Cytogenetics is the study of chromosome structure and function. Chromosome analysis is an integral part of diagnosing hematologic malignancies and is important for determining prognosis (or risk category) and/or treatment. According to the WHO classification of leukemias and lymphomas from 2008,¹ there are several categories of leukemias and lymphomas that are defined by their specific clonal chromosome abnormalities. Chromosome and/or fluorescence in situ hybridization (FISH) analyses are considered “standard of care” for diagnosing and following patients with most hematologic malignancies. Array analyses of these same malignancies, using either array comparative genomic hybridization (CGH) or single nucleotide polymorphism (SNP) array analysis, have been used for identification of smaller, more subtle or complex anomalies not appreciated by chromosome analysis and/or FISH; however, the diagnostic and prognostic value of this technology is just beginning to emerge.

Cytogenetic analysis of hematologic malignancies involves at least two, if not three, different approaches. The first is “classic chromosome analysis” (CCA). The second is FISH using DNA probes labeled with fluorochrome(s). Whether FISH is performed on metaphase cells (metaphase chromosomes) or interphase cells (resting, non-dividing nuclei), this technique can be used to ask very specific questions. However, to determine the correct interrogating probe, one must know the question to be answered. The advantage of using interphase FISH is that mitotic cells are not necessary for analysis, as they are for CCA and metaphase FISH. In addition, even formalin-fixed paraffin-embedded tissue can be studied in this fashion. The third approach involves arrays, either CGH arrays, utilizing primarily oligonucleotides spaced throughout the genome, or SNP arrays, with heterozygous SNPs positioned throughout the genome. Both of these array types can be used to determine DNA copy number (one vs. two vs. three); however, loss of heterozygosity (LOH) can only be detected if one uses SNP arrays. In addition, balanced rearrangements (copy neutral), i.e., translocations, inversions, etc., cannot be appreciated by either array method. Malignancies are often made up of both normal and abnormal cells, as well as varying percentages of abnormal clones. This heterogeneity, which can be seen as low levels of mosaicism, typically below 15% to 20%, cannot be detected by array analysis.

Chromosome analysis can be performed on numerous types of dividing tissue and can help to render diagnoses for constitutional (germ line) chromosome conditions (i.e., trisomy 21), as well as acquired (somatic) chromosome abnormalities associated with different types of cancer. Chromosome analysis requires living, dividing cells which are arrested in metaphase by using a substance that inhibits spindle fiber formation during mitosis (e.g., Colcemid, Velban). Typically, leukemias and lymphomas are studied by preparing short-term cultures (direct, 24-, 48-, or 72-hour) grown in suspension. The cells are then “harvested” using a hypotonic solution (sodium chloride or sodium citrate) and fixed using a mixture of methanol and acetic acid. Slides are made by dropping the cell suspension on the slides and drying them. The slides are aged and then banded using trypsin (or pepsin) and Giemsa (or Wright’s or Leischman’s) stain. This produces the G-banding pattern widely used to recognize and identify chromosomes and their abnormalities (Fig. 3.4). Chromosome analysis is performed by individuals who have been trained to recognize the banding patterns of each individual chromosome pair, chromosomes #1 through #22 and the X and Y sex chromosomes. Banded metaphases are identified and analyzed using a light microscope equipped with high resolution objectives (typically 10× oculars, with 63× or 100× objectives—enlarged up to 1000× their normal size). Images are then acquired using a CCD (charged coupled device) camera, and proprietary software is used to create the karyotype. A karyotype is the chromosomal makeup of a particular cell or individual. The software enables a technologist to create the digital karyotype, but the software/computer cannot completely recognize nor interpret the karyotype; this must be done interactively by the trained technologist. Chromosome analysis of leukemias and lymphomas requires complete analysis of a minimum 20 metaphase cells if possible.¹⁶ This is not a random process. Technologists (or automated imaging systems) scan the slides looking for abnormal metaphases. Often, there are normal metaphases that are part of the milieu. These are typically avoided, if there are abnormal metaphases present. All cells are completely analyzed—matched band-by-band, chromosomeby-chromosome pair, looking for any inconsistencies or abnormalities, be they structural or numerical. Clonal abnormalities are described and documented. Clonal anomalies are defined as two or more cells with the same structural abnormality or same extra chromosome, while loss of a chromosome must be observed in three or more cells in order to be considered clonal.¹⁷ Karyotypes are then created as both an analytical and a documentary tool and the chromosome diagnosis rendered.

