Molecular Diagnostics || Framework for Quality Assurance in Molecular Diagnostics

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition

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1. INTRODUCTION

A framework for ensuring laboratory quality was laid downby Congress in the Clinical Laboratory Improvement Act of1967. Because of public concerns about the quality of clinicallaboratory testing in the United States, Congress passed theClinical Laboratory Improvement Amendments of 1988(CLIA’88) setting forth uniform quality standards for all labora-tories performing tests for health purposes on human specimens.Laboratories must register with the Department of Health andHuman Services (HHS) to obtain and maintain a current CLIAcertificate regardless of whether they receive payment fromMedicare or Medicaid programs. The Centers for Medicare &Medicaid Services (CMS), formerly the Health Care FinancingAdministration (HCFA), is a federal agency within HHS and ischarged with the implementation of the CLIA’88 regulations.CMS working with the Public Health Services (PHS), specifi-cally the Centers for Disease Control and Prevention (CDC) andthe Food and Drug Administration (FDA), developed standardsfor laboratory certification and set forth criteria for categorizingtests based on the level of complexity to perform the test. Tests,therefore, are categorized as waived, moderate complexityincluding the subcategory of provider-performed microscopy, orhigh complexity. Waived tests employ methods that are simpleand are assumed to pose no risk or harm to the patient if the testis not performed correctly. Laboratories performing waived testsmust follow manufacturers’ instructions for test performance andare not subject to routine inspections. Tests categorized as mod-erate and high complexity must meet such CLIA’88 require-ments as: (1) maintaining optimal patient specimen integrity andidentification throughout the testing process, (2) specifying theresponsibilities and qualification for personnel performing thetest, (3) establishing and following written Quality Control pro-cedures and (4) having a comprehensive Quality Assurance pro-gram in place. The laboratories performing moderate- andhigh-complexity testing must participate in a proficiency testingprogram for each specialty, analyte, or test for which they are

certified and are subject to inspections by CMS or other pri-vate accrediting organizations. The major differences betweenmoderate- and high-complexity testing are in the quality controland personnel standards. On February 29, 1992, these revisedCLIA’88 regulations were published in the Federal Register (1)FFand it is recommended that each laboratory have a copy.

On January 24, 2003, almost 11 yr later, CMS and the CDCpublished a final rule in the Federal Register (2)FF revising thequality control and personnel standards for laboratory servicesand changing the consensus requirements for grading profi-ciency tests. The rule specifies a common set of quality control(QC) standards for both moderate and high complexity testing(now known as nonwaived testing) and reduces the frequencywith which QC is to be done in most specialty and subspecialtyareas. Under the new rules, agencies providing proficiency test-ing programs must grade a laboratory’s sample in cases wherethere is 80%, not the current 90%, agreement among the partic-ipants or referee laboratories. This will ensure that more labo-ratories will be graded and ultimately improve the accuracy oflaboratory testing. The CLIA rules now put the onus on the lab-oratory to review and validate nongraded samples, especiallywhen the results do not agree with the intended response.Another change in the regulations is that directors of high-complexity testing are to be board certified unless they areeligible to be grandfathered under the phase-in requirements.

The CLIA’88 standards were designed to enhance patientsafety while making it easier for laboratories to read, under-stand, and comply with the requirements. Subpart J (PatientTest Management), Subpart K (Quality Control), and Subpart P(Quality Assurance) were consolidated and reorganized into anew Subpart J (Facility Administration for Nonwaived Testing)and a new Subpart K (Quality Systems for Nonwaived Testing).Subpart J clarifies requirements for facility space, utilities,safety, transfusion services, as well as record and specimenretention. Subpart K pertains to the total testing process. Thereorganization is written to parallel the flow of a patient speci-men through the laboratory from the acquisition of a specimen

Framework for Quality Assurancein Molecular Diagnostics

MARLENE SABBATHSS -SOLITARE, SE ELWYN J. BAPTISTBB ,TTAND TERESITATT CUYEGKENG REDONDO

Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press, a part of Springer usiness Media, LLC 2006Science+B

with the test request, to test performance and reporting ofresults. Each phase of the testing process now has its own stan-dards. In this way, laboratories have a better chance to identifyand prevent errors. This subpart of the rules also incorporates therequirements of quality assurance (QA) (renamed quality assess-ment to more clearly reflect the activities performed) under eachappropriate section, namely: General Laboratory Systems, Pre-analytic Systems, Analytic Systems, and Post-analytic Systems,to ensure that quality services are provided throughout the test-ing process. The essential component of the QA program is forlaboratories to ensure continuous improvement of their perform-ance and services through monitoring and evaluating the effec-tiveness of their policies and procedures for each phase of thetesting system. They are to identify problems and take correctiveaction, to revise policies to prevent recurrences of problems, andto assess the adequacy and competency of the laboratory staff.Essential to the assessement activities is documentation.

There are quality system standards for 19 different non-waived testing subspecialties that have been set forth in SubpartK of the final CLIA’88 regulations. No standards exist specifi-cally for molecular testing, except for cytogenetics. Becauselaboratories that test human specimens must comply withCLIA’88 regulations, there are agencies reviewing the currentregulations to determine which are applicable or should beamended to cover molecular testing. Until such time, molecu-lar testing laboratories must follow good laboratory practiceguidelines and participate in external quality assessment pro-grams because they are considered in the nonwaived testingcategory. Both federal and state agencies (CLIA and New YorkState, Department of Health) and professional organizations(College of American Pathologists, American College ofMedical Genetics and National Committee for ClinicalLaboratory Standards) have available guidelines, recommenda-tion, and checklists pertaining to quality control, quality assur-ance, proficiency testing, and personnel requirements. Many ofthe recommendations of the American College of MedicalGenetics (ACMG) in the third edition of Standards andrrGuidelines for Clinical Genetics Laboratories (3) have beenendorsed and adopted by the College of American Pathologists(CAP). Jointly, the ACMG and CAP in their proficiency testingprogram offer surveys that are designed to emphasize the pre-analytic and postanalytic phases of molecular genetic testingand molecular oncology testing (4). The CAP also offers on-site laboratory inspection and a certification program. CAP hasbeen given deemed status by CMS as a laboratory certifyingagency under CLIA’88 and is also recognized by the JointCommission on Accreditation of Healthcare Organization(JCAHO). As such, any laboratory providing molecular testingcan seek certification through the CAP LaboratoryAccreditation Program as a means to comply with CLIA’88regulations. CLSI (National Committee for Clinical LaboratoryStandards) is a voluntary organization that develops consensusrecommendations for the standardization of test methodologieswithin the healthcare field. Its Molecular Diagnostic Methodsfor Genetic Diseases; Approved Guidelines (5) covers allphases of operation of a molecular genetics diagnostic labora-tory, including: nomenclature, laboratory safety, specimen han-dling and testing, quality assurance and results reporting.

