7 Special Studies

The pathologist’s H&E is like the clinician’s H&P (history and physical) - basic exams to be performed on every patient or specimen forming the cornerstone of diagnosis. However, the pathologist is no longer limited to the H&E; there are a wide variety of special studies available to evaluate pathologic processes, from simple histochemical stains to global gene expression patterns. Pathologists are now clinical cell biologists. Familiarity with the types of special studies available is important as the initial processing of the gross specimen may limit the types of studies that can be performed.

HISTOCHEMISTRY

Almost all histochemical stains are suitable for formalin-fixed tissues. Common stains and their uses are listed in Table 7-1. However, numerous other types of stains and modifications are used and pathologists must be aware of individual laboratory practices.

TABLE 7–1 HISTOCHEMICAL STAINS

STAIN	COMPONENTS STAINED	POSSIBLE USES AND COMMENTS
AFOG (acid fuschin orange G; modified Masson’s trichrome)	Nuclei – brown Connective tissue – blue Basement membrane – blue Proteins, fibrin, readsorption droplets in cells, immune complexes – red/orange/yellow RBCs – yellow	Evaluation of renal biopsies
Alcian blue	Acid mucins – blue (e.g., normal intestinal glands) Nuclei – red Cytoplasm – pink	Sometimes used to identify mucosubstances in mesotheliomas or intestinal metaplasia. Affected by pH. Hyaluronidase digestion can be used to identify hyaluronic acid.
Alcian blue/PAS	Intestinal metaplasia – dark purple Normal stomach – pink	Demonstrates both acid and neutral mucins
Alcian yellow	Free mucus – yellow Bacteria – dark blue	Identification of H. pylori in gastric biopsies
Acid-fast bacilli stains (Fite-Faraco, Ziehl-Neelson, Kinyoun)	TB – red and beaded MAI – red Nocardia – pink Tissue – blue	Identification of mycobacteria. Modifications are used to demonstrate M. leprae or Nocardia. Carnoy’s fixed tissues cannot be used and B-5 is suboptimal. Slides must be examined under oil.
Alizarin red S	Calcium – orange red, polarizes	Identifies calcium in tissues
Bile	Bile – dark green on a yellow background	Identification of bile
Bodian’s	Nerve fibers and neurofibrils – black Nuclei – black Tissue – blue	Neural tumors, identification of axons
Chloroacetate esterase (Leder; CAE)	Mature myeloid cells, mast cells – red granules Nuclei – blue	Evaluation of leukemias Identification of mast cells Cannot be used for tissue fixed in Zenker’s or B-5.
Congo red	Amyloid – orange red with apple green birefringence after polarization Nuclei – blue	Detection of amyloid. Immunoperoxidase studies can be used to identify specific types. Overstaining can result in false positives.
Dieterle	Spirochetes, Legionella, other bacteria – brown to black Tissue – pale yellow or tan	Infectious lesions Melanin, chromatin, formalin pigment, and foreign material may also stain
Diff Quik (a modified Giemsa stain)	H. pylori – dark blue Other bacteria – blue Nuclei – dark blue Cytoplasm – pink	Evaluation of chronic gastritis
Elastic stains (Verhoeff–van Gieson)	Elastic fibers – blue black to black Nuclei – blue to black Collagen – red Other tissue – yellow	Identification of arteries and veins, vasculitis, invasion of lung tumors into visceral pleura, abnormal elastic fibers in elastofibromas
Fibrin (see Phosphotungstic acid hematoxylin or Mallory PTAH)		To demonstrate fibrin in renal biopsies
Fontana-Masson	Melanin, argentaffin granules, chromaffin granules, some lipofuschin – black Nuclei – red	Identification of melanin in melanomas and secretory granules in neuroendocrine tumors This stain has largely been replaced by IHC
Giemsa (May-Grunwald)	Bacteria (e.g., H. pylori) – blue Parasites (Leishmania, Plasmodium) – blue Mast cells – red to purple granules Nuclei – blue Cytoplasm of leukocytes – pink to blue depending on cell type and differentiation	Lymphoproliferative disorders (good nuclear and cytoplasmic detail) Identification of bacteria, rickettsias, and Toxoplasma gondii
Gram (Brown-Hopps, Brown-Brenn)	Gram-positive bacteria – blue Gram-negative bacteria – red Nuclei – red Tissue – variable	Identification of bacteria, some cases of actinomycetes, Nocardia, coccidioidomycosis, blastomycosis, cryptococcosis, aspergillosis, rhinosporidiosis, and amebiasis
Grimelius	Argentaffin and argyrophil granules – dark brown to black Nuclei – red Background – pale yellow-brown	Evaluation of neuroendocrine tumors (largely replaced by the use of immunohistochemistry for chromogranin)
Hematoxylin and eosin (H&E)	Nuclei – dark blue or purple Cytoplasm – pink to red	Standard stain for the routine evaluation of tissues
Iron (colloidal iron)	Ferric iron (e.g., hemosiderin) – blue Nuclei – red Background – pink	Bone marrow (iron stores, myelodysplasias), liver (hemochromatosis) Chromophobe renal cell carcinomas are positive
Jone’s silver methenamine	Basement membrane – dark maroon to black	Evaluation of renal biopsies
Melanin bleach		Removes melanin from tissue, usually for IHC Melanin can be difficult to distinguish from IHC positivity
Methyl green-pyronin Y	DNA (nuclei) – green to blue-green RNA - red Goblet cells - mint green Plasma cell and immunoblast cytoplasm - pink to red Mast cells - orange Background - pale pink to colorless	Plasma cell lesions (largely replaced by IHC) Does not work well on tissues decalcified with formic acid.
Mucicarmine (Mayer)	Mucin - deep rose to red Capsule of Cryptococcus - deep rose to red Nuclei - black Tissue - blue or yellow	Identification of adenocarcinomas, identification of Cryptococcus
Oil red O	Fat - red Nuclei - blue	Requires frozen sections (lipids are dissolved by most fixatives or during processing). Tissue fixed in formalin can be used if tissue is frozen.
Periodic acid – Schiff (PAS)	Glycogen - red Basement membranes (BM) - red Mucins - red Colloid - red Fungi - red	Classification of tumors with glycogen (e.g., Ewing’s/PNET, rhabdomyosarcoma, renal cell carcinoma), glomerular diseases (BM), identification of adenocarcinomas (mucin), fungal diseases (especially in argentophic areas – neutrophils and debris), spironolactone bodies in adrenal adenomas treated with this drug
Periodic acid – Schiff with diastase digestion (PAS-D)	As above except glycogen that has been digested will not be stained	Identification of glycogen in tumors Identification of fungus in glycogen-rich tissue (e.g., skin) PAS-D resistant deposits in liver are present in alpha-1-antitrypsin deficiency
Phosphotungstic acid hematoxylin (Mallory PTAH)	Glial fibers - blue Nuclei - blue Neurons - salmon Myelin - blue Skeletal muscle cross striations - blue Fibrin - blue Collagen - red brown	Identification of neural lesions Skeletal muscle differentiation (Zenker’s fixative is preferred). This stain has been replaced by IHC for muscle markers.
Reticular fibers (Gomor’s reticulin, Gordon and Sweets, Snook)	Reticulin - black Mature collagen, type 1 – brown Immature collagen, types 3 and 4 - black	Bone marrow (myelophthisis), liver (fibrosis, veno-occlusive disease), carcinoma vs. sarcoma (reticular network) (but largely replaced by IHC)
Silver stain (Grocott methenamine–silver nitrate – GMS)	Fungi - black Pneumocystis - black Mucin - taupe to gray Tissue - green	Evaluation of infectious diseases Bacteria will also stain black.
Steiner	Spirochetes, H. pylori, Legionella, other bacteria - dark brown to black Tissue - light yellow	Evaluation of infectious diseases
Sulfated Alcian blue	Myocytes – yellow Connective tissue – red-purple Amyloid – sea-foam green	Identification of amyloid in cardiac biopsies
Toluidine blue	Mast cells - deep violet Background - blue	Mast cell diseases, chronic cystitis
Trichrome (Gomori, Masson)	Mature collagen, type 1 – dark blue Immature collagen, types 3 and 4 - light blue Mucin - green or blue Nuclei - black Cytoplasm, keratin, muscle fibers - red	Liver (fibrosis)
Trichrome - modified	Viable myocardium - magenta to brick red Nonviable myocardium -dusky gray to mauve	Evaluation of myocardial biopsies
Von Kossa calcium	Calcium - black Tissue - red	Demonstration of phosphate and carbonate radicals with calcium in tissues, identification of malakoplakia (Michaelis-Gutmann bodies)
Warthin Starry	Spirochetes - black Other bacteria - black (including Bartonella sp.) Tissue - pale yellow to light brown	Infectious lesions (including syphilis, cat scratch fever, and bacillary angiomatosis)
Wright’s	Eosinophilic granules - pink Neutrophilic granules - purple Lymphocytic cytoplasm - blue Nuclei -blue to purple	Blood smears

The WebPath section of the University of Utah site (http://library.med.utah.edu/webpath) has useful descriptions of special stains and illustrative photographs.

IMMUNOPEROXIDASE STUDIES

The development of methods to detect antigens on tissue sections with antibodies was a major advance in surgical pathology. Immunohistochemical (IHC) studies are most frequently used for the following purposes:

• Classification of tumors (e.g., carcinoma vs. lymphoma, B cell vs. T cell lymphoma).

• Identification of in situ lesions vs. invasive carcinomas (e.g., myoepithelial markers in breast cancers, basal cell markers in prostate).

• Prognostic factors (e.g., Ki-67 in glioblastomas).

• Predictive factors to guide specific therapy (e.g., c-KIT, estrogen and progesterone receptors, HER2/neu).

• Identification of extracellular material (e.g., β-2 microglobulin amyloid).

