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From: Medical Biomethods HandbookEdited by: J. M. Walker and R. Rapley © Humana Press, Inc., Totowa, NJ

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Immunocytochemistry

Patricia A. Fetsch

1. IntroductionImmunocytochemistry (aka immunohistochemistry), a technique used for the localization of

specific cellular antigens, was introduced to diagnostic tumor pathology about 20 yr ago. Theprocedure allows for the visualization of antigens in tissue samples via the sequential applica-tion of a specific antibody to the antigen (primary antibody), a secondary antibody to the pri-mary antibody, an enzyme complex, and a chromogenic substrate. The enzymatic activation ofthe chromogen results in a visible reaction product at the antigen site, which is interpretedusing a light microscope. Immunocytochemistry is routinely used in hospital laboratories todiagnose cancers, identify infectious organisms, differentiate malignant from benign processes,and reveal prognostic indicators.

2. BackgroundA 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 tumorshave the capability to metastasize (i.e., spread from one organ to another not directly connectedwith it). Metastasis of cancers can occur by seeding of body cavities whenever the neoplasmpenetrates into a natural open field, such as the pleural cavity, by transport through the lym-phatic system, or by dissemination through the peripheral blood.

In many cases, tumors can be identified based on their histologic characteristics using rou-tine hematoxylin and eosin–stained tissue sections. However, malignant tumors of diverse ori-gin might sometimes resemble each other. At times, inflammatory (reactive) lesions might bedifficult to distinguish from cancerous ones. In addition, many cancer patients present withmetastases. In some, the primary tumor site is obvious on the basis of clinical or radiologicfeatures, 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 tis-sue-specific or organ-specific antigens in the biopsy specimen of the primary tumor or meta-static deposit can lead to the identification of the tumor source. For example, prostate-specificantigen is a marker of tumors of the prostate gland. Thus, immunocytochemical stains are oftenemployed as a means of improving diagnostic accuracy in cases where the diagnosis based onhistologic appearance alone is uncertain.

3. Procedure3.1. Sample Types

In routine hospital practice, when tissues are removed from a patient, they are either snap-frozen or placed in formaldehyde. Prior to immunocytochemical staining, formaldehyde-fixed

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samples must be processed. Tissue processing includes dehydration, paraffin (wax) embed-ding, and sectioning (cutting) of tissues. Both paraffin-embedded and frozen tissues are rou-tinely cut in 4- to 5-µm sections and mounted on microscopic glass slides; paraffin-embeddedslides are then dried in a 60°C oven for at least 1 h. One slide is typically stained with hema-toxylin and eosin for microscopic assessment of the tissue sample.

Cytologic samples can also be submitted to the laboratory for pathologic interpretation, andthese include cervical smears (i.e., Pap smears), fine-needle aspirates (i.e., samples collectedvia the aspiration of cells from a lesion with a small bore needle), and body fluids. Processingof cytologic samples most often involves preparations such as blood smears, cytospins (a cellpreparation system that uses centrifugal force to deposit cells on a microscope slide in a mono-layer), or liquid-based, thin-layer preparations (ThinPreps™) (1–6). When the sample volumeis sufficient, the fluid can be centrifuged to form a pellet, which can then be placed in formal-dehyde and processed in a manner similar to that for a tissue sample. This is referred to as a cellblock.

3.2. Fixation

Fixation prevents autolysis and preserves the antigens of excised tissues. As previouslystated, the most common tissue fixative is 10% neutral-buffered formaldehyde. Other fixativesroutinely used, particularly for frozen tissues and cytologic preparations, include acetone, alco-hol, and paraformaldehyde (1,7). There is often a fine balance between good morphology andthe need for antigen preservation. In fact, some fixatives might not be optimal for the immuno-cytochemical detection of certain antigens. Thus, when introducing a new antibody to the labo-ratory, 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 indetecting certain cellular antigens. Formaldehyde fixation of tissue specimens is typically12–24 h, whereas cytospin samples are fixed in acetone or alcohol for 5–10 min or formalde-hyde/paraformaldehyde for 10–15 min (1).

