Steven L. Gersen and Martha B. Keagle (eds.)The Principles of Clinical Cytogenetics3rd ed. 201310.1007/978-1-4419-1688-4_6© Springer Science+Business Media New York 2013
6. Quality Control and Quality Assurance
(1)
Department of Allied Health Sciences, College of Agriculture and Natural Resources, University of Connecticut, 358 Mansfield Road, Unit 2101, Storrs, CT 06269, USA
Abstract
Upon receiving news that results of a chromosome analysis are abnormal (and even sometimes that they are normal), a patient will frequently ask: “How do I know that the lab didn’t make a mistake? How do I know that the sample they reported on was really mine? How can I be certain that this is all correct?” Most would be surprised to learn of the myriad of checks and balances that exist in clinical cytogenetics laboratories. Based on the consensus of professionals and on common sense, The American College of Medical Genetics (ACMG) Standards and Guidelines for Clinical Genetics Laboratories (ACMG 2009 Edition/Revised 01/2010; www.acmg.net/StaticContent/SGs/Section_E_2011.pdf) are the basis for oversight by regulatory agencies and are intended to prevent clinical and clerical errors. These comprise the area of laboratory medicine known as quality assurance and quality control (QA/QC). They are supplemented by both total quality management (TQM) and complete quality improvement (CQI) programs that seek to minimize errors when the laboratory interfaces with referring physicians and their patients.
Introduction
Upon receiving news that results of a chromosome analysis are abnormal (and even sometimes that they are normal), a patient will frequently ask: “How do I know that the lab didn’t make a mistake? How do I know that the sample they reported on was really mine? How can I be certain that this is all correct?” Most would be surprised to learn of the myriad of checks and balances that exist in clinical cytogenetics laboratories. Based on the consensus of professionals and on common sense, The American College of Medical Genetics (ACMG) Standards and Guidelines for Clinical Genetics Laboratories are the basis for oversight by regulatory agencies and are intended to prevent clinical and clerical errors [1]. These comprise the area of laboratory medicine known as quality assurance and quality control (QA/QC). They are supplemented by both total quality management (TQM) and complete quality improvement (CQI) programs that seek to minimize errors when the laboratory interfaces with referring physicians and their patients.
The nature of clinical cytogenetics is such that it includes both quantitative and qualitative components of tests. Some aspects are generic to practices in laboratories of any kind, while others are specific to cytogenetics laboratory tests.
A proper QA/QC program requires that policies for validation of protocols and reagents, training and credentials of individuals performing cytogenetic analysis, sample identification, safety for laboratory staff, and other compliance issues must all be in place. Laboratories are inspected periodically by various state and national entities, and most have institutional and internal regulations and guidelines as well.
There are many steps that occur between obtaining a specimen for chromosome analysis and the generation of a final clinical report. After collection of the specimen itself, accessioning, culturing, harvesting, slide preparation and staining (probe hybridization for fluorescence in situ hybridization [FISH]), microscopic analysis, electronic imaging, karyogram production, creation of a final report, and actual reporting of results are the path that specimens follow as they progress into and out of the cytogenetics laboratory. During this process, many variables can subject a specimen or data to a variety of conditions that must be managed for a proper diagnosis to ultimately be reached.
Central to any QA/QC program is the laboratory’s standard operating procedure (SOP) manual. This often formidable document contains the policies and procedures that must be followed in order for the laboratory to perform chromosome analysis. It includes requirements of physical space and mechanical systems, specimen requirements and collection procedures, transport requirements, personnel experience and credentials, and safety and protection for personnel. It includes sections on training and compliance with the various regulatory agencies that monitor and inspect laboratories, and, finally, it may contain a section pertaining to quality assurance and quality control. The majority of these issues pertain to the analytic component of testing.
With the rapid growth of knowledge and expansion of genetic testing, the laboratory has become increasingly involved in ensuring that the pre-analytic and post-analytic aspects of testing are also designed to ensure the appropriate use of tests and their results. These commonly include issues of analytical test validation, documentation of clinical validity, interpretation of test results, and educational materials that allow the laboratories’ clients to interface with it. These aspects are commonly encompassed in a complete quality improvement program.
Entire books could be written that address each of these issues in detail; entire chapters could be devoted to labels alone! Such detail is beyond the scope of this book, however. This chapter will provide an overview of the ways in which laboratories deal with many of these steps in order to ensure proper patient care.
