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FOURTH EDITION
Gattuso’s
Differential
Diagnosis in
Surgical Pathology
Vijaya B. Reddy, MD, MBA Daniel J. Spitz, MD
The Harriet Blair Borland Chair of Chief Medical Examiner
Pathology Macomb and St. Clair Counties,
Professor and Chairperson Michigan
Department of Pathology Clinical Assistant Professor of
Rush University Medical Center Pathology
Chicago, Illinois Wayne State University School of
United States Medicine
Detroit, Michigan
Odile David, MD United States
Associate Professor and Director of
Cytopathology Meryl H. Haber, MD
University of Illinois at Chicago Borland Professor and Chairman
Chicago, Illinois of Pathology, Emeritus
United States Rush Medical College of Rush
University
Chicago, Illinois
United States
iii
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GATTUSO’S DIFFENENTIAL DIAGNOSIS IN SURGICAL PATHOLOGY,

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Printed in the United States.
Last digit is the print number: 9 8 7 6 5 4 3 2 1
Contributors
SYLVIA ASA, MD, PhD BYRON CRAWFORD, MD

Professor Professor and Chair
Pathology Pathology
Case Western Reserve University LSU School of Medicine
Consultant in Endocrine Pathology Shreveport, Louisiana
Pathology United States
University Hospitals Cleveland
Cleveland, Ohio KOSSIVI DANTEY, MD
United States Assistant Professor
Consultant in Endocrine Pathology Drexel University College of Medicine
Pathology Pathology
University Health Network Allegheny Health Network
Toronto, Ontario Pittsburgh, Pennsylvania
Canada United States
ELIZAVETA BELYAEVA, MD VIRGINIA E. DUNCAN, MD, MS

Assistant Professor of Pathology and Laboratory Assistant Professor
Medicine Pathology
Tulane University School of Medicine University of Alabama at Birmingham
New Orleans, Louisiana Birmingham, Alabama
United States United States
PINCAS BITTERMAN, MD ADEL K. EL-NAGGAR, MD

Professor Emeritus Professor
Departments of Pathology and Obstetrics and pathology
Gynecology The University of Texas MD Anderson Cancer Center
Rush University Medical Center Houston, Texas
Chicago, Illinois United States
United States
MARK F. EVANS, PhD
DUSTIN E. BOSCH, MD, PhD Assistant Professor
Fellow Pathology and Laboratory Medicine
Department of Laboratory Medicine and Pathology Larner College of Medicine
University of Washington School of Medicine University of Vermont
Seattle, Washington Burlington, Vermont
ELIZABETH J. COCHRAN, MD HUMA FATIMA, MD

Professor Associate Professor
Department of Pathology Pathology
Medical College of Wisconsin University of Alabama at Birmingham
Milwaukee, Wisconsin Birmingham, Alabama
KUMARASEN COOPER, MBChB, DPhil

Professor of Pathology
Department of Pathology
Hospital of University of Pennsylvania (HUP)
Philadelphia, Pennsylvania
United States
vii
viii Contributors
SANDRA E. FISCHER, MD CRISTINA MAGI-GALLUZZI, MD, PhD

Associate Professor Director
Laboratory Medicine and Pathobiology Anatomic Pathology
University of Toronto Department of Pathology
Staff Pathologist Professor of Pathology
Laboratory Medicine Program Pathology
University Health Network University of Alabama at Birmingham
Clinician Investigator Section Head
Princess Margaret Cancer Centre Genitourinary Pathology
University Health Network Director
Toronto, Ontario Genitourinary Pathology Fellowship Program
Canada The C. Bruce Alexander Endowed Professorship in
Pathology
JULIA T. GEYER, MD Birmingham, Alabama
Associate Professor of Pathology and Laboratory United States
Medicine
Weill Cornell Medicine MEERA MAHALINGAM, MD, PhD, FRCPath
New York, New York Professor of Dermatology
United States Tufts University School of Medicine
Senior Lecturer
RICHARD J. GROSTERN, MD Pathology
Section Director Harvard Medical School
Ophthalmic Pathology Dermatopathology Section Chief
Department of Ophthalmology Department of Pathology and Laboratory Medicine
Rush Medical College of Rush University VA Integrated Service Networks (VISN1), New England
Chicago, Illinois West Roxbury, Massachusetts
RALPH H. HRUBAN, MD MARIA J. MERINO, MD

Professor Chief
Pathology and Oncology Translational Surgical Pathology
The Johns Hopkins University School of Medicine NCI
Baltimore, Maryland Bethesda, Maryland
ALIYA N. HUSAIN, MBBS IRA MILLER, MD, PhD

Professor Director of Orthopedic Pathology
Pathology Department of Pathology
University of Chicago Rush University Medical Center
Chicago, Illinois Chicago, Illinois
ALEXANDRA N. KALOF, MD ATTILIO ORAZI, MD, FRCPath

Associate Professor Pofessor of Pathology
Pathology and Laboratory Medicine Department of Pathology
Larner College of Medicine TexasTech University Health Sciences Center
University of Vermont El Paso, Texas
Burlington, Vermont United States
United States
HREEM N. PATEL, MD
NIKOLAJ P. LAGWINSKI, MD Assistant Professor
Associate Pathologist Ophthalmology
GI Insitute Rush University Medical Center
Ameripath Cleveland Chicago, Illinois
Oakwood Village, Ohio United States
United States
Contributors ix
SUNNY B. PATEL, BS, MD† JEFREE SCHULTE, MD

Resident Physician Assistant Professor
Department of Ophthalmology Department of Pathology and Laboratory Medicine
Rush University Medical Center University of Wisconsin School of Medicine and Public
Chicago, Illinois Health
United States Madison, Wisconsin
United States
ROBERT E. PETRAS, MD
Associate Clinical Professor of Pathology DAVID SUSTER, MD
Northeast Ohio Medical University Assistant Professor
Rootstown, Ohio Pathology
United States Rutgers University
New Jersey Medical School
MICHAEL R. PINS, MD Newark, New Jersey
Professor and Chair of Pathology United States
Pathology
Chicago Medical School of Rosalind Franklin University SAUL SUSTER, MD
of Medicine and Science Professor and Chairman Emeritus
North Chicago, Illinois Department of Pathology
United States Medical College of Wisconsin
Chairman of Pathology Milwaukee, Wisconsin
Pathology United States
Advocate Lutheran General Hospital and Advocate
Children’s Hospital PAUL E. SWANSON, MD
Park Ridge, Illinois Professor
United States Department of Laboratory Medicine and Pathology
University of Washington School of Medicine
SONAM PRAKASH, MBBS Seattle, Washington
Clinical Professor United States
Laboratory Medicine
University of California San Francisco CARMELA D. TAN, MD
San Francisco, California Associate Professor
United States Cleveland Clinic Lerner College of Medicine of Case
Western University
VIJAYA B. REDDY, MD, MBA Cleveland Clinic
The Harriet Blair Borland Chair of Pathology Cleveland, Ohio
Professor and Chairperson United States
Rush University Medical Center ELIZABETH THOMPSON, MD, PhD
Chicago, Illinois Assistant Professor
United States Pathology
The Johns Hopkins University School of Medicine
E. RENE RODRIGUEZ, MD Baltimore, Maryland
Professor United States
Cleveland Clinic Lerner College of Medicine of Case
Western University MICHELLE D. WILLIAMS, MD
Cleveland, Ohio Professor
United States Pathology
The University of Texas MD Anderson Cancer Center
JOHN J. SCHMIEG, MD, PhD Houston, Texas
Staff Hematopathologist United States
The Joint Pathology Center
Silver Spring, Maryland LEI YAN, MD, PhD
United States Assistant Professor
Rush University Medical Center
Chicago, Illinois
United States
†Deceased
x Contributors
MATTHEW M. YEH, MD, PhD MING ZHOU, MD, PhD

Professor Chair and Pathologist-in-Chief
Department of Laboratory Medicine and Pathology Pathology and Laboratory Medicine
University of Washington School of Medicine Tufts Medical Center
Adjunct Professor Tufts University School of Medicine
Department of Medicine Boston, Massachusetts
University of Washington School of Medicine United States
Seattle, Washington
United States
To Swathi, my shining star
VIJAYA B. REDDY
To my wife, Nancy, and my children, Vincent and Francesca

PAOLO GATTUSO
To my family, to whom I owe my appreciation of life and learning

ODILE DAVID
To my parents, for setting me on the right track, and to my wife, Jodi, for her continuous
support and encouragement
DANIEL J. SPITZ
To all of my former students, residents, fellows, and physician associates

from whom I have always learned more than I have been able to teach
MERYL H. HABER
Acknowledgments
We thank all our contributors for their expertise, knowl- Elsevier, and Michael Houston, Executive Content Strate-
edge, and invaluable role in the continued success of this gist, for his support and encouragement in the produc-
book. tion of a fourth edition. A special thanks to Ann Ruzycka
The editors also gratefully acknowledge the work of Anderson, Senior Content Development Specialist, and
authors who have contributed to this book in its previous Sharon Corell, Senior Project Manager, for their patience
editions. and competence in keeping the book on course over the
We thank Irma Parker for her assistance and persis- past year.
tence in contacting the contributors and ensuring the
timely submissions. We are thankful to our publisher, Vijaya B. Reddy, MD
xiii
Preface
The concept of this textbook was conceived just over 20 illustrating particular diagnostic characteristics continue
years ago by attending pathologists and residents in the to be a hallmark.
Department of Pathology of Rush Medical Center in Chi- In this current edition, 5 years since the previous, each
cago. This, the fourth edition, has gradually evolved into section has been reviewed and revised especially with the
a widely used textbook for practicing surgical patholo- application of diagnostic biomarkers. As in the third edi-
gists, residents, and others of the medical field interested tion, Dr. Vijaya Reddy oversaw these additions and revi-
in diagnostic pathology. Conceptually, it began as and sions, monitored the selection of new authors, prompted
remains a nonencyclopedic compendium of a wide vari- previous authors, added photomicrographs, and pursued
ety of surgical pathology specimens with which patholo- overall development. This volume is now more encom-
gists are confronted daily. No attempt was made to copy passing than before. It includes new and valuable diag-
or emulate already existing textbooks of pathology. nostic findings and, even though we have attempted to
Instead, the author’s goals were to produce a usable text make it more concise, it remains more than a thousand
in outline format with succinct text discussions and clear pages. We are gratified that over the past score of years
descriptions of differential diagnoses of pathologic enti- this textbook has been a helpful aid to pathologists in sur-
ties. A “Pearls” line or two is included to facilitate prompt gical pathology laboratories around the world. Selected
and accurate diagnosis. Each discussed entity is accom- updated references remain included, as are the many
panied by numerous carefully selected high-quality color “Pearl” paragraphs.
photographs, both macro and microscopic, of commonly
encountered specimens surgically removed. These images Meryl H, Haber, MD
xi
Chapter 1
Special Diagnostic Techniques in
Surgical Pathology
ALEXANDRA N. KALOF • MARK F. EVANS • KOSSIVI DANTEY • KUMARASEN COOPER
Chapter Outline
Light Microscopy 1 Cytogenetic Analysis 12
Tissue Processing Overview 1 Molecular Pathology Methods 17
Fixation 1
Introduction 17
Histologic Stains 3
Nucleic Acid Extraction Methods 17
Fluorescence Microscopy 6 Tissue Microdissection Methods 23
Electron Microscopy 7 Gel Electrophoresis Methods 26
Blot Hybridization Methods 27
Technical Overview 7
Amplification Methods 29
Ultrastructure of a Cell 7
Nanostring Technology 33
Immunohistochemistry 9 Microarray Technology 34
Introduction 9 Nucleic Acid Sequencing 35
Technical Overview 9 In Situ Hybridization 36
Flow Cytometry 11 Protein Analytic Methods 37
Introduction 11 Emerging Developments 39
Technical Overview 11 Resources 40
• S ectioning
LIGHT MICROSCOPY • Embedded in paraffin, which is similar in density to
tissue, tissue can be sectioned at anywhere from 3 to
TISSUE PROCESSING OVERVIEW
10 μm (routine sections are usually cut at 6 to 8 μm)
• F ixation • Staining
• Preserves tissues in situ as close to the lifelike state as • Allows for differentiation of the nuclear and cyto-
possible plasmic components of cells as well as the intercel-
• Ideally, fixation will be carried out as soon as possible lular structure of the tissue
after removal of the tissues, and the fixative will kill • Cover-slipping
the tissue quickly, thus preventing autolysis • The stained section on the slide is covered with a
• Dehydration thin piece of plastic or glass to protect the tissue from
• Fixed tissue is too fragile to be sectioned and must be being scratched, to provide better optical quality for
embedded first in a nonaqueous supporting medium viewing under the microscope, and to preserve the
(e.g., paraffin) tissue section for years
• The tissue must first be dehydrated through a series
of ethanol solutions
FIXATION
• Clearing
• Ethanol is not miscible with paraffin, so nonpolar • T
here are five major groups of fixatives, classified
solvents (e.g., xylene, toluene) are used as clearing according to mechanism of action: aldehydes, mercuri-
agents; this also makes the tissue more translucent als, alcohols, oxidizing agents, and picrates
• Embedding • Aldehydes
• Paraffin is the usual embedding medium; however, • Formalin
tissues are sometimes embedded in a plastic resin, • Aqueous solution of formaldehyde gas that pen-
allowing for thinner sections (required for electron etrates tissue well but relatively slowly; the stan-
microscopy [EM]) dard solution is 10% neutral buffered formalin
• This embedding process is important because the • A buffer prevents acidity that would promote
tissues must be aligned, or oriented, properly in the autolysis and cause precipitation of formol-
block of paraffin heme pigment in the tissues
1
2 Chapter 1 — Special Diagnostic Techniques in Surgical Pathology
• T issue is fixed by cross-linkages formed in the formalin- heme pigment that appears as black,
proteins, particularly between lysine residues polarizable deposits in tissue
• This cross-linkage does not harm the struc- • Common buffers include phosphate, bicarbonate,
ture of proteins greatly, preserving antigenicity, cacodylate, and veronal
and is therefore good for immunoperoxidase • Fixative solutions penetrate at different rates, depend-
techniques ing on the diffusibility of each individual fixative
• Glutaraldehyde • In order of decreasing speed of penetration: form-
• The standard solution is a 2% buffered glutaraldehyde, acetic acid, mercuric chloride, methyl
aldehyde and must be cold, buffered, and not alcohol, osmium tetroxide, and picric acid
more than 3 months old • Because fixation begins at the periphery, thick sec-
• Fixes tissue quickly and therefore is ideal for EM tions sometimes remain unfixed in the center, com-
• Causes deformation of α-helix structure in pro- promising both histology and antigenicity of the
teins and therefore is not good for immunoperoxi- cells (important for immunohistochemistry [IHC])
dase staining • It is important to section the tissues thinly (2 to 3 mm)
• Penetrates poorly but gives best overall cytoplas- • Should be at least a 10:1 ratio of fixative to tissue
mic and nuclear detail • Increasing the temperature, as with all chemical
• Tissue must be as fresh as possible and preferably reactions, increases the speed of fixation
sectioned within the glutaraldehyde at a thick- • Hot formalin fixes tissues faster, and this is often
ness of no more than 1 mm to enhance fixation the first step on an automated tissue processor
• Mercurials • Formalin is best at 10%; glutaraldehyde is gener-
• B-5 and Zenker ally made up at 0.25% to 4%
• Contain mercuric chloride and must be disposed • Formalin should have 6 to 8 hours to act before the
of carefully remainder of the processing is begun
• Penetrate poorly and cause tissue hardness but • Decalcification
are fast and give excellent nuclear detail • Tissue calcium deposits are extremely firm and do not
• Best application is for fixation of hematopoietic section properly with paraffin embedding because of the
and reticuloendothelial tissues difference in densities between calcium and paraffin
• Alcohols • Strong mineral acids such as nitric and hydrochloric
• Methyl alcohol (methanol) and ethyl alcohol acids are used with dense cortical bone because they
(ethanol) remove large quantities of calcium at a rapid rate
• Protein denaturants • These strong acids also damage cellular morphology
• Not used routinely for tissue because they dehy- and thus are not recommended for delicate tissues
drate, resulting in the tissues becoming brittle such as bone marrow
and hard • Organic acids such as acetic and formic acid are
• Good for cytologic smears because they act quickly better suited to bone marrow because they are not
and give good nuclear detail as harsh; however, they act more slowly on dense
• Oxidizing agents cortical bone
• Permanganate fixatives (potassium permanga- • Formic acid in a 10% concentration is the best all-
nate), dichromate fixatives (potassium dichro- around decalcifier
mate), and osmium tetroxide cross-link proteins
• Cause extensive denaturation PEARLS
• Some of these have specialized applications but are
• P rolonged fixation can affect immunohistochemical
used infrequently
results owing to alcohol precipitation of antigen at the cell
• Picrates
surface; to optimize antigenicity of the tissue for IHC, the
• Bouin solution has an unknown mechanism of action
American Society of Clinical Oncology/College of American
• It does almost as well as mercurials with nuclear
Pathologists (ASCO/CAP) guidelines recommend fixation
detail but does not cause as much hardness
of tissue destined for IHC in neutral buffered formalin for
• Picric acid is an explosion hazard in dry form
a minimum of 6 hours and a maximum of 48 hours (see
• Recommended for fixation of tissues from testis,
Wolff et al., 2007)
gastrointestinal tract, and endocrine organs
• Urate crystals are water soluble and require a nonaqueous
• Factors affecting fixation
fixative such as absolute alcohol
• Buffering
• If tissue is needed for immunofluorescence (e.g., kidney or
• Penetration
skin biopsies) or enzyme profiles (e.g., muscle biopsies),
• Volume
the specimen must be frozen without fixative; enzymes
• Temperature
are rapidly inactivated by even brief exposure to fixation
• Concentration
• For rapid intraoperative analysis of tissue specimens,
• Time interval
tissue can be frozen, and frozen sections can be cut with
• Fixation is optimal at a neutral pH, in the range of
a special freezing microtome (“cryostat”); the pieces of
6 to 8
tissue to be studied are snap-frozen in a cold liquid or cold
• Hypoxia of tissues lowers the pH, so there must
environment (−20°C to −70°C); freezing makes the tissue
be buffering capacity in the fixative to prevent
solid enough to section with a microtome
excessive acidity; acidity causes formation of
Chapter 1 — Special Diagnostic Techniques in Surgical Pathology 3
Figure 1.2 Masson trichrome stain. Cirrhosis of the liver charac-

