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Immunocytochemistry

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Immunocytochemistry
Patricia A. Fetsch

1. Introduction Immunocytochemistry (aka immunohistochemistry), a technique used for the localization of specific cellular antigens, was introduced to diagnostic tumor pathology about 20 yr ago. The procedure allows for the visualization of antigens in tissue samples via the sequential application of a specific antibody to the antigen (primary antibody), a secondary antibody to the primary antibody, an enzyme complex, and a chromogenic substrate. The enzymatic activation of the chromogen results in a visible reaction product at the antigen site, which is interpreted using a light microscope. Immunocytochemistry is routinely used in hospital laboratories to diagnose cancers, identify infectious organisms, differentiate malignant from benign processes, and reveal prognostic indicators. 2. Background A tumor (neoplasm) is an abnormal mass of tissue in which the growth is uncontrolled and progressive. Neoplasms can be benign (noninvasive) or malignant (invasive). Malignant tumors have the capability to metastasize (i.e., spread from one organ to another not directly connected with it). Metastasis of cancers can occur by seeding of body cavities whenever the neoplasm penetrates into a natural open field, such as the pleural cavity, by transport through the lymphatic system, or by dissemination through the peripheral blood. In many cases, tumors can be identified based on their histologic characteristics using routine hematoxylin and eosinstained tissue sections. However, malignant tumors of diverse origin might sometimes resemble each other. At times, inflammatory (reactive) lesions might be difficult to distinguish from cancerous ones. In addition, many cancer patients present with metastases. In some, the primary tumor site is obvious on the basis of clinical or radiologic features, but often, the origin of the tumor is ambiguous. All cells in the human body, whether normal or malignant, express proteins (antigens) which, in many cases, are specific to that particular cell type. Immunocytochemical detection of tissue-specific or organ-specific antigens in the biopsy specimen of the primary tumor or metastatic deposit can lead to the identification of the tumor source. For example, prostate-specific antigen is a marker of tumors of the prostate gland. Thus, immunocytochemical stains are often employed as a means of improving diagnostic accuracy in cases where the diagnosis based on histologic appearance alone is uncertain. 3. Procedure 3.1. Sample Types In routine hospital practice, when tissues are removed from a patient, they are either snapfrozen or placed in formaldehyde. Prior to immunocytochemical staining, formaldehyde-fixed
From: Medical Biomethods Handbook Edited by: J. M. Walker and R. Rapley Humana Press, Inc., Totowa, NJ

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samples must be processed. Tissue processing includes dehydration, paraffin (wax) embedding, and sectioning (cutting) of tissues. Both paraffin-embedded and frozen tissues are routinely cut in 4- to 5-m sections and mounted on microscopic glass slides; paraffin-embedded slides are then dried in a 60C oven for at least 1 h. One slide is typically stained with hematoxylin and eosin for microscopic assessment of the tissue sample. Cytologic samples can also be submitted to the laboratory for pathologic interpretation, and these include cervical smears (i.e., Pap smears), fine-needle aspirates (i.e., samples collected via the aspiration of cells from a lesion with a small bore needle), and body fluids. Processing of cytologic samples most often involves preparations such as blood smears, cytospins (a cell preparation system that uses centrifugal force to deposit cells on a microscope slide in a monolayer), or liquid-based, thin-layer preparations (ThinPreps) (16). When the sample volume is sufficient, the fluid can be centrifuged to form a pellet, which can then be placed in formaldehyde and processed in a manner similar to that for a tissue sample. This is referred to as a cell block.

3.2. Fixation
Fixation prevents autolysis and preserves the antigens of excised tissues. As previously stated, the most common tissue fixative is 10% neutral-buffered formaldehyde. Other fixatives routinely used, particularly for frozen tissues and cytologic preparations, include acetone, alcohol, and paraformaldehyde (1,7). There is often a fine balance between good morphology and the need for antigen preservation. In fact, some fixatives might not be optimal for the immunocytochemical detection of certain antigens. Thus, when introducing a new antibody to the laboratory, a variety of fixatives might need to be examined to determine optimal immunoreactivity. Fixation time is also critical, as longer exposure to fixatives could decrease sensitivity in detecting certain cellular antigens. Formaldehyde fixation of tissue specimens is typically 1224 h, whereas cytospin samples are fixed in acetone or alcohol for 510 min or formaldehyde/paraformaldehyde for 1015 min (1).

