Safety of Laboratory Staff
and
Quality Assurance Programmes
Lecture
Module 11
IAEA
International Atomic Energy Agency
General introduction
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Laboratory safety
Two basic objectives:
• To ensure safety of laboratory staff
• To ensure that no hazardous materials leave
laboratory
These are both achieved by well controlled
safe working practices
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Background information
• WHO Laboratory Biosafety Manual 3rd
edition
• In addition, staff should conform to their
national and institutional legislation on
regulations regarding safe working generally
in laboratories
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Specifics
There are some particular features concerning
safety in cytogenetics laboratories that are
worth highlighting.
• Risk of blood borne infections
• Risk from use of ultra-violet light
• Risk from use of specialised chemicals
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Infection risk
Two broad principles:
• Containment of materials Universal precautions
• Staff health preventative measures Vaccinations
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Optical risk
Ultraviolet lamps may be used in:
• Sterilizing interior of class 2
microbiological safety cabinets
• Exposing slides during FPG staining
procedure
• UV fluorescence microscopes
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Chemicals classically used in
biological dosimetry
•
•
•
•
Certain fine chemicals and
pharmaceuticals are used
routinely in procedures of
biological dosimetry
When present in cultures or used
in staining procedures, they are
mostly used in small volumes and
in dilutions that generally present
no health hazard
They are, however, made up and
stored in concentrated stock
solutions
Main reagents of concern and their
internationally agreed risk phrases
(R numbers) are listed here
Benzylpenicillin
R 42; 43
Bromodeoxyuridine
R 20; 21;
22; 46; 61
Calyculin A
R 23; 24;
25; 38
Colcemid
R 25; 63
Cytochalasin B
R 26; 27;
28; 63
Formamide
R 37; 38;
41; 61
Giemsa stain
R 20; 21;
22; 40; 41
Heparin
R 36; 37;
38
Hoechst stain
R 23; 24;
25; 36; 37; 38
Hypaque
R 42; 43
Okadaic acid
R 23; 24;
25; 38
Pepsin
R 36; 37;
38; 42
Phytohaemagglutinin R 20; 21;
22, 43
Ribonuclease A
R 20; 21;
22; 38
Streptomycin
sulphate
R 20; 21;
22; 61
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WHO Laboratory Biosafety Manual, part VI
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Chemical risk : meanings of internationally
agreed risk phrases (R numbers)
R20 Harmful by inhalation
R21 Harmful in contact with skin
R22 Harmful if swallowed
R23 Toxic by inhalation
R24 Toxic in contact with skin
R25 Toxic if swallowed
R26 Very toxic by inhalation
R27 Very toxic in contact with skin
R28 Very toxic if swallowed
R36 Irritating to eyes
R37 Irritating to respiratory system
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R38 Irritating to skin
R40 Possible risk of irreversible effects
R41 Risk of serious damage to eyes
R42 May cause sensitization by inhalation
R43 May cause sensitization by skin contact
R46 May cause heritable genetic damage
R61 May cause harm to the unborn child
R63 Possible risk of harm to the unborn child
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Quality programmes and the ISO
standards
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Overview of the technical validations
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2002 INTERNATIONAL INTERCOMPARISON
Lab 14; 0,5 Gy/min
Yield of aberrations
1,6
Lab 2; 0,45 Gy/min
1,4
Lab 6; 0,5 Gy/min
1,2
Lab 7; 0,24 Gy/min
1
Lab 12; 0,5 Gy/min
Lab 13; 0,5 Gy/min
0,8
Lab 15; 1 Gy/min
0,6
Lab 4; 0,7 Gy/min
0,4
Lab 11; 0,09 Gy/min
0,2
0
0
Polynomial (Lab 15; 1
Gy/min)
Polynomial (Lab 14; 0,5
Gy/min)1
2
3
Polynomial (Lab 12; 0,5
Dose (Gy)
Gy/min)
Polynomial (Lab 6; 0,5
Gy/min)
Polynomial (Lab 13; 0,5
Gy/min)
Polynomial (Lab 7; 0,24
Gy/min)
Polynomial (Lab 2; 0,45
Gy/min)
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5
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Standards used
• IAEA, EPR-Biodosimetry, 2011: Cytogenetic dosimetry: applications in
•
•
•
•
preparedness for and response to radiation emergencies
IAEA, Safety Guide GS-G-3.2: The management system for technical
services in radiation safety
ISO/CEI 17025:2005 General requirements for the competence of
testing and calibration laboratories
ISO 19238:2004 Radiation protection- performance criteria for service
laboratories performing biological dosimetry by cytogenetics
ISO 21243:2008 Radiation protection — Performance criteria for
laboratories performing cytogenetic triage for assessment of mass
casualties in radiological or nuclear emergencies — General principles
and application to dicentric assay
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Standard must cover these aspects
•
•
•
•
•
What you are trying to achieve
What you do
How do you make sure it is right
What do you do when it is not right
And, lastly, how do you check that
system works
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ISO Standard general approach
• Why a quality assurance approach ?
