Liquid-delivery quality assurance in clinical laboratories: navigating regulations and standards for liquid-delivery verification.
Now, consider the consequences if this liquid-delivery instrumentation operates out of specification for one day. One week. One month. How much uncertainty can medical laboratories legally tolerate? And, even more importantly, how much uncertainty should medical laboratories tolerate?
While there are federal regulations mandating that laboratories follow liquid-delivery quality-control processes, each organization has the flexibility to make scientifically valid decisions about methods for and frequency of equipment calibration. Due to the ambiguity of current regulations and the difficulty in keeping up with technological advancements, regulations are incomplete; and a variety of different guides and standards have been brought forward. This article is intended to help readers navigate current regulations, standards, and guidance regarding sufficient calibration procedures, emphasizing the need for more stringent liquid-delivery quality assurance.
Liquid handling in the clinical laboratory
Medical laboratories routinely prepare samples for testing, producing results that are used to identify toxins, diagnose illnesses, and make decisions about patient care. Many applications in clinical laboratories are likely to include one or more liquid-handling processes, and errors in liquid delivery can result in the following scenarios:
* False-negative results: Dispensing samples in volumes inaccurate by even miniscule amounts can alter concentration and prevent the identification of disease or harmful substances in samples. These false negatives may result in improper diagnoses, the consequences of which cannot be fairly measured. For example, imagine the consequences of a missed diagnosis of avian flu.
* False-positive results: False reactions caused by malfunctioning liquid-delivery devices can also lead to incorrect diagnoses, unnecessary therapeutic treatment, and improper patient care.
Although some tests do not rely on accurate volumes, many are highly quantitative. Some examples of volume-dependent, quantitative tests include:
* PSA testing: This test measures the level of prostate-specific antigen (PSA) in the blood of men, as high levels of PSA can possibly indicate cancer, infection, or inflammation of the prostate. Malfunctioning pipettes can lead to false diagnoses and unnecessary, painful biopsies or delay treatment of a malignant disease.
* qPCR testing: Because quantitative PCR (qPCR) methodologies rely on the accurate addition of samples in controlled, minute quantities for proper analysis, liquid-delivery quality assurance is essential. In addition, because PCR reagents are often added in submicroliter quantities, even a slight discrepancy in delivered volumes can alter the concentration and ratio of reagents in the master mix and skew assay results.
* Lead testing: When analyzing blood for lead poisoning, testing procedures measure the level of lead concentration, obviously contingent upon volume.
Liquid-delivery quality requirements
In light of the severe consequences that can arise from improperly functioning liquid-handling instrumentation, most people would conclude that federal regulations must tightly govern equipment-performance verification and quality-control systems. Unfortunately, this is not the case. Although regulations do exist, they are very broad and open to interpretation--most have not caught up with advancements in liquid-handling technology.
For example, Clinical Laboratory Improvement Amendments (CLIA), codified through 42 CFR [section]493, require that equipment be calibrated and records properly maintained. These regulations, however, do not specifically address liquid handling.
Compliance with 42 CFR [section]493 is monitored by a variety of organizations, and one is the College of American Pathologists (CAP), which was granted regulatory powers by the Center for Medicare and Medicaid Services (CMS). CAP has released numerous checklists used by inspectors to govern the laboratory-accreditation process. The guidelines leave it up to laboratories, however, to determine the actual calibration standard operating procedures (SOPs), from which pipettes are to be checked and how often.
Additional guidance is presented in CAP's "Laboratory Instrument Evaluation, Verification and Maintenance Manual" (5th edition, 1999), which recommends that pipettes be checked "on a routine schedule at least monthly."
Lastly, it is important to note 21 CFR [section]58 regulates laboratories conducting studies for food, drugs, and other products monitored by the United States Food and Drug Administration (FDA). These laboratories must adhere to good laboratory practices, or GLP, which state that measuring equipment "shall be adequately tested, calibrated and/or standardized," that "written standard operating procedures" shall be used, and that "remedial action be taken in the event of failure or malfunction of equipment." In addition, section 63 (21 CFR [section]58.63) requires that "written records shall be maintained" of all calibration activities.
Guidelines emerge to outline U.S. best practices
Liquid-handling processes, like many laboratory technologies, have continually advanced more quickly than corresponding regulations. For example, the transition from glass to hand-held manual action pipettes created the need for new standards and the need for preventive-maintenance polices in liquid-delivery devices. Similarly, the present trend toward advanced automation in clinical laboratories has driven the need for new calibration methods as well as the need to select from and standardize the best of these new methods. Because regulations provide inadequate guidance for modern laboratories, independent organizations release standards and guidelines to improve industry operations and promote best practices.
With a long history of importance and responsibility in the world of testing and metrology is the American Society for Testing and Materials (now ASTM International), which develops market-relevant standards on a global scale. ASTM International Standard E 1154 states that liquid-delivery device calibration should be performed every three months with 10 data points, while a "quick check" verification should be performed every month with four data points. This standard is not required practice in the clinical industry; but it does provide a point of reference for laboratories evaluating internal programs, since it is one of the few standards that make specific recommendations for both frequency and number of data points in pipetting calibration.
International organizations are emerging to provide border-spanning guidance to clinical laboratories. For example, the International Organization for Standardization (ISO) emerged as a key global guiding body for a range of industries, particularly laboratories. A non-governmental organization, this network identifies and adopts relevant standards that can improve practices and ensure quality in products and services. These standards are highly useful for global laboratories to coordinate operations and maintain consistent quality programs worldwide.
