Figure 4-3 Flow cytometry analyzes particles or cells moving in single file in a fluid stream (a). Fluorochrome-labeled specific antibodies bind to the surface of cells, and a suspension of labeled cells is sequentially illuminated by a laser (b). As the excited fluorochrome returns to its resting energy level, a specific wavelength of light is emitted (c), which is sorted by wavelength (d) and received by a photodetector (e). This signal is then converted to electronic impulses, which are in turn

analyzed by computer software. (Courtesy of Patricia Chévez-Barrios, MD.)

In addition, multiple antibodies and cellular size can be analyzed, and the relative percentages of cells may be displayed. For example, 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 advantage of this method is that it actually shows the percentages of particular cells in a specimen. Disadvantages are the failure to show the location and distribution of these cells in tissue and the possibility of sampling errors. Depending on the number of cells in the sample and on clinical information, the flow cytometrist chooses the panel of antibodies to be tested. Flow cytometric data should therefore be used as an adjunct to morphologic H&E and sometimes immunohistochemistry interpretation. Flow cytometric analysis is particularly useful for the evaluation of lymphoid proliferations.

Molecular Pathology

Molecular biology techniques are used increasingly in diagnostic ophthalmic pathology and

extensively in experimental pathology (Table 4-2). More recently, their use has expanded to include prognostication of disease and determination of treatment. Molecular pathology is used to identify tumor-promoting or tumor-inhibiting genes (CGH, PCR, array CGH), such as the retinoblastoma gene; and viral DNA or RNA strands, such as those seen in herpesviruses and Epstein-Barr virus (PCR, in situ hybridization [ISH]). The evolution of molecular pathology techniques has made it possible not only to recognize the presence or absence of a strand of nucleic acid but also to localize specific DNA sequences within specific cells (FISH, ISH). Two major techniques have markedly advanced our knowledge of developmental biology and tumorigenesis: PCR (and its variations) and microarray (and its subtypes).

Figure 4-4 Flow cytometry scatter graphs showing a clonal population of CD19+ kappa restricted lymphocytes. Note that most of the CD19+ (red in left graph) cells fail to express lambda light chains; however, the cells do exhibit strong kappa

expression (red in right graph). (Courtesy of Patricia Chévez-Barrios, MD.)

Table 4-2

Polymerase chain reaction

A common molecular biology technique is the polymerase chain reaction (PCR), which amplifies a single strand of nucleic acid across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence (Fig 4-5). The PCR method relies on thermal cycles of repeated heating and cooling of the DNA sample for DNA melting and enzymatic replication. Primers, which are short DNA fragments containing sequences complementary to the target region, DNA polymerase, and nucleotides are the components required in order for selective and repeated amplification to occur. The selectivity of PCR is due to the use of primers that are complementary to the DNA region targeted for amplification.

Figure 4-5 A, A polymerase chain reaction (PCR) starts with a denaturing step where DNA samples are heated to 95°C to separate the target DNA into single strands. B, Next, the temperature is lowered to 55°C to allow the primers to anneal to their complementary sequences. The primers are designed to bracket the DNA region to be amplified. C, The temperature is raised to 72°C to allow Taq polymerase to attach at each priming site and extend or synthesize a new DNA strand between primer sequences producing 2 new DNA molecules. D, Step C is repeated multiple times to generate thousands of copies.

(Courtesy of Theresa Kramer, MD.)

The techniques of PCR have advanced considerably in recent years, and there are now approximately 20 variations on PCR, including real-time and quantitative real-time PCR. The variations center around quantification; increased definition of the sequence amplified (allele, single nucleotide, generation of long sequences with overlap technology, intersequences, flanking sequences

and genomic inserts, methylation-specific sequences, conserved sequences, simultaneous multiple gene amplification); isothermal amplification methods for use in living cells (PAN-AC); and increased resolution of the sequence (multiplex ligation-dependent probe amplification, MLPA). MLPA permits multiple targets to be amplified simultaneously with only a single primer pair and is becoming important in prognostication of tumors. The clinical relevance of detecting a PCR product depends on numerous variables, including the primers selected, the laboratory controls, and the demographic considerations. Thus, for the clinician making a clinicopathologic diagnosis, PCR should be used mainly to derive supplementary information. See also Part III, Genetics, in BCSC Section 2, Fundamentals and Principles of Ophthalmology.

Microarray

Microarrays are used to survey the expression of thousands of genes in a single assay, the output of which is called a gene expression profile. Using microarray technology, scientists and clinicians can attempt to understand fundamental aspects of growth and development, as well as to explore the molecular mechanisms underlying normal and dysfunctional biological processes and elucidate the genetic causes of many human diseases. DNA microarray, microRNA microarray (MMChips), protein microarray, tissue microarray (Fig 4-6), cellular (or transfection) microarray, antibody microarray, and carbohydrate (glycoarray) microarray are some of the different types of microarrays available.

Although there are a variety of DNA microarray platforms, the basic process underlying all of them is straightforward: A glass slide or chip is spotted or “arrayed” with oligonucleotides or DNA fragments that represent specific gene coding regions (called probes). Fluorescently or chemiluminescently labeled purified cDNA or cRNA (called target) is hybridized to the “arrayed” slide/chip. After the chip is washed, the raw data are obtained by laser scanning, entered into a database (some public, others mined), and analyzed by statistical methods.

An example of one of these microarray platforms is the DualChip low-density microarray. These DNA microarrays were developed as a flexible tool capable of reliably quantifying the expression of a limited number of genes of clinical relevance, but DualChip technology has also been applied to tumor diagnosis and tumor-acquired drug resistance. Validation of the results of microarray experiments is a critical step in the analysis of gene expression. Quantitative real-time PCR is the method of choice for validation of gene expression profiling.

Figure 4-6 Tissue microarrays are constructed with small core biopsies of different tumors/tissues. A core is obtained from the donor paraffin block of the tumor (a). A recipient paraffin block is prepared, creating empty cores (b). The cores are incorporated into the slots (c) until all are occupied (d). Glass slides are prepared and stained with a selected antibody (e). Microscopic examination reveals the different staining patterns of each core (f). (Courtesy of Patricia Chévez-Barrios, MD.)

Clinical use of PCR and microarray

Routine clinical use of PCR and microarray is limited to the diagnosis of leukemias, lymphomas, soft-tissue tumors, and tumors with nondiagnostic histopathology results. Commercial microarray and PCR platforms now exist that can be used for assigning biopsy-sized tumor samples to 1 of 2 distinct molecular classes, based on gene expression analysis that distinguishes low-grade tumors from high-grade tumors. The selection of commercially available microarray and PCR kits is growing rapidly.

Leukemias, lymphomas, and soft-tissue tumors represent a heterogeneous group of lesions whose classification continues to evolve as a result of advances in cytogenetic and molecular techniques. In the 1990s, traditional diagnostic approaches were supplemented by the successful application of these newer techniques (see Table 4-2) to formalin-fixed, paraffin-embedded tissue, making it possible to subject a broader range of clinical material to molecular analysis. Thus, molecular genetics has already become an integral part of the workup of tumors, such as pediatric orbital tumors