, irregular-staining patches of cytoplasm seen near the edge of the cell in granulocytes in certain conditions. Electron microscopic examination reveals that they are composed of aggregates of rough endoplasmic reticulum remaining from an earlier stage of development. The Döhle 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.

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Unusually Darkly Staining Döhle Bodies

Unusually dark-staining Döhle bodies are seen in the cytoplasm of this cell, along with bacteria, an infrequent finding. The bacteria are indicated by the red arrows and stain almost black. The Döhle bodies are patches of dark-blue stained material, indicated by the blue arrows.

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May-Hegglin Anomaly

This blood smear was taken from a patient with the May-Hegglin anomaly. A May-Hegglin Dohle body is indicated by the arrow near the edge of the cytoplasm at the top of the neutrophil. In addition, notice the giant platelet that is indicated by the red arrow, another characteristic of May-Hegglin anomaly.

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Auer Rod

An Auer rod is indicated by the arrow in this blast cell.

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Cytoplasmic Vacuolation

Vacuoles are areas of the cytoplasm which do not stain with Wright's stain and appear as holes in the cytoplasm. Their composition may vary; some will contain remnants of bacterial digestion, autodigestion in an aging cell, while others may contain fat. It is not possible to differentiate the various types of vacuoles on Wright stained smears using light microscopy. Vacuoles may be seen occasionally in an aging granulocyte (degenerative vacuolation), but are seen more frequently and are significant in cases of bacterial infection and septicemia.

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Smaller Vacuoles

The vacuoles seen in the cytoplasm of this cell are somewhat smaller and several are located near the lower edge of the cytoplasm.

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Inherited Anomalies

Several rare inherited anomalies show atypical granulation in the cytoplasm of peripheral blood cells.

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Which of the following inclusions may be seen in the cytoplasm of myeloblasts? View Page
Auer rods are significant when they are seen in the cytoplasm of blast cells because they are diagnostic for:View Page
Chediak-Higashi anomaly is characterized by which of the following? View Page
Auer Rods

Auer rods are red staining, needle-like bodies seen in the cytoplasm of myeloblasts, and/or progranulocytes in certain leukemias. Auer rods (see arrow in image) are cytoplasmic inclusions which result from an abnormal fusion of the primary (azurophilic) granules. Single or multiple Auer rods may be seen in the cytoplasm of a cell. If more than one is present, they are frequently close together and may even be overlapping. Their identification is very important because, if found, they can confirm the presence of myeloblasts indicating the presence of a non-lymphocytic (myeloid) leukemia. They can also be seen in myeloid blast crisis in chronic granulocytic leukemia. Auer rods are never seen in lymphoblasts. This differentiation is important because the treatment of lymphoblastic and myeloblastic leukemia are different. Auer Rods are always classified as pathological.

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Auer Rods, continued

Two Auer rods can be seen in the cytoplasm of this cell.

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Döhle Bodies, continued

Döhle bodies are seen in a number of conditions, including:infections burns measles leukemia chemotherapyDöhle bodies are only present when the body is responding to unusually severe stress or stimulus. This severe stress may cause the cytoplasm of some cells to mature improperly. Their presence does not aid in the diagnosis of the disorders in which they are found, but they are frequently seen along with toxic granulation and/or vacuoles in cases of infection or burns.

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More on May-Hegglin

May-Hegglin bodies stain blue and appear to have a more definite shape than Dohle bodies. When examined under electron microscopy, they appear to be aggregates of thread-like structures in a crystal-like arrangement. May-Hegglin inclusions are RNA material believed to be derived from the endoplasmic reticulum. May-Hegglin bodies can be seen in monocytes and platelets as well as in all mature granulocytes. The platelets in May-Hegglin anomaly are very bizarre in appearance and thrombocytopenia is usually noted. When examining a slide that has bizarre platelets and blue-appearing bodies in the cytoplasm, thought should be given to the possibility of the May-Hegglin anomaly.

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One Auer Rod

One Auer rod can be seen in the cytoplasm of this cell at approximately 11 o'clock. Auer rods are easily missed. When many blasts are present on a slide, you should look for them carefully.

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Toxic Granulation

Toxic granulation is manifested by the presence of large granules in the cytoplasm of segmented and band neutrophils in the peripheral blood. The color of these granules can range from dark purplish blue to an almost red appearance. Toxic granules are actually azurophilic granules, normally present in early myeloid forms, but are not normally seen at the band and segmented stages of neutrophil maturation. These granules contain peroxidases and hydrolases. Toxic granulation is seen in cases of severe infection, as a result of denatured proteins in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is the most frequent cause of toxic granulation. This phenomenon may be seen in cells which also contain Döhle bodies and/or vacuoles. Cells containing toxic granules may have decreased numbers of specific granules. Note: Cells containing only a few specific granules, with or without toxic granules, are said to be degranulated. The nucleus in degranulated cells may often be round-bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will disintegrate soon. Increased basophilia of azurophilic granules simulating toxic granules may occur in normal cells with prolonged staining time or decreased pH of the stain. The blue arrow in the image points to a neutrophil with toxic granulation. Döhle bodies are also present in the cell, indicated by the red arrows.

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Dohle Body

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.

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Cytoplasmic Vacuolation

There are two large vacuoles (unstained areas in the cytoplasm) present in this cell.

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Band Neutrophil Showing Toxic Granulation

This image shows a band neutrophil with toxic granulation. The granules scattered throughout the cytoplasm are larger, more numerous and darker than those of normal neutrophils.

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Chediak-Higashi Anomaly

Chediak-Higashi anomaly is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules. These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes. These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections. An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.