• Identification of infectious agents (e.g., CMV or HSV).

Use of Immunohistochemistry.

A differential diagnosis is generated after examination of the H&E-stained slides. IHC is then used to gain evidence for or against diagnostic possibilities. “Trolling” cases through an immunohistochemistry laboratory by ordering numerous antibody studies without a clear reason in mind is more likely to lead to misguided diagnosis due to aberrant immunoreactivity than to provide an unexpected correct diagnosis.

Panels.

There are no absolute rules for immunoreactivity in cells and tissues. Aberrant immunoreactivity or loss of immunoreactivity is occasionally observed for all antibodies, either due to biologic variability (e.g., occasional keratin-positive melanomas) or technical factors (e.g., impure antibodies, cross-reaction with other antigens, failure to preserve antigenicity). Thus, immunohistochemical markers are used most effectively as panels of markers with interpretation based on an immunohistochemical profile.

Slides for Immunohistochemistry.

Tissue is often dislodged from normal glass slides during the treatments required for IHC. Thus, slides must be coated (e.g., with glue, poly-L-lysine, gelatin, albumin) or special commercial slides must be used. If slides are being prepared by another laboratory, the type of glass slide to be used must be specified.

Factors Affecting Immunogenicity.

There are numerous variables that can affect antigenicity. The most common are listed below. Each laboratory must optimize its procedures for each antibody used. Studies on tissues or slides not prepared in the routine fashion for a laboratory must be interpreted with caution.

• Type of fixative. Some fixatives destroy some antigens (e.g., Bouin’s diminishes ER immunoreactivity, keratins are not well preserved in B5).¹ Most studies are based on formalin-fixed tissue. Results cannot be assumed to be equivalent for other fixatives.

• Length of time of fixation in formalin causes protein cross-linking, and antigenicity generally decreases with fixation times over 24 hours. To some extent, this effect can be reversed using antigen retrieval methods. Antigenicity can also be reduced if the tissue is fixed for too short a period (e.g., less than 6 hours).

• Prior decalcification in hydrochloric acid. Decreases antigenicity of some epitopes (predominantly nuclear) but not others (predominantly cytoplasmic).² Decalcifying agents using EDTA did not alter immunogenicity.

• Decreased: estrogen receptor (ER), progesterone receptor (PR), Ki-67, p53, BerEp4 (tumor cells), H blood group.

• Not affected: calcitonin, chromogranin, GCDFP-15, HMB 45, thyroglobulin, S100, prostate-specific antigen (PSA), keratins (CK 20, CAM5.2, AE1/AE3), A and B blood groups, others.

• Temperature of baking the slide.

• Length of time since the glass slide was cut. The immunoreactivity of the majority of antigens declines over days to weeks with potential complete loss at one month.^3,⁴ The loss may be due to exposure of tissue to air with oxidation of amino acids, as the immunogenicity of tissue deeper in the block can be preserved for many years. Antigen retrieval methods do not completely restore the antigenicity of old slides. Coating slides with paraffin, storing the slides in a nitrogen desiccator, and/or storing at lower temperatures can partially preserve antigenicity. However, studies should be performed on newly cut slides, if possible.

• Antigen retrieval procedures (e.g., proteolysis, heating [microwave, steam], special incubation fluids). To some extent these methods reverse the effects of formalin fixation. Variable effects are observed for different antibodies.

• Type of antibody (polyclonal vs. monoclonal vs. mixture of different monoclonals, epitope detected, mouse vs. rabbit). Very different results can be obtained with different antibodies to the same protein or different commercial sources of the same antibody.

• Incubation time, incubation temperature, dilution of antibody.

• Methods of signal amplification.

Controls.

Controls are essential for the appropriate interpretation of immunohistochemical studies and ensure that all steps of this complicated procedure have been performed adequately.

Positive controls consisting of tissues known to be immunoreactive should be included each time an antibody is used for a test case. Internal positive controls should always be evaluated when present, as they control not only for the technique used but also for the antigenicity of the tissue under investigation. The immunoperoxidase table lists normal cells that are generally immunoreactive for each antibody. Some laboratories have used vimentin as a control for immunogenicity as almost all tissue should demonstrate positivity.⁵ Given the wide and non-specific distribution of vimentin, smooth muscle alpha actin may be more useful in this context as pericytes, vascular smooth muscle, and myoepithelial cells present in most tissues are immunoreactive.

Examples of internal controls:

• S100: Normal nerves, melanocytes, and Langerhans cells in epidermis, cartilage, some myoepithelial cells, skin adnexa

• Estrogen and progesterone receptors: Normal luminal cells in ducts and lobules of the breast

• CD31, FVIII: Vascular endothelium

• c-KIT (CD117): Mast cells

• Smooth muscle alpha actin: Blood vessel walls, myoepithelial cells in the breast

• Vimentin: Blood vessels, stromal cells

• High molecular weight (MW) keratin: Squamous epithelium

• Low MW keratin: Glandular epithelium

• CD15: Polymorphonuclear leukocytes

Negative controls usually consist of replacing the primary antibody with non-immune animal serum diluted to the same concentration as the primary antibody. No positive reaction should be present. If multiple primary antibodies are used reactive with different target antigens, then they may serve as negative controls for each other. Although the best negative control would be to use antibody preabsorbed against the target antigen, this is rarely practical in a diagnostic laboratory. Diagnostic slides should also be evaluated for internal negative controls. Aberrant immunoreactivity of tissues that should not be positive is indicative that the immunoreactivity is nonspecific and the study should not be used for interpretation.

Evaluation of Studies

The following features must be taken into consideration when evaluating studies:

Location of Immunoreactivity

Antigens are present in specific sites. Some antigens may be present in more than one location or be extracellular.

Nonspecific positivity should be suspected when immunoreactivity is present in atypical locations:

• Background: Suspect nonspecific positivity if normal cells or noncellular components are positive. This can occur with suboptimal performance of the assay or suboptimal antibodies.

• Edge artifact: Antibodies can pool at edges or holes in tissue. True positivity should also be present in the center of the tissue.

• Necrosis or crushing of cells: Nonspecific positivity can be seen in disrupted cells. Although keratin is generally reliable in necrotic tumors, other markers generally should not be interpreted.

• Inappropriate location (e.g., cytoplasm instead of nucleus): Occasionally ER or PR are present in the cytoplasm instead of the nucleus. This is not interpreted as a positive result. Plasma cells have large amounts of cytoplasmic immunoglobulins that can crossreact with many antibodies.

• In rare cases, immunoreactivity in an unusual location is of diagnostic importance:

• TTF-1: Cytoplasmic (instead of nuclear) positivity in hepatocellular carcinomas.

• Ki-67 (MIB1): Cytoplasmic and membrane (instead of nuclear) positivity in trabecular hyalinizing adenomas of the thyroid and sclerosing hemangiomas of the lung.

• Beta-catenin: Nuclear (instead of cytoplasmic) positivity in solid pseudopapillary tumors of the pancreas and pancreatoblastomas. Both nuclear and cytoplasmic positivity is seen in the majority of colon carcinomas. Nuclear positivity is present in about 20% of endometrioid endometrial carcinomas and 70% of cases of desmoid-type fibromatosis.

• ALK: The pattern of immunoreactivity correlates with the different types of chromosomal translocations in anaplastic large cell lymphomas.

• NPM (nucleophosmin) shuttles between the cytoplasm and nucleus. NPM1 mutations occur in about 30% of adult AML and cause aberrant cytoplasmic expression of NPM (“NPMc+ AML”). These cases have a specific gene expression profile and distinctive clinical and prognostic features.

Examples of the normal location of antigens are shown in Figure 7-1.

Figure 7–1 Location of immunoreactivity (indicated in blue).

Identification of Immunoreactive Cells

Immunoreactivity of tumor cells must be distinguished from immunoreactivity of normal entrapped cells (e.g., desmin [+] skeletal muscle cells infiltrated by tumor, S100 [+] Langerhans cells in tumors, smooth muscle alpha actin [+] blood vessels, etc.). Plasma cells have large amounts of cytoplasmic immunoglobulin and can react nonspecifically with many antibodies.

Intensity of Immunoreactivity

Some weak immunoreactivity may be present as a nonspecific finding. It is important to compare positive cells with control slides and with normally nonimmunoreactive cells to determine whether the immunoreactivity is significant.

Number of Immunoreactive Cells

In some cases, the number of positive cells may be important as a criterion for positivity or as a prognostic marker (e.g., markers of proliferation such as Ki-67, HER2/neu). In other cases, rare weakly positive cells must be distinguished from intermingled normal cells or just nonspecific immunoreactivity.

Criteria for a “Positive” Result

Specific criteria for evaluating IHC have been developed for a few antibodies (see Tables 7-12 to 7-16). However, criteria do not exist for most antibodies or are not universally used by all pathologists. The significance of immunoreactivity varies with the type of lesion, the antibody, and the specific assay. Strong positivity in the majority of cells is easily interpreted as positive. As the number of positive cells decreases, and the intensity of immunoreactivity weakens, the lower threshold of a “positive” result becomes more difficult to determine.

Time

Alkaline phosphatase chromogens (red color) fade over time. DAB (a brown color) is more permanent. This is not a problem in evaluating current pathology specimens. However, if slides are reviewed after a period of time, some chromogens may fade and once positive results may appear to be negative.

Common Panels for Immunohistochemical Studies

The following tables include information from the literature as well as the personal experiences of the staff at Brigham and Women’s Hospital. Because of the many differences in specific antibodies, laboratory assays, and criteria for considering a result “positive,” results may vary for different institutions. The results have been divided into five categories for general markers and four categories for hematopathology markers. Note that “%” refers to the number of tumors reported to be positive, not the number of cells positive within a tumor (Table 7-2).