3.3. Pretreatment
Many antigens are altered during formaldehyde fixation and processing, because crosslinks are formed to preserve protein structure. Antigenic determinants (epitopes) might be destroyed, denatured, or masked, which could diminish or nullify their detection. Thus, pretreatment steps are required in some instances to visualize certain antigens in formalin-fixed material. These pretreatment steps include proteolytic digestion (i.e., trypsin, pepsin, proteinase K) and heatinduced epitope retrieval, in which tissue sections are subjected to high-temperature heating in a buffer solution using microwave irradiation, autoclaving, pressure cookers, or steaming in an effort to unmask relevant antigens (813).

3.4. AvidinBiotin Complex


The avidinbiotin complex (ABC) procedure is the most universally used immunocytochemical technique, with a high sensitivity and low background (14). In this method, the antigen in the specimen is recognized by a primary antibody. These primary antibodies are typically commercially manufactured and are most often anti-human antibodies produced in mice or rabbits. Next, a secondary antibody that is conjugated to biotin is applied and it attaches to the primary antibody. An avidinbiotin complex is then added. The affinity between the protein avidin and vitamin biotin is very strong. Avidin has four sites capable of binding to biotin. Three of the four binding sites are attached to biotin plus a peroxidase complex; the fourth site binds to the biotin attached to the secondary antibody. Thus, this procedure amplifies the level of staining intensity by increasing the number of potential binding sites. A chromogen is introduced that reacts with peroxide in the ABC reagent to produce a colored reaction product representing the presence of the antigen. The most commonly utilized chromogen, 3,3'-diaminobenzidene, produces a permanent brown stain and is often used with a hematoxylin (blue) counterstain. Table 1 presents a simplified step-by-step immunocytochemical procedure.

Immunocytochemistry 3.5. Interpretation

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When analyzing an immunostained specimen, deposits of colored chromogen indicate the presence of the antigen and represent positive staining. Depending on the cellular location of the antigen, the pattern of cell staining can be cytoplasmic, nuclear, or membrane, and focal or diffuse.

3.6. Quality Control


A positive tissue or cell preparation should be used as a control for every separate primary antibody in a run. This control is known to contain target elements of the antibody and is ideally prepared in the same manner as the patient sample from fixation through processing whenever possible. Although every immunocytochemical run must include positive controls for each antibody, a negative control for each sample is imperative for assessing nonspecific background staining (1519). For the negative control, a duplicate slide from the case under study is tested, and an irrelevant antibody or nonimmune serum from the sample animal (i.e., mouse or rabbit) is applied in place of the primary antibody (2,16). This slide is then used to assess nonspecific staining of the sample as a result of crossreactivity of immunogens other than those for which the primary antibody is testing. This positive staining, which is not a result of antigenantibody binding, is termed nonspecific background stain. The most common cause of this is the attachment of protein to highly charged collagen in the specimen. In particular, tissues such as the kidney and liver and tumors arising from them contain a high level of endogenous biotin, which can result in background staining that is often difficult to interpret without appropriate pretreatment steps. Therefore, it is imperative to consistently include a negative specimen control on all test samples for background staining assessment. True immunoreactivity in the test samples must be of a greater intensity than that seen on the negative control sample. 4. Applications 4.1. Differential Diagnosis of Tumors There are hundreds of antigens identifiable by the immunocytochemical technique, and a practical approach should be taken when using immunostains to help narrow the differential diagnosis. When dealing with a neoplasm of unknown etiology, it is advisable to work up the case in a sequential manner, using discriminating antibodies first to categorize the malignancy as to the cell type of origin (see Table 2), followed by testing with confirmatory antibodies (see Table 3). There is often overlapping immunoreactivity in many tumors and expression of antigens in metastatic malignancies is often heterogenous, so it is best to use a number of antibodies to obtain the appropriate diagnosis. Monoclonal antibodies (produced in mice) provide the greatest specificity and produce the least amount of background staining compared to polyclonal antisera, which are usually made in rabbits. The following is a brief description of the most commonly used markers in clinical laboratories.