Of particular relevance is ISO 15189 entitled "Medical Laboratories--Particular Requirements for Quality and Competence," which was created to apply ISO 17025 (General Requirements for the Competence of Testing and Calibration Laboratories) to medical-testing laboratories and has been recognized as fulfilling CLIA requirements. This standard and its associated documents are silent on the particulars of liquid handling but do require that all equipment that can contribute significant error be calibrated using traceable means. For nearly all analytical methods, liquid handling undoubtedly falls into this equipment category. While the standards do not specifically address liquid-delivery devices, they do recommend that standard calibration and check methods be used, as they are more easily validated and less expensively defended in audits.
To provide additional guidance regarding liquid handling, ISO Technical Committee 48 released a seven-part series, ISO 8655, defining accepted liquid-delivery performance and calibration practices.
Parts 1 through 5 define and specify minimum performance requirements for accuracy and precision in liquid handling, including details on accepted metrological requirements and maximum permissible error.
The next two sections of ISO 8655 provide guidance regarding accepted methods for verifying performance of liquid-delivery devices. Part 6, released in 2002, discusses gravimetric calibration, which verifies liquid volumes by measuring weight on a balance.
New guidelines recommend photometry for performance assessment
Due to advancing technologies that overcome several limitations of gravimetry, such as susceptibility to evaporation errors, difficulty in verifying the performance of individual channels in multi-channel devices, and the requirement of a temperature-and-humidity-controlled environment for accurate results, ISO added Part 7 to its standard in 2005. Here, ISO formally approved photometry for assessment of liquid-delivery equipment performance. Relying on known light absorption properties at specific wavelengths, photometric calibration can provide strong assurance of data integrity, quickly and conveniently.
Two specific variants of photometric calibration are highlighted by the standard: single dye and dual dye. As its name implies, single-dye photometry measures light absorption in one colorimetric solution to verify volume. The dual-dye approach to calibration, called ratiometric photometry, employs two highly characterized solutions to combat accuracy problems typically associated with single-dye absorbance measurements and yields results with uncertainty of less than 1% for volumes as low as 0.1 microliters.
As the clinical industry continues to evolve, and laboratories are faced with new challenges and new solutions, it is likely that ISO will continue to advance its standards. Currently, for example, ISO 8655 is clearly applicable to hand-held pipettes. When these standards were prepared, ISO's focus did not extend to robotic pipettors, explaining the exclusion of these liquid-handling devices. The scope of the committee now includes a broader range of laboratory equipment and is likely to move in one of two directions: write additional standards to guide calibration of automated liquid handlers or revise existing standards to include them. It is important to note that 8655 Part 7 does approve "vertical-beam photometry," which is very useful in verifying robotic pipettors that dispense to microtiter plates.
Laboratory trends emphasizing greater process control
Clinical laboratories are being heavily challenged to evaluate their processes from start to finish by the increasing number of regulations and standards, and the industry-wide focus on quality control evident in the advancing CLIA recommendations and tightening state standards.
The high failure rate of liquid-handling instrumentation is cause for concern, especially in light of this growing focus on quality, the decreasing volumes in common tests, and the drastic consequences of failure. Forward-looking laboratories are taking measures to verify accuracy and precision and maintain liquid-handling quality control to facilitate compliance and produce quality test results.
Table 1. Standards recommending liquid-delivery calibration best practices Organization Standard title Description CMS (CLIA) 42 CFR [section]493: Laboratory Equipment must be Requirements calibrated and records maintained. Perform and document calibration procedures. Include acceptable limits (tolerances) and a scheduled frequency of calibration. CAP Laboratory Instrument Evaluation, Check pipettes on Verification and Maintenance a routine Manual schedule at least monthly. FDA 21 CFR [section]58: Good Calibrate Laboratory Practice for routinely using Non-Clinical Laboratory Studies SOPs. Keep written records and perform remedial action when equipment is found to be out of tolerance. ASTM ASTM E 1154: Standard Provides general International Specification for Piston- or specifications Plunger-Operated Volumetric and guidance for Apparatus pipettes. Recommends pipette calibration every three months with 10 data points, and a quick check verification every month with four data points. ISO ISO 15189: Medical Silent on the Laboratories--Particular particulars of Requirements for Quality and liquid handling, Competence but requires that all equipment that can contribute significant error be calibrated using traceable means. ISO ISO 17025: General Requirements Calibrate using for the Competence of Testing and traceable Calibration Laboratories equipment with stated uncertainty. Prefers standard calibration and check methods. No specific guidance on liquid handling in this document (see AOAC version). ISO ISO 8655-2: Piston-Operated Establishes Volumetric Apparatus, Part 2 - minimum Piston Pipettes performance specifications (accuracy and precision) for hand-held pipettes. Compliance with these specifications must be met for the pipette to carry the CE mark. ISO ISO 8655-6: Piston-Operated Describes Volumetric Apparatus, Part 6 - gravimetric Gravimetric Methods for the method for Determination of Measurement calibration and Error testing of pipettes. ISO ISO 8655-7: Piston-Operated Describes Volumetric Apparatus, Part 7 - photometric and Non-Gravimetric Methods for the titration methods Assessment of Equipment for calibration Performance and verification of pipettes.
By George Rodrigues, PhD
George Rodrigues, PhD, is senior scientific manager at ARTEL, a company with more than 22 years of research, development, and manufacturing experience in the scientific instrumentation industry including its liquid-delivery quality-assurance system. Rodrigues can be contacted at 207-854-0860 or firstname.lastname@example.org
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|Publication:||Medical Laboratory Observer|
|Date:||Dec 1, 2006|
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