3: การประเมินความเสี่ยง (risk assessment ... level... · acceptable levels 5. Complete BioRisk Assessment 1 2 ... When is hazard/threat identification

1

3: การประเมนิความเสีย่ง (risk assessment) และระดบัความปลอดภยัทางชวีภาพ

(biosafety level): งานวจิยัดา้นจลุนิทรยี ์

ศ.ดร. ศรสีนิ คสูมทิธิ ์

คณะเวชศาสตรเ์ขตรอ้น มหาวทิยาลยัมหดิล

Biosafety is the application of safety precautions that

reduce a laboratory personnel's accidental exposure risk of

exposure to a potentially infectious agents or toxins and limit

contamination of the work environment and, ultimately, the

community.

Biosecurity describes "the protection, control and

accountability for valuable biological materials within

laboratories, in order to prevent their unauthorized access,

loss, theft, misuse, diversion or intentional release to pursue

bioterrorism or biological weapons proliferation.”

Biosafety vs Biosecurity

Laboratory acquired infection (LAI) = an infection obtained through laboratory or laboratory-related activities as a result of work with infectious biological agents, which may be either symptomatic of asymptomatic

Factors contributing to LAI

20% are caused by equipment failure

80% of which are caused by human factors

Lack of knowledge of biological agent

Lack of proper emergency responses

Lack of training, passing down of bad habits

Carelessness, ignorance in work practices

Laboratory Acquired Infections (LAI)

MMWR Supplements 2012/61(01);1-101

Practices

20%

Parental inoculations with syringe, needles or

other contaminated sharps;

Spillages & splashes onto skin and mucous

membrane

Ingestion or exposure through mouth

Pipetting or touching mouth or eyes with fingers

or contaminated objects

80%

MMWR Supplements 2012/61(01);1-101

Laboratory Acquired Infections (LAI)

Infectious aerosols (and droplets)-directly or

hand contamination

Purpose

Able to correctly identify & evaluate the risks associated

with handling biological agents; and to put in place

appropriate control measures to mitigate / control / manage

such risks

Risk Assessment & Management

To ensure that risks to employees,

the public, or the environment

are consistently minimize to an acceptable levels

5

Complete BioRisk Assessment

1

2

• Personnel Factor: Personnel supervising & personnel using the material: experience

3

4

5

• Environment Factors: laboratory, facility, community

• Working activity factor: Experimental protocols & lab practices, SOP

• Equipment factors

Agent characteristics & biological materials (RG/BSL etc)Include Biological, chemical, radiological, liquid nitrogen, flammable

6

When is hazard/threat identification done

Whenever you start a job/task

Throughout the life cycle of the job/task

Whenever there is significant change in the

job/task

Whenever there is new knowledge that affects

the job/task

If there has been an accident

Periodically depending on the local legislation

and/or institutional policy

Hazard/Threat identification

7

Risk Assessment/Evaluation

: http://www.austrac.gov.au/elearning_amlctf_programcourse/mod4/module_4_risk_16.html

A process used to estimated risk against a given risk criteria to

determine the significance of the risk

Risk = Severity x Likelihood

8

Factors Should Be Considered in A Risk

Assessment for Pathogen

Virulence/Pathogenicity of the organism

Route of transmission

inhalation/intranasal:infectious aerosol;

ingestion: gastrointestinal tract (eg Salmonella); (e.g.

contaminated fingers in mouth eg mouth pipetting;

eating, drinking in the lab);

contact (mucous (mouth, nose)/eyes/ percutaneous),

skin abrasions/cuts; Parental (needle sticks);

Formites-contaminated environmental surfaces eg

BBP, HIV-virus, Hepatitis B,C; through wound eg

Staphylococcus

: http://www.austrac.gov.au/elearning_amlctf_programcourse/mod4/module_4_risk_16.html 9

Factors Should Be Considered in A Risk

Assessment for Pathogen

Host range (human, broad/multi- host eg human, animals,

plant fish, environmentally or agriculturally important)

Stability: -Agent: prolonged viability (spore formation).

- Survival in environment eg. Rickettsia, B.

anthracis

Infectious dose (low eg. M. tuberculosis , Francicella

tularensis etc.