TABLE 7–2 EVALUATION OF POSITIVITY OF IMMUNOHISTOCHEMICAL STUDIES

The actual markers used to evaluate a case will depend upon the differential diagnosis based on the H&E appearance. In some cases, an initial panel, which is often used for typical cases, has been suggested. Not all markers listed would be used for all cases and some markers are included to indicate when they would not be useful for distinguishing the tumors listed in the table.

POSITIVE is defined as the presence of immunoreactive cells and NEGATIVE as the absence of immunoreactive cells. Unfortunately, “positive” has also been used in some studies to mean “absence of immunoreactivity” when this finding supports a diagnosis. For example, the absence of SMAD4 (DPC4) has been reported as a “positive” result for pancreatic carcinoma, as this marker is absent in the majority of these tumors. This method of reporting results becomes confusing as “positive” and “negative” are dependent on the expected diagnosis. It is preferable to report the findings (positive = immunoreactivity present; negative = immunoreactivity absent) and then interpret them as supporting, or not supporting, the diagnoses in the differential diagnosis.

Cytokeratin 7 and cytokeratin 20 tables

The combination of these two cytokeratins have been found to be useful to divide carcinomas into four main groups (Ck7+/Ck20+, Ck7+/Ck20-, Ck7-/Ck20+, Ck7-/Ck20-).

In Tables 7-3 to 7-7, other commonly used antibodies have been included to show differences within each group. The most useful additional antibodies will depend on the specific differential diagnosis.⁶^-⁸

TABLE 7–3 PREDOMINANTLY CK7+/CK20+

TABLE 7–4 NO DOMINANT CK7/CK20 PATTERN OR PATTERN UNKNOWN

TABLE 7–5 PREDOMINANTLY CK7+/CK20−

TABLE 7–6 CK7−/CK20+

TABLE 7–7 PREDOMINANTLY CK7−/CK20–

Small blue cell tumors

See Table 7-8.

TABLE 7–8 SMALL BLUE CELL TUMORS

Spindle cell lesions, soft tissue lesions, and sarcomas

See Table 7-9.

TABLE 7–9 SPINDLE CELL/SOFT TISSUE LESIONS/SARCOMAS

Metastatic tumors of unknown origin

Pathologists frequently receive specimens with metastatic tumors. Often, the site of origin is known to the clinician, but this information is not provided to the pathologist. A good clinical history is frequently more successful for correct classification than a battery of studies.

The Ck7/Ck20 pattern is generally helpful to narrow down the potential site of origin of carcinomas (see Tables 7-3 to 7-7). Additional studies can then be used to identify specific types of carcinoma.

The most important tumors to identify are those with specific therapeutic treatments for cure or palliation (Table 7-10).¹⁰

TABLE 7–10 METASTATIC TUMORS OF UNKNOWN ORIGIN

Poorly differentiated tumors

See Table 7-11.

TABLE 7–11 POORLY DIFFERENTIATED TUMORS

TYPE OF TUMOR	IMMUNOHISTOCHEMICAL MARKER	COMMENTS
Carcinoma	Broad spectrum keratins AE1/AE3 or PANK (MNF-116)	Some carcinomas may express unusual keratin sub-types. If negative, try other keratin types (e.g., CAM5.2). The Ck7/Ck20 pattern may be helpful in determining the likely site of origin. Some non-carcinomas can have an epithelioid appearance and strongly express keratins (e.g., epithelioid angiosarcoma, epithelioid sarcoma, mesothelioma).
Melanoma	S100 protein	S100 is strongly positive in the vast majority of melanomas. Some carcinomas (especially breast) and sarcomas are also positive for S100 and additional markers may be required. HMB-45 and MART-1 are expressed by most epithelioid melanomas but may be focal or absent in non-epithelioid melanomas (e.g., spindle cell or desmoplastic melanomas).
Lymphoma	Leukocyte common antigen (LCA)	Present in almost all non-Hodgkin’s lymphomas. May be absent in 30% of anaplastic (Ki-1) large cell lymphomas. These lymphomas are keratin negative but may express EMA. These tumors will be positive for CD30 (Ki-1) and ALK.

Estrogen and progesterone receptor evaluation

Hormone receptors are routinely determined on all invasive breast carcinomas and DCIS. ER and PR are weak prognostic markers and are more useful to predict the likelihood of response to hormonal therapies.

Many different methods are currently used to report the results of IHC studies for ER and PR. One method that has been used in multiple studies is the Allred score method (Table 7-12).

TABLE 7–12 ER AND PR—ALLRED SCORE

Patients with carcinomas that scored 3 (<1% of cells with intermediate intensity or 1% to 10% of cells with weak intensity) or above had improved disease-free survival when treated with endocrine therapy.¹¹ Patients with carcinomas with a total score of 2 (<1% weakly positive cells) had a survival rate similar to ER-negative carcinomas (total score of 0).

About 80% of DCIS cases are positive for ER using the same method of scoring. Women with ER-positive DCIS were shown to experience fewer local recurrences, contralateral recurrences, and distant recurrences when treated with tamoxifen (NSABP B24 study).

With optimization of IHC using newer antigen retrieval methods, 99.2% of carcinomas will have scores of 0, 7, or 8.¹² Therefore, many laboratories report results as positive or negative. The value of further subdividing cases by percent positive cells, H-score, or image analysis for either prognosis or to predict response to tamoxifen has not been demonstrated. Intensity of immunoreactivity is difficult to evaluate as most cases show strong reactivity with optimal assay methods and most carcinomas show considerable variability in intensity.

A possible method for reporting results is shown in Table 7-13. The same system can be used for reporting progesterone receptor results. The use of both ER and PR may be helpful for determining the likelihood of response to tamoxifen, as has been shown with data using the biochemical assay (Table 7-14). Presumably, the presence of the ER-regulated gene product PR is more predictive of an intact ER regulatory pathway.

TABLE 7–13 REPORTING RESULTS OF ER AND PR EVALUATION

TABLE 7–14 RESPONSE TO TAMOXIFEN

STATUS OF CARCINOMA	% OF CARCINOMAS	% OF PATIENTS RESPONDING TO TAMOXIFEN
ER+ PR+	63%	75% to 80%
ER+ PR–	15%	25% to 30%
ER– PR+	5%	40% to 45%
ER– PR–	17%	<10%

Recent national guidelines for reporting ER and PR have been released and should be consulted for additional information about performing and interpreting these studies (Hammond ME, et al, American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer, J Clin Oncol 2010 Apr 19).False negative results, and to a lesser extent, false positive results, can also be problems. False negative results may be due to a large number of causes including:

• Low sensitivity of the assay

• Errors in performing the assay

• Delayed fixation of tissue

• Over- or underfixation of tissue

• Overheating of tissue (e.g., with cautery during surgery)

• Decalcification of tissue

Most cases of false negativity can be suspected, as the normal breast tissue will also be negative. In such cases, the assay should be repeated on the same block, a different block from the same case, or blocks from another case, if available. If the normal tissue remains negative, the possibility of loss of antigenicity in the tissue can be mentioned in the report.

False positive results are quite unusual, as the antibody should not crossreact with other antigens.

• Entrapped normal ducts or lobules misinterpreted as carcinoma — this can be a difficult issue for DCIS as some ducts or lobules may be only partially involved by DCIS.

• Control placed on same slide misinterpreted as the carcinoma

• Sclerosing adenosis or myofibroblastoma (or other benign lesions) misinterpreted as invasive carcinoma

HER2/neu score

The HER2/neu immunoreactivity scoring system in Table 7-15 was recommended by an expert panel.¹³ Other panel suggestions:

• If cytoplasmic positivity obscures the membrane pattern, repeat the assay or perform FISH.

• If normal ducts and lobules show definitive positivity, repeat the assay.

• In cases of invasive carcinoma, only the areas of invasion should be scored. In some cases the associated DCIS can show stronger immunoreactivity.

• Fixation must be in neutral buffered formalin and should, ideally, be between 6 and 48 hours for excisions, and at least 1 hour for needle biopsies. However, any effect from longer fixation has not been shown.

• Unstained slides should not be used if prepared >6 weeks earlier. Loss of antigenicity has been shown for other antigens, but not specifically for HER2.

TABLE 7–15 HER2-IHC SCORING

Only membrane immunoreactivity is scored. Marked cytoplasmic immunoreactivity may make interpretation difficult. FISH studies may be preferred for such cases (Table 7-16).

TABLE 7–16 FISH RESULTS

RESULT	CRITERIA	comment
Positive for amplification	>6.0 gene copies or >2.2 ratio
Equivocal for amplification	4.0 to 6.0 genes or 1.8 to 2.2 ratio	Guidelines suggest counting additional cells for FISH, retesting, or performing IHC
Negative for amplification	<4.0 genes or <1.8 ratio

Patients with a ratio of 2.0 or greater have been eligible for Herceptin trials.

In >90% of carcinomas with protein overexpression, the HER2/neu gene has been amplified. In 3% to 5% of cases, protein overexpression can occur due to other mechanisms. In <5% of cases, there may be gene amplification without protein overexpression. In general, there is a 20% to 40% response to Herceptin alone in patients with cancers showing gene amplification by FISH, and <5% of patients respond if the gene is not amplified. Therefore, FISH studies may be helpful for cases with 2+ positivity or difficult to interpret cases (e.g., with variable positivity or cytoplasmic positivity).

Well- and moderately differentiated lobular carcinomas are rarely positive (<5%). However, in some cases there may be edge enhancement of individual tumor cells that may be difficult to interpret. FISH studies may be helpful.

In rare cases, DCIS overexpresses HER2/neu but the accompanying invasive carcinoma does not. This is a source of potential false positive results for IHC or FISH.

Myoepithelial markers in breast carcinoma

Myoepithelial markers can be useful for the evaluation of breast lesions (Table 7-17):

• Invasive carcinoma vs. sclerosing adenosis (frequently involved by DCIS, LCIS, or apocrine metaplasia).

