molecular pathology checklist · molecular pathology checklist 09/25/2012 edition the following...

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Molecular Pathology Checklist

CAP Accreditation Program

College of American Pathologists 325 Waukegan Road Northfield, IL 60093-2750 www.cap.org

09.25.2012

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Molecular Pathology Checklist 09.25.2012

Disclaimer and Copyright Notice If you are enrolled in the CAP's Laboratory Accreditation Program and are preparing for an inspection, you must use the Checklists that were mailed in your application or reapplication packet, not those posted on the Web site. The Checklists undergo regular revision and Checklists may be revised after you receive your packet. If a Checklist has been updated since receiving your packet, you will be inspected based upon the Checklists that were mailed. If you have any questions about the use of Checklists in the inspection process, please e-mail the CAP ([email protected]), or call (800) 323-4040, ext. 6065. The checklists used in connection with the inspection of laboratories by the Laboratory Accreditation Program of the College of American Pathologists have been created by the College and are copyrighted works of the College. The College has authorized copying and use of the checklists by College inspectors in conducting laboratory inspections for the CLA and by laboratories that are preparing for such inspections. Except as permitted by section 107 of the Copyright Act, 17 U.S.C. sec. 107, any other use of the checklists constitutes infringement of the College’s copyrights in the checklists. The College will take appropriate legal action to protect these copyrights. All Checklists are ©2012. College of American Pathologists. All rights reserved.

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Molecular Pathology Checklist

TABLE OF CONTENTS

SUMMARY OF CHANGES .................................................................................................................................. 5 UNDERSTANDING THE 2010 CAP ACCREDITATION CHECKLIST COMPONENTS ..................................... 8 HOW TO INSPECT USING R.O.A.D INSPECTION TECHNIQUES................................................................... 9 INTRODUCTION ............................................................................................................................................... 10 DEFINITION OF TERMS ................................................................................................................................... 10 APPLICABILITY ................................................................................................................................................. 12 QUALITY MANAGEMENT AND QUALITY CONTROL ..................................................................................... 12

GENERAL ISSUES ........................................................................................................................................ 12 PROCEDURE MANUAL ................................................................................................................................ 13 ASSAY VALIDATION ..................................................................................................................................... 13 COLLECTION, TRANSPORT, PREPARTION, AND STORAGE OF SPECIMENS...................................... 17 QUANTITATIVE ASSAYS: CALIBRATION AND STANDARDS ................................................................... 22 REAGENTS ................................................................................................................................................... 27 CONTROLS ................................................................................................................................................... 28 AMPLIFICATION ............................................................................................................................................ 33 METHODS AND INSTRUMENT SYSTEMS.................................................................................................. 34

Restriction Endonucleases ......................................................................................................................... 34 Sequencing ................................................................................................................................................. 35 Next Generation Sequencing ..................................................................................................................... 36

Analytical Wet Bench Process ................................................................................................................ 37 Bioinformatics Pipeline for NGS ............................................................................................................. 39

Electrophoresis ........................................................................................................................................... 42 Polymerase Chain Reaction (PCR) ............................................................................................................ 43 Arrays ......................................................................................................................................................... 44 Parentage and Forensic Identity Testing.................................................................................................... 45 Fluorescence and Non-Fluorescence In Situ Hybridization (FISH, ISH) ................................................... 47 Brightfield In Situ Hybridization .................................................................................................................. 50 Spectrophotometers ................................................................................................................................... 50 Signal Detection Instruments ..................................................................................................................... 51 Film Processing/Photographic Equipment ................................................................................................. 52 Pipettes ....................................................................................................................................................... 53 Thermometers and Temperature-Dependent Equipment .......................................................................... 53 Instrument Maintenance ............................................................................................................................. 54

POST ANALYSIS ........................................................................................................................................... 56 Results Reporting ....................................................................................................................................... 56 Records ...................................................................................................................................................... 60

PERSONNEL ..................................................................................................................................................... 61 LABORATORY SAFETY ................................................................................................................................... 62

RADIATION SAFETY ..................................................................................................................................... 63

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SUMMARY OF CHECKLIST EDITION CHANGES Molecular Pathology Checklist

09/25/2012 Edition

The following lists of requirements provide information on what has changed in this edition of the checklist, or in the previous edition. This information is provided in three categories:

1. New — requirements that have been added 2. Revised — requirements listed in this section fall into two categories:

● A major change to a requirement or a note that would necessitate a change in procedure for the laboratory

● A change to the Phase 3. Deleted/Moved/Merged — requirements listed in this section fall into three categories:

● Deleted — requirements that have been removed

● Moved — requirements that have been relocated from this checklist into another checklist, or have been moved within this checklist and given a new checklist requirement number (resequenced)

● Merged — requirements that have been combined with a similar requirement in the checklist If this checklist was created for an on-site inspection or self-evaluation, it has been customized based on the laboratory's activity menu. The listing below is comprehensive; therefore, some of the requirements included may not appear in the customized checklist. Such requirements are not applicable to the testing performed by the laboratory. Note: For the detail of the changes, refer to the "Changes Only" document which may be found on the CAP website through e-LAB Solutions (Laboratory Accreditation Program Master and Custom Checklists). To access this document select "Changes Only" from the Checklist Type drop-down menu. The "Changes Only" document contains the text of new and deleted checklist requirements, major and minor requirement revisions, and changes to explanatory text. These changes are presented, in order, as they appear in the checklist. Major requirement revisions will display a "Revised" flag. Minor revisions will not display a "Revised" flag and are defined as those editorial changes that are not likely to affect your laboratory operations, but are worded to better convey the intent of the requirement. Changes appear in redline/strikeout format that compares the previous checklist edition to this edition. Requirements that have been moved or merged will appear at the end of that file. NEW Checklist Requirements Requirement Effective Date MOL.31710 07/11/2011 MOL.34220 07/31/2012 MOL.34934 07/31/2012 MOL.34936 07/31/2012 MOL.34938 07/31/2012 MOL.34940 07/31/2012 MOL.34944 07/31/2012 MOL.34946 07/31/2012 MOL.34948 07/31/2012 MOL.34952 07/31/2012 MOL.34954 07/31/2012 MOL.34956 07/31/2012 MOL.34958 07/31/2012

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MOL.34960 07/31/2012 MOL.34962 07/31/2012 MOL.34964 07/31/2012 MOL.34966 07/31/2012 MOL.34968 07/31/2012 MOL.34970 07/31/2012 MOL.34972 07/31/2012 REVISED Checklist Requirements Requirement Effective Date MOL.20550 07/31/2012 MOL.30670 07/11/2011 MOL.30785 07/31/2012 MOL.31245 07/31/2012 MOL.31705 07/31/2012 MOL.32370 07/11/2011 MOL.32430 07/11/2011 MOL.32435 07/11/2011 MOL.33778 07/11/2011 MOL.34003 07/11/2011 MOL.34229 07/31/2012 MOL.34495 07/11/2011 MOL.34900 07/11/2011 MOL.39288 07/11/2011 MOL.39323 09/25/2012 MOL.39393 09/25/2012 MOL.46258 07/11/2011 MOL.49550 07/11/2011 MOL.49575 07/11/2011 MOL.49600 07/11/2011 MOL.49615 07/11/2011 MOL.49640 07/11/2011 MOL.54570 07/11/2011 DELETED/MOVED/MERGED Checklist Requirements Requirement Effective Date MOL.05075 07/10/2011 MOL.10150 07/10/2011 MOL.10160 07/10/2011 MOL.10170 07/10/2011 MOL.10200 07/10/2011 MOL.13466 07/10/2011 MOL.16732 07/10/2011 MOL.20000 07/30/2012 MOL.20050 07/30/2012 MOL.29290 07/30/2012 MOL.29405 07/10/2011 MOL.29520 07/10/2011 MOL.29750 07/10/2011 MOL.29865 07/10/2011 MOL.29980 07/10/2011 MOL.30095 07/10/2011 MOL.30210 07/10/2011

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MOL.30325 07/10/2011 MOL.30680 07/10/2011 MOL.30957 07/10/2011 MOL.31475 07/10/2011 MOL.31935 07/10/2011 MOL.32050 07/10/2011 MOL.32300 07/10/2011 MOL.33050 07/10/2011 MOL.33819 07/10/2011 MOL.34065 07/30/2012 MOL.34106 07/30/2012 MOL.34147 07/30/2012 MOL.34850 07/10/2011 MOL.34930 07/10/2011 MOL.35942 07/10/2011 MOL.38862 07/10/2011 MOL.39572 07/30/2012 MOL.40666 07/30/2012 MOL.43928 07/10/2011 MOL.45792 07/10/2011 MOL.47190 07/10/2011 MOL.48122 07/10/2011 MOL.50000 07/10/2011 MOL.56380 07/10/2011

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UNDERSTANDING THE CAP ACCREDITATION CHECKLIST COMPONENTS

To provide laboratories with a better means to engage in and meet their accreditation requirements, the CAP has enhanced the checklist content and updated its design. New components containing additional information for both the laboratory and inspectors include Subject Headers, Declarative Statements and Evidence of Compliance. See below for a definition of each new feature as an example of how they appear in the checklists.

Using Evidence of Compliance (EOC)

This component, which appears with several checklist requirements, is intended to:

1 Assist a laboratory in preparing for an inspection and managing ongoing compliance 2 Drive consistent understanding of requirements between the laboratory and the inspector 3 Provide specific examples of acceptable documentation (policies, procedures, records, reports,

charts, etc.) Evidence of Compliance suggests ways to document compliance with checklist requirements. Other types of documentation may be acceptable. Whenever a policy/procedure/process is referenced within a requirement, it is only repeated in the Evidence of Compliance if such statement adds clarity. All policies/procedures/processes covered in the CAP checklists must be documented. A separate policy is not needed for each item listed in EOC as it may be referenced in an overarching policy.

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HOW TO INSPECT USING R.O.A.D INSPECTION TECHNIQUES (Read, Observe, Ask, Discover)

CAP has streamlined the inspection approach used during onsite inspections and is now offering guidance to inspectors by providing assessment techniques to facilitate a more efficient, consistent, and effective inspection process. Specific inspector instructions are listed at the beginning of a grouping of related requirements. Rather than reviewing each individual requirement, CAP inspectors are encouraged to focus on the Inspector Instructions for a grouping of related requirements. Once an area of concern has been identified through "Read," "Observe," "Ask," "Discover," or a combination thereof, inspectors are encouraged to "drill down" to more specific requirements, when necessary and review more details outlined in the Evidence of Compliance statements. If a requirement is non-compliant, circle the requirement number to later list on the Inspector Summation Report. Inspectors may also make notes in the margins of the checklist document. Inspector Instructions and Icons used to evaluate a laboratory's performance now appear in several areas throughout the Inspector Checklists. Please note that all four R.O.A.D elements are not always applicable for each grouping, or sections of related requirements.

Inspector Instructions:

READ/review a sampling of laboratory documents. Information obtained from this review will be useful as you observe processes and engage in dialogue with the laboratory staff. (Example of the complimentary inspector instructions for Quality Management/Quality Control General Issues section appearing across checklists):

● Sampling of QM/QC policies and procedures

● Incident/error log and corrective action

OBSERVE laboratory practices by looking at what the laboratory personnel are actually doing and note if practice deviates from the documented policies/procedures. (Example)

● Observe the settings/QC range limits established in the laboratory LIS/HIS to ensure that the laboratory's stated ranges are accurately reflected

ASK open-ended, probing questions that start with phrases such as "tell me about..." or "what would you do if..." This approach can be a means to corroborate inspection findings that were examined by other techniques, such as Read & Observe. Ask follow-up questions for clarification. Include a variety of staff levels in your communication process. (Example)

● As a staff member, what is your involvement with quality management?

● How do you detect and correct laboratory errors?

DISCOVER is a technique that can be used to "drill down" or further evaluate areas of concern uncovered by the inspector. "Follow the specimen" and "teach me" are two examples of Discovery. Utilizing this technique will allow for the discovery of pre-analytic, analytic, and post-analytic processes while reviewing multiple requirements simultaneously. (Example)

● Select several occurrences in which QC is out of range and follow documentation to determine if the steps taken follow the laboratory policy for corrective action

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INTRODUCTION

Inspection of a molecular pathology laboratory requires special knowledge of the science. Ideally, the inspector should be an actively practicing molecular scientist familiar with the Checklist and possessing the technical and interpretive skills necessary to evaluate the quality of the laboratory's performance. If the team leader's laboratory performs similar molecular pathology services as the inspected lab, the inspecting laboratory's molecular pathology section director or section supervisor is a qualified inspector. If the team leader has no such resource, the list of qualified regional inspectors included in the Inspector's Inspection Packet should be consulted.

An inspection of a laboratory section, or department will include the discipline-specific checklist(s), the Laboratory General Checklist, and the All Common Checklist. In response to the ongoing request to reduce the redundancy within the Accreditation Checklists, the CAP accreditation program is introducing the All Common Checklist (COM). The purpose of the All Common Checklist is to group together those requirements that were redundant in Laboratory General and the discipline-specific checklists. Therefore, the CAP centralized all requirements regarding: proficiency testing, procedure manuals, test method validations, and critical results into one checklist, the COM checklist. Note for non-US laboratories: Checklist requirements apply to non-US laboratories unless the checklist items contain a specific disclaimer of exclusion.

DEFINITION OF TERMS

Analytical measurement range (AMR) validation - the process of confirming that the assay system will correctly recover the concentration or activity of the analyte over the AMR Annual - every 12 calendar months Biennial - every 24 calendar months Calibrator, historical - the set of archived results of a single-point calibrator that demonstrates stability of the assay over time Credentialing - the process of obtaining, verifying, and assessing the qualifications of a practitioner to provide care in a health care organization Digital image analysis - the computer-assisted detection or quantification of specific features in an image following enhancement and processing of that image, including immunohistochemistry, DNA analysis, morphometric analysis, and in situ hybridization Examination - in the context of checklist requirements, examination refers to the process of inspection of tissues and samples prior to analysis. An examination is not an analytical test. FDA - in the context of checklist requirements, FDA should be taken to mean the national, state, or provincial authority having jurisdiction over in vitro diagnostic test systems High complexity - rating given by the FDA to commercially marketed in vitro diagnostic tests based on their risks to public health. Tests in this category are seen to have the highest risks to public health.

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Moderate complexity - rating given by the FDA to commercially marketed in vitro diagnostic tests based on their risks to public health Nonwaived - tests categorized as either moderately complex (including provider-performed microscopy) or highly complex by the US Food and Drug Administration (FDA), according to a scoring system used by the FDA Reagent - any substance in a test system other than a solvent or support material that is required for the target analyte to be detected and its value measured in a sample Semiannual - every 6 calendar months Telepathology - the practice in which the pathologist views digitized or analog video or still image(s), and renders an interpretation that is included in a formal diagnostic report or document in the patient record Test system - the process that includes pre-analytic, analytic, and post-analytic steps used to produce a test result or set of results. A test system may be manual, automated, multi-channel or single-use and can include reagents components, equipment or instruments required to produce results. A test system may encompass multiple identical analyzers or devices. Different test systems may be used for the same analyte. Waived - a category of tests defined as "simple laboratory examinations and procedures which have an insignificant risk of an erroneous result." Laboratories performing waived tests are subject to minimal regulatory requirements.

