Recommendations for the validation of flow cytometric testing during drug development: I instrumentation

Journal of Immunological Methods 363 (2011) 104–119

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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

Research paper

Recommendations for the validation of flow cytometric testing during drug development: I instrumentation Cherie L. Green a,⁎, Lynette Brown b, Jennifer J. Stewart b, Yuanxin Xu c, Virginia Litwin d, Thomas W. Mc Closkey e a b c d e

Department of Clinical Immunology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 9132, USA Flow Contract Site Laboratory, LLC, 13029 NE 126th PL, Unit A229, Kirkland, WA 98034, USA Genzyme Corporation, One The Mountain Road, Framingham, MA 01701, USA Laboratory Science, Covance, 8211 Scicor Dr, Indianapolis, IN 46214, USA ICON Central Laboratories, 123 Smith Street, Farmingdale, NY 11735, USA

a r t i c l e

i n f o

Article history: Received 13 April 2010 Received in revised form 12 July 2010 Accepted 12 July 2010 Available online 22 July 2010

Keywords: Flow cytometry Instrument qualification Instrument validation Drug development Biomarker

a b s t r a c t Flow cytometry is a powerful and flexible analytical tool used during all stages of drug development. While substantial effort is invested in development and validation of analytical methods, instrument validation is often neglected. Flow cytometers are evolving at a pace that surpasses the protracted timeline of drug discovery and development. Therefore, it becomes fundamentally important to the success of the study to document the validated state of the flow cytometer and verify data integrity at the time of study conduct. It is important to bear in mind that validation strategies are critical components of the entire process involved in bringing new therapeutic options to the medical community; drugs which eventually manifest as successful new treatments for those individuals afflicted with disease. Formal industry guidance is provided through Good Laboratory Practices [GLP], which require validation of all computerized systems and equipment used to support pre-clinical studies for regulatory submissions. Key elements of instrument validation processes have been delineated through guidance documents published by regulatory agencies and industry working groups to support the rigorous compliance needs of GLP. However, most testing to support drug development is conducted in less strict regulatory environments. Such comprehensive validation efforts may not be appropriate for laboratories supporting early discovery or basic research, however, laboratories involved in regulated stages of development, such as pre-clinical and clinical phases, should consider these recommendations. This paper presents a consensus methodological approach that the authors have used successfully to ensure data integrity in flow cytometric studies conducted during drug development. © 2010 Elsevier B.V. All rights reserved.

“The man of science has learned to believe in justification, not by faith, but by verification.” — Thomas H. Huxley (1825–95) English biologist.

⁎ Corresponding author. Tel.: + 1 805 447 7008. E-mail addresses: [email protected] (C.L. Green), [email protected] (L. Brown), [email protected] (J.J. Stewart), [email protected] (Y. Xu), [email protected] (V. Litwin), [email protected] (T.W. Mc Closkey). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.07.004

1. Introduction Flow cytometry is a laser based technology used to perform fluorescence associated measurements of cells and other particles (e.g. bead-based immunoassays) that began approximately four decades ago, initially residing in research laboratories (Fulwyler, 1965) (Kamentsky et al., 1965) (Herzenberg et al., 1976). However, applications of flow cytometry in clinical laboratories soon developed with DNA content analysis (Darzynkiewicz et al., 1977) (Crissman et al., 1978); (Barlogie et al., 1980) and later with immunophenotyping used

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for both diagnosis and monitoring of hematological malignancies (Fernandes et al., 1979) (Wormsley et al., 1981) (Barr and Toh, 1983) and HIV infection (Prince and John, 1986;PlaegerMarshall et al., 1987); (Giorgi et al., 1987). This powerful cellular analysis platform has progressed from research settings to clinical diagnostics and most recently to those laboratories involved in studies developing and testing investigational drugs. The clinical motivation to develop therapeutic candidates that perturb specific cell types in an effort to intervene in disease and the increasing capabilities of the instruments and availability of commercial novel reagents have led to the increased utilization of flow cytometry. An additional factor driving the incorporation of flow cytometric testing in drug development is the change in the nature of therapies used to treat human disease. The mainstays of therapy in the 20th century were small molecule drugs, while the class of drugs exhibiting the most growth during the 21st century is a new type of therapeutic compound, biologics. Biologics are manufactured in living systems such as microorganisms or plant or animal cells and can be large molecular weight, complex molecules such as antibodies or cytokines which require the use of both ligand binding assays and cell-based testing methodologies to support their development (Maggon, 2007); (Giezen et al., 2009). The past decade has witnessed tremendous growth in biotherapeutics and the co-emergence of sophisticated cellbased experiments asking questions of higher complexity with flow cytometry instruments that are incorporating improved technology to keep pace. Flow cytometry has become an extremely valuable tool in the drug development laboratory, as applications in high content analysis (Robinson, 2010), pre-clinical drug development (Lappin, 2010), imaging cytometry (Zoog et al., 2009) phospho-specific drug screening (Krutzik and Nolan, 2006), and receptor pharmacology (Sklar et al., 2007) are currently being employed. The use of biomarker and safety testing to quantitatively measure pharmacodynamic changes in cellular composition and function in subjects treated with investigational drugs has become an essential component for bringing new therapies to patients. As pharmaceutical development becomes more dependent upon flow cytometric data to support filings for novel therapeutic drugs, validation of the instruments producing this data will become increasingly important. Understanding the regulatory requirements which govern the various phases of drug development may seem daunting but is essential for appropriately allocating a laboratory's resources to ensure compliance with regulatory and industry standards (Appendix A). It is important to note that these regulations do not detail the process of instrument validation but rather state the requirement. Equipment used to support regulated testing must be qualified for intended use. 2. Rationale for validation of flow cytometers in drug development Validation of flow cytometers used to support decisions made during the drug development process provides assurance to regulatory agencies, administration, sponsors, auditors, and scientists themselves, that the output generated on these instruments is reliable and precise. The gestation of a new therapeutic drug from discovery to commercialization may take a decade or longer. Advance-