Disease outcomes (pathogenicity of the organism / severity

(morbidity / mortality)

Availability of effective preventive measure (e.g., vaccines)

Availability of effective treatment (e.g., antibiotics)

Whether the pathogen is indigenous to the country

: http://www.austrac.gov.au/elearning_amlctf_programcourse/mod4/module_4_risk_16.html 10

Classification of Infectious Microorganism by Risk Group

WHO Laboratory Biosafety Manual 3rd Edition (2004)

Risk

Group 1

no or low

individual and

community risk

A microorganism that is unlikely to cause human disease or animal disease

eg. non-pathogenic organisms , non-pathogenic E. coli-K12, B. subtilis;

well established animal tissues and cell lines; well established human cell

line- history of safe

Risk

Group 2

moderate

individual risk,

low community

risk

A pathogen that can cause human or animal disease but is unlikely to be a

serious hazard to laboratory workers, the community, livestock or the

environment. Laboratory exposures may cause serious infection, but

effective treatment and preventative measures are available and the risk of

spread of infection is limited.Eg. Bacillus cereus, Clostridium spp, Campylobacter, Most wild type E. coli

eg E.coli O157 , Pseudomonas aeruginosa, Proteus vulgaris, Staph aureus,

Dengue, Hepatitis A, B & C , Adeno virus, CMV, Vaccinia virus,

Toxoplasma, human blood & blood products, clinical samples,

Risk

Group 3

high individual

risk, low

community risk

A pathogen that usually causes serious human or animal disease but does

not ordinarily spread from one infected individual to another. Effective

treatment and preventive measures are available.Serious respiratory agents Eg. Bacillus anthrasis , Mycobacterium

tuberculosis, Rickettsia spp, HIV (culture), Yersenia pestis (resistant

strains), prion

Risk

Group 4

high individual

and community

risk

A pathogen that usually causes serious human or animal disease and

that can be readily transmitted from one individual to another, directly

or indirectly. Effective treatment and preventive measures are not

usually available.Eg. Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever

Titer/Concentration and Volume of material used Higher concentrations increase the risks of working withthat agent. Working with large volumes of concentrated infectious material also increases the risks, sinceadditional handling of the materials is often required.

What are the laboratory factors? (Lab procedures withrisk of exposure: e.g., potential for splashes, vortexing, centrifugation, sharps, needles or injection, animals; skillsand training level of investigators.

Health status of investigator: personal factorsinfluencing risk e.g. health status, immune state: immunosuppression, pregnancy, vaccination status, allergies etc.

External factors to be considered in a risk

assessment include…………….

12

Risk Assessment: Large Scale and High

Concentration

Small scale or bench scale: < 10 liters

Eg. BSL1, bench-top experiment

BSL2, PPE required experiment

Large scale: > 10 liters

High concentrations: 103 versus 1010 have different requirements

Modified from http://ehs.unc.edu/training/self_study/bsl2_dlam/container.php?page=66&x=15&y=13

Risk Assessment: Blood and Body Fluids

Risk associated with blood and bodily fluids is the potential exposure to BBP which may be present in contaminated blood and bodily fluids and are capable of causing disease in exposed individuals. The pathogens of greatest concern are

Hepatitis B virus (HBV): It can be transmitted not only by percutaneous exposures, but also via mucous membranes. The incubation period for hepatitis B is 45 to 160 days.

Hepatitis C virus (HCV): the interval between exposure and seroconversion is approximately 8 to 10 weeks.

HIV: It is difficult to become infected with HIV through a needle stick injury or other exposure to blood or other body fluids. The risk depends on the amount of virus to which one is exposed and the titre of HIV viral RNA. There is no vaccine for HIV, but drugs are available which reduce the risk of becoming infected with the virus.

14

Examples: Blood-borne Pathogens

Hepatitis B virus (HBV)/Hepatitis C virus

Human immunodeficiency virus (HIV)

Staphylococcus and Streptococcus

Salmonella and Shigella

Syphilis

TB

Treponema pallidum (syphilis)

Plasmodium spp. (malaria)

Brucella spp.

Leptospira interrogans

Arboviruses

Hemorrhagic fever viruses

etc

15

Blood Borne Pathogen:

Transmission Potential

Contact with another person’s blood or bodily fluid that may contain blood

Mucous membranes: eyes, mouth, nose

Non-intact skin

Contaminated sharps/needles

16

Risk Assessment of Fungal Agents

Infections are not communicable, but require common exposure from a point source.

Some fungi have dimorphic: yeast form and mold form

Yeast forms may be present in the tissues of infected animals and in clinical specimens, identifying isolates, and processing animal tissues.

Mold form cultures containing infectious conidia or spore may pose a hazard of aerosol exposure.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are recommended for activities with clinical materials, animal tissues, yeast-form cultures, and infected animals eg. Crytococcus neoformans, yeast form of Blastomycesdermatitidis.

BSL-3 practices, containment equipment, and facilities are required for or propagating and manipulating sporulating mold-form cultures eg. Blastomyces dermatitidis, eg. Coccidioidesspp, Histoplasma capsulatum etc.

BMBL 5th Edition (2010) 17

Risk Assessment

Cells, Tissues and Cell Lines

The origin of the cells or tissues (species and tissue type) :

infectious for humans are most likely to arise from cells of

human or primate origin but other mammalian, avian and

invertebrate cell lines may also present risks

The source (recently isolated or well- characterized).