4.1.1. Mesothelial Markers


Mesothelial cells are flattened epithelial cells that line the serous body cavities (i.e., abdomen, pericardium, thorax). Spontaneous accumulation of fluid in a serous cavity above the normally small amount is referred to as an effusion and might be caused by inflammation, fluid overload, or a malignant neoplasm. When an effusion develops in one of these cavities, mesothelial cells could exfoliate and large numbers might be found in the fluid. These cells can be benign, as in the case of a reactive/inflammatory effusion, or malignant. Malignant effusions can be the result of metastatic tumor cells or malignant mesothelial cells. Malignant mesotheliomas are tumors derived from these types of cells.
1. Epithelial membrane antigen (EMA). EMA belongs to a group of proteins known as human milk-fat globule membrane proteins. It is present in a wide variety of epithelia of both normal

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Table 1 Simplified Immunocytochemical Staining Procedure

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and neoplastic types (2025). A distinctive EMA pattern in malignant mesothelioma shows a characteristic thick cell membrane staining in the periphery of cell clusters, which highlights the long microvillus projections associated with these types of cells (26). 2. Calretinin. Calretinin is a neuron-specific-calcium binding protein that is strongly expressed in neural tissues and certain non-neural cell types, including mesothelium (2734). Antibodies to this protein are strongly immunoreactive with malignant and benign mesothelial cells, as evident by both a cytoplasmic and nuclear type of staining pattern, described by one group as a fried-egg appearance (3538). It is of great value in differentiating effusions because of mesothelial cells origin (typically calretinin positive) from those caused by metastatic tumor cells (typically calretinin negative).

Immunocytochemistry Table 1 (continued)

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*Digestion/heat induced epitope retrieval is performed if indictated PBS, phosphate buffered saline.

4.1.2. Carcinoma Markers


Carcinomas are malignant neoplasms derived from epithelial cells. Adenocarcinomas are a subtype of carcinoma in which the cells are in a glandular or glandlike pattern. Examples include breast, colon, ovarian, prostate and some lung cancers.
1. Cytokeratins (CKs). Cytokeratins are intermediate-sized (10-nm) monofilaments found in the cytoplasm of almost all true epithelial cell types and form part of the cytoskeletal complex in the epidermis and in most other epithelial tissues (20,3941). The tissue-specific distribution

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Table 2 Initial Screening Markers for Various Malignancies
Neoplasm Category Carcinoma Germ cell Lymphoid Melanoma Mesothelioma Neuroendocrine Sarcoma In general, immunoreactive with antibodies to Cytokeratin (CK) Placental alkaline phosphatase Leukocyte common antigen (CD45) S-100 protein Calretinin Neuron-specific enolase Vimentin