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ment of technology and staff turnover may occur during the time period when data are awaiting review. Ensuring longitudinal fidelity of the data to make scientifically sound decisions and facilitating traceability of data by an independent 3rd party such as the FDA is critical to bring new therapies to the public. Unlike clinical diagnostic testing which is often used statically to diagnose a patient, pharmacodynamic measurements must be interpreted in the context of a subject's baseline (pre-treatment) and is generally monitored as change from baseline. The timeframe for seeing therapeutic activity in downstream cellular populations may be extended based upon disease and mechanism of action of the therapeutic drug. Therefore, all stages of the analytical process should be controlled. The level of regulatory rigor that is applied to the testing environment is dependent upon the stage of drug development, with non-clinical studies initially requiring the most stringent regulations. GLP requires a formal demonstration that equipment used to generate data to support regulatory filing of a new therapeutic drug is suitable for the intended purpose. Formal instrument validation, based on predicate rules (passing or failing as a measure of predefined criteria) can demonstrate the suitability of the instrument and ensure longitudinal integrity of the data. The principles of analytical instrument validation have been well described in regulatory and industry guidance documents. The code of Federal Regulations Part 11 provides guidance on the management of the computer system and associated data from inception to archival. From a laboratory users standpoint the most relevant elements include; testing to verify that an instrument is installed properly and performs as intended, identifying potential risks to the data, establishing controlled procedures for maintenance, calibration, system access and security, electronic data handling, training of personnel, and implementing processes to assess and document changes to the system in a controlled manner. While a comprehensive approach is required in a GLP setting, these processes are increasingly becoming standard practice throughout all regulated stages of drug development. When making the decision to allocate resources for instrument validation, it is important not to deviate from these well proscribed procedures. In fact, deviating from these principles may elicit inquiry during a regulatory audit. 3. The instrument Flow cytometry systems vary widely in function, complexity, and regulatory status (e.g. IVD approved, part 11 compliant, CE mark approved). There are many manufacturers of flow cytometers (Smith et al., 2010; Chapman, 2000), including a growing number of vendors which have released instruments with applications to support in vitro diagnostic (IVD) testing for lymphocyte subset quantification, stem cell enumeration, and histocompatibility testing (Table 1). Flow cytometers are complex, flexible instruments, with unique validation needs. Unlike other lab methods, there are no accuracy standards in the strictest definition (Wu et al. this issue). This lack of standards increases the challenge when verifying the performance of the flow cytometer. Additional layers of complexity are added when considering the output

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Table 1 Summary of Currently Available Flow Cytometry Cell Analyzers*. Instrument

Excitation sources

Parameters

Key features

1 laser

2 fluorescent 2 light scatter (FSC/ SSC)

• Dedicated for CD4/CD8 counts for HIV/AIDS • Minimal user intervention

COULTER® EPICS XL™/ and XL-MCL™ 1 laser (488 nm) (Beckman Coulter)

4 fluorescent 2 light scatter (FSC/ SSC)

• Digital processing • HTS carousel option • Single laser simplicity

FACSCalibur™ (BD Biosciences)

2 (488;635 nm)

4 fluorescent 2 light scatter (FSC/ SSC)

• Analog instrument • Carousel option

Cytomics FC 500 (Beckman Coulter)

2 (488;635 nm)

5 fluorescent 2 light scatter (FSC/ SSC)

• 5 color detection off blue line • Digital compensation • Plate-based HTS option b

BD FACSCanto (BD Biosciences)

2 to 3 lasers (488, 633 nm)

Up to 8 fluorescent 2 light scatter (FSC/ SSC)

• Carousel and HTS options • Fixed laser alignment

BD FACSCanto II (BD Biosciences)

2 to 3 lasers (405, 488, 633 nm)

Up to 8 fluorescent 2 light scatter (FSC/ SSC)

• Carousel and HTS options • Fixed laser alignment

CyFlow® SL (Partec GmbH)

1 laser (488 nm, with option for others)

3 fluorescent 2 light scatter (FSC/ SSC)

• Mobile, can run on 12 V DC • Volumetric counting • Sub-micron detection

CyFlow® ploidy analyzer (Partec GmbH)

1 laser/ 1 LED (UV LED and 532 nm) 2 fluorescent 1 light scatter (SSC)

Guava EasyCyte™ Mini (Millipore)

1 laser (blue)

3 fluorescent 1–2 light scatter (FSC/ optional SSC)

• Compact Benchtop • Absolute counting • Microcapillary flow cell

Guava PCA (Millipore)

1 laser (green)

2 fluorescent 1 light scatter (FSC)

• Compact Benchtop • Absolute counting • Microcapillary flow cell

Guava PCA-96 Base System (Millipore)

1 laser (green)

2 fluorescent 1 light scatter (FSC)

• Compact Benchtop • Automated MTP sampling • Microcapillary flow cell

Guava EasyCyte 8HT (Millipore)

2 lasers (red, blue)

6 fluorescent 2 light scatter (FSC/SSC)

• Compact Benchtop • Automated MTP sampling • Microcapillary flow cell

C6 Flow Cytometer (Accuri Cytometers, Inc.)

2 lasers (488, 640 nm diode)

4 fluorescent 2 light scatter (FSC/SSC)

• Benchtop • 6 log dynamic range • Low-pressure fluidics

Attune™ Acoustic Focusing (Applied Biosytems)

2 lasers (red, blue)

6 fluorescent 2 light scatter (FSC/SSC)

• Small Benchtop footprint • Acoustic focusing • Optimized for rare event detection

Cyan ADP (Beckman Coulter)

2 to 3 lasers (488, 642, 405 nm)

7–9 fluorescent 2 light scatter (FSC/SSC)

• Small Benchtop footprint • Automated compensation • Walk-up operation

Gallios (Beckman Coulter)

2 to 3 lasers (blue, red and violet)

6–10 fluorescent 2 light scatter (FSC/SSC)

• Optimized with reagents • Boulevard light path • Easy change filters

MoFlo™ XDP (Beckman Coulter)

3 lasers (from 355, 405, 488, 635 nm)

12 fluorescent 2 light scatter (FSC/SSC)

• Multiple sort gates • 4-way sorting • 5-log dynamic range

Approved for clinical diagnostic use a BD FACSCount™ Instrumentation System, (BD Biosciences)

Basic and clinical research

• Compact with integrated GUI c • Genome sizing/ploidy • Plant, animal, microbes

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Table 1 (continued) Instrument

Excitation sources

Parameters

Key features

CyFlow® space (Partec GmbH)

3 lasers (375, 405, 488, 635 nm)

7 fluorescent 2 light scatter (FSC/ SSC)

• Compact Benchtop • Volumetric Counting • Sub-micron detection

ImageStreamX (Amnis, Corp)

Up to 5 lasers (405, 488, 560, 592, 658 nm)

Up to 10 fluorescent Brightfield and darkfield

• Imaging cytometer • Dots are also images • Phenotype and Morphology

BD LSRFortessa™ (BD Biosciences)

5 lasers (from 11 wavelengths)

18 fluorescent 2 light scatter (FSC/SSC)

• Reduced footprint (cf. LSR II) • Upgradeable • Optimizes excitation/emission

BD LSR™ II (BD Biosciences)

4 to 7 lasers (special order, from 11 Up to 18 fluorescent wavelengths) 2 light scatter (FSC/SSC)

• Fully flexible platform • Upgradeable • Optimizes excitation/emission

Basic and clinical research

Basic and clinical research, drug discovery and industrial 3 fluorescent 1 light scatter (SSC) Coulter Volume

• Absolute cell counts • HTS with sample prep(with plate loader option) • Sample track and 21 CFR part 11 compliant

Cell Lab QUANTA* SC (Beckman Coulter)

Arc lamp and 1 laser (488 nm )

CyFlow® ML(Partec GmbH)

5 UV lamp/LED (375, 405, 488, 638, 13 fluorescent • Compact Benchtop 532, 561) 3 light scatter (2 FSC/SSC) • Volumetric counting • Sub-micron detection

MACSQuant (Miltenyi Biotec GmbH)

3 lasers (405, 488, 635 nm)

7 fluorescent 2 light scatter (FSC/SSC)

• Compact benchtop design • Couples to magnetic bead column • Integrated fluidics and GUI

S1000 Flow (Stratedigm, Inc.)