This will give an indication of potential contaminants and

potential for expression/reactivation of latent viruses.

Type of cell line: primary cell cultures present the

greatest risk of carriage and followed by continuous cell

lines unless known to be persistently infected

18

Potential Harzadous Human Cell Lines

Potential laboratory hazards associated with human cells and

tissues include

Blood borne pathogens HBV, HIV, HCV, EBV, HPV and CMV

M. tuberculosis that may be present in human lung tissue. Other

primate cells and tissues also present risks to laboratory

workers.

Cells immortalized with viral agents such as SV-40, EBV

adenovirus or human papilloma virus (HPV), as well as cells

carrying viral genomic material also present potential hazards to

laboratory workers.

Tumorigenic human cells also are potential hazards as a result of

self-inoculation.

Laboratory workers should never handle autologous cells or

tissues, blood, lymphoid and neural tissues and should always

be considered potentially hazardous 19

Primary Cell Culture/Cell Lines vs RG/BSL

Primary cultures of human, primate, mammalian or insect cells

from a source known to NOT contain infectious agents (RG 1)

should be treated using BSL-1 practices and procedures

Untransformed mammalian cell lines - Risk Group 1

MCF-7 (Human breast carcinoma cell line)

NIH 3T3 (Mouse fibroblast cell line)

All primary human cell cultures (explants) and subsequent in vitro passages fall under the blood-borne pathogen standard (BSL2)

Human cell lines are considered to be potentially infectiousunless the specific cell line has been characterized to be free ofhepatitis viruses, HIV, EBV, human papilloma viruses and otherrecognized blood borne pathogens (BSL2)

20

Primary Cell Culture/Cell Lines vs RG/BSL

Transformed mammalian cell lines – Risk Group 2 -HeLa cell lines

Primary cultures of insect or mammalian cells from a source where infection status is UNKNOWN (or known to be infected) are considered to be potentially infectiousand should be handled using BSL-2 practice and containment. All work should be performed in a BSC (BMBL 5 th edition(CDC)

Any injections with human cell lines into animals will be conducted in a BSC and handled at ABSL-2.

21

The risk assessment for recombinant DNA should

include:

source of the DNA to be transferred (host)

ability of vector to survive outside the laboratory

(vector)

interaction between transferred gene and host

(product)

When assessing the risk group and containment level for a genetic

engineered protocol, if one of the components is potentially

hazardous, a risk level appropriate to the known hazard is

assigned.

Risk Assessment: Recombinant DNA

22

What are the vectors (viral vector) / host / product?

1. Host /recipient system:

Intrinsic characteristics of host/recipient (risk group)

Source of DNA, nature of insert gene

- Is the gene or sequence (including synthetic) from RG-2,

RG-3, or RG-4 agent, biological toxin, or select agent?

- Is there any risks associated w/ the gene or sequence

such as: up-regulation/silencing expression, regain of

function, oncogenes, virulence factors, toxins, or

expanded host range?

- Does the gene or sequence change sensitivity to

antibiotics, herbicides, pesticides, or insecticides that

would be used to control the host?

Factors Should Be Considered in a Risk

Assessment for Recombinant Research

WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III-D of the NIH Guidelines 23

Factors Should Be Considered in a Risk

Assessment for Recombinant Research

2. Vector system

Intrinsic characteristics (risk group)

Replication competence

Residual viral gene expression

3 End product/Gene product effects:

Expression of a foreign gene

Possible introduction or increase of virulence

Toxicity (Gene code for toxin)

Increased ability to evade immune system

Allergenicity

Pharmaceutical activity eg. antibiotic resistance

WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III-D of the NIH Guidelines 24

Viral Vector Considerations

1. Route of Transmission

Blood-borne

Lentivirus

Direct contact

Vaccinia

Respiratory

Adenovirus, Adeno-associated virus (>150 pfu

intranasal)

Influenza virus (790 pfu nasopharyncheal)

WHO Laboratory Biosafety Manual 3rd Edition (2004)25


2. Host Range

Based on human pathogens

Replication incompetent eg Lentivirus

Potential for oncogenesis through insertional

mutagenesis eg Lentivirus

Non-pathogenic viruses eg. Adeno-associated

virus

Based on non-human pathogens

Reduced pathogenicity (Vaccinia, Avipox)

Non-pathogenic (Baculovirus)

Tropism and host range

Change in cell type or species affected


26

3. Contamination

Viral vectors, e.g. adenovirus may be contaminated

with replication-competent viruses, generated by

rare spontaneous recombination events in the

propagating cell lines, or may derive from

insufficient purification.

These vectors should be handled at the same

biosafety level as the parent adenovirus from which they are derived.