3.3. Pretreatment

Many antigens are altered during formaldehyde fixation and processing, because crosslinksare formed to preserve protein structure. Antigenic determinants (epitopes) might be destroyed,denatured, or masked, which could diminish or nullify their detection. Thus, pretreatment stepsare required in some instances to visualize certain antigens in formalin-fixed material. Thesepretreatment steps include proteolytic digestion (i.e., trypsin, pepsin, proteinase K) and heat-induced epitope retrieval, in which tissue sections are subjected to high-temperature heating ina buffer solution using microwave irradiation, autoclaving, pressure cookers, or steaming in aneffort to unmask relevant antigens (8–13).

3.4. Avidin–Biotin Complex

The avidin–biotin complex (ABC) procedure is the most universally used immunocy-tochemical technique, with a high sensitivity and low background (14). In this method, theantigen in the specimen is recognized by a primary antibody. These primary antibodies aretypically commercially manufactured and are most often anti-human antibodies produced inmice or rabbits. Next, a secondary antibody that is conjugated to biotin is applied and it attachesto the primary antibody. An avidin–biotin complex is then added. The affinity between the pro-tein 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 sitebinds to the biotin attached to the secondary antibody. Thus, this procedure amplifies the levelof staining intensity by increasing the number of potential binding sites. A chromogen is intro-duced that reacts with peroxide in the ABC reagent to produce a colored reaction product repre-senting 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.

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3.5. Interpretation

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

3.6. Quality Control

A positive tissue or cell preparation should be used as a control for every separate primaryantibody in a run. This control is known to contain target elements of the antibody and is ideallyprepared in the same manner as the patient sample from fixation through processing wheneverpossible.

Although every immunocytochemical run must include positive controls for each antibody,a negative control for each sample is imperative for assessing nonspecific background staining(15–19). For the negative control, a duplicate slide from the case under study is tested, and anirrelevant antibody or nonimmune serum from the sample animal (i.e., mouse or rabbit) isapplied in place of the primary antibody (2,16). This slide is then used to assess nonspecificstaining of the sample as a result of crossreactivity of immunogens other than those for whichthe primary antibody is testing. This positive staining, which is not a result of antigen–antibodybinding, is termed nonspecific background stain. The most common cause of this is the attach-ment of protein to highly charged collagen in the specimen. In particular, tissues such as thekidney and liver and tumors arising from them contain a high level of endogenous biotin, whichcan result in background staining that is often difficult to interpret without appropriate pretreat-ment steps. Therefore, it is imperative to consistently include a negative specimen control onall test samples for background staining assessment. True immunoreactivity in the test samplesmust be of a greater intensity than that seen on the negative control sample.

4. Applications4.1. Differential Diagnosis of Tumors

There are hundreds of antigens identifiable by the immunocytochemical technique, and apractical approach should be taken when using immunostains to help narrow the differentialdiagnosis.

When dealing with a neoplasm of unknown etiology, it is advisable to work up the case in asequential manner, using discriminating antibodies first to categorize the malignancy as to thecell 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 meta-static malignancies is often heterogenous, so it is best to use a number of antibodies to obtainthe appropriate diagnosis. Monoclonal antibodies (produced in mice) provide the greatest speci-ficity 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., abdo-men, pericardium, thorax). Spontaneous accumulation of fluid in a serous cavity above thenormally small amount is referred to as an effusion and might be caused by inflammation, fluidoverload, or a malignant neoplasm. When an effusion develops in one of these cavities, mesothe-lial 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 theresult of metastatic tumor cells or malignant mesothelial cells. Malignant mesotheliomas aretumors derived from these types of cells.

1. Epithelial membrane antigen (EMA). EMA belongs to a group of proteins known as humanmilk-fat globule membrane proteins. It is present in a wide variety of epithelia of both normal

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and neoplastic types (20–25). A distinctive EMA pattern in malignant mesothelioma shows acharacteristic “thick” cell membrane staining in the periphery of cell clusters, which highlightsthe long microvillus projections associated with these types of cells (26).