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APPLICABILITY

The Molecular Pathology Checklist covers clinical molecular genetic testing in the areas of oncology, hematology, inherited disease, HLA typing, forensics and parentage applications. The inspection of laboratories performing such molecular testing requires the Molecular Pathology checklist, except that the Cytogenetics or Anatomic Pathology checklist (as appropriate) may be used to inspect fluorescence in situ hybridization (FISH), when such testing is performed in the cytogenetics, cytopathology or anatomic pathology section. Also, the Anatomic Pathology checklist may be used to inspect in situ hybridization (ISH), when ISH testing is performed in the anatomic pathology or cytopathology section. The Microbiology checklist must be used to inspect laboratories that perform molecular testing for infectious disease (both for FDA-cleared/approved tests, and non-FDA-cleared/approved tests).

QUALITY MANAGEMENT AND QUALITY CONTROL

GENERAL ISSUES


● Sampling of TAT records

MOL.20300 Turnaround Time Phase I

There is evidence that the laboratory monitors sample turnaround times and that they are appropriate for the intended purpose of the test.

NOTE: Appropriate turnaround times will vary by test type and clinical application. There are certain clinical situations in which rapid completion is essential. For example, inappropriate delays in completing a prenatal diagnosis test can cause unacceptable emotional stress for the parents, make ultimate pregnancy termination (if chosen) much more difficult, or even render the results of the test unusable.

Evidence of Compliance:

✓ Written policy defining turnaround time and mechanism for monitoring AND

✓ Records showing that times defined in the policy are routinely met

**REVISED** 07/31/2012 MOL.20550 Test Result Statistics Phase I

When appropriate, statistics on molecular pathology test results (e.g. percentages of normal and abnormal findings) are maintained, and appropriate comparative studies performed.

NOTE: Periodic review of test result statistics can be used to identify changes in test performance. This process may detect systemic errors.

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✓ Written procedure for calculating statistics AND

✓ Records of statistical data, evaluation and corrective action if indicated

PROCEDURE MANUAL


● Representative sample of procedures for completeness. Current practice must match contents of procedures/policies.

MOL.30440 Calculations for Quantitative Tests Phase II

For quantitative molecular tests, methods for calculating quantitative values adequately are described and units clearly documented.

NOTE: Quantitative testing requires that the dynamic range of the assay be defined and assay performance tested with controls in each run, including a negative, low positive, and high positive control. When melting curves are generated, there should be criteria for interpreting results.

MOL.30555 Analytic Interpretation Guidelines Phase II

There are written guidelines for analytic interpretation of results.

NOTE: For a qualitative assay, the procedure manual should describe, for example, the expected band pattern, melting temperature, or numeric cutoff to distinguish a positive from a negative result. For a quantitative assay, the manual should describe, for example, the criteria for verifying test performance characteristics of the run (e.g. assay sensitivity and linearity are within pre-established range, there is no significant inhibitor of the patient reaction, the calculated value appears reasonable from visual inspection of raw data) prior to releasing the quantitative result.

**REVISED** 07/11/2011 MOL.30670 Intended Use of Assay Phase II

The procedure manual describes the intended use of the assay for ordering the tests in patient management, with pertinent literature references.

REFERENCES 1) ISO 14971:2007(E). Medical devices - Application of risk management to medical devices. 2nd Edition. 2007-03-01

ASSAY VALIDATION

The analytical performance characteristics of the assay must be verified or established in accordance with the requirements in the Test Method Validation section of the All Common Checklist. This includes accuracy, precision, reference range, reportable range, analytical sensitivity, and analytical specificity.

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Analytic sensitivity refers to the ability of an assay to detect a given analyte (i.e. the lower limit of detection; for example, the ability to detect a clonal immunoglobulin gene rearrangement comprising only 1-5% of the cell population). Analytic specificity refers to the false-positive rate in addition to the degree to which interfering substances are not detected by an assay. The laboratory may also need to establish other performance characteristics such as linearity, specimen stability or carryover, when applicable. In addition, the laboratory should determine (or be able to cite literature that addresses) the clinical/diagnostic sensitivity and specificity of the assay. Clinical sensitivity (also called diagnostic sensitivity) refers to the ability of an assay to detect a disease or clinical condition, while clinical specificity refers to the degree to which an assay is negative when disease is absent. Diagnostic sensitivity and specificity must be determined relative to some "gold standard" (e.g. biopsy findings, clinical findings, etc.). The sensitivity of an assay equals [TP/(TP+FN)] X 100 and the specificity of an assay equals [TN/(TN+FP)] X 100. (TP=true positive, TN=true negative, FN=false negative, FP=false positive.) Determinations of sensitivity and specificity should be done in a "blinded" fashion (i.e. without prior knowledge of the patient's disease status). For some disorders, it may not be possible to identify large numbers of positives (i.e. patients with the disorder) to establish the diagnostic sensitivity of the assay. In such instances, the laboratory should procure as many positive cases as is reasonably possible for method validation and in addition cite any publications that have investigated the diagnostic sensitivity of the assay. A laboratory-developed test (LDT) is defined as follows: A test used in patient management that has all of the following characteristics:

● The test is performed by the clinical laboratory in which the test was developed

● The test is neither FDA-cleared nor FDA-approved, or is an FDA-cleared/approved test modified by the laboratory (sample types or the use of collection devices not listed in manufacturer instructions constitute modifications, by this definition)

● The test was first used for clinical testing after April 23, 2003 A laboratory is considered to have developed a test if the test procedure was created by the laboratory performing the testing, irrespective of whether fundamental research underlying the test was developed elsewhere or reagents (including ASRs), equipment, or technology integral to the test were purchased, adopted, or licensed from another entity. Reference: Lawrence Jennings, Vivianna M. Van Deerlin, Margaret L. Gulley (2009) Recommended Principles and Practices for Validating Clinical Molecular Pathology Tests. Archives of Pathology & Laboratory Medicine: Vol. 133, No. 5, pp. 743-755.


● Sampling of assay validation studies, including comparisons and appropriate sample types

● Sampling of assay validation studies for LDTs introduced since last on-site inspection

● Sampling of laboratory-developed patient test reports including methodology, statement and performance characteristics

● How does your laboratory validate assay performance prior to test implementation?

● How does your laboratory validate clinical claims made by the laboratory about LDT's?

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**REVISED** 07/31/2012 MOL.30785 Validation Studies - LDTs or Modified FDA-Approved/Cleared Tests Phase II

There is documentation that the laboratory has performed analytic validation studies to establish the performance characteristics (including accuracy, precision, reportable range, reference range, analytic sensitivity, and analytic specificity).

NOTE: A summary of the validation data for all laboratory-developed tests introduced since the last on-site inspection should be available to the inspector. The inspector should also check validation data for assays introduced prior to the last on-site inspection.


✓ Written procedure for performing validation studies AND

✓ Validation records for molecular LDTs REFERENCES 1) Association for molecular pathology statement. Recommendations for in-house development and operation of molecular diagnostic

tests. Am J Clin Pathol. 1999;111:449-463 2) Department of Health and Human Services, Centers for Medicare & Medicaid Services. Clinical laboratory improvement amendments

of 1988; final rule. Fed Register. 2003(Jan 24): [42CFR493.1253(b)(2)] 3) Lawrence Jennings, Vivianna M. Van Deerlin, Margaret L. Gulley (2009) Recommended Principles and Practices for Validating

Clinical Molecular Pathology Tests. Archives of Pathology & Laboratory Medicine: Vol. 133, No. 5, pp. 743-755 4) Halling KC, Schrijver I, Persons DL (2012) Test Verification and Validation for Molecular Diagnostic Assays: Archives of Pathology &

Laboratory Medicine: Vol. 136, No. 1, pp 11-13

MOL.30900 Validation Studies - Specimen Selection Phase II

Validation studies include specimens representing each of the possible reportable results (genotypes).

NOTE: Assays for genetic disorders with a limited number of possible genotypes (e.g. hereditary hemochromatosis) should confirm the ability of the assay to detect these genotypes. Assays for genetic disorders with considerable allelic heterogeneity and/or significant numbers of private mutations (e.g. cystic fibrosis or hereditary nonpolyposis colorectal cancer) should confirm the accuracy of the methodology used to ensure that the assay will detect a high percentage of the possible genotypes. However, it will not be possible to document that such assays can detect every possible genotype.


✓ Records of validation studies

MOL.31015 Validation Studies - Specimen Types Phase II

Validation studies with an adequate number and representative (reasonable) distribution of samples are performed for each type of specimen expected for the assay (e.g. blood, fresh/frozen tissue, paraffin-embedded tissue, prenatal specimens).


✓ Records of validation studies

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MOL.31130 Validation Study Comparison Phase II

The results of each validation study are compared to another valid assay, such as comparison to another test method or specimen exchange with a laboratory performing the same type of test.

NOTE: There may not be a closely related assay to be used for comparison. In such cases, the assay performance (sensitivity and specificity) should be assessed in relation to the patient's clinical diagnosis and in addition assessed by exchanges of specimens with a laboratory that performs the assay in a similar fashion.


✓ Records of comparison of each validation study to another test method OR records of comparison using specimen exchange with another laboratory

**REVISED** 07/31/2012 MOL.31245 Reference/Reportable Range - Qualitative Phase II

For qualitative assays, the reference and reportable ranges are defined.

NOTE: For qualitative assays (e.g. assays for germline mutations), the laboratory must define the reference value (normal versus abnormal result) and reportable outcomes (e.g. homozygous wild type, heterozygous or homozygous mutant). If the reference value depends on the clinical situation, then a plan for interpreting the patient result must be defined.


✓ Written procedure defining reference and reportable ranges for each test REFERENCES 1) American College of Medical Genetics Laboratory. Standards and Guidelines for Clinical Genetics Laboratories, 3rd ed. Bethesda,

MD: ACMG, 2003. Available at: http://www.acmg.net Accessed 2006 2) NCCLS. Fluorescence In Situ Hybridization (FISH) Methods for Medical Genetics; Approved Guideline. NCCLS document MM7-A

(ISBN 1-56238-524-0). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 1908-1898 USA, 2004

MOL.31360 Reference/Reportable Range - Quantitative Phase II

For quantitative assays, the reference and reportable ranges are defined.

NOTE: Reference and reportable ranges are pertinent to quantitative assays (e.g. assays for tumor burden, or chimerism). The “reference range” is the range of results expected in the “normal” population, while the “reportable range” encompasses the full range of reported values. The laboratory must define the analytic measurement range (AMR) as described in the “Quantitative Assays; Calibration and Standards” section of the checklist. The laboratory must also determine how to handle positive patient results below or above the AMR, since numerical values outside the AMR may be inaccurate. For example, these may be reported a <x or>y, or they may be reported as “low positive” or “ high positive” along with an explanation that values outside the linear range cannot be quantitated, or the sample may be concentrated or diluted and rerun to calculate an accurate value within the reportable range.


✓ Written procedure defining reference and reportable range for each test

MOL.31590 Clinical Performance Characteristics Phase II

The clinical performance characteristics of each assay are documented, using either literature citations or a summary of internal study results.

NOTE: The clinical performance characteristics of a test relate to its diagnostic sensitivity and

http://www.acmg.net/

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specificity, and its positive and negative predictive values in the (various) target population(s). Issues that affect the clinical interpretation of a test which should be considered include (1) the clinical setting in which the test is used, (2) genotype/phenotype associations when these vary with particular mutations or polymorphisms, and (3) genetic, environmental or other factors which modify the clinical expression of the genetic alteration detected.

Establishing clinical validity may require extended studies and monitoring that go beyond the purview or control of the individual laboratory. The laboratory should perform clinical validation in-house, except in the case of very rare conditions, in which case data from the literature can be used, or in the case of very common conditions for which the clinical validity is well-established in the literature. For FDA-approved/cleared tests, literature or manufacturer data can be used in all cases. It is essential that directors use their professional judgment in evaluating the results of such studies and in monitoring the state-of-the-art worldwide as it applies to newly discovered gene targets and potential new tests, especially those of a predictive or incompletely penetrant nature. REFERENCES 1) American College of Medical Genetics, Standards and Guidelines for Clinical Genetics Laboratories, Third Edition, 2002, Section C8 2) Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Genetic Diseases; Approved Guideline—

Second Edition. CLSI document MM1-A2 (ISBN 1-56238-615-8). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006

**REVISED** 07/31/2012 MOL.31705 LDT Reporting Phase II

Prior to implementing a laboratory-developed test (LDT), reports are designed to contain a description of the method, a statement that the assay was developed by the laboratory, and appropriate performance characteristics.

NOTE: General guidelines for reports are given in the Results Reporting section of this checklist. Laboratories subject to US regulations often include LDT disclaimer as follows: "This test was developed and its performance characteristics determined by <insert laboratory/company name>. It has not been cleared or approved by the FDA. The laboratory is regulated under CLIA as qualified to perform high-complexity testing. This test is used for clinical purposes. It should not be regarded as investigational or for research." REFERENCES 1) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical

and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2011

**NEW** 07/11/2011 MOL.31710 LDT Clinical Claims Validation Phase II

All clinical claims made by the laboratory about a laboratory-developed test are validated.

NOTE: Clinical claims may include statements about a test's diagnostic sensitivity and specificity, ability to predict the risk of a disease or condition, clinical usefulness, or cost-effectiveness. Clinical claims may be found on the test report or in other information distributed by the laboratory (websites, test catalogues, newsletters, memoranda, advertisements, etc.). Laboratories are not required to make clinical claims about a test, but any claims made by the laboratory must be validated. In general, the laboratory should validate claims through a clinical study, but for rare conditions or well-accepted uses of a test, reference to published peer-reviewed literature is acceptable.


✓ Records of clinical studies performed by the laboratory OR peer-reviewed literature that reasonably substantiate all claims made by the laboratory about a test

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COLLECTION, TRANSPORT, PREPARATION, AND STORAGE OF SPECIMENS


● Sampling of requisition forms for completeness

● Sampling of nucleic acid extraction policies and procedures

● Sampling of nucleic acid measurement records

● Sampling of RNA assessment records/false negative rate records

● Sampling of molecular pathology specimen processing, handling, aliquoting, storage, and retention policies and procedures

● Sampling of specimen rejection records/logs

● Sampling of molecular pathology specimens (labeling)

● What is your course of action when you receive unacceptable molecular pathology specimens?

● How does your laboratory ensure RNase-free conditions are maintained?

● How does your laboratory ensure sample adequacy?

● How does your laboratory ensure specimen identity throughout all phases of analysis?

MOL.32350 Requisition Information Phase II

Test requests are accompanied with a pedigree and/or racial/ethnicity, when appropriate (e.g. for linkage analysis).


✓ Specimen requisitions/collection forms REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7162 [42CFR493.1241(c)]

MOL.32355 Specimen ID Phase II

There is a system to positively identify all patient specimens, specimen types, and aliquots through all phases of the analysis, including specimen receipt, nucleic acid extraction, nucleic acid quantification, hybridization, detection, documentation, and storage.