Pre-analytical Testing Components
Before a test specimen arrives in the laboratory, there are a number of things that must be done correctly to ensure that an accurate and useful test result is provided. Laboratories often develop and provide materials to their clients 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.
Test Validation
Prior to initiating testing, there should be evidence of clinical validation of the test. This may be done by the laboratory developing the test or may be apparent from the scientific literature and merely documented. With the advent of the 1992 modifications to the 1988 Clinical Laboratory Improvement Amendments (“CLIA ‘88”) regulations, laboratories are required to validate all tests being introduced into service whether they were newly developed or long used in other laboratories [2]. Further, all new tests must be revalidated every 6 months. Approaches to validation vary for quantitative versus qualitative tests. Classical concepts such as sensitivity (the ability to detect a target when it is present) and specificity (the ability to not detect a target when it is not present) are common measures of analytical validity for quantitative tests. These are most often applied to FISH (particularly when interphase based) and microarray tests, (see Chaps. 17 and 18) but also are important when mosaicism is under consideration. Requirements for validation may vary with the regulatory status of a product. When a test is approved by the US Food and Drug Administration (FDA), the laboratory is expected to demonstrate that the test operates within the performance characteristics described by the manufacturer. When tests are not FDA approved or have been modified, the laboratory is expected to demonstrate their validity independently. For the more qualitative classical chromosome analysis, laboratories commonly validate their ability to process particular specimen types, to perform particular tests, or to detect a particular abnormality by testing samples from individuals with those abnormalities.
Specimen Submission
Specimens are almost always collected by individuals who rely upon the laboratory to provide a requisition form and instructions for specimen collection and transport. Thus, quality assurance and quality control begins by interactions with the health-care providers who will collect and submit specimens for chromosome analysis.
Collection Protocol
A collection protocol from the cytogenetics laboratory is of critical importance, as it establishes the collection guidelines for individuals who are not intimately familiar with the operating procedures of the laboratory. A collection protocol should include:
Ideal volume of specimen for collection.
Suitable transport containers, anticoagulants, or media.
Transport temperature and the maximum permissible transport time to ensure optimum specimen growth.
Confirmation of the identification of the patient from whom the specimen was collected.
Specimen container labeling and requisition form requirements.
Laboratory hours, phone numbers, contact individuals, and after-hours procedures.
Once established, it is important to keep copies of this protocol anywhere a specimen might be collected, including a hospital’s general laboratory, departmental clinics and operating room suites, and outpatient clinics and referring physician’s offices. It is also a good idea to routinely discuss collection protocols with the appropriate individuals, especially those who do not frequently submit samples to the laboratory. Regular interaction helps promote a complete understanding of collection requirements, as well as general expectations for samples submitted for cytogenetic analysis. It also provides an opportunity to discuss questions, concerns, or suggested improvements of collection or submission procedures.
Specimen Labeling and Requisition Forms
Accurate specimen identification is one of the most important policies to implement. Specimen labels should include at least two sources of identification, such as patient name, date of birth, or a unique patient-specific number, for proper identification in the event of a labeling error.
The requisition form is equally important, as it supplies the laboratory with the patient and clinical data associated with the specimen. When Medicare is to be billed for laboratory tests and the physician believes that a portion of the laboratory charges may not be covered, the requisition (or an accompanying document) must also include an advanced beneficiary notification (ABN), which informs the patient that he or she will be billed should Medicare deny payment. Certain states or other regulatory agencies also require that informed patient consent be part of, or accompany, the requisition form.
For obvious reasons, it is desirable to have a properly completed requisition (paper or electronic) accompany each specimen submitted to the laboratory, but it is also important for the laboratory to develop a policy for dealing with specimens that are not accompanied by a requisition, or for requisitions that have not been filled out completely. Of special importance are those requests for chromosome and/or FISH analysis that are made verbally with the laboratory. In these instances, it is important for the laboratory to obtain written or electronic authorization for the study. The provision of sufficient clinical information to ensure that appropriate tests and analyses have been requested is a valuable cross-check.
Rejection Criteria
It is very important for individuals to clearly understand the minimum requirements for submission of a specimen for chromosome analysis, FISH, or arrays, and what circumstances would prevent a laboratory from performing analysis. The collection protocol and requisition forms should clearly state these requirements. Although extremely rare, circumstances can arise that prevent a laboratory from accepting a specimen for analysis.
