Dna Synthesis

The synthesis of a new molecule of DNA is called replication. This process requires many enzymes and cofactors. The first step of the process involves breakage of the hydrogen bonds that hold the DNA strands together. DNA helicases and single-strand binding proteins work to separate the strands and keep the DNA exposed at many points along the length of the helix during replication. The area of DNA at the active region of separation is a Y-shaped structure referred to as a replication fork....

QBanding Quinacrine Banding

Q-banding is a fluorescent technique and was the first banding method developed for human chromosomes. Certain fluorochromes, such as quinacrine dihydrochloride, will bind to DNA and produce Fig. 2. G-Banding (Giemsa banding). Note the light and dark bands along the length of each chromosome. (Image provided by Alma Ganezer.) Fig. 2. G-Banding (Giemsa banding). Note the light and dark bands along the length of each chromosome. (Image provided by Alma Ganezer.) distinct banding patterns of...

Robotic Harvesters

As described in Chapter 4, harvesting of mitotic cells for cytogenetic analysis involves exposing the cells to a series of reagents that separate the chromosomes, fix them, and prepare them for the banding and staining process. This traditionally involves pelleting the cells by centrifugation between steps, in order to aspirate one reagent and add another, a process that, by its very nature, is not amenable to any form of automation. However, the in situ method of culture and harvest of...

Polyploidy

Polyploidies are numerical chromosome abnormalities with changes in the number of complete sets of chromosomes. They are usually incompatible with fetal survival and are extremely rare in liveborns. The chromosome number in triploidy is 3n 69 (see Fig. 9). It is estimated to occur in approximately 1 of all human conceptions and is found in 17-18 of all chromosomally abnormal abortuses (175,176). Only very rarely do triploid conceptuses survive to term. Two distinct phenotypes have been...

Brightfield Microscopy

The brightfield microscope is arguably the most important piece of equipment in the cytogenetics laboratory. Knowledge of its component parts and their proper use is fundamental. The basic components of a brightfield microscope are illustrated in Fig. 1. The Transmitted Light Source and Power Supply A transmitted light source is found in the base of a microscope, often in the rear, but occasionally in the front of the scope. The bulb housings of many microscopes will automatically center the...

G11 Banding Giemsa at pH

This technique specifically stains the pericentromeric regions of all chromosomes, the heterochro-matin regions of chromosomes 1, 9, and 16 and the distal Yq, and the satellites of the acrocentric chromosomes. An alkaline treatment of the chromosomes causes loss of the Giemsa binding sites. Optimal results are achieved at pH 11.6. At this high alkaline pH, only the azure component of Giemsa binds with the majority of the chromosomes, staining them light blue. The eosin component of Giemsa binds...

Human Chromosomes

Of the 46 chromosomes in a normal human somatic cell, 44 are autosomes and 2 are sex chromosomes. The autosomes are designated as pairs 1-22. The numbers are assigned in descending order of the length, size, and centromere position of each chromosome pair. In a normal female the sex chromosomes are XX, and in a normal male, they are XY. Until the advent of certain specialized staining techniques, arbitrary identification of individual chromosome pairs was based on the size and position of the...

Numerical Abnormalities Of Chromosomes

The term numerical abnormality refers to gain or loss of chromosomes. As outlined above, all such abnormalities are presented in numerical order with the exception of the X and Y, which are always listed first. To designate an additional or a missing chromosome plus (+) and minus (-) signs are placed before the specific chromosome number. Thus, -7,+18 would mean a missing chromosome 7 and an extra chromosome 18. Note that these abnormalities are presented in numerical order, regardless of...

Drying Chambers

Again, as described in Chapter 4, the typical end product of the cytogenetic harvest is a centrifuge tube with fixed cells, both mitotic and nonmitotic. Spreading of chromosomes is achieved by placing Fig. 2. Multiprep robotic harvester. This device was designed specifically for cytogenetics laboratories, with enhancements such as automatic fixative mixing, integral fume extraction, multiple dispensing and aspiration probes to reduce the risk that blockage will ruin a harvest, and on-board...

Advantages of the In Situ Method over the Flask Method

The primary advantage of using the in situ method is that it provides information about the colony of origin of a cell. This is important when deciding whether an abnormality seen in some but not all cells represents true mosaicism (constitutional mosaicism) or an artifact of tissue culture (pseudo-mosaicism). True mosaicism is said to be present when there are multiple colonies from more than one culture with the same chromosomal abnormality. Pseudomosaicism is suggested if a single colony...

Culture Failure

As described in Chapter 4, the basic procedure for producing chromosomes for analysis from any tissue type requires living cells that can somehow be coaxed into active division. Without mitosis, there can be no chromosomes to process and examine. There are several possible reasons for cell culture failure The sample did not contain any living cells. In some cases, this is clinically not surprising it is frequently the case with products of conception obtained from fetal demise or in necrotic or...

Designation of the Number of Signals

When designating interphase ish results, the abbreviation nuc ish is followed by a space, the chromosome band to which the probe is mapped, and then, in parentheses, by the GDB locus designation, a multiplication sign, and the number of signals detected One copy of the X centromere probe DXZ1 is detected, as is one copy of the Y chromosome probe DYZ3. This implies the presence of one X and one Y chromosome, suggesting an XY sex chromosome complement. No other information is presented, and so...

Cell Harvest

After the cell cultures have grown for the appropriate period of time and there is a sufficient number of dividing cells, the cells are harvested. Harvest is the procedure of collecting the dividing cells at metaphase, their subsequent hypotonic treatment and fixation, and the placement of the chromosomes on glass slides so they can be stained and microscopically examined. The basic steps of cell harvest are the same for all specimen types, with minor variation. An example is shown in Fig. 1. A...