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of these intermediate filaments is generally well preserved in neoplasms and forms the basis for the use of antibodies to keratins (40,41). Antibodies to CK are used to identify normal and malignant cells of epithelial origin, with a cytoplasmic staining pattern. Most adenocarcinomas as well as mesotheliomas show intense immunoreactivity with CKs (33). Cytokeratin-specific antibodies that are directed toward epitopes expressed in a limited number of epithelial cells can assist in identifying the site of primary or metastatic neoplasms. Antibodies to CK7 and CK20 are the most widely studied and used in this context, especially when used in combination (42). Tumor-associated glycoprotein (B72.3). B72.3 is an antibody that recognizes a tumor-associated oncofetal antigen. It is present in a wide variety of adenocarcinomas, including those originating from lung, gastrointestinal tract, pancreas, breast, endometrium, and ovary (33,43). It is not expressed by leukemias, lymphomas, sarcomas, melanomas, or benign tumors (20,21,25,44,45). The antigen is present on normal secretory endometrium, but not on other normal tissues. Positive immunoreactivity is indicated by a membrane staining pattern. Human epithelial antigen (BerEP4). BerEP4 is an antibody prepared by the immunization of mice with cells from the MCF7 breast cancer cell line and reacts with two glycoproteins present on the surface and in the cytoplasm of epithelial cells (20,21,45). BerEP4 stains the majority of adenocarcinomas, but in contrast to all other antiepithelial antibodies, the antibody does not label mesothelial cells (33,46,47). The antibody does not react with nerve, glial, muscle, or mesenchymal tissue, including lymphoid tissue (33). Positive reactions with BerEP4 can be evidenced by membrane staining, which can be in conjunction with cytoplasmic staining. Carcinoembryonic antigen (CEA). CEA was first described as a specific marker for colon cancer (20,21,25,44,45,48,49). It is highly specific for adenocarcinoma cells and is typically negative in benign, reactive, and malignant mesothelial cells (21,33). There is little or no reactivity with nonmalignant tissues, except for granulocytes. Immunoreactivity with this antibody, indicated by a cytoplasmic staining pattern, is associated with colorectal carcinomas, as well as some lung, breast, and gastric cancers (33). Thyroid transcription factor-1 (TTF-1). TTF-1 is a tissue-specific transcription factor expressed in the thyroid and lung (50). Immunoreactivity with this antibody, indicated by a nuclear staining pattern, is associated with thyroid and primary lung cancers (33,51). Prostate-specific antigen (PSA)/prostatic acid phosphatase (PAP). PSA is a serine protease present in high levels in semen; PAP is an isoenzyme of acid phosphatase found in large amounts in the prostate and seminal fluid. Antibodies to PSA and PAP react with normal and neoplastic prostate tissue and are, thus, useful in the diagnosis of prostate cancer (33,52). Cancer Antigen 125 (CA 125). CA 125 is a marker most strongly associated with epithelial ovarian tumors, although it is not a specific marker of ovarian cancer and can be detected immunocytochemically in breast and lung cancers as well as mesotheliomas (33,5355). However, the morphological distinction between primary ovarian cancer and metastatic adenocarcinoma to this location can be difficult, and the addition of CA 125 to a panel of antibodies can be of benefit for that distinction.

Immunocytochemistry
Table 3 Confirmatory Markers for Various Malignancies
Carcinomas Breast Colon Lungnon-small-cell Ovarian Prostate Renal cell Thyroid Lymphoid B-Cell lymphomas T-Cell lymphomas Hodgkins Lymphoma vs reactive lymph node Hyperplasia (reactive) Malignant lymphoma Melanocytic Melanoma Germ cell Choriocarcinoma Embryonal Seminoma Yolk sac Mesothelial Reactive Malignant mesothelioma Neuroendocrine Carcinoid Small-cell-lung carcinoma Islet cell tumor of the pancreas Paraganglioma Sarcoma Ewings/primitive neural ectodermal tumor Gastrointestinal stromal tumor Leiomyosarcoma Rhabdomyosarcoma Synovial Vascular Miscellaneous Infectious Prognostic

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CK7+, CK20, Ber-EP4+, B72.3+, ER+, PR+ CK7, CK20+, CEA+, 19-9+, Ber-EP4+, B72.3+ CK7+, CK20, TTF-1+ CK7+, CK20+/, B72.3+, CA125+ CK7, CK20, PSA+, PAP+ CK7, CK20, EMA+, CD10+, vimentin+ CK 7+, CK 20, TTF-1+ CD20+, CD79a+ CD3+ CD15+, CD30+ Bcl-2, no clonality Bcl-2+, shows either kappa or lambda clonality HMB45+, MART-1+, tyrosinase+ CK+, AFP-, HCG+ CK+, CD30+, CK7+, CK20 CK, AFP, HCG, CD30 CK+, AFP+, CD30 EMA+/, BerEP4, calretinin+ EMA + (thick membrane), BerEP4, calretinin+ Synaptophysin+, chromogranin+, CK+, CK7, CK20 Synaptophysin+, CK+, CEA+, TTF-1+ Synaptophysin+, chromogranin+, CK+, glucagon+, insulin+ Synaptophysin+, chromogranin+, S100+, CK S100, CD99+ CD34+, CD117+ S100, actin+, desmin+ S100+/, actin+, desmin+, myogenin+ S100, CK+, EMA+ S100, CD31+, CD34+, factor VIII+, CK+/ CMV, SV40, HHV8, EBV LMP Ki-67, p53, Her2/neu, ER/PR,CD117