2. Calretinin. Calretinin is a neuron-specific-calcium binding protein that is strongly expressedin neural tissues and certain non-neural cell types, including mesothelium (27–34). Antibodiesto this protein are strongly immunoreactive with malignant and benign mesothelial cells, asevident by both a cytoplasmic and nuclear type of staining pattern, described by one group asa “fried-egg” appearance (35–38). It is of great value in differentiating effusions because ofmesothelial cells origin (typically calretinin positive) from those caused by metastatic tumorcells (typically calretinin negative).

Table 1Simplified Immunocytochemical Staining Procedure

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4.1.2. Carcinoma Markers

Carcinomas are malignant neoplasms derived from epithelial cells. Adenocarcinomas are asubtype of carcinoma in which the cells are in a glandular or glandlike pattern. Examples in-clude breast, colon, ovarian, prostate and some lung cancers.

1. Cytokeratins (CKs). Cytokeratins are intermediate-sized (10-nm) monofilaments found in thecytoplasm of almost all true epithelial cell types and form part of the cytoskeletal complex inthe epidermis and in most other epithelial tissues (20,39–41). The tissue-specific distribution

Table 1 (continued)

*Digestion/heat induced epitope retrieval isperformed if indictated

PBS, phosphate buffered saline.

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Table 2Initial “Screening” Markers for Various Malignancies

Neoplasm Category In general, immunoreactive with antibodies to

Carcinoma Cytokeratin (CK)Germ cell Placental alkaline phosphataseLymphoid Leukocyte common antigen (CD45)Melanoma S-100 proteinMesothelioma CalretininNeuroendocrine Neuron-specific enolaseSarcoma Vimentin

of these intermediate filaments is generally well preserved in neoplasms and forms the basisfor the use of antibodies to keratins (40,41). Antibodies to CK are used to identify normal andmalignant cells of epithelial origin, with a cytoplasmic staining pattern. Most adenocarcino-mas as well as mesotheliomas show intense immunoreactivity with CKs (33).Cytokeratin-specific antibodies that are directed toward epitopes expressed in a limited num-ber 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, especiallywhen used in combination (42).

2. Tumor-associated glycoprotein (B72.3). B72.3 is an antibody that recognizes a tumor-associ-ated oncofetal antigen. It is present in a wide variety of adenocarcinomas, including thoseoriginating 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 othernormal tissues. Positive immunoreactivity is indicated by a membrane staining pattern.

3. Human epithelial antigen (BerEP4). BerEP4 is an antibody prepared by the immunization ofmice with cells from the MCF7 breast cancer cell line and reacts with two glycoproteins presenton the surface and in the cytoplasm of epithelial cells (20,21,45). BerEP4 stains the majority ofadenocarcinomas, but in contrast to all other antiepithelial antibodies, the antibody does notlabel mesothelial cells (33,46,47). The antibody does not react with nerve, glial, muscle, ormesenchymal tissue, including lymphoid tissue (33). Positive reactions with BerEP4 can beevidenced by membrane staining, which can be in conjunction with cytoplasmic staining.

4. Carcinoembryonic antigen (CEA). CEA was first described as a specific marker for coloncancer (20,21,25,44,45,48,49). It is highly specific for adenocarcinoma cells and is typicallynegative in benign, reactive, and malignant mesothelial cells (21,33). There is little or no reac-tivity with nonmalignant tissues, except for granulocytes. Immunoreactivity with this anti-body, indicated by a cytoplasmic staining pattern, is associated with colorectal carcinomas, aswell as some lung, breast, and gastric cancers (33).

5. Thyroid transcription factor-1 (TTF-1). TTF-1 is a tissue-specific transcription factor expressedin the thyroid and lung (50). Immunoreactivity with this antibody, indicated by a nuclear stain-ing pattern, is associated with thyroid and primary lung cancers (33,51).

6. Prostate-specific antigen (PSA)/prostatic acid phosphatase (PAP). PSA is a serine proteasepresent in high levels in semen; PAP is an isoenzyme of acid phosphatase found in largeamounts in the prostate and seminal fluid. Antibodies to PSA and PAP react with normal andneoplastic prostate tissue and are, thus, useful in the diagnosis of prostate cancer (33,52).