terized by bridging fibrosis (blue) and regenerative nodule formation
(red).
Figure 1.1 Elastin/Alcian blue stain. Aortic cystic medial degen-
eration in Marfan syndrome. Elastin stain highlights fragmentation of
elastic fibers (brown-black) and pooling of mucopolysaccharides (blue)
• E lastic fibers are blue to black; collagen appears red;
within the media.
and the remaining connective tissue is yellow
• Masson trichrome stain
• Helpful in differentiating between collagen fibers
HISTOLOGIC STAINS
(blue staining) and smooth muscle (bright red stain-
• T he staining process makes use of a variety of dyes that ing) (Figure 1.2)
have been chosen for their ability to stain various cel- • Reticulin stain
lular components of tissue • A silver impregnation technique stains reticulin
• Hematoxylin and eosin (H&E) stain fibers in tissue section black
• The most common histologic stain used for routine • Particularly helpful in assessing for alteration in the
surgical pathology normal reticular fiber pattern, such as can be seen in
• Hematoxylin, because it is a basic dye, has an affinity some liver diseases or marrow fibrosis
for the nucleic acids of the cell nucleus • Jones silver stain
• Hematoxylin does not directly stain tissues but • A silver impregnation procedure that highlights
needs a “mordant” or link to the tissues; this is pro- basement membrane material; used mainly in kid-
vided by a metal cation such as iron, aluminum, or ney biopsies (Figure 1.3A)
tungsten
• The hematoxylin-metal complex acts as a basic dye, Fats and Lipids
and any component that is stained is considered to • O il red O stain
be basophilic (i.e., contains the acid groups that bind • Demonstrates neutral lipids in frozen tissue
the positively charged basic dye), appearing blue in • Sudan black stain
tissue section • Demonstrates neutral lipids in tissue sections
• The variety of hematoxylin stains available for use is • Mainly used in hematologic preparations such as
based partially on choice of metal ion used, which peripheral blood or bone marrow aspirations for
can vary the intensity or hue demonstration of primary granules of myeloid
• Conversely, eosin is an acid aniline dye with an affin- lineage
ity for cytoplasmic components of the cell
• Eosin stains the more basic proteins within cells Carbohydrates and Mucoproteins
(cytoplasm) and in extracellular spaces (collagen) • C
ongo red stain
pink to red (acidophilic) • Amyloid is a fibrillar protein with a β pleated sheet
structure
Connective Tissue • Amyloid deposits in tissue exhibit a deep red or
• E
lastin stain salmon color, whereas elastic tissue remains pale
• Elastin van Gieson (EVG) stain highlights elastic pink (Figure 1.4A)
fibers in connective tissue • When viewed under polarized light, amyloid depos-
• EVG stain is useful in demonstrating pathologic its exhibit apple-green birefringence (Figure 1.4B)
changes in elastic fibers, such as reduplication, • The amyloid fibril–Congo red complex demonstrates
breaks or splitting that may result from episodes of green birefringence owing to the parallel alignment
vasculitis, or connective tissue disorders such as Mar- of dye molecules along the β pleated sheet
fan syndrome (Figure 1.1) • The thickness of the section is critical (8 to 10 μm)
A
A
B
B
C
C Figure 1.4 Alzheimer disease. A, Congo red–positive core of Al-
zheimer disease plaque. B, Apple-green birefringence of amyloid core
Figure 1.3 Membranous glomerulopathy. A, Jones silver stain under polarized light. C, Bielschowsky stain highlighting Alzheimer
highlighting basement membrane “spikes” (arrow) along glomerular disease plaque (arrow) and neurofibrillary tangle within neuronal cell
capillary loops corresponding to basement membrane material sur- bodies (arrowhead).
rounding intramembranous immune complexes. B, Direct immu-
nofluorescence showing diffuse, granular staining of the glomerular
capillary basement membranes with goat antihuman immunoglobu-
lin G. This technique requires fresh-frozen tissue sections. C, Electron
microscopy showing intramembranous electron-dense immune com-
plexes within the glomerular capillary basement membranes. (Courte-
sy of Pamela Gibson, MD, University of Vermont/Fletcher Allen Health
Care, Department of Pathology, Burlington, VT.)
• M ucicarmine stain
• Demonstrates epithelial mucin in tissue sections
• Also highlights mucin- rich capsule of Cryptococcus
species
• Periodic acid–Schiff (PAS) stain
• Glycogen, neutral mucosubstances, basement mem-
branes, and fungal walls exhibit a positive PAS (bright
rose)
• PAS with diastase digestion: diastase and amylase act
on glycogen to depolymerize it into smaller sugar
units that are then washed out of the section
• Digestion removes glycogen but retains stain-
ing of other substances attached to sugars (i.e.,
mucopolysaccharides)
• Alcian blue stain
• May be used to distinguish various glandular epithe-
lia of the gastrointestinal tract and in the diagnosis
of Barrett epithelium Figure 1.5 Luxol fast blue stain. Demyelination in multiple sclerosis
• pH 1.0: acid sulfated mucin positive (colonic-like) (colorless regions).
• pH 2.5: acid sulfated mucin (colonic-like) and acid
nonsulfated mucin (small intestine–like) positive
• Neutral mucins (gastric-like) negative at pH 1.0 and
2.5
Pigments and Minerals

• F
erric iron (Prussian blue), bilirubin (bile stain), cal-
cium (von Kossa), copper (rhodanine), and melanin
(Fontana-Masson) are the most common pigments and
minerals demonstrated in surgical pathology specimens
Nerves and Fibers

• B ielschowsky stain
• A silver impregnation procedure that demonstrates
the presence of neurofibrillary tangles and senile
plaques in Alzheimer disease (Figure 1.4C)
• Axons stain black
• Luxol fast blue stain Figure 1.6 Aspergillus organisms in the lung stained by Grocott me-
• Demonstrates myelin in tissue sections thenamine silver stain.
• Loss of staining indicates myelin breakdown second-
ary to axonal degeneration
• Gray matter and demyelinated white matter should • G rocott methenamine silver (GMS) stain
be almost colorless and contrast with the blue- • Demonstration of fungi or Pneumocystis organisms
stained myelinated white matter (Figure 1.5) (fungi may also be demonstrated by PAS- amylase
stain) (Figure 1.6)
Hematopoietic and Nuclear Elements • Warthin-Starry and Steiner stains
• T oluidine blue stain • Silver impregnation technique for spirochetes (e.g.,
• Demonstrates mast cells in tissue Borrelia burgdorferi, Treponema pallidum) in tissue
• Giemsa, Wright, and May-Grünwald stains sections
• For cellular details, including hematopoietic (periph- • Note: all bacteria are nonselectively blackened by
eral blood or bone marrow) and cytology preparations silver impregnation methods such as the Warthin-
• Leder stain (chloracetate esterase) Starry and Steiner stains
• Identification of cytoplasmic granules of granulo- • These methods are more sensitive for small gram-
cytes and myeloid precursors negative bacteria (e.g., Legionella species, Helicobacter
pylori, and Bartonella species) than tissue Gram stain
Microorganisms: Bacteria, Fungi, Parasites • Ziehl-Neelsen method for acid-fast bacteria (AFB)
• B rown and Brenn Gram stain • Detect the presence of acid-fast mycobacteria (bright
• Demonstration of gram- negative (red) and gram- red) in tissue sections (background light blue)
positive (blue) bacteria in tissue (Figure 1.7)
• Giemsa stain • Fite method should be used to demonstrate Mycobac-
• Demonstration of bacteria, rickettsia, and Toxo- terium leprae or Nocardia species, both of which are
plasma gondii in tissue sections weakly acid fast
Selected References FLUORESCENCE MICROSCOPY

Bancroft JD, Gamble M. Theory and Practice of Histochemical Techniques.
5th ed. Philadelphia: Elsevier; 2001. • Tissue is exposed to short- wavelength ultraviolet (UV)
Carson FL. Histotechnology: A Self-Instructional Text. 2nd ed. Chicago: light (2500 to 4000 angstroms) through a mercury or halo-
American Society for Clinical Pathology (ASCP) Press; 1997. gen lamp; the energy is absorbed by molecules that then
Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clini-
cal Oncology/College of American Pathologists guideline recom-
release the energy as visible light (4000 to 8000 angstroms)
mendations for human epidermal growth factor receptor 2 testing • In immunofluorescence techniques, antibodies are
in breast cancer. Arch Pathol Lab Med. 2007;131:18–43. labeled with a fluorescent dye such as fluorescein iso-
thiocyanate (FITC)
• Direct immunofluorescence
• Fluorescein-labeled antihuman globulin primary
antibodies are applied to frozen, unfixed tissue sec-
tions to locate and combine with antibodies, com-
plement, or antigens deposited in tissue
• Indirect immunofluorescence
• Unlabeled primary antibody is applied to the tissue
section, followed by application of an FITC-labeled
antibody that is directed against a portion of the
unlabeled primary antibody
• More sensitive and more expensive
• Primary application in surgical pathology is detec-
tion of autoimmune diseases involving the skin and
kidney (Table 1.1)
Selected References
D’Agati VD, Jennette JC, Silva FG. Non-neoplastic kidney diseases. In:
AFIP Atlas of Nontumor Pathology. Vol. 4. Washington, DC: Armed
Forces Institute of Pathology; 2005.
Figure 1.7 Ziehl-Neelsen stain for acid-fast bacilli. Abundant My- Kalaaji AN, Nicolas MEO. Mayo Clinic Atlas of Immunofluorescence in
cobacterium avian intracellulare organisms (red) within macrophages Dermatology: Patterns and Target Antigens. New York, NY: Informa
in the lung. Healthcare; 2006.
TABLE 1.1 IMMUNOFLUORESCENCE PATTERNS AND DISEASE ASSOCIATIONS

Disease Antibodies Pattern Histologic Manifestation
Skin
Pemphigus vulgaris Antidesmosomal Intercellular chicken-wire IgG in Suprabasal vesiculation
epidermis
Bullous pemphigoid Antiepithelial BM; anti- Linear IgG along BM; in salt-split Subepithelial vesiculation
hemidesmosome (collagen XVII skin, reactivity along roof
[BP180])
Epidermolysis bullosa EBA Ag Linear IgG along BM; in salt-split Subepithelial vesiculation
acquisita (EBA) skin, reactivity along floor
Dermatitis herpetiformis Antigluten Granular IgA, especially in tips of Subepithelial vesiculation
dermal papillae
Kidney
Anti–glomerular basement Anti-GBM COL4-A3 antigen Linear GBM staining for IgG, cor- Crescentic GN
membrane (anti-GBM) responding granular staining
disease for C3
Membranous Subepithelial deposits secondary to in Diffuse, granular GBM staining for Diffusely thickened glomerular
glomerulopathy situ immune complex formation IgG and C3 capillary loops with lacelike
(antigen unknown; associated splitting and “spikes” identi-
with lupus nephritis, hepatitis B, fied on Jones silver stain
penicillamine, gold, malignancy)
IgA nephropathy Deposited IgA polyclonal: possible IgA ± IgG, IgM, and C3 in mesan- Focal proliferative GN; mesangial
increased production in response gium widening
to exposure to environmental
agents (e.g., viruses, bacteria,
food proteins such as gluten)
Membranoproliferative Type I: immune complex Type I: IgG + C3; C1q + C4 Mesangial proliferation; GBM
glomerulonephritis Type II: autoantibody to alternative Type II: C3 ± IgG; no C1q or C4 thickening; splitting
complement pathway
BM, Basement membrane; GBM, glomerular basement membrane; GN, glomerulonephritis; Ig, immunoglobulin.
• N ucleolus
ELECTRON MICROSCOPY
• Dense, rounded basophilic structure that consists of
• T he electron microscope has a magnification range of 80% to 90% protein
1000 to 500,000 diameters (×) (the upper limit of light • Produces most of the ribosomal RNA (rRNA)
microscopy is approximately 1000 diameters), thereby • Mitotically or metabolically active cells have mul-
allowing for analyzing the ultrastructure of a cell tiple nucleoli
• There are two types of EM: • Chromatin
• Transmission EM • Heterochromatin: stainable, condensed regions of
• Scanning EM chromosomes seen as intensely basophilic nuclear
• Two-dimensional (2D) black-and-white image is material in light microscopy
produced • Euchromatin: nonstainable, extended portions of the
• Tissue either transmits electrons (producing chromosomes that consist of genetically active DNA
“lucent” or clear areas in the image) or deflects
electrons (producing electron “dense” or dark Cytoplasm
areas in the image) • P lasma membrane
• Useful in the diagnosis of nonneoplastic diseases • Appears as two electron-dense (dark) layers with an
of the kidney intervening electron-lucent (light) layer
• Three-dimensional (3D) black-and-white image • Basement membrane = basal lamina (lamina densa +
results as an electron beam sweeps the surface of lamina lucida) + lamina reticularis + anchoring fibrils +
the specimen and releases secondary electrons microfibrils
• Lower resolution than transmission EM and used • Lamina densa
primarily in the research setting to study cell sur- • Electron-dense membrane made up of type
face membrane changes IV collagen fibers coated by a heparan sulfate
• Application in surgical pathology: EM is a useful proteoglycan
diagnostic technique to supplement morphologic, • Approximately 30 to 70 nm thick with an underly-
immunohistochemical, cytogenetic, and molecular ing network of reticular collagen (type III) fibrils,
analysis of tissues which average 30 nm in diameter and 0.1 to 2 μm
• Immunoperoxidase techniques have largely replaced in thickness
EM for tumor diagnosis in surgical pathology • Mitochondria
• EM is useful in • The energy-producing component of the cell; these
• Renal, skin, myocardial, nerve, and muscle biopsies membrane-bound organelles undergo oxidative reac-
• Undifferentiated or poorly differentiated neoplasms tions to produce energy
• Diagnosis of lysosomal storage disorders • Energy generation occurs on the cristae, which are
• Ciliary dysmorphology composed of the inner mitochondrial membrane
• Visualization of infectious agents • Most cells contain shelflike mitochondrial cristae
• Steroid-producing cells (i.e., adrenal cortex) contain
tubular cristae
TECHNICAL OVERVIEW • Mitochondrial crystals are always pathologic
• T he main fixative used for EM is glutaraldehyde, which • Hürthle cell change occurs when the cytoplasm of a
penetrates tissues more slowly than formalin; cubes of cell becomes packed with mitochondria
tissue 1 mm or smaller are needed • Ribosomes
• Processing post fixation with osmium tetroxide, which • Sites of protein synthesis
binds to lipids in membranes for better visualization; • Usually responsible for the basophilic staining of the
dehydration with graded alcohols; infiltration with pro- cytoplasm on H&E-stained sections
pylene oxide and epoxy resin; embedding in epoxy resin • Endoplasmic reticulum
• 1-μm sections (semithin) are cut and stained with tolu- • Membrane-bound channels responsible for the trans-
idine blue to verify that the area of interest has been port and processing of secretory products of the cell
selected for EM • Granular or rough endoplasmic reticulum is abun-
• 100-nm sections (ultrathin) are cut and collected on dant in cells that actively produce secretory prod-
copper grids ucts (e.g., plasma cells producing immunoglobulin
• Tissues are stained with heavy metals (uranyl acetate [Ig] and pancreatic acinar cells producing digestive
and lead citrate) enzymes); the granular appearance is due to attached
• Electron dense: darker in color as a result of heavy ribosomes
impregnation with heavy metal • Smooth endoplasmic reticulum is abundant in cells
• Electron lucent: lighter in color that synthesize steroids (i.e., adrenal cortex, Sertoli-
Leydig cells) and in tumors derived from these types
ULTRASTRUCTURE OF A CELL of cells
• Golgi apparatus
Nucleus • Concentrates and packages proteins into secretory
• N
uclear membrane vesicles for transport to the cell surface
• N uclear pore • Prominent in cells that secrete proteins
TABLE 1.2 CYTOPLASMIC GRANULES

Type Size Morphology Product Cell Type/Tumor
Mucigen 0.7–1.8 μm Electron lucent Glycoprotein Mucin secreting
Serous, zymogen 0.5–1.5 μm Electron dense Proenzyme/enzyme Example: acinar cells of
pancreas
Neuroendocrine 100–300 nm Dense core Example: biogenic amines Neuroendocrine cells
Figure 1.9 Electron microscopy. Birbeck granules (arrow) in Lang-

Figure 1.8 Electron microscopy. Neuroendocrine granules in small erhans cell histiocytosis. (Photo courtesy of Janet Schwarz, Senior
cell carcinoma of the lung. Research Technician, Microscopy Imaging Center, University of Ver-
mont, Burlington, VT.)
Single Membrane–Bound Structures

TABLE 1.3 FILAMENTS AND TUBULES
• C ytoplasmic granules are classified based on size and
morphology (Table 1.2) Component Diameter Location
• Lysosomes Microfilaments (actin, 6–8 nm Cytoskeleton of all
• Contain enzymes that assist in digesting material to nonmuscle myosin) cells
be disposed of in the cell Intermediate filaments 10 nm
• Endogenous and exogenous pigments can be col- Cytokeratin >19 proteins Epithelial cells
lected in lysosomes; can be large and filled with 40–68 kD
undigested cellular components in lysosomal storage Glial fibrillary acid 55 kD Astrocytes
protein
disorders
Neurofilament 68, 160, 200 kD Neural tissue
• Dense core granules: seen in cells and tumors with neu-
Vimentin 57 kD Mesenchymal tissues
roendocrine differentiation (Figure 1.8) Desmin 53 kD Muscle
• Melanosomes and premelanosomes are specific single Microtubules 25 nm Neural derivatives (e.g.,
membrane–bound structures neuroblastoma)
• Weibel-Palade bodies are specific for endothelial cells
• Birbeck granules are seen in Langerhans cell histiocyto- kD, kilodaltons; nm, nanometers; 50 kD = ∼4 nm.
sis (Figure 1.9)
• T erminal web and rootlets in villi are seen in foregut
Filaments and Tubules derivatives (e.g., colon)
• F ilaments are classified based on size (Table 1.3) • Junctions are seen in virtually all cells except those of
• Microtubules are seen in association with the mitotic hematopoietic origin
spindle and in cells or tumors of neural origin (e.g., • Basal lamina is seen surrounding all endodermal and
neuroblastoma) ectodermal derivatives; cells with muscle differen-
tiation also may have a basal lamina, which may be
Cell Surface incomplete
• C ell processes are seen in cells that are capable of move-
ment; some tumors, such as schwannomas and menin- Extracellular Matrix
giomas, demonstrate interdigitating processes • C
ollagen shows a regular structure amyloid
• Villi are prominent and regular in cells or tumors of • Fibrils measuring approximately 10 nm in diameter,
glandular origin (Figure 1.10) with an electron-lucent core
• V arious techniques may unmask antigens, such as

digestion by enzymes (e.g., trypsin) or antigen retrieval
using heat, metallic mordants, or alkaline buffers
• Commonly used enzymes include peroxidase, alka-
line phosphatase, and glucose oxidase
• Most commonly used chromogen substrates pro-
duce brown 3,3’-Diaminobenzidine (DAB), or red
3-Amino-9-Ethylcarbazole (AEC) reaction products
• Definition of terms
• Polyclonal antibody: conventional antiserum pro-
duced by multiple plasma cells of an animal that had
been injected with an antigen; a polyclonal antibody
may have multiple determinants (binding sites)
• Monoclonal antibody: produced by fusion of a malignant
cell with a plasma cell producing antibody to a specific
Figure 1.10 Electron microscopy. Short villi lining an intracytoplas-
epitope; antibodies may be grown in tissue culture
mic lumen in adenocarcinoma of the breast. • Antibodies for the detection of cellular components
• Intermediate filaments (see Table 1.3)
• Other cellular and tissue components: (e.g., α1-
antitrypsin, myeloperoxidase, synaptophysin and
chromogranin, myoglobin)
• F
ibrils are straight, nonbranching, and arranged • Leukocyte antigens and Ig components commonly
randomly used in paraffin-embedded tissues
• T-cell
Selected References • CD1a: thymocyte; also marks Langerhans cells
Ghadially FN. Diagnostic Electron Microscopy of Tumors. Boston: • CD3: Pan–T-cell marker that shows cytoplasmic and
Butterworth-Heinemann; 1986. membrane staining
Ghadially FN. Diagnostic Ultrastructural Pathology. 2nd ed. Boston: • CD5: Pan–T-cell marker also expressed by some B-cell
Butterworth-Heinemann; 1998.
lymphomas
Ghadially FN. Ultrastructure of the Cell and Matrix. 4th ed. Boston:
Butterworth-Heinemann; 1997. • CD43: Pan–T- cell marker also expressed by some
B-cell lymphomas
• CD45RO (UCHL-1), CD4, CD8: T-cell markers
IMMUNOHISTOCHEMISTRY • B-cell
INTRODUCTION • CD20: Pan–B-cell marker
• Ig heavy and light chains: used for demonstration of
IHC combines anatomic, immunologic, and biochemical clonality in B-cell neoplasms
techniques to identify specific tissue components using • Myeloid
a specific antigen-antibody reaction labeled with a vis- • CD15 (Leu-M1): pan-myeloid antigen that also marks
ible reporter molecule. This binding is then visualized Reed-Sternberg cells of Hodgkin lymphoma
through the use of various enzymes that are coupled to • Monocyte and histiocyte
the antibodies being used. The enzyme acts on a chromo- • CD163, CD68
genic substrate to cause deposition of a colored material • Natural killer cell
at the site of antibody-antigen bindings. Hence IHC per- • CD57 (Leu-7)
mits the visualization and localization of specific cellu- • CD56 (neural cell adhesion molecules, NCAM,
lar components within a cell or tissue while importantly Leu-19)
preserving the overall morphology and structure of the • Megakaryocyte
tissue section. Key improvements in protein conjugation, • CD41
antigen preservation and antigen retrieval methods, and • Factor VIII–von Willebrand factor (vWF)
enhanced immunodetection systems have enshrined IHC • Ulex europaeus agglutinin-1 (UEA-1)
as a major adjunctive investigative tool for both surgical • Hormones and hormone receptors
and cytopathology. IHC is not only critical for the accu- • Presence may have prognostic significance
rate diagnosis of malignancies but also plays a pivotal role • Estrogen and progesterone receptors in breast
in prognostic evaluation (e.g., estrogen and progesterone carcinomas
receptors in breast cancer) and treatment strategies (e.g., • Androgen receptors
c-kit protein for gastrointestinal stromal tumors and HER- • Infectious agents
2-neu in certain breast cancers). • Oncogenes and oncogene products
• May correlate with prognosis
TECHNICAL OVERVIEW • bcl-1, bcl-2, bcl-6 in lymphoid neoplasms
• HER-2-neu and C-erbB2 in breast carcinomas (Figure
• F
ormalin cross- links proteins in tissues; success of 1.11)
immunohistochemical staining depends on the avail- • p53 tumor suppressor gene: mutations are seen in a
ability of an antigen after fixation variety of malignant tumors
Figure 1.11 Immunohistochemistry for HER-2-neu in a breast adeno- Figure 1.12 Immunohistochemistry for HepPar-1 highlighting strong
carcinoma showing (3+) membranous staining. cytoplasmic staining of normal hepatic parenchyma.
GROUND RULES FOR • A void preordering an IHC panel of antibodies before