In the event of a problem with a sample, the laboratory should make immediate contact with the individual submitting the specimen, either to obtain clarification of the specimen identity or to discuss potential difficulties in obtaining a result. In most instances, both parties will elect to proceed, knowing that the success of the analysis may be impacted. In some instances, the problems are insurmountable, and a repeat sample is needed. When this occurs, it is a requirement for the lab to carefully document the reason for rejection or failure, as well as disposition of the specimen in the patient report and appropriate log.
Analytical Testing Components
The analytical phase of testing includes the actual processing and analysis of the specimen. Although specimen accessioning are often considered pre-analytical, it is included here because labeling and tracking of specimens through a test is among the most common causes of error in clinical laboratory testing. This phase usually ends when a laboratory test result is apparent.
Specimen Accessioning
Once a specimen has been received, an accession process is used to log it into the laboratory and to prepare it for analysis. During this time an accession and/or laboratory number is assigned to a specimen, relevant patient and clinical data are entered into a logbook and/or database, and the culture and analysis requirements for the studies requested are identified.
Assessing the Condition of the Specimen and Requisition
After receipt of the specimens in the laboratory, the individual responsible for accessioning specimens must check the sample and requisition for the appropriate labels, transport reagents (medium, anticoagulants, etc.), specimen condition (color, clotted, adequate sample size, transport temperature, etc.), and date of collection. When a problem is detected, the individual should follow the laboratory procedure for informing the “submitter” of the specimen and take appropriate actions. Problems with the specimen and action taken might also be documented.
Accession Numbers and Patient Database
It is important to assign a unique identifier to each specimen as it enters the laboratory, distinguishing it from other specimens, as well as from a patient’s previous studies. The lab number, patient data, and clinical information are then often transferred into a logbook or electronic database, creating a patient record that can be tracked and cross-referenced against previous and/or future studies. In addition, other data can be entered into a database record as a study progresses, allowing the laboratory to track:
Culture conditions
Results
Turn around times (TATs)
Dates of specimen receipt, processing, and report
Individual(s) issuing reports
Cytogenetic results versus the findings of patients with similar histories or abnormalities (interpretation of results)
Culture failures, labeling errors, transcription errors, misdiagnoses, and actions taken
Incidence of submission problems
Electronic databases need to be managed within the laboratory to ensure the accuracy of the data as well as patient confidentiality.
Once a specimen has been logged into the cytogenetics laboratory, it must be prepared for cell culture. This may include notification of appropriate individuals of its receipt, creation of culture records and container labels, and creation of a patient folder or file for paper records. If the sample is not set up immediately, it needs to be stored under appropriate conditions.
There should be a system for identifying specimens that require special handling such as an accelerated study, a preliminary report, or a completion by a certain date to meet anticipated turn around times. These requirements should be clearly indicated on all appropriate forms and/or computer fields, and all individuals involved with the study should be notified.
Specimen Labels
The accuracy of any laboratory result requires correct specimen labeling. After the initial accessioning process, a number of items need to be labeled, including a culture worksheet; culture flasks, tubes, or Petri dishes; microscope slides; a microscope analysis worksheet; metaphase prints; karyograms; FISH images; and reports. The laboratory labeling policy should allow patient identification to be cross-checked in the event of a labeling error.
Specimen Culture, Harvesting, Slide Preparation, and Staining
All equipment and supplies used for culture and harvesting of cells, preparation of slides, and banding and staining of chromosomes should be monitored in order to provide high-quality analyses.
Cell Culture
Whenever possible, duplicate or independently established cultures should be created for all samples, and these should be placed in separate incubators, each equipped with its own power, CO2 source (if utilized), and emergency alarm. A backup procedure must also be created that ensures that cultures will be maintained in the event of a power (emergency generator) or CO2 (automatic gas tank supply change) failure.
Precautions to prevent contamination should be taken when a specimen is added to culture medium, a culture is transferred between containers, or reagents are added to a specimen culture. Working with specimens within the area of a laboratory designated for biological hazardous materials and using sterile technique in laminar flow hoods will greatly reduce the risk of bacterial contamination of the specimen and exposure of staff to biohazards. In addition, using latex gloves, cleaning work surfaces with alcohol before and after use, and exposing container openings, pipettes, or other measuring devices to a flame will reduce the likelihood of contamination.
