IMPLEMENTATION OF MOLECULAR IN THE LABORATORY: OPTIMIZING SPACE AND WORKFLOW

Transcription

1 IMPLEMENTATION OF MOLECULAR IN THE LABORATORY: OPTIMIZING SPACE AND WORKFLOW Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC WS2-2

2 Objectives of Talk Discuss the design and workflow of a molecular diagnostic laboratory Define space and resource options Provide insight into the physical location of the molecular laboratory based on choice of technology and test menu Describe the selection and implementation of good laboratory practices in the molecular laboratory

3 Why Perform Molecular? Extensive growth and development over decades Significant advantages over more conventional methods Industry is totally driven by technology Steady growth fueled by new technologies, innovations, expanded test menu Broad usage in ID, genetics, oncology, hematology, pharmacology

4 Implementing a Molecular Test Many Obstacles Juice Worth The Squeeze

5 Implementing a Molecular Assay Simply another technique Similar implementation plan A little more homework, time, energy, expense, and thought depending on whether you build your own assays or go commercial Similar to any other clinical test, primary goal is to provide reliable and timely results for patient care and management

6 Implementing a Molecular Assay Basically ask the same general questions: Why do this test? Does it meet a need? Will the assay be better than what we currently offer? Is there a commercial assay system or do I need to build my own? How will the new test impact the laboratory? Will there be buy-in from department, institution, providers, payers?

7 Practicality in Laboratory What specimen(s) and how to collect, handle, and process? Does test require special equipment? Can test be performed on all needed shifts? What is the turnaround time? What are the personnel requirements? Are quality control and proficiency test material available? To what extent will quality control be done? Is there adequate space? Can test be automated to reduce labor? Does system have indication for all uses of interest? What are the regulatory/compliance issues?

8 Many Things to Consider Test Menu Clinical Utility Choice of Technology Standardization Resources Facilities Finance Research/Development Verification/Validation QC/QA Staffing Requirements Training/Education Proficiency Regulation/Legislation

9 Lots to Worry About

10 Start Off Slow and Simple Bringing in the first test is the hardest Take a hard look at your laboratory Define what works and what can be improved Molecular can be applied to all areas of CM Bring in test that allows other molecular assays to be added without additional capital, personnel, or space Facilitate personnel training so that the next test is an easy setup Establish a QA/QC infrastructure that satisfies CLIA and CAP

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12 Menu Considerations Look at existing test menu; decide which tests might be useful to bring in-house If you don t know, engage key clinical players; identify medical/icp needs Clinical necessity Cost avoidance Screening programs Improved performance/tat Assay Economics Match test selection with technical capabilities and real-world clinical needs in terms of test volume, required TAT, associated costs, revenue, reimbursement

13 Clinical Utility Know your patient population Special populations transplant, HIV, cancer, cardiopulmonary, fetal/newborn, genetic/metabolic/ hematologic disorders, GI/liver, adolescents Community-based infections Hospital-acquired infections Screening programs (HAIs, STDs, HIV/Hepatitis) Ask how they could benefit from molecular testing Ask what impact molecular will have on patient care & management Know/understand your current testing limitations

14 Clinical Value of Molecular Assays Qualitative Tests Early detection of infectious agents, virulence factors, resistance genes Identify new pathogens Detection of infectious agents that are: Uncultivable Fastidious or slow-growing Too dangerous to amplify in culture Nonviable or present in extremely low numbers or in small specimen volumes Molecular epidemiology Quantitative Tests Associate infection with disease Monitor efficacy of therapy Predict treatment failure and emergence of drug resistance Assess disease progression Facilitate understanding of natural history and pathogenesis of organisms

15 Choice of Technology Laboratory-Developed Assays Normally best option for experienced molecular diagnostic laboratories Less expensive; allows for broad menu Requires extensive verification/validation of performance Analyte-Specific Reagents (ASRs) FDA-Cleared Commercial Assays can get tests online sooner; menu is growing May require multiple assay/platform combinations Can modify for off-label use

16 Implementation of LDAs Do I really want to do this? Do I really need to do this? Can I really do this? What will be the impact on my laboratory? What is the clinical relevance? What about labor, cost, time, technical burden, throughput, workflow, training, competency, QC, QA, PT? What s out there and available to me?