NOTE: Each specimen container must identify the patient uniquely. Identification may be text-based, numeric, bar-coded, etc. The form of this system is entirely at the discretion of each laboratory, so long as all primary collection containers and their aliquots have a unique label which one can trace back to full particulars of patient identification, collection date, specimen type, etc.

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MOL.32360 Specimen Handling Phase II

Procedures are documented to prevent specimen loss, alteration, or contamination.

REFERENCES 1) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


MOL.32365 Specimen Preservation/Storage Phase II

There is a documented procedure describing methods for specimen preservation and storage before testing, consistent with good laboratory practice.

REFERENCES 1) Schultz CL, et al. A lysis, storage, and transportation buffer for long-term, room-temperature preservation of human clinical lymphoid

tissue samples yielding high molecular weight genomic DNA suitable for molecular diagnosis. Am J Clin Pathol. 1999;111:748-752 2) Makowski GS, et al. In situ PCR amplification of Guthrie card DNA to detect cystic fibrosis mutations. Clin Chem. 1996;41:471-479 3) Farkas DH, et al. Specimen stability for DNA-based diagnostic testing. Diagn Mol Pathol. 1996;5:227-235 4) Kaul K, et al. Amplification of residual DNA sequences in sterile bronchoscopes leading to false-positive PCR results. J Clin Microbiol.

1996;34:1949-1951 5) Kessler HH, et al. Effects of storage and types of blood collection tubes on hepatitis C virus level in whole blood samples. J Clin

Microbiol. 2001;39:1788-1790 6) Tsui NMY, et al. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem. 2002;48:1647-1653 7) Rainen L, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem. 2002;48:1883-1890 8) Pahl A, Brune K. Stabilization of gene expression profiles in blood after phlebotomy. Clin Chem. 2002;48:2251-2253 9) Clinical and Laboratory Standards Institute (CLSI), Collection, Transport, Preparation, and Storage of Specimens for Molecular

Methods; Approved Guideline. CLSI document MM13-A (ISBN 1-56238-591-7). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005

**REVISED** 07/11/2011 MOL.32370 Specimen Rejection Criteria Phase II

There are written criteria for rejection of unacceptable or possibly commingled specimens.

NOTE: Unacceptable specimens include those that are mislabeled, submitted in the wrong anticoagulant or fixative, or are a specimen type that has not been validated by the laboratory. In addition, because of the high sensitivity and potential for contamination in molecular testing involving amplification of DNA, the laboratory must be alert to the possibility of commingled specimens. An example of a potentially commingled specimen is one that is received after the specimen container was entered by a sampling device that enters multiple samples, albeit with rinses in between specimens. If such samples must be tested by molecular methods, the results should be interpreted with caution, considering the potential for contamination. In such situations, the laboratory should include a note in the report stating the issue.


✓ Records of rejected specimens

MOL.32375 Physician Notification Phase II

The submitting physician (or requester) is promptly notified when a specimen is inadequate or if insufficient nucleic acid is isolated.


✓ Records of physician notification of inadequate specimen in patient record or log

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MOL.32380 Disposition of Unacceptable Specimens Phase II

The disposition of all unacceptable specimens is documented in the patient report and/or quality management records.

NOTE: This information is essential to proper patient test management and to the laboratory quality management program.

MOL.32385 Specimen Aliquots Phase II

If aliquoting of specimens is performed, there is a written procedure to prevent any possible cross-contamination of the specimens.

NOTE: Although in some cases it may be appropriate to aliquot a specimen, the laboratory must have a policy that no aliquot is ever returned to the original container.

MOL.32390 Specimen Processing/Storage Phase II

Patient samples are processed promptly or stored appropriately to minimize degradation of nucleic acids.


✓ Written procedure for processing and storage of specimens REFERENCES 1) Farkas DH, Kaul KL, Wiedbrauk DL, et al. Specimen Collection and Storage for Diagnostic Molecular Pathology Investigation. Arch

Pathol Lab Med. 1996;120:591-596 2) Kiechle FL, Kaul KL, Farkas DH. Mitochondrial Disorders: Methods and Specimen Selection for Diagnostic Molecular Pathology. Arch

Pathol Lab Med. 1996;120:597-603 3) Farkas DH, Drevon AM, Kiechle FL, et al. Specimen Stability for DNA-based Diagnostic Testing. Diag Molec Pathol. 1996;5(4):227-

235

MOL.32395 Neoplastic Cell Content Phase II

For paraffin embedded tumor specimens from which DNA is extracted for analysis (e.g. microsatellite instability, KRAS or KIT analysis), there is documentation of histological assessment of neoplastic cell content.

NOTE: In addition to confirming the presence or absence of neoplastic cells by a pathologist, it may be necessary for some assays to assess neoplastic cellularity to insure that the percentage of neoplastic cells exceeds the limit of detection for the assay.

A corresponding H&E section from the same tissue block used for DNA extraction may be used to assess sample adequacy. Alternatively, a stain such as toluidine blue may be used to stain the slide that is being used for DNA extraction. When assessment of sample adequacy is performed outside of the testing laboratory, documentation of such assessment should accompany the sample.

MOL.32425 Nucleic Acid Extraction/Isolation/Purification Phase II

Nucleic acids are extracted, isolated, and purified by methods reported in the literature, by an established commercially available kit or instrument, or by validation of a method developed in-house.

NOTE: Extraction procedures may combine purification or isolation of nucleic acids according to the level of purity needed for downstream applications.


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✓ Records to support nucleic acid extraction/purification is performed by a validated method REFERENCES 1) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


**REVISED** 07/11/2011 MOL.32430 Nucleic Acid Quantity Phase II

The quantity of nucleic acid is measured, when appropriate.

NOTE: The quantity of nucleic acid must be measured prior to use in a procedure whose success generally depends on accurately determining the concentration/quantity of the nucleic acid.


✓ Written procedures defining conditions under which quantity of nucleic acid is measured AND

✓ Records of nucleic acid measurement

**REVISED** 07/11/2011 MOL.32435 Nucleic Acid Quality Phase II

The integrity and purity of nucleic acid is assessed, when appropriate.

NOTE: RNA in specimens is highly labile because RNase is ubiquitous and difficult to inhibit. For human RNA targets, RNA quality must be assessed. An appropriate “housekeeping” mRNA should be assessed as an internal control for RNA integrity. However, depending on the target, it may not be necessary for all specimens to be assessed for RNA quality.


✓ Records of nucleic acid quality assessment REFERENCES 1) Tsui NBY, Ng EKO, Lo YMD. Stability of Endogenous and Added RNA in Blood Specimens, Serum and Plasma. Clin Chem 48:1647-

1653,2002 2) Farrell R. Gel electrophoresis based assessment of cellular RNA quality may also be used (RNA Isolation Strategies). In: RNA

Methodologies: A Laboratory Guide for Isolation and Characterization. Academic Press, 1998

MOL.32440 Ribonuclease-Free Conditions Phase I

Ribonuclease-free conditions are maintained for all assays that detect RNA or use an RNA probe.

NOTE: RNA is extremely susceptible to degradation by ribonucleases that are ubiquitous in the environment. To ensure preservation of target RNA or RNA probes, special precautions are needed.


✓ Written procedure defining environmental requirements for RNase-free conditions AND

✓ Records that RNase-free conditions are maintained (i.e. wipe test in event of contamination incident) with corrective action if conditions are not met

REFERENCES 1) Gulley ML, et al. Guidelines for interpreting EBER in situ hybridization and LMPI immunohistochemical tests for detecting Epstein-

Barr virus in Hodgkin lymphoma. Am J Clin Pathol. 2002;117:259-267

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MOL.32445 Concentration Techniques Phase I

Concentration techniques for quantitative tests are verified.

NOTE: Techniques used to concentrate specimens for analysis must be verified at specified, periodic intervals (not to exceed one year or manufacturer's recommendations).


✓ Written procedure for verifying the accuracy of concentration techniques AND

✓ Records of concentration technique verification documented at defined frequency

MOL.33150 Specimen Storage Phase II

Stored specimens are maintained in a way to allow prompt retrieval for further testing.

MOL.33250 Specimen Retention Phase II

Specimens are retained in compliance with applicable laws and regulations.

NOTE: CAP retention guidelines may be found in the Quality Management section of the Laboratory General checklist, and also at http://www.cap.org/. However, state or local laws and/or regulations may be more stringent than CAP guidelines.

Retention of fluorochrome-stained slides should be determined by the laboratory director.


✓ Written retention policy

QUANTITATIVE ASSAYS: CALIBRATION AND STANDARDS

CALIBRATION is the set of operations that establish, under specified conditions, the relationship between reagent system/instrument response and the corresponding concentration/activity values of an analyte. During the validation of a quantitative assay, calibrators are used to generate a calibration curve that spans the analytical measurement range (AMR) to assess accuracy, linearity, limit of detection (LOD) and limit of quantification (LOQ). Unlike standards used to generate a standard curve, calibrators must have a matrix appropriate for the clinical specimens assayed by that method. For example, an assay that measures copies of RNA transcript would require calibrators that consist of RNA target in an appropriate matrix such as total RNA. After validation, it is necessary to periodically verify the accuracy and AMR of quantitative assays. This could be done by running a calibration curve or more simply by testing 2 or more calibrators. By choosing calibrators at or near the limits of detection/quantification, the AMR can also be verified. (For Laboratories subject to US regulations, note that the term "calibration verification," as used in the Checklists, carries a more restrictive meaning that in CLIA. As defined in the January, 2003 revision of CLIA, "calibration verification" refers to 2 distinct processes: 1) verification of correct method calibration and 2) validation of the reportable range. This checklist restricts the use of the term "calibration verification" to the first process. The checklist uses a different term, "analytical measurement range (AMR) validation" to refer to the second process). Recalibration or calibration/AMR verification must be performed at least once every six months. Successful calibration verification certifies that the calibration is still valid; unsuccessful calibration verification requires remedial action, which usually includes recalibration. The performance of recalibration or a calibration/AMR-verification procedure resets the calendar to a new maximum 6-month interval before the next reassessment. Methods that are recalibrated more frequently than every 6 months do not require a separate calibration/AMR verification procedure.

http://www.cap.org/

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In addition to the every 6 month requirement, laboratories must perform calibration or calibration/AMR verification at changes in major system components and at changes of chemically or physically active reagent lots (e.g. new master mix) unless the laboratory can demonstrate that changing reagent lot numbers does not affect the accuracy and AMR used to report patient/client test results. Manufacturers' instructions should be followed when provided. The laboratory should establish other criteria, as appropriate, for recalibration or calibration/AMR verification. These include, but are not limited to, failure of quality control to meet established criteria and major maintenance or service to the instrument. Each laboratory must define limits for accepting or rejecting results of calibration/AMR verification. Materials for calibration, calibration verification, and/or AMR must have a matrix appropriate for the clinical specimens assayed by that method, and target values appropriate for the measurement system. Materials may include, but are not limited to:

1. Calibrators used to calibrate the analytical measurement system 2. Materials provided by the analytical measurement system vendor for the purpose of calibration

verification 3. Previously tested unaltered patient/client specimens 4. Primary or secondary standards or reference materials with matrix characteristics and target values

appropriate for the method 5. Third party general purpose reference materials may be suitable for validation of calibration

following reagent lot changes if the material is documented in the package insert or by the method manufacturer to be commutable with patient specimens for the method. A commutable reference material is one that gives the same numeric result as would a patient specimen containing the same quantity of analyte in the analytic method under discussion; e.g. matrix effects are absent. Commutability between a reference material and patient specimens can be demonstrated using the protocol in CLSI EP14-A2

6. Proficiency testing material or proficiency testing validated material with matrix characteristics and target values appropriate for the method

7. QC material if (and only if) the material has matrix characteristics appropriate for the method and a peer group established mean value based on interlaboratory comparison that is method specific and includes data from at least 10 different laboratories.


● Sampling of calibration and AMR policies and procedures

● Sampling of calibration/calibration verification records

● Sampling of AMR validation records

● Biannual instrument correlation records

● Sampling of calibration materials (labeling, storage, grade quality)

● What is your course of action if calibration is unacceptable?

● When was the last time you performed a calibration procedure and how did you verify the calibration?

● What is your course of action when you receive calibration materials for non-FDA cleared

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assays?

● What is your course of action when preparing controls and calibrators in-house?

● Further evaluate the responses, corrective actions, and resolutions for unacceptable calibration and unacceptable calibration verification

MOL.33655 Calibration Procedures Phase II

Calibration procedures for each method are adequate, and the calibration results are documented.

REFERENCES 1) Department of Health and Human Services, Centers for Medicare & Medicaid Services. Clinical laboratory improvement amendments

of 1988; final rule. Fed Register. 1992(Feb 28):7165 [42CFR493.1217] 2) Department of Health and Human Services, Centers for Medicare & Medicaid Services. Medicare, Medicaid and CLIA Programs;

Laboratory Requirements Relating to Quality Systems and Certain Personnel Qualifications; final rule. Fed Register. 2003(Jan 24):3707 [42CFR493.1255]

3) Kroll MH, Emancipator K. A theoretical evaluation of linearity. Clin Chem. 1993;39:405-413 4) Clinical and Laboratory Standards Institute. Evaluation of matrix effects; approved – guideline – second edition. Clinical and

Laboratory Standards Institute document EP14-A2 (ISBN 1-56238-561-5). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2005

5) Miller WG. “Quality control.” Professional Practice in Clinical Chemistry: A Companion Text, ed. DR Dufour. Washington, DC: AACC Press, 1999:12-1 to 12-22

6) Kroll MH, et al. Evaluation of the extent of non linearity in reportable range studies. Arch Pathol Lab Med. 2000;124:1331-1338

MOL.33696 Calibration Materials Phase II

High quality materials with method- and matrix-appropriate target values are used for calibration and calibration verification whenever possible.


✓ Written procedure defining the use of appropriate calibrators AND

✓ Records of calibration REFERENCES 1) Clinical and Laboratory Standards Institute. Evaluation of Matrix Effects; Approved Guideline—Second Edition. Clinical and

Laboratory Standards Institute document EP14-A2 (ISBN 1-56238-561-5). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005

MOL.33737 Calibration Materials Phase II

All calibration materials used for non-FDA cleared assays are documented as to quality.

NOTE: Commercial standards used to prepare calibrators require certificates of quality from the vendor, or a quality check as part of the initial assay validation process. The laboratory should document the accuracy of a new lot of calibrators by checking the new lot against the current lot.

**REVISED** 07/11/2011 MOL.33778 Calibration Material Labeling Phase II

All calibration materials are properly labeled as to content, calibration values, date placed in service, and expiration date (if applicable).

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NOTE: Complete values need not be recorded directly on each vial of calibrator material, so long as there is a clear indication where specific values may be found for each analyte tested and each analyzer used by the laboratory.

The dates may be recorded in a log (paper or electronic), rather than on the containers themselves, providing that all containers are defined so as to be traceable to the appropriate data in the log.


✓ Written procedure defining elements required for labeling of calibration material

MOL.33860 Calibration/Calibration Verification Criteria Phase II

Criteria are established for frequency of calibration or calibration verification, and the acceptability of results.