27

Classification of Recombinant DNA Activities by Risk Group


Risk

Group

1

no or low

individual and

community risk

-rDNA activities holding no or a negligible risk eg. rDNA

activities using such non-pathogenic organisms as hosts for

the expression of genes incorporated into bacterial plasmids or

low risk viral vectors eg E. coli K-12, Saccharomyces

cerevisiae or Saccharomyces uvarum, baculovirus or Adeno

Associated Virus; vector from commercial supplier RG1

Risk

Group

2

moderate

individual risk,

low community

risk

-recombinant DNA activities using viral vector systems such as

Adenoviruses and some Retroviral vectors, particularly

Lentiviral vectors, and expression of recombinant DNA in

BSL-2 organisms.

Risk

Group

3

high individual

risk, low

community risk

Recombinant DNA activities using genetic material from BSL-

3 organisms or such organisms as host cells, cell lines

modified using DNA from Risk Group 3, cell lines contains a

toxin with an LD50 of less than 100 nanograms per kg/kg

body weight

Risk

Group

4

high individual

and community

risk

Recombinant DNA activities using genetic material from

BSL-3 organisms or such organisms as host cells, cell lines

modified using DNA from Risk Group 428

Mitigation Control Measures

Engineering Controls (EC)

Physical changes to work stations, equipment, materials,

production facilities, or any other relevant aspect of the

work environment that reduce or prevent exposure to

hazards (lab design)

Administrative Controls (AC)

Policies, standards and guidelines use to control risks

Practices & Procedures (P&P)

Processes and activities that have been shown in

practice to be effective in reducing risks

Personal Protective Equipment (PPE)

Devices worn by the worker to protect against hazards in the laboratory

Summary of Biosafety Level Requirements (physical structure/ airflow system/equipment/waste management system)

BSL1 BSL2 BSL3 BSL4

Isolation1 of laboratory No No Yes Yes

Room sealable for decontamination No No Yes Yes

Ventilation

- inward airflow

- controlled ventilating system

- HEPA-filtered air exhaust

No

No

No

Desirable

Desirable

No

Yes

Yes

Yes/No2

Yes

Yes

Yes

Double-door entry No No Yes Yes

Airlock No No No Yes

Airlock with shower No No No Yes

Anteroom No No Yes -

Anteroom with shower No No Yes/No3 No

Effluent treatment No No Yes/No3 Yes

Autoclave

- on site

- in laboratory room

- double-ended

No

No

No

Desirable

No

No

Yes

Desirable

Desirable

Yes

Yes

Yes

Biologicalsafety cabinet No Desirable Yes Yes

Personnel safety monitoring capacity4 No No Desirable Yes


Biosafety Level 1 (BSL1)

http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

Biosafety Level 1 is suitable for work involving

well-characterized agents not known to consistently

cause disease in immunocompetent adult humans, and present minimal potential hazard to

laboratory personnel and the environment

Practices -Standard microbiological practices /aseptic

technique

- Sharps” precautions

- Red bag waste disposal

Safety Equipment

(Primary barriers)

- No primary barriers required

- PPE: laboratory coat, gloves, eye and

face protection

Facilities

(Secondary barriers)

- Laboratory bench, sink, eye-wash, fume hood

emergency shower outside the area31


Biosafety Level 2 is suitable for work involving agents that

pose moderate hazards to personnel and the environment.

Practices BSL1++ practice plus:

- Limited access

- Door posts biohazard warning sign

-“Sharps” precautions

- Biosafety manual defining any needed waste

decontamination or medical surveillance policies

Safety Equipments

(Primary barriers)

-Class I or II BSCs or other physical containment device used

for all open manipulations of agents that cases

splashes or aerosols of infectious materials

-PEE: Laboratory cloths, gloves, gown, face and eye protection

Facilities

(Secondary

barriers)

-BSL-1 facilities with the addition of: accessible autoclave,

directional airflow, no air recirculation, disinfection/

decontamination procedures in place.


Biohazard Warning Sign


7. A sign incorporating the universal

biohazard symbol must be posted at

the entrance to the laboratory when

infectious agents are present.

The sign may include the name of

the agent(s) in use, and the name and

phone number of the laboratory

supervisor or other responsible

personnel. Agent information should

be posted in accordance with the

institutional policy.

BSL2-4

33

Risk Management

Hazard Communication

Biohazard labels shall be placed on:

the surface of all equipment (freezers, incubators,

refrigerators) which may be contaminated with

biohazardous materials.

sample transport outer containers.

medical waste bins

Biohazard signs shall be placed on:

the outer door of BL-2 labs.

medical waste storage areas

34

Biosafety Level 3 is applicable to special clinical and

diagnostic services, research, or production facilities

where work is performed with indigenous or exotic agents

that may cause serious or potentially lethal disease through

the inhalation route of exposure.