Working with one specimen at a time and disposing of all used pipettes or containers that come into contact with a specimen (before moving onto the next) will greatly reduce the likelihood of cross contamination or improper identification. It is also important to note that the transfer of reagents into a culture should be performed using a fresh pipette when there is any risk of contact with a specimen or specimen aerosol.
Culture Protocols
Cell and tissue culture begins with a protocol that outlines tested and reproducible steps to produce cells and metaphase chromosomes for analysis. The quality control of new reagent lots and changes in established protocols should be completed prior to their use with patient specimens. For critical reagents that may be of variable quality from manufacturer to manufacturer or from lot to lot (such as serum), prepurchase testing of multiple lots can ensure that the highest-quality reagent is available to the laboratory. The methods of QC testing should be appropriate to the reagent and method being tested and may include parallel testing of the current validated reagents/devices against the new lots of reagents/devices using nonclinical control specimens or reference materials. It is also important to track the history of protocol modifications, allowing a comparison of past culture techniques and successes. The format of a culture protocol should comply with the requirements of the agency used for laboratory accreditation.
Equipment Maintenance
Consistency and reliability of laboratory procedures cannot be accomplished without well-maintained equipment, and there are many regulations that reflect this.
Refrigerators, freezers, and water baths should be closely monitored daily for temperature and cleaned following regular schedules. Centrifuges should be monitored for accurate speed semiannually. Laminar flow hoods should be cleaned before and after use and be equipped with an antibacterial light or cover to prevent contamination during periods of nonuse. Biological safety cabinets also should be checked and certified annually for airflow and bacterial contamination, and pH meters should be cleaned and calibrated regularly. Balances should be kept clean of laboratory reagents and calibrated regularly to ensure proper weight measurements. Ovens need to be monitored daily for temperature. Trays for slide preparation and storage should be kept clean to reduce chemical contamination of staining reagents.
Incubator temperature and gas (CO2) concentration should be monitored continuously and documented daily. Incubators should be on a regular cleaning schedule and, as discussed earlier, should also be equipped with separate power and gas sources, as well as emergency alarms. Incubator gas and power supplies should also have a backup in the event of a failure, and the laboratory should maintain an emergency plan in the event of complete incubator failure. Records of equipment monitoring and maintenance should be documented in an equipment log.
Automated harvesting procedures are used by many cytogenetics laboratories as a way of increasing laboratory productivity and improving consistency (see Chap. 7). However, automation does not imply “carefree.” Laboratories that utilize such technology must strictly follow the manufacturer’s recommended operational guidelines and closely monitor the equipment for acceptable performance. A procedure for the use of automated equipment that details the procedural steps for operation, appropriate reagents, calibration and cleaning requirements, and preventive maintenance must be prepared. It is also important for individuals operating the equipment to receive proper training before using it on clinical specimens.
Harvesting, Slide Making, and Staining
The transition from cell/tissue culture to microscopically analyzable chromosomes is achieved by harvesting the dividing cells (which involves mitotic arrest, osmotic swelling of cell membranes, and fixation), spreading of the chromosomes on microscope slides, and staining the chromosomes with one of various methods which produce an appropriate banding pattern (see Chap. 4). Each of these steps must be optimized to facilitate correct diagnoses.
Protocols
After cells have been successfully cultured, the techniques of harvesting, slide making, and banding/staining will determine the ultimate quality of the metaphase chromosomes available for analysis. Following validated protocols is very important for these procedures, but frequent modifications may be required to address changing laboratory conditions. It is important to note that these procedures can be especially sensitive to individual technique, particularly fixation and slide making, and that mastery of these skills requires individuals to observe and document minor variations in procedure or laboratory conditions that improve or detract from chromosome morphology.
New protocols, procedural changes, introduction of new reagents, reagent concentrations, microscope slides, etc. must be validated under controlled conditions. The method of validation should be one that is appropriate for the reagent or technique being tested and may include parallel testing of current versus new, testing on nonclinical control specimens, or direct analysis using reference materials. It is also important to track the history of harvesting, slide preparation, and staining protocol modifications in order to allow a comparison of past techniques to present successes. Documentation of proactive and reactive factors from these procedures is important to ensure quality metaphase chromosomes, as well as to identify and track problems that reduce specimen quality.