228 SECTION V / QUALITY ASSURANCE IN THE MOLECULAR DIAGNOSTICS LABORATORY

Even though components of a quality assurance program(preanalytical, analytical, and postanalytical) are well estab-lished and consistent among published guidelines, there areconcerns that safeguards are not in place to ensure high-qualityservice in molecular testing laboratories. A report byMcGovern (6) concluded that a number of molecular genetictesting laboratories had suboptimal quality assurance practicesand suggested that both personnel qualifications and laboratorypractice standards need improvement. This was not substanti-ated by Hofgartner and Tait (7), who inferred that the majorityof molecular genetic laboratories were accredited by the CAP,therefore, rigorous quality standards to improve the quality ofgenetic testing exist.

A framework for a quality assurance program as promul-gated by the new CLIA’88 regulations and as guidelines offeredby professional organizations as the American College ofMedical Genetics (ACMG), College of American Pathologists(CAP), and CLSI (Clinical and Laboratory Standards Institute,formerly NCCLS or National Committee for ClinicalLaboratory Standards) is discussed in this chapter. Emphasiswill be placed on how to apply quality assurance standards tolaboratories to molecular diagnostic testing.

2. QUALITY ASSURANCE (QUALITY ASSESSMENT)A quality assurance (QA) program that is established, imple-

mented, and maintained by a laboratory can help ensure high-quality results are provided in clinically relevant turnaroundtimes. Its major goal is to minimize laboratory errors by contin-ual assessment and subsequent improvement of the servicesprovided. Such a program is to cover all steps of the testingoperation, including the preanalytical, analytical, and postana-lytical processes, which must be continuously monitored andassessed. A successful QA program should identify and moni-tor key indicators, preferably of high-risk, high-volume activi-ties, on a regular basis, to assess quality and to detect possibleways to improve processes and/or products. The documentdescribing the policies and procedures of a QA program shouldcontain the elements of why there is a need to assess, what is tobe monitored, how and when this is to be done, and by whom.It should also include criteria to assure the competency of thelaboratory personnel and provide a means of assessing labora-tory activities through proficiency testing and peer review.

2.1. PREANALYTIC PHASE The preanalytical phase of aquality assurance program addresses activities that occur priorto testing the patient’s specimen. This phase of the testingprocess has not received the attention it should. The final rulesof the Federal Register (2) state that all laboratories arerequired to identify ways to monitor, assess, and when indi-cated, correct problems that might occur when tests arerequested and during specimen collection and handling. Thus,a vital link in the quality assurance process is the proper iden-tification of patients and their specimens, because a mistake ineither of these areas might affect and subsequently influencetest results. Each laboratory, therefore, should establish clearlydefined criteria for monitoring the proper collection, labeling,preservation, transportation, and storage of a specimen to betested as part of their QA program. Included in the program arethe reasons for the rejection of an unacceptable specimen and

what corrective action is to be taken. A well-designed manual orelectronic test request form should be prepared by the laboratory,so as to obtain relevant information required to provide accurateresults and appropriate report interpretations. If results areentered into a recording or laboratory information system, thelaboratory must ensure the information is correct and provide asystem for timely correction of both clerical and analyticalerrors. There should be written criteria determining whether aninformed consent is a requirement for specific molecular assays.

2.1.1. Test Requests Manual or electronic test requestforms should be designed to allow for sufficient identification ofthe patient (name and address, date of birth, gender) and physi-cian (name, address, and phone number), the test(s) requested,and pertinent clinical information. Date and time of collectionas well as type of specimen collected should be included. Forsome assays, such as molecular genetic tests, the requisitionmight require racial/ethnicity data or a pedigree (e.g., for link-age analysis). Specific requirements for parentage/forensicidentity testing can be found in detail by referring to the CAPMolecular Pathology Checklist and the American Association ofBlood Banks Standards. Completed manual or electronic testrequests must accompany the patient’s specimen before a labo-ratory accession number is assigned. Multiple specimens fromthe same patient are each to have their own accession number.

One of the important considerations in molecular testing isto ensure that the clinical indications are appropriate for the testrequested. This is the responsibility of the laboratory director orother members of the medical laboratory staff. If clarificationof a clinical condition or any additional information regardingthe test request is required, it is to be discussed with the refer-ring physician. If there is a change in the test requested afterconsultation, it is to be documented and kept as part of thepatient’s file.

2.1.2. Specimen Collection and Handling The qualityof test results is influenced to a high degree by the proper col-lection and handling of specimens. Ideally, laboratories shouldhave designated reception areas to ensure that specimens arereceived and stored to minimize the chances of errors and ormix-ups. Procedures should be in place to include details ofproper labeling of specimens, method of collection of speci-mens from all sources, specimen preservation (especially ifprocessing is delayed), and their proper transportation to thelaboratory. The protocols must be consistent with good labora-tory practice and in compliance with all relevant safety codesas stipulated by the Occupational Safety and HealthAdministration (OSHA) and their institution (3,4).

When received in the laboratory, a specimen should havebeen properly labeled with patient name, a unique identifyingnumber (e.g., medical record), source of specimen, date andtime collected. At this time, the accession process begins wherethe condition of the specimen (sample volume sufficient foranalysis, is sample clotted, is container intact), the requestform, and the referral data are reviewed for completeness. Thedate and time of receipt and the laboratory accession numberare recorded onto a label firmly attached to each specimen con-tainer. If there is insufficient information for the laboratory touniquely identify the specimen, CLSI recommends that speci-mens be rejected. However, the sample still could be tested if

CHAPTER 18 / QUALITY ASSURANCE IN MOLECULAR DIAGNOSTICS 229

the demographics are incomplete, provided the informationwill be obtained by fax or phone in a timely manner. It is advis-able that the action taken to obtain required data be docu-mented by laboratory personnel and placed in the patient’s file.

