QUALITY CONTROL AND QUALITY ASSURANCE FOR MOLECULAR INFECTIOUS DISEASE LABS

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1 QUALITY CONTROL AND QUALITY ASSURANCE FOR MOLECULAR INFECTIOUS DISEASE LABS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC

2 Objectives Describe pre-analytical, analytical, and postanalytical phases of QC/QA Discuss guidelines for test requests and specimen collection, handling and processing Explain lab design and best lab practices Define reagent prep and use and instrument maintenance and calibration Summarize proficiency testing, training and competency assessment Discuss interpreting, recording and reporting test results

3 Quality Control refers to the measures that must be included during each assay run to verify that the test is working properly Quality Assurance defined as the overall program used to ensure that the final results being reported by the laboratory are correct

4 Quality Assessment Quality Assessment (Proficiency Testing) means to determine the quality of the results generated by the laboratory. Quality assessment is a challenge to the effectiveness of the QC and QA programs that are in place.

5 Variables Affecting Quality of Results Educational background and training of personnel Selection and condition of specimens Controls used in test runs Reagents Equipment Interpretation, recording and reporting of results

6 Landscape of Laboratory Testing Diagnostic lab testing single highest volume activity in healthcare Drives 70% of decision-making across medicine 12.8 billion tests performed each year in U.S. Large scale analysis of 1.6 million results from 46 of 50 most commonly ordered laboratory tests On average, 30% of all tests are probably unnecessary Overutilization and underutilization are wide spread

7 Molecular ID Testing Plays major role in clinical care of patients with infectious diseases Identify specific pathogens Determine and optimize appropriate therapy Monitor response to therapy Assess prognosis Provide disease surveillance Accurate and timely diagnosis required

8 Laboratory Testing Errors

9 Quality Use of Microbiology Tests Using the right test At the right time For the right patient With the right clinical condition At the right price With the right outcome

10 Molecular QC/QA One of the largest stumbling blocks for traditional Clinical Microbiology Laboratories that start performing molecular testing Molecular QC/QA more closely resembles Clinical Chemistry QC/QA Newer CAP Microbiology Checklists have been refined and full sections now devoted to Molecular Microbiology Many changes in QC/QA; many are new to traditional microbiologists

11 Molecular QC/QA Program Written clearly defined program that ensures quality throughout the preanalytical, analytical, and post-analytical phases of testing Capable of identifying systems problems and opportunities for system improvement The laboratory must be able to develop plans for corrective/preventative action based upon data from its QC/QA system

12 Monitoring Testing Process

13 Importance of Quality Control Ensure accuracy and reproducibility of lab test results used in patient care Ensure integrity and confidence in test results Provide safety in management of patients Comply with local, regional, and national regulations and lab accreditation requirements Maintain staff morale and reputation of lab

14 Types of Molecular Assays Unmodified FDA-Cleared/Approved Tests Modified FDA-Cleared/Approved Tests Laboratory-Developed Tests Qualitative versus Quantitative Tests

15 Pre-Analytical Phase Addresses activities that occur prior to testing Test requests/ordering Patient identification Specimen collection and transport Specimen handling and processing Rejection of specimens

16 Test Requests/Ordering Develop well-designed order entry (EMR or manual) Allow for sufficient identification of patient and ordering physician, the test requested, and pertinent clinical information Should include date and time of specimen collection and specimen type; information on collector Should accompany patient s specimen to the laboratory Ensure that clinical indications are appropriate for test(s) requested

17 Specimen Collection Specimen collection, transport, handling and processing can have a significant effect on final test results; integrity of target nucleic acid sequence must be maintained Establish clearly defines criteria for monitoring proper specimen collection, labeling, preservation, transportation, and storage of specimens Establish educational programs Develop firm (but fair) rejection policy

18 Guidelines for Specimens Clearly defined method(s) of collection of specimens from all sources Sample type and quantity of specimen Preparation of collection site Timing of collection during disease Specimen quality/adequacy Collection and transport devices Identification of specimens Appropriate transport conditions Storage time, temperatures, and conditions

19 Specimens for RNA Detection Specimens for RNA detection require special collection and handling conditions RNA may be degraded by RNases Use method that stabilizes RNA until specimen can be processed and stored appropriately

20 Analytical Phase Written Procedure Manual Nucleic Acid Extraction and Storage Contamination Control Laboratory Design Laboratory Practices Testing Controls Test Performance Equipment Maintenance Personnel Competency Proficiency Testing Accreditation

Procedure Manual Written instructions that contain sufficient detail that qualified laboratory personnel can perform tests consistently and accurately Must include: Principle of Test Clinical