Slide Preparation
The chromosomes present in harvested metaphases must be spread apart so that they can be microscopically analyzed. They must lie flat so that staining is uniform and all chromosomes are in a single plane of focus, and they must be aged (literally or artificially) in order for most banding and staining procedures to work properly.
Even when all else has gone well with the tissue culture and harvesting procedures, poor slide preparation can result in scarce, poorly spread, or improperly aged metaphase spreads for staining and microscope analysis. The following variables should be considered:
Harvesting method (centrifuge tubes vs. in situ processing) (see Chap. 4)
The humidity and temperature of the laboratory or drying chamber utilized (see Chap. 7)
The number of fixations and the method of fixing the specimen
The slide temperature
Wet or dry slides? How much water?
The angle of the slide during specimen application
The method of applying the specimen
The method of drying the slide
The slide-aging technique
Each of these factors can significantly contribute to the success of slide preparation. As these can be variable from day to day and between individuals, close observation and documentation of technique may allow the highest proficiency of these skills.
Banding and Staining
While slide preparation and aging are important factors contributing to the lab’s ability to successfully stain a specimen, adjustments to solution concentrations, the time slides are left in the staining solution, etc., can also influence successful staining of cytogenetic samples. Careful preparation of reagents and documentation of adjustments made to staining procedures help the laboratory personnel to refine their techniques.
The shelf life and storage conditions of banding and staining reagents are important considerations and should also be documented in a staining log. As reagents arrive in the laboratory, lot numbers should be recorded and compared with previous lots used. Reagent containers should be labeled with the reagent name, quantity, concentration, storage requirements, date received, and expiration date. Reagents that require refrigeration should have minimum and maximum permissible temperatures documented, and these should not be exceeded. Existing supplies of reagents should be rotated so that they are depleted before new supplies are used.
Although good specimen staining is critical for optimal microscope analysis, it is also necessary to consider the microscope on which a specimen will be analyzed and the staining requirements of the recording medium. When a laboratory has a variety of microscopes, each may have a light source, contrast or interference filters, objectives, or other lenses that produce images with a unique set of visual characteristics. Additional variables, such as excitation and barrier filters, are introduced with the use fluorescence microscopy, and features such as the numerical aperture of lenses or bulb intensity may be critical (see Chap.5). Individual preference is also an important factor in identifying a staining intensity that is well suited for microscope analysis.
When accessing the quality of banding in G-banded images, it is important to identify staining intensities that produce:
Chromosome pale ends that contrast well against background areas
A wide range in mid-gray intensity
Dark bands in close proximity that appear as distinct bands
Comparing the requirements of the individual performing the microscope analysis against the requirements of the recording media and documentation of ideal conditions in a staining log will help laboratories gain control of the many variables of a staining procedure.
Specimen Analysis
Any chromosome analysis begins by identifying the specific requirements for the specimen type being examined. Following this, the basic steps are: the microscope analysis itself (location of metaphase spreads suitable for analysis, counting the chromosomes and determining the sex chromosome complement, and analysis of the band pattern of the individual chromosomes), imaging of the metaphase spreads, preparation of karyograms, and documentation and reporting of results. The procedure begins with a protocol that must be accessible and thoroughly understood by all individuals performing chromosome analysis. An analogous process is required for FISH studies.
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 and the sex chromosome complement identified and analyzed in detail (band-for-band) must be clearly stated, including whether each type of examination is to occur at the microscope, on an image, or via a karyogram. 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 things, 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 create karyograms from, 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 and retention of images, both during analysis and once analysis has been completed.
Personnel Requirements
The experience level, credentials, and workload of each technologist are all 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 turn around time and the number of cases to be completed in a week, is an important aspect 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 in the training an individual receives on the components of a microscope and their proper use. Any protocol for microscopy should therefore include training of personnel in the use of microscopes, quality checks to identify equipment in need of service or adjustment, and identification of individuals in need of additional training.
The selection of microscopes 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 users need to understand the limiting factors of any given scope. “Newer” does not necessarily imply “better,” and many “veteran” microscopes produce excellent images. It is often the resolution of the objective (lens), not extraneous accessories, that is the key to image clarity. Also, good images are more likely to come from well-prepared 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 Chap. 5.
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