quality managementin flow cytometry–requirementsofglp ... · attests that a laboratory has been...

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Quality management in flowcytometry – requirements of GLP,

GMP, ISO, and clinical studies

Prof. Dr. Ulrich SackInstitut für Klinische Immunologie

Medizinische Fakultät&

Translational Centre for Regenerative MedicineUniversität Leipzig

Disclosure of Conflicts of Interest

There are no conflicts of interest in thisfield to declare.

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What is accreditation

a third-party recognition of competence to perform specific tasks

attests that a laboratory has been successful in meeting the requirements of international accreditation standards (www.ukas.org)

confirms the successful implementation of elements of a comprehensive quality management system into medical laboratories [EN ISO 15189:2007; RL 90/385/EWG, 93/42 EWG, 98/79/EG and DIN EN ISO/IEC 17025].

Coming soon: ISO 15189:2012; few relevant changes

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http://www.ukas.org/

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Regulatory rules for diagnostic laboratories

1. Analysis in pharmaceutical research, in drug safety testing: GLP (regulated by laws)

2. Analysis for later use in drug development, for monitoring of patients in clinical studies and under similar conditions (but avoid „GLP-like conditions“)

3. Analysis as quality control for pharmaceutical production: GMP (national implemented according to ATMP, drug licensing processes, transfusion law etc)

4. Analysis in medical diagnostic laboratories1. Germany: Guidelines of the medical association (RiLiBÄK):

compulsory, structure adapted to DIN/ISO/EN 151892. ROW: Accreditation (DIN/ISO/EN 15189): yet voluntary, but

European Accreditation Law existent

5. Quality control in studies (GCLP)

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X

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When is GLP applicable?

Unless specifically exempted by national legislation, these Principles of Good Laboratory Practice apply to all non-clinical health and environmental safety studies required by regulations for the purpose of registering or licensing pharmaceuticals, pesticides, food and feed additives, cosmetic products, veterinary drug products and similar products, and for the regulation of industrial chemicals.

i.e. GLP in Europe must not mean accreditation and does not cover diagnostic labs. It is different to CAP in U.S.!

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X

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QC systems are about documentation

Most of us in the lab take notes on scraps of paper, paper towels or forget to record when which experiment was finished.

In industry however, lab notes can and do turn into patents, inventions and capital returns. Most science students do not receive adequate training in how or what industry expects. The key words here are … document, document, document.

If it's not recorded, then you did not do it, or you did not do it right!

• Improved quality of records for thesis and publication

• Developed documentation for patent • Experience that may be valuable to a future

employer

This leads to:

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What must be documented?

• Training records (were the personnel properly trained?)

• Equipment and instrument logbooks (was the instrument functioning correctly?)

• Sample and chemical unique ID numbers (was the correct sample and/or chemical used?)

• Master schedule (how are the various studies scheduled?)

• Facility floor plan, organizational chart, facility inspections (is the facility adequate?)

• Expiration dates of all chemicals

• Validity of balance with certified weights

• Use of instruments and any routine maintenance or problems …

• Cytometry: follow MiFloCyte-Guidelines!

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X

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When is GMP applicable?

GMP is required for

All processes included into compound or drug production for therapeutic use

Requirements are directly defined by authorities according to drug safety and performance requirements

Requests for GMP conform quality controls are defined for every singular compound dependent on the manufacturing process.

See previous presentation

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Special requirements under GLP/GMP policy

use of qualified equipment

Enhanced requirements for quality management activities: Planning with definition of demands

Documentation of the process

Reporting and assessment of results

Permanent documentation of processes belonging to the drug production or compound investigation process.

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X

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Common features of all qualitymanagement systems

Definition of Structure

Resources Staff, rooms, equipment

Processes Communication, contracts, pre-analytics, analytics, post-

analytics, report writing, documentation

Management Quality, documents, strategy

Who, when, where, what, how, …

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Documents in a laboratory

Mostly one central core document, a so-called Quality manual

Scientific publications, handbooks Contracts, organisation charts, legal documents Information for clients, patients, … Standard operating procedures as daily help for

laboratory work, written in an easy-to-understand style and in locally used language

Documents describing establishment of diagnostic procedures

Panel of external documents included in QMS

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5. Quality control in studies (GCLP)1. By WHO; fits well accreditation; accepted?

X

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What is important for flow cytometry?

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Everything.