• DCIS vs. DCIS with microinvasion – Double immunolabeling with p63 (brown nuclear) and cytokeratin (AE1/AE3 – red cytoplasm) can be useful to highlight small nests of tumor cells lacking myoepithelial cells. A double stain with SMMHC and cytokeratin AE1/AE3 can also be helpful.

• DCIS vs. carcinoma invading as circumscribed tumor nests vs. lymphovascular invasion.

• Microglandular adenosis is the only “benign” breast lesion that lacks myoepithelial cells. However, this lesion may be a form of well-differentiated non-metastasizing invasive carcinoma. The cells are negative for ER and PR and strongly positive for S100.

TABLE 7–17 MYOEPITHELIAL MARKERS IN BREAST CARCINOMA

Epidermal lesions of the nipple

See Table 7-18.

TABLE 7–18 EPIDERMAL LESIONS OF THE NIPPLE AND PAGET DISEASE AT OTHER SITES

Breast carcinoma in males versus metastatic prostate carcinoma

See Table 7-19.

TABLE 7–19 BREAST CARCINOMA IN MALES VERSUS METASTATIC PROSTATE CARCINOMA

Signet ring cell carcinomas of the stomach and breast (lobular carcinoma)

See Table 7-20 and Fig. 7-2.

TABLE 7–20 SIGNET RING CELL CARCINOMAS OF THE STOMACH AND BREAST (LOBULAR CARCINOMA)

Figure 7–2 Metastatic lobular carcinoma of the breast can morphologically resemble primary signet ring cell gastric carcinomas. Both typically lack e-cadherin expression. In addition, lobular breast carcinomas can be clinically occult or can present as distant metastases many years after the initial presentation. Signet ring cells associated with breast carcinoma more commonly have a central mucin vacuole with a targetoid appearance (cell A). Gastric signet ring cells usually have many small vacuoles giving the cytoplasm a foamy appearance (cell B). These criteria are not reliable in distinguishing these two carcinomas. However, the presence of the first type of signet ring cell in a biopsy from the gastrointestinal tract should raise the possibility of metastatic breast carcinoma.¹⁴ The majority of lobular breast carcinomas will be ER positive, and this is a reliable marker to exclude gastric carcinoma. In the minority of ER-negative cases, PR, GCDFP, MUC1, CDX2, and Hep Par may be helpful markers.¹⁵

Fibroblastic/myofibroblastic lesions of the breast

See Table 7-21.

TABLE 7–21 FIBROBLASTIC/MYOFIBROBLASTIC LESIONS OF THE BREAST

Ovarian carcinoma versus mesothelioma

See Table 7-22.

TABLE 7–22 OVARIAN CARCINOMA VERSUS MESOTHELIOMA

Primary ovarian carcinoma versus metastatic carcinomas

See Table 7-23.

TABLE 7–23 PRIMARY OVARIAN CARCINOMA VERSUS METASTATIC CARCINOMAS

Endocervical carcinoma versus endometrial carcinoma

See Table 7-24.

TABLE 7–24 ENDOCERVICAL CARCINOMA VERSUS ENDOMETRIAL CARCINOMA

Endometrial stromal sarcoma versus leiomyosarcoma

See Table 7-25.

TABLE 7–25 ENDOMETRIAL STROMAL SARCOMA VERSUS LEIOMYOSARCOMA

Trophoblastic lesions

See Table 7-26.

TABLE 7–26 TROPHOBLASTIC LESIONS

Metastatic adenocarcinomas in the abdomen

See Table 7-27.

TABLE 7–27 METASTATIC ADENOCARCINOMAS IN THE ABDOMEN

CNS neoplasms

See Table 7-28.

TABLE 7–28 CNS NEOPLASMS

Hemangioblastoma versus metastatic renal cell carcinoma

See Table 7-29.

TABLE 7–29 HEMANGIOBLASTOMA VERSUS METASTATIC RENAL CELL CARCINOMA

Tumors of germ cells and sex-cord stromal tumors

See Table 7-30.

TABLE 7–30 TUMORS OF GERM CELLS AND SEX-CORD STROMAL TUMORS

Adrenal and kidney tumors

See Table 7-31.

TABLE 7–31 ADRENAL AND KIDNEY TUMORS

Tumors of bladder, prostatic, and renal origin

See Table 7-32.

TABLE 7–32 TUMORS OF BLADDER, PROSTATIC, AND RENAL ORIGIN

Prostate carcinoma versus other lesions

See Table 7-33.

TABLE 7–33 PROSTATE CARCINOMA VERSUS OTHER LESIONS

Hepatic tumors

See Table 7-34.

TABLE 7–34 HEPATIC TUMORS

Thyroid and parathyroid lesions

See Table 7-35.

TABLE 7–35 THYROID AND PARATHYROID LESIONS

Differential diagnosis of epithelial mesothelioma and lung adenocarcinoma

See Table 7-36.

TABLE 7–36 DIFFERENTIAL DIAGNOSIS OF EPITHELIAL MESOTHELIOMA AND LUNG ADENOCARCINOMA

Initial panel: AE1/AE3, calretinin, WT-1 (clone 6F-H2), CEA, Leu-M1, and TTF-1 with additional studies ordered in difficult cases.

Other antibodies generally reported as negative in epithelial mesotheliomas and positive in lung adenocarcinomas include the following: MOC-1, B72.3, Ber-EP4, and BG-8. Cytokeratins 5/6 are reported to be positive in mesotheliomas and negative in lung carcinomas. However, in our experience, these markers have proven less useful than the ones listed earlier. The use of EMA is controversial. Strong membrane positivity is characteristic of epithelial mesothelioma, whereas cytoplasmic positivity is characteristic of adenocarcinomas.

Less is known about the immunophenotype of pure sarcomatoid mesotheliomas. The spindle cells are positive for cytokeratin, but are less frequently positive for the other markers as compared to the epithelioid cells. Tumors that can, on occasion, resemble mesotheliomas are generally negative for cytokeratins, with the notable exceptions of some cases of angiosarcoma, epithelioid hemangioendothelioma, synovial sarcoma, epithelioid sarcoma, and leiomyosarcoma (see Table 7-9).¹⁶

Lung carcinoma

See Tables 7-37 and 7-38.

TABLE 7–37 LUNG CARCINOMAS

TABLE 7–38 DIFFERENTIAL DIAGNOSIS OF LUNG CARCINOMAS

DIFFERENTIAL DIAGNOSIS	MOST USEFUL MARKERS
Adenocarcinoma vs. squamous cell carcinoma	Keratin 7, keratin 20, TTF-1, p63
Small cell carcinoma vs. basaloid squamous cell carcinoma	P63, TTF-1
Small cell carcinoma vs. carcinoid tumor	Mitoses, necrosis, amount of cytoplasm
Large cell neuroendocrine carcinoma vs. carcinoid tumor	Mitoses, necrosis
Mucinous lung carcinoma vs. metastatic colon cancer	TTF-1, CDX2 (mucinous BAC can be focally positive for CDX2)
Lung carcinoma vs. metastatic breast carcinoma	TTF-1, compare ER/PR/HER2 pattern in primary breast carcinoma and lung tumor

B-cell neoplasms

See Table 7-39.

TABLE 7–39 B-CELL NEOPLASMS

T-cell neoplasms

See Table 7-40.

TABLE 7–40 T-CELL NEOPLASMS

Hodgkin lymphoma

See Table 7-41.

TABLE 7–41 HODGKIN LYMPHOMA

Amyloid

Amyloidosis (Greek for amylon = starch plus eidos = resemblance) is seen in many different clinical settings and is associated with many diseases. Pathologists can narrow down the differential diagnosis considerably to help guide clinical decision making. Finding an amyloid deposit in any tissue is similar to finding metastatic carcinoma in a lymph node – in both settings clinical information (e.g., history, physical examination, radiology studies, results of laboratory tests) is essential in arriving at the correct interpretation. A little immunohistochemistry and a lot of clinical judgment by the pathologist can help establish the cause with a greater degree of certainty.¹⁷

Finding and characterizing amyloid deposits:

1. Examine the H&E slide for noncellular material in the correct location for the suspected disease (Table 7-42).

2. Amyloid deposits will be orange-pink on Congo Red stains or sea-foam green on Sulfated Alcian blue stains. Amyloid may be more apparent on these stains. HOWEVER, beware of overcalling cases in which there is not a histologic correlate for amyloid in the stained tissue. If there is background positivity in normal tissue due to overstaining, the slide cannot be interpreted. Positive controls must show appropriate specific positivity.

3. Congo red-positive amyloid should become an apple green color when viewed under polarized light. This may require the high-quality polarizers that are built in to the microscope. Lower quality polarizers (i.e., the cut squares of polarizing material) may not be adequate. Collagen (silver when H&E is polarized) and fibrin (does not polarize) may mimic amyloid.

4. The amyloid deposits can be further characterized using immunohistochemistry or immunofluorescence (see Table 7-42) based on the clinical information, the organ or structures involved, and the distribution of amyloid deposits in the tissue. Amyloid can also be identified using EM (non-branching fibrils, 7.5 to 10 nm width and up to 1 micron in length).

5. A firm diagnosis is not always possible. The final diagnosis should be based on a combination of histologic, immunohistochemical, and clinical data.

TABLE 7–42 AMYLOID

Antibodies for immunohistochemistry

See Tables 7-43 and 7-44.