NOTE: Criteria typically include:

1. At changes of reagent lots, unless the laboratory can demonstrate that the use of

different lots does not affect the accuracy of patient/client test results and the range used to report patient/client test data

2. QC fails to meet established criteria 3. After major maintenance or service 4. When recommended by the manufacturer 5. At least every 6 months


✓ Written procedure defining the method, frequency and limits of acceptability of calibration verification for each instrument/test system AND

✓ Records of calibration verification documented at defined frequency REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):3707[42CFR493.1255(b)(3)] 2) Miller WG. “Quality control.” Professional Practice in Clinical Chemistry: A Companion Text, ed. DR Dufour. Washington, DC: AACC

Press, 1999:12-1 to 12-22

MOL.33901 Recalibration Phase II

The system is recalibrated when calibration verification fails to meet the established criteria of the laboratory.


✓ Written procedure defining criteria for recalibration AND

✓ Records of recalibration, if calibration or calibration verification has failed

MOL.33942 AMR Validation Phase II

Validation of the analytical measurement range (AMR) is performed with matrix-appropriate materials that include the low, mid and high range of the AMR, and the process is documented.

NOTE: If the materials used for calibration or for calibration verification include low, midpoint, and high values that are near the stated AMR, and if calibration verification data are within the laboratory's acceptance criteria, the AMR has been validated; no additional procedures are required. If the calibration and/or calibration verification materials do not span the full AMR, or the laboratory extends the AMR beyond the manufacturer's stated range, the AMR must be validated

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by assaying materials reasonably near the lowest and highest values of the AMR.

Calibration, calibration verification, and validation of the analytical measurement range (AMR) are required to substantiate the continued accuracy of a test method. The CLIA regulations use the term "calibration verification" to refer to both verification of correct method calibration and validation of the analytical measurement range. This Checklist uses separate terms to identify two distinct processes that are both required for good laboratory practice.

The AMR is the range of analyte values that a method can directly measure on the specimen without any dilution, concentration, or other pretreatment that is not part of the usual assay process. Validation of the AMR is the process of confirming that the assay system will correctly recover the concentration or activity of the analyte over the AMR.

The materials used for validation must be known to have matrix characteristics appropriate for the method. The test specimens must have analyte values that as a minimum are near the low, midpoint, and high values of the AMR. Guidelines for analyte levels near the low and high range of the AMR should be determined by the laboratory director. Factors to consider are the expected analytic imprecision near the limits, the clinical impact of errors near the limits, and the availability of test specimens near the limits. It may be difficult to obtain specimens with values near the limits for some analytes. In such cases, reasonable procedures should be adopted based on available specimen materials. The method manufacturer's instructions for validating the AMR should be followed, when available. Specimen target values can be established by comparison with peer group values for reference materials, by assignment of reference or comparison method values, and by dilution ratios of one or more specimens with known values. Each laboratory must define limits for accepting or rejecting validation tests of the AMR.

The AMR must be revalidated at least every 6 months, and following changes in major system components or lots of analytically critical reagents (unless the laboratory can demonstrate that changing reagent lot numbers does not affect the range used to report patient test results, and control values are not adversely affected).


✓ Written procedure for AMR validation defining the types of materials used and acceptability criteria consistent with manufacturer's instructions

REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):3707 [42CFR493.1255]

MOL.33983 AMR Validation Criteria Phase II

Criteria are established for validating the analytical measurement range, and compliance is documented.

NOTE: If the materials used for calibration or for calibration verification include low, midpoint, and high values that are near the stated AMR, and if calibration verification data are within the laboratory's acceptance criteria, the AMR has been validated; no additional procedures are required. If the calibration and/or calibration verification materials do not include the full AMR, or the laboratory extends the AMR beyond the manufacturer's stated range, the AMR must be validated by assaying materials reasonably near the lowest and highest values of the AMR.


✓ Written procedure defining the method, frequency and acceptability criteria for AMR validation

**REVISED** 07/11/2011

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MOL.34003 Comparability of Instrument/Method Phase II

If the laboratory uses more than one instrument/method to test for a given analyte, the instruments/methods are checked against each other at least twice a year for correlation of results.

NOTE: This requirement applies to tests performed on the same or different instrument makes/models or by different methods. This comparison must include all nonwaived instruments/methods. The laboratory director must establish a protocol for this check.

Quality control data may be used for this comparison for tests performed on the same instrument platform, with both control materials and reagents of the same manufacturer and lot number.

Otherwise, the use of human samples, rather than stabilized commercial controls, is preferred to avoid potential matrix effects. The use of pooled patient samples is acceptable since there is no change in matrix. In cases when availability or pre-analytic stability of patient/client specimens is a limiting factor, alternative protocols based on QC or reference materials may be necessary but the materials used should be validated (when applicable) to have the same response as fresh human samples for the instruments/methods involved.

This checklist requirement applies only to instruments/methods accredited under a single CAP number.


✓ Written procedure for performing instrument/method correlation including criteria for acceptability AND

✓ Records of correlation studies reflecting performance at least twice per year with appropriate specimen types

REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Medicare, Medicaid and CLIA programs;

CLIA fee collection; correction and final rule. Fed Register. 2003(Jan 24):5236 [42CFR493.1281(a)] 2) Podczasy JJ, et al. Clinical evaluation of the Accu-Chek Advantage blood glucose monitoring system. Lab Med. 1997;28:462-466 3) Ross JW, et al. The accuracy of laboratory measurements in clinical chemistry: a study of eleven analytes in the College of American

Pathologists Chemistry Survey with fresh frozen serum, definitive methods and reference methods. Arch Pathol Lab Med. 1998;122:587-608

4) Miller WG, Erek A, Cunningham TD, et al. Commutability limitations influence quality control results with different reagent lots. Clin Chem. 2011;57:76-83

MOL.34024 Calibrator Preparation Phase II

Calibrators and controls are prepared separately.

NOTE: In general, calibrators should not be used as QC materials. If calibrators are used as controls, then different preparations should be used for these two functions. For example, when using commercial calibrators and controls, the lot number for calibration should be different than the lot number used for QC, whenever possible.


✓ Written policy for preparation of controls and calibrators in-house REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):3708 [42CFR493.1256(d)(9)]

REAGENTS


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● Sampling of probe/primer information

Additional requirements are in the REAGENTS section of the All Common Checklist. MOL.34188 Probe Characteristics Phase II

Sufficient information is documented regarding the nature of any probe or primer used in an assay to permit interpretation and troubleshooting of test results.

NOTE: Items of importance where appropriate include: the type (genomic, cDNA, oligonucleotide or riboprobe) and origin (human, viral, etc.) of the probe or sequence; the oligonucleotide sequence and complementary sequence or gene region recognized; an appropriate restriction enzyme map of the DNA; known polymorphisms, sites resistant to endonuclease digestion, and cross-hybridizing bands; the labeling methods used and standards for adequacy of hybridization or amplification. For linkage analysis, recombination frequencies and map positions must be documented. Loci should be designated as defined by the Human Gene Mapping Nomenclature Committee. For inherited disease tests, additional information such as chromosomal location of the target, allele frequencies of the mutation in various ethnic groups, and recombination frequencies (for linkage probes) may be required. Sequence and size data may not be available for commercially-obtained tests when this information is considered proprietary. REFERENCES 1) McAlpine PJ, et al. The Catalog of mapped genes and report of the nomenclature committee. Human gene mapping. Cytogenet Cell

Genet. (most recent version)

CONTROLS

Controls are samples that act as surrogates for patient/client specimens. They are processed like a patient/client sample to monitor the ongoing performance of the entire analytic process in every run. Molecular tests typically include positive and negative controls and, in some instances, a sensitivity control to show that low level target is detectable. An internal control, extraction control, and a contamination control may be indicated. A single control may be able to serve multiple purposes. Quantitative tests typically include at least two (2) levels of control at relevant decision points to verify that calibration status is maintained within acceptable limits.


● Sampling of QC policies and procedures

● Sampling of QC records, including monthly monitoring of imprecision

● Sampling of control material (storage)

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● How do you determine when QC is unacceptable and when corrective actions are needed?

● How does your laboratory verify the cut-off value used to distinguish positive from negative results?

● What is your course of action when monthly statistical data changes significantly from the previous month's data?

● Select several occurrences in which QC is out of range and follow documentation to determine if the steps taken follow the laboratory policy for corrective action

**NEW** 07/31/2012 MOL.34220 Daily QC - Nonwaived Tests Phase II

Controls are run daily for quantitative and qualitative tests.

NOTE 1: Controls should verify assay performance at relevant decision points. The selection of these points may be based on clinical or analytical criteria.

NOTE 2: Except for tests meeting the criteria in Note 3, below, daily external surrogate sample* controls must be run as follows:

● For quantitative molecular tests, 3 controls must be run daily—a negative control, a low positive control, and a high positive control, unless a different requirement is specifically required by this checklist

● For qualitative tests, a negative control and a positive control (when available) must be run daily.

Control testing is not necessary on days when patient testing is not performed.

NOTE 3: Daily controls may be limited to electronic/procedural/built-in (e.g. internal, including built-in liquid) controls for tests meeting the following criteria:

1. For quantitative tests, the test system includes 2 levels of electronic/procedural/built-

in internal controls that are run daily 2. For qualitative tests, the test system includes an electronic/procedural/built-in internal

control run daily 3. The system is FDA-cleared or approved, and not modified by the laboratory** 4. The laboratory has performed studies to validate the adequacy of limiting daily QC to

the electronic/procedural/built-in controls. Validation studies must include daily comparisons of external controls to built-in controls for at least 20 consecutive days. For validation of multiple identical devices, the minimum of 20 consecutive daily comparisons applies to the initial device; the laboratory director is responsible for determining the extent of the validation studies for the other devices. Acceptable validation is required before daily quality control can be limited to built-in controls. The laboratory director is responsible for determining criteria for acceptability, and

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other details of the validation. Validation records must be retained while an instrument is in service, and for two years afterwards. The requirement for 20 consecutive daily comparisons is effective for validation studies performed after 1/31/2012.

5. External surrogate sample controls are run for each new lot number or shipment of test materials; after major system maintenance; and after software upgrades.*** Regarding external controls for qualitative tests, best practice is to run a weak positive control, and in the case of drug testing, also a high negative control (e.g. 25% below cutoff) to maximize detection of problems with the test system.

6. External surrogate sample controls are run as frequently as recommended by the test manufacturer, or every 30 days, whichever is more frequent.

*A "surrogate sample" is a specimen designed to simulate a patient sample for quality control purposes. For example, traditional external liquid control materials are considered surrogate sample controls. Some surrogate sample controls may not be external, but may be contained within an instrument (e.g. in a cartridge); systems using these built-in controls must meet the requirements in Note 2, above.

**Sample types (or use of collection devices) not listed in manufacturer instructions are acceptable, if validated by the laboratory.

***Repetition of the initial validation study is not required when running external surrogate sample controls with new lots/shipments of test materials, after system maintenance or software upgrades, or in accordance with paragraph 6 in the Note.

NOTE 4: Controls must assess adequacy of extraction and amplification. If the internal control does not go through the extraction method, a separate extraction control is needed for each run (positive controls fulfill this requirement).


✓ Records of QC results including external and electronic/procedural/built-in control systems AND

✓ Records documenting in-house validation of electronic/procedural/built-in control systems, if used

REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Medicare, Medicaid and CLIA programs;

CLIA fee collection; correction and final rule. Fed Register. 2003(Jan 24):5232 [42CFR493.1256(d)(3) (i, ii)] 2) Steindel SJ, Tetrault G. Quality control practices for calcium, cholesterol, digoxin, and hemoglobin. A College of American

Pathologists Q-Probes study in 505 hospital laboratories. Arch Pathol Lab Med. 1998;122:401-408 3) Voss EM, et al. Determining acceptability of blood glucose meters. Statistical methods for determining error. Lab Med. 1996;27:601-

606 4) Clinical and Laboratory Standards Institute (CLSI). Statistical Quality Control for Quantitative Measurement Procedures: Principles

and Definitions; Approved Guideline—Third Edition. CLSI document C24-A3 (ISBN 1-56238-613-1). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006

5) Ye JJ, et al. Performance evaluation and planning for patient/client-based quality control procedures. Am J Clin Pathol. 2000;113:240-248

6) LaBeau KM, et al. Quality control of test systems waived by the clinical laboratory improvement amendments of 1988. Perceptions and practices. Arch Pathol Lab Med. 2000;124:1122-1127

**REVISED** 07/31/2012 MOL.34229 Controls - Qualitative Assays Phase II

For qualitative tests, positive, negative and sensitivity controls are run for each assay, when appropriate.

NOTE: Ideally, one should use a positive control for each analyte in each run. However, in some circumstances such as in a large mutation panel for cystic fibrosis, this procedure is not practical. One way to address this situation is to rotate positive controls in a systematic fashion and at a frequency determined by the director. When appropriate, the sensitivity control is used to verify detection of low-level target sequences.

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✓ Records of QC results REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical Laboratory Improvement

Amendments of 1988; Final Rule. Fed Register. 2003(Jan 24);7166 [42CFR493.1256(D)(3)(II)] 2) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


MOL.34270 Controls - Quantitative Assays Phase II

For quantitative tests, control materials at more than one concentration (level) are used in every run.

NOTE: Controls should verify assay performance at relevant analytic and clinical decision points.


✓ Records of QC results REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Statistical Quality Control for Quantitative Measurement Procedures: Principles


2) Ye JJ, et al. Performance evaluation and planning for patient/client-based quality control procedures. Am J Clin Pathol. 2000;113:240-248.

MOL.34311 Tolerance Limits - Controls Phase II

Tolerance and acceptability limits are defined for all control procedures, control materials and standards.

NOTE: These controls must be appropriate for the range of sensitivities tested and should, ideally, focus on result ranges that are near clinical decision points.


✓ Records of defined tolerance limits for control range verification of each lot

MOL.34352 QC Verification Phase II

The results of controls are verified for acceptability before reporting of results.

NOTE: It is implicit in quality control that patient test results will not be reported when controls are unacceptable.


✓ Written policy/procedure stating that controls are reviewed and acceptable prior to reporting patient results AND

✓ Evidence of corrective action taken when QC results are not acceptable REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 1992(Feb 28):7166 [42CFR493.1218(e)]

MOL.34393 QC Corrective Action Phase II

There is documentation of corrective action when control results exceed defined acceptability limits.

NOTE: Patient test results obtained in an analytically unacceptable test run or since the last acceptable test run must be evaluated to determine if there is a significant clinical difference in

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patient results. Re-evaluation may or may not include re-testing patient samples, depending on the circumstances.

Even if patient samples are no longer available, test results can be re-evaluated to search for evidence of an out-of-control condition that might have affected patient results. For example, evaluation could include comparison of patient means for the run in question to historical patient means, and/or review of selected patient results against previous results to see if there are consistent biases (all results higher or lower currently than previously) for the test(s) in question). REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Oct 1):1046[42CFR493.1282(b)(2)]

MOL.34434 QC Handling Phase II

Control specimens are tested in the same manner and by the same personnel (including specimen preparation) as patient samples.

