The Principles of Clinical Cytogenetics - Extra Materials - Springer

78 Martha Keagle and Steven Gersen
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 α-fetoprotein (AFP) testing. Other tests were available, but rare. The cytogenetic contribution
to hematology/oncology essentially involved whether a bone marrow specimen was “positive or negative”
for the “Philadelphia chromosome.” Constitutional chromosome analysis from peripheral blood
implied that the patient had to be an adult or a child.
Today’s prenatal caregivers and their patients must chose among traditional amniocentesis, early
amniocentesis, chorionic villus sampling, or, sometimes, percutaneous umbilical blood sampling (see
Chaper 12). A decision concerning whether ploidy analysis via FISH is warranted must be made, and
acetylcholinesterase (AChE) is often a factor in the diagnosis of certain open fetal lesions, but AFP
and AChE cannot be performed on all sample types. Many disorders can be also diagnosed by biochemical
or molecular methods, and ethical dilemmas surround the potential to prenatally diagnose
late-onset disorders such as Huntington’s disease. Screening for increased potential or predisposition to
develop certain cancers or other diseases will create new moral and ethical pitfalls. Each of these
might ultimately affect the number of cells available for chromosome analysis, and all of these issues
can play a role in the timing and choice of sampling procedure.
Today, the cytogenetics laboratory provides indispensable information for the diagnosis, prognosis,
or monitoring of patients with a wide variety of hematological disorders and other neoplasms, using not
only bone marrow, but in some cases blood, lymph node biopsies or tumor tissue or aspirates. Treatment
decisions often rest on the results of a chromosome analysis, but some tissue types are only appropriate
under certain conditions, and an incorrect selection here can delay a vital diagnosis.
Instead of a child or an adult suspected of having a constitutional chromosome abnormality, a
blood sample, therefore, could also be from a patient with leukemia or a fetus. These must all be
handled differently, and the information they provide is unique in each circumstance.
Procedure
After all of the appropriate laboratory manipulations and staining procedures have been performed,
there are several steps involved in the clinical analysis of chromosomes. These begin with the microscope,
where selection of appropriate metaphases begins the process. Although technologists are
trained to recognize well-spread, high-quality cells under low-power magnification, they must also
remember to examine some poor quality metaphases when analyzing hematological samples, as these
often represent abnormal clones.
Under high power, the chromosome morphology and degree of banding (resolution) are evaluated.
If these are appropriate, the number of chromosomes is counted, and the sex chromosome constitution
is typically determined. The microscope stage coordinates of each metaphase are recorded, and
in many laboratories, an “identifier” of the cell is also noted. This is typically the position of one or
more chromosomes at some reference point(s) and serves to verify, should there be a need to relocate
a cell, that the correct metaphase has been found. Any other characteristics of the metaphase being
examined, such as a chromosome abnormality or quality of the banding and chromosome morphology
are also noted.
In the United States, certifying agencies such as the College of American Pathologists (CAP)
require that a minimum number of metaphases be examined for each type of specimen, barring technical
or clinical issues that can sometimes prevent this (see Chapter 6). There are also requirements
for a more detailed analysis (typically band by band) of a certain number of cells, as well as standards
for the number of metaphases from which karyotypes are prepared. Regulations notwithstanding, it is
clearly good laboratory practice to analyze every chromosome completely in several cells, and even
more important to check all chromosomes in certain situations, such as when analyzing cancer specimens.
Depending on the results obtained and/or initial diagnosis, additional cells might be examined
in order to correctly identify all cell lines present.