The Principles of Clinical Cytogenetics - Extra Materials - Springer

100 Michael Watson and Steven Gersen
Analysis Protocols
An analysis protocol must identify the general requirements for each specimen type. The protocol
should identify normal parameters and normal variants and should distinguish between true abnormality
and artifact. The number of cells from which chromosomes are to be counted, sex chromosomes
examined, and bands analyzed in detail must be clearly stated, including whether each type of
examination is to occur at the microscope, on an image, or via a karyotype. A protocol should set
standards for the selection of suitable metaphase spreads, as well as the number of cultures (and
colonies, when applicable) from which cells should be examined. When an abnormality is detected,
the appropriate steps to take should be specified. Other items, such as an appropriate banding resolution
level, maximum allowable number of overlapping chromosomes, random chromosome loss, and
dealing with metaphases in close proximity, might also be included.
A protocol should identify the procedures used to document each metaphase, as well as the data to
be recorded on a microscope analysis worksheet, requirements for imaging, the number of cells to be
karyotyped, the number of individuals who should take part in performing the analysis, and the individual
who should verify the results.
Finally, a protocol should establish the policies for the storage of microscope slides, both during
analysis and once analysis has been completed.
Personnel Requirements
In addition to understanding specimen analysis requirements, it is important to identify personal
criteria to be met for and by individuals performing the analysis. The experience level, credentials,
and workload of each technologist are important considerations and the laboratory must be appropriately
staffed to allow for complete, accurate, and timely results of all samples received. When possible,
it is often recommended to split the analysis of a specimen between two individuals in some
way, increasing the potential for detection of a subtle abnormality.
Establishing goals for individuals or groups to meet, such as turnaround time and the number of
cases to be completed in a week, are important aspects of effective laboratory management. The
quality of analysis should not, however, be sacrificed in the attainment of these goals, and performance
monitors should include frequent statistical analysis of failure rates and percentage of abnormal
cases.
Microscopy
A significant part of quality microscopy lies within the training an individual receives on the
components of a microscope and their proper use. One of first subjects detailed in any protocol for
microscopy should, therefore, be training, including quality checks to identify equipment in need of
service or adjustment or individuals in need of additional training.
The selection of a microscope for analysis and documentation of results (image production) is also
a very important consideration. It is not unusual for a laboratory to have microscopes of various
quality grades, and individuals need to understand the limiting factors of any given scope. “Newer”
does not necessarily imply “better” in this instance, as many “veteran” microscopes can produce
excellent images, and it is often the resolution of the objective (lens), not extraneous accessories, that
is the key to image clarity. Also, keep in mind that good images are more likely to come from wellprepared
microscope slides. Controlling the slide preparation process and using a microscope with
the appropriate lenses and features will promote quality cytogenetic analysis and image documentation.
For additional details on microscopy, see Chapter 5.
General Analysis Requirements
Analysis requirements have evolved as a mix of “conventional wisdom” and statistically validated
needs for specific types of study. Professional organizations have developed consensus-based standards
for different types of analysis (ACMG, 2003) and regulatory bodies have typically used these