Special Testing and

Procedures in Pathology
AAO READING ROO PAGE 29-40

JIHAN FILDZAH NADIA

 HIGHLIGHT

• Special procedures such as immunohistochemistry and flow cytometry, as well as

molecular genetic and cytogenetic techniques, have led to improvements in the di-
agnosis of eye diseases.
• The ophthalmic surgeon is responsible for submitting tissue in a manner that allows
special studies to be performed when needed. Appropriate submission requires
preoperative consultation with the pathologist.
• Frozen section pathology should be used when the pathologic results would alter
the surgical procedure, as with evaluation of surgical margins when a malignancy is
excised.
 INTRODUCTION

• New methodologies in pathologic analysis of tissue specimens have contributed to

improvements in the diagnosis of infectious agents, dystrophies, degenerations, and
neoplasms, as well as to the classification of neoplasms, particularly the non-
Hodgkin lymphomas and sarcomas.

• Refinements in immunohistochemical (IHC) staining and in flow cytometric,

molecular genetic, and cytogenetic techniques lead to more accurate diagnoses and
to more precise identification of biomarkers of value in risk stratification,
prognostication, and targeted therapeutics.
 Imunohistochemistry (IHC)
• Express specific antigens.
• To identify the cell type or cell of origin, typically in
tumors.
• In the IHC stains commonly used in ophthalmic
pathology, a primary antibody binds to a specific
antigen in or on a cell, and a secondary antibody linked
to a chromogen (a compound that produces a particular
color as a result of a chemical reaction) then binds to the
primary antibody.

• A specific antigen can be identified using this method,

and depending on the antigen profile, the specific cell
type that expresses this antigen(s) can be ascertained.
• Many antibodies are routinely used for diagnosis,
treatment, and prognosis.
 Flow Cytometry

• Pathologists use flow cytometry to analyze the physical and chemical properties of cells moving in
single file in a fluid stream.
• The most common use in clinical practice -> immunophenotyping hematopoietic proliferations.
• Performed on ocular adnexal tissue, aqueous humor, or vitreous.
• One example -> leukocyte immunophenotyping.
• The cells need to be fresh (unfixed, unfrozen).
• An advantage -> shows the proportion of particular cells in a specimen in histogram format. In
addition, multiple antibodies and cellular size can be analyzed simultaneously. For example, the
proportion of CD4 (helper T cells), CD8 (suppressor T cells), both CD4+ and CD8+, or either CD4+
or CD8+ may be displayed for a given lymphocytic infiltrate.
• The disadvantages -> its failure to show the location and distribution of these cells in tissue and the
possibility of sampling error.
 Molecular Pathology

• Molecular biology techniques  diagnostic ophthalmic pathology and extensively in

experimental pathology.
• Disease prognostication and treatment selection.
• To identify tumor-promoting or tumor-inhibiting genes, such as the retinoblastoma gene.

• Not only to recognize the presence or absence of a strand of nucleic acid but also to localize
precise DNA sequences within specific cells (eg, via fluorescence in situ hybridization [FISH]
or ISH).

• Two major techniques of developmental biology and tumorigenesis :

PCR (and its variations) and microarray (and its subtypes).
 Polymerase chain reaction

• A common molecular biology technique  PCR method, which amplifies a single strand of
nucleic acid by several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
• This method relies on thermal cycles of repeated heating and cooling of the DNA sample for
thermal denaturation (DNA melting) and enzymatic replication.
• The components required for selective and repeated amplification are primers [short DNA
fragments that contain sequences complementary to the target region (cDNA)], DNA
polymerase, and nucleotides.
• PCR techniques  20 PCR variants.
• The clinical relevance of detecting a PCR product depends on numerous variables (primers
selected, laboratory controls, and demographic considerations.)
• PCR  as an adjunct to routine pathologic diagnostic techniques.
 Microarrays

• To survey the expression of thousands of genes in a single assay, the output of which is
called a gene expression profile (GEP).

• Different types of microarrays are available, including DNA microarrays (the most
common type), microRNA microarrays (MMChips), protein microarrays, tissue
microarrays, cellular (or transfection) microarrays, antibody microarrays, and
carbohydrate(glycoarray) microarrays.
• The basic process  is straightforward: a glass slide
or chip is spotted or “arrayed” with oligonucleotides
or DNA fragments (called probes) that represent
specific gene- coding regions. Fluorescently or
chemiluminescently labeled purified cDNA or cRNA
(called target) is hybridized to the arrayed slide or
chip.

• After the chip is washed, the raw data are obtained

by laser scanning, entered into a database, and
analyzed with statistical methods.

• Quantitative (real-time) PCR is the preferred method

for validating gene expression profiling.
 Clinical use of PCR and microarray

1. To the diagnosis of leukemias, lymphomas, soft-tissue neoplasms, and tumors with

nondiagnostic histopathology results.

2. The detection of infectious agents (eg, the herpesvirus family), in tumor prognostication
(eg, uveal melanoma), and in detection of genetic alterations that are amenable to targeted
therapies (eg, cutaneous melanoma and hematologic malignancies).

• However, the cost of these testing modalities is often significantly higher than that of other,
more traditional, diagnostic modalities and should be discussed with patients before tests
are ordered.
 Special Procedures – FNAB (Fine Needle Aspiration Biopsy

• Intraocular FNAB  to distinguishing between primary

uveal tumors and metastases.
• The procedure is performed under direct visualization
through a dilated pupil, transvitreally or transsclerally
• Special cytology fixatives, typically alcohol-based
• The cells obtained through FNAB can be processed using
various cytopathologic techniques, such as a cytospin
preparation, in which cells are centrifuged onto a glass
slide, and processed into a paraffin block (cell block).
• A cell block allows the pathologist to employ special
stains, IHC, ISH, microarray, and gene expression
profiling if needed and as cellular material permits
• Intraocular FNAB has been postulated to increase the risk of tumor spread
outside the eye, although this is controversial. In general, when properly
performed, FNAB does not pose a major risk for tumor seeding.

• Some orbital surgeons have used FNAB to diagnose orbital lesions, especially
optic nerve tumors and presumed metastases to the orbit
 FROZEN SECTION

• Tissue that is snap-frozen and immediately sectioned in a cryostat, is indicated when the results
of the study will affect intraoperative management of the patient.
• Permanent sections, tissue that is processed into paraffin prior to sectioning, are always
preferred in ophthalmic pathology because of the inherent small size of the samples and the
superior morphological preservation achieved with this technique. If the tissue sample is too
small, it could be lost during frozen sectioning.
• Indication for a frozen section  to determine whether the resection margins are free of tumor,
especially in eyelid carcinomas. When tissue is submitted for margin evaluation, appropriate
orientation of the specimen, correlated with documentation (through drawings of the excision
site, labeled margins, or margins of the excised tissue that are tagged with sutures or other
markers), is crucial;
 • Two techniques can be used for
assessing the margins in eyelid
carcinomas (eg, basal cell carcinoma,
squamous cell carcinoma):

1. Routine frozen sections

2. Mohs micrographic
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