17 Implementation of LDAs First, identify the analyte(s) and clinical condition(s) for which tests would be useful in your laboratory Give serious consideration to reagents and platforms that are available to use Impossible and/or impractical to evaluate them all Envision what you would like your molecular testing to be; do your homework, make informed decisions, and select wisely based on anticipated workload Development of assays is not a one time thing; will constantly need to maintain, change and adapt Start small and build on successes

18 Choice of Technology Real-time PCR has reached greatest maturity Multiple platforms: large and small, automated for extraction, amplification, detection Many reagents and chemistries Closed systems Improved quantitation Novel assay formats Continues to rapidly change

19 Assay Design and Development Specimen selection, collection and processing Reagent selection and preparation Extraction method and platform Assay setup and platform for amplification and detection/quantification Result interpretation and reporting Optimize each step of the analytical process

20 Levels of Service Provided Level 1 Labs with little experience with molecular diagnositics Lack resources to conduct extensive test validation Perform only FDA-cleared/approved tests Level 2 Labs with more experience with molecular diagnostics Greater available resources for test validation Perform FDA-cleared/approved and use ASRs for LDT developed by others Level 3 Labs with the highest level of experience with molecular diagnostics Sufficient resources to design and validate LDTs Serve as resources for Level 1 and 2 labs FS Nolte Creating a molecular microbiology laboratory. Clin. Microbiol. Newsl. 27:75-78

21 Compact NA Extraction Systems Roche MagNA Pure Compact QIAGEN BioRobot EZ1 & EZ1 Advanced/Advanced XL Beckman Coulter SPRI-TE NorDiag Arrow Promega Maxwell-16

22 Larger NA Extraction Platforms Roche MagNA Pure LC 2.0 BioMerieux NucliSens EASYMAG Abbott Molecular m2000 Roche MagNA Pure 96 QIAGEN QIAsymphony SP Hologic Gen-Probe Panther Roche AmpliPrep

23 ABI Real-Time PCR Instruments 7500/Fast/Fast Dx ViiA 7 StepOne/StepOne Plus QuantStudio 12K Flex/Dx

24 Roche Real-Time PCR Instruments Light Cyclers LC 1.5 LC 2.0 COBAS TaqMan 48 COBAS TaqMan 96 Light Cycler 96 Light Cycler 480 Light Cycler 1536 Light Cycler Nano

25 Other Selected Real-Time Instruments BioMerieux NucliSens Easy Q Cepheid SmartCycler Abbott Molecular m2000 rt Stratagene Mx 3005P & Mx 3000P QIAGEN Rotor-Gene Q 3M Integrated Cycler

26 Compact Specimen In-Result Out Systems Roche Cobas Liat BioFire FilmArray Cepheid GeneXpert Alere i System Cepheid GeneXpert Omni

27 Vendor Support Criteria Test Menu Technical support Pricing Kits/reagents Consideration or comments Specimen types Current and future tests Good working relationship with manufacturer Reasonable time frame for troubleshooting and repairs Remote access capability, if applicable Initial cost of instrument(s) Reagent/kit costs Other reagent and consumable costs Cost of other equipment required but not supplied by manufacturer Kit configuration Ability to sequester lots PS Mitchell, JJ Germer, JD Yao Laboratory Design and Operations in Molecular Microbiology: Diagnostic Principles and Practice, 2 nd ed., ASM Press, Washington, DC

28 Instrument Functionality Criteria Workflow Reliability Flexibility Ease of use Consideration or comments Routine maintenance Hands-on and hands-off time Distribution of labor over the run time Time to first result and/or run completion Specimen throughput Hardware Software Run failure rate Specimen failure rate (invalid results) Batch size options Sample input volume options Capability of performing LDTs (open platform) Reporting unit options for quantitative tests Time required and complexity of routine maintenance Run-ready reagents PS Mitchell, JJ Germer, JD Yao Laboratory Design and Operations in Molecular Microbiology: Diagnostic Principles and Practice, 2 nd ed., ASM Press, Washington, DC