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General requirements: Staff

Qualified and competent personnel Duties Documentation regarding history, knowledge,

competencies Adequate number of co-workers Authorizations Plan for qualification, documentation of

qualification Documentation of settling-in periods Periodic assessment of competencies

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General requirements: Rooms

Suitable rooms Adequate environmental conditions Risk evaluation Suitable conditions for outpatients contact (blood

withdrawal) Supervision, regulation and registration of environmental

conditions (temperature, insulation, humidity, noise…) Exclusion of cross contamination (molecular biology,

microbiology) Room cleaning Storage, waste disposal according to regulations

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General requirements: Equipment

Necessary equipment must be present Regular surveillance and calibration of equipment Plan & reports for maintenance and calibration Manufacturer notes, handbooks, further documents Labeling Authorization of lab members Rules for system malfunction Rules for handling, transportation, storage and use

Rules for special conditions such as shared use of flow cytometers by diagnostic and research groups (Universities!)

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General requirements: pre-analytic

procedures

Request form, internet representation Special requirements for blood withdrawal and sample

transportation Rules for handling of wrong samples (lymphocyte subpopulations

out of serum samples) Control of unambiguous identity of samples Surveillance of sample transportation (blood withdrawal time,

transportation time, temperature, contact to frozen samples…) Documentation of exact time-points: blood withdrawal, lab

entrance, analysis, report writing Rules for emergency inquiries Rules for oral communication (broadening of analysis according to

first results) Sample storage

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General requirements: analysis

Use of adequate and state-of-the-art methods Validated procedures Initial and intermediate control and clearance of

diagnostic procedures by responsible staff members Documentation of all methods (SOP; can include

manufacturers notes) Integration of short protocols into the SOPs. Regular monitoring of test kit information for relevant

changes Regular control of reference values Information of customers/clients about

methodic changes or altered normal values

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General requirements: Post-analytic

procedures

Control and release of reports by responsible staff members (physicians?)

Storage of reserve samples

Waste disposal according to local rules

Storage of raw data

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General requirements: Reports

Responsibility of the management

Format, content, speed and transfer to be negotiated with clients

Clear indications of incorrect samples (pre-analysis)

Storage of reports

Procedures for critical results (rules and documentation)

Procedures for handling of preliminary reports

Clear rules for modification of reports

Documentation of date, time, signature

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Factors affecting flow cytometricanalysis

Methodologies

Preparation

Ficoll vs Lysis

Lyse & Wash vs Lyse-No-Wash

Fluorochromes

2- / 3- / 4- … Color Methods

Gating

Lymphogate vs Panleukogate

CD4+ Gating

Absolute Counting

Dual-Platform vs Single-Platform

Determinants

Regulatory Guidelines

National GLP Guidelines

IVD-Directive

Reimbursement Policies

Clinical Concepts

Diagnostic andtherapeutic guidelines

Validation of methods in clinical studies

Scientific Progress

Identification ofprognostic phenotypes

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General requirements: Quality

assurance

Control systems (internal and external) Daily internal use of control blood preparations

Half-yearly external controls

Calculation of measurement uncertainty

Calibration of all systems

Data presentation in SI systems

Comparison of methods performed with various methods, machines or at different sites Adequate number of relevant samples: high positive, decision

range, additional matrix effects (lipemia, hemolysis)

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Validation of tests

In diagnostic labs, only minimal requirements for CE-labeled tests (only familiarization, must be documented)

Sometimes no possible (few samples, no standards, no reference method)

Can be replaced by published data, but must be explained!

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In house production of in vitro diagnostics

Diagnostic kits or reagents, that are produced or arranged in the lab for use in the lab

For exclusive use in the lab

Only allowed for public laboratories or labs with direct contact to patients

Dependent on number of performed tests

Otherwise: CE labelled tests ONLY.

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200 300 400 500 600 700 800

AVERAGE of Mini Wright 1 and Wright 2

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(Min

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Mean

2,6

-1.96 SD

-22,0

+1.96 SD

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Bland and Altman Plot

100 200 300 400 500 600 700

Wright 1

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600

500

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300

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Min

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Passing and Bablock Regression

Correct calculation and graphicpresentation of method

comparisons

Flow cytometry validation

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• Every method applied in the laboratory must be validated or verified, especially if “in-house” methods are being used. Like any diagnostic laboratory, flow cytometry laboratories must run validated tests. • In contrast to most tests in clinical chemistry, only a minority of diagnostic panels can be introduced to flow cytometric laboratories without adaptations. • Many tests have been developed and scientifically validated for flow cytometry without any gold standard references.• Any deviation from the manufacturers’ instructions must be documented and fully validated and these records must be retained. • A flow cytometry laboratory applying for ISO 15189 accreditation must validate four areas:

• instrument validation (ISO 15189 4.6.2, 5.3.2), • method validation (ISO 15189 5.5.2), • Laboratory Information System validation, and • validation of results (ISO 15189 5.8.13).