4.1.3. Germ Cell Markers


Germ cell tumors are derived from the cells responsible for reproduction (i.e., ovum and spermatozoon). These tumors occur primarily in the ovary and testes and include seminomas, dysgerminomas, embryonal carcinomas, yolk sac tumors, choriocarcinomas, and teratomas. Placental alkaline phosphatase (PLAP) is a membrane-bound enzyme normally produced by primordial germ cells and syncytiotrophoblasts of the placenta, and the detection of its expression has been useful in the diagnosis of germ cell tumors (33,5658). -fetoprotein (AFP), a major fetal serum protein normally produced by the fetal yolk sac, can be detected immunocy-

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tochemically in nonseminomatous germ cell tumors (33,59). Human chorionic gonadotropin (-HCG) is synthesized by syncytiotrophoblastic cells in choriocarcinoma, and antibodies to HCG are useful in that diagnosis (33,60).

4.1.4. Malignant Melanoma Markers


Malignant melanomas are neoplasms derived from cells capable of forming the pigment melanin, and they arise most commonly in the skin or eye. S-100 protein is a calcium-binding protein found in glial cells, melanocytes, chondrocytes, and adnexal glands of the skin (6165). Many malignant neoplasms express S-100 protein, and practically all malignant melanomas show immunoreactivity in the nucleus and cytoplasm. Various antibodies can be used for the sensitive and specific diagnosis of malignant melanoma. These melanoma-associated antigens include the following: HMB45, an antibody directed against a premelanosome glycoprotein (gp100); MART-1 (melanoma antigen recognized by T-cells), a melanocyte-specific transmembrane protein; and tyrosinase, the rate-limiting enzyme in melanin synthesis (33,6467). Interestingly, these melanoma-associated antigens are recognized by both CD4+ and CD8+ T-cells in a human leukocyte antigen-restricted fashion and can evoke powerful immune responses. Peptides derived from these antigens are currently being utilized as a target for T-cells in numerous melanoma immunotherapy protocols (68).

4.1.5. Lymphoid Markers


Lymphoid neoplasms are malignant proliferations of white blood cells. These tumors include leukemias (an abnormal proliferation of leukocytes in the blood and bone marrow) and lymphomas (neoplasms of lymphoid tissue, arising as discrete tissue masses). Lymphocytes are white blood cells produced by lymphoid tissue and are classified as either B-cell or T-cell. B-cell lymphomas include Burkitt, follicular, diffuse large cell and mantle cell lymphoma; T-cell lymphomas include adult T-cell leukemia/lymphoma and anaplastic large-cell lymphoma. The cell surface antigens of leukocytes have been assigned cluster of differentiation (CD) designations. There are over 200 human leukocyte differentiation antigens. CD45 (leukocyte common antigen) is a membrane protein found on all leukocytes (69). Markers of B-cell lineage include CD19, CD20, CD22, and CD79a (7073), whereas markers of T-cell lineage include CD3, CD4, CD5, CD7, and CD8 (74). ReedSternberg cells of Hodgkins disease are marked by CD15 and CD30 (7577).