Practices BSL1+2++ practice plus:

- Full training (Worker certification)

- PEE: Protective laboratory clothing

(Wrap around disposable clothing is required), gloves, gown, face

shield or other splatter guard, eye protection (goggles), respiratory

protection (N95 Respirator) , shoe covers, boots

- Restricted access (Entry and exiting procedures to cover)

- Personnel safety monitoring capacity eg. window, closed circuit television

two-way communication; escort requirements

- Decontamination of laboratory clothing before laundering.

- Decontamination of all waste

- Emergency response



35

Biosafety Level 3 is applicable to special clinical and

diagnostic services, research, or production facilities

where work is performed with indigenous or exotic agents

that may cause serious or potentially lethal disease through

the inhalation route of exposure.

Safety Equipment

(Primary barriers)

-Class I or II Biological Safety Cabinet (BSC) or equivalent

containment for all open manipulations of agents

- Safety centrifuge cups and rotors

- Double door autoclave or equivalent


Biosafety Level 3 (BSL3)(continued)

Facilities

(Secondary

barriers)

-2 doors in a series to access lab and anteroom

-Special exhaust ventilation

(Not re-circulated, airflow (HVAC)

-Negative pressure

-Sinks, doors, floor, airlock…

-Able to wash entire lab

36

Biosafety Level 4 is required for work with dangerous and exotic agents

that pose a high individual risk of aerosol-transmitted laboratory infections

and life-threatening disease that is frequently fatal, for which there are no

vaccines or treatments, or a related agent with unknown risk of transmission.

Practices BSL3 practice plus:

- Laboratory staff must have specific and thorough training

in handling extremely hazardous infectious agents

-Clothing change before entering

-Shower on exist

-All material decontaminated on exist from facility

-Special Disposal

Safety Equipment

(Primary barriers)

-All procedures conducted in Class III BSCs or

in combination with full-body, air-supplied,

positive pressure suit

Facilities

(Secondary barriers)

BSL3 plus:

- Separate building or isolated zone

- Design specifications are extremely stringent

- Lab. must be sealable

- Suit Laboratory or Cabinet Laboratory/ Air lock entry /Shower exit

- Dedicated supply and exhaust, vacuum, and decontamination systems

- Other requirements outlines in the text

- No such facilities exist in Thailand



37

Standard Microbiological Practices


1. Control access to the laboratory

2. A documented safety) manual must be available for all staff

3. Personal Protective Equipment (PPE) to be used

for laboratory procedures: PPE acts as a barrier to

minimize the risk of exposure to aerosols, splash

and accidental inoculation

4. Proper foot wear (No sandals in lab)

5. Tie long hair back to prevent catching in the

equipment or catching fire over Bunsen burner flame

6. Eating, drinking, smoking, handling contact lenses,

applying cosmetics, and storing food for human

consumption must not be permitted in laboratory areas

7. Mouth pipetting is prohibited.

8. Perform procedures to minimize splashes or aerosolseg. Pipette carefully; Transfer biological solutions with caution


9. Decontaminate work surface before leaving the Lab

10. Leave your lab coat in the lab

11. Remove gloves before touching door handles phones etc.

12. Wash your hands with soap and water before leaving lab

13. Safe storage of waste materials

14. Decontaminate all cultures, stocks, and other potentially

infectious materials before disposal using an effective method.

a. Materials to be decontaminated outside of the immediate

laboratory must be placed in a durable, leak proof container and

secured for transport.

b. Materials to be removed from the facility for decontamination

must be packed inaccordance with applicable

local, state, and national regulations.

15. When possible use plastic instead of glass

39


16. Safe handling of sharps, such as needles, scalpels, pipettes, and

broken glassware must be developed and implemented.

Avoid manipulating needles after use:

a. Needles must not be bent, sheared, broken, recapped, removed from

disposable syringes, or otherwise manipulated by hand before

disposal.

b. Used disposable needles , syringes, glass pipette and other

contaminated sharps must be carefully placed in conveniently

located puncture-resistant containers used for sharps disposal

(Closable, puncture resistant, leak proof on sides and bottom,

Red / fluorescent orange container or orange-red BioHaz label

a. Non-disposable sharps must be placed in a hard walled

container for transport to a processing area for decontamination,

preferably by autoclaving.

d. Broken glassware must not be handled directly. Instead, it

must be removed using a brush and dustpan, tongs, or forceps.

e. Plastic ware should be substituted for glassware whenever possible.

