Multicolor flow cytometry analysis (FCA) is an invaluable laboratory tool for the characterization of hematolymphoid malignancies. It permits multiparametric measurement of cellular properties that include size, cytoplasmic complexity, and antigen expression. A typical flow cytometer is composed of a laminar flow cell transport system, one to several laser lights, photodetectors, and a computer-based data management system. The intricate design of flow cytometers ensures that cells flowing in a fluid sheath are hydrodynamically focused to intercept laser light at a specific frequency. The interaction of the laser light with the cell results in light scatter and, in the presence of bound fluorochrome-tagged antibodies, excitation and resultant emission of light at a different wavelength. These events are captured by sensitive photodetectors and converted to measurable parameters. Scattered light captured by a detector positioned at a right angle (90°) from the laser source measures cytoplasmic complexity whereas scattered light captured by detectors along the original trajectory of the laser beam (180°) measures cell size.

Flow cytometry analysis is a robust tool to simultaneously assess coexpression of multiple antigens expressed by cells (Fig. 1.2). This is useful to many clinical assays including cell lineage determination, biomarker detection, minimal residual disease assessment, enumeration of cell subsets (e.g., stem cells, T-cell subsets), and measurement of proliferation and apoptosis.

For such applications, antibodies with covalently linked fluorescent molecules (fluorochromes) are used to identify target antigens and provide a means for qualitative and quantitative assessment of antigen expression. This ability to perform multiparametric analysis on an individual cell offers FCA a distinct advantage over immunohistochemistry particularly in hematolymphoid disorders [8]. On the other hand, the use of FCA to evaluate solid tumors remains technically limited.

Conventional cytogenetic analysis (cytogenetics) is a laboratory discipline that involves the study of chromosomes, also known as karyotyping. Chromosomal alterations are common in cancer and are broadly categorized into recurrent and nonrecurrent abnormalities. Tumors arising in the pediatric age group are more likely than those arising in adults to harbor recurrent cytogenetic abnormalities. Frequently, such recurrent cytogenetic abnormalities are integral elements of pediatric cancer pathogenesis and their detection has emerged as an important adjunct for diagnostic evaluation.

For conventional cytogenetic analysis of tissue samples viable fresh cells are required for analysis. The average viable human cell divides once every 24 h and certain cell types, such as lymphocytes, do not divide at all, which mandates special culture techniques and growth stimulation of the cell of interest to increase the yield of analyzable material. Bone marrow is typically cultured for 24–48 h whereas lymphocytes from tissue may require 3–4 days in culture medium containing proliferation inducers for maximum yield. Cultured cells are then subjected to metaphase arrest before being processed to prepare chromosome spreads. Chromosomes are then stained, most commonly with Giemsa or Wright stains, for visualization of the characteristic banding patterns. Positively charged dyes in stains bind to the negatively charged DNA in chromosomes.