29 Instrument Specifications Criteria Compatibility/placement Space requirements Utilities Consideration or comments Compatibility of instrument with existing lab Environmental factors that may adversely affect instrument Impact of instrument on nearby work areas (eg airflow, noise) Compatibility with LIS Footprint, floor or bench space requirements Space requirements for proper ventilation Space requirements for supporting equipment (eg computer, monitor, keyboard, printer) Space for in-lab storage or reagents and consumables Number and locations of bench top work areas required Power requirements (110 or 220V and number of outlets) UPS requirements Instrument to instrument electrical connections Data ports or phone jacks Proximity to sinks and drains Water quality PS Mitchell, JJ Germer, JD Yao Laboratory Design and Operations in Molecular Microbiology: Diagnostic Principles and Practice, 2 nd ed., ASM Press, Washington, DC

30 Molecular Laboratory Operation Collection Processing Extraction Storage Preanalytic Analytic Master Mix Prep Loading Amplification Detection Analyses Interpretation Reporting Consultation Post Analytic Design and operation have much in common with other areas of clinical laboratories with some unique features that will be discussed

31 Facilities Requirements Appropriate space, design and workflow are major considerations How much square footage is needed or available for use? Where will the space be located (new or existing)? Should it be centralized or decentralized? Will I need space for future expansion (say yes!)? What about choices of assays and platforms? What about workflow, issues of safety, quality control and storage?

32 Facilities Requirements Historically, physical plan for molecular assays has centered firmly on the fear of and need to prevent amplicon contamination Necessitated distinct and separate work spaces or individual rooms With evolution of molecular technology over the years, traditional blueprint is giving way to design options with adaptable physical features

33 Space Needs for MDx Laboratory Dependent on a Number of Factors Level and Type(s) of Service Anticipated Volume of Samples Received Desired/Expected Turn-Around Times Fastest TAT require redundancy of equipment and additional space Types and Technical Complexity of Assays and Systems/Platforms Selected Open vs. Closed FDA-cleared IVD tests vs. LDTs using home-made reagent mixes or store-bought ASRs Retention of Specimens and/or Extracted Products Reagent Storage

34 Location of Molecular Services Fully Integrated into Microbiology Laboratory Separate Decentralized Molecular Section Centralized Multi-Disciplinary Molecular Pathology Laboratory Genetic Diseases Hematopathology Infectious Diseases Pharmacogenomics Oncology Major argument for centralization is efficiency achieved by sharing space, facilities, equipment, personnel, reagents, supplies/materials Mosammaparast, N., A. McAdams, F.S. Nolte, and P. Gilligan Point-Counterpoint: Molecular testing for infectious diseases should be done in the clinical microbiology laboratory. J. Clin. Microbiol. 50:

35 Molecular Laboratory Design Exquisite analytical sensitivity of nucleic acid amplification techniques presents unique challenges for lab design and workflow Contamination from specimens, controls and amplicons a huge concern Laboratory design and workflow must take into account current and future use of open and closed systems

36 Open vs. Closed Testing Formats Open Platforms Traditional end-point PCR with manipulation of amplified products Risk of amplicon contamination is GREAT Closed Systems Real-time PCR Risk of amplicon contamination is SUBSTANTIALLY REDUCED or ZERO

37 Minimizing Contamination Use of separate work areas Implementation of engineering controls Unidirectional flow Meticulous sample handling and pipetting techniques PPE Chemical and/or enzymatic methods to prevent/remove contaminating nucleic acid Use of appropriate negative/reagent controls

38 Contamination Specimen/Target Template Where target template itself is the source of contamination Sample processing and extraction areas Now days, BIGGEST CONCERN Amplicon PCR product contamination associated with post PCR manipulation/analysis Closed systems help avoid amplicon contamination Aerosols often cannot be seen, felt, or detected before the contamination happens

39 Uracil-N-Glycosylase (UNG) Enzyme used to eliminate carryover PCR products In a new reaction, any residual products from previous PCR amplifications will be digested and prevented from amplifying dttp is substituted with dutp, and UNG is included in reaction mix Any uracil-containing nucleic acid is quickly destroyed by UNG in the initial PCR cycle