4 lasers (372, 405, 488, 532, 640 nm)

8 fluorescent 2 light scatter (FSC/SSC)

• Benchtop design • Temp-controlled HTS loader • User-replaceable modules

Adapted from Smith et al., 2010. a With the exception of the FACSCount the instruments approved for clinical diagnostics may also be used for basic and clinical research using non-locked or user-designed protocols. b High throughput screening. c Graphic User Interface.

associated with flow cytometers. It is common for acquisition and analysis templates to be customizable, and “data” consists of many outputs, of which some are primary; raw listmode files, and some which are manipulated (e.g. analyzed files, statistics export, experimental templates, and compensation files). Such complexity can be addressed by thinking of the system as a whole and necessitates the involvement of personnel with expertise in flow cytometry in the validation process. 4. Defining the strategy Industry working groups such as American Association of Pharmaceutical Scientists (AAPS), United States Pharmacopeia (USP) and International Society for Pharmaceutical Engineering (ISPE) have published useful recommendations for qualification of instruments and validation of computerized systems (Bansal et al., 2004). Each recommendation document presents classification categories to assist the user in determining the complexity of instrumentation and resources needed to complete the validation. While some recommendation documents focus on the qualification of the instrument, others emphasize computerized systems. In

reality, one cannot exist without the other (e.g. instrument can't generate data without the computer). When establishing the validation strategy, it is important to consider capacity planning. While an institution may initially purchase only one instrument, additional instruments may be obtained in the future, based on laboratory needs and resources. Using a “family of systems” approach to validation may streamline the process for subsequent instruments. A group of instruments sharing identical configuration and functionality can be considered a family. Using this strategy, common documents are applied to the “system” (validation plan, specifications, SOPs) while subsequent systems require only installation, operational verification and inter-instrument comparability testing. The validation process outlined in this paper can be separated into three distinct phases; 1) Planning, 2) Testing, and 3) Implementation. A schematic of each phase and associated activities is shown in Fig. 1. Prior to committing resources for a validation project, the laboratory should assess the intended use and applicable regulatory compliance needs of the system. Drafting a system description will guide the decision as well as streamline the subsequent validation process.

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Fig. 1. Phases of Instrument Validation Activities and Associated Documentation.

5. Planning phase 5.1. Flow cytometry system description 5.1.1. Instrument configuration With the ever-improving technology incorporated into new instrument design, it is tempting to purchase the most advanced technology to provide maximum analytical flexibility. However, from a validation perspective, flow cytometers with fixed configurations present less difficulty when approaching a formal validation process. Instruments with flexible designs that are biased towards research applications are more challenging to validate and require more detailed testing. Instruments with fixed configurations can be tested against vendor's specifications, whereas, custom configurations require the user to define the specifications. The ability to modify a flow cytometer for a particular research application, while providing increased scientific horsepower to the investigator, also results in additional complexity in instrumentation, reagents, and analysis. The intent of this paper is to provide recommendations on the validation process for flow cytometry analyzers with fixed configurations. 5.1.2. Software Validation of software should be approached within the overall design of the flow cytometry system. If for example, the software is needed to operate the instrument (QC applications, acquisition applications); it should be incorporated within the overarching instrument validation plan. If

multiple software applications exist on a single instrument, the validation team should assess if all software should be included in the validation and the extent of the testing required to verify performance of the software. For example, IVD assays are generally performed using regulated software applications associated with the instrument to acquire, analyze, and report data. These applications typically have enhanced security functions and specific templates for acquisition and analysis, thus, verification of functionality and confirmation of data integrity may be sufficient for validating the software. It has become increasingly common to use stand alone software applications to perform post acquisition analysis such as FCS Express (DeNovo Software), FlowJo (Treestar, Inc.), and WinList (Verity Software House). In this case, it is important to consider the use and interdependence of each component. If data will be generated using multiple instruments but analyzed with a single stand alone software application, the validation of such software should be addressed separately. Stand alone software can also be used to support the validation of instrument software. An example of this would be using the stand alone software to verify functionality of instrument software (e.g., CD3+ lymphocytes displayed in both software applications). Other factors that should be considered are the procedures for exporting data (listmode files, exported statistics, analysis templates, and experiments) from one system to another and the subsequent manipulation through post

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Fig. 2. Example Process Flow Diagram for Acquisition and Analysis of Flow Cytometry Data.

acquisition analysis. The integrity of all data should be confirmed at each step of the export and analysis. For example, manual verification should be performed to ensure that data obtained from the flow cytometer, from the stand alone analysis software, and from the database are identical. 5.1.3. Environment The environment in which the system will be maintained and used should be considered. Ideally, instruments should be segregated by intended use and regulatory compliance needs. However, it is more common for instruments to reside in shared use (core) facilities, supporting both regulated and non-regulated testing. Management of instruments in core facilities that support both research and regulated use is challenging and controversial. If instruments must be shared among users for different purposes (based on stage of drug development) it is important to assess whether the instrument is an appropriate candidate for validation. Based on a documented rationale, applying principles of regulated testing such as placing the system under change control, establishing standardized procedures for operation, maintenance, data management, and limiting access to regulated templates may be sufficient to ensure data integrity. However, it is important to recognize that these steps alone do not demonstrate instrument validation as outlined in this paper. 5.1.4. Manufacturer It is the responsibility of the manufacturer to have quality systems in place for developing and manufacturing the system, including functional and design specifications and assembly processes of the hardware and testing of associated software. The ability of a vendor to provide accurate and

timely system maintenance, technical support, and training should also be a factor when choosing an instrument. The manufacturer should test the assembled instrument prior to shipment and should make available the results of final instrument and software tests. Typically, the vendor who is frequently the manufacturer, may offer to conduct and document the installation and operational qualifications. Finally, the manufacturer should notify users when issues are discovered or updates are offered after a product's release, offer user training, and installation support, and respond to user audits as necessary.

5.2. The validation team Once the flow cytometer has been chosen and the compliance need has been defined, the validation team can be assembled. Establishment of the validation team is critical for success of a flow cytometry instrument validation project. Each person assigned should be highly qualified to do their part. Qualification of each member should be documented. Key members include: scientific management, system owner, users, information technology (IT), and quality assurance (QA). Scientific Management is the entity with the overall responsibility for assigning the team members which includes designating the system owner. This also includes providing the necessary resources for the equipment and validation project. Management should monitor the scheduled timeline for project completion, make sure all applicable regulations are complied with and provide review and approval for the flow cytometry system validation documents.

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Fig. 3. Example of Validation Plan.