4.1.6. Neuroendocrine Markers


Neuroendocrine tumors are neoplasms that share morphologic and biochemical features with cells of the neuroendocrine system. These tumors include carcinoids, small-cell carcinomas of the lung, and islet cell tumors of the pancreas. Neuron-specific enolase (NSE) is found in a variety of normal and neoplastic neuroendocrine cells and predominates in the brain. Antibodies to NSE react with neuroendocrine as well as a variety of tumors, including melanomas, astrocytomas, glioblastomas, and gangliomas (33,78,79). Other neuroendocrine markers include chromogranin, a major constituent of neuroendocrine secretary granules (80), and synaptophysin, a calcium-binding glycoprotein that is the most abundant integral membrane protein constituent of synaptic vesicles of neurons (81,82). Islet cell tumors of the pancreas and carcinoids typically express both chromogranin and synaptophysin (33,83,84).

4.1.7. Sarcoma Markers


Sarcomas are connective tissue neoplasms formed by the proliferation of mesodermal cells and are usually highly malignant. Vimentin is an intermediate filament protein that is ubiquitous in soft tissues, although not considered to be cell-type-specific (85). Desmin and actin are markers of striated and smooth muscle cells and are used in the diagnosis of tumors of muscle origin, such as rhabdomyosar-

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coma (33,86). Endothelial cell markers such as CD31 and factor VIII-related antigen are often expressed by vascular tumors such as angiosarcomas (33,87). Ewings sarcoma is characterized by high MIC2 cell surface antigen expression, a glycoprotein detected by anti-CD99 (33,88,89).

4.2. Therapy-Linked Diagnostics/Prognostic Indications


The prognosis, or outcome of a disease, is dependent on many factors, including tumor type, histologic size and grade, and lymph node status. In addition, independent biologic markers have been identified that are often important prognostic determinants. All of these factors can be used to make management decisions, which direct the course of treatment for the cancer patient. Many prognostic biologic markers can be identified immunocytochemically, and the results of these tests often have direct, immediate therapeutic implications for many types of cancer. The antigens are usually evaluated semiquantitatively, both in terms of the proportion of tumor cells showing positivity and the strength of the reaction. Hormone receptors such as estrogen and progesterone receptors on breast cancer cells can be assessed in this manner, and the presence of these receptors is a favorable prognostic indicator, because women whose tumors are estrogen and progesterone positive have a higher rate of response to endocrine therapy (90,91). Detection of the overexpression of proto-oncogenes such as CD117 (c-Kit) and c-erb-B2 (HER-2/neu) also have prognostic value as predictors of those tumors more likely to respond to adjuvant chemotherapy. For example, antibodies to human epidermal growth factor receptor-2 (HER-2) are currently used immunocytochemically for the assessment of breast cancer patients for whom Herceptin (trastuzumab) treatment is being considered (9294). CD117 is an epitope on the extracellular domain of the transmembrane kinase receptor Kit, the product of the protooncogene c-kit, and can be detected on the cell surface of various malignant cell types. Imatinib mesylate (Gleevec) is a tyrosinase kinase inhibitor that selectively acts on mutated Kit and is currently used as targeted molecular therapy in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors (9598).

4.3. Identification of Infectious Agents


Immunocytochemistry can be used for the diagnosis of infectious disease, most often in cases of viral infections such as hepatitis B, cytomegalovirus, human polyoma BK virus/simian virus 40 (SV-40), and herpesviruses (HHV8, EBV LMP). For example, EpsteinBarr virus latent membrane protein (EBV LMP) is a viral protein that serves as a marker of EBV infection, which, interestingly, is also associated with the etiology of Hodgkins lymphoma (99). Another example of this use of immunocytochemistry is in cases of BK viral infections, which can occur in immunocompromised patients. In the kidney, this infection is associated with mononuclear interstitial inflammatory infiltrates and tubular atrophy, findings that can be difficult to distinguish from acute transplant rejection (100). Immunocytochemistry can provide rapid and sensitive results in this scenario to demonstrate BK virus infections using antibodies to SV-40 (101). 5. Conclusion Therapeutic procedures can alter the course of disease and life-span of the cancer patient. In most instances, obtaining a correct diagnosis and appropriate treatment as early in the disease as possible improves the patients prognosis and quality of life. In current practice, a diagnostic decision is based on tumor morphology, as well as clinical and radiologic findings. In cases where cell morphology is not definitive, the pathologist might base the diagnosis on whether a certain protein is expressed or not by the cells in question. The availability of specific monoclonal antibodies has greatly facilitated the identification of cell proteins and, thus, immunocytochemistry has become indispensable in the practice of diagnostic pathology. In fact, the combined results of morphologic and immunocytochemical examinations form the basis of most tumor classifications today.