QUALITY APPLICATION OF previewing the morphology; remember that IHC is
IMMUNOHISTOCHEMISTRY IN SURGICAL an ancillary or adjunctive technique to the quality
PATHOLOGY practice of surgical pathology and not vice versa
• Interpretation
• T echnique • Interpretation of IHC should always be made in the
• It is imperative that the pathologist work closely context of the known subcellular localization or dis-
with the immunohistotechnologist to optimize, vali- tribution of the targeted antigen (e.g., membranous,
date, and interpret the IHC assay for any particular cytoplasmic, nuclear, or perinuclear “Golgi pattern”
antibody reagent of immunoreactivity) (Figures 1.12 and 1.13)
• Adequate fixation of tissue or specimen in 10% • Controls
buffered formalin is essential to high-quality IHC; it • Finally, the importance of adequate incorporation
is probably better to overfix (because modern anti- of appropriate tissue and reagent (both positive
gen retrieval systems can unmask epitopes) rather and negative) controls in every run of IHC cannot
than underfix (because inadvertent alcohol fixa- be overemphasized; this is ultimately the highest
tion during tissue processing precipitates and masks form of quality control of the IHC assay and should
epitopes) be reviewed daily to avoid false-positive and false-
• It is best to use a polymer-based detection system, negative interpretation
which has the advantage of being avidin- biotin
free, thereby avoiding false immunoreactivity with
endogenous biotin A PRACTICAL TABULAR APPROACH TO
• Appropriate antigen retrieval systems should be USING IMMUNOHISTOCHEMISTRY FOR
optimized for each antibody (noting that different COMMON DIAGNOSTIC PROBLEMS
antibodies require unique systems, and some require
• B
ecause a complete technical overview of IHC and com-
none)
prehensive listing of available antibodies is beyond the
• Antibody choice
scope of this chapter, our goal is to provide a practical
• A generic screening panel of antibodies should be
approach to IHC application in surgical pathology; the
chosen initially, followed algorithmically by a spe-
following tables are presented as guidelines to assist with
cific panel to further characterize a neoplasm
the choice of an antibody panel when confronted with
• Avoid using a single antibody in isolation (because
certain differential diagnoses (Tables 1.4 through 1.36)
this may result in a potentially erroneous diagnosis),
and always use more than one antibody to target a PEARLS
specific antigen
• The choice of a panel of antibodies to target a spe- • T umors are not 100% specific or sensitive to a particular
cific antigen should always be made in the con- immunoreagent; interpretation of these tables should be
text of the morphology and clinical presentation used in this context to avoid diagnostic pitfalls
of any neoplasm; avoid use of the “buckshot” • Always target the IHC panel in the context of the
approach in hope that an IHC assay returns a posi- morphologic differential diagnosis
tive reaction
Selected References FLOW CYTOMETRY

Dabbs D. Diagnostic Immunohistochemistry. 2nd ed. Philadelphia:
Churchill Livingstone; 2006. INTRODUCTION
Jagirder J. Immunohistochemistry: then and now. Arch Pathol Lab Med.
2008;132:323–509. • F low cytometry is widely used to immunophenotypi-
Leong AS-Y, Leong TY-M. Newer developments in immunohistology. J cally detect clonal hematopoietic populations (e.g.,
Clin Pathol. 2006;59:1117–1126. leukemia and lymphoma)
Yaziji H, Barry T. Diagnostic immunohistochemistry: what can go
wrong? Adv Anat Pathol. 2006;13:238–246.
• When performed on peripheral blood, bone marrow,
and lymph nodal tissue, single- cell suspensions are
required
• Manipulation of solid tissue samples into single-cell
suspensions can sometimes compromise the integrity
of the cell surface
TECHNICAL OVERVIEW
• S ingle-cell suspension is split into multiple tubes
• Various fluorescent- labeled antibodies against differ-
ent cell surface antigens (each with a different attached
fluorochrome) are added to each tube
• One by one, the cells are run through the flow cytom-
eter; as the cells pass through the counting chamber,
multiple data points are collected
• Degree of forward light scatter (FSC): indicator of cell
size
• Degree of 90-degree light scatter or side scatter (SSC):
indicator of nuclear complexity and cytoplasmic
granularity
A
• Intensity of fluorochrome on the cell surface: detects
expression of cell surface antigens (e.g., CD45, leuko-
cyte common antigen)
• Gating: the cells of interest are digitally selected for
interpretation; for example, if lymphocytes are to be
examined, one would “gate” around the cells that
exhibit low side scatter (SSC) (little cytoplasmic granu-
larity) and strong CD45 (leukocyte common antigen)
expression (Figure 1.15)
• Typical findings in mantle cell lymphoma would
include a CD20-positive population (B-cells) exhibit-
ing coexpression of CD19 and CD5 (narrowing the dif-
ferential to small lymphocytic lymphoma and mantle
cell lymphoma), with light chain restriction support-
ing clonality. Lack of CD23 expression helps to exclude
small lymphocytic lymphoma, which would have an
immunophenotype similar to that of mantle cell lym-
B phoma, except for CD23 expression and dimmer light
Figure 1.13 Immunohistochemistry for TTF-1. A, Nuclear immu- chain expression. Follicular lymphoma would also
noreactivity in normal thyroid parenchyma. B, Nuclear immunoreac- consist of a population of CD20-positive B-cells that
tivity in pulmonary adenocarcinoma. express CD10 and lack CD5
TABLE 1.4 IMMUNOHISTOCHEMISTRY APPROACH TO UNDIFFERENTIATED TUMORS

Pan-CK EMA S-100 SALL4 LCA CD138
Carcinoma + + − −/v − −
Melanoma −/v − + − − −
Germ cell v − − + − −
Lymphoma − − − − + −
Anaplastic plas- − + − − −/+ +
macytoma/
myeloma
EMA, Epithelial membrane antigen; LCA, leukocyte common antigen; Pan-CK, pan-cytokeratin; SALL4, sal-like4; v, variable; +, positive; −, negative; −/+,
rarely positive.
Selected Reference • A fter staining of the chromosomes, specific chromo-

Carey JL, McCoy P, Keren DF. Flow Cytometry in Clinical Diagnosis. 4th
somal abnormalities can be detected
ed. Chicago: ASCP Press; 2007. • In surgical pathology practice at University of Ver-
mont Medical Center we routinely submit fresh
tissue in Hanks solution for cytogenetics in the fol-
CYTOGENETIC ANALYSIS
lowing cases
• T
echnical overview • All renal tumors (except for urothelial carcinomas of
• Fresh tissue is incubated in short-term culture, and the renal pelvis)
metaphase chromosomes are spread on glass slides • Any soft tissue tumor larger than 5 cm (including
adipocytic neoplasms) (Figure 1.16)
TABLE 1.5 IMMUNOPHENOTYPIC DISTRIBUTION • In addition, a portion of fresh tissue (1 cm3, if avail-
OF CYTOKERATINS 7 AND 20 able) is snap-frozen for potential molecular analy-
ses for tumor-specific translocations or for potential
Carcinoma Typea CK7 CK20
treatment protocols
Colorectal and Merkel cell − + • Oncogenes (Table 1.37) and tumor suppressor genes
Hepatocellular − − (Table 1.38) of importance in surgical pathology
Salivary gland + − • Cytogenetic abnormalities of importance in surgical
Lung, non–small cell carcinoma + −
pathology (Table 1.39)
Lung, neuroendocrine carcinoma − −
Breast, ductal + −
Ovarian, serous, and endometrioid + − Selected References
Endometrial and endocervical + − Gersen SL, Keagle MB. The Principles of Clinical Cytogenetics. 2nd ed.
Renal cell − − Totowa: Humana Press; 2004.
Prostatic − − Korf B. Molecular medicine: molecular diagnosis (part I). N Engl J Med.
Urothelial + + 1995;332:1218–1220.
Pancreas +/− +/− Korf B. Molecular medicine: molecular diagnosis (part II). N Engl J Med.
1995;332:1499–1502.
Mesothelioma + −
Richmond JA, Tang M, Cooper K. Cytogenetic and clinicopatho-
logic analysis of benign lipomatous tumors. Arch Pathol Lab Med.
CK, Cytokeratin; +, positive; −, negative; +/−, variably positive.
aOnly approximately 70% to 90% of these tumors follow the given 2005;129:553.
CK7/20 immunoprofile; therefore reliance solely on this profile to
determine the primary site of carcinomas is not recommended.
TABLE 1.6 SPECIFIC ANTIBODY REAGENTS TO IDENTIFY PRIMARY SITE OF METASTATIC CARCINOMA
Carcinoma Type Antibody Signal Localization Other Tumors Identified
Breast GCDFP-15 Cytoplasmic Salivary, sweat gland
Breast Mammaglobin Cytoplasmic Salivary, sweat gland
Breast GATA3 Nuclear Urothelial, Salivary glands
Colon CDX2 Nuclear Subset of pancreas, gastric
Hepatocellular HepPar-1 Ag Cytoplasmic Hepatoid carcinomas of
stomach, ovary
Hepatocellular pCEA or CD10 Bile canaliculi Hepatoid carcinomas
Hepatocellular GPC-3 Membranous and Melanoma, a subset of
cytoplasmic chronic active hepatitis
Lung and thyroid except mucinous TTF-1 Nuclear Neuroendocrine carcinoma
adenocarcinoma in situ (formerly extrapulmonary
mucinous BAC)
Lung squamous cell carcinoma p40 Nuclear —
Ovarian serous WT-1, p16 Nuclear Mesothelioma (WT-1)
Prostate NKX3.1 Nuclear
Prostate PSA, PAP Cytoplasmic
Squamous, urothelial, thymic p63 Nuclear Salivary gland, neuroendo-
crine, subset prostate
Thyroid Thyroglobulin Cytoplasmic —
Urothelial Uroplakin III Membranous —
Renal, clear RCC Membranous
BAC, Bronchoalveolar carcinoma; GATA3, GATA Binding Protein 3; GCDFP-15, gross cystic disease fluid protein-15; GPC-3, glypican 3; NKX3.1, NK3
Homeobox 1; PAP, prostatic acid phosphatase; pCEA, polyclonal carcinoembryonic antigen; PSA, prostate-specific antigen; RCC, renal cell carcinoma;
TTF-1, thyroid transcription factor-1; WT-1, Wilms tumor gene protein 1.
Modified from Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus meta-
static neoplasms. Arch Pathol Lab Med. 2007;131:1648–1654; Bishop JA, Teruya-Feldstein J, Westra WH, et al. p40 (ΔNp63) is superior to p63 for the
diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 2012;25:405–415.
From Conner JR, Hornick JL. Metastatic carcinoma of unknown primary: diagnostic approach using immunohistochemistry. Adv Anat Pathol.
2015;22(3):149–167.
From Miettinen M, McCue PA, Sarlomo-Rikala M, et al. GATA3: a multispecific but potentially useful marker in surgical pathology: a systemic analysis of
2500 epithelial and nonepithelial tumors. Am J Surg Pathol. 2014;38(1):13–22.
TABLE 1.7 IMMUNOHISTOCHEMISTRY PANEL FOR INTERPRETATION OF LUNG MESOTHELIOMA AND

ADENOCARCINOMA
Epithelioid Mesothelioma Sarcomatoid Mesothelioma Adenocarcinoma (Percentage
Antibody (Percentage Positive) (Percentage Positive) Positive)
Epithelial Marker
Naspin A Negative Negative 83 (lung)
mCEA 3 — 81
Ber-Ep4 10 0 80
B72.3 7 0 80
CD15 (Leu-M1) 7 0 72
MOC-31 7 0 93
TTF-1 Negative 0 72 (lung)
Mesothelial Marker
Cytokeratin 5/6 83 13 15
Calretinin 82 88 15
WT-1 77 13 4
D2-40 86–100 0 36 (weak)
Mesothelin 100 0 —
BAP1 (loss of nuclear expression) 55.4 41.7 __
BAP1, BRCA1-associated protein 1; mCEA, monoclonal carcinoembryonic antigen; TTF-1, thyroid transcription factor-1; WT-1, Wilms tumor gene
protein 1.
Modified from Marchevsky AM. Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med. 2008;132:397–
401; Bishop JA, Sharma R, Illei PB: Naspin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney,
thyroid, and malignant mesothelioma. Hum Pathol. 2010;41:20–25.
From Erber R, Warth A, Muley T, et al. BAP1 loss is a useful adjunct to distinguish malignant mesothelioma including the adenomatoid-like variant from
benign adenomatoid tumors. Appl Immunohistochem Mol Morphol. 2019 Jan 11. doi: 10.1097 [Epub ahead of print].
TABLE 1.8 IMMUNOHISTOCHEMISTRY PANEL FOR LUNG ADENOCARCINOMA AND BREAST

ADENOCARCINOMA
Immunostain Lung Adenocarcinoma (Percentage Positive) Breast Adenocarcinoma (Percentage Positive)
TTF-1 77 0
Naspin A 83 0
Mammaglobin 17 85
GCDFP-15 2 53
ER 4 72
ER, Estrogen receptor; GCDFP-15, gross cystic disease fluid protein-15; TTF-1, thyroid transcription factor-1.
Data from Takeda Y, Tsuta K, Shibuki Y, et al. Analysis of expression patterns of breast cancer-specific markers (mammaglobin and gross cystic disease
fluid protein 15) in lung and pleural tumors. Arch Pathol Lab Med. 2008;132:239; Striebel JM, Dacic S, Yousem SA. Gross cystic disease fluid protein
(GCDFP-15): expression in primary lung adenocarcinoma. Am J Surg Pathol. 2008;32:426; Bishop JA, Sharma R, Illei PB. Naspin A and thyroid transcrip-
tion factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol. 2010;41:20–25.
TABLE 1.9 IMMUNOHISTOCHEMISTRY COMPARISON OF SPINDLE CELL AREAS IN METAPLASTIC CARCINOMA,

PHYLLODES TUMOR, AND FIBROMATOSIS OF THE BREAST
CD34 SMA 34βe12 Pan-CK β-catenin Desmin p63
Metaplastic carcinoma − +/− +/− −/+ − −/+ +
Phyllodes +/− +/− − − − −/+ −
Fibromatosis − +/− − − + − −
Myofibroblastoma + +/− − − − + −
Myoepithelial tumor − +/− +/− + − −/+ +/−
Pan-CK, Pan-cytokeratin; SMA, smooth muscle actin; +, positive; −, negative; +/−, often positive; −/+, rarely positive.
Modified from Dunne B, Lee AH, Pinder SE, et al. An immunohistochemical study of metaplastic spindle cell carcinoma, phyllodes tumor and fibromatosis
of the breast. Hum Pathol. 2003;34:1009–1015.
TABLE 1.10 USEFUL ANTIBODY PANEL TO DEMONSTRATE MYOEPITHELIAL AND BASAL CELLS IN BREAST
LESIONS TO DISTINGUISH BENIGN (+) FROM INVASIVE (−) CARCINOMA
Myoepithelial/Basal Cells Stromal Myofibroblasts
Smooth muscle heavy-chain myosin + (Cytoplasmic) −/+
p63 + (Nuclear) −
α-SMA + (Cytoplasmic) +/−
S-100 + (Nuclear and cytoplasmic) v
Calponin + (Cytoplasmic) −/+
D2-40a −/+ −
SMA, Smooth muscle actin; v, variable; +, positive; −, negative; −/+, rarely positive.
aD2-40 is a useful marker to highlight lymphatic endothelium in lymphovascular invasion (LVI) by carcinoma but may in addition occasionally stain myo-
epithelial and basal cells—hence the use of D2-40 to demonstrate that LVI should always be accompanied by p63/SMHCM immunohistochemistry.
Modified from Rabban JT, Chen YY. D2-40 expression by breast myoepithelium: potential pitfalls in distinguishing intralymphatic carcinoma from in situ
carcinoma. Hum Pathol. 2008;39:175–183.
TABLE 1.11 IMMUNOHISTOCHEMICAL PANEL APPROACH TO DIFFERENTIAL DIAGNOSIS OF

HEPATOCELLULAR CARCINOMA
Inhibin/
Arginase-1 HepPar-1 CK19 MOC-31 GPC-3 pCEA CDX-2 TTF-1 RCC Melan-A/D2-40
Hepatocellular carcinoma + + − −/+ + + − −a − −
Cholangiocarcinoma −/+ − +/− +/− − − − − −
Metastatic adenocarcinoma −
Colon − − − − + − − −
Thyroid, lung − − − − − + − −
Tumors with polygonal cells −
RCC − − + − − − + −
Adrenocortical carcinoma − − − − − +
Neuroendocrine tumorsb − − + − v
Hepatoid carcinoma (e.g., gastric, − +
ovary)
CK, Cytokeratin; p-CEA, canalicular pattern of staining; RCC, renal cell carcinoma; TTF-1, thyroid transcription factor-1; v, variable; +, positive; −, negative;
+/−, often positive; −/+, rarely positive.
aCertain TTF-1 antibody reagents may highlight the cytoplasm of liver cells (only nuclear immunoreactivity should be interpreted as being of thyroid or
lung origin in the correct clinical setting).

bStrong synaptophysin and chromogranin support neuroendocrine tumor; TTF-1 may notoriously highlight extrapulmonary neuroendocrine tumors.
Modified from Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus
metastatic neoplasms. Arch Pathol Lab Med. 2007;131:1648–1654; Yan BC, Gong C, Song J, et al. Arginase-1: a new immunohistochemical marker of
hepatocytes and hepatocellular neoplasms. Am J Surg Pathol. 2010;34:1147–1154.
TABLE 1.12 IMMUNOHISTOCHEMISTRY PANEL INTERPRETATION FOR GASTROINTESTINAL AND ABDOMINAL

SPINDLE CELL TUMORS
ALK WT-1 DOG1 CD117 CD34 STAT6 SMA Desmin S-100 Protein β-Catenin
Leiomyoma −/+ − − + + −
Leiomyosarcoma (LMS)a +/− − −/+a + + −

Inflammatory myofibroblastic tumor + − − − +/− − −
Inflammatory fibroid polyp − − + +/− − −
Solitary fibrous tumor − − + + − − −
Desmoid fibromatosis − − − + −/+ − + (Nuclear)
Gastrointestinal schwannoma − − − − − +
Metastatic melanoma − +/− − − − +
Desmoplastic small round cell tumor + − − − − + −
GIST + + + +/− −/+ −/+
ALK, Anaplastic lymphoma kinase; GIST, gastrointestinal stromal tumor; SMA, smooth muscle actin; STAT6, Signal transducer and activator of transcription
(STAT) 6; +, positive; −, negative; +/−, often positive; −/+, rarely positive.
aRetroperitoneal LMS may be positive.
Modified from Miettinen M, Sobin LH, Sarlomo-Rikala M. Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with
a reference to CD117 (KIT). Mod Pathol. 2000;13:1134–1142; Sah SP, McCluggage WG. DOG1 immunoreactivity in uterine leiomyosarcomas. J Clin
Pathol. 2013;66:40–43; Hill DA, Pfeifer JD, Marley EF, et al. WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing
sarcoma/primitive neuroectodermal tumor: an immunohistochemical and molecular diagnostic study. Am J Clin Pathol. 2000;114:345–354.
From Coffin CM, Hornick JL, Fletcher CD. Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical
features including ALK expression in atypical and aggressive cases. Am J Surg Pathol. 2007;31(4):509–520.
Doyle LA, Vivero M, Fletcher CD, et al. Nuclear expression of STAT6 distinguishes solitary fibrous tumor from histologic mimics. Mod Pathol.
2014;27(3):390–395.
TABLE 1.13 IMMUNOPHENOTYPE OF PRIMARY OVARIAN AND METASTATIC COLORECTAL ADENOCARCINOMA