Standard Microbiological Practices

40

41

Biosafety Guidelines for Infectious Microorganisms

Laboratory Biosafety Manual

3rd Edition (2004)

Australian/New Zealand Standard Safety in laboratories

Part 3: Microbiological safety and containment

Canadian Laboratory Safety Guidelines (2004)

NIH Guidelines for Research

Involving Recombinant DNA Molecules (2013)

Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition (2010)

Guidelines from Department of Medical Science, Ministry of

Public Health

National Guidelines for Pathogens

42

1. Biosafety Guidelines for Work Related to Modern

Biotechnology or Genetic Engineering (2004, 2009,

2011)

National Guidelines for Biotechnology

2004 20112009

43

Biological Safety Cabinet (BSC)

http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixA.pdf

Source: Appendix A of the Biosafety in Microbiological and Medical Laboratories (BMBL); Primary

Containment for Biohazards: Selection, Installation, and Use of Biosafety Cabinets

Selection of a safety cabinet through risk assessment

BSLProtection Provided

BSC ClassPersonnel Product Environment

BSL 1-2 Yes No Yes I

BSL 1-3 Yes Yes Yes II (A1, A2, B1,

B2)

BSL 4 Yes Yes Yes III; II – When

used in suit room

with suit

44


http://www.escoglobal.com/resources/pdf/BSC-Types-Classes.pdf45

Biological Safety Cabinet (BSC) Class I

http://canadianbiosafetystandards.collaboration.gc.ca/cbsg-nldcb/assets/longdesc/figure-11-eng.php#figure-11-1a

Supply

ambient

air

Intake

75ft/min

- Open-fronted safety

cabinets

- Negative-pressure

ventilated cabinet

- Inward airflow protect

workers, but NOT

product protection

- Exhausted to outside

100%, re-circulated

0%

- Not for use with

tissue culture

Exhausted HEPA filter

Exhaust plenum

Contaminated

air

46

Biological Safety Cabinet (BSC) Class II


- Type A: 30% exhausted through HEPA is discharged into the room (A1 and

A2), 70% recirculated through HEPA

- Negative-pressure ventilated cabinet

- The air is drawn from the room into

the work opening and it passes

the operator and the product

before it leaves the

cabinet via HEPA filter

normally ducted

to atmosphereSupply air

Intake

–75 ft/min

(A1)

-100

ft/min

(A2)

Exhaust HEPA filter air30%

<Supplied HEPA filter


blower

47

Type B1: 70% exhausted to outside re-circulated= 30%

-Negative-pressure ventilated cabinet

-Class II B: HEPA filtered

air is discharged out

of the room

-Minute amount

of volatile

chemicals

allowed

Biological safety cabinet (BSC) Class II


Intake

100ft/min

blower


Supplied HEPA filter

Exhaust HEPA filter air70%

48

Type B2: 100% exhausted to outside, re-circulated = 0%

Biological Safety Cabinet (BSC) Class II


Intake

100ft/min

Supply air

exhaust air pulled

through dedicated

exhaust duct into

facility exhaust

system;

100FPM intake

all ducts and plenums

are under negative

pressure

Exhausted air

49


Completely sealed, ventilated, closed front air-tight cabinet

Ventilated cabinet - totally enclosed: use for work with risk group ¾ agents

Operations are conducted through attached rubber gloves.

Both supply and exhaust air are HEPA-filtered

Provides both personnel and product

protection

Biological Safety Cabinet (BSC) Class III

100% exhausted to

outside, recirculated= 0%

Negative pressure

Double-

HEPA filter

Room

Supply air

HEPA filter

50


Biosafety in Microbiological and Biomedical Laboratories, 5th Edition

Clean Dirty

Ideal BSC Work Area (SOP)

work from a “clean” to a “dirty” side

Never use a Bunsen burner, since this may create turbulence in the airflow

Do not store excess equipment in cabinet

(Work with Cell Lines and Cultures during aspiration of

infectious fluids) (SOP)

Waste solutions collected properly

•

• All waste solutions and unused cells must be decontaminated for 24 hours before autoclaved

– 10% bleach solution (Sodium hypochlorite) at final concentration

– bleach with 5.25% available hypochlorite

(sodium hypochlorite is effective against all types of micro-organisms)

Disinfectant

Hydrophobic filter

HEPA filter

Ideal Vacuum Line Protection

Source: Biosafety in Microbiological and Biomedical Laboratories, 5th Edition52

Waste Management

www.uottawa.ca/services/ehss/docs/BiomedicalWasteDisposalProceduresSept07.pdf

Types of Laboratory Wastes

General Waste: All general waste that does not contaminated

with biological agent, chemical and/ or radioactive material

Biohazardous Waste: All waste that contaminated with

infectious materials , other potentially infectious materials ,

blood or blood products, pathological waste, microbiological

waste, sharps and animal Waste

Chemical Waste: A waste that is made from harmful

chemicals, decomposed chemicals or expired chemicals.

Radioactive Waste: All general waste is contained radioactive material.

53

Types of Biological Waste

Solid

– Labware (flasks, tubes, plates, bottle, vials)

– Lab waste (pathogens, stocks, specimens, biological toxin, recombinant DNA organisms, cultures, swabs, tissue, plastic pipettes or pipette tips,

– Contaminated gloves, apparel, wipes etc.