NOTE: It is implicit in quality control that control specimens be tested in the same manner as patient specimens. Moreover, QC specimens must be analyzed by personnel who routinely perform patient testing. This does not imply that each operator must perform QC daily, so long as each instrument and/or test system has QC performed at required frequencies, and all analysts participate in QC on a regular basis. To the extent possible, all steps of the testing process must be controlled, recognizing that pre-analytic and post-analytic variables may differ from those encountered with patients.


✓ Records reflecting that QC is run by the same personnel performing patient testing REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; Final rule. Fed Register. 1992(Feb 28):7166 [42CFR493.118(c)]

MOL.34475 QC Statistics Phase II

For quantitative assays, quality control statistics are performed at least monthly to define analytic imprecision and to monitor trends over time.

NOTE: The laboratory must use statistical methods such as calculating SD and CV at specified intervals to evaluate variance in numeric QC data.


✓ QC records showing monthly monitoring and corrective action, as applicable REFERENCES 1) Mukherjee KL. Introductory mathematics for the clinical laboratory. Chicago: ASCP, 1979:81-94 2) Barnett RN. Clinical laboratory statistics, 2nd ed. Boston, M; Little, Brown, 1979 3) Weisbrodt IM. Statistics for the clinical laboratory. Philadelphia, PA: JB Lippincott, 1985 4) Matthews DF, Farewell VT. Understanding and using medical statistics. NY, NY: Karger, 1988 5) Department of Health and Human Services, CMS. Clinical laboratory improvement amendments of 1988; final rule. Fed Register.

2003(Jan 24):7146 [42CFR493.1256(d)(10)(i) 6) Ross JW, Lawson NS. Analytic goals, concentrations relationships, and the state of the art of clinical laboratory precision. Arch Pathol

Lab Med. 1995;119:495-513 7) Clinical and Laboratory Standards Institute (CLSI). Statistical Quality Control for Quantitative Measurement Procedures: Principles


8) Brooks ZC, et al. Critical systematic error used of varied QC rules in routine chemistry. Clin Chem. 2000;46:A70

**REVISED** 07/11/2011 MOL.34495 Monthly QC Review Phase II

Quality control data are reviewed and assessed at least monthly by the laboratory director

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or designee.

NOTE: The QC data for tests performed less frequently than once per month should be reviewed when the tests are performed.


✓ Records of QC review with documented follow-up for outliers, trends or omissions

MOL.34516 Qualitative Cut-Off Phase I

For qualitative tests that use a cut-off value to distinguish positive from negative, the cut-off value is established initially, and verified with every change in lot or at least every 6 months.

NOTE: The threshold value that distinguishes a positive from a negative result should be established or verified when the test is initially placed in service, and verified with every change in lot (e.g. new master mix), instrument maintenance, or at least every six months thereafter. Note that a low-positive control that is close to the threshold value can satisfy this checklist requirement, but must be external to the kit (e.g. weak-positive patient sample or reference material prepared in appropriate matrix).


✓ Written procedure for initial establishment and verification of the cut-off value AND

✓ Records of initial establishment and verification of cut-off value documented at defined frequency

MOL.34557 Control Storage Phase I

Controls are stored in a manner that maintains their integrity.

AMPLIFICATION


● Sampling of amplification policies and procedures

● Physical containment practices (frequent glove change, separate manipulation of pre- and post-specimens, dedicated pipettes)

● How does your laboratory distinguish a true negative from a false negative result?

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MOL.34875 Carryover Phase II

Nucleic acid amplification procedures (e.g. PCR) are designed to minimize carryover (false positive results) using appropriate physical containment and procedural controls.

NOTE: This item is primarily directed at ensuring adequate physical separation of pre- and post-amplification samples to avoid amplicon contamination. The extreme sensitivity of amplification systems requires that the laboratory take special precautions. For example, pre- and post-amplification samples should be manipulated in physically separate areas; gloves must be worn and frequently changed during processing; dedicated pipettes (positive displacement type or with aerosol barrier tips) must be used; and manipulations must minimize aerosolization. In a given run, specimens should be ordered in the following sequence: patient samples, positive controls, negative controls (including “no template” controls in which target DNA is omitted and therefore no product is expected). Enzymatic destruction of amplification products is often helpful, as is real-time measurement of products to avoid manual manipulation of amplification products.


✓ Written procedure that defines the use of physical containment and procedural controls as applicable to minimize carryover

REFERENCES 1) Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-238 2) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


**REVISED** 07/11/2011 MOL.34900 Internal Controls - NAA Phase II

In all nucleic acid amplification procedures, internal controls are run to detect a false negative reaction secondary to extraction failure or the presence of an inhibitor, when appropriate.

NOTE: The laboratory should be able to distinguish a true negative result from a false negative due to failure of extraction or amplification. Demonstration that another sequence can be successfully amplified in the same specimen should be sufficient to resolve this issue. For quantitative amplification assays, the effect of partial inhibition must also be addressed.

The internal control should not be smaller than the target amplicon.


✓ Written procedure defining use of internal controls OR records of assay validation and monitoring statistics for test result trends

METHODS AND INSTRUMENT SYSTEMS

Restriction Endonucleases


● Sampling of RE digestion records

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MOL.34907 Restriction Endonuclease Digestion Confirmation Phase II

The completeness and accuracy of restriction endonuclease digestion are confirmed, when appropriate.

NOTE: The treatment of DNA with restriction endonucleases (RE) must be performed for an appropriate amount of time and under appropriate reaction conditions, i.e. to guard against non-specific activity. The efficacy of RE digestion must be established for each new lot of enzyme and in each run. Buffers must be used before their expiration date and properly stored.


✓ Written procedure defining conditions under which RE should be used AND

✓ Records of confirmation of efficacy of RE digestion with each new lot of enzyme and in each run

Sequencing


● Sampling of sequencing policies and procedures

● How does your laboratory ensure individual nucleotides are visualized adequately?

● How does your laboratory interpret sequence variation?

MOL.34914 Gene Information Phase II

There is adequate information about the gene being tested regarding its wild type sequence and reported mutations and polymorphisms.

NOTE: DNA sequencing assays should be reserved for those disease genes that have been adequately characterized in the literature and in genomic databases so that the complete wild type sequence of the target region is known, as well as the identity and location of both clinically silent and clinically important mutations and polymorphisms.


✓ Records of literature references for wild type sequence and reported mutations and polymorphisms

REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI), Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved

Guideline. CLSI document MM9-A (ISBN 1-56238-558-3). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004

MOL.34921 Sequencing Assay Optimization Phase II

Sequencing assays are optimized to ensure a readable signal throughout the length of the target region and ready detection of sequence variants, especially those in heterozygous state.

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NOTE: Sequencing assays differ from most other molecular pathology assays in that many targets (individual nucleotides) are examined at once, rather than addressing a discrete nucleotide mutation site. Assay procedures must assure that each of these targets is visualized adequately to produce an unequivocal sequence readout, whether this is done by manual or automated methods. Heterozygous point mutations in particular may be overlooked if the signals are low or unequal. One approach to preventing this problem is to perform sequencing in both directions (opposite strands).


✓ Written procedure for performing sequencing assays

MOL.34929 Sequencing Data Criteria Phase I

Criteria are established for the acceptance and interpretation of primary sequencing data.

NOTE: Criteria for acceptance and interpretation of sequencing data must include correct assignments for non polymorphic positions, definition of the sequencing region, criteria for peak intensity, baseline fluctuation, signal-to-noise ratio and peak shapes.

MOL.34931 Sense/Antisense Sequence Phase I

The sequence of sense and antisense strands are determined for heterozygous templates, rare alleles or rare combinations of alleles.

NOTE: The sequence of sense and antisense strands must be determined for rare alleles and rare combinations of alleles. The sequence of sense and antisense strands should be determined for heterozygous templates. If only one strand is sequenced for heterozygous templates, validation must show that sequencing of only one strand will consistently yield accurate sequence assignments. If assignments are routinely based on only one strand, periodic confirmation of complementary strands is recommended.


✓ Written procedure detailing how the sequence of sense and antisense strands are determined for heterozygous templates, rare alleles or rare combinations of alleles AND

✓ Records showing sequencing of sense and antisense strand OR records of validation for sequence assignments based on one strand

MOL.34932 Sequence Interpretation Guidelines Phase I

The laboratory follows professional guidelines for interpretation of sequence variation.

NOTE: The laboratory should have an algorithm for decision-making in interpretation of pathogenic variants, benign variants and variants of unknown clinical significance.

REFERENCES 1) ACMG Standards and Guidelines for Clinical Laboratories, http://www.acmg.net

Next Generation Sequencing

Next Generation Sequencing (NGS) incorporates two processes: (1) the analytical wet bench process of sample and library preparation and sequence generation and (2) the bioinformatics process or pipeline of sequence alignment, annotation and variant calling. These two processes are inextricably linked as the output from each process supports the optimization of the other. The large volumes of data produced by NGS platforms put substantial demands on laboratories in terms of the requirements for documentation, validation, quality control


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and assurance, monitoring, data storage, as well as assessment and implementation of new technology and software releases.


● Sampling of next generation sequencing policies and procedures

● Records of wet bench processing and bioinformatics process validation

● QA program records with corrective action for component failure

● Sampling of exception log records

● What is your course of action when processes deviate from standard operating procedures?

Analytical Wet Bench Process **NEW** 07/31/2012 MOL.34934 Wet Bench Process Documentation Phase I

The laboratory documents the analytical wet bench process used to generate next generation sequencing data, including sample and library preparation as well as sequence generation.

NOTE: Documentation to support the clinical operation must include:

● Methods for converting DNA or RNA into a next-generation sequencing library, if applicable

● Methods and enrichment reagents for targeted sequencing of multi-gene panels and exomes, if applicable

● Methods for indexing (barcoding) and sample pooling, if applicable

● Sequencing platform and manufacturing (or vendor) versions of sequencing reagents flow cells or chips

● Analytical run parameters including base quality per read position, read lengths and read configuration (example, pair end or mate pair)

● "Expected" analytical performance characteristics per vendor specifications and actual laboratory experience (example, minimal thresholds for base quality per read position and numbers of reads passing filters)

● Metrics and QC parameters for optimal performance


✓ Policy or procedure that describes the laboratory's method for such documentation AND

✓ Documentation that complies with the policy or procedure

**NEW** 07/31/2012 MOL.34936 Wet Bench Process Validation Phase I

The laboratory validates the analytical wet bench process and revalidates after changes or upgrades to any components used to generate next generation sequencing data.

NOTE: Validation of the analytical wet bench process can be method-based or analyte-specific and must describe required performance characteristics of the entire process and individual process steps. Revalidation may cover all or a subset of steps in the process depending on the

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extent of the modification. Acceptance criteria for analytical runs must be established.

● Validations must include information on the analytical target (examples, exons, genes, exomes, genomes, and transcriptomes). The ability of the analytical process to sequence the target (e.g. percentage of target adequately sequenced) must be described.

● Validations must determine and document analytical sensitivity, specificity, reproducibility, repeatability and precision for the types of variants assayed (e.g. single nucleotide variants, insertions and deletions, homopolymer or repetitive sequences).

● Interference by clinically relevant pseudogenes and other sequences highly homologous to the target must be determined and documented.

● Sequencing error rates (i.e. false positives and false negatives) for variants assayed must be determined and documented using an alternative method which may include an alternate NGS chemistry.

● Indexing (barcoding) and sample pooling methods must be validated to ensure that individual sample identity is maintained throughout the analytical wet bench process.


✓ Records of validation and revalidation studies

**NEW** 07/31/2012 MOL.34938 Quality Assurance Program Phase I

The laboratory has a documented Quality Assurance (QA) program for the analytical wet bench process used to support the analysis, interpretation and reporting of next generation sequencing based results.

NOTE: Procedures are in place for corrective measures should one or more components in the analytical wet bench process fail laboratory-determined quality assurance standards.


✓ Records of Quality Assurance program results and corrective measures, as required

**NEW** 07/31/2012 MOL.34940 Confirmatory Testing Phase I

The laboratory has a policy for when confirmatory testing of identified or reported variants will be determined by an alternative method.

NOTE: The laboratory maintains an ongoing record of the sensitivity, specificity, false positives, false negatives, reproducibility and repeatability of results and compares these with data obtained during the validation process.


✓ Policy or procedure that describes the indications for confirmatory testing

**NEW** 07/31/2012 MOL.34944 Patient Reports Phase I

The specific methods, instrument(s) and reagents are traceable for each patient report.

**NEW** 07/31/2012 MOL.34946 Exception Log Phase I

The laboratory maintains an exception log for patient samples where steps used in the

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analytical wet bench process deviate from laboratory standard operating procedures.

NOTE: The log file must include documentation of each deviation and reason(s) for undertaking with sign-off by the laboratory director or designee(s). The exception log should retain links to the patient sample. There should be regular documented review of the exception log by the laboratory director with comment on any issues or corrective actions taken as a result of these reviews.

**NEW** 07/31/2012 MOL.34948 Monitoring of Upgrades Phase I

The laboratory has a policy for monitoring and implementing upgrades to instruments, sequencing chemistries and reagents or kits used to generate next generation sequence data.


✓ Documentation that demonstrates compliance with the policy or procedure

**NEW** 07/31/2012 MOL.34952 Sequence Variants - Interpretation/Reporting Phase I

Interpretation and reporting of sequence variants takes into consideration professional organizations' recommendations and guidelines.

NOTE: The laboratory should have a decision making process for classifying and interpreting pathogenic variants, potential pathogenic variants, benign variants, and variants of unknown clinical significance.


✓ Policy or procedure that describes the process used for assessment of sequence variations for potential pathogenicity

**NEW** 07/31/2012 MOL.34954 Clinically Significant Genetic Findings Phase I

The laboratory has a policy regarding reporting of clinically significant genetic findings unrelated to the clinical purpose for testing.

NOTE: Gene panel(s), exome, transcriptome, and genome sequencing may yield unexpected (or incidental) clinically significant genetic findings unrelated to the disorder for which the patient is undergoing testing.

**NEW** 07/31/2012 MOL.34956 Alternative Performance Assessment Phase I

Alternative Performance Assessment for Next Generation Sequencing is part of the All Common Checklist: See COM.01500 Alternative Performance Assessment.

NOTE: For sequencing based testing (including Sanger, pyrosequencing or next generation) alternative assessment may be performed by method and specimen type.

Bioinformatics Pipeline for NGS

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**NEW** 07/31/2012 MOL.34958 Process/Pipeline Documentation Phase I

The laboratory documents the bioinformatics process or pipeline(s) used to support the analysis, interpretation, and reporting of next generation sequencing based results.

NOTE: A bioinformatics process or pipeline includes all algorithms, software, scripts, database packages, reference sequences, and databases whether in-house developed, vendor-developed and/or supported or open source. Flow diagrams may be helpful in providing a graphical overview of processes.

Documentation used to support clinical operations must include:

● The individual applications used, with versions and appropriate command line flags or other configuration items that deviate from the standard, baseline installation

● Additional scripts or steps used to connect discrete applications

● Description of input and output data files or information in each process step

● Metrics and QC parameters for optimal performance

● Criteria for variant calling


✓ Policy or procedure that describes the laboratory's method for such documentation

**NEW** 07/31/2012 MOL.34960 Bioinformatics Process - Validation/Revalidation Phase I

The laboratory has validated the bioinformatics process or pipeline(s) and revalidates after any changes or upgrades are made to any components used to generate next generation sequencing data.