TABLE 7–43 ANTIBODIES FOR IMMUNOHISTOCHEMISTRY

TABLE 7–44 ALTERNATIVE NAMES FOR ANTIGENS

LOOKING FOR?	FIND IT UNDER:
1D5	Estrogen receptor (G)
6F/3D	Beta-amyloid
12E7	CD99 (G, H)
34βE12	Keratins (G)
38.13	CD77(H)
70 kD NF	Neurofilaments (G)
200 kD NF	Neurofilaments (G)
903	Keratins--34βE12 (G)
A (blood group antigen)	Blood group antigens (G)
A (Ig heavy chain alpha)	Heavy chain immunoglobulins (H)
A32 antigen	CD146 (G)
A103	MELAN-A (G)
AAT	Alpha 1-antitrypsin (G, H)
ACH	Alpha-1 antichymotrypsin (H)
AE1/AE3	Keratins (G)
AFP	Alpha-fetoprotein (G)
Alpha 1-antitrypsin	Alpha 1-antitrypsin (G, H)
Alpha 1-antichymotrypsin	Alpha 1-antichymotrypsin (H)
Alpha 1-fetoprotein	Alpha fetoprotein (G)
Alpha fetoprotein	Alpha fetoprotein (G)
Alpha-methylacyl-CoA racemase	AMACR (G)
Alpha smooth muscle actin	Alpha smooth muscle actin (G)
AMACR	AMACR (G)
Amyloid	Beta-amyloid (G)
Androgen receptor	Androgen receptor (G)
Apolipoprotein J	Clusterin (H)
AR	Androgen receptor (G)
B (blood group antigen)	Blood group antigens (G)
B1	CD20 (H)
B2	CD21 (H)
B4	CD19 (H)
B72.3	B72.3 (G)
bcl-1	Cyclin D1 (H)
bcl-2	bcl-2 (H, G)
B-cell specific activator protein	BSAP (H)
BER-EP4	BER-EP4 (G)
BERH2	CD30 (G, H)
Beta-amyloid	Beta-amyloid (G)
Beta-catenin	Beta-catenin (G)
Beta-2 microglobulin	Beta-2 microglobulin (G)
BG8	BG8 (G)
B-HCG	Human chorionic gonadotropin (G)
BLA.36	CD77 (H)
BL-CAM	CD22 (H)
Blood group antigens	Blood group antigens (G, H)
BR-2	Gross cystic disease fluid protein-15 (G)
BRST-2	Gross cystic disease fluid protein-15 (G)
C3b/C4bR	CD35 (H)
C5b-9	C5b-9 (G)
c-kit	CD117 (G)
CA 15-3	Epithelial membrane antigen (G, H)
CA 19-9	CA 19-9 (G)
CA 27.28	Epithelial membrane antigen (G, H)
CA 72-4	B72.3 (G)
CA125	CA125 (G)
CA19-9	CA19-9 (G)
Calcitonin	Calcitonin (G), Hormones (G)
Caldesmon	Caldesmon (G)
Calgranulin	MAC 387 (G)
CALLA	CD10 (G, H)
CALP	Calponin (G)
Calponin	Calponin (G)
Calprotectin	MAC 387 (G)
Calretinin	Calretinin (G)
CAM5.2	Keratins (G)
Carbohydrate antigen 19-9	CA19-9 (G)
Carcinoembryonic antigen	Carcinoembryonic antigen (G)
CD1a	CD1a (H)
CD2	CD2 (H)
CD3	CD3 (H)
CD4	CD4 (H)
CD5	CD5 (G, H)
CD7	CD7 (H)
CD8	CD8 (H)
CD10	CD10 (G, H)
CD11b	CD11b (H)
CD11c	CD11c (H)
CD13	CD13 (H)
CD15	CD15 (G, H)
CD16	CD16 (H)
CD19	CD19 (H)
CD20	CD20 (H)
CD21	CD21 (H)
CD22	CD22 (H)
CD23	CD23 (H)
CD25	CD25 (H)
CD30	CD30 (G, H)
CD31	CD31 (G)
CD33	CD33 (H)
CD34	CD34 (G, H)
CD35	CD35 (H)
CD38	CD38 (H)
CD43	CD43 (H)
CD44v3	CD44v3 (G)
CD45	CD45 (H)
CD45RA	CD45RA (H)
CD45Ro	CD45Ro (H)
CD56	CD56 (H)
CD57	CD57 (G)
CD61	CD61 (H)
CD68	CD68 (G, H)
CD74	CD74 (H)
CDw75	CDw75 (H)
CD77	CD77 (H)
CD79a	CD79a (H)
CD79b	CD79b (H)
CD95	CD95 (H)
CD99	CD99 (G, H)
CD117	CD117 (G)
CD141	CD141 (G)
CDX	CDX (G)
CDKN2	p16 (G)
CDP	Gross cystic disease fluid protein-15 (G)
CEA	Carcinoembryonic antigen (G)
c-erbB2	HER-2/neu (G)
Chromogranin A	Chromogranin A (G)
c-kit	CD117 (G)
CLA	CD45 (H) or HECA-452 (H)
CLDN1	Claudin (G)
Clusterin	Clusterin (H)
Collagen IV	Collagen IV (G)
Common acute leukemia antigen	CD10 (G, H)
Complement lysis inhibitor	Clusterin (H)
CR1	CD35 (H)
Cyclin D1	Cyclin D1 (H)
Cystic fibrosis antigen	MAC 387 (G)
D (Ig heavy chain delta)	Heavy chain immunoglobulins (H)
DBA.44	DBA.44 (H)
Desmin	Desmin (G)
DF3	Epithelial membrane antigen (G, H)
DPB	CD45RA (H)
E2 antigen	CD99 (G, H)
EBERS	Epstein-Barr virus (G, H)
EBNA	Epstein-Barr virus (G, H)
E-cadherin	E-cadherin (G)
EGFR	EGFR (G)
EM ACT	HHF-35 (G)
EMA	Epithelial membrane antigen (G)
E-MEL	HMB-45 (G)
Endothelial cell antigen	HECA-452 (H)
Ep-CAM	BER-EP4 (G)
Epidermal growth factor receptor	EGFR (G)
Epithelial membrane antigen	Epithelial membrane antigen (G, H)
Epithelial specific antigen	BER-EP4 (G)
Epstein-Barr virus	Epstein-Barr virus (G, H)
ER	Estrogen receptor (G)
erbB2	HER-2/neu (G)
ESA	BER-EP4 (G)
Estrogen receptor	Estrogen receptor (G)
Ewing’s sarcoma marker	CD99 (G, H)
Factor VIII related antigen	Factor VIII (G)
FVIII:RAg	Factor VIII (G)
Factor XIIIa	Factor XIIIa (G)
Fascin	Fascin (H)
Fast myosin	Myosin heavy chain (G)
Fibronectin	Fibronectin (G)
Fli-1	Fli-1 (G)
FMC7	FMC7 (H)
FMC 29	CD99 (G, H)
Friend leukemia integrin-site 1	Fli-1 (G)
FVIII:g	Factor VIII (G)
G (Ig heavy chain gamma)	Heavy chain immunoglobulins (H)
Gal-3	Galectin-3 (G)
Galectin-3	Galectin-3 (G)
Gastrin	Hormones (G)
GCDFP	Gross cystic disease fluid protein-15 (G)
GFAP	Glial fibriallary acidic protein (G)
Glial fibrillary acidic protein	Glial fibriallary acidic protein (G)
Glucagon	Hormones (G)
Glucose transporter 1	GLUT-1 (G)
GLUT-1	GLUT-1 (G)
GPIIIa	CD61 (H)
gp80	Clusterin (H)
gp200	RCC (G)
GPA	Glycophorin A (H)
Granzyme B	Granzyme B (H)
Gross cystic disease fluid disease-15	Gross cystic disease fluid protein-15 (G)
H (blood group antigen)	Blood group antigens (G)
H222	Estrogen receptor (G)
Hb	Hemoglobin (H)
HBME-1	HBME-1 (G)
h-caldesmon	Caldesmon (G)
H-CAM	CD44v3 (G)
HCG	Human chorionic gonadotropin (G)
HCL	DBA.44 (H)
HBME-1	HBME-1 (G)
Heavy chain immunoglobulins	Heavy chain immunoglobulins (H)
HECA-452	HECA-452 (H)
Hematopoietic progenitor cell, class 1	CD34
Hemoglobin	Hemoglobin (H)
HepPar-1	HepPar-1 (G)
Hepatocyte paraffin-1	HepPar-1 (G)
HER-2/neu	HER-2/neu (G)
HHF-35	HHF-35 (G)
HHV8	HHV8 (H)
HLA-DR	HLA-DR (H)
HMB-45	HMB-45 (G)
HMFG	Epithelial membrane antigen (G, H)
hMLH1	hMLH1 (G)
hMSH2	hMLH1 (G)
HNK-1	CD57 (G)
HP1	HepPar-1 (G)
HPCA-1	CD34 (G, H)
HPL	Human placental lactogen (G)
HuLy-m6	CD99 (G, H)
Human chorionic gonadotropin	Human chorionic gonadotropin (G)
Human herpes virus 8	HHV8 (G, H)
Human mutL homologue 1	hMLH1 (G)
Human mutS homologue 2	hMLH1 (G)
Human placental lactogen	Human placental lactogen (G)
IL-2 receptor	CD25 (H)
Inhibin-alpha subunit	Inhibin-alpha subunit (G)
Insulin	Hormones (G)
J5	CD10 (G, H)
JOVI 1	TCR (H)
K (Ig lighit chain kappa)	Light chain immunoglobulins (H)
Keratin 5/6	Keratins (G)
Keratin 7	Keratins (G)
Keratin 20	Keratins (G)
Keratins	Keratins (G)
Ki-1	CD30 (G, H)
Ki-67	Ki-67 (G)
kip2	p57 (G)
Kit	CD117 (G)
KP-1	CD68 (G, H)
L (Ig light chain lambda)	Light chain immunoglobulins (H)
L1 antigen	MAC 387 (G)
L26	CD20 (H)
L60	CD43 (H)
Laminin	Laminin (G)
LCA	CD45 (H)
Leu 1	CD5 (H)
Leu 2	CD8 (H)
Leu 3	CD4 (H)
Leu 5a + b	CD2 (H)
Leu 7	CD57 (G, H)
Leu 9	CD7 (H)
Leu16	CD20 (H)
Leu 22	CD43 (H)
Leukocyte common antigen	CD45 (H)
Leu-M1	CD15 (G, H)
Light chain immunoglobulins	Light chain immunoglobulins (H)
LFA-2	CD2 (H)
LMP-1	Epstein-Barr virus (G, H)
LN1	CDw75 (H)
LN2	CD74 (H)
Lysozyme	Lysozyme (H, G)
M (Ig heavy chain mu)	Heavy chain immunoglobulins (H)
Mac-1	CD11b (H)
MAC 387	MAC 387 (G)
Mac-M	CD68 (G, H)
MART 1	MELAN A (G)
Mast cell tryptase	Mast cell tryptase (H)
mb-1	CD79a (H)
MCAM	CD146 (G)
ME491	CD63 (G)
MELAN-A	MELAN-A (G)
Melanoma antigen recognized by T cells	MELAN-A (G)
Melanoma-associated antigen	CD63 (G)
Melanoma cell adhesion molecule	CD146 (G)
Melanoma-specific antigen	HMB-45 (G)
MELCAM (or Mel-CAM)	CD146 (G)
MIB-1	Ki-67 (G)
MIC-2	CD99 (G, H)
MLH1	hMLH1
MN-4	CD146 (G)
MNF-116	Keratin--Pan-K (G)
MPO	Myeloperoxidase (H)
MRF4	Myf-4 (G)
MSA	HHF-35 (G)
MSH2 or MSH6	hMLH1
MTS1	p16 (G)
MUC1	Epithelial membrane antigen (G, H)
MUC18	CD146 (G)
Muscle common actin	HHF-35 (G)
Muscle specific actin	HHF-35 (G)
My 7	CD13 (H)
My 9	CD33 (H)
Myeloperoxidase	Myeloperoxidase (H)
Myf-4	Myf-4 (G)
MyoD1	MyoD1 (G)
Myogenin	Myf-4 (G)
Myoglobin	Myoglobin (G)
Myosin heavy chain	Myosin heavy chain (G)
NCAM	CD56 (H)
Neprilysin	CD10 (G, H)
NEU N	NEU N (G)
Neurofilaments	Neurofilaments (G)
Neuron specific enolase	Neuron specific enolase (G)
NFP	Neurofilaments (G)
NKI-betab	HMB-45 (G)
NKI/C3	CD63 (G)
NSE	Neuron specific enolase (G)
O13	CD99 (G, H)
OC125	CA125 (G)
Oct2	Oct2 (H)
Octomer transcription factor	Oct2 (H)
p16	p16 (G)
p27^kip1	p27^kip1 (G)
p53	p53 (G)
p57	p57 (G)
p63	p63 (G)
P504S	AMACR (G)
PAN-K	Keratins (G)
PAP	Prostate acid phosphatase (G)
PECAM-1	CD31 (G)
PEM	Epithelial membrane antigen (G, H)
Perforin	Perforin (H)
PGM1	CD68 (G, H)
PgR	Progesterone receptor (G)
PK antigen	CD77 (H)
Placental alkaline phosphatase	Placental alkaline phosphatase (G)
PLAP	Placental alkaline phosphatase (G)
Platelet glycoprotein IIIa	CD61 (H)
PMS2	hMLH1
Podoplanin	D2-40
PR	Progesterone receptor (G)
PRAD1	Cyclin D1 (H)
PrAP	Prostate acid phosphatase (G)
Prealbumin	Prealbumin (G)
Progesterone receptor	Progesterone receptor (G)
Prostate acid phosphatase	Prostate acid phosphatase (G)
Prostate specific antigen	Prostate specific antigen (G)
PSA	Prostate specific antigen (G)
QBEnd10	CD34 (G, H)
Renal cell carcinoma marker	RCC (G)
ret	ret (G)
RCC	RCC (G)
rT3	CD2 (H)
S-100	S-100 (G)
S-Endo-1	CD146 (G)
SGP-2	Clusterin (H)
SMA	Alpha smooth muscle actin (G)
SM-ACT	Alpha smooth muscle actin (G)
Smad4	DPC4 (G)
SM-MHC	Myosin heavy chain (G)
Somatostatin	Hormones (G)
SP40	Clusterin (H)
Stem cell factor receptor	CD117 (G)
Synaptophysin	Synaptophysin (G)
Syndecan-1	CD138 (H)
Synuclein-1	Synuclein-1 (G)
T3	CD3 (H)
T4	CD4 (H)
T6	CD1a (H)
T8	CD8 (H)
T11	CD2 (H)
T64	Clusterin (H)
TAG-72	B72.3 (G)
Tau	Tau (G)
T cell antigen receptor	TCR (H)
T cell intracellular antigen	TIA-1 (H)
TCR	TCR (H)
TdT	Terminal deoxytransferase (H)
TE	CD2 (H)
Terminal deoxytransferase	Terminal deoxytransferase (H)
TH	CD4 (H)
Thrombomodulin	CD141 (G)
Thyroglobulin	Thyroglobulin (G)
Thyroid transcription factor 1	TTF-1 (G)
TIA-1	TIA-1 (H)
TM	CD141 (G)
traf-1	traf-1 (H)
Transthyretin	Prealbumin (G)
TRPM2	Clusterin (H)
TTF-1	TTF-1 (G)
TTR	Prealbumin (G)
Tumor-associated glycoprotein 72	B72.3 (G)
Tumor necrosis factor receptor–associated factor	traf-1 (H)
UCHL-1	CD45Ro (H)
UEA 1	Ulex (G)
Ulex	Ulex (G)
Vimentin	Vimentin (G)
von Willebrand’s factor	Factor VIII (G)
VWF	Factor VIII (G)
Wilms’ tumor 1 protein	WT1 (G)
WT1	WT1 (G)