Mucinous Ovarian Tumors
Intestinal Type Endocervical Type Endometrioid Adenocarcinoma Metastatic Colorectal Adenocarcinoma
CK7 +++/+ +++ +++ −
CK20 −/+/+++ − − +++
mCEA + − − ++
STAB2 +++
CDX2 + − −/+ ++
ER − + + −
ER, Estrogen receptor; mCEA, monoclonal carcinoembryonic antigen; STAB2, Special AT-rich sequence binding-protein 2 (SATB2); +++, diffusely positive;
+, focally positive; −, negative.
Modified from McCluggage WG: My approach to and thoughts on the typing of ovarian carcinomas. J Clin Pathol. 2008;61:152–163.
From Conner JR, Hornick JL. Metastatic carcinoma of unknown primary: diagnostic approach using immunohistochemistry. Adv Anat Pathol.
2015;22(3):149–167.
TABLE 1.14 IMMUNOHISTOCHEMISTRY PANEL FOR PRIMARY AND METASTATIC ADENOCARCINOMA

OF THE OVARY
PAX2 PAX8 CK7 CK20 CDX2 DPC4
Primary mucinous, intestinal type − − + + +/− +
Primary endometrioid + + + − − +
Metastatic colorectal − − − + + +
Metastatic pancreas − − +/− +/− − −
Metastatic thyroid +
Metastatic renal +
Metastatic thymic +
CK, Cytokeratin; DPC, deleted in pancreatic carcinoma; +, positive; −, negative; +/−, often positive.
Modified from Ji H, Isacson C, Seidman JD, et al. Cytokeratins 7 and 20, Dpc4, and MUC5AC in the distinction of metastatic mucinous carcinomas in the
ovary from primary ovarian mucinous tumors: Dpc4 assists in identifying metastatic pancreatic carcinomas. Int J Gynecol Pathol. 2002;21:391–400;
Ozcan A, Liles N, Coffey D. PAX2 and PAX8 expression in primary and metastatic müllerian epithelial tumors: a comprehensive comparison. Am J Surg
Pathol. 2011;35:1837–1847.
TABLE 1.15 IMMUNOHISTOCHEMISTRY: HIGH-GRADE SEROUS CARCINOMA AND POORLY DIFFERENTIATED

ENDOMETRIOID ADENOCARCINOMA OF THE OVARY AND ENDOMETRIUM
WT-1 p53 p16 β-Catenin
Serous +++ +++ +++ Membranous
Endometrioid −/+ −/+/+++a −/+ Membranous/nuclear
WT-1, Wilms tumor gene protein 1; +++, diffusely positive; +, focally positive; −, negative.
aThe +++ expression corresponds to some high-grade carcinomas.
Data from McCluggage WG. My approach to and thoughts on the typing of ovarian carcinomas. J Clin Pathol. 2008;61:152–163.
TABLE 1.16 IMMUNOHISTOCHEMISTRY APPROACH TO OVARIAN SEX CORD–STROMAL TUMORS AND

ENDOMETRIOID ADENOCARCINOMA
FOXL2 Inhibin Calretinin CD99 EMA Pan-cytokeratin
Granulosa cell tumor + + + + − −/+
Sertoli-Leydig cell tumor +/− + + + − +/−
Endometrioid − − − + +
adenocarcinoma
EMA, Epithelial membrane antigen; +, positive; −, negative; +/−, often positive; −/+, rarely positive.
Modified from Mount SL, Cooper K. Tumours with divergent müllerian differentiation of the uterine corpus. Curr Diagn Pathol. 2005;11:349–355; Al-Agha
OM, Huwait HF, Chow C, et al: FOXL2 is a sensitive and specific marker for sex cord-stromal tumors of the ovary. Am J Surg Pathol. 2011;35:484-494.
TABLE 1.17 IMMUNOHISTOCHEMISTRY APPROACH TO ENDOCERVICAL ADENOCARCINOMA AND

ENDOMETRIOID ENDOMETRIAL ADENOCARCINOMA
mCEA Vimentin ER/PR p16 HPV DNA
Endocervical + − − + +
Endometrial − + + −/+ −
ER/PR, Estrogen/progesterone receptor; HPV, human papillomavirus; mCEA, monoclonal carcinoembryonic antigen; +, positive; −, negative; −/+, rarely positive.
Modified from Staebler A, Sherman ME, Zaino RJ, Ronnett BM. Hormone receptor immunohistochemistry and human papillomavirus in situ hybridization
are useful for distinguishing endocervical and endometrial adenocarcinomas. Am J Surg Pathol. 2002;26:998–1006.
TABLE 1.18 IMMUNOHISTOCHEMISTRY IN THE DIFFERENTIAL DIAGNOSIS OF SQUAMOUS AND GLANDULAR

LESIONS OF THE UTERINE CERVIX
p16a MIB-1 (Ki-67)
LSIL (CIN I) +/− Increased
HSIL (CIN II-III) + Increased (full thickness)
Adenocarcinoma in situ + +
Atypical immature metaplasia − −/+
Reactive squamous or glandular atypia − +
Tubal metaplasia +/− −
CIN, Cervical intraepithelial neoplasia; HSIL, high-grade squamous intraepithelial neoplasia; LSIL, low-grade squamous intraepithelial neoplasia; +, positive;
−, negative; +/−, often positive; −/+, rarely positive. MIB-1, monoclonal antibody directed against the Ki-67 antigen, a nuclear antigen expressed by all
human proliferating cells.
aExpression of p16 (nuclear and cytoplasmic) is a surrogate marker for high-risk human papillomavirus (HPV), for example, HPV-16 and HPV-18. In LSIL,
the p16 expression may be confined to the lower one third or one half of the squamous epithelium or show focal immunoreactivity (the latter being
a pattern of expression, albeit cytoplasmic only, that may be seen in reactive squamous epithelia). HSIL p16 immunoexpression usually involves two-
thirds or full thickness of the squamous epithelium.
Modified from Kalof AN, Cooper K. p16INK4a immunoexpression: surrogate marker of high-risk HPV and high-grade cervical intraepithelial neoplasia.
Adv Anat Pathol. 2006;13:190–194.
TABLE 1.19 P57KIP2 IMMUNOREACTION AND HER-2 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ANALYSIS
IN MOLAR PREGNANCY
Villous Cytotrophoblasts Villous Stroma Syncytiotrophoblasts HER-2 FISH Analysis
Complete hydatidiform − − + Diploid
molar pregnancy
Partial hydatidiform mo- + + + Triploid
lar pregnancy
Hydropic abortion + + + Diploid
Note: p57KIP2 is a paternally imprinted, maternally expressed gene protein. Hence, complete moles comprising only paternal genes will not express this
protein.
Modified from Hoffner L, Dunn J, Esposito N, et al. p57KIP2 Immunostaining and molecular cytogenetics: combined approach aids in diagnosis of
morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions. Hum Pathol.
2008;39:63; and LeGallo RD, Stelow EB, Ramirez NC, et al. Diagnosis of hydatidiform moles using p57 immunohistochemistry and her2 fluorescent in
situ hybridization. Am J Clin Pathol. 2008;129:749.
TABLE 1.20 IMMUNOHISTOCHEMICAL APPROACH FOR TROPHOBLASTIC LESIONS

Trophoblastic Lesion CK18 p63 hPL MIB-1 LI (%)
Exaggerated placental site +++ − +++ <1
Placental site trophoblastic tumor +++ − +++ >1
Placental site nodule +++ +++ −/+ <10
Epithelioid trophoblastic tumor +++ +++ −/+ >10
Choriocarcinoma +++ −/+ ++
Note: Expression of p63 highlights mononucleated trophoblasts corresponding to cytotrophoblasts, and human chorionic gonadotropin selectively stains
syncytiotrophoblasts; this combination is indicative of choriocarcinoma. CK, Cytokeratin; hPL, human placental lactogen; LI, labeling index; MIB-1, Ki-
67 proliferation marker; +++, diffusely positive; ++, focally positive; −, negative; −/+, rarely positive.
Modified from Shih IM, Kurman RJ. p63 Expression is useful in the distinction of epithelioid trophoblastic and placental site trophoblastic tumors by profil-
ing trophoblastic subpopulations. Am J Surg Pathol. 2004;28:1177–1183.
TABLE 1.21 IMMUNOHISTOCHEMISTRY FOR SELECTED GERM CELL TUMORS

SOX2 SALL4 c-kit OCT3/4 CD30 AFP GPC-3 D2-40 β-hCG
Germinoma − + + + − − − + −a
Embryonal carcinoma + + − + + − − − v
Yolk sac tumor − + − − − v + − −
Choriocarcinoma − +/− − − − − − − +
AFP, α-fetoprotein; β-hCG, β-human chorionic gonadotropin; GPC-3, glypican-3; SALL4, sal-like4; SOX2 (also known as SRY [sex determining region Y]-
box2); v, variable; +, positive; −, negative; +/−, often positive. Cao D, Li J, Guo CC. SALL4 is a novel diagnostic marker for testicular germ cell tumors.
Am J Surg Pathol. 2009;33:1065–1077; Gopalan A, Dhall D, Olgac S, et al. Testicular mixed germ cell tumors: a morphological and immunohisto
chemical study using stem cell markers, OCT3/4, SOX2 and GDF3, with emphasis on morphologically difficult-to-classify areas. Mod Pathol 2009;22:
1066–1074.
aExcept for syncytiotrophoblastic giant cells in seminoma.
Modified from Ulbright TM. The most common, clinically significant misdiagnoses in testicular tumor pathology, and how to avoid them. Adv Anat
Pathol. 2008;15:18–27; and Young RH. Testicular tumors: some new and a few perennial problems. Arch Pathol Lab Med. 2008;132:548–564.
TABLE 1.22 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH RENAL CELL NEOPLASMS

Melan
RCC CD10 CK7 AMACR CA IX A or HMB45 TFEB TFE3 CD117 PAX2 PAX8
Clear cell carcinoma +/− + − − + − − + +
Chromophobe − − +/− − − − + − +
carcinoma
Papillary carcinoma +/− −/+ +/− + − − − −/+ +
Oncocytoma − − −/+ − − − +/− − +
Xp11/TFE3 + − + + −
translocation
renal carcinoma
RCC with t(6,11) + +
AMACR, α-methylacyl coenzyme A racemase (P504S); CA IX, carbonic anhydrase 9; CK, cytokeratin; PAX2, paired box gene-2; PAX8, paired box gene-8;
RCC, renal cell carcinoma; TFE3, transcription factor E3; +, positive; −, negative; +/−, often positive; −/+, rarely positive.
Modified from Truong LD, Shen SS. Immunohistochemical diagnosis of renal neoplasms. Arch Pathol Lab Med. 2011;135:92–109; Ozcan A, de la Roza G,
Ro JY, et al. PAX2 and PAX8 expression in primary and metastatic renal tumors: a comprehensive comparison. Arch Pathol Lab Med. 2012;136:151–154;
Suárez-Vilela D, Izquierdo-García F, Méndez-Álvarez JR, et al. Renal translocation carcinoma with expression of TFEB: presentation of a case with distinc-
tive histological and immunohistochemical features. Int J Surg Pathol. 2011;19:506–509.
TABLE 1.23 SELECTED IMMUNOHISTOCHEMISTRY TABLE 1.24 IMMUNOHISTOCHEMISTRY

TO DISTINGUISH VASCULAR NEOPLASM APPROACH TO ATYPICAL GLANDULAR
Neoplasm IHC
PROLIFERATIVE LESION IN THE PROSTATEa
Basal Cell Markers
Angiosarcomaa CD31, CD34, ERG
(HMWCK 34βE12, AMACR
Epithelioid CD31, CD34, ERG, CAMTA1
Lesion CK5/6, p63) (p504S)
hemangioendothelioma
Pseudomyogenic AE1/AE3, FLI1, FOSB (96%) Atrophic glands + −
hemangioendothelioma Post–atrophic hyperplasia + −
Kaposi sarcoma HHV8, CD31, CD34, ERG Basal cell hyperplasia + −
Epithelioid hemangioma CD31, CD34, ERG, FOSB (54%) Atypical adenomatous +/− (patchy) −/+
hyperplasia (adenosis)
CAMTA1, Calmodulin binding transcription activator 1; ERG, ERG (Erythro- Prostatic intraepithelial + +
blast transformation-specific [ETS]-related gene); FLI1, Friend leukemia neoplasia
integration-1; FOSB, FBJ Murine Osteosarcoma Viral Oncogene Homo-
Prostate carcinoma −a,b +
log B; IHC, immunohistochemistry.
aAngiosarcoma is also cytokeratin and EMA positive (+). Al-Abbadi MA,
AMACR, α-methylacyl coenzyme A racemase; CK, cytokeratin; HMWCK,
Almasri NM, Al-Quran S, Wilkinson EJ. Cytokeratin and epithelial mem-
high-molecular-weight cytokeratin; +, positive; −, negative; +/−, often
brane antigen expression in angiosarcomas: an immunohistochemical
positive; −/+, rarely positive.
study of 33 cases. Arch Pathol Lab Med. 2007;131:288–292. aSee Figure 1.14.
Miettinen M, Wang ZF, Paetau A, et al. ERG transcription factor as an bRarely, p63 may demonstrate immunoreactivity in prostate carcinoma
immunohistochemical marker for vascular endothelial tumors and
(see Ali TZ, Epstein JI. False positive labeling of prostate cancer with
prostatic carcinoma. Am J Surg Pathol. 2011;35:432–441.
high molecular weight cytokeratin: p63 a more specific immunomarker
Doyle LA, Fletcher CD, Hornick JL. Nuclear expression of CAMTA1 distin-
for basal cells. Am J Surg Pathol. 2008;32:1890–1895).
guishes epithelioid hemangioendothelioma from histologic mimics. Am
Modified from Paner GP, Luthringer DJ, Amin MB. Best practices in di-
J Surg Pathol. 2016;40(1):94–102.
agnostic immunohistochemistry: prostate carcinoma and its mimics in
Hung YP, Fletcher CD, Hornick JL. FOSB is a useful diagnostic marker
needle core biopsies. Arch Pathol Lab Med. 2008;132:1388–1396.
for pseudomyogenic hemangioendothelioma. Am J Surg Pathol.
2017;41(5):596–606.
MOLECULAR PATHOLOGY METHODS NUCLEIC ACID EXTRACTION METHODS

Background
INTRODUCTION
• T he extraction of nucleic acids from pathology sam-
Molecular tests are widely relied upon as standard of ples involves cell lysis followed by selective DNA or
care assays in the diagnosis of a wide variety of patho- RNA isolation, and a quantity and quality assessment
logic conditions. Ongoing advances in molecular pathol- relative to the requirements of the end- diagnostic
ogy, genomics, epigenetics, proteomics, and infectious test
diseases research, as well as technological developments • Pathology samples: biopsy or surgical material (fresh
continue to expand the potential of molecular assays to or formalin-fixed, paraffin-embedded [FFPE] tissue),
improve disease characterization and patient care. This body fluids (amniotic, saliva, stools, urine), buccal cell
section provides an overview of the molecular techniques scrapes, cervical scrapes, fine-needle aspiration, hair
applicable to pathology practice. root, peripheral blood, primary cell culture.
TABLE 1.25 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH PROSTATE AND UROTHELIAL CARCINOMAS

GATA3 CK7 CK20 NKX3.1 PSA Uroplakin p63
Prostate carcinoma − −/+ −/+ + + − −/+
Urothelial carcinoma + +/− +/− − − +/− +
Notes: Only CK7/20 negativity (prostate carcinoma) and CK7/20 positivity (urothelial carcinoma) reliably distinguish between these two carcinomas.
Any other permutation is unreliable. Uroplakin is highly specific for urothelial carcinoma but has a low sensitivity, being focally present in approxi-
mately 50% to 60% of tumors. Expression of p63 is used more often to highlight basal cells in benign prostate glands but may rarely be positive in the
prostate carcinoma itself (see Ali TZ, Epstein JI. False positive labeling of prostate cancer with high molecular weight cytokeratin: p63 a more specific
immunomarker for basal cells. Am J Surg Pathol. 2008;32:1890–1895).
CK, Cytokeratin; PSA, prostate-specific antigen; +, positive; −, negative; +/−, often positive; −/+, rarely positive. GATA3, GATA binding protein 3.
Modified from Hammerich AH, Ayala GE, Wheeler TM. Application of immunohistochemistry to the genitourinary system (prostate, urinary bladder,
testis, and kidney). Arch Pathol Lab Med. 2008;132:432–440; Chang A, Amin A, Gabrielson E, et al. Utility of GATA3 immunohistochemistry in dif-
ferentiating urothelial carcinoma from prostate adenocarcinoma and squamous cell carcinomas of the uterine cervix, anus, and lung. Am J Surg Pathol.
2012;36:1472–1476.
TABLE 1.26 RECOMMENDED ANTIBODY PANEL FOR COMMON PLEOMORPHIC CUTANEOUS SPINDLE CELL
TUMORS
Cytokeratins Melanocytic Smooth
(Pan, HMW, (HMB-45, Muscle Endothelial
CD10 p63 CK5/6) S-100 Protein Melan-A) Actin Desmin (CD31, CD34)
Sarcomatoid − + + − − − − −
squamous cell
carcinoma
Melanoma − − −/+ + +/− − −/+ −
Atypical fibroxanthoma + −/+ − − − −/+ − −
Leiomyosarcoma − − −/+ − + +/− −/+
Angiosarcoma − − −/+ − − − − +
+, positive; −, negative; +/−, often positive; −/+, rarely positive.