Fig. 5641. A 74-yr-old male presented with fluid (effusions) in his left and right pleural cavities. The clinical differential included reactive vs malignant etiology. The pleural fluid was tapped for diagnostic evaluation, and cytologic analysis showed an atypical mesothelial cell proliferation. Formalin-fixed, paraffin-embedded cell block sections prepared from the pleural fluid were stained with antiepithelial membrane antigen (EMA). A strong, thick membrane staining pattern is seen in the atypical cells. This distinctive pattern is often characteristic of malignant mesothelial cells and highlights their long microvillus projections. Diagnosis: malignant mesothelioma. Fig. 2. A 60-yr-old female newly diagnosed with ovarian cancer and undergoing treatment developed a moderate right pleural effusion. The clinical differential included benign/reactive vs malignant etiology. A tap of the effusion showed red blood cells, reactive mesothelial cells, lymphocytes, and numerous atypical cells. Formalin-fixed, paraffin-embedded cell block sections were stained with anticalretinin. A nuclear and cytoplasmic staining pattern is shown in the reactive mesothelial cells whereas the atypical cells are negative. This case highlights the role of calretinin immunoreactivity in discriminating mesothelial cells (typically positive) from adenocarcinoma cells (typically negative) using antibodies to calretinin. Diagnosis: adenocarcinoma with ovarian primary. Fig. 3. A 72-yr-old female with a history of colon cancer presented with a pleural effusion. The clinical differential included infectious vs metastatic disease. The fluid was tapped and showed numerous atypical cells. Formalinfixed, paraffin-embedded cell block sections were stained with anti-B72.3. B72.3 is an antigen expressed by a wide variety of adenocarcinomas, including 8595% of colon cancers. A membrane staining pattern is seen in the atypical cells. Diagnosis: colorectal carcinoma with metastatic pleural effusion.

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Fig. 4. A 38-yr-old female presented with a neck mass. The clinical differential included reactive vs malignant etiology. A fine-needle aspirate of the mass was performed, and cytologic examination showed red blood cells, lymphocytes, neutrophils, and atypical cells singly and in clusters. An alcoholfixed cytospin prepared from the aspirate was stained with anti-cytokeratin. Positive staining with cytokeratin indicates an epithelial origin of the atypical cells. A cytoplasmic staining pattern is seen in the atypical cells. Diagnosis: poorly differentiated carcinoma.

Note: All figures in this chapter are stained with the diaminobenzidene chromogen (brown stain/ positive immunoreactivity) and hematoxylin (blue counterstain).

Immunocytochemistry

Fig. 5. A 70-yr-old female presented with a565 left adnexal mass. The differential diagnosis included colon vs ovarian cancer. The 14-cm mass was removed and showed histologic features characteristic of serous carcinoma of the ovary. Formalin-fixed, paraffin-embedded tissue sections were stained with anti-CA125. The malignant cells show a membrane staining pattern. This case highlights the role of CA125 immunoreactivity in discriminating between primary and metastatic carcinomas of the ovary (typically positive) and intestine (typically negative). Diagnosis: serous carcinoma of the ovary.