In situations where molecular testing laboratories are a partof a hospital-based department or reference laboratory, anaccession number specific for molecular tests could be com-puter generated (8). This molecular accession number andpatient name is carried throughout the testing process and helpsto reduce transcription errors. Only authorized laboratory per-sonnel will have access to a password-protected computer sys-tem and to accession patient information. Folders can becreated for each patient, at this time, to include a worksheetspecific for the test requested, billing information, and a filecard with patient name/accession number/test results/date final-ized. These can function as a backup if the computer systemshould malfunction. The final report and supporting test datawill eventually be included in the folder. A log of all acces-sioned patient specimens that were stored must be maintainedto allow prompt retrieval for repeat or additional testing (4).

The proper collection of the patient sample will depend onthe clinical diagnosis and the test requested. Molecular analy-sis of neoplasia, infectious diseases, or genetic disordersrequire specimens from tumor tissue, body fluids, or any nucle-ated cells. The use of amplification techniques now rendersextremely small samples, such as buccal scrapings, urine spec-imens, dried blood spots, and thin slices of paraffin-embeddedtissue, adequate for testing. Laboratories must have availablewritten requirements for the proper collection of patient speci-mens. For example, molecular testing of blood and bone mar-row samples are to be collected in nuclease-free EDTA or ACD(acid citrate dextrose) tubes, not sodium heparin, to inhibit clot-ting. Heparin reportedly acts as an inhibitor in amplificationassays (9). If a sample is received in a heparinized tube, it isrecommended that the white blood cells be separated andwashed with a physiological buffer before the nucleic acidextraction. This is especially true for RNA analysis, asheparinized plasma has not proven to be suited for quantitativereverse transcription–polymerase chain reaction (RT-PCR) test-ing, as exemplified by poor quality results (10). Fresh tissuesamples for molecular testing should be frozen soon after col-lection to prevent DNA degradation and sent to the molecularlaboratory immediately. If not frozen, it can be placed in aholding media and stored at 4°C for a maximum of 48 h.ffTissues that have been paraffin-embedded or specimens pre-served in ethanol are stable at room temperature, with minimalDNA degradation. However, tissues fixed in Zenker’s, B5, orBouin’s produce extensive DNA damage, making their useinappropriate for molecular studies.

Blood and bone marrow specimens for DNA analysis, if col-lected properly, can be stored at 4°C for 3–5 days and still yieldffhigh-quality DNA for any amplification assay or Southern blotanalysis. Farkas et al. (11) evaluated the quality and stability ofDNA within clinical specimens such as blood (white blood cellswere isolated before storing) and solid tissue (placenta) whenstored at several different temperatures for various lengths oftime. It was concluded that leukocytes and solid tissues kept at4°C or frozen are best for short- to intermediate-term storage. In

our laboratory, all blood and body fluid samples are refrigeratedaupon arrival (usually up to 3 d), whereas tissue is immediatelystored at –70°C, until ready for processing.

Specimens for RNA analysis require specific collection andhandling conditions. One of the common causes of the failureto obtain results is due to insufficient stabilization of RNA inthe specimen prior to dispatch to the laboratory. If chaotropicagents, such as guanidium isothiocyanate, are added immedi-ately after collection, the specimen, under these conditions, canbe stored for 7 d at ambient temperature before testing. Theseagents are known to remove proteins and denaturing RNase,which would otherwise degrade RNA. If these agents are notavailable, consider freezing the specimen to keep the RNA sta-ble. It has been demonstrated that blood samples requiringRNA testing can be stored at 4°C for up to 48 h if the plasmaffis separated (12).

2.1.3. Rejection of Specimens Specimen rejection maybe justified if there is improper handling or a delay in trans-portation to the laboratory. Unacceptable specimens for testingwould be ones that are received thawed in the laboratory whenthey should have been kept frozen, collected in the wrong anti-coagulant, clotted, contaminated, or contain insufficient quan-tity. However, sub-optimal collection or storage conditions ofpatient samples should be reviewed on a case-by-case basis.For example, small, improperly handled specimens may besuitable for amplification assays, yielding interpretable molec-ular results, but not suitable for Southern blotting where largequantities of high-quality DNA are required. In general, it isleft to the discretion of the laboratory staff and director to deter-mine whether or not the specimen should be rejected.

If a specimen is rejected, the laboratory personnel must doc-ument the occurrence. The documentation should include thedate, time, and condition the specimen was received, notifica-tion of the referring physician, and whether another specimenwas obtainable. This record is kept in the patient file and docu-mented as part of the QA records.

2.1.4. Informed Consent For some molecular genetictests, informed consent might be required by federal, state, orlocal laws before initiating the test. This ensures that the patientvoluntarily agrees to testing and has some understanding of thereasons for this test. The level of consent depends on whetherthe genetic test is used for predictive or diagnostic purposes.The laboratory can be of assistance in determining the appro-priate level of informed consent and refer to established guide-lines (4). The New York State Department of Healthrecommends consent from all patients who are having a genetictest; however, the referring physician could sign the test requi-sition indicating that she/he conveyed the required informationto the patient (13).

2.2. ANALYTICAL PHASE In the analytical phase, labo-ratories performing nonwaived testing must meet applicablerequirements as set forth in the CLIA’88 regulations andestablished by CAP’s Laboratory Accreditation Program. Suchrequirements include the availability of a procedure manual,specimen storage, criteria to obtain accurate and reliable testresults, and a system to maintain patient records (2). Particularattention will be given to the following: nucleic acid extraction,contamination affecting amplification assays, use of controls,


validation of tests, maintenance of equipment, documentationof competency of personnel, and the laboratory’s participationin internal and external assessment programs.

