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. 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.
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.
Once the appropriate number of mitotic cells has been examined and analyzed, a representative sample must be selected for imaging and ultimate preparation of karyotypes. This will involve either traditional photography and manual arrangement of chromosomes (becoming increasingly rare) or computerized image capture and automated karyotyping (see Chapter 7). Many laboratories also image additional cells, to be included as references in the patient chart. Ultimately, summary information (patient karyotype, banding resolution, number of cells examined, analyzed and imaged, etc.) is recorded in the patient's file and is used, either manually or via computer, in the clinical report.
The final steps of the process typically involve a clerical review of all relevant clinical, technical, and clerical data, examination of the patient's chart and karyotypes by the laboratory director (often preceded by the supervisor and/or other senior laboratory personnel), and generation of the formal clinical report. In addition to the appropriate physician and patient demographic information, this should include the number of metaphases that were examined microscopically, the banding resolution obtained for the specimen, the number of cells analyzed in detail, the number of karyotypes prepared, the patient's karyotype, and a clinical interpretation of the results, including, where appropriate, recommendations for additional testing and/or genetic counseling.
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