NOTE: Validation of the bioinformatics process or pipeline(s) must describe required performance characteristics of the entire bioinformatics process or pipeline(s) and individual process steps. Revalidation may cover all or a subset of steps in the process or pipeline(s), depending on the extent of the modification. Acceptance criteria for the bioinformatics process or pipeline(s) must be established.

● The validation report should include test scripts, test datasets, and other elements used in the validation.

● Validation must describe the analytical target sequence (examples: exons, genes, exomes, genomes, and transcriptomes). The ability of the bioinformatics process or pipeline(s) to assemble or align the target sequence and generate a variant data set must be described.

● Validations must determine and document analytical sensitivity, specificity, reproducibility and precision for the types of variants assayed (e.g. single nucleotide variants, insertions and deletions, homopolymer or repetitive sequences).

● Interference by clinically relevant pseudogenes and other sequences highly homologous to the target must be determined and documented.

● Bioinformatics process or pipeline(s) error rates (i.e. false positive and false negatives) for variants assayed must be determined and documented.

Indexing (bar coding) and sample pooling methods must be validated to ensure that individual sample identity is maintained throughout the bioinformatics process or pipeline(s).


✓ Records of validation and revalidation studies

**NEW** 07/31/2012

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MOL.34962 Bioinformatics Process/Pipeline - QA Program Phase I

The laboratory has a documented Quality Assurance (QA) program for the bioinformatics process or pipeline used to support the analysis, interpretation and reporting of next generation sequencing based results.

NOTE: Procedures are in place for corrective measures should one or more components in the bioinformatics process or pipeline fail laboratory-determined quality assurance standards.


✓ Records of Quality Assurance program results and corrective measures

**NEW** 07/31/2012 MOL.34964 Bioinformatics Process/Pipeline - Updates Phase I

The laboratory has a policy for monitoring and implementing patch-releases, upgrades, and other updates to the bioinformatics process or pipeline.


✓ Procedure defining process for monitoring patch-releases, upgrades and updates AND

✓ Documentation of monitoring activities AND

✓ Dates of implementation, as appropriate

**NEW** 07/31/2012 MOL.34966 Input Storage Phase I

The laboratory has a policy regarding the storage of input, intermediate and final data files generated in the bioinformatics analyses performed on patient samples.

NOTE: This policy must include: which input, intermediate and final data files are retained and which may be purged after a period of time after a final report has been generated. Selection of files for retention should support primary results generated and re-analysis at a later date if requested or required. This policy must be in accordance with local, state or national requirements for storage of data.

**NEW** 07/31/2012 MOL.34968 Version Traceability Phase I

The specific version(s) of the bioinformatics process or pipeline(s) used to generate next generation sequencing data are traceable for each patient report.

NOTE: Details of the version of the bioinformatics process or pipeline used to generate each patient report may utilize a laboratory-specific system that refers to the entire process or pipeline, as well as changes to the versions of the individual components within the process or pipeline.

**NEW** 07/31/2012 MOL.34970 Exception Log Phase I

The laboratory maintains an exception log for patient cases where steps used in the bioinformatics process or pipeline deviate from laboratory SOPs.

NOTE: The log file must include documentation of each deviation and reason(s) for undertaking, with sign-off by the laboratory director or designee(s). The exception log should retain links to the patient case.


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✓ Documentation of review of the exception log by the laboratory director AND

✓ Documentation of any issues and corrective actions taken as a result of these reviews

**NEW** 07/31/2012 MOL.34972 Data Transfer Confidentiality Policy Phase I

There are procedures in place to ensure that internal and external storage and transfer of sequencing data provides reasonable confidentiality and security, and conforms to patient confidentiality requirements.

NOTE: It is recognized that laboratories may transfer data to external laboratories and other service providers for storage and analysis. This may include data storage and analysis through cloud-based computing.

Procedures to ensure confidentiality might include message security, system and user authentication, activity logs, encryption, and access restrictions.

Electrophoresis


● Sampling of electrophoresis policies and procedures

● Autoradiographs/gel photographs (sufficient resolution/quality)

● How does your laboratory prevent degradation of the nucleic acid sample used for electrophoresis?

MOL.34974 Loading Nucleic Acids Phase II

Standard amounts of nucleic acid are loaded on analytical gels, when possible.

MOL.35050 Molecular Weight Markers Phase II

Known molecular weight markers that span the range of expected bands are used for each electrophoretic run.


✓ Records of appropriate markers documented with each run

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MOL.35100 Visual/Fluorescent Markers Phase II

Visual or fluorescent markers are used to determine the endpoint of gel electrophoresis.

MOL.35150 Autoradiograph/Electrophoretic Gel Interpretation Phase I

Autoradiographs or electrophoretic gels are interpreted using objective criteria.


✓ Written procedure including interpretive criteria for autoradiographs or gels REFERENCES 1) American Association of Blood Banks. Standards for parentage testing laboratories. Bethesda, MD: AABB, 1998:7.100, 7.500 2) Cossman J, et al. Gene rearrangements in the diagnosis of lymphoma/leukemia. Guidelines for use based on a multiinstitutional

study. Am J Clin Pathol. 1991;95:347-354 3) NCCLS. Immunoglobulin and T-cell receptor gene rearrangement assays; approved guideline – second edition. NCCLS document

MM2-A2 (ISBN 1-56238-466-X). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002

MOL.35175 Audioradiograph/Gel Photograph Resolution Phase II

The autoradiographs and gel photographs are of sufficient resolution and quality (low background, clear signal, absence of bubbles, etc.) to permit the reported interpretation.

Polymerase Chain Reaction (PCR)


● Sampling of PCR policies and procedures

● Sampling of PCR calibrator results

● Sampling of new fluorescent oligonucleotides validation records

MOL.35370 Melting Temperature Phase I

For tests that generate a result based on a Tm, appropriately narrow temperature ranges (≤ +/- 2.5°C) are defined and monitored.

MOL.35414 PCR Calibrators Phase I

For quantitative real-time PCR tests, calibrator results fall within a specified range for each run.

MOL.35458 Fluorescent Oligonucleotide Reagents Phase I

New lots of fluorescent oligonucleotide reagents are tested against control targets concurrent with or before being placed into service.


✓ Written policy for the validation of new reagent lots AND

✓ Records of validation of new fluorescent oligonucleotides

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Arrays

Arrays include a variety of reverse and forward hybridization formats. Reverse hybridization arrays use multiple unlabeled probes on a solid support to interrogate a patient sample that carries a label, either direct (fluorescent or radioactive) or indirect (affinity labels such as biotin, digoxigenin, etc.). Another form of array involves multiple real-time amplification assays to measure multiple targets simultaneously. Controls for arrays monitor those steps carried out by the laboratory (sample preparation and labeling, hybridization and detection) and by the manufacturer (assay preparation, detection and hybridization reagents). Manufacturers also contribute to QC by producing products under good manufacturing procedures (GMP), providing control material for each analyte, and by providing sequence information or confirmatory tests to resolve ambiguous results.


● Sampling of array quality verification and lot-to-lot comparison records

MOL.35722 Integrity/Labeling Verification Phase I

Patient nucleic acid integrity and labeling are verified.

NOTE: In many current applications, the sample is labeled during a PCR or RT-PCR reaction. Some arrays include a control feature that targets an endogenous positive target. Other possible controls include visualizing the material on electrophoretic gels/capillaries or by detection of label. Addition of an exogenous spiked control during labeling will monitor efficiency of labeling but will not control for quality of sample nucleic acid.


✓ Written procedure detailing process for verifying nucleic acid integrity/labeling AND

✓ Records of verification REFERENCES 1) CLSI. Diagnostic Nucleic Acid Microarrays: Approved Guideline; CLSI document MM12 (ISBN 1-56238-6085). Clinical and

Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2011

MOL.35766 Array Quality Phase I

The quality of the arrays is verified and lot-to-lot comparisons are done.

NOTE: Manufacturer quality control specifications should be available as a reference. Additional verification by the laboratory should include:

● Verification of each probe for each lot. This can be achieved in part by using labeled oligonucleotides that hybridize to all probes, a mixture of labeled oligonucleotides specific for each probe, or a mixture of control samples that hybridize to each probe.

● For quantitative assays such as gene dosage, pathogen load, or expression levels, a positive control near limiting dilution (low positive) for one or more probes should be included in each run. These controls should be rotated to include all analytes. A separate array for template blanks (i.e. no RT control or water) should also be included to monitor for inadvertent contamination.

● Function checks on software used to analyze array data points.

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✓ Written procedure defining process for verifying array quality and lot-to-lot comparison of arrays AND

✓ Records of verification and lot-to-lot comparison

Parentage and Forensic Identity Testing


● Sampling of parentage/forensic policies and procedures

● Sampling of paternity/forensic data for completeness

● Sampling of requisition/collection forms (include transfusion/transplant history)

● Sampling of information/label verification records

● Sampling of patient reports for completeness

● Limited-access secured area for specimens

● What is your laboratory's course of action when evaluating closely spaced alleles?

MOL.37442 Paternity/Forensic Data Phase II

For paternity/forensic identity testing, the following data are obtained.

1. Place and date of specimen collection 2. Identity of person collecting the specimen 3. Photograph or photocopy of a picture identification card for each individual

tested 4. Signed record of information (including name, race, relationship) for each

individual tested 5. Date of birth of child 6. Synopsis of case history/investigation, sample source 7. Documentation of informed consent

REFERENCES 1) Standards for Parentage testing laboratories. American Association of Blood Banks. Standards for parentage testing laboratories.

Bethesda, MD: 2003:5.2.4

MOL.37584 Transfusion/Transplantation History Phase II

For parentage/forensic identity testing, the laboratory seeks a history of transfusion in the preceding three months or a history of allogeneic hematopoietic progenitor cell transplantation at any time.


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✓ Specimen requisition/collection forms with transfusion/transplant history REFERENCES 1) Standards for Parentage testing laboratories. American Association of Blood Banks. Standards for parentage testing laboratories.

Bethesda, MD: 2003:5.2.4.6

MOL.37726 Specimen Labeling - Paternity/Forensic ID Phase II

For paternity/forensic identity testing, the information about each individual and the accuracy of the sample label are verified by that individual or the legal guardian.


✓ Records of information and label verification by patient or legal guardian

MOL.37868 Specimen Verification Phase II

The condition of the specimen is documented upon receipt in the laboratory including any evidence of tampering, adequacy of volume, and a firmly attached label bearing a unique identification.

MOL.38010 Secured Parentage/Forensic Records Phase II

For legal parentage and forensic testing, specimens are maintained in a limited access, secured area and appropriate documentation of chain-of-custody maintained.


✓ Written policy addressing restricted access to parentage and forensic specimens and records AND

✓ Records of authorized personnel with access AND

✓ Documentation of chain-or-custody on patient reports

MOL.38152 Report Content Phase II

The report includes the individual paternity index for each genetic system, the combined paternity index, the probability of paternity as a percentage, prior probability of paternity used in calculations and the population for used for comparison.

REFERENCES 1) Standards for parentage testing laboratories. Bethesda, MD: AABB, 2003:6.4 (6R-B)

MOL.38294 DNA Results Interpretation Phase II

For parentage testing, DNA results (RFLP, STR, SNP) are interpreted twice, independently.


✓ Written policy stating the requirement for a second, independent interpretation of DNA results AND

✓ Patient records/worksheets REFERENCES 1) Standards for parentage testing laboratories. Bethesda, MD: AABB, 2003:5.4.2.1-3

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MOL.38436 Exclusion Evaluation Phase II

For parentage testing, exclusions based on closely spaced alleles (usually defined as less than one tandem repeat apart) are evaluated by co-electrophoresis or other methods.


✓ Written procedure addressing method of evaluation for closely spaced alleles AND

✓ Records of evaluation by secondary method REFERENCES 1) Standards for parentage testing laboratories. Bethesda, MD: AABB, 2003:5.4.2.1-2

MOL.38578 Forensic ID Testing Requirements Phase II

For forensic identity testing, laboratory methods, test validation, and interpretation and reporting of results meet current guidelines.

NOTE: For laboratories subject to US regulations, these guidelines are provided by the DNA Advisory Board Standards and SWGDAM. REFERENCES 1) DNA Advisory Board. Quality assurance standards for DNA testing laboratories. Forensic Science Communications 2000:2.2 2) Short tandem repeat (STR) interpretation guidelines. Ibid 3) Guidelines for mitochondrial DNA (mtDNA) nucleotide sequence interpretation. Ibid. 2003:5

Fluorescence and Non-Fluorescence In Situ Hybridization (FISH, ISH)


● Sampling of FISH/ISH policies and procedures

● Sampling of HER2 assay validation studies

● Sampling of probe validation records

● Sampling of QC records

● Sampling of patient reports for completeness including ASCO/CAP scoring when applicable

● What is your course of action if a probe does not produce an internal control signal?

● What is your laboratory's course of action when negative HER2 and/or negative results by IHC are obtained and the fixation time was not appropriate?

MOL.39004 FISH Scoring Phase II

There are documented procedures for scoring FISH results, including the number of cells scored, and all analyses are scored according to these procedures.

REFERENCES 1) American College of Medical Genetics Laboratory. Standards and Guidelines for Clinical Genetics Laboratories, 3rd ed. Bethesda,



http://http/www.acmg.net

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MOL.39146 FISH Controls Phase II

Control loci (internal or external) are used with and documented for each FISH analysis.

NOTE: When normal chromosome targets are expected to be present within a sample, an internal control for that target should be used during each hybridization (i.e. a locus specific probe at a different site on the same chromosome and/or a normal locus on the abnormal homolog). If a probe is used that does not produce an internal control signal (e.g. a Y chromosome probe in a female), another sample that is known to have the probe target should be run in parallel with the patient sample. REFERENCES 1) American College of Medical Genetics Laboratory. Standards and Guidelines for Clinical Genetics Laboratories, 3rd ed. Bethesda,



MOL.39155 Morphologic Interpretation Phase II

For in situ hybridization studies, the morphologic interpretation and correlation of results are performed by a qualified anatomic or clinical pathologist as appropriate.

NOTE: In situ hybridization requires evaluation of the histopathology or cytopathology in the hybridized slide to ensure that the hybridization signal is located to the appropriate lesional cells. For example, for HER2, FISH or ISH, a corresponding H&E slide must be evaluated by a pathologist to ensure that the studies are performed on invasive tumor cells. REFERENCES 1) Unger ER, Lee DR. In situ hybridization: principles and diagnostic applications in infection. J Histotechnol. 1995;18:203-209.

**REVISED** 07/11/2011 MOL.39288 Retention - Images Phase II

Photographic or digitized images are retained for documentation of all FISH assays (at least one cell for assays with normal results and at least two cells for assays with abnormal results).

NOTE: Images of FISH assays for neoplastic disorders must be retained for 10 years; images of FISH assays for constitutional disorders must be retained for 20 years. Brightfield ISH slides or images must be retained for the same time periods.