2.2.1. Procedure Manual The procedure manual mustcontain sufficient details of all laboratory assays written so thatqualified laboratory personnel can perform them consistentlyand accurately. The procedure is to include the principle of thetest, pertinent clinical significance, specimen requirements,including collection, storage, preservation, and transportationto laboratory, criteria for specimen acceptability and rejection,the reagents needed, the steps to perform the test, quality con-trol measures, verification ranges, interpretation of results, andreferences and pretinent notes. The style and format of the pro-cedure manual, noted in the CAP Molecular PathologyChecklist, is at the discretion of the Laboratory Director; how-ever, CLSI provides guidelines in their G6P42-A4 publication(14). Electronic procedure manuals are acceptable as long asthey are available to all personnel. They are subject to the samecontrols as the paper version in that they are to be reviewedannually by the laboratory director (or designee). All new pro-cedures or revisions of existing ones are also to be reviewed bythe laboratory director (or designee) prior to implementation. Ifa procedure is discontinued, a copy, must be maintained for aminimum of 2 yr. The initial date of use and retirement datemust be clearly indicated on the procedure.

2.2.2. Nucleic Acid Extraction and Specimen StorageThe extraction of nucleic acid, DNA, or RNA, is a crucial partof sample handling. Any errors that occur cannot be rectified atlater stages of the testing process. Considerable attentionshould be given by well-trained technologists to adhering to theprocedure at this stage of the assay. Methods that rapidly isolatehigh-quality DNA suitable for analysis are widely available. Kitsexist that provide all the components and procedures necessaryfor isolating DNA from whole blood, bone marrow samples, cul-tured cells, and so forth (15). However, automated instrumentsthat extract nucleic acid limit the hands on part of the extractionprocedure, thereby reducing the chances that an error will occurduring the extraction process.

Once DNA or RNA are extracted, the conditions underwhich these nucleic acids are stored to prevent any significantloss in quality or quantity is important. This is especiallyimportant if they are to be used in other molecular assays orretested. The storage conditions for DNA and RNA differ. DNAcan be stored for years if kept at 4°C in a buffered solution,such as Tris-EDTA. Diluting and storing in water will easilydegrade DNA after a few weeks and it no longer will be usefulfor any molecular assay. RNA suspended in water must befrozen at –70°C soon after extraction and can be stored longterm as such. Freezing and thawing RNA more than three timesaffects its stability and recovery (10).

All nucleic acid samples must carry the laboratory’s acces-sion number, including the date of preparation. This uniqueidentifier would assist in tracing the sample through the testingprocedure. There must be a schedule for retaining nucleic acidspecimens as stated in the CAP Molecular Pathology Checklist.

2.2.3. Contamination A major concern for any diagnos-tic molecular laboratory performing nucleic acid amplificationmethods is the occurrence of false-positive results due to

sample-to-sample contamination of nucleic acid during testingor the “carry-over” of DNA from a previous amplification ofthe same target. Poor laboratory technique and lack of attentionto detail by technologists create situations in which amplifiedproducts contaminate areas of the laboratory. To minimize theoccurrence of false-positive results, laboratories should imple-ment specific practices and procedures including universalstandards. An immediate plan of action when a contaminationproblem is observed would be to cease all testing, discardpotentially contaminated reagents and supplies, and appropri-ately clean work surfaces and equipment. Testing would onlyresume if new aliquots or lot numbers of reagents, sterilizedglassware, pipette tips, filtered buffers, etc., are available foruse. The source of the contamination and the process used in itselemination must be documented and become part of the labo-ratory’s QA program. Avoiding or minimizing laboratory con-tamination requires an appropriate laboratory design and goodlaboratory practices.

2.2.3.1. Laboratory designrr Each laboratory shoulddevelop its own unidirectional workflow to avoid the possibil-ity of amplicon contamination. Ideally, a laboratory performingamplification assays should be divided into three separate workareas: (1) a reagent preparation area or room, (2) an area forspecimen preparation, and (3) an area for amplification anddetection (16).

The reagent preparation area (area 1) must be kept veryclean and away from any amplified products or patient speci-mens. The specimen processing area (area 2) should be locatedas far away from the amplification and detection area (area 3).This separation reduces the possibility of aerosols fromextracted specimens to contaminate amplified products leadingto false-positive results. Personnel traveling from the specimenpreparation to the reagent preparation or the amplification anddetection areas must always change their laboratory coats andgloves. Dedicated laboratory coats should be available whengoing in and out of each area. Laboratory coats or gloves wornin area 3 must never be worn in areas 1 or 2. Careless adher-ence to these recommendations can lead to carryover contami-nation from amplified products. However, with the introductionof commercially licensed tests and newer methodologies, someof the above requirements may be reduced.

Prior to the preparation of a specimen for amplification,bench tops and pipettes are to be wiped down with 10% sodiumhypochlorite (bleach) and rinsed with 70% alcohol and water toremove biohazardous agents and extraneous nucleic acids.Contaminating amplicons remaining on workbenches can bedestroyed by ultraviolet (UV) irradiation from germicidal bulbsthrough the creation of thymine dimers resulting in nucleic acidunsuitable for analysis. The use of Class II Biological safetycabinets with UV bulbs and HEPA-filtered air or bench topdead-air boxes with UV light attachments provide a clean dust-free worktop. These units are ideal for areas where specimensare prepared for extraction and where reagents are prepared.The UV light should be turned on at least one-half hour beforemanipulating any nucleic acid at the workbench and turned offbefore placing hands in working area.

It is recommended that there be a dedicated set of pipetteswith plugged (aerosol-barrier) tips or positive-displacement tips


for each of the three areas. The exclusive use of pipettes and tipsffwill eliminate cross-contamination of samples. Prior to usepipette tips can be autoclaved, while pipette barrels, test tuberacks, and mini centrifuges wiped with 70% ethanol. They allcan remain under the hood when the UV light is on. Equipmentsuch as gel apparatus and combs, centrifuges, microtome bladesfor cutting paraffin-embedded tissue, etc., should also becleaned with 70% ethanol to minimize cross contamination.