7. Cancer Antigen 125 (CA 125). CA 125 is a marker most strongly associated with epithelialovarian tumors, although it is not a specific marker of ovarian cancer and can be detectedimmunocytochemically in breast and lung cancers as well as mesotheliomas (33,53–55). How-ever, the morphological distinction between primary ovarian cancer and metastatic adenocar-cinoma to this location can be difficult, and the addition of CA 125 to a panel of antibodies canbe of benefit for that distinction.

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Table 3Confirmatory Markers for Various Malignancies

CarcinomasBreast CK7+, CK20–, Ber-EP4+, B72.3+, ER+, PR+Colon CK7–, CK20+, CEA+, 19-9+, Ber-EP4+, B72.3+Lung–non-small-cell CK7+, CK20–, TTF-1+Ovarian CK7+, CK20+/–, B72.3+, CA125+Prostate CK7–, CK20–, PSA+, PAP+Renal cell CK7–, CK20–, EMA+, CD10+, vimentin+Thyroid CK 7+, CK 20–, TTF-1+

LymphoidB-Cell lymphomas CD20+, CD79a+T-Cell lymphomas CD3+Hodgkin’s CD15+, CD30+

Lymphoma vs reactive lymph nodeHyperplasia (reactive) Bcl-2–, no clonalityMalignant lymphoma Bcl-2+, shows either kappa or lambda clonality

MelanocyticMelanoma HMB45+, MART-1+, tyrosinase+

Germ cellChoriocarcinoma CK+, AFP-, HCG+Embryonal CK+, CD30+, CK7+, CK20–Seminoma CK–, AFP–, HCG–, CD30–Yolk sac CK+, AFP+, CD30–

MesothelialReactive EMA+/–, BerEP4–, calretinin+Malignant mesothelioma EMA + (thick membrane), BerEP4–, calretinin+

NeuroendocrineCarcinoid Synaptophysin+, chromogranin+, CK+, CK7–,

CK20–Small-cell-lung carcinoma Synaptophysin+, CK+, CEA+, TTF-1+Islet cell tumor of the pancreas Synaptophysin+, chromogranin+, CK+,

glucagon+, insulin+Paraganglioma Synaptophysin+, chromogranin+, S100+, CK–

SarcomaEwing’s/primitive neural ectodermal tumor S100–, CD99+Gastrointestinal stromal tumor CD34+, CD117+Leiomyosarcoma S100–, actin+, desmin+Rhabdomyosarcoma S100+/–, actin+, desmin+, myogenin+Synovial S100–, CK+, EMA+Vascular S100–, CD31+, CD34+, factor VIII+, CK+/–

MiscellaneousInfectious CMV, SV40, HHV8, EBV LMPPrognostic 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 andspermatozoon). 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 byprimordial germ cells and syncytiotrophoblasts of the placenta, and the detection of its expres-sion has been useful in the diagnosis of germ cell tumors (33,56–58). α-fetoprotein (AFP), amajor 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(β-HCG) is synthesized by syncytiotrophoblastic cells in choriocarcinoma, and antibodies toHCG are useful in that diagnosis (33,60).

4.1.4. Malignant Melanoma Markers

Malignant melanomas are neoplasms derived from cells capable of forming the pigmentmelanin, 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 (61–65). Many malignant neoplasms express S-100 protein, andpractically 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 directedagainst a premelanosome glycoprotein (gp100); MART-1 (melanoma antigen recognizedby T-cells), a melanocyte-specific transmembrane protein; and tyrosinase, the rate-limit-ing enzyme in melanin synthesis (33,64–67). Interestingly, these melanoma-associated anti-gens are recognized by both CD4+ and CD8+ T-cells in a human leukocyte antigen-restrictedfashion and can evoke powerful immune responses. Peptides derived from these antigens arecurrently being utilized as a target for T-cells in numerous melanoma immunotherapy proto-cols (68).