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• Method validation is potentially the most difficult and time consuming. • In section 5.5.3, linearity, precision, accuracy, limit of detection, sensitivity, and specificity are listed as performance parameters for validation. • Therefore each flow cytometry laboratory needs to design, plan, and perform these validations for each test they report for patients. • Once implemented and become routine for the users, methods should be reviewed on a periodic basis as dictated by the quality management system (i.e., quality control/assessment and/or audits or repeated untoward incidents) and should be monitored by the quality manager.• Furthermore, often, the object of flow cytometric analysis is to measure the diversity and variability of a widely heterogeneous cell population.


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• Leukaemia/lymphoma testing is especially characterized by examining the profile of antigen expression and comparing this to normal populations. • The complexity of leukaemia diagnostics is dynamic and the technical approaches to undertaking this test are constantly being reviewed in accordance with the latest opinion/data, particularly with the introduction of new instruments that are capable of multi-parametric analysis. • There are many working groups attempting to establish international consensus documents, but the challenges are numerous and include: validation of novel tests, generation of normal values, establishing quality control mechanisms (both internal and external), development of international standards, and the harmonisation of reporting results.


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• Instruments have to be calibrated and standardised in clearly defined terms. Calibrated results obtained from standardised instruments can be compared objectively and quantitatively with those from other laboratories. • Synthetic or biological calibration particles can be used for calibration. Synthetic particles are manufactured from polymers (beads, plastic-beads, latex-beads, micro-beads). Biological particles can be stained with the same fluorochromes used for cell staining. For standardisation of cytometers, both synthetic and biological particles are applicable according to the IVD specification given by manufacturers with all results plotted on a Levy-Jennings type plots to allow the identification of early problems.

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Special requirements for flow cytometry

Definition and control of upper and lower limits for cell populations

Detection of viable cells in samples (especially, when more than 24h old)

Daily control of process integrity by control samples Sample processing during 24 h following blood withdrawal Protocols avoiding shedding Daily calibration and compensation Gating, negative controls Defined gating strategies Age-matched normal ranges Report writing by help of original histograms

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Common quality problems

Blood withdrawal time

Participation in external quality controls

Calculation of measurement uncertainty

Definition of clinical indications for every analysis

Rules for restricted access to labs and equipment

Analysis of research samples

Qualification of staff members, continued process of education

Premixed reagents

Gating strategies

Non-CE tests

Immune function depends on daytime

A circadian clock in macrophages controls inflammatory immune responses. Keller M, Mazuch J, Abraham U, Eom GD, Herzog ED, Volk HD, Kramer A, Maier B. Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21407-12.

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• CD4+ T-cells („Immune Profile“)

• CD34+ precursor cells• HLA-B27• Residual leukocytes in

blood products• CD38 on T-cells• HLA-DR on monocytes

• Leukemic blast crisis• Minimal residual disease• Premalign diseases• Inborn immune defects• CVID• Cross match-analysis• Antigen-specific

T-cells

• Dendritic cells• Allo- and autoantibodies• Basophile activation• Prothrombotic syndromes• Transplantation monitoring• Multi-Drug-Resistance

Accreditation Consensus Formation Optimization and „Proof of Principle“

Strategies for the Expansion of the Diagnostic Repertoire

Integration into the analytical process

Multicentric validation of assays

Publication of protocols

Certified Tests Clinically Established Methods Clinical Research

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Conclusion

1. Today, GLP-relevant flow cytometric tests are extremely rare. But, GLP-conform work enhances efficacy and transparency.

2. Requests for GMP conform quality controls are defined for every singular compound dependent on manufacturing process.

3. Rules as defined by DIN/ISO/EN 15189 must be followed in every diagnostic laboratory.

4. Good work depends on general laboratory organization.

5. Diagnostic work includes a whole process chain.

6. Every test must be validated.

7. Novel tests will improve diagnostic but must fulfil high quality standards.

QC & Education in Flow Cytometry

European approach

Co-operation with ISAC, IFCC, and national societies

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14th Euroconference of clinical cell analysis, Lisboa, 15.-17.9.2014

Thank you!