2.2.3.2. Laboratory practicesrr Reagents used in amplifi-cation assays once prepared should be divided into aliquots,and stores in an are separate from where specimens are pre-pared or amplified. Aliquoting minimizes the number ofrepeated sampling from the same test tube and reduces thepotential for cross-contamination. Lot numbers and storedquantities of reagents should be recorded so that if carry-overdoes occur the source can be easily identified. All componentsof an amplification assay, such as dNTPs, primers, buffers, Taqpolymerase, and RNAse/DNAse-free water, should be added tothe reaction tube in the reagent preparation area and securelycapped. Each tube should be capped after the addition of thesample, before proceeding to the next tube. It is recommendedthat the tubes be subject to a quick centrifugation before uncap-ping to prevent aerosolization.

Carry-over of nonspecific PCR products can be preventedby chemical modification of amplified fragments. For example,inactivation of an amplified product can be done by substitut-ing dUTP for dTTP, thereby generating uracil-containing frag-ments in the reaction mixture (17). The bacterial enzymeuracil-N-glNN ycosylase (UNG) is added to the reaction and incu-bated prior to amplification. All uracil-containing DNA carriedover from previous PCR reactions are enzymatically destroyed.Another example that is a major problem in isolating high-qualityRNA is contamination by ribonuclease (RNAse). Sources ofthese highly resistant RNA-degrading enzymes are glasswareand the hands of laboratory personnel. The treatment of glass-ware with an RNAse inhibitor, such as diethylpyrocarbonate(DEPC), followed by autoclaving or baking in a 250°C oven for4 h will inactivate RNAse. Sterile disposable pipette tips andEppendorf tubes are reported to be RNAse free, but autoclavingagain will further reduce the chances of contamination.

2.2.4. Controls A variety of controls must be included foreach test system to ensure that the results obtained are valid. Theselection, number, and preparation of controls for proceduresinvolving nucleic acid extraction, restriction enzyme digestion,electrophoresis, etc., are determined by the laboratory. Controlsthat must be included for each assay system are negative, posi-tive, sensitivity controls and a molecular weight marker.

A negative control, meaning a reagent control to which notemplate has been added, must be included in every amplifica-tion assay. This negative control assesses the quality of thereagents by detecting any contaminant or increased backgroundsignal due to a few molecules of contaminating sequences. Apositive control, which also contains all reagents required foramplification, provides proof that the assay works and specificevidence of amplification for each mutation or genotypeincluded in the test system. In a Southern blot assay, a generearrangement (bands other than germline) must be observedfor each restriction enzyme digest. These tests need to be

repeated. In RT-RNA assays, a “no reverse transcriptase” con-trol is added each time to the test. If a positive result is observedwhen reverse transcriptase is omitted from a reaction tube, itindicates that either the reagents or the sample is contaminated.Molecular weight markers used with gel electrophoresis mustspan the range of expected bands for that specific primer set orprobe locus.

Each molecular assay should also incorporate a sensitivityor analytical control to determine the lowest level of an analytethat can be detected by that specific methodology. This sensi-tivity control is especially important in Southern blot andamplification assays. For the Southern blot technique the sensi-tivity control corresponds to above 5% of the tumor cell popu-lation and is to be included with each electrophoresis run. Foramplification assays, a known positive sample for that primerset should be serially diluted to the lowest limit for detection ofa monoclonal population and included with each analysis.

To determine whether the nucleic acid is amplifiable, a con-trol using primers directed toward a second target within theDNA template is recommended. A positive result indicates thatthe template is amplifiable and no reaction inhibitors are pres-ent. In some instances, these primers can be incorporated intoan assay (multiplexing), saving both time and reagents.

Positive control specimens may not always be readily avail-able for use in various assays. There are a number of sources inwhich such specimens can be obtained. The American TypeCulture Collection (ATCC) and other commercial sources offermany tumor cell lines, DNA, and viruses for purchases. Seethese websites for details: http://ccr.coriell.org/nigms andwww.atcc.org. Other sources for obtaining controls are throughinterlaboratory exchanges, proficiency testing samples, orpatient samples that have been tested in parallel with alternativetechnology (14). There is a need to establish a disease consor-tia so that the samples for controls (especially positive ones)can be obtained for testing. The Association for MolecularPathology (AMP) through its website (www.amp.org) hasproven to be a valuable source for technical information andeducation in the applications of molecular diagnostics and amedia for exchange of methodologies and ideas.

2.2.5. Test validation Test validation should be con-TTducted before a new test is introduced for clinical diagnosis.Each laboratory must document that they have validated thetests they are offering. Validation ensures that the test meetsacceptable performance standards and is appropriate for thepopulation for whom it is intended. Because requirements forthe proper in-house validation of a test are not always clearlydefined, the Association for Molecular Pathology (AMP) pro-vides recommendations for in-house development of molecularassays (18) and the College of American PathologistsMolecular Pathology checklist refers to standards for test vali-dation. Under CLIA’88 each laboratory that introduces anFDA-cleared or -approved test must demonstrate that it canobtain performance specifications, such as accuracy and preci-sion, comparable to those established by the manufacturer. Thelaboratory must verify that the manufacturer’s normal valuesare appropriate for their patient population. Assays developedin-house often use reagents purchased under the analyte-specific reagent (ASR) rule. Before introducing an ASR-based


test, the laboratory must establish the following performancecharacteristics prior to reporting patient test results (2).

• accuracy (verified by using split patient samples withanother laboratory already performing the test and by usinga recognized national agency that provides proficiency testsample, i.e., CAP)

• precision (can be established by using different levels ofcontrols to determine within-run and between run and totalimprecision)

• reportable range of the test results• reference (normal) ranges• analytic sensitivity (or lowest detectable limit)• analytic specificity (the ability of the test to only detect the

measurable quantity)• clinical sensitivity (the percentage of positive tests when

the clinical disorder is present)• clinical specificity (the percentage of negative tests when

the clinical disorder is absent)• the positive predictive value (the likelihood that the clini-

cal disorder is present when the test is positive) and• the negative predictive value (the likelihood that the clinical

disorder is absent when the test is negative).

The actual experiment performed to establish analytical andclinical performance characteristics vary and are dependent onwhether it is a qualitative, semiquantitative, or quantitative typeof test. Guidance for these experiments/evaluations can beobtained from some of the NCCLS documents (ww.nccls.org)or from the ACMG Standards and Guidelines specifically formolecular technique (3).