4.1.5. Lymphoid Markers

Lymphoid neoplasms are malignant proliferations of white blood cells. These tumors includeleukemias (an abnormal proliferation of leukocytes in the blood and bone marrow) and lympho-mas (neoplasms of lymphoid tissue, arising as discrete tissue masses). Lymphocytes are whiteblood cells produced by lymphoid tissue and are classified as either B-cell or T-cell. B-celllymphomas include Burkitt, follicular, diffuse large cell and mantle cell lymphoma; T-cell lym-phomas 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 (leukocytecommon antigen) is a membrane protein found on all leukocytes (69). Markers of B-cell lin-eage include CD19, CD20, CD22, and CD79a (70–73), whereas markers of T-cell lineageinclude CD3, CD4, CD5, CD7, and CD8 (74). Reed–Sternberg cells of Hodgkin’s disease aremarked by CD15 and CD30 (75–77).

4.1.6. Neuroendocrine Markers

Neuroendocrine tumors are neoplasms that share morphologic and biochemical features withcells of the neuroendocrine system. These tumors include carcinoids, small-cell carcinomas ofthe lung, and islet cell tumors of the pancreas.

Neuron-specific enolase (NSE) is found in a variety of normal and neoplastic neuroendo-crine cells and predominates in the brain. Antibodies to NSE react with neuroendocrine as wellas a variety of tumors, including melanomas, astrocytomas, glioblastomas, and gangliomas(33,78,79). Other neuroendocrine markers include chromogranin, a major constituent of neu-roendocrine secretary granules (80), and synaptophysin, a calcium-binding glycoprotein that isthe most abundant integral membrane protein constituent of synaptic vesicles of neurons(81,82). Islet cell tumors of the pancreas and carcinoids typically express both chromograninand synaptophysin (33,83,84).

4.1.7. Sarcoma Markers

Sarcomas are connective tissue neoplasms formed by the proliferation of mesodermal cellsand are usually highly malignant.

Vimentin is an intermediate filament protein that is ubiquitous in soft tissues, although notconsidered to be cell-type-specific (85). Desmin and actin are markers of striated and smoothmuscle 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 oftenexpressed by vascular tumors such as angiosarcomas (33,87). Ewing’s sarcoma is character-ized 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 markershave been identified that are often important prognostic determinants. All of these factors canbe used to make management decisions, which direct the course of treatment for the cancerpatient.

Many prognostic biologic markers can be identified immunocytochemically, and the resultsof 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 tumorcells showing positivity and the strength of the reaction. Hormone receptors such as estrogenand progesterone receptors on breast cancer cells can be assessed in this manner, and the pres-ence of these receptors is a favorable prognostic indicator, because women whose tumors areestrogen 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 toadjuvant chemotherapy. For example, antibodies to human epidermal growth factor receptor-2(HER-2) are currently used immunocytochemically for the assessment of breast cancer patientsfor whom Herceptin® (trastuzumab) treatment is being considered (92–94). CD117 is an epitopeon the extracellular domain of the transmembrane kinase receptor Kit, the product of the proto-oncogene c-kit, and can be detected on the cell surface of various malignant cell types. Imatinibmesylate (Gleevec®) is a tyrosinase kinase inhibitor that selectively acts on mutated Kit and iscurrently used as targeted molecular therapy in the treatment of chronic myelogenous leukemiaand gastrointestinal stromal tumors (95–98).

4.3. Identification of Infectious Agents

Immunocytochemistry can be used for the diagnosis of infectious disease, most often incases of viral infections such as hepatitis B, cytomegalovirus, human polyoma BK virus/simianvirus 40 (SV-40), and herpesviruses (HHV8, EBV LMP). For example, Epstein–Barr viruslatent membrane protein (EBV LMP) is a viral protein that serves as a marker of EBV infec-tion, which, interestingly, is also associated with the etiology of Hodgkin’s lymphoma (99).Another example of this use of immunocytochemistry is in cases of BK viral infections, whichcan occur in immunocompromised patients. In the kidney, this infection is associated withmononuclear interstitial inflammatory infiltrates and tubular atrophy, findings that can be diffi-cult to distinguish from acute transplant rejection (100). Immunocytochemistry can providerapid and sensitive results in this scenario to demonstrate BK virus infections using antibodiesto SV-40 (101).