21 Procedure Manual Written instructions that contain sufficient detail that qualified laboratory personnel can perform tests consistently and accurately Must include: Principle of Test Clinical Significance Specimen Requirements Reagents Needed Procedural Steps Quality Control Calibration Reference Ranges Calculations Result Reporting Test Limitations References Yearly review by Director or designee; review all new procedures and revisions prior to implementation Electronic or paper procedures acceptable

22 Nucleic Acid Isolation Adequate procedures for release and isolation of target nucleic acid Keep process simple and minimize number of manipulations Maintain integrity of target while inactivating or removing inhibitory or interfering substances Yield, purity, intactness can be measured Ideally, isolated nucleic acid should be tested immediately; if delay, store under appropriate conditions until testing can be completed

23 Nucleic Acid Isolation/Preparation

24 Contamination Control During specimen collection and transport During initial specimen handling and processing by lab During nucleic acid extraction During amplification During product detection Contamination of reagents

Laboratory Design Molecular space should be divided into at least 3 separate work areas: Area 1 Reagent preparation area/room Area 2 An area/room for

25 Laboratory Design Molecular space should be divided into at least 3 separate work areas: Area 1 Reagent preparation area/room Area 2 An area/room for specimen processing/nucleic acid extraction Area 3 An area/room for amplification and detection *Area 4 For preparation of controls, calibrators, standards

Laboratory Practice 1 Dedicated supplies and reagents for each work area Use plugged aerosol resistant pipette tips Use closed systems or methods to control

26 Laboratory Practice 1 Dedicated supplies and reagents for each work area Use plugged aerosol resistant pipette tips Use closed systems or methods to control product carryover (e.g., uracil-n-glycosylase (UNG) to inactivate amplified product) Use appropriate positive and negative controls Meticulous cleaning is a must!

27 Laboratory Practice 2 Follow unidirectional flow from pre-pcr to post- PCR Pulse-spin reagents, samples, controls; Add reagents to vials before samples Use gloves and protective clothing which are dedicated to each area Decontaminate pipettes and instruments and wipe work surfaces with 10% bleach; rinse with 70% ethanol Meticulous cleaning is a must!

28 Laboratory Practice 3 Carefully open and close all tubes to minimize aerosolization of contents Keep all non-essential tubes closed during sample addition Order of preparation and loading of samples for PCR should be actual clinical specimens first, followed by positive and then negative controls Hoods and/or glove boxes (with UV light) are often necessary for nucleic acid isolation and set up of amplification reactions Meticulous cleaning is a must!

General Rules of Thumb Traffic can only go from clean to dirty area Objects can only go from clean to dirty area Reagents and supplies needed in a clean area need to be prepared and

29 General Rules of Thumb Traffic can only go from clean to dirty area Objects can only go from clean to dirty area Reagents and supplies needed in a clean area need to be prepared and stored in a clean area Equipment used in clean area cannot be moved and used in dirty area Personal safety equipment cannot move with you from dirty to clean area Meticulous cleaning is a must!

General Rules of Thumb Protocol books, worksheets, sample tubes, test data and results cannot move from dirty area to clean area Can employ color

30 General Rules of Thumb Protocol books, worksheets, sample tubes, test data and results cannot move from dirty area to clean area Can employ color coded lab coats, pipettes, safety glasses, racks, etc. to monitor traffic Have respect for RNases if working with RNA Meticulous cleaning is a must!

Reagents & Solutions All test reagents and controls should be stored properly and in a manner which minimizes contamination and degradation Prepare and divide into single-use aliquots Store in area

31 Reagents & Solutions All test reagents and controls should be stored properly and in a manner which minimizes contamination and degradation Prepare and divide into single-use aliquots Store in area that is separate from specimen preparation and post amplification Do not store in frost-free freezers Dedicated equipment and supplies should be used Reagents can be premixed into single-use, master mixes Meticulous cleaning is a must!

32 Reagents Test new lots for purity, functionality, concentration, and contamination before use Compare performance of new reagents to ones currently in use Test new reagents against reference materials; monitor quality with use Keep meticulous QC records

33 QC for Molecular ID Assays Qualitative Controls (match matrix) Amplification: Positive for each analyte; Negative (reagent blank or specimen) and Sensitivity Every run, establish range values, and monitor Internal (inhibition) for extraction and amplification Calibration verification (if use cutoff to distinguish positive/negative) weak positive Every 6 mo; after any major system component change and QC failure Quantitative Controls (match matrix) Amplification: Positives at 2 levels and Negative Every run, establish range values, and monitor Internal (inhibition) for extraction and amplification Calibration verification (verify cutoff) Analytical measurement range verification (low, mid, high) Every 6 mo; after any major system component change and QC failure Adapted from EM Burd 2010 Clin. Microbiol. Rev. 23:

34 Daily QC

35 QC of Multiplex Tests Ideally, include a positive control for each of the nucleic acid targets included in test for each run Often impractical if number of targets is large Multi-target control or rotate single target controls

Example of Multiplex Controls FilmArray RP Panel M210 (Maine Molecular Controls) ANALYTE M211 M212 Adenovirus Positive Negative Coronavirus 229E Negative Positive Coronavirus HKU1 Negative Positive

36 Example of Multiplex Controls FilmArray RP Panel M210 (Maine Molecular Controls) ANALYTE M211 M212 Adenovirus Positive Negative Coronavirus 229E Negative Positive Coronavirus HKU1 Negative Positive Coronavirus NL63 Negative Positive Coronavirus OC43 Negative Positive Human Metapneumovirus Positive Negative Human Rhinovirus/ Enterovirus Entero 1 Positive Negative Entero 2 Positive Negative HRV1 Positive Negative HRV2 Positive Negative HRV3 Positive Negative HRV4 Positive Negative Influenza A H FluA-H Positive Negative Influenza A H3 FluA-H1-pan Positive Positive FluA-H3 Positive Negative FluA-pan1 Negative Positive FluA-pan2 Positive Negative Influenza B Negative Positive Parainfluenza Virus 1 Positive Negative Parainfluenza Virus 2 Negative Positive Parainfluenza Virus 3 Negative Positive Parainfluenza Virus 4 Positive Negative Respiratory Syncytial Virus Negative Positive Bordetella pertussis Negative Negative Chlamydophila pneumoniae Negative Negative Mycoplasma pneumoniae Negative Negative M211 and M212 Main Molecular Controls run periodically and with each new lot. No need for negative control. Assay contains internal RNA process and second stage DNA PCR controls.

37 QC Acceptance Limits & Statistics

38 Inhibition Assessment

39 Distribution of QC Results W. Greg Miller Chapter 10, Quality Control in Henry s 22 nd ed., Elsevier, Philadelphia, PA

40 Types of Errors Random Systematic Random & Systematic BLUE = True Value Average GREEN = Sample Value Average

41 Westgard QC Evaluation Rules Used to define specific performance limits for a particular assay Can detect both random and systematic errors Warning rules and mandatory rules have been applied over the years Violation of warning rules triggers a review of test protocol, reagent performance and equipment calibration Violation of mandatory rules triggers rejection

42 Levey-Jennings Plots Warning Rules Mandatory Rules

43 Westgard Sigma Multirule QC

44 Primers/Probes - LDTs

Commercial Reference Materials Quality control material may be difficult to find for less common pathogens Number of companies now provide reference material for a

45 Commercial Reference Materials Quality control material may be difficult to find for less common pathogens Number of companies now provide reference material for a growing list of pathogens Acrometrix Advanced Biotechnologies American Type Culture Collection (ATCC) Main Molecular Quality Controls, Inc. Seracare Life Sciences Zeptometrix

46 International Standards WHO, NIST (US) and NIBSC (UK) have been active in establishing IS for viruses HIV-2, HIV-2, HAV, HBV, HCV, Parvovirus, HPV-16, HPV-18, CMV, and EBV Primary (reference) standards are assigned quantity values accepted as gold standards Provide means to normalized inter-laboratory and method to method variability Secondary standards are larger pools of material that are quantified by direct comparison to the primary standards Calibrators should be traceable to IS if available

47 Equipment & Instruments Ensure equipment and instrument function Establish system for monitoring Detection of drift, instability, or malfunction before problem affects test results Regular preventive maintenance and cleaning If multiple instruments, functions must be check against each other every 6 months or after repair Keep meticulous maintenance, service and repair records Establish service contracts with vendor

49 Quality Assurance/Monitoring A continuous process Documents that a test which has already been verified/validated is repeatedly giving the expected results as test is performed over time Confirms that test continues to perform according to laboratory s requirements and its intended use

50 Quality Monitoring Process Personnel competency assessment Meticulous record keeping Vigilance in laboratory Quality control monitoring Quality improvement Internal and external proficiency testing Correlation with clinical findings; interacting with healthcare providers Trend analysis Storage of specimens for follow-up testing/evaluations Integral part of a labs QA program

51 Quality Control Monitoring Have system in place to continuously monitor performance of positive and negative controls, standards, and calibrators Perform wipe testing can reinforce need to disinfect the environment daily Monitor prevalence rates of specific viral diseases