Samples for chromosome analysis—fresh bone marrow, bone core biopsy, or peripheral blood—are transported at room temperature in either a sodium heparinized green-topped tube or in transport media with sodium heparin added to prevent clotting. These samples should be received within 24 hours of collection, if possible. Some bone marrow samples are particularly finicky and the abnormal clonal cells are fragile (e.g., acute lymphocytic leukemia). Lymphomas are minced and placed into short-term suspension culture.

It is important for the hematopathologist to have a basic understanding of cytogenetic nomenclature, as he/she is often asked to incorporate this data into an integrated or comprehensive report including all clinical laboratory analytic data on individuals with hematologic malignancies (i.e., flow cytometry, molecular and cytogenetic analytic data). The International System of Cytogenetic Nomenclature (ISCN)¹⁸ is the accepted method of describing the karyotype of an individual or tumor. There are very specific rules for how this information is presented. This is the internationally accepted cytogenetic language that, using alpha/numeric/symbolic string text allows one laboratory to describe what was observed in the karyotype and another laboratory to understand what that means. Every few years this system of nomenclature is updated. The most recent update was in 2009. ISCN first came into existence in 1978; however, there were several conferences held from 1960 until then to codify the human karyotype, with banded ideograms first introduced in 1971. An ideogram is a scientific representation of the light and dark bands, sub-bands and sub-sub-bands observed by metaphase chromosome analysis. Each chromosome has its own particular set of recognized bands, which allows it to be identified as such (Fig. 3.5). For instance, all human chromosome #1’s look very similar to one another, having the same pattern of light and dark bands, with the exception of a known variant region near the centromere. Chromosomes are divided into short arm and long arm by the centromere or primary constriction, which mediates attachment to the spindle fiber apparatus in mitosis. Bands in the short arm are labeled “p,” while bands in the long arm are labeled “q.” Each chromosome arm has landmark bands which demarcate the regions of the chromosome arm (this is the first number indicated after the p or q designation). These regions are then divided into bands, and possibly sub-bands or sub-sub-bands. Bands are numbered in increasing order starting at the centromere and proceeding toward the end of the chromosome arm (or telomere). The total number of chromosomes observed is stated first, with the sex chromosome designation given following a comma. There are normally no spaces between the numbers, letters, and punctuations that make up the karyotype designation. As an example, a female patient with the Philadelphia chromosome would have a karyotype written as “46,XX,t(9;22)(q34;q11.2)[18]/46,XX[2],” meaning that she has the Ph or t(9;22) in 18 of her metaphases (18 in []), a slash designating a second normal cell line with 46,XX (or normal chromosomes) in two metaphases (2 in []). The breakpoint in chromosome #9 is at band 9q34 (long arm or q arm, region 3, band 4 or band three four, not thirty-four) and the breakpoint in chromosome #22 is at sub-band 22q11.2 (long arm or q arm, region 1, band 1, sub-band .2 or band one one point two, not eleven point two). There are rules as well for describing both interphase and metaphase FISH (Table 3.2).

In situ hybridization was first described by Gall and Pardue in 1969,¹⁹ when they hybridized radioactively labeled probes to highly repetitive sequences in mouse and Drosophila. In 1981 Harper and Saunders²⁰ used a similar technique using tritiated (³H) nucleotides to label probes and autoradiographic methods to map human genes. Also in 1981, Langer et al.²¹ introduced biotinlabeled probes for gene mapping purposes, which then could be detected with streptavidin conjugated antibodies which had been fluorescently tagged. In 1988, Pinkel et al.²² described chromosome painting probes, while Kallioniemi et al. in 1992²³ introduced CGH using metaphase chromosomes as the interrogator.