• To satisfy the requirements of professionalism
• To point out its qualification and its competence
• To develop the competence by inquiring and informing
• Quality assurance approach can be divided into two parts:
• First, internal mechanisms such as “ internal audit” or “total quality
management”, which are processes of professional self-review and quality
awareness, respectively.
• Second, certification (accreditation) which applies to processes more under
the direct control of managers and operated by an external agency, in other
words an external standards approach.
• How ISO can help to achieve the standardization aims ?
• Large consensus from scientists to industrials
• Participation on a voluntary basis
• To develop the competence by inquiring and informing
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Biodosimetry ISO documents
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Interaction steps in handling numbers
of cases
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General organisation
Management process
Measure and continual system
improvement
Quality objectives, Management
review
Corrective and preventive actions / Internal
audits
Client satisfaction measurement
Client
Technical
process
A satisfied
client
Resources management
Quality management system
Documents control / Quality and technical records
control
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Personnel department/
Purchases
Supplies equipments and
reagents
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Management process
Quality objectives, Management review
• One review of the system every year
Check of all the documents to ensure current accuracy
Quality objective checks
• Each staff member has their own function
The head of the lab formulates the objectives and defines staff functions
One technician is in charge of the quality assurance system
One scientist is in charge of the laboratory material
One technician is in charge of purchases
One scientist is in charge of documents management
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Measure and continual system improvement
Corrective and preventive actions / Internal audits
• Establishment of a complaints book:
Difficult to fill in regularly
Direct benefit for the lab
• Audit:
To evaluate the system periodically
To progress improvements
Conducted by external consultants to have an original point of
view
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Quality management system
Documents control / Quality and technical records control
• A quality assurance manual + others
• Traceability
Of products
Of the sample
• Equipment maintenance
• According to the critical points of the technique:
Microscopes
The laboratory (safety, sterility…)
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Resources management
Personnel department/ Purchases Supplies
equipments and reagents
• General institutional processes
Recruiting
Purchases: for some products only one supplier
• Materials management
Identification of key products
Guaranteed supply of ready-to-use products
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Client
Satisfied
client
Technical
process
• Lab must ensure that it can be contacted 24 / 7
• All contacts from potential clients should be followed up
• Results can be conveyed to client electronically to ensure speed
• Written report then follows and is endorsed by medical doctor
• The client is invited to give feed-back to laboratory
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List of documents linked to case
investigation
• When medical doctor contacts the lab
• Fill in contact details form
• Send instructions sheet for blood sampling and transport
conditions
• Send questionnaire for explaining accident circumstances
(contract)
• When sample(s) arrive(s)
• Record of details of sample and its coding
• Record of culture details
• Record reagents specifications e.g., batch numbers
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Technical validations
Client
TECHNICAL PROCESS AND MAJOR VALIDATION STEPS
Images analysis system
validation
Reference curve
curve
Reference
validation
validation
by statistic
statisticapproach
approach
by
Dicentrics
Dicentrics
number
number
Comparisonof automatic scoring
/ manual scoring (mitotic index
and dicentric frequency )
Cell culture
A satisfied
client
Cytogenetic dose
dose
Cytogenetic
(Gy)
(Gy)
Mean dose
dose
Mean
received
received
by the
the
by
body
body
Method validation
• by experimental approach , different
parameters were tested
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•other aspects :
Intra comparison
Inter comparison
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Approach through audits
• Uncertainty arises from:
Products
Material
Operator
Image analysis system
Number of dicentrics
Number of cells scored
Yield of
dicentrics
Dose
Number of cells scored
Curve fitting
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Dose effect
curve
Dicentrics
number
Dose +/95 % CI
dose
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Images analysis system
validation
Comparison of automatic scoring
/ manual scoring ( mitotic index
and dicentric frequency )
•Aim: scoring 500, 1000 or 2000 cells as
accurately and fast as possible
2 ways:
• Step 1 : Metaphase finder
• Step 2 : Manual scoring
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•Step 1 : Metaphase finder
•Step 2 : High magnification
acquisition
•Step 3 : Screen scoring
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Images analysis system
validation
Comparison of automatic scoring
/ manual scoring (mitotic index
and dicentric frequency )
• The metaphase finder validation
Comparison of the number of mitoses found by the
system and the “real” number of metaphases
10 % error accepted
• High magnification validation
Evaluation of scorable mitoses on the system
Comparison of dicentrics yield by the system vs manual
10 % error accepted
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Method validation by experiments (1)
Identification of potential influencing factors
Identification of tested interval (GUM)
Uncertainty on sample
manipulation, dilution …
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Method validation by experiments (2)
Parameters to measure
• The mitotic index
Number of mitoses
Total number of lymphocyte s
• The dicentric frequency
and the associated dose
Number of dicentrics
Number of cells scored
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Example of study of factors assessed for their
impact on yield of dicentrics
FACTORS
INFORMATION
Potential
impact
Yes
No
Medium : RPMI
Foetal Calf Serum
Nutritious medium required for lymphocytes to be maintained in vitro
Lymphocytes can divide without serum
PenicillinStreptomycin
Sodium Pyruvate
The antibiotics avoid bacterial proliferation but do not interfere with lymphocyte division No
This sugar is not necessary it is just a complement to the one present in the medium
L-Glutamine
No
This amino acid has a short storage life and so is added to the medium just prior to
use.
