Nucleoli Information and Courses from MediaLab, Inc.

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.

This is an example of a Nucleolar ANA pattern.This pattern is characterized by staining in the nucleoli of the interphase cells (a). The nucleolar staining can display subtle variations in staining inside the nucleoli including smooth, speckled and clumpy. All are reported as ANA positive, Nucleolar. In this sample staining is present in the chromosomal area of the metaphase mitotic cells (b) along with some staining in the area outside of the chromosomal area. The staining of the mitotics can be different with different anti-nucleolar antibodies. Remember the ANA pattern is determined by staining in the interphase cells and the mitotics are used to assist in interpretation.Follow-up testing for anti-nucleolar antibodies is very limited and in most cases is not done. There are assays for identification of anti-RNP polymerase antibodies. Anti-nucleolar antibodies are primarily seen in patients with systemic sclerosis.This pattern is reported as ANA positive, Nucleolar; titering is necessary.Nucleolar:Interphase cells Staining only of the nucleoli Clumpy Smooth Speckled Metaphase mitotic cells Can demonstrate variable staining depending on the antigen involved

This is an example of an unusual ANA pattern called nuclear membrane.This pattern is characterized by smooth staining in the nucleus of the interphase cells (a). The nucleoli may or may not stain. Notice however the chromosomal area of the metaphase mitotic cells is negative (b). Upon closer examination also notice the staining of folds in the nuclear membrane (c).Follow-up testing is not required for this pattern. Possible antigens include nuclear lamins A, B or C, nuclear pore complex and gp210. These antibodies are generally seen in autoimmune liver diseases.This pattern is reported as ANA positive, Nuclear Membrane; titering is necessary.Unusual Homogeneous - Nuclear Membrane (Envelope):Interphase cells Smooth staining of entire nucleus Brighter staining at the periphery of the nucleus May notice wrinkles in nuclear envelopeMetaphase mitotic cells No staining in the chromosome region Region outside of the chromosome area will stain with variable intensity

This is an example of a homogeneous ANA pattern.This pattern is characterized by smooth staining in the nucleus of the interphase cells (a). The nucleoli may or may not stain. Notice the smooth staining in the chromosomal area of the metaphase mitotic cells (b).Follow-up testing for anti- dsDNA, histone or chromatin antibodies is recommended. These antibodies are primarily seen in patients with systemic lupus erythematosus (SLE).This pattern is reported as ANA positive, Homogeneous; titering is necessary.Homogeneous:Interphase cells Smooth staining of entire nucleus Nucleoli may or may not stainMetaphase mitotic cells Smooth staining of the chromosomal region No staining outside of the chromosomal area

This is an example of a Speckled ANA pattern called Nuclear Matrix.This pattern is characterized by coarse granular speckled staining in the nucleus of the interphase cells (a). The nucleoli do not stain and the speckles tend to outline the nucleoli. Notice the absence of staining in the chromosomal area of the metaphase mitotic cells (b) and some speckling in the area outside of the chromosomal area.Follow-up testing to rule-out antibodies to the extractable nuclear antigens (ENA's) such as Sm or U1-RNP is recommended. Nuclear Matrix antibodies are not specific for a single SARD.This pattern is reported as ANA positive, Speckled; titering is necessary.Nuclear Matrix:Interphase cells Coarse speckles outline nucleoliMetaphase mitotic cells No staining in the chromosome region Region outside of the chromosome area will stain with variable intensity

In this sample we see the Speckled ANA pattern, granular/speckled staining in the nucleus of the interphase cells, nucleoli do not stain, the absence of staining in the chromosomal area of the metaphase mitotic cells, and some speckling in the area outside of the chromosomal area (a), along with the hyperexpressing cells demonstrating the SSA/Ro pattern (b).In cases of mixed speckled and SSA/Ro follow-up testing for anti-ENA antibodies is necessary.This pattern is reported as ANA positive, Speckled and SSA/Ro pattern, anti-SSA/Ro antibodies present; titering is necessary.Speckled and SSA/Ro:Interphase cells Speckled staining of the entire nucleus (speckled) Hyperexpressing SS-A/Ro pattern in 10-15% Non-hyperexpressing interphase cellsMetaphase mitotic cells No staining in the chromosomal region Region outside of the chromosome area will stain with variable intensity

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.

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.

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.

Most technologists working in the clinical hematology setting are familiar with the morphology of blood cells found in peripheral blood smears. Many of the same blood cells found in the peripheral blood are also found on cytospin preparations of body fluids. While the morphologies are similar between the two sources, there are changes to the "comfortable/familiar" peripheral blood morphology that are introduced by the cytospin technique. It is paramount to fully conceptualize the appearance of both normal and abnormal blood cell morphologies in body fluids in order to accurately perform fluid differentials.The cytopsin process works by wicking fluid into a filter while fluids samples are spun into a central column and deposited in a mono-layer onto a defined area of a slide. This allows the cells to be concentrated for appropriate identification. This technique can cause pronounced changes to morphology and staining characteristics, as well as cellular destruction if the cytospin malfunctions.The cytospin technique is known to stretch and distort cellular and nuclear morphology and allow nucleoli to appear more prominent than what one would normally see in peripheral smears. Cytospinning, however, does not change nuclear:cytoplasmic ratios nor does it alter relative chromatin textures or clumping patterns. Though this technique can make cells appear larger that on the peripheral blood smear, it can not change cytoplasmic textures and granulation. Focusing on these steadfast features can make cytospin morphology less intimidating.

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.

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 chromatoin and a slightly more promanent nucleoli. The cytoplasm is also more basophilic, and the vacuoles are atypical. They are not the typical round vacuoles seen in macrophages/histocytes; 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.

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.

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.

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.

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.

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.

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

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

The 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.