5. ConclusionTherapeutic 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 diseaseas possible improves the patient’s prognosis and quality of life. In current practice, a diagnosticdecision is based on tumor morphology, as well as clinical and radiologic findings. In caseswhere cell morphology is not definitive, the pathologist might base the diagnosis on whether acertain protein is expressed or not by the cells in question. The availability of specific mono-clonal antibodies has greatly facilitated the identification of cell proteins and, thus, immunocy-tochemistry has become indispensable in the practice of diagnostic pathology. In fact, thecombined results of morphologic and immunocytochemical examinations form the basis ofmost tumor classifications today.

564 FetschFig. 1. A 74-yr-old male presented with fluid (ef-fusions) in his left and right pleural cavities. Theclinical differential included reactive vs malignantetiology. The pleural fluid was tapped for diagnosticevaluation, and cytologic analysis showed an atypi-cal mesothelial cell proliferation. Formalin-fixed,paraffin-embedded cell block sections prepared fromthe pleural fluid were stained with antiepithelialmembrane antigen (EMA). A strong, thick mem-brane staining pattern is seen in the atypical cells.This distinctive pattern is often characteristic of ma-lignant mesothelial cells and highlights their longmicrovillus projections. Diagnosis: malignant me-sothelioma.

Fig. 3. A 72-yr-old female with a history ofcolon cancer presented with a pleural effusion.The clinical differential included infectious vsmetastatic disease. The fluid was tapped andshowed numerous atypical cells. Formalin-fixed, paraffin-embedded cell block sectionswere stained with anti-B72.3. B72.3 is an anti-gen expressed by a wide variety of adenocarci-nomas, including 85–95% of colon cancers. Amembrane staining pattern is seen in the atypi-cal cells. Diagnosis: colorectal carcinoma withmetastatic pleural effusion.

Fig. 4. A 38-yr-old female presented with a neckmass. The clinical differential included reactive vsmalignant etiology. A fine-needle aspirate of themass was performed, and cytologic examinationshowed red blood cells, lymphocytes, neutrophils,and atypical cells singly and in clusters. An alcohol-fixed cytospin prepared from the aspirate was stainedwith anti-cytokeratin. Positive staining withcytokeratin indicates an epithelial origin of the atypi-cal cells. A cytoplasmic staining pattern is seen inthe atypical cells. Diagnosis: poorly differentiatedcarcinoma.

Fig. 2. A 60-yr-old female newly diagnosed withovarian cancer and undergoing treatment developeda moderate right pleural effusion. The clinical dif-ferential included benign/reactive vs malignant eti-ology. A tap of the effusion showed red blood cells,reactive mesothelial cells, lymphocytes, and numer-ous atypical cells. Formalin-fixed, paraffin-embed-ded cell block sections were stained withanticalretinin. A nuclear and cytoplasmic stainingpattern is shown in the reactive mesothelial cellswhereas the atypical cells are negative. This casehighlights the role of calretinin immunoreactivity indiscriminating mesothelial cells (typically positive)from adenocarcinoma cells (typically negative) us-ing antibodies to calretinin. Diagnosis: adenocarci-noma with ovarian primary.

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

Immunocytochemistry 565Fig. 5. A 70-yr-old female presented with a leftadnexal mass. The differential diagnosis includedcolon vs ovarian cancer. The 14-cm mass was re-moved and showed histologic features characteristicof serous carcinoma of the ovary. Formalin-fixed,paraffin-embedded tissue sections were stained withanti-CA125. The malignant cells show a membranestaining pattern. This case highlights the role ofCA125 immunoreactivity in discriminating betweenprimary and metastatic carcinomas of the ovary(typically positive) and intestine (typically nega-tive). Diagnosis: serous carcinoma of the ovary.

Fig. 6. A 28-yr-old male presented with multiplethyroid nodules and enlarged palpable cervicallymph nodes; he had no other previous symptoms.The clinical differential included benign goiter/re-active lymph node vs malignant thyroid/reactivelymph node vs malignant lymphoma. A fine-needleaspirate of the thyroid showed numerous lympho-cytes and red blood cells, as well as large atypicalcells. Formalin-fixed, paraffin-embedded cell blocksections were stained with anti-CD30. CD30 is anantigen expressed on the Reed–Sternberg cells ofHodgkin’s lymphoma. A strong membrane stainingpattern is seen in this atypical cell. Diagnosis: Clas-sical Hodgkin lymphoma.