The assigned System Owner should have a very good understanding of the flow cytometry system and its associated applications. This person is responsible for ensuring that the flow cytometry system operates as intended and is maintained in a validated state according to applicable regulations. The system owner is the natural designee to draft the overall quality control and maintenance procedures and associated SOPs. Users include analysts and their supervisors who are ultimately responsible for the instrument operations and data quality. Users should be adequately trained in the instrument's use, and their training records should be maintained as required by the applicable regulations. Consultants, validation specialists, and quality assurance personnel can advise and assist as needed, but the final responsibility for validating instruments lies with the users and the validation team. It is the

responsibility of the user to maintain the validated state of the instrument by complying with procedures set forth in the validation plan including routinely performing and documenting quality control (QC) and maintenance. Typically, IT assures that the validation of the computerized system and associated secure data storage locations fits into the current and future IT infrastructure. IT is responsible for performing installations of the computer network and maintenance of that network, providing the needed items for data archiving and training of users on the use of the network. IT also manages the security of the system by regulation of user's rights by the implementation of user names and passwords. QA, the independent reviewer, has the responsibility to review the validation documentation and inspect the validation activities for compliance with applicable regulations. QA

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personnel should work with the system owner and users in order to understand the instrument validation process and the intended application. Finally, QA should review the validation documentation for internal accuracy and consistency and to determine whether it meets applicable regulatory requirements. The bottom line is that QA personnel are best suited to ensure regulatory compliance, thus they should be closely involved throughout the installation and validation phases. 5.3. Standard operating procedures and system documents GLP regulations require SOPs for the establishment and routine use of validated computerized systems to address the computer life cycle topics: Operation, Maintenance, Security, Change Control, Data Management, Contingency Plan and Disaster Recovery, Archival, and Internal Audits. Additional procedures such as guidance for training and documenting certification of staff to operate and maintain the instrument and periodic review should be established. SOPs are controlled documents which need to be clearly written with adequate detail. The “intent” of an SOP is that laboratory personnel, following adequate training, should be able to replicate a particular procedure. Particularly in the case of a laboratory with multiple instruments, or with instruments in multiple locations, the SOP serves as the standardization vehicle. SOPs should be written from a “real-world” perspective as they should detail how procedures are actually performed. A key point regarding SOPs is that it is critical to ensure that instrument SOPs are followed, as laboratory technical personnel may have different approaches towards performing the same task. Ensuring that all staff follows detailed, step-by-step SOPs provides the uniformity necessary for flow cytometric analysis in the context of regulated analytical testing. 5.3.1. Operation, maintenance, and calibration The purpose of this SOP is to describe how the instrument and associated software will be used, maintained, and calibrated for its intended purpose. These procedures should be based on guidance set forth in the instrument manufacturer or vendor user's manual but also may specify additional requirements defined by the user. Procedures and associated schedules for routine and non-routine maintenance should be outlined and should be in alignment with vendor recommendations. Establishing clear forms in which users can document routine and non-routine maintenance and calibration (including user initials and date activity was performed) aid in tracking compliance with procedures set forth in the SOPs. All forms, memos, and associated QC reports and trending graphs should be considered controlled documents and maintained in a logbook that is routinely reviewed and archived. 5.3.2. Security The purpose of this SOP is to outline security measures to protect people, property, and sensitive information by the ability to identify, deter, detect, observe, report, and respond to conditions that present a threat to these items. This includes both physical security of the premises by locks and security systems and logical security by regulation of access

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privileges (administrator or user) by the implementation of user names and passwords. 5.3.3. Change control This SOP describes the procedure for making changes to validated systems. A change request should be formally approved by the system owner and documented. The risk of the change should be assessed for its potential impact on the performance of the system. It is important to carefully assess the impact of a change made to the instrument or its related software, thus, planning for such a change is an important component. Ideally, an experimental design which includes “before” and “after” functional testing for comparison should be implemented. 5.3.4. Data management SOPs should be in place to define the procedures for all backup and restoration of electronic and paper data, applications, system utilities, and the operating systems. This includes the frequency and period of retention as well as minimum requirements for media management and off-site storage. Ideally, data should automatically be transferred to a secure server that is automatically backed up at regular intervals. This ensures that should a catastrophic computer failure occur, all previous data is already backed up. This type of approach minimizes the level of human manipulation to archive data. However, many instrument vendors discourage saving data directly to a network server and may design their software applications to save data directly to the work station hard drive. Moreover, security of the network server itself may limit or deny direct transfer of files, depending on how security privileges are established on the network. In this case, procedural controls should be clearly identified and fully tested during validation to ensure data integrity at each checkpoint. Flow cytometry data (listmode files) generally requires additional analysis using software applications to represent the data in readable format. Many laboratories find themselves working with both paper and digital documents, operating in a hybrid environment that blends the ever growing volume of electronic information with the paper world. This blending of paper and electronic systems, while each has their own benefits, together creates significant organizational complexity and costs. One approach to complex intermingling of these two systems differentiates short-term versus long-term usage. 5.3.5. Contingency plan and disaster recovery This SOP should describe a contingency plan outlining the procedures to be followed in case of system failure or unavailability with a detailed plan for recovery. Appropriate tests should be performed and documentation of results obtained should be maintained. 5.3.6. Internal audits This SOP describes the implementation of risk-based IT audit procedures based on a formal risk assessment methodology to determine the appropriate frequency and extent of work. Audit procedures will vary depending upon the sophistication of the data center and end-user systems. The

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audit procedures may include manual testing processes or computer-assisted audit programs. 5.3.7. Training Clearly defined guidance on performing and documenting training of users will ensure that the instrument is maintained and used as intended. Activities should include a combination of reading associated SOPs and demonstrating comprehension through instructor-led exercises. The use of desktop sharing software to train flow cytometry operators located at different sites is a useful tool in achieving harmonization of instrument related procedures among different laboratories. 5.3.8. System specific documents Installation and user manuals, release notes, and vendor audit reports are all examples of supporting documents that may be provided by the vendor. The installation manual is a set of instructions that defines the minimum hardware and software requirements for the system to perform properly and should be followed when the system is first installed. The user manual is the Manufacturer/Vendor supplied document outlining proper use of the system. Release notes contain updated information on changes to a software package compared to a previous version. The vendor audit report contains the results of the vendor inspection concerning the development of the system or software. It is common practice for vendors to provide these documents in electronic form only now so procedures must be outlined for how to maintain access to current documents only and archive previous versions. However the documents are provided, they should be readily accessible to the user.

ria for demonstrating that the system has been successfully validated should be included as well as procedures for communicating the release of the system into regulated use. The plan should also refer to any applicable SOPs and define the timeline for approval. An example validation plan is shown in Fig. 3. 5.5. User requirements and functional specifications The flow cytometer is intended to acquire and analyze cells or other particles such as beads using light scatter and fluorescence based measurements. The users for the system are those trained for such processes. The user requirements outline what processes the operator requires the flow cytometer with associated software to perform. The functional specifications identify expected capabilities of the system and are guided by the specific user requirements (Table 2). 5.6. System compliance and functional risk analysis Prior to executing the validation testing, it is important to identify any risks associated with the system in relationship to the process flow. The validation team should determine if the identified risk(s) can be mitigated and if there is a specific action to reduce risk this should be tested during the operational qualification. For risks that cannot be mitigated through system function, procedural controls must be clearly defined in the SOP. For system compliance an example would be: If an instrument is unable to save data directly to a secure server, procedural controls should be in place to instruct the operator to perform step-by-step procedures for saving and backing up data, thus minimizing risk to the greatest extent possible. For examples of functional risk analysis see Table 3.