G, general markers; H, hematopathology markers.

Results

The results of studies are incorporated into the surgical pathology report. The following information is included:

1. The type of tissue studied: formalin-fixed (or other fixatives) tissue, cryostat sections, cytology preparations, etc.

2. The type of immunoagents used, being as specific as possible. For example, do not just list keratin but specify the type of keratin (e.g., AE1/AE3).

3. The results of the studies in great enough detail to allow interpretation. For example the type of cell that is immunoreactive (e.g., tumor vs. nontumor), intensity of immunoreactivity (e.g., weak, strong) and/or the number of cells immunoreactive (e.g., focal vs. diffuse).

4. Integration of the results into the final diagnosis specifying whether they confirm or support a diagnosis, make one diagnosis more likely than others, or exclude one or more diagnoses.

ELECTRON MICROSCOPY

Indications for EM Studies

• Diagnostic renal biopsies for glomerular disease

• Adenocarcinoma versus mesothelioma (see Table 7-36)

• Difficult to classify tumors (Tables 7-45 and 7-46)

• Nerve (e.g., toxic or drug-induced neuropathy) and muscle biopsies (e.g., inclusion body or nemaline myopathy)

• Bullous skin diseases (e.g., epidermolysis bullosa)

• Ciliary dysmorphology (primary ciliary dyskinesia or Kartagener syndrome)

• Endomyocardial biopsies (e.g., adriamycin toxicity, amyloid, nemaline myopathy)

• Liver biopsies for microvesicular fat in acute fatty liver of pregnancy

• Small bowel biopsies to look for pathogens (e.g., Whipple disease)

• Congenital, inherited, and metabolic diseases (e.g., ceroid lipofuscinoses)