Modified from Folpe AL, Cooper K. Best practices in diagnostic immunohistochemistry: pleomorphic cutaneous spindle cell tumors. Arch Pathol Lab Med.
2007;131:1517; Hultgren TL, DiMaio DJ. Immunohistochemical staining of CD10 in atypical fibroxanthomas. J Cutan Pathol. 2007;34:415–419; Dotto
JE, Glusac EJ. p63 is a useful marker for cutaneous spindle cell squamous cell carcinoma. J Cutan Pathol. 2006;33:413–417.
TABLE 1.27 MIB-1(KI-67) MEMBRANOUS AND CYTOPLASMIC STAINING

MIB-1 (Ki-67); Clone MIB-1, 1:700; Dako, Glostrup, Denmark Staining Pattern
Hyalinizing trabecular tumor of the thyroid Cytoplasmic staininga
Pulmonary sclerosing hemangiomas Membrane and cytoplasmic stainingb
aFrom Hirokawa M, Carney JA. Cell membrane and cytoplasmic staining for MIB-1 in hyalinizing trabecular adenoma of the thyroid gland. Am J Surg
Pathol. 2000;24:575–578.
bFrom Kim BH, Bae YS, Kim SH, et al. Usefulness of Ki-67 (MIB-1) immunostaining in the diagnosis of pulmonary sclerosing hemangiomas. APMIS.
2013;121:105–110.
TABLE 1.28A IMMUNOHISTOCHEMISTRY PANEL FOR THE INTERPRETATION OF LOW-GRADE (SMALL) B-CELL
LYMPHOMA
CD23 (%) CD5 (%) Cyclin D1 (%) CD10 (%) LEF1 (%) SOX11 (%)
SLL/chronic lymphocytic 85 80 0 0 92
leukemia
Mantle 2 80 75–100 2 94
Marginal zone 8 0 0 2
Lymphoplasmacytic 0–30 5 0 3
Follicular 0–25 0 0 85
Extranodal marginal 0 0 0 0
SLL, Small lymphocytic lymphoma; LEF1, lymphocyte enhancer-binding factor 1; SOX11, SRY (sex-determining region Y)–box 11.
Modified from http://surgpathcriteria.stanford.edu.
Ek S, Dictor M, Jerkeman M, et al. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood.
2008;111(2):800–805.
Menter T, Dirnhofer S, Tzankov A. LEF1. A highly specific marker for the diagnosis of chronic lymphocytic B cell leukaemia/small lymphocytic B cell lym-
phoma. J Clin Pathol. 2015;68(6):473–478.
TABLE 1.28B BASIC IMMUNOPHENOTYPIC APPROACH TO LYMPHOMAS OF SMALL B-CELL TYPE (CD20+ AND
LOW KI-67)
CD5
+ –
CD23/FMC-7 CD10
+/– –/+ – +
Chronic lymphocytic Mantle cell Marginal zone Follicular

leukemia/Small lymphoma lymphoma/ lymphoma
lymphoblastic (Cyclin D1+, lymphoplasmacytic
lymphoma (LEF1+) SOX11+) lymphoma
TABLE 1.29 ANTIBODY PANEL FOR DIFFERENTIAL DIAGNOSIS OF HODGKIN LYMPHOMA

CD30 CD15 CD20 CD45 (LCA) ALK
NLPHL (Nodular lymphocyte predominant Hodgkin − − + + −
lymphoma)
ALCL + − − −/+ +
DLBCL −/+ − + + −
ALCL, Anaplastic large cell lymphoma; ALK, alkaline kinase; DLBCL, diffuse large B-cell lymphoma; LCA, leukocyte common antigen; +, positive;
−, negative; −/+, rarely positive.
TABLE 1.30 IHC CLASSIFICATION OF DIFFUSE • T

here is increasing interest in the potential of liquid
LARGE B-CELL LYMPHOMAS INTO GERMINAL biopsy (blood/plasma/serum) samples for use in molec-
CENTER B-LIKE CELL (GCB) AND NON-GCB ular diagnostics. Diagnostic signals may be shed into
the circulation in the form of cell-free DNA or RNA
CD 10
(cfDNA/cfRNA) of diseased/normal tissue origin; cell-
free fetal DNA (cffDNA); circulating tumor cells (CTCs)/
circulating tumor DNA (ctDNA)/coding or noncoding
circulating tumor RNA (ctRNA), in particular RNA asso-
+ – ciated with exosomes
GCB BCL6 DNA Extraction Methods

• C
lassical methods were time consuming (∼3 days) and
required relatively large quantities of tissues (100 mg
to >1 g)
+ –
•
Extraction kits are now available that use glass- fiber
filters that selectively bind DNA following tissue treat-
MUM 1 NON–GCB
ments with a protease and chaotropic buffers (which
disrupt protein and DNA secondary structures); the
glass fibers, typically loaded in mini- columns, are
+ – washed and centrifuged to rinse away cellular debris,
extraction solution reagents, and pathology tissue pro-
NON–GCB GCB cessing chemicals; the DNA is then recovered from
the resin/glass-fiber by low-salt buffer rinses and cen-
Modified from Hans CP, Weisenburger DD, Greiner TC. Confirma-
tion of the molecular classification of diffuse large B-cell lym-
trifugation; pure DNA recovery from diverse pathol-
phoma by immunohistochemistry using a tissue microarray. Blood. ogy samples is possible within several hours by these
2004;103:275–282. procedures.
TABLE 1.31 IMMUNOPROFILE OF SMALL ROUND CELL TUMORS

FLI-1 NKX2-2 Pan-CK CD99 Desmin Myogenin WT-1 CD56
Ewing sarcoma, primitive + + v + − − − v
neuroectodermal tumor
Rhabdomyosarcoma − − − v + + − +
Poorly differentiated syno- − − + + − − − +
vial sarcomaa
Desmoplastic small round + − + v + − + v
cell tumor
Neuroblastoma − − − − − − − +
Lymphoblastic + − − + − − − −
lymphomab
Wilms tumor + − v v + vc + +
FLI-1, Friend leukemia virus integration-1; NKX2-2, Homeobox protein Nkx-2.2; Pan-CK, pan-cytokeratin; WT-1, Wilms tumor gene protein 1; +, positive;
−, negative; v, variable.
Folpe AL, Hill CE, Parham DM, et al. Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on
CD99-positive mimics of Ewing’s sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 2000;24:1657–1662.
aEpithelial membrane antigen is frequently positive.
bFrequently leukocyte common antigen negative.
cIn rhabdomyomatous Wilms tumor.
Modified from Barami A, Truong LD, Ro JY. Undifferentiated tumor: true identity by immunohistochemistry. Arch Pathol Lab Med. 2008;132:326–348.
From Hung YP, Fletcher CD, Hornick JL. Evaluation of NKX2-2 expression in round cell sarcomas and other tumors with EWSR1 rearrangement: imperfect
specificity for Ewing sarcoma. Mod Pathol. 2016;29(4):370–380.
TABLE 1.32 IMMUNOHISTOCHEMISTRY PANEL TABLE 1.33 IMMUNOHISTOCHEMISTRY TO DETECT

TO DISTINGUISH FOLLICULAR VARIANT OF THE GAIN OR LOSS OF PROTEIN EXPRESSION
PAPILLARY THYROID CARCINOMA FROM
Loss of SMARCB1 (INI-1)
FOLLICULAR ADENOMA
Expression (%)
HBME1 (%) CK19 (%) Galectin-3 (%)
Medullary carcinoma of kidney 100
FVPTC 96 91–100 98 Malignant rhabdoid tumor 98
FA 7–11 44–68 30 (Glypican 3+)a
Epithelioid sarcoma (classic and 90
Note: The combination of HBME1 and CK19 has the greatest utility in proximal type)
differentiating FVPTC from benign follicular lesions.
Epithelioid MPNST 50
FA, Follicular adenoma; FVPTC, follicular variant of papillary thyroid carci-
noma.
Loss of Succinate De-
Data from Erickson LA, Lloyd RV. Utility of a panel of immunohistochemi-
cal markers in the diagnosis of follicular variant of papillary thyroid
hydrogenase B (SDHB)
carcinoma. Adv Anat Pathol. 2008;15:59–60. Expressionb
Succinate dehydrogenase-deficient − (no expression)
wildtype GISTs, paragangliomas,
• Inclusive nucleic acid extraction is a feature of most
and pheochromocytomas
automated molecular diagnostic platforms. Sporadic GISTs (association with KIT + (expression)
or PDGFRA mutations)
RNA Extraction Methods
• C lassical methods required the rapid homogeniza- Kohashi K, Nakatsura T, Kinoshita Y, et al. Glypican 3 expression in
tion of large quantities of fresh tissues in protease/ tumors with loss of SMARCB1/INI1 protein expression. Hum Pathol.
2013;44:526–533.
guanidinium thiocyanate solution to denature ubiq- GIST, Gastrointestinal stromal tumor; MPNST, myxoid malignant periph-
uitous endogenous RNases that otherwise degrade eral nerve sheath tumor.
cellular RNA aGlypican 3 positive in malignant rhabdoid tumor and negative in epithe-
• Current methods allow the relatively rapid (1-day) recov- lioid sarcoma.
bBarletta JA, Hornick JL. Succinate dehydrogenase-deficient tumors: diagnos-
ery of RNA again following tissue homogenization in a
tic advances and clinical implications. Adv Anat Pathol. 2012;4:193–203.
chaotropic guanidinium salt solution that leaves RNA Modified from Hollmann TJ, Hornick JL. INI1-Deficient Tumors: Diagnostic
contained in an aqueous phase and protein and DNA Features and Molecular Genetics. Am J Surg Pathol. 2011; 35(10):e47–e63.
in an organic phase. Admixture of the aqueous phase
with nucleic acid binding glass filters allows the recov-
ery of pure total RNA by elution from the glass filters require dedicated extraction reagents for extraction
with a low-salt buffer. Total RNA can be used for mRNA from tissue or plasma/serum/exome sources
isolation (purification by passage through oligo[dT]
cellulose spin columns) or mRNA assays (polymerase DNA and RNA Quantification, Purity, and
chain reaction [PCR], microarray, sequencing, etc.) and Integrity Assay
long noncoding RNA (lncRNA; >200 nucleotides) assays • H
igh-integrity nucleic acids are best extracted from fresh
(PCR, microarray, sequencing, etc.). MicroRNA (mRNA) tissue specimens. Extraction from tissues preserved in
species are approximately 18 nucleotides in length and liquid nitrogen is the next best option. Commercially
TABLE 1.34 IMMUNOHISTOCHEMISTRY TO DETECT THE GAIN OR LOSS OF PROTEIN EXPRESSION

MSH2 MSH6 MLH1 PMS2
Tumor positive for microsatellite instability (highly suspicious for Lynch Present Loss Present Present
syndrome); germline mutation of MSH6
Tumor positive for microsatellite instability (highly suspicious for Lynch Loss Loss Present Present
syndrome); germline mutation of MSH2
Tumor positive for microsatellite instability (highly suspicious for Lynch Present Present Present Loss
syndrome); germline mutation of PMS2
Tumor positive for microsatellite instability; majority of colon cancers with Present Present Loss Loss
this pattern of protein loss are associated with somatic changes
(not inherited mutation); follow-up testing should include a BRAF
mutational analysis or methylation of MLH-1 testing
—Presence of the BRAF V600E mutation and MLH1 promoter hypermethylation
provide confirmation of a sporadic MSI tumor (loss of MLH1 secondary
to promoter hypermethylation, not due to inherited mutation)
—Absence of BRAF V600E and negative MLH1 promoter hypermethylation is
highly suggestive of a germline mutation of MLH1
Tumor negative for microsatellite instability; germline mutation unlikely Present Present Present Present
(however, up to 10% of Lynch syndrome patients may have retained
expression of these 4 MMR proteins)
Loss, negative nuclear staining; Present, positive nuclear staining.

Courtesy of Rebecca Wilcox, MD, University of Vermont/Fletcher Allen Health Care.
TABLE 1.35 IMMUNOHISTOCHEMISTRY FOR A270 are indicators of contamination with organics (e.g.,
GENE PRODUCTS DETECTION FROM SPECIFIC guanidinium salts) or phenol, respectively. Contamina-
MUTATIONS tion with proteins can be inferred from an A280 reading,
Antibodies Against Gene Products from Specific Mutations whereat peak protein absorbance occurs. Particulate
Gene Malignancy
matter contamination can be gauged from an A320 read-
ing. Typically, an A(260–320): A(280–320) ratio is calculated;
BRAF V600E Melanoma (37/38)a a value of 1.7 to 2 indicates pure DNA or RNA
Hairy cell leukemia (32/32)b
• Nucleic acid integrity can be estimated by comparing
Papillary thyroid carcinoma (72/144)c
EGFR Non–small cell lung cancerd
nucleic acid fragment size against a molecular weight
ladder following agarose gel electrophoresis. High-
aFrom Long GV, Wilmott JS, Capper D, et al. Immunohistochemistry is integrity genomic DNA extracted from fresh or frozen
highly sensitive and specific for the detection of V600E BRAF mutation tissues or cultured cells demonstrates a band greater
in melanoma. Am J Surg Pathol. 2013;37:61–65. than 30 to 40 kilobase pairs (kb) in size. The presence
bFrom Andrulis M, Penzel R, Weichert W, et al. Application of a BRAF
V600E mutation-specific antibody for the diagnosis of hairy cell leuke- of a smear extending to smaller sized fragments indi-
mia. Am J Surg Pathol. 2012;36:1796–1800. cates degraded DNA. DNA extracted from FFPE tissues
cFrom Koperek O, Kornauth C, Capper D, et al. Immunohistochemical
typically appears as a smear extending from approxi-
detection of the BRAF V600E-mutated protein in papillary thyroid carci- mately 1 kb to less than 100 base pairs (bp). Total RNA
noma. Am J Surg Pathol. 2012;36:844–850.
dFrom Hasanovic A, Ang D, Moreira AL, Zakowski MF. Use of mutation integrity is gauged in terms of the presence of 28S (∼5
specific antibodies to detect EGFR status in small biopsy and cytology kb) and 18S (∼2 kb) rRNA. Discrete 28S and 18S bands,
specimens of lung adenocarcinoma. Lung Cancer. 2012;77:299–305. with minimal smearing, indicate intact RNA species,
whereas partial or absent bands and smeared rRNA
indicate a degraded sample
available storage reagents (e.g., RNAlater, Ambion, • Instruments such as the Agilent 2100 Bioanalyzer (Agi-
Inc., Foster City, CA) preserve tissue morphology and lent Technologies, Inc., Santa Clara, CA) and Qubit
nucleic acids integrity. Disaggregated cells/cell cultures Fluorimeter (Thermo Fisher Scientific, Waltham, MA)
can be stored at −20°C in 70% ethanol with efficient have facilitated rapid DNA and RNA quantitation and
nucleic acid preservation. DNA and RNA extracts from purity analyses, and RNA integrity assays
FFPE tissues tend to be degraded. In general, the quality
of nucleic acids extractable from FFPE blocks decreases Nucleic Acids Storage
with block age • D NA is generally stored at 4°C for assays performed
• The concentration of extracted nucleic acids is assessed within 1 week to 1 month of extraction, and in aliquots
spectrophotometrically. Both DNA and RNA absorb at −20°C or −80°C for longer- term storage; repeated
UV light with peak absorbance at a wavelength of 260 freeze-thawing may lead to DNA degradation
nm; an absorbance (A260) reading of 1.0 demonstrates a • RNA is more labile than DNA and is susceptible to
DNA concentration of 50 μg/mL or an RNA concentra- degradation by RNases that are a pervasive laboratory
tion of 40 μg/mL hazard. For short-term use, RNA is stored at −20°C,
• The purity of extracted DNA or RNA is also determined and at −80°C or under liquid nitrogen for longer-term
spectrophotometrically. Readings taken at A230 and at storage
TABLE 1.36 SELECTED SOFT TISSUE TUMORS WITH

CORRESPONDING IMMUNOHISTOCHEMISTRY
CD34+ Spindle Cell Tumors
Cellular angiofibroma (ER/PR +)
Angiomyofibroblastoma (ER/PR +)
Superficial myofibroblastoma (ER/PR +)
Solitary fibrous tumor (CD99 +, STAT6 +)
Spindle cell lipoma (Rb IHC, loss of nuclear expression)
Dermatofibrosarcoma protuberans
Kaposi sarcoma (HHV8 +)
IHC for Liposarcomas (LPSs)
Myxoid LPS S-100 protein + (mature fat
cells and lipoblasts)
Well-differentiated/atypical MDM2 +
lipomatous tumor CDK4 +
p16 +
Dedifferentiated liposarcoma MDM2 + A
CDK4 +
IHC for Myxoid Spindle Cell Tumors
Myxofibrosarcoma Vimentin +
Low-grade fibromyxoid MUC4 +
sarcoma Vimentin +
Myxoid LPS S-100 protein + (mature fat
cells and lipoblasts)
Extraskeletal myxoid S-100 protein + (17%)a
chondrosarcoma Vimentin +
Chordoma Brachyury +
CK +
EMA +
S-100 protein +
Myxoid leiomyosarcoma Desmin +
h-Caldesmon +
DOG1 −/+
Myxoid malignant peripheral SMA + (50%)
nerve sheath tumor (MPNST) S-100 + (50%) focal
CD34 +/− B
Myxoid dermatofibrosarcoma CD34 +
protuberans (DFSP) Apolipoprotein D +
Myxoid solitary fibrous tumor STAT6+
(SFT) CD99 +
CD34 +
Myxoid monophasic synovial Keratin +
sarcoma CD99 +
TLE1 +
+, positive; −, negative; +/−, often positive; −/+, rarely positive. MUC4

(mucin 4 gene): Doyle LA, Möller E, Dal Cin P, et al. MUC4 is a highly
sensitive and specific marker for low-grade fibromyxoid sarcoma. Am
J Surg Pathol. 2011;35:733–741. MDM2, CDK4, and p16: Thway K,
Flora R, Shah C, et al. Diagnostic utility of p16, CDK4, and MDM2 as
an immunohistochemical panel in distinguishing well-differentiated
and dedifferentiated liposarcomas from other adipocytic tumors. Am J
Surg Pathol. 2012;36:462–469. Apolipoprotein D: West RB, Harvell J,
Linn SC, et al. Apo D in soft tissue tumors: a novel marker for dermato-
fibrosarcoma protuberans. Am J Surg Pathol. 2004;28:1063–1069. TLE1
(transducer-like enhancer of split 1): Foo WC, Cruise MW, Wick MR, C
Hornick JL. Immunohistochemical staining for TLE1 distinguishes synovi-
al sarcoma from histologic mimics. Am J Clin Pathol. 2011;135:839–844. Figure 1.14 Immunohistochemistry in prostate adenocarcinoma.
aOliveira AM, Sebo TJ, McGrory JE, et al. Extraskeletal myxoid chondrosar- Both p63 (A) and 34βE12 (B) highlight an intact basal cell layer sur-
coma: a clinicopathologic, immunohistochemical, and ploidy analysis rounding benign glands and loss around small acini of invasive adeno-
of 23 cases. Mod Pathol. 2000;13:900–908. carcinoma. C, P504S immunohistochemistry shows strong, granular
From Chen BJ, Mariño-Enríquez A, Fletcher CD, Hornick JL. Loss of retino- luminal staining in invasive adenocarcinoma and prostatic intraepithe-
blastoma protein expression in spindle cell/pleomorphic lipomas and lial neoplasia. Normal glands are negative.
cytogenetically related tumors: an immunohistochemical study with
diagnostic implications. Am J Surg Pathol. 2012 Aug;36(8):1119–1128.
From Jambhekar NA, Rekhi B, Thorat K, et al. Revisiting chordoma with
brachyury, a “new age” marker: analysis of a validation study on 51
cases. Arch Pathol Lab Med. 2010;134:1181–1187.
CD45 Pos
1023
Grans
CD45 Neg
SS Lin Monos
Other
Lymphs
0
100 101 102 103

CD45 PC7
A
103 103 103
F1 F2 A1 A2 C1 C2
102 102 102

Kappa
FMC7
CD19
101 101 101
100 100 100

F3 F4 A3 A4 C3 C4
100 101 102 103 100 101 102 103 100 101 102 103
CD5 Lambda CD23
B
Figure 1.15 Flow cytometry. A, Gating for lymphocytes (CD45 versus side scatter, linear scale [SS Lin]) shows the relative locations of granulocytes
(Grans), monocytes (Monos), and lymphocytes (Lymphs) (arrow). B, Mantle cell lymphoma. Flow cytometric analysis of a lymph node specimen
shows that nearly all of the lymphocytes express CD19, CD5, and kappa immunoglobulin light chains. A subset coexpresses FMC7, whereas the
cells are negative for CD23. Expression of CD20 is not dim (data not shown). This immunophenotypic profile fits with involvement by mantle cell
lymphoma. (Courtesy of Michael R. Lewis, MD, MBA, Department of Pathology, University of Vermont Medical Center Burlington, VT.)
TISSUE MICRODISSECTION METHODS scraped off the slides using a syringe needle. DNA/RNA
extraction is by mini-column applications
Background • Laser capture microdissection (LCM) (Figure 1.17) uses
• M
icrodissection allows the targeted collection of mini- specialized microscopy apparatus such as the ArcturusXT
mal numbers of cells or tissues from slide mounted system (Thermo Fisher Scientific, Waltham, MA)
cytologic specimens, or frozen or FFPE tissue sections. • The procedure involves overlaying the tissue of interest
Sample tissues may be treated for nucleic acids or pro- with a thermoplastic film contained in a cap. LCM can
tein extraction be applied to frozen or FFPE tissues, or to blood smears,
or cytologic or cell culture samples. Tissues can be
Methods unstained or histochemically or immunohistochemi-
• In manual microdissection, lightly stained tissue sec- cally stained (chromogenic or fluorescence). A pulsed
tions are viewed by dissecting microscope and damp- laser beam is targeted against the selected cells, which
ened with 70% ethanol; targeted tissues are selectively fuses them to the thermoplastic film. The cap is then
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18
Figure 1.16 Well-differentiated

liposarcoma. Karyotype analysis
of a deep retroperitoneal lesion re- 19 20 21 22 X Y
vealed a giant ring chromosome.
(Courtesy of Mary Tang, MD, Cy-
togenetic Laboratory, University ring chromosome
of Vermont/Fletcher Allen Heath
Care, Burlington, VT.) A
TABLE 1.37 ONCOGENES OF IMPORTANCE IN SURGICAL PATHOLOGY