Hepes
Controls the pH of the medium. pH change is detected by a colour change in the
medium.
Bromodeoxyuridine
This is a thymidine analogous, it allows the scoring of dicentrics in first division
(BrdU)
metaphases only. It has an impact on the cell cycle duration. The number of
metaphases can vary according to its concentration.
Phytohemagglutinine This is antigenic mitogen required to stimulate T cells into cycling.
(PHA)
Colcemid
Required to block cells in the metaphase stage of the cell cycle
Hypotonic shock
Required for cell membrane lysis.
(KCl)
Acetic acid Required to fix the cells and to have chromosomes spread on the slide. It has no
Methanol
effect on the number of metaphases neither on the number of dicentrics.
No
Blood
The number of lymphocytes in the blood sample can affect the quality of the culture
Yes
Culture duration
KCl incubation
duration
Incubator
temperature
Can have an impact on the number of metaphases.
Some variations are found in the literature, therefore the impact is negligible
Yes
No
From IAEA Manual it should be of 37  .0.5°C but difficult to control
Yes
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No
Yes
Yes
Yes
No
No
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Parameters to test
• Experimental range :
Parameters
Low value 1
Usual value
high value 2
BrdU
94 mg
100 mg
106 mg
PHA
146 µl
150 µl
154 µl
Colcemid
duration
44 h
46 h
48 h
Colcemid
concentration
48 µl
50 µl
52 µl
Temperature
36°C
37°C
38°C
Blood volume
0.4 ml
0.5 ml
0.6 ml
Medium volume
4.75 ml
5 ml
5.25 ml
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Screening plan of Plackett et Burman
EXP
1
2
3
4
5
6
7
8
9
10
11
12
BrdU
2
2
1
2
2
2
1
1
1
2
1
1
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PHA
1
2
2
1
2
2
2
1
1
1
2
1
Tps Col
2
1
2
2
1
2
2
2
1
1
1
1
Col
1
2
1
2
2
1
2
2
2
1
1
1
KCl
1
1
2
1
2
2
1
2
2
2
1
1
Tps KCL
1
1
1
2
1
2
2
1
2
2
2
1
Sang
2
1
1
1
2
1
2
2
1
2
2
1
Milieu
2
2
1
1
1
2
1
2
2
1
2
1
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Results for mitotic index
0,080
0,075
mitotic index
0,070
0,065
0,060
0,055
BUdr
PHA
Medium
Blood
Colcemid
colcemid
duration
temperature
operator
0,050
Parameter tested
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Parameters which affect the result
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Results for dicentric frequency
0,035
Dicentric yield
0,033
0,031
0,029
Operator
Colcemid
duration
Blood
PHA
Colcemid
0,027
Temperature
Medium
BUdr
0,025
Parameter tested
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No significant parameter
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Summary of mitotic and dicentric
indices
• Some parameters affect number of mitoses: BrdU
concentration, medium volume, the blood volume,
duration of culture, incubator temperature
• No parameter affects dicentric frequency
• No operator effect was measured for both mitotic
index and dicentric yield
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Staff annual scoring proficiency check
• One slide scored (a low dose first year, a high dose
second year)
• No more than 20 % variation
Operator
a
b
c
d
e
f
g
h
nb cells
129
131
127
141
105
123
138
129
nb aber
131
147
109
117
123
121
124
112
Yield
1.02
1.12
0.86
0.83
1.17
0.98
0.90
0.87
Var
0.016
SD
0.127
Mean
- 20 % : 0.77
CV
0.968
+20 %: 1.16
0.132
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Final thought: convince staff to follow
QA&QC programme
The main difficulties :
• to convince people of importance of such project
• to change their way of working
• all staff members have to be involved in project
• the method validation is time consuming
• generation of many documents
To have efficient system it is important to build it as light as possible
The benefits are improvement in :
• technical process quality
• raising standing of service in eyes of requestors and any legal outcomes
Whole process is constantly evaluated and changes are made for
improved efficiency
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