Once the test is validated, it is recommended that some gen-eral quality assurance parameters be applied. These wouldinclude monitoring the effect the test results has no patient care,determining how useful the test results are for clinical manage-ment, and determining whether the cost of doing the test justifiesthe result in saving through more rapid and accurate diagnosis.

2.2.6. Maintenance of Equipment An organized systemrfor maintaining and monitoring all equipment and instrumentsmust be established by the laboratory to check the critical oper-ating characteristics so as to provide consistent and reliable testresults. Periodic maintenance and function checks must be donein accordance with the manufacturer’s specifications. They are tobe documented and kept at the workbench along with any serv-ice or repair records. For an instrument without specified func-tion checks it is the laboratory’s responsibility to establishmaintenance and function checks. This system should be in com-pliance with CLIA’88 standards [see Standard: Maintenance andfunction checks 493.1254 in Federal Register (2)FF ] and as statedin the CAP Molecular Pathology Checklist (4).

2.2.7. Competency of Personnelcc The competency oftechnologists who perform clinical laboratory tests is to beassessed annually. The Laboratory Director, or designee, selectsthe process to be measured, indicates the reason for the selec-tion, and describes the details of the task. Evaluation couldeither entail observing a specific aspect of a laboratory proce-dure, the operation of an instrument, or important laboratorysafety procedures. It could also involve discussions with thetechnologist regarding a clinical procedure to assess their levelof understanding or a review of issues pertaining to the control

and prevention of contamination when performing amplificationassays in the laboratory. A specific example would be monitoringhow a technologist in a molecular laboratory extracts DNA fromperipheral blood using a specific manufacturer’s kit.

The text of the evaluation should indicate the methodologyused and the criteria anticipated for a satisfactory performance.If the competency is less than 100%, an explanation of theproblem is to be provided, including any corrective or addi-tional training measures to be implemented within a realistictimeframe. The evaluation is to be documented as well assigned and dated by the evaluator.

2.2.8. Proficiency Testing and Accreditation The goalof proficiency testing (PT) is to evaluate a laboratory’s per-formance compared to other laboratories (its peers) participat-ing in the PT program. It is a process for assessing laboratoryperformance by which multiple laboratories analyze similarsamples that are collected and evaluated by an outside entity(19). All CLIA laboratories performing nonwaived testing mustparticipate in organized PT programs when available. Whenone does not exist for the test, there should be an external orinternal PT program established by the laboratory. Applicableexternal procedures include the splitting of samples for analy-ses with a reference or other laboratory certified to perform thetest or participating in an ungraded PT program, as organizedby the Association for Molecular Pathology. For the internalprogram, some examples are to retest specimens previouslyassayed by laboratory personnel using pseudonym identifiers(blind study) or to perform clinical validation by chart review.In the blind-study PT method, the Laboratory Director selectsthe specimen, evaluates the results, and documents the outcomeindicating that it is part of the internal PT program.

The College of American Pathologists requires laboratoriesto participate in proficiency testing or a CAP-approved pro-gram of graded interlaboratory comparison testing as part of itsaccreditation process. The laboratory must enroll in programswith analytes matching those tests that are performed for clini-cal testing. For molecular diagnostics, ACMG/CAP providessurveys for Molecular Genetics and Molecular Pathology. Thelaboratories are sent four to six specimens twice a year for eachsurvey. The survey samples must be integrated within the rou-tine laboratory workload using the same method as for patientsamples and rotated among all laboratory personnel. A detailedquestionnaire accompanies the samples and is used to recordresults and interpretation. The results are anonymouslyreviewed and analyzed. The final graded report, when issued toeach of the participants, is an indication of the laboratory’s per-formance compared to other laboratories performing the sameprocedures using identical or similar methods.

Unacceptable responses might be the result of improper han-dling of the PT sample, incorrect recording of results, or theincompetency of the testing personnel. Therefore, if an unaccept-able result is received by the laboratory, the findings must be eval-uated and corrective action taken. This action might entailpersonnel training and reviewing of test procedures. Both CLIAand CAP require documentation of action of unacceptable results.

Through the PT process, participants can identify proce-dural problems and take corrective action before patient resultsare affected. It can be used to document personnel competency


and identify problems that might be resolved with additionaltraining and monitoring. Proficiency testing provides continu-ing medical education for the laboratory.

2.3. POSTANALYTICAL PHASETT The postanalytic phaseissues involve assuring the accuracy and reliability of testresults, reports designed to provide patient laboratory data effec-tively, and maintain patient confidentiality. The final reportshould provide an appropriate summary of the methods, theobjective findings, and a clinical interpretation. Laboratory testresults must be readily available to the laboratory and releasedto the requesting physician in a timely manner.

2.3.1. Laboratory Test Reports The laboratory reportshould include the name and address of the patient, a uniquepatient identifier, name and location of the laboratory, the datethe test was performed, and description of the test methodol-ogy. The report should also include the source of the specimenand any information regarding the condition of the specimensif it did not meet the laboratory’s criteria of acceptance. All lab-oratory reports must be clear, concise, accurate, and fully inter-pretative. The results should be brief and unambiguous. If theyare complex, results may be presented in table format ratherthan text form. A statement interpreting the data, includingclinical implications, follow-up recommendations, and the lim-its of the assay, must be written so that it is understandable to anonmedical professional. The final report is to be signed by thelaboratory director or other authorized individual and indicatethe date it was finalized.

Molecular testing laboratories must include as part of thereport the federally required clarifying statement for tests usinganalyte-specific reagents. The mandatory language is “This testwas developed and its performance characteristics determinedby (laboratory name). It has not been cleared or approved by theU.S. Food and Drug Administration.” The CAP recommends theaddition of “The FDA has determined that such clearance orapproval is not necessary. This test is used for clinical purposes.It should not be regarded as investigational or for research. Thislaboratory is certified under the Clinical LaboratoryImprovement Amendments of 1988 (CLIA’88) as qualified toperform high complexity clinical laboratory testing” (4). It isour suggestion that FDA approved tests also be recognized assuch in final reports.