Mode of Activation/
Category Proto-Oncogene Location (Chromosome) Association
Signal Transduction Proteins
Nonreceptor tyrosine kinase ABL Translocation/9q34 Chronic myeloid leukemia/acute lymphoblastic
leukemia. t(9;22)(q34;q11) (Philadelphia
chromosome) forming bcr-abl fusion protein
GTP-binding KRAS Point mutation/12p12 Colon, lung, pancreatic carcinomas
NRAS Point mutation/1p22 Melanomas, hematologic malignancies
HRAS Point mutation/11p15 Bladder and kidney tumors
RAS signal transduction BRAF Point mutation/7q34 Melanoma
WNT signal transduction Beta-catenin Point mutation Hepatoblastomas, hepatocellular carcinoma
bcl-1 (PRAD-1) 11q13 Parathyroid adenomatosis; mantle zone
lymphomas with translocation to 14q32
bcl-2 18q21 Block of apoptosis; translocation to 14q in follicular
lymphomas
bcl-6 3q27 Diffuse large cell lymphoma
Growth Factor Receptors
erbA 17 Erythroleukemia
erbB1 (EGFR) Overexpression/7p11-12 Squamous cell carcinoma of lung, gliomas
neu (erbB2, HER-2) Overexpression/17q11-12 Breast carcinoma, gastric and esophageal
carcinoma
KIT Point mutation Gastrointestinal stromal tumors, seminomas,
leukemias
PDGFRB Overexpression, translocation Gliomas, leukemias
Ret 10q11.2 Medullary and papillary thyroid carcinomas
FLT3 Amplification Breast and ovarian carcinomas
Nuclear-Regulatory Proteins
Transcriptional activators L-MYC Amplification/1p32 Small cell carcinoma of lung
N-MYC Amplification/2p23-24 Neuroblastoma, small cell carcinoma of lung
C-MYC Translocation/8q24 Burkitt lymphoma
Cell Cycle Regulators
Cyclins Cyclin D Translocation Mantle cell lymphoma
Amplification Breast and esophageal carcinomas
Cyclin E Overexpression Breast carcinoma
Cyclin-dependent kinase CDK4 Amplification or point Glioblastoma, melanoma, sarcoma
mutation
TABLE 1.38 TUMOR SUPPRESSOR GENES OF IMPORTANCE IN SURGICAL PATHOLOGY
Gene Location (Chromosome) Association
RB (retinoblastoma) 13q14 Retinoblastoma, childhood osteosarcoma
p53 17p13.1 Mutations in cancers of colon, breast, lung, leukemia, sarcoma; progression to diffuse large
cell lymphoma (germline mutation of p53 forms the basis for Li-Fraumeni syndrome)
WT-1 11p13 Wilms tumor; desmoplastic small round cell tumor
EWS 22q12 Ewing/primitive neuroectodermal tumor, soft tissue clear cell sarcoma, desmoplastic small
round cell tumor, myxoid liposarcoma, acute myelogenous leukemia
BRCA1 and BRCA2 17q21 Breast and ovarian carcinomas
APC/B-Catenin 5q21 Familial adenomatous polyposis coli; carcinomas of colon, stomach, pancreas
PTEN 10q23 Endometrial and prostate cancers/Cowden syndrome
NF1 17q11 Schwannomas, neuroblastomas, neurogenic sarcomas
NF2 22q12 Central schwannomas, meningiomas
TABLE 1.39 CYTOGENETIC ABNORMALITIES OF IMPORTANCE IN SURGICAL PATHOLOGY

Tumor Chromosomal Abnormality Fusion Transcript, Involved Genes
Hematopoietic Neoplasms
Acute myelogenous leukemia (AML)
—AML-M1 t(9;22) BCR-ABL
—AML-M2 t(8;21) (favorable) CBFα-ETO
—AML-M3 t(15;17) RARα/PML
—AML-M4eo inv(16) (favorable) CBFβ/MYH11
Chronic myelogenous leukemia t(9;22)(q34;q11) BCR-ABL
B-cell acute lymphoblastic leukemia t(12;21) CBFα-ETV6
Chronic lymphocytic leukemia Trisomy 12, deletions of 11q, 13q and 17p
Burkitt lymphoma t(8;14), t(8;22), t(4;8) Involving c-myc and immunoglobulin (Ig) loci
Follicular lymphoma t(14;18) BCL2 gene
Mantle zone lymphoma t(11;14) BCL1 (cyclin D1) and IgH
Primitive Precursor Cell Neoplasms
Ewing sarcoma/primitive neuroectodermal tumor t(11;22)(q24;q12) EWS-FLI1 (or EWSR1-ERG, EWSR1-PATZ1, FUS-ERG)
CIC-Rearranged Sarcoma t(4;19)(q35;q13.1) CIC-DUX4 (CIC-DUX4L or CIC-FOX04)
BCOR-Rearranged Sarcoma inv(X)(p11p11) BCOR-CCNB3 (BCOR-MAML3 or ZC3H7B-BCOR)
Medulloblastoma del 17q
Neuroblastoma del 1p (poor prognosis); double minute N-myc amplification
chromosomes
Retinoblastoma del 13q (band q14)
Wilms tumor del 11p (band p13)
Epithelial Neoplasms
Colorectal carcinoma del 17p
Mesothelioma del of 1p, 3p, 22p
Renal cell carcinoma (RCC)
Clear cell carcinoma del 3p
Papillary RCC Trisomy 7 and 17
Chromophobe RCC Loss of chromosome 1, 2, 6, or 10
Oncocytoma Loss of chromosome 1; translocation
involving 11q13
Small cell carcinoma del 3p
Soft Tissue Neoplasms
Alveolar soft part sarcoma t(X;17)(p11;q25) TFE3-ASPL fusion
Chondrosarcoma, extraskeletal myxoid t(9;22)(q22;q12) EWS-NR4A3 fusion
Clear cell sarcoma t(12;22)(q13;q12) EWSR1-ATF1 fusion
Desmoplastic small round cell tumor t(11;22)(q24;q12) EWSR1-WT-1 fusion
Dermatofibrosarcoma protuberans Ring form of chromosomes 17 and 22 COL1A1-PDGFB fusion
Fibrosarcoma, infantile t(12;15)(p13;q26) ETV6-NTRK3 fusion
Hibernoma Translocation at 11q13
Inflammatory myofibroblastic tumor t(1;2)(q22;p23) TPM3-ALK fusion
Leiomyoma t(12;14), del 7q
Leiomyosarcoma del 1p
Lipoma Rearrangement of 12q15 HMGIC fusion
Liposarcoma (myxoid) t(12;16)(q13;p11) TLS/CHOP
Liposarcoma (well differentiated) Ring chromosome 12
Rhabdoid tumor Deletion of 22q INI1 inactivation
Rhabdomyosarcoma (alveolar) t(2;13)(q35;q14) PAX3-FKHR
Rhabdomyosarcoma (embryonal) Trisomies 2q, 8, and 20
Synovial sarcoma t(X;18)(p11;q11) SYT-SSX1/SYT-SSX2
Central Nervous System Neoplasms
Atypical teratoid rhabdoid tumor Deletion of 22q INI1 inactivation
Oligodendroglioma del 1p, 19q (improved response to
chemotherapy)
Schwannoma Deletion of 22q NF-2 inactivation
Place cap on tissue Pulse laser at target cells
Figure 1.17 Laser capture technol-

ogy. Target tissues are overlaid with a
cap using microscope guidance. Cells
are adhered to the thermoplastic film
of the cap by laser pulsing. Lifting the
cap removes the target cells for nucleic
acids or protein extraction. (Courtesy of Extract molecules from target cells Remove cap with adhered target cells
Molecular Devices, Sunnyvale, CA.)
removed from the tissue section surface and nucleic concentration of the agarose determines the gel pore
acids are recoverable from the cells adhered thereto size, which in turn determines the size range of DNA
after cell lysis treatments applied directly to the cap fragments that can be resolved. The higher the agarose
film concentration, the smaller the pore size. At low aga-
rose concentrations, small DNA fragments pass rapidly
Applications through the gel and cannot be resolved, whereas large
• L
CM it is useful in surgical pathology practice when there DNA molecules are size sorted; at high agarose concen-
is a suspicion of patient sample cross- contamination. trations, the mobility of large DNA molecules through
PCR-based microsatellite sequence identity testing com- the gel is limiting and large fragment sizes cannot
paring the known patient sample with the queried tissue be resolved, whereas the resistance to the passage of
supports verification of patient identity smaller fragments is sufficient for their resolution (e.g.,
0.5% agarose gel resolves DNA fragment sizes in the
GEL ELECTROPHORESIS METHODS range ∼0.7 to 25 kb; a 2% agarose gel resolves DNA frag-
ment sizes in the range ∼0.05 to 2 kb)
Background • The gel is poured into a horizontal casting tray, and a
• G el electrophoresis, as a method for separating, identi- comb is inserted at one end to mold wells
fying, or purifying nucleic acids • When set, the “slab” gel is placed in an electrophoresis
• Nucleic acids are negatively charged at neutral pH tank and immersed under electrophoresis buffer
due to the phosphate in the sugar-phosphate back- • DNA sample is combined with a gel-loading dye. The
bone of DNA or RNA. Accordingly, in the presence of loading dye (typically bromophenol blue containing
an electric field, nucleic acids will migrate from the sucrose and/or glycerol) ensures the DNA sample sinks
cathode to the anode; migration through a sieving into the well and indicates how far the samples have
matrix (gel) depends on the size of the nucleic acid migrated during electrophoresis. Electrophoresis is gen-
molecule, its conformation (secondary folding), net erally conducted at 5 V/cm measured as the distance
charge (dependent on the pH of the gel buffer), and between the negative and positive electrodes
gel pore size • A DNA ladder is also run so that sample DNA fragment
• Agarose gel and polyacrylamide gel are the basic sizes can be estimated
forms of electrophoresis. Variations on these methods • DNA is most commonly visualized in agarose gels by
include pulsed-field gel electrophoresis (PFGE), capil- staining with ethidium bromide, which intercalates
lary gel electrophoresis (CGE), denaturing gradient gel double-stranded DNA and fluoresces under UV illu-
electrophoresis (DGGE), and temperature gradient gel mination. Less toxic (noncarcinogenic) alternatives to
electrophoresis (TGGE) ethidium bromide, such as SYBR, EvaGreen, or GelStar
dyes, stain DNA and with higher sensitivity
Agarose Gel Electrophoresis • Applications
• A
garose is manufactured from seaweed such as Rho- • Agarose gel electrophoresis is commonly used for
dophyta. It consists of multiple linked repeat units the analysis of end-point PCR/reverse transcription
of the disaccharide agarobiose (D- galactose and (RT)-
PCR assays where the presence or absence of
3,6-anhydro-L-galactose) amplicons defines the interpretation of the test; for
•
Agarose gel is prepared by heating agarose pow- example, the detection of a fusion transcript or a
der to near boiling in electrophoresis buffer. The pathogen
• The analysis of restriction fragment length poly- when one primer is labeled with a radioisotope; the
morphism (RFLP) assays (see RFLP section) generally gel is dried then fastened with an x-ray film in a cas-
requires agarose gel electrophoresis sette. Gel/image analysis apparatuses fitted with a laser-
• The technique is used routinely in molecular biology induced fluorescence (LIF) detector can be used to
for the analysis of recombinant DNA experiments analyze PCR amplicons that include a fluorophore-
and can be used for the purification of probes for in labeled primer
situ hybridization (ISH) and blot hybridization by • Applications
excision of DNA fragments from a gel followed by • End- point PCR fragment analysis where fragment
mini-column purification size differences are slight
• The discrimination of PCR fragments of identical size
Pulsed-Field Gel Electrophoresis (bp) but containing different sequences (mutations or
• P FGE is an electrophoretic method for the improved variants) can be performed by several polyacrylamide
resolution of high- molecular- weight DNA. Standard gel–based techniques, including single- stranded
agarose gel electrophoresis separates DNA under a con- conformational polymorphism (SSCP) analysis, and
stant and uniform electric field. Under these condi- DGGE
tions, DNA greater than 50 kb is poorly distinguished • Polyacrylamide slab gels are used for sequencing
from DNA in the size range of 30 to 50 kb assays and for microsatellite marker- based assays
• The improved resolution of PFGE is accomplished by using autoradiography or fluorescently labeled
alternating the direction of the electrical field. In the fragments
simplest approach, the direction of field is constantly
reversed so that the DNA spends some time moving Capillary Gel Electrophoresis
backward. More refined techniques alternate the field so • C GE supports automated DNA sequencing and frag-
that the DNA moves through the gel in a zigzag pattern ment analyses
• Applications • The technique involves the electrophoresis of DNA
• PFGE can be used for the identification of microor- molecules through a solution phase polyacrylamide-
ganismal strains such as Escherichia coli O157:H7, based gel matrix contained within a 20-to 50- inch
Salmonella, Shigella, Listeria, or Campylobacter. High- silica capillary with a 25-to 100-μm bore
molecular-weight DNA extracts (from culture) are • Proximal to the anode end of the capillary is a LIF detec-
digested with a restriction enzyme (RE) (see the tor. The light emissions from fluorescence labeled DNA
Southern blotting section). The PFGE electrophoretic fragments (e.g., by way of PCR using a fluorescence-
DNA “fingerprint” helps to identify the infective labeled primer) are registered by the LIF detector, which
strain. The Centers for Disease Control and Preven- can detect up to four different emission wavelengths
tion (CDC) maintains databases of PFGE standard- • Given the LIF detector location, all DNA fragments
ized molecular subtypes for the identification of are sieved through the complete length of gel prior to
microorganisms detection. This supports the accumulation of data hav-
• In combination with Southern blot analysis, PFGE ing high resolution; single base differences can be dis-
can be used in the evaluation of autosomal domi- tinguished. Emission data are recorded using dedicated
nant ataxia computer software that integrates the data collected
during the time course of the electrophoresis. The final
Polyacrylamide Gel Electrophoresis data are presented in terms of peak heights and areas
• P olyacrylamide is produced from monomers of acryl- (relative to a fluorescence emissions scale [representing
amide in a reaction initiated by free radicals gener- DNA amplicons/fragments]) and with reference to a
ated by reduction of ammonium persulfate by TEMED DNA size marker
(N,N,N′,N′-
tetramethylene diamine). These linear • Applications
strands of polyacrylamide form into a gel after cross- • CGE is widely used for sequencing and microsatellite
linkage by N,N′-methylenebisacrylamide. The higher assay data analyses
the concentration of acrylamide the finer the resolu-
tion of DNA fragments. The advantage of polyacryl- BLOT HYBRIDIZATION METHODS
amide over agarose is that size differences at the base
pair bases can be distinguished (e.g., 5% polyacryl- Southern Blotting
amide gel resolves DNA fragment sizes in the range ∼80 • B
ackground
to 500 bp; a 15% polyacrylamide gel resolves DNA frag- • Dr. E.M. Southern developed the Southern blot tech-
ment sizes in the range ∼25 to 150 bp) nique in 1975 as a method for transferring DNA out
• Polyacrylamide “slab” gels are set between glass plates of an agarose slab gel onto a solid support (a nitrocel-
and run under a buffer in a vertical gel apparatus. Sam- lulose or nylon membrane)
ples and DNA ladder plus loading dye, are loaded into • The method involves the use of restriction endo-
wells and DNA fragment progression is estimated from nucleases to cut (restrict) genomic DNA into differ-
the dye migration ently sized fragments that are size- fractionated by
• Polyacrylamide gels may be stained for DNA with ethid- gel electrophoresis. After transfer, the membrane is
ium bromide, SYBR dyes and the like, or silver nitrate. hybridized with a labeled probe specific to the target
PCR amplicons can be detected by autoradiography sequence of interest
Genomic 1 2 3 Stack of
DNA Large Membrane
paper towels
Gel
Restriction Gel DNA

enzyme electrotransfer to
digest phoresis membrane
Figure 1.18 Southern blot analysis. Small

Following agarose gel electrophoresis Membrane (blot) Salt solution
of restriction endonuclease-treated 1 2 3 1 2 3
genomic DNA, alkali-denatured DNA
is transferred onto a nylon membrane Hybridization Bands
with labeled represent
by capillary action. The recovered probe Wash
membrane is screened for target gene
Probe location
sequences by hybridization with a detection
labeled probe. (Modified from Leon-
ard DGB, ed. Diagnostic Molecular Pa-
thology. Philadelphia: WB Saunders;
2003.) Film
• C an be used to detect chromosomal rearrangements, competition from normal cells in a sample will mask
DNA amplifications, deletions, and loss of heterozy- the detection of an anomaly by PCR
gosity and to assess clonal status • The detection of clonality by Ig gene rearrange-
• The technique (Figure 1.18) generally requires rela- ments in B-cell lymphoproliferative disorders can
tively large quantities of high- molecular-
weight aid the diagnosis of minimal residual disease. PCR
DNA (5 to 10 μg per restriction endonuclease-treated tests for B-cell clonality may have a false-negative
sample) rate of up to 30%, and the “gold standard” test for
• Applications the detection of Ig clonal rearrangements may be
• The Southern blot method is widely used in RFLP Southern blot analysis. Clonality can be inferred
applications. The number of restriction sites for a from a comparison of normal and B- cell tumor
given restriction endonuclease in the site of a gene tissue restriction fragment sizes following hybrid-
may vary because of normal (polymorphic) variation ization with a region of the Ig gene. The normal
between individuals or due to sequence mutations. sample will demonstrate a single band representing
These differences can result in altered restriction the germline Ig gene, whereas a B-cell tumor also
fragment patterns. Altered fragment sizes between demonstrates a unique band size as a consequence
individuals may also result when the restriction frag- of gene rearrangement and altered restriction site
ment contains a variable number of tandem repeat position(s)
(VNTR) sequences. VNTR regions contain microsat- • Southern blotting can also have an advantage over
ellite or minisatellite repeats comprising approxi- PCR in the detection of fragile X syndrome. PCR can
mately less than 6 bp or approximately 10 to 100 be used in this diagnosis by designing primers that
bp repeat sequences, respectively. Differences in the include the (CGG)n repeat unit within the amplicon.
number of these repeat units may be detectable as However, especially when the expansion involves
altered fragment sizes (see Figure 1.18). The size dif- hundreds to thousands of (CGG) repeats, PCR ampli-
ferences may be simple polymorphisms or can reflect fication can be problematic and may fail
a disease condition. For example, the (CGG)n micro- • Southern blotting can be combined with PCR.
satellite trinucleotide in the FMR1 gene is repeated Hybridization with a target- specific probe can be
approximately 5 to 44 times among normal individ- used to confirm. PCR amplicons represent the tar-
uals, whereas in patients with fragile X syndrome the get and are not anomalous products resulting from
number of repeats is expanded more than 230 up to incidental primer annealing events. PCR amplicon
thousands of times. The detection of this expansion RFLP analyses may also be performed by Southern
by Southern blot analysis also involves the use of a blotting
methylation-sensitive restriction endonuclease that
fails to cut a restriction site that is unmethylated in Northern Blotting
normal individuals but methylated in fragile X syn- • N
orthern blotting is used in the analysis of mRNA
drome patients expression. mRNA constitutes up to 5% of the total cel-
• Despite the requirement for relatively large quantities lular RNA. Extracted mRNA is denatured with formalde-
of DNA and time-consuming procedures, Southern hyde or glyoxal to prevent the formation of secondary
blotting may have advantages over PCR in certain RNA structures. Digestion of the RNA into smaller frag-
applications. For example, when available sequence ments is not required as native mRNA fragment sizes
data are insufficient to design PCR primers specific to range from approximately 300 to 12,000 nucleotides;
the site of a chromosomal rearrangement or where the average size is 1000 to 3000 nucleotides. Following
agarose gel electrophoresis, RNA is transferred to a 5 3