Fig. 7. A 73-yr-old male presented with enlarge-ment of the testes. A right orchiectomy was per-formed and a 10-cm mass was removed. Thedifferential diagnosis based on histologic examina-tion included seminoma, melanoma, and carcinoma.Formalin-fixed, paraffin-embedded tissue sectionswere stained with anti-CD20. CD20 is an antigenacquired late in the pre-B-cell stage of maturationand remains on cells throughout most of their differ-entiation; it is present in almost all mature B-celllymphomas. The atypical cells show a strong mem-brane staining pattern. Diagnosis: malignant lym-phoma, B-cell type. Note: Malignant lymphoma isthe most common testicular tumor in older individu-als.

Fig. 8. A 32-yr-old male presented with acute on-set of scrotal pain and swelling. An orchiectomy of aright testicular mass was performed. Based on thegross and histologic features, the differential diag-nosis included germ cell tumor, lymphoma, andmetastatic tumors such as melanoma and poorly dif-ferentiated carcinoma. Formalin-fixed, paraffin-em-bedded tissue sections were stained withantiplacental alkaline phosphatase (PLAP). PLAP isan oncofetal antigen found to be expressed by ma-lignant tumors of germ cell origin. A strong cyto-plasmic staining pattern is seen in the atypical cells.Diagnosis: germ cell tumor/seminoma. Note: Semi-noma is the most common form of testicular germcell tumor in younger individuals.

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Fig. 12. A 42-yr-old female with a history ofbreast cancer, who 50 d after bone marrow trans-plant, presented with a new lesion below an existingwound (which was at the previous surgical resectionsite). The clinical differential included metastatic vsinfectious etiology. A fine-needle aspirate of theright shoulder lesion showed blood elements andatypical cells occurring in clusters. Formalin-fixed,paraffin-embedded cell block sections prepared fromthe aspirate were stained with anti-HER-2/neu. Theoverexpression of this oncoprotein correlates with abetter response to Herceptin therapy. A full mem-brane staining pattern is seen in the atypical cells.Diagnosis: metastatic breast cancer.

Fig. 9. A 45-yr-old male with a history of malig-nant melanoma presented with a suspicious noduleon the back. A skin biopsy was performed, and ac-etone-fixed frozen-tissue sections were stained withanti-HMB45. HMB45 is an antibody that reacts withan antigen present in premelanosomes and is a sensi-tive marker for melanoma. Cytoplasmic staining isseen in both the malignant cells in the dermis as wellas the normal melanocytes in the epidermis of theskin. Diagnosis: metastatic malignant melanoma.

Fig. 10. A 20-yr-old female with a history ofEwing’s sarcoma (primary site: left pelvis) presentedwith a new right inguinal nodule. The clinical differ-ential included benign/reactive vs metastatic etiol-ogy. A fine-needle aspirate of the nodule showedblood elements and numerous atypical cells. Forma-lin-fixed, paraffin-embedded cell block sections pre-pared from the aspirate were stained with anti-CD99.CD99 is a cell surface antigen expressed in the ma-jority of Ewing’s sarcomas. A membrane and cyto-plasmic staining pattern is seen in the atypical cells.Diagnosis: progressive Ewing’s sarcoma/primitiveneuroectodermal tumor (PNET).

Fig. 11. A 43-yr-old Asian female presented witha 3-wk history of fever, abdominal pain, and diar-rhea. A colonoscopy was done and revealed findingssuggestive of carcinoma of the colon. A biopsy wasthen performed, and histologic examination showedscattered atypical cells as well as an inflammatoryinfiltrate. Formalin-fixed, paraffin-embedded tissuesections from the colon were stained withanticytomegalovirus (CMV). CMV is an importantopportunistic pathogen in immunocompromised pa-tients, and CMV colitis is not uncommon in patientswith acquired immune deficiency syndrome (AIDS).The CMV inclusions in the large atypical cells scat-tered throughout the specimen show a nuclear stain-ing pattern. Diagnosis: CMV colitis.

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