A copy of the final report, including all supporting test data,are to be kept in the patient’s file. Requests for copies to be sentto other health care providers are to documented by a writtenrequest from the patient. The reports, when finalized, are oftendistributed to the requesting physician via standard mail, or fax,or by hand.

Any questionable findings must be resolved by additionalanalysis (reprobing of blot or digestion of another sample, etc.)before reporting the results to the referring physician. If thereis a lack of correlation of molecular results to other laboratoryfindings, such as histopathologic diagnosis, it is recommendedthat these cases be investigated to resolve the discrepancies. Itcould be just a problem with tissue sampling. Guidelines stan-dardizing the reporting of results, interpretations, when torepeat a test, recommendations as to secondary testing, and cor-relation of results with other clinical data should all be devel-oped as part of the postanalytic quality assurance process.

As per CLSI guidelines (5), all patient laboratory recordsshould be accessible and easily retrieved. They should be cross-referenced by both the patient name and by a second uniqueidentifier (e.g., medical record member or laboratory accessionnumber). The records are retained for a period of time asrequired by applicable federal or state regulations. CLIA‘88recommends that copies of patient records should be kept for aminimum of 10 yr, or as required by state law (20). Electronicrecords are acceptable. However, guidelines for specimenretention time have not yet been agreed upon.

2.3.2. Timeliness of Reporting One of the importantcommitments in a laboratory’s operation is providing testresults within a time period suitable for prompt patient manage-ment decisions. This involves establishing limits for turnaroundtime (TAT) of tests offered. Turnaround time is defined as thetime (e.g., number of hours or days) it takes to issue a resultfrom receipt of the specimen in the laboratory to when the finalreport is resulted. What is acceptable will depend on the type ofsample, the test requested, and the laboratory workload.

It is each laboratory’s responsibility to check, at regularintervals, TAT against the threshold set for each of the assaysoffered. This can be monitored by establishing an indicator thatwould tabulate the percentage of reports completed by thestated TAT for the test(s). Prolonged TAT is to be investigatedand corrective action taken perhaps by improving or changingthe methodology. It is the laboratory director’s responsibility tomonitor TAT, including the volume of samples analyzed todetect trends, systematic errors, or local population variationsthat might have an effect on tests results or interpretation.

2.3.3. Correction of Errors Despite care and protectivemeasures, errors do occur during the accessioning process. Asstated in the CAP Molecular Pathology checklist, there must bea system for timely correction of both clerical and analyticalerrors. Clerical errors, such as incorrect spelling of a patient’sname, incorrect address, wrong medical record number, orwrong date of birth, can occur during the accessioning of apatient specimen or as a result of misinformation provided bythe referring clinician. These are to be corrected as soon as theybecome evident. Errors in the reported test results or misinter-pretation of laboratory findings, must be corrected immediately.The referring physician is to be promptly notified and anamended report issued. A copy of this amended report is placedin the patient’s file, including documentation of the error and thecorrective action taken. To reduce clerical and analytical errors,laboratory results and patient demographics should be checkedindependently by a qualified member of the laboratory staffprior to signing out of the report by the laboratory director.

Errors occurring during the testing process, such as mislabel-ing a test tube, mixing two different patient specimens, or record-ing incorrect data on worksheets, must be corrected promptly. Thetechnologist who has made the error(s) must document the eventand indicate how it was corrected. In some cases, the test mighthave to be repeated at the laboratory’s expense. In general, iden-tifiable errors must be corrected immediately, because they couldlater translate into significant problems and potentially compro-mise the quality of patient care (8).

2.3.4. Patient Confidentiality Records should be main-tained in a manner to preserve patient confidentiality. All


reports are issued to the referring physician or genetic profes-sional, not the patient. They can be released to other healthcareprofessionals only with appropriate “authorization for release”from the patient. The laboratory must have a policy in place toprotect the confidentiality of the test results reporting. Forexample, if a request for test results made by telephone fromthe referring physician’s office, the laboratory personnelreceiving the call must verify that it is the physician’s officecalling. The name and telephone number of the caller isobtained and patient’s laboratory folder is reviewed to verifythis is the referring physician. The event is documented andremains part of the patient file. If discrepancies exist, the mat-ter should be referred to the laboratory director or manager.

Recently, new patient rights and healthcare provider regula-tions mandated by The Health Insurance Protability &Accountability Act (HIPAA) (21) were instituted. The regula-tions require all healthcare providers and their staff to restrictthe use and disclosure of medical information and provide pri-vacy rights for all their patients. Since each patient has the rightto know who has access to and where their test results weresent, laboratories should incorporate into their QA policies howthey are maintaining patient confidentiality. In New Jersey, thelaboratory is legally required to share certain patient informa-tion, outside the realm of treatment, with agencies concernedwith payment of services or healthcare, i.e., the New JerseyDepartment of Health and local health departments, that havenot been authorized by the patient. To comply we developed acomputer program to allow tracking of this information effi-ciently upon request of the patient.

3. PERSONNEL QUALIFICATIONSUnder the CLIA’88 rule, there is a change in the personnelAA

standards for directors of high complexity laboratories. Theymust hold a doctoral degree in a chemical, physical, biological,or clinical laboratory science from an accredited institution andbe certified and continue to be certified by a board approved byHHS (2). The rule “grandfathers” in current experienced direc-tors without board certification, and those who have served asdirectors of a laboratory performing high complexity testingprior to February 24, 2003 and have 2 yr of laboratory trainingand experience directing high complexity testing.

In addition, the American College of Medical Geneticsrequires the following to be met for laboratory directors (3).They must have a PhD or MD, have at least 2 yr of experienceor postdoctoral training in a clinical laboratory subspecialty, aswell as being certified by specialty Boards. Board certificationby the American Board of Medical Genetics or CanadianCollege of Medical Geneticists documents that this individualhas both training and experience suitable for a director ofmolecular testing laboratory. The American Board of Pathology(ABP), the American Board of Clinincal Chemistry (ABCC),and the American Board of Bioanalysts (ABB) also providerecognized board certifications in this subspecialty. The labora-tory director must be on site regularly or a accessible to the lab-oratory. He/she must ensure that the laboratory is in compliancewith all regulations and maintains an ongoing quality assementprogram. The director determines the appropriate tests to beperformed, the techniques to follow and the equipment and

reagents to be used. He/she reviews, interprets, and reports alllab results and assures their accuracy.