5.4. Validation plan 6. The testing phase The Validation Plan should define the system description, state the purpose and scope of the project, identify roles and responsibilities of the team, and establish a strategy for completing the project. A process flow diagram helps define the validation scope by visualizing the data flow, including associated interfaces with external systems and data life cycles (Fig. 2). The plan should outline the deliverables of the project including; User Requirements, Functional Specifications, Risk Analysis, IQ/OQ/PQ, and Validation Report. Acceptance crite-

Test protocols defining the test specifications, instructions for testing and expected result should be drafted in the planning phase with execution of the testing performed during the testing phase. 6.1. Installation and operational qualification (IQ, OQ) The purpose of the IQ and OQ is to provide evidence that instrument hardware and associated software are installed and

Table 2 Examples of User Requirements and Functional Specifications. User requirement

Functional specification

System shall have procedures for monitoring quality control

Quality control application within the software provides performance checks and trending reports

System shall acquire and analyze data

Software acquires sample with specific event stop count and creates dot plots, histograms and gates

System shall compensate fluorescent spillover

Software allows for automatic and/or manual compensation procedures, describe specific process

System shall provide batch data analysis and statistic export into spreadsheets

Describe process for batch analysis and statistic export

System shall provide 21 CFR Part 11 compliance for security and audit trials

Describe each functional specification for security and audit trails. If procedural controls are needed when part 11 is not available, describe the procedural controls

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Table 3 Examples of Functional Risk Analysis. Functional specification

Risk analysis

Mitigation strategy

Test with expected outcome

Quality control application in the software

Failure to perform instrument quality control procedure could result in the inability to demonstrate data integrity on day of acquisition

Implement secondary QC procedure and test. Risk is also reduced by each assay having its own control which would not be tested here but within the assay validation.

Perform secondary QC procedure. Results should pass established acceptance criteria.

Compensate fluorescent spillover

Failure to perform compensation for fluorescent spillover prior to data acquisition could contribute to erroneously analyzed data

Implement procedural practices for performing compensation. Software may have feature for postacquisition compensation which should be tested.

Perform post-acquisition compensation and compare results to data compensated prior to acquisition. Verify compensation is as expected.

functioning per manufacturer's specifications and user's requirements. This can be performed by the instrument vendor, by internal staff, or by contracted external consultants. During the IQ, environmental and utilities requirements such as space, temperature, and electrical components are verified against vendor's minimal specifications, which can be found in the instrument manual (Table 4). During the OQ, tests are developed with acceptance criteria to verify that functional parameters are operational and adhere to vendor's specifications or user requirements (Table 5). This includes both positive and negative (stress tests) outcome assessment for the system. If there are specific actions stated in the risk assessment that attempt to reduce risk, this action is tested here. Many vendors now provide formal IQ/OQ packages which can be leveraged to expedite the validation process. Using the vendor to perform the IQ and/or OQ has many merits including; ensuring vendor accountability by addressing corrective actions for defective components at the time of installation, leveraging resources in a constrained environment, and providing a declaration of successful installation.

Additional benefits of having the vendor perform the OQ is that a certified technician will perform the OQ using calibrated equipment and vendor specified testing materials that may not be routinely maintained by the laboratory (e.g., laser power meter, pressure testing equipment, etc.). Disadvantages of vendor-performed IQ/OQ include increased cost to the initial resource investment and still require oversight by the validation team to ensure that testing is comprehensive and that documentation practices are in compliance with corporate policy. Since the vendor-performed IQ/OQ can be very costly, the IQ/OQ can alternatively be performed in-house by qualified staff. In-house validations can be used for instruments that have already been installed but no formal installation qualification was performed by the vendor at the time of installation. The main advantage of performing the IQ/OQ inhouse is that qualified staff has the expertise to understand the intended use of the system. Vendor testing may prove insufficient for complete verification of the system performance. Tests can then be designed with this in mind to

Table 4 Examples of Installation Qualification Parameters and Associated Testing and Documentation. Installation parameter

Test

Documentation

Notes

Environment

Verify Benchtop and associated lab space meets vendor specification

Checklist with detailed dimensions and positive notation of Pass/Fail and initial and date

Consider space requirements for instrument/ computer footprint and additional clearance for future maintenance

Utilities

Verify temperature and humidity of lab space meets vendor specification

Checklist with detailed dimensions and positive notation of Pass/Fail and initial and date

Equipment used to perform verification should be documented in report

Electrical

Verify electrical requirements meet vendor's specifications

Checklist with detailed specifications and positive notation of Pass/Fail and initial and date

Equipment used to perform verification should be documented in report

Hardware

Verify all components are installed and free of damage

Document instrument specifications (model, serial number, manufacturer date), including automated sample acquisition modules

Include, if any, external hard drives used for data backup and uninterrupted power supplies (UPS)

Document workstation specifications (computer model, serial number, software version

Printers should also be included and identified as non-networked or networked

Verify workstation and Computer system and associated software are associated installed software

Demonstrate computer system communicates to instrument by acquiring data Checklist with detailed specifications and positive notation of Pass/Fail and initial and date

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Table 5 Examples of Operational Qualification Parameters and Associated Testing and Documentation. Operational parameter

Tests

Documentation

Notes

Software Perform automated system functions functionality (startup, QC)

Screenshot and/or report with positive If maintenance procedures are an automated software notation of Pass/Fail, initial and date function include here.

System alerts

Document warnings displayed with screenshots, initial and date

Stress the system to demonstrate that system detects problems and displays appropriate warnings Example: Attempt to acquire data with low fluid levels or disconnected computer cable

Visual cues can also be used to prompt user to change fluids Example: Fluidics icons change color when low levels are detected System should have warning and not allow further acquisition until fluidics issues are addressed

Sample flow rate

Demonstrate sample flow rate is within Checklist with detailed specifications vendor's specifications and positive notation of actual result, Pass/Fail and initial and date

Equipment used to perform verification should be documented in report

Optical precision

Run calibration beads to verify %CVs and Checklist with detailed specifications and positive notation of actual result, laser power output meets vendor Pass/Fail and initial and date specifications

