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Cytospin Information and Courses from MediaLab, Inc.

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

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Body Fluid Differential Tutorial
Infectious Organisms in Body Fluids

Under normal circumstances, fluids collected from any enclosed body space such as a cerebrospinal fluid (CSF), pleural, peritoneal, pericardial or synovial fluids, should be sterile. When an infection occurs within a body cavity, the fluid that is collected from that site for diagnostic purposes will potentially have infectious organisms present on the cytospin. There can be several types of organisms demonstrated on the Wright stained preparation.Bacteria and fungus are the most common but it is possible to demonstrate the presence of protozoan parasites as well.Spirochetes and acid fast bacilli (AFB) such as mycobacteria will not stain with Wright stain so they will not be detected even if they are present.Since bronchial alveolar lavages, or BALs, are collected through an open airway it is normal to observe respiratory flora, however yeast and hyphae are never normal in a BAL.It is standard practice before reporting the presence of bacteria in a fluid to correlate/confirm the findings with the microbiology lab.

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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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Retinoblastoma in Cerebrospinal Fluid

Retinoblastoma is a tumor arising from retinal tissues of the eye, which can be inherited or spontaneous; the inherited form is associated with a mutation in the Rb1 gene. Retinoblastoma is known to spread into the optic nerve and CSF. This photo shows a retinoblastoma tumor clump. Notice the smaller size of the tumor cells. They are similar in appearance to Acute Lymphocytic Leukemia (ALL), L1 lymphoblasts. Also, it is important to note the clustering of these cells which form a three dimensional aggregate on the cytospin. While a L1 ALL might have similar nuclear:cytoplasmic proportions with similar chromatin and cytoplasmic characteristics, L1 ALL will typically show a liquid distribution pattern on the cytospin with a more even distribution and without clumping.

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Metastatic Melanoma in the Cerebrospinal Fluid

Melanoma is the most deadly form of skin cancer and is the third most common form of cancer to spread to the brain.In patients with metastatic melanoma, approximately 30% will experience involvement of the brain and cerebrospinal fluid. Once melanoma has spread to the brain it is no longer curable.This photo shows a cerebrospinal fluid cytospin from a teenage patient who presented with headaches, mental status changes and new onset of seizures. There had been no prior recognition of a melanoma and there was no evidence of such on the physical exam. The cerebrospinal fluid showed a few cells with the "classic" look of melanoma - a "shaggy" mesothelial cell with blue-black melanine inclusions.This is very typical of metastatic melanoma. Sometimes the melanin granules can be so sparse that they are difficult to find.

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Medulloblastoma in Cerebrospinal Fluid

Medulloblastoma is a malignant brain tumor that originates in the cerebellum. It is included in the family of primitive neuroectodermal tumors, or PNETs. It is a highly invasive brain tumor which is known to spread through the brain and cerebrospinal fluid.Patients with meduloblastoma will typically have spinal taps performed after resection to evaluate for the presence of metastasis. These samples are then evaluated in the laboratory.This cytospin shows multiple large bizarre tumor clumps. Note that there are also several tumor cells that appear to have ingested other tumor cells (see arrows).

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Primitive Neuroectodermal Tumor (PNET) in Cerebrospinal Fluid

Primitive Neuroectodermal Tumor, or PNET, is a soft tissue tumor that arises from primitive neural crest tissue. It shares the same cytogenetic translocation as Ewings sarcoma; a tumor of the bone. When present in cerebrospinal fluid cytospins, PNET cells tend to occur as singlets or doublets rather than large tumor clumps.Notice how these two cells are "stuck" to each other. In a cytospin with this low number of cells, it would be unusual for two cells to be touching each other unless they were actually fused together- this is an important and distinctive feature of this condition.

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Peripheral Morphology vs Cytospin Morphology

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.

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Bone Marrow Contamination in Cerebrospinal Fluid

When a spinal tap is performed correctly, the cell types found in the cytospin present a "snapshot" of the cerebrospinal fluid cell content. When there is difficulty obtaining a clean tap, the cell types present will reflect the type of fluids that are co-mingled with the cerebrospinal fluid. In traumatic taps, the cell types and proportions of red and white blood cells present will mirror that found in the patient's peripheral blood, as there has been a contamination of peripheral blood (from the trauma) mixed into the cerebrospinal fluid during the sample collection.In patients with softer bones (infants and osteopenic patients), it is not uncommon for the contamination to come, not from peripheral bleeding, but from bone marrow found inside the softened spinous process that was penetrated inadvertently during the procedure. When this occurs, any or all of cell types found within the bone marrow can be present in the cytospin.In the image shown here there are early nucleated RBC and WBC precursors, typical of contamination with bone marrow.

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Normal Lymphocytes

This image demonstrates the normal range of variation for lymphocytes on a cytospin. The clover-leafing of the nuclei is a cytospin effect and is normal as long as the chromatin is mature, the nuclear:cytoplasmic ratio is normal, and overall size is normal. Notice how the larger cells seem flattened and a bit more open in appearance than they would be on a peripheral smear.

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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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Neutrophils

Neutrophils in a cytospin will look the same as in peripheral blood. The cytospin technique will accentuate the segmentation in the nucleus. Toxic vacuolation and toxic granulation will be the same as on a peripheral smear.