Fig. 6. A 28-yr-old male presented with multiple thyroid nodules and enlarged palpable cervical lymph nodes; he had no other previous symptoms. The clinical differential included benign goiter/reactive lymph node vs malignant thyroid/reactive lymph node vs malignant lymphoma. A fine-needle aspirate of the thyroid showed numerous lymphocytes and red blood cells, as well as large atypical cells. Formalin-fixed, paraffin-embedded cell block sections were stained with anti-CD30. CD30 is an antigen expressed on the ReedSternberg cells of Hodgkins lymphoma. A strong membrane staining pattern is seen in this atypical cell. Diagnosis: Classical Hodgkin lymphoma. Fig. 7. A 73-yr-old male presented with enlargement of the testes. A right orchiectomy was performed and a 10-cm mass was removed. The differential diagnosis based on histologic examination included seminoma, melanoma, and carcinoma. Formalin-fixed, paraffin-embedded tissue sections were stained with anti-CD20. CD20 is an antigen acquired late in the pre-B-cell stage of maturation and remains on cells throughout most of their differentiation; it is present in almost all mature B-cell lymphomas. The atypical cells show a strong membrane staining pattern. Diagnosis: malignant lymphoma, B-cell type. Note: Malignant lymphoma is the most common testicular tumor in older individuals. Fig. 8. A 32-yr-old male presented with acute onset of scrotal pain and swelling. An orchiectomy of a right testicular mass was performed. Based on the gross and histologic features, the differential diagnosis included germ cell tumor, lymphoma, and metastatic tumors such as melanoma and poorly differentiated carcinoma. Formalin-fixed, paraffin-embedded tissue sections were stained with antiplacental alkaline phosphatase (PLAP). PLAP is an oncofetal antigen found to be expressed by malignant tumors of germ cell origin. A strong cytoplasmic staining pattern is seen in the atypical cells. Diagnosis: germ cell tumor/seminoma. Note: Seminoma is the most common form of testicular germ cell tumor in younger individuals.

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Fig. 9. A 45-yr-old male with a history of malignant melanoma presented with a suspicious nodule on the back. A skin biopsy was performed, and acetone-fixed frozen-tissue sections were stained with anti-HMB45. HMB45 is an antibody that reacts with an antigen present in premelanosomes and is a sensitive marker for melanoma. Cytoplasmic staining is seen in both the malignant cells in the dermis as well as the normal melanocytes in the epidermis of the skin. Diagnosis: metastatic malignant melanoma.

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Fig. 10. A 20-yr-old female with a history of Ewings sarcoma (primary site: left pelvis) presented with a new right inguinal nodule. The clinical differential included benign/reactive vs metastatic etiology. A fine-needle aspirate of the nodule showed blood elements and numerous atypical cells. Formalin-fixed, paraffin-embedded cell block sections prepared from the aspirate were stained with anti-CD99. CD99 is a cell surface antigen expressed in the majority of Ewings sarcomas. A membrane and cytoplasmic staining pattern is seen in the atypical cells. Diagnosis: progressive Ewings sarcoma/primitive neuroectodermal tumor (PNET). Fig. 11. A 43-yr-old Asian female presented with a 3-wk history of fever, abdominal pain, and diarrhea. A colonoscopy was done and revealed findings suggestive of carcinoma of the colon. A biopsy was then performed, and histologic examination showed scattered atypical cells as well as an inflammatory infiltrate. Formalin-fixed, paraffin-embedded tissue sections from the colon were stained with anticytomegalovirus (CMV). CMV is an important opportunistic pathogen in immunocompromised patients, and CMV colitis is not uncommon in patients with acquired immune deficiency syndrome (AIDS). The CMV inclusions in the large atypical cells scattered throughout the specimen show a nuclear staining pattern. Diagnosis: CMV colitis. Fig. 12. A 42-yr-old female with a history of breast cancer, who 50 d after bone marrow transplant, presented with a new lesion below an existing wound (which was at the previous surgical resection site). The clinical differential included metastatic vs infectious etiology. A fine-needle aspirate of the right shoulder lesion showed blood elements and atypical cells occurring in clusters. Formalin-fixed, paraffin-embedded cell block sections prepared from the aspirate were stained with anti-HER-2/neu. The overexpression of this oncoprotein correlates with a better response to Herceptin therapy. A full membrane staining pattern is seen in the atypical cells. Diagnosis: metastatic breast cancer.

Immunocytochemistry
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