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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.)

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.

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.

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.

The image to the right is a higher power field view of an ependymal clump. Notice how the nuclei are all nearly identical in size and have strikingly similar staining characteristics. Despite the fact that this is actually a clump of cells, there is actually little, if any, indication of individual cells. This multinucleated giant cell appearance is characteristic of ependymal clumps.

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 monocytes clefting.

Mesothelial cells are frequently found in clusters and clumps. In Image 1 below, the individual cells in this clump still maintain the round nuclus 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.

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.

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.

One of the normal roles of the marrow macrophage is to remove cellular debris. In a normal bone marrow, this includes the engulfment of extruded RBC nuclei and old, non-nucleated RBCs at the end of their lifespan. In some patients, macrophages lose their ability to distinguish self from non-self (i.e., invader/ pathogen) and good cells from old/senescent cells. This can happen because of an inborn error in the macrophages or by an infection-mediated transformation. When this change occurs, any cell in the vicinity of a defective macrophage become a target for engulfment. This term is called hemophagocytosis.The top image on the right shows two macrophages that have ingested several different cell types (see red arrows). There is the normally ingested non-nucleated RBC, but also the abnormally ingested segmented neutrophil and at least one early nucleated RBC precursor. Viable bone marrow precursors are not the usual diet of macrophages.The lower image on the right shows an even more impressive macrophage with at least a dozen or more ingested RBCs as well as three segmented neutrophils and a lymphocyte.

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.

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.

Four blast cells are seen in this field. Notice the smooth chromatin pattern, nucleoli, high NC ratio and irregularly shaped nuclei. These blasts were observed in a spinal fluid sample from a patient with acute lymphocytic leukemia.

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.

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.

Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides. Fixative: 10% Neutral Buffered FormalinStepReagentTimeTechnical Notes1Weigert's Iron Hematoxylin Working Solution½ Solution A (Hematoxylin and 95% Alcohol) and ½ Solution B (Distilled Water, Hydrochloric Acid and 29% Ferric Chloride Solution)7 MinutesBe careful not to overstain with Hematoxylin as this can obscure the carminophilic tissue elements.If Gills hematoxylin is used in place of Weigert's Iron Hematoxylin the mucin may have a bluish cast.2Running Tap Water10 Minutes3Working Mucicarmine Solution10mL Mucicarmine Stock Solution and 40mL Distilled Water60 MinutesStock Mucicarmine should be stored refrigerated to slow down deterioration of the solution. Expiration of the solution should be closely monitored. Do not freeze solution to avoid frequent freeze-thaw cycles that may cause deterioration.4Distilled Water1-2 Changes5Metanil Yellow0.25% Solution30 Seconds to 1 MinuteBe careful no to overstain with Metanil Yellow as this can obscure carminophilic tissue elements. Tartrazine can be used in place of Metanil Yellow. Expected ResultsMucin = Deep Rose Red Capsule of Cryptococcus = Deep Rose Red Nuclei = Black Other tissue elements = Blue or yellowPost Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped. Mucicarmine stock solution should be stored refrigerated Room temperature storage will accelerate the chemical breakdown of the solution. Frozen storage is not recommended to avoid repeated freeze-thaw cycles that may also affect the chemical reactivity of the stain.

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.

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.

Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slidesPreferred fixative: 10% neutral buffered formalin (NBF)Control: Artery or skinReagentTimeTechnical NotesVerhoeff (iron) hematoxylin30 minutesMake fresh; save solution until staining is complete.The ferric chloride in this particular hematoxylin is required as a mordant. Running water washUntil solution drains clear2% ferric chloride solutionDifferentiate until black fibers are well defined and background is grayReview slides microscopically to ensure differentiation is complete.If differentiation is not complete, return to solution for 30 seconds and repeat.Running water washUntil solution drains clear5% hypo (sodium thiosulfate)1 minute Removes iodineRunning water wash1 minuteVan Gieson's solution5 minutesCounterstainPost staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols followed by two changes of xylene and then coverslip.Expected results:Elastic fibers and nuclei - BlackCollagen - RedOther tissue elements - Yellow

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.

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.

The mononuclear leukocytes consist of two cell types: lymphocytes and monocytes. In contrast to the granulocytes, these cells have rounded nuclei, some with indentations or folds. Granules are not prominent.

As time progresses, the band nucleus gradually develops constrictions, resulting in the formation of nuclear lobes. In this way, the band neutrophil matures into a segmented neutrophil. Notice, in the image to the right, that the cell in the upper right-hand corner has segmented nuclei. This cell is a segmented neutrophil. The cell in the center of the image is a band neutrophil. This band has not yet matured enough for its nucleus to segment. However, bands to eventually mature into segmented neutrophils with time.

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.

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.

The granulocytes found in normal peripheral blood are neutrophils, eosinophils and basophils.Most have segmented nuclei, and are therefore classified as being at the "segmented" stage of development. Some that are a little less mature have unsegmented nuclei. These are classified as "bands." Generally, we differentiate between the band and segmented forms of neutrophils, but since eosinophils and basophils are present in such low numbers, and since their nuclei are often obscured by cytoplasmic granules, we usually don't concern ourselves with designating the band forms.Since hematologists and textbooks use several different terms for these cells, synonyms for each term will be given and then may be used interchangeably throughout the course.

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

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.

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.

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.

Steps for Regressive H&E Staining:1. Hydrate slides2. Overstain in hematoxylin3. Tap water rinse4. Differentiate5. Tap water rinse6. Blue7. Running tap water rinse8. Eosin counterstain9. Dehydrate10. Clear11. CoverslipSteps for Progressive H&E Staining:1. Hydrate slides2. Stain in hematoxylin3. Tap water wash for 15 minutes (helps to blue nuclei)4. Proceed with steps 8 - 11

TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs. The minimal infectious inoculum may be as low as one viable organism.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body; the individual has no symptoms and is noninfectious.Most persons infected with M. tuberculosis do not experience clinical illness and are noninfectious. About 5-10% of persons who are infected and are not treated will develop active TB during their lifetime. The risk for progression is highest during the first several years after infection.Most often, M. tuberculosis infects the lungs. However, it can infect almost any organ in the body, including bones and joints. Tuberculosis meningitis is a TB infection that occurs outside the lungs with devastating consequences, most often in young children and patients with AIDS.

A normal bone marrow smear stained with Wright/Giemsa stain is captured in this photograph.Note the normal maturation sequence beginning with myelocytes (the two large cells in the left upper corner)through metamyelocytes, band neutrophils,and multi-lobed segmented neutrophils.The small cells with darkly staining, centrally placed nuclei are normoblasts (three are clustered in the left lower field).Absent in this field are eosinophils, basophils and megakaryocytes.A normal M:E ratio of 2.4:1 is calculated from the twelve myeloid cells and five normoblasts. Two lymphocytes are identified, one left center, the other left upper.

Pelger-Huet anomaly is a congenitally acquired condition of nuclear segmentation that has no clinical significance. There is no loss of cellular function.The condition can be suspected if typical bilobed, "pince-nez" nuclei are observed (section A in the composite image). Band neutrophils usually have two distinct lobes, connected by a relatively short but thick bridge as illustrated in sections B and D. Monolobulated cells may also be encountered, especially if a homozygous inheritance is present, as illustrated in section C. If 70% or more of the segmented neutrophils on the differential possess these nuclear morphologies, the possibility of a homozygous Pelger-Huet should be considered.