✓ Written retention policy REFERENCES 1) American College of Medical Genetics Laboratory. Standards and Guidelines for Clinical Genetics Laboratories, 3rd ed. Bethesda,

MD: ACMG, 2003. Available at: http://www.acmg.net Accessed 2006

**REVISED** 09/25/2012 MOL.39323 HER2 Assay Validation Phase I

If the laboratory tests for HER2 gene amplification by in situ hybridization (e.g. FISH, CISH*, SISH*, etc.), the laboratory has documented appropriate validation for the assay(s).

NOTE: Initial test validation must be performed on a minimum of 25 cases (recommended 25-100). Validation may be performed by comparing the results of testing with a validated alternative method (i.e. IHC vs. FISH) either in the same laboratory or another laboratory, or with the same validated method performed in another laboratory; validation testing must be done using the same set of cases in both labs. If the same validated method is used as the



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comparison method for validation, the laboratory must also compare its method with the alternative method (whether IHC or (F)ISH to define which cases should be reflexed to the alternative method).

The validation data should clearly show the degree of concordance between methods for each possible result (i.e. for IHC: 0, 1+, 2+, 3+; for FISH, CISH, SISH: positive, negative, and indeterminate (if applicable), as defined by the cut-offs listed in the latest version of the CAP/ASCO guideline).

The characteristics of the cases used for validation should be similar to those seen in the laboratory's patient population (i.e. core biopsies vs. open biopsy material, primary vs. metastatic tumor, etc.).

The validation documentation should identify the comparative test method(s) used.

Samples used for validation must be handled in conformance with the guidelines in this checklist. If specimens are fixed in a medium other than 10% neutral buffered formalin, the validation study must show that results are concordant with results from formalin-fixed tissues.

If significant changes are made in testing methods (e.g. probe, pretreatment protocol), revalidation is required.

This checklist item applies to laboratories that perform the technical testing of specimens for HER2 amplification. Patient specimens should be fixed in the same manner as the specimens used for the validation study(ies).

(*CISH = chromogenic in-situ hybridization; SISH = silver-enhanced in-situ hybridization)


✓ Documentation of validation data including criteria for concordance

MOL.39358 HER2 by ISH - Fixation Phase I

If the laboratory assesses HER2 gene amplification by in situ hybridization (e.g. FISH, CISH, SISH), the laboratory has a documented procedure for ensuring appropriate length of fixation of specimens tested.

NOTE: Specimens subject to HER2 testing should be fixed in 10% neutral buffered formalin for at least 6 hours and no longer than 48 hours. The volume of formalin should be at least 10 times the volume of the specimen. Decalcification solutions with strong acids should not be used. While fixation outside these time limits is not an absolute exclusion criterion for HER2 testing, laboratories should qualify any negative results for specimens fixed for less than 6 or longer than 48 hours.

Laboratories testing specimens obtained from another institution should have a policy that addresses time of fixation. Information on time of fixation may be obtained by appropriate questions on the laboratory's requisition form.

**REVISED** 09/25/2012 MOL.39393 HER2 by ISH/FISH - Scoring Phase I

If the laboratory interprets HER2 gene amplification by in situ hybridization (e.g. FISH, CISH, SISH), the laboratory reports the results using the ASCO/CAP scoring criteria.

NOTE: For HER2 gene status determined by in-situ hybridization, positive (amplified) cases are those with ratios of HER2 to CEP17 of > 2.2. Negative cases are defined as those with ratios of < 1.8. Equivocal cases are those with a ratio of 1.8 – 2.2.* For test systems without an internal control probe, positive (amplified) cases are those with an average HER2 gene copy number > six signals/nucleus, negative cases are those with < four signals/nucleus, and equivocal cases

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are those with an average HER2 gene copy number of four to six signals/nucleus. Careful attention should be paid to the recommended exclusion criteria for performing or interpreting in situ hybridization for HER2 (e.g. signal obscured by background; for FISH, difficulty in defining areas of invasive carcinoma under UV light; see table 6 in reference 1, below).

*For FDA-approved/cleared test systems that do not provide an equivocal category, the manufacturer's instructions should be followed. REFERENCES 1) Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline

recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007;131:18-43.

MOL.39398 ISH Interpretation Phase I

Appropriate interpretation of ISH results are provided in the report.

NOTE: ISH reports should include both an analytical and clinical interpretation.

Brightfield In Situ Hybridization


● Sampling of brightfield ISH policies and procedures

● Sampling of QC records

MOL.39430 Assay Conditions/Tissue Pretreatment Verification Phase II

Assay conditions and conditions of tissue pretreatment are verified for each sample, using an appropriate positive control probe(s) against endogenous targets.

NOTE: The laboratory must document that assay conditions and tissue pretreatment allow for detection of the intended target sequence. Nucleic acid preservation and accessibility vary with fixation and processing. Adjusting assay conditions to demonstrate the signal for an endogenous target with a positive control probe allows negative results with the test probe to be interpreted. Negative results with the endogenous positive control probe allow inadequately preserved samples to be eliminated. The positive control probe may be directed to any target known to be in the sample.


✓ Records of positive control probes for endogenous targets for each sample REFERENCES 1) Unger ER, Lee DR. In situ hybridization: principles and diagnostic applications in infection. J Histotechnol. 1995;18:203-209

Spectrophotometers


● Spectrophotometer policies/procedures

● Sampling of manufacturer required system checks

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● How does your laboratory verify calibration curves?

MOL.44394 Filter Photometers Phase II

Filters (filter photometers) are checked at least annually to ensure they are in good condition (e.g. clean, free of scratches).


✓ Records of filter checks documented at defined frequency

MOL.44860 Wavelength Calibration Phase II

Spectrophotometer wavelength calibration is checked regularly with appropriate solutions, filters or emission line source lamps, if so required by the manufacturer.


✓ Records of wavelength calibration documented at defined frequency

MOL.45326 Calibration Curves Phase II

For procedures using calibration curves, all the curves are rerun regularly and/or verified after servicing or recalibration of instruments.


✓ Records of calibration curves rerun and/or verification documented at defined frequency

Signal Detection Instruments

The following requirements apply to scintillation counters, luminometers, densitometers, etc.


● Sampling of background checks

**REVISED** 07/11/2011 MOL.46258 Background Level Criteria Phase II

Background levels are compared on each day of use with established criteria for

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acceptability.


✓ Records of background checks and documentation of corrective action when levels are unacceptable



MOL.46491 Bleed-Through Signal Phase I

For test platforms measuring multiple fluorochromes, precautions are taken to identify and correct for bleed-through signal from one channel to another.


✓ Written procedure defining steps taken to identify and correct bleed-through REFERENCES 1) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


Film Processing/Photographic Equipment


● Sampling of film processing maintenance records

● Use of UV protective shielding, if applicable

MOL.46724 Film-Processing Phase II

Film-processing (developing) equipment is routinely serviced, repaired, and appropriately replenished with reagents, if maintained by the laboratory.

NOTE: If the laboratory uses another department's film processing equipment, the quality of the autoradiographs produced must be monitored and the appropriate personnel notified if corrective action is required.


✓ Records of maintenance and repairs documented at defined frequency

MOL.47656 UV Protection Phase I

If ultraviolet light sources are used, proper protective shielding is available to users.


✓ Written policy including precautionary measures when UV light source are utilized

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Pipettes


● Automatic pipette calibration procedure

● Sampling of pipette/dilutor checks

MOL.48588 Automatic Pipettes Phase II

Automatic pipettors that are used for quantitative dispensing of material are checked for accuracy and reproducibility before being placed in service and at least annually, and these results are documented.

NOTE: Such checks are most simply done gravimetrically. This consists of transferring a number of measured samples of water from the pipette to a balance. Each weight is recorded, the weights are converted to volumes, then means (for accuracy), and SD/CV (for imprecision) are calculated. Alternative approaches include spectrophotometry or (less frequently) the use of radioactive isotopes, and commercial kits are available from a number of vendors. Computer software is useful where there are many pipettes, and provide convenient documentation. REFERENCES 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin

Chem. 1995;41:S183 4) NCCLS. Laboratory Statistics—Standard Deviation; A Report. NCCLS document EP13-R (ISBN 1-56238-277-2). NCCLS, 940 West

Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1995 5) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995 6) CLSI. Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guideline. CLSI Document GP31-A. (ISBN 1-

56238-697-2). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898, USA, 2009 7) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14 8) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22 9) Skeen GA, Ashwood ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479

Thermometers and Temperature-Dependent Equipment


● Sampling of temperature logs (refrigerator, freezer, incubator, oven, water bath, heat block, thermocycler)

● How does your laboratory ensure the individual wells of the thermocycler are maintaining accurate temperature?

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MOL.49054 Temperature Checks Phase II

Temperatures are checked and recorded daily for the following types of equipment.

1. Water baths 2. Heating blocks 3. Incubators and ovens (where temperature control is necessary for a

procedure) 4. Refrigerators and freezers

NOTE: Temperature-dependent equipment containing reagents and patient specimens must be monitored daily, as equipment failures could affect accuracy of patient test results. Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing.

The two acceptable ways of recording temperatures are: 1) recording the numerical temperature, or 2) placing a mark on a graph that corresponds to a numerical temperature (either manually, or using a graphical recording device). The identity of the individual recording the temperature(s) must be documented (recording the initials of the individual is adequate).

The use of automated (including remote) temperature monitoring systems is acceptable, providing that laboratory personnel have ongoing immediate access to the temperature data, so that appropriate corrective action can be taken if a temperature is out of the acceptable range. The functionality of the system must be documented daily.

MOL.49520 Thermocycler Temperature Checks Phase I

Individual wells (or a representative sample thereof) of thermocyclers are checked for temperature accuracy before being placed in service and at least annually thereafter.

NOTE: A downstream measure of well-temperature accuracy (such as productivity of amplification) may be substituted to functionally meet this requirement.


✓ Written procedure for verification of thermocycler accuracy AND

✓ Records of thermocycler verification REFERENCES 1) Saunders GC, et al. Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA

analyses. Clin Chem. 2001;47:47-55

Instrument Maintenance


● Sampling of instrument(s) policies and procedures

● Sampling of instrument maintenance logs and service/repair records

● Sampling of manufacturer required system checks

● Instrument records (promptly retrievable)

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MOL.49525

Instrument Operation Phase II

There are documented standard procedures for set-up and normal operation of instruments.

NOTE: All laboratory instruments and equipment must be maintained in a manner consistent with safe and reliable testing. The laboratory should have an organized system for monitoring and maintaining all instruments. The procedures and schedules for instrument maintenance must be as thorough and as frequent as specified by the manufacturer. Since some equipment has no standard frequency or extent of maintenance, each laboratory should establish a maintenance schedule that reasonably reflects the workload and specifications of its equipment. All servicing and repairs must be documented.

MOL.49530 Routine Maintenance Schedule Phase II

There is a regular schedule or system for checking the critical operating characteristics for all instruments in use.

NOTE: Function checks should be designed to evaluate the critical operating characteristics to detect drift, instability or malfunction before the problem affects patient results.

MOL.49535 Function Checks Phase II

Appropriate function checks are performed for all instruments prior to testing patient samples.

NOTE: There must be a schedule and procedure at the instrument for appropriate function checks. These may include (but are not limited to) electronic, mechanical and operational checks. The procedure and schedule must be as thorough and as frequent as specified by the manufacturer. Function checks should be designed to check the critical operating characteristics to detect drift, instability or malfunction, before the problem is allowed to affect test results. All servicing and repairs must be documented.

MOL.49540 Instrument Tolerance Limits Phase II

Tolerance limits for acceptable function are documented for specific instruments, when appropriate.

MOL.49545 Instrument Troubleshooting Phase II

Instructions are provided for minor troubleshooting and repairs of instruments (e.g. manufacturer's service manual).

**REVISED** 07/11/2011

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MOL.49550 Instrument Service Records Phase II

Instrument maintenance, service, and repair records (or copies) are promptly available to, and usable by the technical staff operating the equipment.

NOTE: The effective utilization of instruments by the technical staff depends upon the prompt availability of maintenance, repair and service documentation (copies are acceptable). Laboratory personnel are responsible for the reliability and proper function of their instruments and must have access to this information. Off-site storage, such as with centralized medical maintenance or computer files, is not precluded if the records can be promptly retrieved.

POST ANALYSIS

Results Reporting


● Sampling of molecular genetic test reporting policies and procedures

● Sampling of patient test reports for completeness

● Sampling of patient test reports performed with Class I ASRs including appropriate disclaimer

● What is your laboratory's course of action when discrepancies exist between the preliminary and final reports?

● What is your laboratory's course of action when molecular results are discrepant with other clinicopathologic findings?

● How does your laboratory ensure patient confidentiality when releasing/transmitting patient reports?

● Follow a molecular genetic test report from specimen receipt and processing to evaluation, interpretation, identification and reporting

MOL.49555 Preliminary Reports Phase II

Preliminary reports are promptly generated, when indicated.

MOL.49560 Preliminary/Final Report Discrepancies Phase II

Discrepancies between preliminary and final reports are investigated and documented.

MOL.49565 Discrepant Results Phase I

Discrepancies between the molecular pathology laboratory's final results, other laboratory findings, and the clinical presentation are investigated and documented, along with any necessary corrective action.

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NOTE: When molecular results are questioned because they are discrepant with other clinicopathologic findings, an investigation should be carried out and documented along with any corrective action.

MOL.49570 Final Report Criteria Phase II

The final report includes an appropriate summary of the methods, the loci or mutations tested, the analytic interpretation (i.e. test result), and clinical interpretation if appropriate.

NOTE: Laboratory reports should be designed to convey patient results effectively to a non-expert physician. This includes documentation of the analytic procedure used or the commercial kit version accompanied by an interpretation of the findings.

“Analytic interpretation” means examining the raw data to reach a conclusion about the quality or quantity of the analyte. “Clinical interpretation” means reaching a conclusion about the implications of the result for the patient. The clinical interpretation may be stated in general terms, or may be based on specific knowledge of the patient's situation. REFERENCES 1) Margaret L. Gulley, Rita M. Braziel, Kevin C. Halling (2007) Clinical Laboratory Reports in Molecular Pathology. Archives of

Pathology & Laboratory Medicine: Vol. 131, No. 6, pp. 852-863

**REVISED** 07/11/2011 MOL.49575 Mutation Database Phase I

The laboratory's database for known mutations, benign polymorphisms and variants of undetermined significance are documented and updated as needed, when applicable.

NOTE: The database of mutations must be documented and updated in a timely fashion after new mutations have been reported or verified in the published literature.

MOL.49580 ASR Disclaimer Phase II

If patient testing is performed using Class I analyte-specific reagents (ASRs) obtained or purchased from an outside vendor, the patient report includes the disclaimer required by federal regulations.

NOTE: ASRs are antibodies, both polyclonal and monoclonal, specific receptor proteins, ligands, nucleic acid sequences, and similar reagents which, through specific binding or chemical reaction with substances in a specimen, are intended for use in a diagnostic application for identification and quantification of an individual chemical substance or ligand in biological specimens.

An ASR is the active ingredient of a laboratory-developed test system.

This checklist requirement concerns Class I ASRs. Class I ASRs are not subject to preclearance by the US Food and Drug Administration (FDA) or to special controls by FDA. Most ASRs are Class I. Exceptions include those used by blood banks to screen for infectious diseases (Class II or III), or used to diagnose certain contagious diseases (e.g. HIV infection and tuberculosis) (class III).