membrane by a capillary, vacuum, or electrotransfer 3 5
process and the membrane is hybridized with a labeled DNA denaturation (95°C)
probe to the gene target. The resulting data indicate 3
5
whether the gene is overexpressed or underexpressed,
3
or if an abnormally sized transcript is expressed. The 5
method requires relatively large amounts of high integ- Primer annealing (40°−60°C)
rity RNA, is time consuming, and requires a high level 5 3
Cycle 1
of laboratory skill, all of which limits the clinical utility 3 5
5 Primer 1 3 Primer 2
of northern blotting 3 5
DNA synthesis (72°C)
Dot Blotting 5 3
• D ot blot hybridization involves spotting denatured 3
Primer 2
5
Primer 1
DNA or RNA onto a membrane for hybridization with 5 3
a labeled probe. The method allows confirmation that 3 5
a genomic DNA or RNA sample or a PCR product is Repeat cycle 30−50 times
positive for the probe target. The method can also be 109 −1015
used semiquantitatively to assess or compare target PCR
sequence load within a sample products
• Reverse-line dot blot hybridization
• An alternative approach to the standard dot-blot is
to fix an array of unlabeled probes onto the mem-
brane and hybridize this with labeled nucleic acids/ Cycle 2 Cycle 3 30−50 cycles
PCR products Figure 1.19 Polymerase chain reaction (PCR). A PCR cycle consists
• Applications of denaturation, primer annealing, and DNA synthesis or extension
• A variety of “line probe assays” (LiPAs) have been steps. Following the first PCR cycle, there is (theoretically) a per-cycle
developed. These include screening tests for ApoE doubling in the number of copies of the PCR product. (Modified from
Leonard DGB, ed: Diagnostic Molecular Pathology. Philadelphia: WB
mutations, cystic fibrosis mutations, hepatitis B
Saunders; 2003.)
virus (HBV) and human papillomavirus (HPV)
genotyping, human leukocyte antigen (HLA) typ-
ing, and mycobacteria detection • I n digital PCR (dPCR; third-generation PCR), approx-
• Outside the United States, Conformité Européenne imately 40 cycles of PCR are performed on a reaction
(CE)-marked LiPA tests are available for PCR-based tube containing 20,000 droplet partitioned reac-
HPV clinical screening tions; each droplet is then assessed individually for
• The SPF10-INNO LiPA HPV Genotyping Test (Inno- fluorescent signal generated from PCR amplicons
genetics, Ghent, Belgium) allows the specific geno- • Basic PCR method
typing of 25 different HPV types • During the denaturation stage, sample specimen
• The Roche Linear Array (LA) HPV genotyping test DNA is rendered to a single-stranded form by heat-
(Roche Molecular Systems, Inc., Branchburg, NJ) ing to 94°C to 98°C
detects 37 different HPV types • In the annealing step, oligonucleotide primers
• With both systems, biotinylated PCR product is hybridize with the target sequences they have been
hybridized with a membrane strip affixed with designed to complement. The annealing temperature
a line of HPV genotype- specific probes. Detec- depends on the deoxyribonucleoside triphosphate
tion of the PCR product label indicates the HPV (dNTP) composition of the primers and is typically
genotype(s) for which the patient is positive in the 40° to 60°C range
• During the extension step (72°C), the annealed
AMPLIFICATION METHODS primer/target DNA seeds the (5′ → 3′) synthesis by
thermostable DNA polymerase of a new DNA strand
The Polymerase Chain Reaction • DNA amplification is accomplished by repetition of
• B
ackground the denaturation, annealing, and extension cycle, 30
• PCR (Figure 1.19) is a method for the in vitro ampli- to 50 more times
fication of DNA involving automated cycles of • The time period for each of the denaturation, anneal-
denaturation, annealing, and extension/synthesis ing, and extension steps can vary from 10 seconds to
performed in a thermocycler greater than 1 minute and depends on reaction volume
• End-point PCR (first-generation PCR) consists of per- size, amplicon base composition and length, thermo-
forming 30 to 50 cycles of amplification followed by stable DNA polymerase activity (∼1000 bp are extended
analysis of the PCR product per minute), and thermal cycler hardware specifications
• In real-time quantitative (q) PCR (second-generation • The essential ingredients in a PCR include the
PCR) continuous cycle-by-cycle monitoring of prod- following:
uct accumulation is performed by measuring fluores- • DNase/RNase free pure water: final PCR reaction
cent signal generated from the amplicons volumes typically vary from 10 μL to 50 μL
• B uffer: pH is typically maintained using a TRIS-HCl– • C onsensus PCR can be used to amplify a single target
based buffer. Other ingredients include KCl, which that has variable sequences or multiple targets that
can aid primer template annealing (K+ ions offset have similar (common) sequences
the repulsive force between negatively charged DNA • Degenerate PCR is also used in the amplification of a
strands), nonionic detergents, and bovine serum variable sequence target and involves a primer design
albumin (BSA) to aid Taq DNA polymerase enzyme that incorporates alternative sequences at a particu-
stability lar primer base sequence
• Magnesium cations: Mg2+ is an essential ingredi- • Nested PCR is a method for improved PCR sensitivity
ent and stabilizes the interaction between the oli- and specificity. Standard PCR is performed; the PCR
gonucleotide primer, template DNA, and Taq DNA product obtained is then used as DNA template for a
polymerase enzyme second round of PCR with nested primers
• dNTPs: 2′-deoxyadenosine 5′-triphosphate (dATP), • Amplification refractory mutation system (ARMS)/
2′-deoxycytidine 5′- triphosphate (dCTP), 2′- allele-specific PCR (AS-PCR)/PCR amplification of spe-
deoxyguanosine 5′-triphosphate (dGTP), and thymi- cific alleles (PASA) supports sequence-specific PCR by
dine 5′-triphosphate (TTP also referred to as dTTP) designing the 3′-base at the end of a primer to match
• Oligonucleotide primers: typically 18 to 25 bases critical target
in length • LA PCR stands for long and accurate PCR and allows
• Template DNA: the amount of sample in a reaction the amplification of sequences 5 to greater than 20 kb
can range from 1 ng to 1 μg, with approximately in length
100 ng representing a standard quantity for many
applications Reverse Transcription Polymerase Chain
• Thermostable DNA polymerase enzymes such Reaction
as Taq DNA polymerase extracted from Thermus • RT-PCR (Figure 1.20) allows the investigation of RNA
aquaticus isolated from a hot springs–dwelling bac- expression via PCR. Thermostable DNA polymerases
terium of the Deinococcus-Thermus phylum require DNA as a substrate; the first step in RT PCR is
• PCR efficiency thus the conversion of (DNA-free) total RNA or mRNA
• Optimization experiments are required to ensure into single-stranded complementary DNA (cDNA).
PCR test efficiency approaches ideal efficiency and The two most commonly used reverse transcriptase
to avoid false-negative data. Potentially, each com- enzymes are the avian myeloblastoma virus (AMV)
ponent of the PCR setup can be manipulated for or Moloney murine leukemia virus (M-MuLV) reverse
improved PCR sensitivity and specificity transcriptases
• In addition to the general determinants of standard
Polymerase Chain Reaction Contamination PCR success, RT-PCR efficiency depends on RNA sam-
Control ple quality and the effectiveness of the reverse tran-
• T he sensitivity of PCR incurs the potential defect of scriptase step
false-
positive data due to the amplification of cross-
contaminating DNA from an exogenous source. Strict
Real-Time Quantitative Polymerase Chain
measures are required from patient sample collection Reaction
through to PCR assay to ensure authentic data and to • In end-point PCR, the final product obtained after 30
avoid false-positive data to 50 PCR cycles is the object of data interpretation.
• Ideally, laboratory space should be arranged such that Although end-point PCR can be semiquantitative, it is
DNA sample extraction, PCR setup, and post- PCR essentially a qualitative assay. qPCR/qRT-PCR is used
manipulations all occur in physically distinct areas, for the accurate quantification of a DNA or RNA (via
and using PCR-grade reagent aliquots, dedicated equip- cDNA) target in a sample
ment, and laboratory coats specific for each area. PCR • Fluorescent DNA- binding dyes (e.g., SYBR green) or
products from previous rounds of PCR represent the amplicon-specific probes that hybridize to accumulat-
major potential source for contamination ing PCR product are used to monitor amplicon accu-
• Substituting dTTP with dUTP results in PCR amplicons mulation at each cycle of PCR; these strategies involve
that are degraded by uracil N-glycosylase (UNG) allows the use of TaqMan, Molecular Beacons, or Scorpions
the inclusion of a pre-PCR incubation step with UNG. probes
This prevents carry-over of PCR amplicons from prior
PCR assays (the major source of contamination) lead- Digital Polymerase Chain Reaction
ing to false-positive data • d PCR or droplet digital PCR (ddPCR) allows absolute
quantification of a target sequence. This is in contrast
Polymerase Chain Reaction Method to qPCR, where the quantification is relative to stan-
Variations dard curve data derived from assays performed using
• PCR technique is a highly adaptable technique, known amounts of target copies
enabling its applicability in a wide range of research • The procedure involves setting up a 20-μL PCR, which
and clinical niches is then partitioned into 20,000 droplets (i.e., 20,000
• Modifications centered on primer design/usage: self-contained individual PCRs in one tube). Target and
• Multiplex PCR supports the simultaneous detection of nontarget sequences are randomly distributed among
more than one target by the use of multiple primer the droplets. After 40 cycles of PCR, droplets are ana-
pair sets lyzed one by one for the detection of fluorescence
Primer 1
3 5
5 3 RNA
Reverse cDNA synthesis by
transcription reverse transcriptase
3 5 RNA:cDNA
5 3 hybrid
Primer 1
Denature RNA : cDNA hybrid
Anneal primer
Synthesize second cDNA strand
3 5 cDNA
PCR Cycle 1
5 3
Primer 2 Denature
Anneal
Synthesize
3 5
5 3
PCR Cycle 2 3 5
5 3
30−50 PCR cycles