Liquid

– Sera, body fluids etc.

Sharps

– Anything with a point or edge capable of piercing or cutting (razors, needles, syringes, glass slides) etc.

54

Solid biological waste (except sharp)

Collect in a rigid leak resistant container with a lid and labeled with the Biohazard Symbol that is lined with a “RED” bag and labeled with: The words “Biohazard, “Biomedical Waste”,

or “Infectious Waste” must be on the container

Must have International Biohazard symbol

Generator Address Labeling (Location where the waste is generated, (room number, name of department), name of responsible person, Telephone number, date of disposal

No liquid waste in red bags!

Put in the box , sealed and labeled

Biohazardous Waste

chfm.ufl.edu/files/2011/09/BMW-2012

Liquid biological waste:

Store liquid waste in closable, puncture-resistant

containers

Autoclave all liquid biohazardous materials (such as

human blood, bacterial cultures in liquid media, body

fluids of animals experimentally infected with

pathogens, etc.), if applicable.

Treat with appropriate chemical disinfectant for the

sufficient contact time.

Biohazardous Waste Handling

56

Sharps

Must not contain any liquid.

Never Re-Cap Needles or Scalpels

Don’t bend, break, or detach from syringe

Discard directly into a rigid, puncture-proof, leak-proof container before being placed in a biowaste box

Must have International Biohazard symbol and the word: “Biohazard”

Label container with the date, PI name, location (building/room #), and phone #

Replace container when ¾ full

Never attempt to re-open a closed sharps container

Biohazardous Waste

chfm.ufl.edu/files/2011/09/BMW-2012.

Decontamination and Waste Management

Sterilization methods

Disinfectants

Gaseous decontamination of room (BSL3,

BSL4)

Irradiation

Waste disposal system / procedure

(regulated by law)

Treatment of mixed waste

Training

58

Materials to be autoclaved eg.

Culture and stocks of infectious agents

Contaminated Solids (paper towel, cloth, plastic pipette

tips, glassware etc.)

Discarded live attenuated vaccines

Plant and animal specimens

Animal tissues

Animal cage waste

Pathogenic plant matter

What should not be autoclaved?

Corrosive chemicals

Flammable chemicals

Combustible

Explosive

Radioactive materials

Procedure of Biological Waste Handling

59

Risk ManagementSelection of Appropriate Detergents and

Disinfectants

BG+ BG- MycoB Spores Yeast Virus Prions

Alcohol 70° ++ ++ ++ 0 + + 0

Aldehydes +++ +++ ++ + +++ ++ 0

Ammonium IV +++ + 0 0 + + 0

Anilides + 0 NP NP 0 NP 0

Chlorhexidine +++ ++ 0 0 + + 0

Cl compounds +++ +++ ++ ++ ++ ++ + (a)

Iodine (+ der.) +++ +++ ++ ++ ++ ++ 0

Hg compounds ++ ++ 0 0 + 0 ou + 0

Phenols : Variable activity depending on components (b)

Hexachlorophene +++ + 0 0 + 0 0

(a) Bleach (6%) during 60 min at 20°C ; (b) discussion on efficacy of phenol on prions

60

General Guidelines for Surface Decontamination

Agent Disinfectant Inactivation time

Recombinant DNA 10% Bleach 20 minutes

Bacterial Spores >6% Hydrogen Peroxide 30 minutes

Vegetative Bacteria 10% Bleach 20 minutes

Viruses & Viroids 10% Bleach 20 minutes

Fungi 10% Bleach 20 minutes

Feline Parvovirus 20% Bleach 30 minutes

Free Living

Cryptosporidium

70% Ethanol 10 minutes

Most Parasites 10% bleach 30 minutes

Prions 50% bleach 60 minutes

61

Class 1 Explosive

Class 2 Gases

Class 3 Flammable liquids

Class 4 Flammable solids

Class 5 Oxidizing agents and Organic peroxide

Class 6 Toxic and Infectious substances

Class 7 Radioactive substances

Class 8 Corrosive substancesClass 9 Miscellaneous (Specify)

UN biological substance category

• UN2814 infectious substance, affecting humans

• UN3373 biological substance

Infectious Substance Transportation

62

Triple Packaging

Main goals

protects the environment, the carrier

protects the sample

arrival in good condition for analysis

The basic triple packaging system

Leak-proof specimen container

Packaged with sufficient absorbent

material to absorb the entire content

of the primary receptacle in case of

breakage

Three layers of protection are needed:

primary receptacle

secondary packaging: are placed in outer shipping

packaging with suitable cushioning material

outer packaging: protects contents from outside

influences, physical damage, while in transit

Smallest overall external dimension 10 x10 cm

Triple pakaging

(WHO, 1997)