If the laboratory performs patient testing using Class I ASRs, federal regulations require that the following disclaimer accompany the test result on the patient report: "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the US Food and Drug Administration."

The CAP recommends additional language, such as "FDA does not require this test to go through premarket FDA review. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory

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Improvement Amendments (CLIA) as qualified to perform high complexity clinical laboratory testing."

The disclaimer is not required for tests using reagents that are sold in kit form with other materials or an instrument, nor reagents sold with instructions for use, nor reagents labeled “for in vitro diagnostic use” (IVD) by the manufacturer.

The laboratory must establish the performance characteristics of tests using Class I ASRs in accordance with the Assay Validation section of this checklist. REFERENCES 1) Department of Health and Human Services, Food and Drug Administration. Medical devices; classification/reclassification; restricted

devices; analyte specific reagents. Final rule. Fed Register. 1997(Nov 21);62243-45 [21CFR809, 21CFR864] 2) Caldwell CW. Analyte-specific reagents in the flow cytometry laboratory. Arch Pathol Lab Med. 1998;122:861-864 3) Graziano. Disclaimer now needed for analyte-specific reagents. Northfield, IL: College of American Pathologists CAP Today.

1998;12(11):5-11 4) U.S. Department of Health and Human Services, Food and Drug Administration. Analyte Specific Reagents; Small Entity Compliance

Guidance. http://www.fda.gov/cdrh/oivd/guidance/1205.html, February 26, 2003 5) Shapiro JD and Prebula RJ. FDA's Regulation of Analyte-Specific Reagents. Medical Devicelink, February 2003.

http://www.devicelink.com/mddi/archive/03/02/018.html

MOL.49585 Report Sign-off Phase II

The final report is reviewed and signed by the director or a qualified designee if there is a subjective or an interpretive component to the test.

NOTE: When diagnostic reports are generated by computer or telecommunications equipment, the actual signature or initials of the director need not appear on the report. Nevertheless, the laboratory must have a procedure that ensures and documents that the report has been reviewed and approved before its release. REFERENCES 1) Standards for parentage testing laboratories. Bethesda, MD: AABB, 2003:6.4 2) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; Standard I.

Northfield, IL: CAP, 1998

MOL.49590 Patient Confidentiality Phase I

Molecular genetic test reports are released and transmitted in a manner adequate to maintain patient confidentiality at a level appropriate for the particular test.

NOTE: In view of the recognized risks of genetic discrimination and stigmatization, confidentiality of molecular test results is an important consideration. Results should be communicated only to the referring physician, genetic counselor, the medical record, or (in some cases) the patient. Potentially non-confidential media (e.g. FAX) should be used with caution. Some patients, aware of the insurability risks, will choose to pay for testing out-of-pocket and request that the results not be recorded in their medical record; such requests should be honored by the laboratory to the extent allowable under applicable laws. Under no circumstances should results be provided to outside parties such as employers, insurers or other family members, without the patient's express consent, despite the fact that there will be cases in which such action would appear to be in the best interest of the patient, family, or society. Laboratory workers must even use caution when publishing or publicly presenting the results of such studies, as some family members have recognized their own pedigrees in published material and thereby derived otherwise confidential information.


✓ Written policy detailing processes for release and transmittance of genetic test results REFERENCES 1) Health Insurance Portability and Accountability Act, 1996

http://www.fda.gov/cdrh/oivd/guidance/1205.html

http://www.devicelink.com/mddi/archive/03/02/018.html

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MOL.49595 Linkage Analysis Criteria Phase II

When linkage analysis is performed, the molecular inherited disease testing report includes an estimate of the risk of false negatives and false positives arising from recombination between the linked probe(s) and the disease allele or mutation.

REFERENCES 1) Keats BJB, et al. Guidelines for human linkage maps. An international system for human linkage maps (ISLM, 1990). Ann Hum

Genet. 1991;55:1-6

**REVISED** 07/11/2011 MOL.49600 Report Criteria Phase II

In genetic testing for complex heritable disease genes with multiple possible mutations, the report includes (where appropriate) an estimate of mutation detection rate and the residual risk of being a carrier for one of the mutations not tested for.

NOTE: Many disease genes, such as those for cystic fibrosis and familial breast/ovarian cancer, are extremely heterogeneous at the molecular level, with hundreds of different mutations reported in different patients and families. Even with gene sequencing, mutation detection is not 100% sensitive, since sequencing will not detect large exons or deletions/duplications or whole gene deletions/duplications. A negative test result, therefore, does not completely rule out the possibility that the patient is a mutation carrier. The test report should convey this information in a fashion understandable to the physician and, when appropriate, the patient. A calculated value for residual risk, based on the known population allele frequencies in the patient's ethnic group, is recommended. REFERENCES 1) Gulley et al. Clinical laboratory reports in molecular pathology. Arch Pathol Lab Med. Vol 131, June 2007

**REVISED** 07/11/2011 MOL.49615 Report Criteria Phase II

At a level appropriate for the particular test, the report includes a discussion of the limitations of the findings and the clinical implications of the detected mutation (or negative result) for complex disorders with regard to recessive or dominant inheritance, recurrence risk, penetrance, severity and other aspects of genotype-phenotype correlation.

NOTE: Because of the complexity of genotype-phenotype correlations for many genetic diseases, simply reporting a molecular genetic test as positive for a mutation is not acceptable since it conveys no information to the referring physician and patient as to the clinical ramifications of the result. Since major and often irreversible surgical or obstetric interventions may be initiated based on the test result, it is essential that the report convey the most current and accurate understanding of penetrance and recurrence risks. REFERENCES 1) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


MOL.49620 Counseling Recommendation Phase I

The report includes a recommendation that patients receive appropriate genetic consultation to explain the implications of the test result, its residual risks and uncertainties, and the reproductive or medical options it raises, to the patient, where

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appropriate.

NOTE: Molecular genetic test results are often extremely complex since they impart a probabilistic risk of disease rather than an objective positive/negative or quantitative answer. Physicians and counselors may require guidance to convey such subtle and emotionally charged information to patients in an understandable manner. In order to derive the most meaningful benefit from this testing, it is recommended that the results and subsequent options from these complex genetic tests be discussed with patients by a trained genetics professional. REFERENCES 1) American Board of Medical Genetics http://www.abmg.org/ 2) National Society of Genetic Counselors http://www.nsgc.org/ 3) American Board of Genetic Counseling http://www.abgc.net/

MOL.49625 Result Correlation Phase II

For assays performed on histology/cytology samples, the interpretive report includes correlation with the morphologic findings, as applicable.

MOL.49630 Standard Nomenclature Phase I

Standard nomenclature is used to designate genes and mutations.

NOTE: Whenever possible, human genes, loci and mutations should be designated according to standard nomenclature as defined in the references below. Where a common name is also in wide use in the medical literature, it may also be given in the report to improve clarity and prevent misunderstanding. One approach is to put the common name in the diagnosis section of the report, and the standard name in the methods section or in an interpretive comment. REFERENCES 1) Wain HM, et al. Guidelines for Human Gene Nomenclature. Genomics. 2002;7:464-470 2) den Dunnen JR, et al. Mutation Nomenclature Extensions and Suggestions to Describe Complex Mutations: A Discussion. Human

Mutation. 1999;15:7-12 3) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical

and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2011 4) http://www.hgvs.org/mutnomen/ accessed 2/8/2012 5) Ogino S, Gulley ML, den Dunnen JT, Wilson RB, Association for Molecular Pathology Training and Education Committee. Standard

mutation nomenclature in molecular diagnostics: practical and educational challenges. J Mol Diagn 2007;9:1-6

Records


● Record retention policy or procedure

● Autoradiographs/gel photographs/in situ hybridization slides (adequately labeled/cross-referenced?

http://www.abmg.org/

http://www.nsgc.org/

http://www.abgc.net/

http://www.hgvs.org/mutnomen/

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MOL.49635 Laboratory Records Phase II

The laboratory record includes sufficient information regarding the individual specimen and assay conditions.

NOTE: Appropriate information may include the quantity and quality of nucleic acid isolated and the amount used in the assay; the lot numbers of the restriction endonucleases, probes or primers used and any assay variables.

**REVISED** 07/11/2011 MOL.49640 Record Retention Phase II

A copy of each final report, all records of results, membranes, autoradiographs, gel photographs, and in situ hybridization slides, are retained in compliance with applicable laws and regulations.

NOTE: CAP requires that test reports for neoplastic conditions be retained for 10 years, and that test reports for constitutional/genetic conditions be retained for 20 years. Electronic versions are acceptable.

MOL.49645 Labeling/Cross-Referenced Phase II

All autoradiographs, gel photographs and in situ hybridization slides are adequately labeled for identification and adequately cross-referenced in the case records.


✓ Written procedure for the labeling of slides and records for cross-reference

PERSONNEL

As applicable, the personnel requirements in the Laboratory General Checklist should be consulted. For optimal patient care, only qualified personnel may be involved with molecular pathology testing.


● Documentation of education and experience

● Continuing education policy

● Sampling of continuing education records

MOL.49650 Director Qualifications Phase II

The director of the molecular pathology laboratory is a pathologist, board-certified physician in a specialty other than pathology, or doctoral scientist in a chemical, physical, or biologic science, with specialized training and/or appropriate experience in molecular pathology.

NOTE: In the case of forensic identity testing, the above or appropriate degree, training, or experience in forensic science is required.


✓ Records of qualifications including degree or transcript, certification/registration, current license (if required) and work history in related field

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MOL.49655 Personnel - Bench Testing Phase II

The person in charge of bench testing of the molecular pathology laboratory is qualified as one of the following.

1. Person who qualifies as a director 2. MB(ASCP), BS, BA or MLS(ASCP)/MT(ASCP) with at least 4 years of

experience (at least 1 of which is in molecular pathology methods) under a qualified director



MOL.49660 Technologist Qualifications Phase II

Persons performing the technical aspects of molecular pathology qualify as one of the following.

1. Experienced in the field under the direct supervision of a qualified director or supervisor, and, for laboratories subject to US regulations, qualified to perform high complexity testing

2. MT(ASCP) certified or equivalent 3. BA or BS degree in biologic sciences with appropriate experience in molecular

pathology methods



MOL.49665 Training/CME Phase I

There is an adequate training program for new technologists, and there is a continuing medical laboratory education program.


✓ Documented training and continuing education program AND

✓ Records of training by the institution or appropriate outside organization AND

✓ Records of continuing education REFERENCES 1) Von Needa P. Keep everyone keen on continuing education. Med Lab Observ. 1979(May):117-126 2) Berry CD. The JCAH approach to continuing education in the clinical laboratory. Lab Med. 1981;12:10-11 3) Umiker W. The role of the pathologist in continuing education programs for laboratory personnel. Lab Med. 1981;12:18-21 4) Yapit MK. Resources and strategies for a successful CE program. Med Lab Observ. 1989(Apr):47-566 5) Matta N. Training new technologists in the basics of molecular pathology. Lab Med. 1993;24:636-641 6) CLSI. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI document MM19-A (ISBN 1-56238-773-1). Clinical


LABORATORY SAFETY

The inspector should review relevant requirements from the Safety section of the Laboratory General checklist, to assure that the molecular pathology laboratory is in compliance. In particular, the Inspector should review the

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use of universal precautions and the handling and disposal of hazardous chemicals such as ethidium bromide, acrylamide, and organic reagents.


● Records of biological safety cabinet certification

● Fume hood/chemical filtration unit

MOL.52760 Fume Hood Phase II

A properly functioning fume hood (or chemical filtration unit) is available for any procedures using volatile chemicals.

**REVISED** 07/11/2011 MOL.54570 Biological Safety Cabinet Phase II

A biological safety cabinet (or hood) is available, when appropriate, and is certified at least annually to ensure that filters function properly and that airflow rates meet specifications.


✓ Maintenance schedule of BSC function checks AND

✓ Records of testing and certification REFERENCES 1) Classification of etiologic agents on the basis of hazard; US Department of Health, Education, and Welfare, PHS, Centers for Disease

Control, Office of Biosafety. Atlanta, GA. Reprinted September, 1976

MOL.58190 Refrigerators Phase II

Refrigerators are free of improper materials (such as food, externally contaminated specimens or unsealed volatile materials).

RADIATION SAFETY


● Sampling of radiation safety policies and procedures

● Sampling of radiation area surveys/wipe tests documentation

● Sampling of radioactive waste disposal documentation

● Sampling of personnel records of radionuclide training

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● Radionuclide storage areas (properly shielded)

● Appropriate signage where radioactive materials are used/stored

● How does your laboratory check the effectiveness of workbench decontamination?

MOL.61050 Radiation Safety Manual Phase II

There is an up-to-date radiation safety manual that includes sections on decontamination and radioactive waste.

NOTE: A radiation safety manual providing procedures for the safe handling of radioactive substances in both routine and emergency situations is required by the Nuclear Regulatory Commission (NRC). Requirements for laboratory safety in nuclear medicine can be found in several references.

MOL.61055 Workspace Decontamination Phase I

Workbenches and sinks are decontaminated each day of use, and the effectiveness checked at least monthly.

REFERENCES 1) U.S. Nuclear Regulatory Commission. Guide for the preparation of applications for medical use programs. Regulatory guide 10.8:

Appendix H - model procedure for area surveys. Washington, DC: USNRC, 1987

MOL.61060 Radionuclides Handling Phase I

There are specific policies regarding authorization or restriction of personnel handling radionuclides.

MOL.61065 Radionuclide Leak Phase II

Policies include procedures for notification if a damaged or leaking radionuclide shipment is received.

NOTE: Procedures must include inspection, monitoring of shipments, and instructions for notification, if leakage or damage is noted in a radionuclide shipment. For laboratories subject to US regulations, this is a Department of Transportation requirement.


✓ Records of inspections and notifications

MOL.61070 Radionuclide Storage Phase II

Radionuclide storage and decay areas are properly shielded, if required for specific

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isotopic materials.


✓ Written procedure defining criteria for radionuclide storage

MOL.61075 Radiation Surveys Phase II

There are regular radiation area surveys and wipe tests, with records maintained.


✓ Written procedure defining frequency of radiation survey and wipe tests to determine exposure rated and detect contamination

MOL.61080 Radioactive Material Sign Phase II

All areas or rooms where radioactive materials are being used or stored are posted to indicate the presence of radioactive materials.

MOL.61085 Radionuclide Training Phase II

Personnel are instructed in decontamination routines and in the safe handling and proper disposal of radionuclides (wastes, syringes, needles, and sponges).

NOTE: Instruction must include decontamination routines and proper disposal of radioactive waste material.


✓ Records of radionuclide training in personnel file

MOL.61090 Radioactive Waste Phase II

Radioactive waste is kept separate from normal trash, stored, and appropriately discarded with documentation.

NOTE: Separate areas must be established for the receipt of radioactive waste and that these areas be properly shielded to reduce radiation levels below maximum permissible limits. Documentation of the radioactive trash disposal must be maintained.


✓ Written procedure defining criteria for proper storage and disposal of radioactive waste

molecular pathology checklist · molecular pathology checklist 09/25/2012 edition the following...

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