All testing reagents should be documented in report

Automated sample acquisition

There is some overlap in PQ, replicate samples could also Checklist with positive notation of Acquire triplicates of testing material (beads or cells) in randomly distributed successful sample acquisition, Pass/Fail be used to demonstrate precision and initial and date locations in carousel or plate

complement vendor's testing. Therefore, vendor testing should be considered a starting place for verification of operational qualification. 6.2. Performance qualification Performance Qualification (PQ) can be the most consuming part of the validation process. Distinguishing PQ strategies of instrument versus assay is often blurred. For example, demonstrating the dynamic range of the instrument is different than establishing the analytical method range of a quantitative fluorescence assay. The dynamic range of the instrument refers specifically to the sensitivity of the optical components of the instrument while the analytical method range takes into account reagent and specimen variables. Beads with 8 peaks of different fluorescent intensities are available (Spherotech, Inc.) and provide a useful tool to assess instrument dynamic range. Validation of flow cytometric assay methods have been reported across multiple applications (Maecker et al., 2008; Hultin et al., 2007). It is helpful to focus validation efforts on representative testing for the intended use. It is interesting to note that this topic is where the authors varied the most in approach. Some laboratories may define the scope of the instrument validation to include testing that demonstrates mechanical and optical performance only, while others may choose to expand the PQ to include comparability of instruments and/or multiple laboratories, in the context of the testing environment. In this section we will discuss some of the possibilities for PQ including; basic instrument performance, inter-instrument and inter-laboratory comparability, and longitudinal performance (Table 6). 6.2.1. Instrument performance Performance of the flow cytometer, including optical alignment and sensitivity should be established prior to embarking on a formal validation of the system and certainly well before the instrument is used to support any regulated testing. Methods for establishing and monitoring optimal instrument performance have been well described (Purvis

and Stelzer, 1998); (Owens et al., 2000). Typically, calibrated particles of varying sizes and fluorescence are used to establish linearity and sensitivity through repeated measurements of fluorescence and % coefficient of variability (CV). The dynamic range and linearity of the system can be measured using calibrated particles with multiple peaks. Characterizing the limits of the instrument and defining a comprehensive quality control monitoring system will alert the user to potential degradation of instrument hardware. Most vendors now provide integrated QC applications thus making it easy to establish optimal instrument settings and to monitor light scatter properties and fluorescence output. If this is not available on the designated system, or if the operator deems the application to be insufficient for monitoring performance for the intended use, manual methods should be established by the user (Hoffman, 2005; Hoffman and Wood, 2007). The starting point for PQ activities should focus on verification of instrument performance including: routine QC monitoring, acquisition precision, carryover to determine impact of samples with high cellular concentrations or sticky reagents, and automated sample collection. Fluorescent measurements and % positive cells and/or absolute cell counts can be verified by using a combination of biological control cells and fluorescently-labeled calibration particles. Additional assay specific functionality or outputs should be addressed during assay validation. While demonstrating performance qualification of a single instrument is essential, in the drug development laboratory it is often necessary to examine multiple flow cytometers for consistency. 6.2.2. Inter-instrument comparability For samples acquired on flow cytometers during drug development studies, it is typical practice to have in place a backup instrument, in order to reduce the impact of an unforeseen instrument breakdown. Comparability should be established between primary and backup instruments. Sideby-side testing with the same samples is usual practice in order to demonstrate that results produced by both

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Table 6 Recommended Performance Qualification Activities. Parameter

Purpose

Factors to consider

Testing

Instrument Performance

Demonstrate instrument performance is optimal and system performs as intended

Optical alignment

Establish QC method including pass/fail criteria and monitor performance using trending, success of qualification will be measured against acceptance criteria of QC method

Sensitivity linearity

Test sensitivity of the system using calibrated multi-peak fluorescent beads of varying sizes, success will be measured by meeting minimum vendor's specifications or user requirements

Acquisition precision, Automated acquisition Acquire replicate samples with automated functionality, functionality success will be measured by demonstration of mechanical operation and precision of replicate samples Acquisition carryover

Acquire samples with varying concentrations of cells to demonstrate carryover across expected range, success will be measured by meeting vendor's minimum specifications

Inter-Instrument Demonstrate comparable Comparability results between instruments

Backup instruments must produce comparable data to reduce introduction of external variation Implement common procedures for instrument setup, calibration, and operation

Acquire beads and/or biological controls on both instruments, success will be measured by meeting predefined acceptance criteria

Inter-lab Comparability

If testing will be performed across multiple sites, must demonstrate robust output-lab to lab variability must be minimized Implement common procedures for instrument setup, calibration, and operation

Use calibrated fluorescent beads to establish target ranges or as an internal assay control

Longitudinal Performance

Demonstrate comparable results between laboratories

Assess longitudinal needs of the system Demonstrate instrument produces consistent data over time

Acquire beads and/or biological controls across labs, success will be measured by meeting predefined acceptance criteria Use calibrated fluorescent beads to establish target ranges or as an internal assay control Acquire beads and/or biological controls over time, success will be measured by meeting predefined acceptance criteria

cytometers are equivalent. Comparisons between multiple flow cytometers require the implementation of common procedures for instrument setup, calibration, and operation in addition to common analytical procedures. Certain steps are essential in this endeavor, including standardization of platform, reagents, methodology, as well as performing specific testing to demonstrate that the flow cytometers are producing comparable results. Comparing outputs from different flow cytometers becomes more challenging when they are located in different laboratories. 6.2.3. Inter-laboratory instrument comparability Along with the new emphasis on biologic therapeutics, two other major trends are notable in the drug development industry over the past decade. First, there have been significant increases in outsourcing laboratory testing during the conduct of pre-clinical and clinical trials and there is increased awareness of the necessity of global testing during drug development. The result of these changes has been the increased role that global central laboratories play during all phases of drug development (Twombly, 2008); (Chadwick, 2001). These changes in trial landscape require extraordinary vigilance by the sponsor to monitor quality. The risk of problems arising in multi-laboratory studies is greater than in those performed in a single site. Lessons have been learned towards achieving harmonization of data generated in multi-site studies in both the HIV (Giorgi et al.,

1990); (Paxton et al., 1989) and stem cell (Levering et al., 2007) arenas, including the use of proficiency sample sendouts to achieve data comparability. Robust measures should be implemented in order to avoid generating vulnerability: data sets in the different laboratories are different due to instrument differences. Many steps can be standardized between laboratories, but the bottom line is that different personnel are running different instruments. Staff not following SOPs or a laser problem at one site which goes unrecognized could result in non-comparable data sets. One approach to mitigating such deleterious outcomes is to assign a single person the task of training staff at all laboratory locations, with an eye towards harmonizing all elements of instrument setup and calibration to the greatest extent possible. Assuring comparability between instruments located at different sites around the world has become of critical importance. Some approaches utilized to achieve standardization between flow cytometers in different geographic locations include; the use of calibrated fluorescent beads to establish target fluorescence channel values on all instruments or the calculation of a standard curve which can be applied to the data. Control reagents (stabilized blood or lyophilized cells) with known, manufacturer-provided ranges such as CD-Chex® Plus (Streck, Inc) and COULTER® CYTOTROL™ Control Cells (Beckman Coulter) can also be used to demonstrate standardization. However, these products are