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Alder Anomaly (Alder-Reilly Anomaly )

Alder anomaly is a rare autosomal recessive 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 inclusions seen in all types of mature white blood cells, and sometimes in earlier cells. 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.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Megakaryocyte in Bone Marrow

The large cell illustrated in this photograph of a Wright/Giemsa-stained bone marrow smear is a megakaryocyte. This megakaryocyte appears mature. The nucleus has at least 8 lobes and the nuclear chromatin is coarse and distinct. Clusters of young platelets are being released from distinct platelet territories at the periphery of the cytoplasm (blue arrows). When mature, each megakaryocyte produces approximately 4000 platelets/day. Production can expand by 8-fold during times of increased demand and under the stimulus of thrombopoietin.

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The neutrophils illustrated in this image are representative of those seen in a female patient's peripheral blood smear. The total WBC was 28.5 X 109/L (reference interval = 4.0 - 11.0). Which of the following BEST describes the condition associated with this WBC count and the white blood cells that are present in the image?View Page
Toxic granulation noted in the neutrophils' cytoplasm reflects an increase in activity of which of the following?(Choose all that apply)View Page
The inclusions noted in the cytoplasm of this white blood cell are most suggestive of which of these conditions?View Page
Alder Anomaly

Alder anomaly is characterized by large azurophilic granules that stain dark-purple and are seen throughout the leukocyte cytoplasm, even covering the nucleus. The inclusions (granules) are seen in the cytoplasm of almost all mature leukocytes i.e., granulocytes, lymphocytes, and monocytes. This distinguishes Alder anomaly inclusions from toxic granulation, which is only observed in neutrophils. Another feature that distinguishes Alder anomaly from toxic changes is the lack of cytoplasmic vacuoles of toxic origin in the neutrophils of Alder anomaly.The background condition in Alder anomaly is mucopolysaccharidosis, collectively, a group of inherited disorders where a deficiency of lysosomal enzymes are lacking that are needed to degrade mucopolysaccharides. The inclusions observed in the leukocytes represent partially degraded mucopolysaccharides within lysosomes. Accompanying conditions are hepatosplenomegaly, corneal opacities, and mental retardation.

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WBC inclusions: Summary

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings. Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Dohle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered. Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower image). Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

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The pale-staining cytoplasmic bodies marked by the arrow in the image may be seen in each of the following conditions except:View Page
May-Hegglin Anomaly

May-Hegglin anomaly is an inherited dominant condition in which large (2 - 5 um) basophilic inclusions, resembling Döhle bodies, are present in granulocytes, including neutrophils, eosinophils, basophils, and monocytes. The inclusions are caused by accumulation of free ribosomes. A May-Hegglin body is indicated by the black arrow in the image on the right. Note that this inclusion is well-defined and there is no evidence of toxic granulation in the cytoplasm. When Döhle-like bodies are identified, May-Hegglin anomaly should be considered in the differential diagnosis, even though this entity is rare. Giant platelets containing few fine granules are also characteristic of May-Hegglin anomaly. The red arrow in the image on the right points to a giant platelet, observed in the same field as a neutrophil containing a May-Hegglin body. Sometimes the platelets have bizarre shapes and variable sizes. Variable degrees of thrombocytopenia complicated by mild bleeding problems and purpura may accompany the aberrant platelets.

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Case History

A 17-year-old female was admitted to the hospital with abdominal pain and a tentative diagnosis of appendicitis. The total white blood count was 14.5 X 109/L with a left shift and neutrophils with changes tagged by the arrow in the image (see blue arrow). The bluish-staining, blurred accumulations in the cytoplasm (Döhle bodies), are located at the cell periphery in neutrophils with toxic changes.Döhle bodies are remnants of endocytoplasmic reticulum and are products of cytokine activity in the induction and shortened activity of neutrophil activation. They are often present in conditions with increased neutrophil lysosomal activity, manifest as toxic granulation.In this case, the presence of Döhle bodies serves as markers for infection-induced leukocytosis and supports the diagnosis of acute appendicitis.

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Eosinophil description

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. The eosinophil's 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.

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Basophils

A basophil and a small lymphocyte are compared in the same field in the upper image, while a single basophil is shown in the lower image.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 image). 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 mast cells (tissue basophils), 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 granulocytes 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. Basophils may be increased in:Myeloproliferative disordersChronic metabolic conditions Myxedema Diabetes mellitusHypersensitivity responses Tuberculosis

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The smudge cells pictured in this image may be found in each of the following situations except:View Page
Hairy cell leukemia

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Case History Two

An 80-year-old man was seen in the emergency room with sudden onset of right-side chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath. His temperature was 101.2°F. A chest X-ray revealed right middle lobe pneumonia. A complete blood count (CBC) was ordered. The results were as follows:CBC ParameterPatient ResultReference IntervalWBC33.0 x 109/L4.0 - 11.0 x 109/LRBC4.5 x 1012/L4.5 - 5.9 x 1012/LHemoglobin15.2 g/dL13.5 - 17.5 g/dLHematocrit44%41 - 53%Platelet200 x 109/L150 - 450 x 109/LSegmented neutrophil6540 - 80%Band neutrophil100 - 5%Lymphocyte 525 - 35%Eosinophil 30 - 5%Basophil 20 - 2%Monocyte252 - 10%A peripheral smear was reviewed based on the elevated WBC and increased monocyte count. A representative field from the Wright-Giemsa stained smear (1000X magnification) is shown on the right. The cells indicated by the blue arrows are atypical monocytes. They have abundant cytoplasm that is more blue than the typical gray-blue cytoplasm of normal monoctes. A few scattered vacuoles are also present. The atypical monocytes, in company with toxic neutrophils (indicated by the red arrow), appeared to be a response to infection. The patient had a past history of tuberculosis, which may account for the monocytosis.

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