40 Building a Basic MDx Laboratory Most do not have opportunity to build the ideal MDx laboratory from scratch Molecular testing is more often integrated into existing laboratory space Significant challenges on what assays and platforms to select and where to place them based on space limitations Successful implementation requires a lot of creative thinking and substantial commitments of resources, time and expertise

41 Molecular Diagnostic Laboratory New Construction Retrofit Old Space

42 CHOP Virology Story Retrofitted molecular assays into traditional virology laboratory Significant growing pains over time Director had no formal training in molecular technology; no personnel with molecular experience Space designed for conventional testing First molecular assay implemented in 1989 End-point PCR for HIV-1 proviral DNA Followed by HSV, enterovirus, and HCV tests Forged ahead, trial by fire, defined best practices as we moved forward Today, molecular LDAs and several FDAcleared tests

43 Basic Workflow for Molecular Test Specimen collection & handling Extraction of nucleic acids Amplification Detection Data analysis Result reporting Clinical Specimens Extraction Amplification Detection Amplification & Detection Results Extraction, Amplification, Detection

44 Molecular Laboratory Design Designed to physically separate rooms or work areas for reagent preparation, nucleic acid extraction, amplification-detection Develop sound work practices/habits to minimize contamination of specimens equipment, instruments, reagents, supplies Ensure unidirectional workflow; move in one direction from PRE to POST amplification throughout testing process Use appropriate PPE for specific work areas

45 Molecular Laboratory Design Molecular space should be divided into at least 2-3 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 Additional area for preparation and manipulation of positive controls, calibrators, standards, and plasmids

46 Molecular Laboratory Design REAGENT PREPARATION ROOM/AREA Dedicated strictly to reagent preparation and storage Maintained under positive air pressure to help minimize/prevent entry of aerosols into the room Should contain dedicated equipment and supplies Staff should wear only PPE devoted to this space No specimens, extracted NA, amplified products, or unqualified equipment or supplies should be brought into this area

47 Molecular Laboratory Design SPECIMEN PREPARATION ROOM/AREA Used for specimen accessioning, sample preparation, NA extraction, and reaction set-up Ideally under negative pressure to retain all aerosols Should contain dedicated equipment and supplies Staff should wear only PPE devoted to this space Movement of personnel is acceptable from reagent preparation room/area into specimen preparation room/area; reverse is discouraged but permitted, if necessary, only with clean PPE Prepared reagents should be contained/covered in spill-free containers to transport into amplificationdetection room/area

48 Molecular Laboratory Design AMPLIFICATION-DETECTION ROOM/AREA Used for nucleic acid amplification and analyses Should contain dedicated equipment and supplies Staff should wear only PPE devoted to this space Generate large amounts of amplicons Risk of contamination is GREAT following postamplification manipulation of amplicons Adding reagents or opening reaction vessels represent main sources of contamination Ideally, room should be under negative pressure to retain all aerosols Use closed system when available to minimize chances of amplicon contamination

49 Additional Room/Area Positive Controls/Standards Prep Room Dedicated to the preparation and manipulation of positive control materials, standards, calibrators, and plasmids Should contain dedicated equipment and supplies Staff should wear only PPE devoted to this space Ideally, room should be under negative pressure to retain all aerosols Defined as DIRTY

50 Clean vs. Dirty Areas WORKFLOW CLEAN ROOMS Preamplification Reagent Prep Sample Prep DIRTY ROOMS Postamplification Amplification Analyses Pos Ctls/Stds Prep

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52 Ideal Molecular Laboratory Design & Workflow Unidirectional Workflow Backflow traffic must be restricted to minimum! Reagent Preparation Room/Area Sample Preparation Room/Area Amplification Room/Area Reagent Storage Amplification Reagent Preparation Master Mix Preparation Specimen Preparation Nucleic Acid Isolation Detection Analysis POSITIVE PRESSURE NEGATIVE PRESSURE NEGATIVE PRESSURE Airflow Outward Permitted workflow (unidirectional) Airflow Inward Airflow Inward Restricted workflow Establishing Molecular Testing in Clinical Laboratory Environments. CLSI document MM19-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.