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generally limited to defined ranges for basic phenotypic markers and cell subsets. Splitting samples for simultaneous testing at multiple sites is also a practice that is employed to provide assurance of comparability, although specimen instability can limit the usefulness of this approach. Establishing standardized, controlled acquisition and analysis templates can also ensure data comparability. Use of well-characterized reference material (stabilized blood, cells, etc) and centralized reagents, where reagents are acquired, titered, and validated at a single location and then distributed to the other sites, is another step towards interlaboratory comparability. Cross-training of analysts such that practices are harmonized also reduces variability. Such training might initially include site visits by analysts, and the use of web-based training to assure continuing competence and compliance in instrument associated procedures. While these performance qualification checks are important, the laboratory involved in flow cytometric testing for drug development should also ensure instrument performance over time, independent of sponsor or study. 6.2.4. Longitudinal performance qualification Maintaining quality performance from flow cytometric instrumentation is important for any user of this technology. However, the consistency over time of flow cytometric output holds enhanced significance during drug development, where quantitative measurements of protein levels or drug receptor occupancy are compared to pre-treatment values. This is especially true for long, protracted, clinical trials which may take place over years, and less so for pre-clinical studies which are generally of shorter duration. Although the principles of ensuring longitudinal instrument performance are universal in all regulated environments, there are additional considerations when using this technology to quantitatively measure fluorescence. One method for standardizing fluorescence over time and across platforms is to implement the use of a standardized curve generated using particles of known fluorescent concentration which can be measured in terms of molecules of equivalent soluble fluorochrome (MESF) (Schwartz et al., 2004); (Wang et al., 2007). MESF beads are currently available in limited fluorochromes therefore alternative methods to standardize cytometric output across platforms have been described. Hard-dyed beads containing mixtures of embedded fluorophores, in addition to biologic calibrators, (Shults et al., 2006) (Wang et al., 2008), have been used to assign relative fluorescence units such as molecules of equivalent fluorescence (MEFL) or equivalent number of reference fluorophores (ERF). In addition, judicious use of control samples can aid in minimizing instrument variation over time as applied to specific drug development studies. For example, a large batch of primary cells may be isolated and cryopreserved in small aliquots. These cells can then be tested at various times over the course of a study, demonstrating longitudinal reproducibility of cytometer output. In a similar manner, a large number of flasks of a particular cell line can be grown up and then frozen in small aliquots. For quantification of fluorescence intensity (MFI) as data output, optimization of assay specific instrument settings (PMT voltages) is recommended. Single or multi-peak beads with fluorescence levels comparable to the expected dynamic

range of the assay can be used to establish target ranges. Using this method, prior to each run, voltages can be adjusted using the beads to meet the target range or value(s). This is one method for controlling the day to day variation that is inherent to the flow cytometer. 7. Implementation phase 7.1. Validation summary report The validation summary report is written when all the testing is complete. The purpose of this report is to summarize validation activities and outcomes and should mirror the Validation Plan. The Validation Report should contain the following elements: • Overview of the system and diagram of technical architecture • Summary of all the validation tests with acceptance criteria and outcome • Requirements traceability matrix to reference requirements to the associated function testing • Summary of test failures or deviations from the Validation Plan and associated resolution • Recommendations • Final authorization statement and signatory page confirming that the system is suitable for use as intended and is capable of generating high quality data that is compliant with all applicable regulations. In our experience, an excellent tool to place early in the report is a summary table of specifications and results. The information in such a table should clearly spell out the tests that were performed during the validation, what was expected, and what results were obtained. In the conclusion, the validation summary report should include the validation status of the instrument and its readiness for operational use. 7.2. System in operation Once all documentation is finalized, a system release report or notification form can be used as a means of signaling that the system is released for production. Additional users of the instrument can then be trained in accordance with SOPs. 7.3. Manage life cycle While following the guidelines for validation of flow cytometers and associated software contained herein is an important first step in ensuring data integrity, as previously mentioned, it is essential that the initial validation is followed by robust monitoring, change control, and proper documentation for consistent performance over time. 7.3.1. Change control To maintain the validated status of the instrument, change control procedures should be effective upon release of the system into production. Establishing procedures for controlling changes throughout the entire lifecycle of a system is essential. Key elements to an effective change control system include: identification of change, assignment of impact level and determination of need for change. If the change is unnecessary

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to the performance of the system and is determined to impact the validation status then changes can be rejected or postponed, such as software upgrades where risk to the validation outweighs the improvement of the software). Defining and executing functional testing to ensure integrity of the system and documenting the outcome completes the process. Examples of impact levels are as follows: • Low: routine change resulting in minimal impact to the system with no cross-system impact. It is helpful to identify anticipated low impact changes in the instrument SOP in order to expedite the change control process Example: replacement of non-routine fluidics components • Moderate: a change that only impacts a single system, and does not require a planned outage exceeding the maintenance window established for the system Example: software upgrades or like-for-like laser replacement • High: a change that has a cross system impact, or requires a planned outage exceeding the maintenance window established for the system, or involves a full version upgrade resulting in a change in functionality Example: upgrading software with new functionality, installation of automated sample acquisition There are many changes that can be anticipated during the life cycle of a flow cytometer. It is helpful to work with the vendor to establish a list of expected changes to the system and document them accordingly in the instrument maintenance SOP. 7.3.2. System retirement When a system is taken out of service, it must be done in a controlled and planned way to minimize risk for regulatory compliance. The retirement should be performed according to a system decommissioning plan and documented. All system documentation (log books, manuals etc.) and the associated software applications should be archived according to the SOP. 8. Conclusions The processes outlined in this paper are those which the authors collectively recommend based on GLP principles and industry guidance documents. Each laboratory is responsible for assessing the compliance needs of their own system, determining the depth of validation activities, and implementing appropriate procedures for verifying instrument operation. It is important to note that even the implementation of only certain components (SOPs, change control, training, and security) of these guidelines may be beneficial, however, with such an approach the instrument would not be considered fully validated. As the role of flow cytometry in the regulated areas of drug development continues to grow, more discussions regarding appropriate application of instrument validation practices will emerge. This paper has drawn upon the authors' collective efforts to ensure data integrity in flow cytometric studies conducted during drug development. Validation of flow cytometers provides assurance that the output generated on these instruments is reliable and precise, thus enhancing the integrity and reconstruction of the data.