Figure 1.20 Reverse transcription polymerase chain
reaction (RT-PCR). Complementary DNA (cDNA) is syn-
3 5
thesized from an RNA sample by a reverse transcriptase
5 3
enzyme; thereafter the cDNA is available for PCR ampli-
3 5 fication. (Modified from Leonard DGB, ed. Diagnostic
5 3 Molecular Pathology. Philadelphia: WB Saunders; 2003.)
released by an amplicon. The fraction of fluorescence [qRT-PCR], Francisella tularensis [qPCR], HBV, hepatitis
positive droplets supports quantification calculation C virus (HCV), HPV, methicillin-resistant Staphylococ-
based on Poisson distribution analyses cus aureus (MRSA) screening [qPCR], West Nile virus
•
The technique enables the highest analytic sensitiv- [qPCR], and Yersinia pestis [qPCR]) and a number of
ity of any molecular method, allowing the detection genetic tests including BRAC1 and BRAC2 mutation
of rare targets in a complex background with sensitiv- screening in ovarian cancer, BRAF V600E mutation
ity down to one target copy in 1015 bases. The method screening in melanoma, EGFR gene deletion an muta-
can be used to investigate gene copy number variation, tion screening in non–small cell lung cancer, and KRAS
mutation detection, gene expression, and viral load mutation screening in colorectal cancer patients
• ddPCR clinical applications include newborn screen-
Polymerase Chain Reaction Tests in ing for severe combined immunodeficiency syndrome
Pathology Practice (SCID), and spinal muscular atrophy carrier screening
• P CR is highly adaptable for use in a wide variety of clin- • Non–FDA-approved tests are also in widespread clinical
ical applications including the following: diagnostics usage including:
• Infectious pathogens detection • Infectious pathogen detection
• Genetic diseases diagnosis • Genetic diseases diagnosis
• Hematologic diseases diagnosis (e.g., chimeric RNA tran- • Bloom syndrome mutation analysis, Fabry dis-
scripts detection such as the bcr-abl translocation pro ease, Factor IX gene, familial amyloidosis, familial
duct characteristic of chronic myelogenous leukemia) factor IX gene familial amyloidosis, familial dys-
• Sarcoma diagnosis by signature gene fusion detec- autonomia, fragile X syndrome, Gaucher disease,
tion (e.g., EWS/FLI1 in Ewing sarcoma/peripheral Fanconi anemia, galactosemia hemochromatosis,
neuroectodermal tumor) Prader Willi/Angelman, spinobulbar muscular
• Solid tumor characterization (e.g., hereditary non- atrophy, Tay-Sachs disease
polyposis colorectal cancer (HNPCC)/Lynch syn- • Tumor characterization/diagnosis
drome mutation analyses) • Adenovirus (qPCR), Bartonella henselae, BK virus,
• Identity testing cytomegalovirus (CMV), enterovirus, HBV,
• Detection of circulating tumor or pathogen nucleic Human Herpesvirus 6 (HHV-6), human meta-
acid signatures pneumovirus (hMPV), JC virus, Legionella RNA,
• PCR-based tests that have been approved and cleared Lyme disease, malaria, parvovirus B19, varicella-
by the US Food and Drug Administration (FDA) include zoster virus
numerous microbial assays (e.g., avian flu [qRT-PCR], • BCR/ABL (qRT- PCR), desmoplastic small-round-
Bacillus anthracis [qPCR], Chlamydia trachomatis, cell tumor (DSRCT) (RT- PCR), Ewing sarcoma (RT-
warfarin sensitivity [qPCR], enteroviral meningitis PCR), HNPCC, JAK2 V617F mutation detection,
microsatellite instability, PML/RARA (qPCR), RET/PTC • Several assays combine PCR with other molecular
rearrangements (RT-PCR), synovial sarcoma (RT-PCR) manipulations
• The Oncotype DX breast cancer assay is a qRT-PCR • Multiplex ligation- dependent probe amplification
array test that screens 21 genes (16 tumor-related (MLPA) is a proprietary technology of MRC-Holland,
genes, 5 reference genes). The test is applicable to Amsterdam, The Netherlands, which allows multiplex
patients with stage I, II, or IIIa invasive breast cancer assaying of disease biomarkers
that is estrogen receptor positive (ER+) and human • The technique involves two steps: ligation followed
epidermal growth factor receptor negative (HER2−). by PCR. First, denatured specimen DNA is hybridized
Based on the gene expression profile, a recurrence overnight with multiple target-specific primer pairs;
score (RS) is calculated indicating prognosis and for example, primers could be specific for a variety of
treatment path. The Oncotype DX DCIS test screens alternative mutations. If two oligonucleotides hybrid-
for seven cancer-related and five reference genes ize directly adjacent to each other, they can be ligated
• Several automated platforms incorporate PCR amplifi- to form one continuous DNA molecule by the enzyme
cation steps DNA ligase. At the 5′ end of the forward oligonucle-
• Roche COBAS 4800 system performs automated otide is a universal PCR primer sequence “X” that does
DNA extraction and is capable of multiplex PCR not hybridize to specimen DNA sequences. At the 3′
(using TaqMan fluorescent probes); for example, the end of the reverse oligonucleotide is a second univer-
FDA-approved COBAS HPV test performs a threefold sal primer sequence “Y.” This second primer also con-
screening assay: HPV-16, HPV-18, and 12 other high- tains a nonhybridizing “stuffer sequence” of variable
risk HPV types; in addition, a β-globin PCR assay is length intermediate between “Y” and the 5′ sequence
performed to confirm amplifiable quality DNA was that binds to target. The length of the “stuffer
extracted from a patient sample. COBAS platforms sequence” is linked to the specific target that the oli-
are also available for HBV, HCV, and human immu- gonucleotide can hybridize with. The total length of
nodeficiency virus (HIV) detection the first oligonucleotide is 50 to 60 bp and the second
• Luminex xTAG technology combines multiplex PCR, oligonucleotide is 60 to 450 bp in length; PCR ampli-
followed by multiplexed primer extension assays fied products range from 130 to 450 bp in length
that incorporates labeled dNTPs. The extended • PCR is then performed on the ligation product using
products are captured on fluorescent microspheres. two primers complementary to X and Y. The length
Coincident dual detection of the microsphere and of the PCR product will depend on which (if any)
extension product fluorescent signatures confirms oligonucleotide pairs initially hybridized and were
detection of a given target in a specimen. The meth- ligated; that is, the length of the “stuffer sequence”
odology is FDA cleared for screening cystic fibrosis, indicates which bio-target was present in the sample
respiratory viruses, gastrointestinal pathogens and • Applications
for polymorphisms associated with CYP2D6 drug • MLPA is applicable for the detection of mutations/
metabolism and has also been developed for numer- SNPs, deletions, and amplifications. Nonamplifica-
ous other applications tion with a particular probe indicates the presence
• AutoGenomics’ BioFilmChip microarray assay mul- of a mutation, Single nucleotide polymorphism
tiplexes ARMS-type PCR of target sequences. Fluo- (SNP), or deletion; “excess” amplification demon-
rescent dCTP is incorporated into PCR amplicons. strates an amplification event
A successfully amplified target is immobilized by • MLPA tests (none are currently FDA cleared/
sequence- specific capture probes embedded in a approved) are available for the diagnosis of a large
fluorescent-labeled hydrogel matrix. Coincident dual variety of pathologic conditions including these:
detection of the hydrogel and amplicon fluorescent • Familial cancers: ataxia telangiectasia, BRCA1 and
signatures confirms detection of a given target in a BRCA2 testing, colon polyposis (APC), MLH1/
specimen. The assay is FDA-cleared for CYP2C19 and MSH1/MSH2/MSH6/PMS2 testing, Li- Fraumeni
warfarin metabolism sensitivity, as well as for coagu- syndrome, multiple endocrine neoplasia, neu-
lation factor (factor II, factor V Leiden) assessments rofibromatosis types 1 and 2, Peutz- Jeghers
• The GenMark Dx eSensor XT- 8 assay involves syndrome, retinoblastoma, von Hippel- Lindau
multiplex PCR of target sequences followed by syndrome, Wilms tumor
incubation with exonuclease III, which results in • Tumor analyses: melanoma (uveal), mismatch
single-stranded amplicon DNA. A hybridization repair genes, neuroblastoma, oligodendroma,
reaction is then performed with a ferrocene labeled PTEN, rhabdoid tumors, tumor suppressor genes
probe; hybridization occurs if the target sequence • Phosphatase and tensin homolog (PTEN) Prena-
has been amplified from the patient specimen. Next, tal and postnatal screening: aneuploidy (Down,
the hybrid solution is pumped through a micro- Edwards, Patau), mental retardation syndromes,
fluidics cartridge containing a series of gold disk microdeletion syndromes (Prader- Willi/Angel-
electrodes. Each electrode is tagged with a unique man; RETT/Xq28 duplication and others)
capture probe. Voltage is applied to the cartridge; • Pharmacogenetics: DPYD deficiency
a current will be detected at the electrode at which • Specific syndromes: cystic fibrosis, Turner/Kline-
probe- hybridized- amplicon has been immobilized felter, typical uremic, Wilson disease
by the capture probe. The assay is FDA cleared for • PCR/oligonucleotide ligation assay (OLA)
investigation of cystic fibrosis, respiratory viruses, • MLPA involves an oligonucleotide ligation step fol-
thrombophilia, and warfarin sensitivity lowed by rounds of PCR, whereas PCR/OLA involves
multiplex PCR (up to 40 cycles) followed by cycles infections. FDA-approved TMA assays for high-risk
(up to 10) of multiplex ligation amplification of oli- type HPV E6/E7 RNA, for HCV, and for gastrointes-
gonucleotides to PCR product tinal infectious agents have also been developed.
• PCR/OLA is FDA cleared for cystic fibrosis gene muta- There is also an FDA-approved test for prostate can-
tion panel screening (CELERA, Alameda, CA) cer gene 3 (PROGENSA PCA3) mRNA detected from
urine samples
NANOSTRING TECHNOLOGY Nucleic Acid Sequence-Based Amplification
• NanoString technology (NanoString Technologies, • N ucleic acid sequence- based amplification (NASBA)
Seattle, WA) is used for RNA expression analyses. Total is an isothermal amplification technique and can be
RNA (extracted from >10,000 cells) is mixed with two used for the amplification of a DNA or RNA target. The
DNA oligonucleotide probes: a 3′ biotin end–labeled technique requires an initial heat denaturation step
target-
specific capture probe, and a target- specific when DNA is the sample to render the target as single-
reporter probe labeled at the 5′ end with a series of dif- stranded sequences
ferently colored fluorescent tags; the tag color order acts • The technique is essentially identical to TMA but uses
as a “bar code” target-specific identifier of the sequence a separate RNase H enzyme and NASBA amplifies its
detected by the probe. If the target sequence is present target by a factor of 109 in a 90-minute reaction at 41°C
in the RNA extract, both probes will hybridize. • Applications: FDA- rated NASBA assays have been
• The tripartite probes/target complex is purified and developed by bioMérieux, Inc. (Durham, NC) for the
then passed across a NanoString cartridge coated with detection of CMV, enterovirus, and HIV RNA
streptavidin. Streptavidin has a high affinity for bio-
tin and immobilizes hybrid targets. A voltage is applied Displacement Amplification
across the cartridge and causes the negatively charged • Several amplification strategies have been developed
nucleic acid hybrids to linearize over the cartridge sur- for use with DNA polymerases that have “strand dis-
face. The detected fluorescent tag codes allow quantifi- placement” activity, e.g., Bst DNA polymerase, (derived
cation of gene target expression in the sample. from Bacillus stearothermophilus) or Phi29 DNA poly-
• The assay is highly multiplexable and can screen for merase (derived from the Bacillus subtilis phage phi29
the detection of up to 800 targets. The assay need not [Φ29])
include amplification steps; if specimen is limiting • As with other DNA polymerases, DNA is synthesized
(<10,000 cells) RNA sample can be amplified prior to in the 5′ → 3′ direction; unlike other polymerases hav-
the assay by RT-PCR ing initiated DNA polymerization from an upstream
• The Prosigna NanoString assay is FDA-cleared for breast primer binding site, these enzymes will displace a
cancer 10-year risk of recurrence (ROR) testing. The test double-stranded DNA region resulting from synthesis
screens for the PAM50 (Prediction Analysis of Micro- initiated at a downstream region. This displacement
array 50) gene set that assesses patient specimens on property supports isothermal DNA amplification as it
the basis of the expression levels of 50 different genes is not necessary to (cyclically) heat-denature DNA to
into the four main molecular subtypes of breast cancer produce a single-stranded template
(luminal A, luminal B, HER2 enriched, and basal-like). • Applications: Two of these approaches have FDA-
On the basis of PAM50 data a 10-year ROR score (0 to cleared proprietary tests
100) is generated • Strand displacement amplification (SDA) tests have
been developed for the detection of C. trachomatis,
OTHER NUCLEIC ACID AMPLIFICATION N. gonorrhoeae, and Legionella pneumophila (BD Pro-
METHODS beTec ET systems for each microorganism; Becton,
Dickinson and Company, Sparks, MD)
Transcription-Mediated Amplification • Loop-mediated isothermal amplification (LAMP)
• T ranscription-mediated amplification (TMA) supports tests are available for a wide range of targets,
the amplification of RNA targets, including species- including Clostridium difficile, groups A and B Strep-
specific rRNA sequences. The method involves an iso- tococcus, and mycoplasma (Meridian Bioscience,
thermal reaction containing the following ingredients: Inc., Cincinnati, OH)
• RNA sample • The method involves generating a target-specific
• A target specific “forward” primer with an RNA poly- sequence using microorganism primers that also
merase promoter sequence at the 5′ end incorporate an RE site into the polymerized prod-
• Reverse transcriptase (RT) with active RNase H activ- uct; exponential amplification of these targets fol-
ity (e.g., avian myeloblastosis virus reverse transcrip- lows triggered by nicks to the amplified DNA at the
tase [AMV-RT]) RE sites
• A target-specific “reverse” primer • In excess of 109 copies of the target may be pro-
• RNA polymerase (e.g., SP6, T3, or T7 RNA polymerase) duced within 15 minutes
• Applications • This self-
contained complex isothermal reaction
• TMA is a proprietary technique of Gen-Probe, Inc. involves the judicious use of multiple primers
(San Diego, CA). FDA-cleared TMA tests are available resulting in the continuous generation of target-
for the detection of C. trachomatis, Neisseria gonor- specific DNA structures. The pyrophosphate ions
rhoeae, Mycobacterium tuberculosis, and Streptococcus released during DNA synthesis bind to magnesium
ions forming a white precipitate of magnesium target DNA sequence with an RNA probe, followed by a
pyrophosphate. The resultant turbidity is mea- signal amplification step
sured by assay instrumentation; the presence or • Applications
absence of threshold turbidity indicates the infec- • The digene HPV Test uses Hybrid Capture 2 (hc2)
tious status of the patient sample technology and is currently the only FDA-approved
HPV screening assay
Helicase Dependent Amplification • FDA-cleared hybrid capture assays are also available
• H elicase dependent amplification (HDA) is an in vitro for the detection/quantitation of CMV, C. trachoma-
isothermal amplification technique that uses DNA heli- tis, and N. gonorrhoeae. Assays are also available for
case to unwind and separate double-stranded helix into HBV and herpes simplex virus (HSV)
single-stranded DNA (ssDNA). ssDNA-binding proteins • The test screens for 13 high-risk HPV genotypes
(SSBs) included in the HDA reaction inhibit ssDNA (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and
reannealing and degradation. Sequence-specific prim- 68)
ers (one labeled) hybridize with the target (if present), • The test requires the majority of cell samples
and DNA polymerase synthesizes a complementary remaining after routine cytology testing and has a
strand. This dsDNA product is rendered single-stranded detection sensitivity of 1000 to 5000 copies of HPV
by the DNA helicase and so on, resulting in an expo- target per sample
nential amplification cascade
• An FDA-approved test for HSV is available from BioHe- Verigene Nanosphere
lix (Beverly, MA) • T he Verigene Nanosphere (Nanosphere, Inc., North-
brook, IL) proprietary instrumentation supports a
Signal Amplification Techniques range of FDA-cleared tests, including ones for respira-
• T he assays described earlier are based on the amplifi- tory viruses, gram- positive and gram-
negative blood
cation of target nucleic acid sequences to a threshold culture, C. difficile, F5/F2/MTHFR, warfarin metabolism,
of detection. Alternatively, an amplification signal can and CYP2C19
be generated from a DNA probe(s) hybridized to target • Automated DNA extraction from patient samples is
sequences followed by sonication to fragment the DNA, which
• Signal amplification techniques may be less suscepti- is then flowed through a cartridge containing target-
ble to false-positive data resulting from patient sample specific capture probes. Gold “nanospheres” coated
cross-
contamination than methods generating mul- with sequence-specific probes are then flowed through
tiple copies or target sequences the cartridge for hybridization to patient DNA immo-
bilized by the capture sequences. Silver ions, hydroxy-
Branch DNA quinone, and an electric current are then passed
• T his method involves the capture of specimen RNA or through the cartridge. Silver ions are reduced to sil-
DNA in a microtiter plate well, followed by a sequential ver atoms on the surface of nanospheres localized to
four-step detection procedure target sequences. The increase in nanosphere diam-
• Multiple “capture” probes specific to the sequence(s) eter due to silver deposition is detected by automated
of interest and lining the microwell hybridize and imaging of the cartridge, allowing inference of target
bind the specimen RNA or denatured DNA. A set of detection
“target” probes is then applied; part of each target • The main advantages of this technique are its rapidity,
probe hybridizes with the captured sequence. “Pream- minimal pipetting steps, and complete absence of any
plifier” sequences are then added; these in turn partly nucleic acid amplification
hybridize with the target probe and an extended
region is available to hybridize with multiple “ampli-
MICROARRAY TECHNOLOGY
fier” constructs applied following the preamplifier.
Finally, alkaline phosphatase-labeled probes bind to • D NA microarrays make up a solid support (a silicon
the amplifier constructs. Alkaline phosphatase activ- chip) imprinted with sequence-specific oligonucleotide
ity is demonstrated by chemiluminescence using a probes. Fluorescence-labeled sample DNA or cDNA is
dioxetane substrate hybridized with the microarray, and the detected emis-
• Branch DNA (bDNA) technology allows highly specific sions demonstrate qualitatively or quantitatively the
and quantitative nucleic acid assays nucleic acid species present in the sample
• Applications • DNA microarrays can be used to examine gene expres-
• FDA- approved bDNA tests are available for HCV sion by simultaneously hybridizing the array with
and for HIV quantification (VERSANT HCV RNA 3.0 cDNA from normal and diseased tissues; each cDNA
Assay and VERSANT HIV-1 RNA 3.0 Assay, respec- preparation is labeled with a different fluorophore.
tively [Siemens Healthcare Diagnostics, Deerfield, Analysis of the intensities of the different labels dem-
IL]). bDNA research applications are available from onstrates genes that are or underexpressed, overex-
Panomics, Inc. (Fremont, CA) pressed, or unchanged in expression
• A similar assay using labeled DNAs and chromosome-
Hybrid Capture specific probes can be performed to infer chromosome
• T
he Hybrid Capture assay (QIAGEN, Germantown, losses or gains. DNA microarrays can also be used to
MD) involves an in vitro solution hybridization of a screen for SNPs
T C T G A T C T T G G T C G C T G G AT A G T C G T C T G T G T T T C T T C G G T G C C C A A
Figure 1.21 Electropherogram showing DNA sequencing data output.
• P otentially, thousands of sequences can be screened lengths of the target and typically yielding sequence
using a single microarray. Limited target (<100) set order data for up to approximately 700 nucleotides.
arrays designed toward cell pathways (e.g., apoptosis, The generated fragments are sieved by CGE. ddNTP
angiogenesis, cell cycle, cytokines, signal transduc- fluorophore emissions are detected as the fragments
tion), or tumor nucleic acid signatures, have also been pass through the gel revealing the base sequence order
developed (Figure 1.21)
• Microarray assay affordability, standardization, and • Applications
clinical interpretability are issues limiting clinical array • DNA sequencing represented the gold standard con-
applications firmation of a mutation; however, the detection of
• Applications a mutation may be compromised in heterogeneous
• There are two FDA- cleared microarray tests; each samples (e.g., tumor cells in a high background of
requires fresh tumor tissue samples normal cells); this issue can be resolvable by next-
• The MammPrint test (Agendia, Inc., Irvine, CA) generation sequencing (NGS) approaches
screens 70 genes to assess the likelihood of recur- • Clinical applications generally involve PCR amplifi-
rence in patients who have undergone breast cancer cation of a defined target region followed by sequenc-
surgery. The expressed gene data indicate patients ing; available tests include the following:
who are at high or low risk of disease recurrence. The • FDA- cleared sequencing assays are available for
test is applicable to lymph node–negative patients HIV drug resistance testing (ViroSeq HIV-1 Geno-
younger than 61 years of age with stage 1 or 2 tumors typing System, Celera Diagnostics, CA, and Tru-
5 cm or less. Gene HIV- 1 Genotyping and Open Gene DNA
• The Pathwork Tissue of Origin Test (Cancer Genetics, Sequencing System, Siemens Healthcare Diagnos-
Inc., Rutherford, NJ) aids identification of the origin tics, Deerfield, IL)
of a tumor. The test measures the expression pattern
of more than 1500 genes in the “uncertain” tumor. Next-Generation Sequencing
This pattern is compared to expression patterns of a • N GS, also referred to as massively parallel sequenc-
panel of 15 known tumor types, representing 60 mor- ing, allows millions up to billions of sequences to be
phologies overall. An objective, probability- based decoded in relatively short time periods (hours to days).
score is generated relative to each of the 15 potential The method involves shearing extracted genomic DNA
tumor types supporting assignment or exclusion of or cDNA (size range ∼500 bp) followed by primer liga-
the uncertain tumor to each panel type tion to the fragment ends. This fragment library is then
amplified (e.g., by PCR) followed by massively parallel
sequencing and alignment of the sequence reads
NUCLEIC ACID SEQUENCING
• Proprietary NGS platforms include Ion Torrent (Thermo
• T
he most widely used nucleic acid sequencing tech- Fisher Scientific, Waltham, MA), Pyrosequencing (Qia-
nique has been the chain termination method origi- gen, Germantown, MD), and Solexa sequencing (Illu-
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to the development of dye terminator sequencing that Fibrosis Clinical Sequencing Assay)
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includes the DNA sample, one PCR primer specific cancer treatment options by screening for sequence
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dNTPs, and four dideoxynucleotides (ddNTPs [ddATP, • FoundationFocus CDxBRCA test (Foundation Medi-
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diester linkage with a distal nucleotide. The proportion Based Next Generation Sequencing Assay (Memorial
of dNTPs and ddNTPs in the reaction mix ensures that Sloan-Kettering Cancer Center, New York, NY). This
DNA fragments are generated representing all possible test detects somatic alterations (point mutations,
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Gattuso's Differential Diagnosis in

Surgical Pathology - eBook PDF

GATTUSO’S DIFFENENTIAL DIAGNOSIS IN SURGICAL PATHOLOGY,

Previous editions copyrighted 2015, 2010, 2002.

Library of Congress Control Number: 2020949417

Executive Content Strategist: Michael Houston

Printed in the United States.

Last digit is the print number: 9 8 7 6 5 4 3 2 1

SYLVIA ASA, MD, PhD BYRON CRAWFORD, MD

ELIZAVETA BELYAEVA, MD VIRGINIA E. DUNCAN, MD, MS

PINCAS BITTERMAN, MD ADEL K. EL-­NAGGAR, MD

ELIZABETH J. COCHRAN, MD HUMA FATIMA, MD

KUMARASEN COOPER, MBChB, DPhil

SANDRA E. FISCHER, MD CRISTINA MAGI-­GALLUZZI, MD, PhD

RALPH H. HRUBAN, MD MARIA J. MERINO, MD

ALIYA N. HUSAIN, MBBS IRA MILLER, MD, PhD

ALEXANDRA N. KALOF, MD ATTILIO ORAZI, MD, FRCPath

SUNNY B. PATEL, BS, MD† JEFREE SCHULTE, MD

MATTHEW M. YEH, MD, PhD MING ZHOU, MD, PhD

To my wife, Nancy, and my children, Vincent and Francesca

To my family, to whom I owe my appreciation of life and learning

To all of my former students, residents, fellows, and physician associates

Figure 1.2 Masson trichrome stain. Cirrhosis of the liver charac-

Pigments and Minerals

Nerves and Fibers

Selected References FLUORESCENCE MICROSCOPY

TABLE 1.1 IMMUNOFLUORESCENCE PATTERNS AND DISEASE ASSOCIATIONS

TABLE 1.2 CYTOPLASMIC GRANULES

Figure 1.9 Electron microscopy. Birbeck granules (arrow) in Lang-

Single Membrane–Bound Structures

• V  arious techniques may unmask antigens, such as

GROUND RULES FOR • A void preordering an IHC panel of antibodies before

Selected References FLOW CYTOMETRY

TABLE 1.4 IMMUNOHISTOCHEMISTRY APPROACH TO UNDIFFERENTIATED TUMORS

Selected Reference • A  fter staining of the chromosomes, specific chromo-

TABLE 1.7 IMMUNOHISTOCHEMISTRY PANEL FOR INTERPRETATION OF LUNG MESOTHELIOMA AND

TABLE 1.8 IMMUNOHISTOCHEMISTRY PANEL FOR LUNG ADENOCARCINOMA AND BREAST

TABLE 1.9 IMMUNOHISTOCHEMISTRY COMPARISON OF SPINDLE CELL AREAS IN METAPLASTIC CARCINOMA,

TABLE 1.11 IMMUNOHISTOCHEMICAL PANEL APPROACH TO DIFFERENTIAL DIAGNOSIS OF

lung origin in the correct clinical setting).

TABLE 1.12 IMMUNOHISTOCHEMISTRY PANEL INTERPRETATION FOR GASTROINTESTINAL AND ABDOMINAL

Leiomyosarcoma (LMS)a +/− − −/+a + + −

TABLE 1.13 IMMUNOPHENOTYPE OF PRIMARY OVARIAN AND METASTATIC COLORECTAL ADENOCARCINOMA

TABLE 1.14 IMMUNOHISTOCHEMISTRY PANEL FOR PRIMARY AND METASTATIC ADENOCARCINOMA

TABLE 1.15 IMMUNOHISTOCHEMISTRY: HIGH-­GRADE SEROUS CARCINOMA AND POORLY DIFFERENTIATED

TABLE 1.16 IMMUNOHISTOCHEMISTRY APPROACH TO OVARIAN SEX CORD–STROMAL TUMORS AND

TABLE 1.17 IMMUNOHISTOCHEMISTRY APPROACH TO ENDOCERVICAL ADENOCARCINOMA AND

TABLE 1.18 IMMUNOHISTOCHEMISTRY IN THE DIFFERENTIAL DIAGNOSIS OF SQUAMOUS AND GLANDULAR

TABLE 1.20 IMMUNOHISTOCHEMICAL APPROACH FOR TROPHOBLASTIC LESIONS

TABLE 1.21 IMMUNOHISTOCHEMISTRY FOR SELECTED GERM CELL TUMORS

TABLE 1.22 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH RENAL CELL NEOPLASMS

TABLE 1.23 SELECTED IMMUNOHISTOCHEMISTRY TABLE 1.24 IMMUNOHISTOCHEMISTRY

MOLECULAR PATHOLOGY METHODS NUCLEIC ACID EXTRACTION METHODS

TABLE 1.25 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH PROSTATE AND UROTHELIAL CARCINOMAS

+, positive; −, negative; +/−, often positive; −/+, rarely positive.

TABLE 1.27 MIB-­1(KI-­67) MEMBRANOUS AND CYTOPLASMIC STAINING

Chronic lymphocytic Mantle cell Marginal zone Follicular

TABLE 1.29 ANTIBODY PANEL FOR DIFFERENTIAL DIAGNOSIS OF HODGKIN LYMPHOMA

TABLE 1.30 IHC CLASSIFICATION OF DIFFUSE • T

GCB BCL6 DNA Extraction Methods

TABLE 1.31 IMMUNOPROFILE OF SMALL ROUND CELL TUMORS

TABLE 1.32 IMMUNOHISTOCHEMISTRY PANEL TABLE 1.33 IMMUNOHISTOCHEMISTRY TO DETECT

TABLE 1.34 IMMUNOHISTOCHEMISTRY TO DETECT THE GAIN OR LOSS OF PROTEIN EXPRESSION

Loss, negative nuclear staining; Present, positive nuclear staining.

PINCAS BITTERMAN, MD ADEL K. EL-NAGGAR, MD

SANDRA E. FISCHER, MD CRISTINA MAGI-GALLUZZI, MD, PhD

TABLE 1.1 IMMUNOFLUORESCENCE PATTERNS AND DISEASE ASSOCIATIONS

TABLE 1.2 CYTOPLASMIC GRANULES

• V arious techniques may unmask antigens, such as

GROUND RULES FOR • A void preordering an IHC panel of antibodies before

TABLE 1.4 IMMUNOHISTOCHEMISTRY APPROACH TO UNDIFFERENTIATED TUMORS

Selected Reference • A fter staining of the chromosomes, specific chromo-

TABLE 1.13 IMMUNOPHENOTYPE OF PRIMARY OVARIAN AND METASTATIC COLORECTAL ADENOCARCINOMA

TABLE 1.15 IMMUNOHISTOCHEMISTRY: HIGH-GRADE SEROUS CARCINOMA AND POORLY DIFFERENTIATED

TABLE 1.20 IMMUNOHISTOCHEMICAL APPROACH FOR TROPHOBLASTIC LESIONS

TABLE 1.21 IMMUNOHISTOCHEMISTRY FOR SELECTED GERM CELL TUMORS

TABLE 1.22 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH RENAL CELL NEOPLASMS

TABLE 1.25 IMMUNOHISTOCHEMISTRY PANEL TO DISTINGUISH PROSTATE AND UROTHELIAL CARCINOMAS

TABLE 1.27 MIB-1(KI-67) MEMBRANOUS AND CYTOPLASMIC STAINING

TABLE 1.29 ANTIBODY PANEL FOR DIFFERENTIAL DIAGNOSIS OF HODGKIN LYMPHOMA

TABLE 1.31 IMMUNOPROFILE OF SMALL ROUND CELL TUMORS

TABLE 1.34 IMMUNOHISTOCHEMISTRY TO DETECT THE GAIN OR LOSS OF PROTEIN EXPRESSION

Figure 1.16 Well-differentiated

TABLE 1.37 ONCOGENES OF IMPORTANCE IN SURGICAL PATHOLOGY

TABLE 1.39 CYTOGENETIC ABNORMALITIES OF IMPORTANCE IN SURGICAL PATHOLOGY

agarose gel electrophoresis, RNA is transferred to a 5 3