52 Training & Competency Establish a complete program of employee training, verification, and competency Verify personnel competency at least semiannually during first year and at least annually thereafter Reverification necessary if change in method or instrumentation

Assay Training Determine magnitude of training and skill level required Each operator must demonstrate proficiency with the method; may require certification Should

53 Assay Training Determine magnitude of training and skill level required Each operator must demonstrate proficiency with the method; may require certification Should include understanding of: Sample collection, handling and storage Reagent handling and storage Proper test protocol How to interpret and report results QC/QA for the system

Competency Assessment Direct observation of routine patient test performance, including specimen handling, processing, testing, instrument maintenance, and function tests

54 Competency Assessment Direct observation of routine patient test performance, including specimen handling, processing, testing, instrument maintenance, and function tests Monitor test result recording and reporting Review worksheets, QC records, PT results, and PM records Wet testing with proficiency samples Assessment of problem-solving skills

commercially-available and alternative proficiency testing programs CAP Proficiency Tests

55 Proficiency Testing/EQA Challenge for PT providers to keep up with pace of new test development and new technology in molecular diagnostics Many laboratories need combinations of commercially-available and alternative proficiency testing programs CAP Proficiency Tests Alternative performance assessment Integrate into the workload Rotated among people at the bench Corrective action on failed PT surveys

56 Proficiency Testing/EQA General principles and rules of PT should be applied PT samples should challenge the diagnostic scope of the test Range of genetic diversity (QUAL) and extremes of measurement ranges (QUANT) Samples should be relevant to current test targets and compatible with available technologies Samples should free of matrix effects and reflect the range of specimens types tested Source of nucleic acid can have a significant effect on participant performance

57 Alternative PT Assessment Used when no commercial PT products available May also be used to challenge pre-analytical and postanalytical processes not addressed by commercially available PT Example - Quality Control for Molecular Diagnostics (QCMD) Participate in ungraded/educational PT Split sample analysis with other laboratories Establish in-house PT Program Use regional pools (can be purchased) Clinical validation by chart review

58 Internal or External Split-Sample PT Minimum of 3-5 samples per testing event At least 2-3 testing events at equal intervals per year Use full range of positive and negative specimens in panel Samples should represent clinical specimens Use of in-house specimens or manufactured reference materials

Corrective Actions Mistakes will occur; avoid assigning blame Clearly document what has happened Thoroughly investigate, take immediate corrective

59 Corrective Actions Mistakes will occur; avoid assigning blame Clearly document what has happened Thoroughly investigate, take immediate corrective action, institute preventive measures Errors often result of larger process issues with reagents, personnel, equipment, workflow Ultimate goal: DO NO HARM

60 Post-Analytical Phase Laboratory Test Reports Timeliness of Reporting Correction of Errors Patient Confidentiality

61 Recording & Reporting Results Laboratory test reports should be clear, concise, accurate, and fully interpretive Verify all results before reporting as final Monitor and report specimen (in)adequacy Final report should include a summary of test method, result(s) and interpretation if appropriate Gene target (biological false positives and negatives) Amplification method Lower limit of detection ASR disclaimer if LDT Quantitative (AMR, LLQ vs. LLD, log 10 conversion, and action thresholds) Name and location of laboratory Good practice to have reports reviewed by 2 nd party

62 Interpretation & Reporting of Results Significance of results must be evaluated with respect to agent, specimen site, and clinical situation Interpretive criteria must include provisions for equivocal results Reporting of results should include as much information as necessary for clinician to properly interpret the results Interact with healthcare providers

Quality Management: Turnaround Times Provide test results in a timely manner to have greatest impact on patient care and management Have system to monitor TATs Are TATs appropriate for the

63 Quality Management: Turnaround Times Provide test results in a timely manner to have greatest impact on patient care and management Have system to monitor TATs Are TATs appropriate for the intended purpose of the test? Consultation with medical staff Batch testing versus on-demand Examples: HSV and enteroviruses in CSF; respiratory viruses; viral load tests; MRSA and C. difficile

64 Quality Management: Statistics

65 Additional Items Correction of Errors Laboratory should have a system in place for timely review and correction of clerical and analytical errors Confidentiality Testing records and reports should be maintained in a manner that preserves patient privacy and confidentiality

66 Total Quality Management Comprehensive organizational approach Focus on continually improving quality and efficiency with which lab operates Includes not only QC and QA, but other technical and administrative considerations that may indirectly influence quality and efficiency

QUALITY CONTROL/QUALITY ASSURANCE IN THE MOLECULAR MICROBIOLOGY LABORATORY

QUALITY CONTROL/QUALITY ASSURANCE IN THE MOLECULAR MICROBIOLOGY LABORATORY Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC