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    Cytogenetics is the branch of genetics that studies chromosome structure and function. Chromosomes (Greek: chromos = colored; soma = body) are present in all nucleated cells and contain DNA with associated acidic and basic proteins. Chromosomes undergo a cycle of condensation and decondensation throughout the cell cycle. Chromosomes are maximally decompacted during interphase and achieve maximum compaction during the metaphase stage of mitosis, just prior to the separation of sister chromatids [Figures 1, 2]. The establishment of the correct diploid chromosome complement (karyotype) of 46 in man (Tjio and Levan, 1956) led to the discovery of major human constitutional chromosomal syndromes and later the observation of acquired chromosome abnormalities in hematologic malignancies and solid tumors. The 46 chromosomes in the normal human karyotype are arranged in decreasing order of size as 23 matching or homologous pairs in a karyogram [Figure 3], with 22 pairs of autosomes and the sex chromosomes (XX female, XY male). One of each pair of autosomes and the X are of maternal origin, while the Y and the remaining autosomes are of paternal origin. Each chromosome is composed of two chromatids joined by a centromere, which is the site of attachment of the spindle fibers. The spindle fibers draw the chromatids to opposite poles during cell division. The position of the centromere is constant for a given chromosome, with three subgroups [Figure 4]:

    Metacentric – centromere in the middle of the chromosome (chromosomes 1, 3, 16, 19, 20) Submetacentric – intermediate position of the centromere (chromosomes 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 17, 18, X) Acrocentric – centromere close to one end of the chromosome (chromosomes 13, 14, 15, 21, 22, Y)

    The centromere divides the chromosome into short (p) and long (q) arms. The telomeres “cap” the p and q arms and are important for structural integrity of the chromosome. Individual chromosomes are identified by their relative size, the position of the centromere and the banding pattern (see banding technique).


    It is important that representative tissue be sent to the laboratory for analysis. Only those cells involved in the neoplastic process will harbor the abnormalities being sought. Specimens should be collected under sterile conditions. Bone marrow aspirate, bone core and peripheral blood (if circulating blast percentage is higher than 10%) samples are routinely analyzed for the diagnosis of acute myeloid leukemia. Heparinized (sodium heparin) samples should be transported (at ambient temperature) to the laboratory in a timely manner (ideally within 24 hours) for tissue culture. Bone marrow smears and touch preparations of the bone marrow core biopsy can also be used for directed fluorescence in situ hybridization (FISH) analyses (see below). Tissue samples may also be analyzed in the setting of extramedullary leukemia (myeloid sarcoma, chloroma). Sample Set-up and Processing

    Conventional cytogenetic analysis requires cells to be actively in cell division. Direct and short- term (24-48 hours) unstimulated cell culture and mitotic arrest with a spindle inhibitor such as Colcemid is followed by treatment with hypotonic saline to facilitate cell membrane disruption and fixation with methanol/acetic acid to create permanent preparations that can be “dropped” onto slides for staining and microscopic analysis. Banding Technique

    Slide preparations are treated chemically (enzymatic digestion) and stained with a DNA-binding dye (Giemsa) to reveal chromosome specific patterns of light and dark bands that are microscopically visible (G-banding). G-band dark bands appear to contain relatively few active genes, are AT-rich and replicate late in S-phase. Light bands contain about 80% of active genes, are relatively GC-rich and replicate early in S-phase. Band Level and Identification

    An International System for Human Cytogenetic Nomenclature (ISCN 2016) describes a standardized numbering pattern for chromosome identification based on the banding pattern. This uniform system of nomenclature is the result of multiple consensus conferences that began in 1960 and continue today. A band is defined as a chromosomal area that is distinguishable from adjacent segments by appearing lighter or darker. A standardized numbering system for bands seen with G-banding is shown diagrammatically in the human idiogram [Figure 5]. This system permits accurate description of breakpoints in chromosomal rearrangements. Each chromosome in the ideogram is divided into a number of chromosome regions using the ends, centromere, and most prominent G-bands as landmarks. Regions or bands are numbered consecutively from the centromere outward along the chromosome arm. In designating a band, four items are required: the chromosome number, the arm symbol, the region number, and the band within that region. These items are given in consecutive order without spacing or punctuation. For example, 5q33 means chromosome 5, long arm, region 3, band 3.