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Neutrophils continued

While the segmented neutrophils are easy to identify in this smear, the large number of cells make it difficult to evaluate this smear for the presence of bacteria or other infectious agents. Remaking the smear with a smaller sample volume or using a larger dilution will make a cytospin that is easier to evaluate.

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The cytospin technique perfectly preserves the morphology of blood cells in a fluid sample.View Page
Acute Monoblastic Leukemia (AMoL)

This cytospin is from a case of acute monoblastic leukemia (AMoL)in a patient with central nervous system involvement.Notice how much larger these blast are compared to the previous patients.The morphology in this smear is consistent with the classification of M5a. M5a stands for "monoblastic without maturation", which is appropriate in this case since there are no promonocytes or monocytes present. When a patient has an established diagnosis of M5b - "monoblastic with maturation", abnormal promonocytes would be evident.It is important to note that each individual patient's morphology will vary depending on the morphology of their original leukemia. If the patient relapses, the morphology observed in both the cerebrospinal fluid and peripheral smears may become more extreme, since therapy selects for the most abnormal and resistant clones.

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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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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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Identification of tuberculosis can be performed by examination of Wright stained cytospin slides of infected body fluids.View Page
Candida albicans - Germ Tube Formation

Candida albicans is known to be a germ tube producer. The elongated, but not quite hyphal, structures that are present in this fungal group are consistent with germ tubes (see arrows).While it is possible to speculate about fungal identity from the cytospin preparation, this is truly the microbiology laboratory's role. This cytospin would be reported as "positive" for yeast and after correlating results with the microbiology laboratory, and according to your laboratory's protocols.

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Hyphae

The white cell clump in this image contains budding yeast and matted branching hyphae contained within it.If you look away from the clump, the yeast and hyphae are not obvious. This is why it is important to always take a close look at any white cell clumps that are present on a cytospin. It is good practice to check fluid differential cell clumps for the presence of infectious organisms.

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Septic Arthritis

This photo is from a patient on chemotherapy who developed septic arthritis due to Haemophilus influenza infection.Notice the extracellular rods and the very rare intracellular rods present in this smear (see arrows). The difficulty in observing these organisms shows the importance of not only a good stain but a well distributed cytospin. Joint fluids are more viscous than other fluids and making a good smear can be difficult. Sometimes making the smear from a saline dilution of the fluid works better.

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Intracellular Diplococci

When intracellular bacteria are present in a body fluid cytospin, they classically appear within neutrophils (see arrows). When the bacterial burden is high, they may also be found in monocytes/histiocytes. It is important to note that monocytes are the less effective phagocytes of these two cell types, so they will never contain intracellular organisms if the neutrophils do not do so as well. If you think you are seeing bacteria in a monocyte/macrophage, and the neutrophils do not display intracellular bacteria, you may actually be observing granules of hemosiderin within the macrophages. Hemosiderin usually stains more green-blue or black than bacteria, and the granules are less regular in size than ingested bacteria. Comparing the Wright stain to the Gram stain, and performing an iron stain, will help with this distinction.

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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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Cerebrospinal Fluid (retired 7/17/2012)
Stained Cytospin Preparations of CSF

All white cells present in a cerebrospinal fluid must be identified. If more than 10 cells/mm3 are present or there is difficulty identifying the few cells that are present, make a cytospin, a filtration, or a sedimentation preparation, stain with Wright-Giemsa, and perform differential count. Cytospins made with a cytocentrifuge are preferred since they are easiest to make and interpret, but filtration and sedimentation methods can also be used to prepare a slide for subsequent staining.

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Cytospin Technique

The cytospin technique uses a high speed centrifuge to concentrate the cells on a slide in a uniform monolayer 6 mm in diameter. The monolayer distribution enhances the morphological appearance of the cells present.Allow the slides to dry in air for several minutes and then stain them with Wright-Giemsa stain. Cytospin slides may be placed in an automatic stainer or stained manually.Perform a 100 or 200 cell differential and record the number of neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, and blasts cells.Pathologists must review any slide which has tumor cells, unidentified cells, or immature stages of cells, such as blasts.Since criteria for review may vary from one laboratory to another, be sure to check the requirements in your laboratory before reporting the differential.

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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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Mature Peripheral Blood Cells

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Up to 2% neutrophils is also considered within normal limits when a cytospin smear is used for the differential. In children, normal CSF cells are 70% monocytes, up to 20% lymphocytes and up to 4% neutrophils. When any of these normal cell abundances are increased, the term pleocytosis is used. Neutrophil pleocytosis is an increase in neutrophils and usually indicates the presence of a bacterial infection.

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True or false: cytospin smears distribute cells in a monolayer.View Page
Neutrophil and Lymphocyte

Two segmented neutrophils and a lymphocyte are present in this field. (The arrow indicates one of the neutrophils.) Occasional neutrophils are considered a normal finding in cytospin smears.

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Introduction to Flow Cytometry: Blood Cell Identification
Sample Preparation

Sample preparationSamples for flow cytometric analysis are prepared based on the following procedural steps. Prepare the sample according to cell type. Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered. Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form. Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components. Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results. Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs). Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form. Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. Centrifuge to precipitate cells. Pour off supernatant. Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant. Resuspend cells in PBS. Analyze cells using flow cytometer.

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