53 Reminders of Unidirectional Flow

54 CHOP Molecular Floor Plan 2 SCHEMATIC OF CHOP CLINICAL VIROLOGY LABORATORY 3 1-Reagent Clean Room 2-Controls/Standards 3-Specimen Processing 4-NA Extraction & Purification 5-Amplification & Detection 4 1 5

55 CHOP Molecular Laboratory Retrofitted into Existing Virology Laboratory Clean Reagent Prep Specimen Processing NA Extraction Amplification-Detection Dirty Reagent Prep

56 Other Equipment Needs Basic Micropipettes Single and Multichannel Fixed and Adjustable Microcentrifuges -80 o C and -20 o C freezers Dead Air Boxes Laminar Flow Hoods Advanced Biophotometer Robotic Workstations Bench Design Fixed Modular

57 If Limited Lab Space Dead Air Boxes Laminar Flow Hoods

58 Modular vs. Fixed Bench Design Desirable to have flexibility, given the variety of footprints for molecular platforms

59 Other Facility Considerations Increased electrical capacity More outlets normally needed in molecular laboratory Ample dedicated electrical lines Emergency backup power outlets Surge protectors

60 Standard Precautions PPE are necessary in the molecular laboratory Laboratory coats, gloves, and safety glasses Protect personnel from chemicals and biohazards and protect patient samples from contamination Use of biological safety cabinets, fume hoods, safety goggles, and face shields may be needed for specific situations Training on appropriate use of PPE is must

61 Movement of Personnel Between rooms/areas should be restricted Ideally, individual(s) should be dedicated to working only in one pre- or post-amp area/room during a normal work day or shift If not possible, tasks can be performed in clean area/room first before working in post-amp room/area without travel back and forth Restriction should also apply to housekeeping personnel-difficult TO DO

62 Cleaning of Work Surfaces KEEP A CLEAN ENVIRONMENT Routine cleaning of work surfaces and equipment IS A MUST Use 10% sodium hypochlorite to decontaminate countertops, laboratory equipment, furniture Degradation of nucleic acids Corrosive to plastic and metal surfaces; need water and/or 70% ethanol rinse Commercial products available to remove DNA and RNA Avoid fabric or porous furniture surfaces; protect countertops with absorbent plasticbacked paper

63 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

64 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

65 Laboratory Practice 3 Carefully open and close all tubes to minimize aerosolization of contents Keep all non-essential tubes closed during sample addition Define order of preparation and loading of samples and controls for PCR 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

66 Reagents & Solutions Prepare and divide into single-use aliquots Store in area that is separate from specimen preparation and post amplification Dedicated equipment and supplies should be used Use gloves and protective clothing which are dedicated to reagent preparation Reagents can be premixed into single-use, master mixes Test new lots for purity, functionality, concentration, and contamination before use Compare performance of new reagents to ones currently in use and reference materials Meticulous cleaning is a must

67 Monitor Laboratory for Contamination All molecular rooms/areas should be monitored for contamination Contamination may come from previous specimens, controls/standards/calibrators, and/or amplicons Can be achieved using swab surveillance testing Collect swab samples from defined work surfaces Elute into liquid, extract NA, and amplify-detect Meticulously decontaminate if contamination is found Retest area after cleaning

68 Personnel Requirements Molecular diagnostic test are classified as either waived, moderately complex or highly complex tests by FDA No defined qualifications for waived testing For Moderate and high complexity testing, personnel must meet the requirements for the level of testing set by CLIA Certification in molecular biology/diagnostics

69 CAP Accreditation Program Use Microbiology Checklist Molecular Microbiology Section Applies to all molecular microbiology tests FDA-cleared/approved tests Modified FDA-cleared/approved tests Laboratory-developed tests Qualitative and quantitative assays; sequencing Does not apply to tests using direct non-amplified probes to identify organisms

70 CPT Coding and Reimbursement CPT coding for molecular diagnostics has evolved over the years Initially all molecular diagnostic tests regardless of the application were coded using procedure-specific, not analyte-specific codes Now have many specific codes based on single- or multi-analyte detection Reimbursement varies by state and public and private payers Emphasis on use of tests with strong clinical utility

71 Future Molecular Laboratory

72 Implementing Molecular Assays Questions & Thank You!!