Chromosome Analysis

Selection of the correct specimen for chromosome analysis and additional tests is not always straightforward, and the submission of an inappropriate sample to the laboratory can create frustration for both patient and clinician. This was not always as complex an issue as it is today. In the 1970s, prenatal diagnosis involved an amniotic fluid specimen, often obtained at exactly 17 weeks of gestation, for chromosome analysis and a-fetoprotein (AFP) testing. Other tests were available, but rare....

Fluorescence And Other In Situ Hybridization

Recent advances in human cytogenetics include the development and application of in situ hybridization (ish) protocols to incorporate and bind labeled, cloned DNA or RNA sequences to cytological preparations. These techniques facilitate the localization of specific genes and DNA segments onto specific chromosomes, ordering the position and orientation of adjacent genes along a specific chromosome, identification of microduplications or microdeletions of loci that lie beyond the resolution of...

Instrumentation For Fish

Metaphase Spreads

Although FISH (see Chapter 17) represents one of the most exciting and clinically significant developments of the last decade, most of the steps involved in preparing samples for analysis are unremarkable and often repetitive and, therefore, lend themselves to automation. When one considers the enormous increase in FISH sample volume most cytogenetics laboratories are experiencing, any device that can reduce the labor component of the process becomes indispensable. Fig. 5. VP 2000 processor....

Structural Chromosome Abnormalities

This category of abnormalities includes several subclasses that will be discussed under separate headings. Again, as previously stated, all chromosomes involved in abnormalities are designated in numerical order, except for the X and Y, which are listed first. When designating an abnormality that is limited to a single chromosome, the abbreviation for that abnormality is used, followed by the chromosome number in parentheses e.g., r(X), del(2), ins(4), dup(5) . If two or more chromosomes are...

Autosomal Monosomies

Triploid Karyotype

As noted in the Introduction, autosomal monosomies are extremely rare in either liveborns or abortuses, reflecting the severity of the genetic imbalance resulting from the loss of an entire chromosome. The only monosomies that have been reported are monosomy 21, mosaic monosomy 22, and a single case reported in an abstract of a possible mosaic monosomy 20 in a 31 2 year old boy with atypical speech language delay, behavior problems, microcephaly, and patchy hypopigmentation of the skin (161)....

Mainline Stemline Sideline and Clonal Evolution

These terms can be confusing and are often misunderstood. The mainline (ml) is the term used to describe the most common clone (i.e., the one represented by the most cells). This is a quantitative issue only. It does not necessarily indicate the most basic clone in tumor progression, which is referred to as the stemline (sl). Clones that evolve from the stemline are referred to as sidelines (sdl) When more than one clone is present but no clear clonal progression is evident, the mainline is...

Culture Initiation

All specimens for chromosome preparation are grown and maintained in an aqueous growth medium. Some media are formulated for specific cell types (e.g., AmnioMax or Chang for amniocytes, Giant Cell Tumor Conditioned Medium for malignancies, PANDIS for breast tumors, etc.), whereas others are appropriate for a broad spectrum of cell types (e.g., RPMI 1640, MEM). All culture media are balanced salt solutions with a variety of additives, including salts, glucose, and a buffering system to maintain...

Preanalytical Testing Components

Before a test specimen arrives in the laboratory, there are a number of steps that must be done correctly to ensure that an accurate and useful test result is provided. Laboratories often develop and provide to their clients materials to guide them in understanding when to test, what to test, and how to order tests. Often considered outside of the day-to-day functioning of the laboratory, these are important to ensuring safe and effective testing. Prior to initiating testing, there should be...

Interpreting A Karyotype Description

Receiving a cytogenetic report that contains the description of a patient's karyotype can create confusion, particularly if complex rearrangements or multiple clones are present. Interpretation of the description of a karyotype can be facilitated by breaking this description into its component parts. First, determine whether more than one cell line is present. This will happen if constitutionally the patient is a mosaic or a chimera as is often the case with acquired cytogenetic abnormalities,...

United States

Accreditation, Inspections and External Proficiency Testing Under the Clinical Laboratory Improvement Amendment of 1988 (CLIA '88), every laboratory performing moderate- to high-complexity testing (i.e., every cytogenetics laboratory) must enroll in Health and Human Services approved external inspection and testing programs. In fact, virtually all clinical laboratories in the United States do so under the auspices of the CLIA-deemed program of the College of American Pathologists (CAP). This...

Trisomy

Big Lips Genetics

Trisomy 8 47,XX or XY,+8 was first reported by Grouchy et al. in 1971 (93). It is rare, with an unknown incidence. More than 100 cases have been reported in the literature (94-97), most of them mosaics 47,+8 46 . The male-to-female ratio is 2-3 1. Growth and the degree of mental deficiency are variable. Mild to severe retardation is seen, and a proportion of patients have normal IQs. Craniofacial dysmorphism (see Fig. 8) includes prominent forehead, deep-set eyes, strabismus, broad nasal...

Partial Autosomal Aneuploidies

Pallister Killian Syndrome

Partial duplication deletion as a result of structural rearrangement is discussed in Chapter 9. Only those partial autosomal aneuploidies that result from the presence of a supernumerary chromosome will be presented in this chapter. Tetrasomy 5p 47,XX or XY, i 5 p10 resulting from the presence of a supernumerary isochromosome for the entire short arm of chromosome 5 is rare and has been reported in only three liveborns, all of whom are mosaics with both normal and abnormal cell lines 205 . The...