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Acknowledgements The authors would like to thank Drs Marian Kelley and Murli Krishna for their sponsorship of the subcommittee, review of the manuscript and guidance throughout the process. This manuscript was prepared by members of the Flow Cytometry Subcommittee of the Ligand Binding Assay Bioanalytical Focus Group (LBAFG) of the American Association of Pharmaceutical Scientists (AAPS). Information from a survey of use of flow cytometric assays was incorporated into this manuscript. For insightful discussion, the assistance of John Ferbas, Steve Zoog, and Carol Rosenthal are gratefully acknowledged. Appendix A. Standards for non-clinical safety studies: GLP and ISO 17025 The Good Laboratory Practice (GLP) regulations refer to a regulatory system of management controls designed specifically to assure the validity and reproducibility of non-clinical (also called pre-clinical) safety studies that support regulatory submissions for product registration. In the U.S. these are administered by the FDA. The guidelines are outlined in Chapter 21 of the Code of Federal Regulations Part 58(http://www. accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart=58, Accessed 21 June 2010). GLP are applied internationally by OECD (Directive67/548/EEC)( http://www. oecd.org, Accessed 21 June 2010). The Commission of the European Communities requires that laboratories carrying out tests on chemical products comply with the GLP regulations. The principles of GLP provide a framework for how laboratory studies are planned, performed, monitored, recorded, reported, and archived. The GLP regulations describe the minimum requirements for the facilities, animal care, specimen and reagent management, personnel (organization, qualification, and training), equipment (design, calibration, maintenance, and validation), standard operating procedures, and for test and control articles. The International Organization for Standardization (ISO) develops standards with the input of experts in a particular field. Although ISO is a non-governmental organization and has no legal authority to enforce the implementation of its standards, many countries have adopted the ISO standards. Like the GLP regulations, ISO 17025 requirements (http:// www.iso.org, Accessed 21 June 2010) were designed to assure data quality. GLP regulations address the aspects of a non-clinical study, whereas; ISO 17025 focuses solely on the laboratory. Differences between the two systems are apparent in the audit and record keeping requirements (Fox, 2003). Standards for conducting clinical trials: GCP The Good Clinical Practice (GCP) regulations are United States regulations that describe the requirements and controls for conducting research with human subjects. The formation of the International Conference on Harmonization (ICH) included representatives from domestic and international regulatory agencies and led to the creation of consolidated guidance documents on GCP (http://www.ich.org/LOB/media/ MEDIA482.pdf. Accessed 21 June 2010). Although not federally mandated, these guidance documents represent international

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best practices to provide ethical and quality standards for conducting clinical trials in humans. GCP focuses on designing, conducting, recording, and reporting clinical trials and defines the roles and responsibilities of clinical trial sponsors, clinical research investigators, and clinical monitors. Standards for manufacturing process: cGMP The current Good Manufacturing Practice (cGMP) regulations describe the requirements and controls for manufacturing, control, packaging, labeling and distribution of pharmaceuticals and other regulated products (http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm064971.htm. Accessed 21 June 2010). The cGMP regulations address issues including; recordkeeping, personnel qualifications, sanitation, cleanliness, equipment verification, process validation, complaint handling and product recalls. These regulations require that manufacturers, processors, and packagers of drugs, medical devices, blood, medical devices, and other regulated products take proactive steps to ensure that their products are safe and effective. Clinical laboratory standards: CLIA, CAP and ISO 15189 In the U.S., the Clinical Laboratory Improvement Amendments (CLIA) of 1988 regulations are administered by the Centers for Medicare and Medicaid Services (http://www.cdc. gov/clia/regs/toc.aspx, Accessed 21 June 2010). These regulations apply to clinical laboratories that receive Federal reimbursement for human specimen testing that is used to diagnose or affect treatment decisions. Testing performed during clinical trials to investigate efficacy and safety of new drugs is not required to be performed under CLIA regulations. The College of American Pathologists (CAP) is the most widely used accreditation body in the U.S. Some states in the U.S. have adopted their own regulatory accreditation programs. The CAP Laboratory Accreditation Program is an internationally recognized program designed to aid clinical laboratories in achieving high standards of excellence in supporting patient care (http://www.CAP.org, Accessed 21 June 2010). The Laboratory General Checklist addresses all areas of the laboratory, including quality management (proficiency testing, quality control); quality assurance (quality plan and program); specimen collection, data handling, and reporting; test method validation (method performance specifications, reference ranges); laboratory computer services; personnel (personnel qualifications, competency, training); physical facilities: laboratory safety; and record keeping. In addition, discipline-specific checklists address instrument installation, calibration, and maintenance. ISO 15189:2007 Medical laboratories — these are the medical laboratory standards for quality and competence, and specify the quality management system requirements for medical laboratories. This standard addresses the collection of patient samples, the interpretation of test results, acceptable turnaround times, how testing is to be provided in a medical emergency and the laboratory role in the education and training of health care staff. ISO 15189:2007 accreditation can be obtained through successful participation in the CAP 15189 program designed specifically to address this quality system.

Clinical research laboratory standards: GCLP As explained above, neither the GLP nor the CLIA regulations are entirely applicable for laboratories conducting the testing for specimens supporting clinical research. Guidelines tailored for clinical research have been proposed. The Good Clinical Laboratory Practice (GCLP) recommendations were designed to fill this gap. They have not been formally endorsed by a regulatory agency to date. GCLP were first published and copyrighted by the British Association of Research Quality Assurance (BARQA) (BARQAGCLP) (Sarzotti-Kelsoe et al., 2009). Subsequently, the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID) and National Institutes of Health (NIH) expanded the existing knowledge on GCLP standards by publishing guidelines on GCLP (NIAID-GCLP) (Ezzelle et al., 2008). GCLP applies the relevant aspects of the GLP, CLIA and GCP regulations and establishes the minimum requirements for optimal laboratory operations. These recommendations will ensure that consistent, reproducible, auditable, and reliable laboratory results are obtained for clinical trials implemented at multiple sites. The GCLP core elements include organization and personnel, laboratory equipment, testing facility operations, quality control program, verification of performance specifications, records and reports, physical facilities, specimen transport and management, personnel safety, laboratory information systems and quality management. Electronic records and signatures requirements: 21 CFR Part 11 In March of 1997, FDA issued the release of final Part 11 regulations that mandated specific criteria for acceptance for electronic records, electronic signatures, and handwritten signatures executed to electronic records as equivalent to paper records and to handwritten signatures executed on paper (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/ CFRSearch.cfm?CFRPart=11, Accessed 21June 2010). These regulations, which apply to all FDA-regulated environments, were intended to permit wide use of electronic technology, compatible with FDA's responsibility to protect the public health. Initially, interpretation of these regulations resulted in much debate on how to demonstrate compliance and in the specific scope of the regulations. FDA published a guidance document in 2003 to assist industry in interpreting a narrower scope of the regulations to minimize controls and costs and could discourage innovation and technological advances. References Bansal, S.K., Layloff, T., Bush, E.D., Hamilton, M., Hankinson, E.A., Landy, J.S., Lowes, S., Nasr, M.M., Jean, P.A., Shah, V.P., 2004. Qualification of analytical instruments for use in the pharmaceutical industry: a scientific approach. AAPS PharmSciTech. 5 (1) (article 22). Barlogie, B., Latreille, J., Freireich, E.J., Fu, C.T., Mellard, D., Meistrich, M., Andreeff, M., 1980. Characterization of hematologic malignancies by flow cytometry. Blood Cells 6, 719. Barr, I.G., Toh, B.H., 1983. Routine flow cytometric diagnosis of lymphoproliferative disorders. Journal of Clinical Immunology 3, 184. Chadwick, G.L., 2001. Clinical trials in the 21st century: a global approach. Research in Nursing & Health 24, 541. Chapman, G.V., 2000. Instrumentation for flow cytometry. Journal of Immunological Methods 243, 3.

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