ACMG also specifies qualifications for other laboratory per-sonal such as the supervisor. They are to hold a Bachelorsdegree and have at least 3 years of experience in medical field.It is recommended that they obtain certification from suchagencies as the American Society for Clinical Pathology(Molecular Pathology) or the National Credentialing Agency inwhich they could become Certified Laboratory specialist inMolecular Biology. Laboratory technologist must hold anundergraduate degree in a biological or chemical field or haveat least five years of relevant laboratory experience.

The ability for laboratory personnel to troubleshoot assaysand handle unexpected laboratory problems involves the con-tinuous update of their knowledge. Continuing education is animportant aspect of quality assement and improvement. Thiscan be handled in several ways such as a journal club in whichselected articles are discussed, reviewing results and interpreta-tions of difficult and interesting patient reports, attendingappropriate seminars and conferences. Such activities are to bedocumented for each laboratory member.

4. CONCLUSIONA rapidly increasing number of laboratories are now estab-

lishing molecular technologies to use for clinical diagnosis.Efforts by professional organizations such as ACMG, AACC,ABB, CAP, AMP, CLSI, and government agencies (exempli-fied in the new CLIA’88 rules) are being directed to the stan-dardization of both the test systems and the laboratoryprocedure. Quality Assurance guidelines specific for molecular


laboratories have not been clearly established yet. What has beenapresented in this chapter is intended to inform and guide suchlaboratory as to what to consider in instituting a QA program.

At our hospital, a plan has been in place for monitoring QAindicators for different laboratory sections. The purpose of theplan is to ensure that the department with all its availableresources implements the mission of the hospital to provide theservice needed to sustain the highest possible standard ofpatient care. The overall responsibility for the QualityAssessment plan lies with the Chairman of the department whoshares reports of QA activity with the QA committees of thehospital. “Key indicators” for each laboratory section areselected at the beginning of each year and monitored monthly.If the indicator selected does not meet the established thresh-old, it is the responsibility of the director of that laboratory sec-tion to formulate an improvement plan and to implement it. Thefollowing is an example of one of the indicators (turnaroundtime) the Diagnostic Molecular Pathology laboratory is moni-toring. It is divided into two parts: the first requires the selec-tion of an indicator to be monitored (Table 1) and the secondoutlines a plan to monitor the indicator selected (Table 2).

Following are suggested key indicators that can be used toassess QA practices in your laboratory. See also the article byMcGovern et al. (6) for other laboratory practice standards thatffcan be used as indicators.

Suggested Indicators for the Preanalytical Phase1. Percentage of specimens received without a requisition2. Percentage of specimens received with inadequate

demographics

Table 1TTIndicator: Turn-Around Time

Important aspect of service to be evaluated Indicators to be monitored Threshold for evaluation

Turnaround time from specimen receipt to result The percentage of specimens for B- and T-cell Less than 90%TTreporting of B- and T-cell gene rearrangements gene rearrangements that are completed withindone by the PCR method 7 d of receipt

Table 2TTPlan to Monitor Turn-Around Time

Indicator to be Source of Frequency of Frequency of monitored collection How collected Who collects collection analysis

The percent of From the data The Molecular Statistics The Molecular On a monthly Statistic analysisspecimens for entered and Test Log program is Statistics Log basis, the is done biannually.B- and T-cell stored in the accessed from the is printed by Section Chief (January to Junegene rearrangements laboratory computer computer system the Pathologist of laboratory and July todone within 7 d system; the laboratory to provide a list of in Charge of the collects, December) of receipt. computer system indicates all accession numbers molecular tabulates, by the Section

the date of receipt and in which B- and T-cell laboratory and reviews Chief and thethe date of report for gene rearrangement (or designee). the data. Pathologist each patient sample. studies were resulted; in Charge of

the total number of molecularcases of B-cell gene laboratory.rearrangementassays and T-cell gene rearrangement assay are obtained.

3. Percentage of specimens with inadequate amounts ofnucleic acid extracted

4. Percentage of specimens received with incorrectpreservatives

5. Percentage of improperly stored specimens6. Percentage of improperly labeled specimens7. Percentage of rejected specimens (broken down by type)8. Monitoring of the average time from specimen collec-

tion to receipt in laboratory9. Percentage of clerical errors (broken down by type)

Suggested Indicators for the Analytical Phase1. Average time from specimen receipt to verification of

result2. Percentage of tests that are repeated (broken down by

reason)3. Percentage of controls that are unacceptable4. Percentage of contaminated runs

Suggested Indicators for Postanalytical Phase1. Percentage of final reports that have significant clerical

or typographic errors2. Percentage of molecular diagnostic reports released

after stated TAT3. Percentage of molecular diagnostic reports confirmed

as delivered electronically

Suggested Indicators for the Entire Testing Process1. Percentage of correlations between two different

methodologies2. Percentage of a specific group of surgical pathology

cases (hyperplastic lymph nodes, ASCUS, etc.) thatwere tested by a molecular method

3. Percent correlation of molecular results and surgicalpathology findings

4. Percent correlation of retrospectively reviewed molecu-lar diagnostic cases

5. Monitoring of the effect the test results has on patientcare (i.e., chart review)

6. Average TAT for molecular diagnostic reports (fromtime of receipt in the laboratory to the time report isresulted)

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2. Medicare, Medicaid and CLIA Programs; Laboratory RequirementsRelating to Quality Systems and Certain Personnel Qualifications;Final Rule (January 24, 2003) Fed. Regi.FF 68:3640, 2003.

3. American College of Medical Genetics (ACMG). Standards andrrGuidelines for Clinical Genetics Laboratories. 3rd ed. Avalable athttp://www.acmg.net.

4. College of American Pathology. Molecular pathology checklist.Avalable at http://www.cap.org.

5. NCCLS. Molecular Diagnostic Methods for Genetic Diseases;Approved Guideline. NCCLS document MM1-A. NCCLS, Wayne,PA, 2000.


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Documents

Molecular Diagnostics || Framework for Quality Assurance in Molecular Diagnostics