63


Summary of Shipping Information (IATA, international Air Transport Association)

Shipment TypeProper Shipping

Name

UN

Number

Packing

Instruction

Max Net qty/pkg

for Passenger

Aircraft

Max Net

qty/pkg for

Cargo Aircraft

Category A infectious

substance, affecting

humans and possibly

animals

Infectious


humans (technical

name)

UN 2814 602 50 ml or 50 g 4 L or 4 kg

Category A infectious

substance, affecting only

animals (not humans)

Infectious


animal (technical

name)

UN 2900 602 50 ml or 50 g 4 L or 4 kg

Category B infectious

substance

Biological

Substance,

Category B

UN 3373 650 4 L or 4 kg 4 L or 4 kg

Diagnostic Specimens Biological

Substance,

Category B

UN 1845 650 4 L or 4 kg 4 L or 4 kg

Non-infectious,

transducing genetically

modified organism or

micro-organism

Genetically

modified micro-

organisms

UN 3245 913 No limit No limit

http://www.ncsu.edu/ehs/dot/Bio_shipping.pdf64

Risk group 3 and 4 and some in Risk group 2Category A

Outer packaging >10 x 10 cm

http://nih.dmsc.moph.go.th/KM/safety19-01-09_1/Packaging&Shipping_of_Hazardous.pdf

UN2814


1.Triple

packaging

3. Declaration

is required

2. Proper

Labeling,

Notification

65

Risk group 1 and 2Category B

http://nih.dmsc.moph.go.th/KM/safety19-01-09_1/Packaging&Shipping_of_Hazardous.pdf

UN3373No biohazard label


Triple

packaging

66

Source: Popattanachai N, DMSC

Follow Pathogen and Animal Toxin Act

B.E. 2525 (1982), amended 2544 (2001)

Thailand

68

Risk Management

Emergency Preparedness

1.Develop emergency action plans (describes the procedures to be followed if

an accident, spill, release, or exposure contaminates personnel or the environment. As

part or the planning, identify and limit conditions that contribute to the risk of spills or

accidents.

2. Train personnel

–Spills (SOP)

–Medical emergencies (SOP)

(Immediate medical assistance)

–Laboratory accidents/exposures (SOP)

–Incident reporting procedures (SOP)

(prompt reporting to the responsible persons/agency:A written report

of any spills, exposure, failures of containment, mechanical

breakdown, and maintenance problems is submitted to the BSO)

3. Emergency scenarios

4. Emergency exercises and simulations

5. Post-exposure management plan68

Spill Clean-Up

Follow clean-up procedures in SOP (training required)

Basic Biological Spill Clean-Up Kit

Nitride gloves (double gloving), splash goggles, shoe

covers

Small disposable broom with dustpan, tongs or forceps

(for picking up sharps)

Paper towels or other absorbent in the lab

Sharps container and/or biohazard waste bags

Disinfectant agent suitable for the agents in the lab

SOP (waterproof copy)

Spill Clean-Up: Blood/Microorganisms

http://www.yale.edu/ehs/onlinetraining/video/bloodspill.htm

Spill out 1. Alert personnel in the area to

hold their breath, leave the

room, and close the door.

2. Post warnings in the area to otify

Public Safety and provide a phone

number where you can be reached.

•Wait thirty minutes to allow airborne

organisms to settle.

3. •Collect all needed spill response

supplies. Don the appropriate PPE

(including a respirator for BSL3.

•Return to lab and clean-up as per

directions. Use forceps, tongs, or broom

to remove broken glass and other items;

place in sharps container or red bag

4. Cover an area twice the size of the

spill with paper towel and apply

bleach or disinfectant soaked-paper

towels as per label directions

5. Soak paper towels with

disinfectant as per label directions as

per label directions70

http://www.yale.edu/ehs/onlinetraining/video/bloodspill.htm

6. Soak the paper towels with

disinfectant (disinfectant

soaked-paper towels)

7. Allow 15 – 20 min contact

time8. Wipe up working towards center.

Remove the paper towels and re-clean

area with disinfectant solution

9. Dry the area with paper

towel

10. Spray disinfectant on used

materials

11. Remove the paper towels and

used materials or equipment and

put into biohazardous waste

container.


: http://www.yale.edu/ehs/onlinetraining/video/bloodspill.htm

13. Wash the hand

14. Inform laboratory personnel when the clean-up is complete

15. Notify lab instructor

12. Remove the PPE and put into biohazardous waste container


72

Report Exposures Immediately

Post-Exposure Evaluation

Confidential medical evaluation

Documenting route of exposure

Documenting circumstances of the incident

Identify source individual

If handle with blood, blood test

for eg. HIV, HBV, etc.

(with individual’s consent)

Provide results to exposed employee

Post exposure prophylaxis and counseling

73

Q&A

Thank you

74

Documents

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