  • In a cytogenetics report, the primary quality indicator is the number of observable bands. The banding level of resolution in hematologic malignancies is in the 300 to 400 band range. Conventional cytogenetic analysis provides a low DNA resolution scan of the entire genome: 100 genes/band, 9 x 106 base pairs/band and 9Mb band width are observed at the 350 band level.

    Number of Cells Counted/Analyzed

    To determine if abnormalities are present, a minimum of 20 cells (metaphase preparations) are analyzed. In each cell, the number of chromosomes is enumerated and each chromosome homolog is identified band for band. Computerized systems for image capture and analysis assist in karyogram preparation and allow for electronic storage of images, however, trained cytogenetic technologists are responsible for this individual cell analysis. A minimum of two karyograms are produced per case, with two additional karyograms per abnormal cell line. ISCN Results

    The ISCN provides specific guidelines for reporting cytogenetic results. In the karyotypic description, the first item recorded is the total number of chromosomes followed by a comma (,). The sex chromosome constitution is given next. Thus, a normal female karyotype is written as 46,XX. The normal male karyotype is 46,XY. Punctuation is very important in cytogenetic nomenclature. All chromosome numbers are followed by a comma and no spaces are used to separate chromosome number from sex chromosome content. To specify structurally altered chromosomes, single and three letter abbreviations are used. The number of the chromosome or chromosome involved in the rearrangement is specified within parentheses immediately following the symbol indicating the type of rearrangement. If two or more chromosomes are altered, a semicolon (;) is used to separate their designations. If one of the rearranged chromosomes is a sex chromosome, this is listed first; otherwise, the rule is that the lowest chromosome number is cited first. The breakpoints, given within the parentheses, are specified in the same order as the chromosomes involved, and a semicolon is again used to separate the breakpoints. Punctuation is never used in intrachromosomal rearrangements, that is, to separate breakpoints in the same chromosome. When reporting results, the number of cells analyzed is given in square brackets at the end of the karyotypic description. If multiple clonal populations are present, the karyotypic description of each clone is separated by a slant line (/). The internationally accepted operational definition states that a clone exists if two or more cells are found with the same structural abnormality or chromosomal gain. If the abnormality is a missing chromosome, the same change must be present in at least three cells analyzed. The stemline indicates the most basic clone in a cell population. All additional deviating clonal findings are termed sidelines.

    The modal number is the most common chromosome number in the population analyzed. The modal number is hypodiploid when the mode is less than 46 chromosomes, and hyperdiploid when the mode is greater than 46 chromosomes. Hypodiploidy and hyperdiploidy are both

  • examples of aneuploidy, where the incorrect number of chromosomes are present. Loss of a chromosome is described as monosomy. Three copies of a chromosome is trisomy, four copies is tetrasomy. Karyotypes with a normal chromosome number, but contain numerical and/or structural abnormalities, are described as pseudodiploid.

    A Glossary of Common ISCN Abbreviations/Punctuation Used to Describe Karyotypes

    add Indicates additional material of unknown origin attached to a chromosome region or a

    band. Latin, additio.


    ~ Approximate sign, indicates uncertainty in chromosome or band designation.  Arrow, meaning from  to [ ] Square brackets. Surrounds number of cells that constitute a clone. c A constitutional anomaly is indicated by the letter c after the abnormality designation.

    48,XX,+8,+21c Tumor cells with a constitutional trisomy 21 and an acquired trisomy 8. : Colon. Used to indicate a chromosome break. :: Double colon. Used to indicate a chromosome break and reunion. , Comma. Separates chromosome numbers, sex chromosomes and chromosome

    abnormalities in the karyotype. cp Composite karyotype. Contains all clonally recurring abnormalities in setting of