• Prion diseases

TABLE 7–45 ELECTRON MICROSCOPIC FEATURES OF POORLY DIFFERENTIATED TUMORS

TABLE 7–46 CELLS, TUMORS, AND STRUCTURES WITH CHARACTERISTIC FINDINGS BY ELECTRON MICROSCOPY

TUMOR	EM FINDINGS	CORRELATIONS AND OTHER DIAGNOSTIC TESTS
Alveolar soft part sarcoma	Rhomboid, rod-shaped, or spiculated crystals in a regular latttice pattern.	The characteristic cytoplasmic crystals are composed of monocarboxylate transporter 1 (MCT1) and its chaperone CD147. These proteins are found in many other cell types and are not specific for this tumor. Cytogenetics: t(X;17) creates a ASPL-TFE3 fusion protein. IHC: TFE3 positive (as well as rare pediatric renal tumors with the same translocation). Nuclear immunoreactivity is not present in other tumors or normal tissues. Histo: The crystals are PAS with diastase positive.
Amyloid	Non-branching fibrils, 7.5 to 10 nm in width and up to 1 micron in length.	May be present associated with plasma cell tumors, medullary carcinoma of the thyroid, Alzheimer’s disease, or as an isolated finding (primary amyloidosis). IHC: Can be used to identify specific types of amyloid (e.g., lambda or kappa chains, beta2 microglobulin, calcitonin, tau)
Bronchioloalveolar carcinoma of the lung (BAL)	Lamellar (surfactant) “myelin-like” granules in the supranuclear cytoplasm (typical of Type II pneumocytes). Clara-like electron-dense granules in supranuclear cytoplasm. Intranuclear inclusions comprised of parallel microtubular arrays. These features can also be seen in metastatic BAL.	Cytogenetics: These carcinomas are less likely to be associated with smoking and have fewer cytogenetic changes. Bronchioloalveolar carcinomas or adenocarcinomas with features of BAL are more likely to respond to Iressa (38%) as compared to other lung carcinomas (14%) due to specific mutations in EGFR. Mucinous BAL has intranuclear inclusions but generally lacks the other EM features.
Chordoma	Desmosomes, large vacuoles, glycogen, dilated ER, cytoplasmic invaginations, and intermediate filaments The physaliphorous (= having bubbles or vacuoles) appearance is due to dilated ER, glycogen, and cytoplasmic invaginations.	IHC: Keratin (corresponds to intermediate filaments), EMA, S100
Clear cell sarcoma	Melanosomes in various stages of development. Glycogen (resulting in clear cytoplasm).	Cytogenetics: t(12;22) EWS;ATF1 fusion protein IHC: S100, HMB45
Dense core granules	Dense core granules (vesicle bound by a single membrane with a dense center – 60 to 300 nm) – cytoplasmic organelles involved in regulated exocytosis of cell products. Examples: Pancreatic beta cells (insulin): angular crystalline inclusions Pheochromocytoma (epinephrine and norepinephrine): Large, pleomorphic, often clear or only partially filled Carcinoid: • Foregut – small, round • Midgut – larger, pleomorphic • Hindgut – mixed	Found in tumors of neuronal or neuroendocrine origin. Vesicles are comprised of granins (predominantly chromogranin A, chromogranin B, and secretogranin II) and various peptide hormones and transmitters, ATP, and biogenic amines IHC: Chromogranin A (most specific). Specific products of tumors can also be detected. Note: Prostate cancers and breast cancers can also show strong chromogranin positivity and can be mistaken for neuroendocrine tumors, particularly at metastatic sites.
Desmoplastic small round cell tumor	Numerous desmosomes and tight junctions, numerous cell processes, large number of organelles (mitochondria and RER), microfilaments, small neurosecretory granules	Cytogenetics: t(11;22) EWS;WT1 fusion protein IHC: Keratin, desmin, WT-1, actin, EMA, NSE
Endothelial cells	Weibel-Palade bodies (cigar-shaped membrane-bound structures filled with tubules in parallel arrays). Intracytoplasmic lumina may be present in normal cells and in epithelioid vascular neoplasms.	Weibel-Palade bodies are frequently absent in tumors arising from endothelial cells (e.g., angiosarcomas). IHC markers are more sensitive to detect endothelial derivation. The membranes are formed by P-selectin and the tubules contain FVIII. IHC: Vascular markers (CD34, CD31, FVIII)
Ewing sarcoma (PNET)	Homogeneous cell population characterized by the lack of specialized features, large pools of glycogen, no organelles, no extracellular matrix, variable numbers of neurosecretory granules and cell processes.	Cytogenetics: t(11;22) EWS;FLI1 fusion protein (and other less common variants) IHC: CD99. FLI1 is also present, but is less specific. Histo: PAS +/− diastase can detect glycogen, but is not currently used for diagnosis.
Granular cell tumor	Numerous lysosomes (filled with tubular, vesicular, and amorphous material), phagosomes, and granules (correlating with the “granular” cytoplasm), reduplicated basal lamina surrounding groups of cells.	IHC: S100, inhibin, CD68, calretinin
Langerhans cell histiocytosis	Birbeck granules (rod-or tennis racket-shaped) structures of variable length with a central periodically striated lamella.	May serve as a reservoir for Langerin (a transmembrane type II Ca2+-dependent lectin) and CD1a in the endosomal recycling compartment. IHC: CD1a, Langerin, S100
Mast cells	Lameller or scroll-like membrane pattern, granules of variable size.	IHC: CD117 (c-kit), mast cell tryptase
Medullary carcinoma of the thyroid	Numerous neurosecretory granules (calcitonin) associated with stromal amyloid (calcitonin).	Cytogenetics: Mutations in the RET gene (sporadic and germline) IHC: Calcitonin (in tumor cells and amyloid), chromogranin
Merkel cell carcinoma	Neurosecretory granules in processes or along cell membranes (subplasmalemmal).	IHC: Chromogranin, NSE, cytokeratin 20
Mesothelioma	Elongated, serpiginous, and branched microvilli (generally 10 to 16 length: 1 width) apical without a glycocalyx or actin rootlets.	Cytogenetics: Characteristic chromosome deletions and loss of 9 and 22 IHC: Calretinin, WT-1
Neuroblastoma	Cellular processes with microtubules (neuropil), dense core granules, Homer-Wright rosettes (the center is comprised of a tangle of cell processes), synaptic vesicles, no glycogen	Cytogenetics: Changes are linked to prognosis IHC: Chromogranin, NSE, NFP, synaptophysin
Oncocytoma	Numerous mitochondria packed in the cytoplasm (correlating with the granular appearance of the cytoplasm). In contrast, chromophobe renal cell carcinoma has fewer mitochondria and more microvesicles.	Cytogenetics: Monosomy with loss of X or Y, 11q13. Chromophobe carcinomas have different cytogenetic changes. IHC: RCC is negative in oncocytomas but positive in 45% to 50% of chromophobe renal cell carcinomas.
Perineurioma	Long cell processes wrapping around adjacent cells	IHC: Claudin-1 (a component of tight junctions), EMA
Rhabdoid tumor of the kidney	Large paranuclear whorls of intermediate filaments (corresponding to cytokeratin and vimentin) and occasional tonofilaments	Cytogenetics: hSNF5/INI1 deletions and mutations on chromosome 22 IHC: Cytokeratin (+), vimentin (+), absence of INI1 nuclear protein
Rhabdomyosarcoma	Parallel thick (12 to 15 cm) and thin (6 to 8 nm) myosin-actin filaments, Z-bands, filament ribosomal complexes. Spider cells may be seen in cardiac tumors (clear cytoplasm divided by cytoplasmic processes and cross striations formed by leptofibrils).	Cytogenetics: Characteristic changes in alveolar and embryonal types IHC: Muscle markers (HHF-35, desmin, myf4)
Schwannoma	Basal lamina prominent, often reduplicated. Luse bodies (long spacing collagen, extracellular), myelin figures, long cell processes wrapping around collagen, may rarely have melanosomes (melanotic schwannoma)	Cytogenetics: Deletion of 2q (NF2 inactivation) IHC: S100

See Tables 7-8, 7-9, 7-22, and 7-47 for additional information.

Method

Ultrastructural details of tissues are lost rapidly. Therefore, fresh tissue must be fixed rapidly and well for EM. Tissues are usually fixed in special fixatives for EM to preserve lipids and glycogen (e.g., 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4).

1. Place a small fragment of tissue in a drop of fixative on a cutting surface.

2. Cut the tissue into multiple tiny fragments, each no greater than 0.1 cm in any dimension.

3. Place the tissue into the vial of fixative. Shake the vial to make sure all the tissue fragments are covered by fixative.

Note: If tissue from a small biopsy is found to be nondiagnostic on H&E, any tissue saved for EM should be retrieved for examination by light microscopy.

Results

A separate electron microscopy report is usually issued. The results should be incorporated into the final diagnosis.¹⁹

SNAP FROZEN TISSUE

Frozen tissue is useful for staining (some antibodies only detect antigens in frozen tissue), enzyme studies (muscle biopsies), and to save tissue for DNA or RNA studies.

Indications

All specimens with a question of a lymphoproliferative disorder, sarcomas, unusual tumors, muscle biopsies.

Methods

Small (approximately 0.5 × 0.5 × 0.3 cm³) portions of tissue are placed in a clean specimen container moistened with a small amount of normal saline until they can be frozen. Specimens should be snap frozen using liquid nitrogen or dry ice and stored at –20°C.

Results

The results of studies on frozen tissue are usually incorporated into the surgical pathology report.

IMMUNOFLUORESCENCE

Like immunoperoxidase studies, immunofluorescence (IF) detects antigens in tissues. However, because amplification of the signal is not used, it is better suited for precise localization of antigen/antibody complexes in tissues or for determining the deposition pattern of immune complexes (e.g., linear vs. granular). Thus, it is most useful for the investigation of diseases related to immune complex deposition such as glomerular diseases and bullous diseases of the skin.

Tissue for IF may be snap frozen (see instructions earlier) or stored in special fixatives for IF. If the specimen is not frozen, special care must be taken to ensure that the biopsy is kept moist in a sealed container.

• Direct IF: Uses antibodies to detect antigens in the patient’s tissues.

• Indirect IF: Uses control tissues to detect antibodies (e.g., anti-BM) in the patient’s serum.

Indications

Some skin biopsies (e.g., lupus, pemphigus, pemphigoid, and dermatitis herpetiformis), all diagnostic nontransplant renal biopsies, some transplant renal biopsies, identification of amyloid in cardiac biopsies, and the evaluation of vasculitis in nerve biopsies.

Method

Tissue must be submitted fresh.

Results

The results of the examination are usually incorporated into the surgical pathology report.

Immunofluorescence of Skin Lesions

• SLE (lupus band test): linear or granular staining along dermal epidermal junction for multiple immunoreactants in about 80% of cases (most commonly IgG and less often IgM or C3). The specificity increases with the number of positive immunoreactants. Uninvolved sun-exposed skin shows positivity in most patients with active systemic lupus. Uninvolved skin in patients with discoid lupus is usually negative for this test.

• Herpes gestationis: perilesional skin shows linear BM zone C3 and sometimes IgG.

• Dermatitis herpetiformis: granular IgA at tips of dermal papillae of uninvolved skin.

• Pemphigus: IgG and C3 between epidermal cells creating a net-like pattern. In pemphigus vulgaris, a split just above the basal cell layer creates a “tombstone” appearance to the row of basal cells at the base of the vesicle. In pemphigus foliaceus and related disorders, the split occurs near the granular cell layer.

• Pemphigoid: Ig and C3 along basement membrane but not between cells. Indirect IF reveals an anti-BM antibody.

MOLECULAR GENETIC PATHOLOGY

Molecular genetic pathology is the newest subspeciality in pathology with board certification. Molecular diagnostics incorporates many types of techniques for the investigation of genetic alterations in cells and viruses (e.g., Southern blotting, PCR analysis, FISH). It has applications in three main areas:

Inherited diseases:

• Identification of inherited diseases (e.g., cystic fibrosis, hemochromatosis, Factor V Leiden, Prothrombin 20210A, Fragile X syndrome).

• Identification of genes conferring susceptibility to diseases (e.g., microsatellite instability [MSI], BRCA1 and 2)

Infectious diseases:

• Detection of organisms

• Identification of specific organisms

• Quantitation of viral infection (e.g., HIV viral load)

Cancer:

• Identification of specific genetic alterations associated with tumors

• Identification of gene mutations associated with susceptibility to treatment (e.g., EGFR mutations in lung cancer, c-kit mutations in GIST)

• Identification of clonality in hematolymphoid proliferations

• Detection of minimal residual disease after treatment

These studies are especially helpful for hematolymphoid proliferations that are difficult to classify because of the frequent and characteristic rearrangements that occur in many of these disorders. Unlike cytogenetics, the cells need not be viable. However, it is preferable that the nucleic acids are relatively intact. Southern blot and RNA-based PCR (RT-PCR) assays are best performed on fresh or frozen tissues. Formalin-fixed, paraffin-embedded tissue is amenable to DNA-based PCR assays. Some fixatives (e.g., Bouin’s) cause extensive breakage of DNA and may preclude genetic analysis of the tissue.

Indications

• B-cell proliferations – clonal rearrangements of the immunoglobulin heavy and light chain genes; specific translocations

• T-cell proliferations – rearrangements of the γ and β T-cell receptor genes

• Leukemias

• Post-transplant lymphoproliferative disorders – clonal populations of EBV-infected cells

• Oligodendrogliomas – PCR-based LOH analysis for 1p/19q deletions.

• Colon cancers possibly associated with hereditary non-polyposis colorectal carcinoma syndrome (HNPCC): microsatellite instability (MSI) testing of colon cancers occurring in patients 50 years of age or younger.

• Human papilloma virus testing: cervical PAP smears, squamous cell carcinomas of the head and neck (see subsequent section).

• GIST – most have mutations in the KIT tyrosine kinase gene. A smaller group (5% to 7%) have mutations in the KIT-homologous tyrosine kinase PDGFRA. About 10% to 15% of GISTs are negative for KIT and PDGFRA mutations (termed “wild-type GISTs”). The specific type of mutation is correlated with prognosis and the respone to specific types of treatment.

• Lung adenocarcinoma – some cancers have mutations in EGFR that predict response to the tyrosine kinase inhibitor gefitinib.

Method of Submitting Tissue

Fresh or frozen tissue (e.g., snap frozen tissue) as well as fluids may be used. Cytologic preparations can be used for FISH. Paraffin blocks can also be used.

Results

The results are usually either reported separately or incorporated into the surgical pathology report.

CYTOGENETICS

Cytogenetic studies have been demonstrated to be useful in several areas important to pathology:

• Tumor classification: Particularly sarcomas (e.g., Ewing’s sarcoma and synovial sarcoma), lymphomas, leukemias, kidney tumors, brain tumors, and other unusual tumors.

• Benign vs. malignant lesions:

• Reactive mesothelial cells vs. mesothelioma

• Lipoma vs. liposarcoma

• Prognosis: Neuroblastoma, oligodendroglioma, multiple myeloma, chronic lymphocytic leukemia.

• Treatment: Amplification of HER2/neu to predict response to Herceptin.

• Research: Translocations are common to many tumors and usually identify genes important to the pathogenesis of the tumor

Cells may be cultured to perform complete karyotype analysis or tissues can be analyzed for specific chromosomal alterations by fluorescence in situ hybridization (FISH).

FISH studies can be performed on cultured cells, cytology specimens, touch preparations, and paraffin-embedded tissues.

Indications

• For karyotype analysis: Soft tissue tumors, mesotheliomas (tissue or pleural fluid), unusual tumors, poorly differentiated tumors, all subcutaneous lipomas >10 cm or of unusual gross appearance, all deep-seated lipomas (subfascial, intramuscular, intraabdominal, retroperitoneal, clinically apparent cord tumors), unusual uterine masses.

• For FISH: Oligodendroglioma, neuroblastoma.

Method for Submitting Tissue

Tissue for karyotyping must be fresh, viable, and relatively sterile. However, tissue may be submitted even if it has not been handled under strictly sterile conditions (contamination is not usually a problem). If specimens are to be held overnight, the tissue should be minced (into 0.1 cm cubes) in a sterile specimen container, covered with culture medium, and held overnight in the refrigerator. Fluids may also be submitted for analysis (especially pleural effusions with a suspicion of mesothelioma).

Results

The results of the cytogenetic analysis should be incorporated into the final diagnosis or reported separately (Tables 7-47 and 7-48).

TABLE 7–47 COMMON CYTOGENETIC AND GENETIC CHANGES IN SOLID TUMORS OF DIAGNOSTIC OR THERAPEUTIC SIGNIFICANCE

TABLE 7–48 COMMON CYTOGENETIC CHANGES IN LYMPHOMAS AND LEUKEMIAS

Tumors and Diseases Associated with Germline Mutations (Tables 7-49 and 7-50)

The following features are suggestive of hereditary susceptibility to cancer:

• Two or more close relatives on the same side of the family with cancer

• Evidence of autosomal dominant transmission

• Early development of cancer in the patient and relatives (in general, <50 years of age)

• Multiple primary cancers

• Multiple types of cancers

• Unusual pathologic features of tumors (Table 7-49)

• A constellation of tumors suggestive of a specific syndrome (Table 7-50)

TABLE 7–49 PATHOLOGIC FEATURES OF TUMORS AND DISEASES SUGGESTIVE OF A GERMLINE MUTATION

TABLE 7–50 HEREDITARY SYNDROMES ASSOCIATED WITH MULTIPLE TUMORS

Pathologists can aid in the detection of hereditary carcinomas by being aware of the types and pathologic characteristics of carcinomas associated with these syndromes. Patients with germline mutations are important to identify in order to:

• Screen patients for other common tumors or other components of the disease

• Consider prophylactic surgery or preventive interventions

• Offer screening to family members at risk and genetic counseling

Although the sporadic forms of cancers, in general, far outnumber cases associated with germline mutations, in some cases the appearance or site of a carcinoma is highly suggestive of a known syndrome and further investigation may be warranted.

CULTURE	HISTOLOGIC SECTIONS
Can be performed on aspirates, swabs, fluids, or tissues.	Requires surgical excision of tissues.
Cultures amplify the number of organisms present, allowing them to be recognized.	Organisms may be rare, or not seen in tissue sections.
The specific organism can be identified and tested for drug susceptibility.	Categories of organisms can be recognized but specific identification may not be possible.
Some organisms cannot be cultured.	Many organisms can be identified that will not grow in culture or that require long culture times (e.g., TB).
It may be difficult to exclude contamination for a positive culture.	Morphologic evidence of an inflammatory response provides evidence for a clinical infection. The location of the infection may be of diagnostic importance (e.g., cellulitis vs. necrotizing fasciitis or superficial colonization of devitalized tissue vs. deep infections involving viable tissues). The use of special studies, such as PCR and other molecular assays, may be warranted if the tissue pattern of injury is indicative of a potential organism despite lack of culture evidence and/or lack of organisms seen on tissue sections.

HISTOCHEMISTRY

IMMUNOPEROXIDASE STUDIES

Use of Immunohistochemistry.

Panels.

Slides for Immunohistochemistry.

Factors Affecting Immunogenicity.

Controls.

Evaluation of Studies

Location of Immunoreactivity

Identification of Immunoreactive Cells

Intensity of Immunoreactivity

Number of Immunoreactive Cells

Criteria for a “Positive” Result

Time

Common Panels for Immunohistochemical Studies

Cytokeratin 7 and cytokeratin 20 tables

Small blue cell tumors

Spindle cell lesions, soft tissue lesions, and sarcomas

Metastatic tumors of unknown origin

Poorly differentiated tumors

Estrogen and progesterone receptor evaluation

HER2/neu score

Myoepithelial markers in breast carcinoma

Epidermal lesions of the nipple

Breast carcinoma in males versus metastatic prostate carcinoma

Signet ring cell carcinomas of the stomach and breast (lobular carcinoma)

Fibroblastic/myofibroblastic lesions of the breast

Ovarian carcinoma versus mesothelioma

Primary ovarian carcinoma versus metastatic carcinomas

Endocervical carcinoma versus endometrial carcinoma

Endometrial stromal sarcoma versus leiomyosarcoma

Trophoblastic lesions

Metastatic adenocarcinomas in the abdomen

CNS neoplasms

Hemangioblastoma versus metastatic renal cell carcinoma

Tumors of germ cells and sex-cord stromal tumors

Adrenal and kidney tumors

Tumors of bladder, prostatic, and renal origin

Prostate carcinoma versus other lesions

Hepatic tumors

Thyroid and parathyroid lesions

Differential diagnosis of epithelial mesothelioma and lung adenocarcinoma

Lung carcinoma

B-cell neoplasms

T-cell neoplasms

Hodgkin lymphoma

Amyloid

Antibodies for immunohistochemistry

Results

ELECTRON MICROSCOPY

Indications for EM Studies

Method

Results

SNAP FROZEN TISSUE

Indications

Methods

Results

IMMUNOFLUORESCENCE

Indications

Method

Results

Immunofluorescence of Skin Lesions

MOLECULAR GENETIC PATHOLOGY

Indications

Method of Submitting Tissue

Results

CYTOGENETICS

Indications

Method for Submitting Tissue

Results

Tumors and Diseases Associated with Germline Mutations (Tables 7-49 and 7-50)

ANALYTICAL CYTOLOGY (FLOW CYTOMETRY)

Indications for Ploidy and S-Phase Analysis

Indications for Cell Surface Marker Analysis

Method for Submitting Tissue

Results

CYTOLOGIC PREPARATIONS FROM SURGICAL SPECIMENS

SPECIMEN RADIOGRAPHY

Indications

Method