Gathering evidence around air quality in IVF labs

Josiane Van der Elst on behalf of EACC

Special thanks to Ronny Janssens, Quality Officer, CRG, AZVUB

Outline
1. 2. Introduction Defining air quality Cleanroom classifications Practical aspects 3. 4. Validation of air quality Where are we today

Introduction
The quality of the air and the environment have always been a matter of concern in IVF labs reduced light no windows limited and controlled access easy to clean no alcohols, no perfumes, no smoking, non toxic painting air conditioning air filtration HEPA positive pressure sterility precautions

We are no maverick cowboys!

Introduction
Now, since 2006, the new EU-Directive 2004/23/EC stipulates quality requirements when human tissues and cells are handled The EU Directive introduces formal requirements that are to be laid down in a national legislation A critical point is clean air

2004/23/EC
Unless otherwise specified in point 4, where tissues or cells are exposed to the environment during processing, without a subsequent microbial inactivation process, an air quality with particle counts and microbial colony counts equivalent to those of Grade A as defined in the current European Guide to Good Manufacturing Practice (GMP),

2004/23/EC
Annex 1 and Commission Directive 2003/94/EC[1] is required with a background environment appropriate for the processing of the tissue/cell concerned but at least equivalent to GMP Grade D in terms of particles and microbial counts. This seems difficult to reconcile with IVF routine but exceptions to these very stringent conditions may apply to IVF
[1] http://pharmacos.eudra.org/F2/eudralex/vol4/home.htm

2004/23/EC
A less stringent environment than specified in point 3 may be acceptable where: a validated microbial inactivation or validated terminal sterilisation process is applied; or, where it is demonstrated that exposure in a Grade A environment has a detrimental effect on the required properties of the tissue or cell concerned;
high air flow can be detrimental in IVF due to temperature and pH fluctuations

Temperature
Pickering S.J., Braude P.R., Johnson M.H. et al. (1990) Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril, 54, 102-108. - 1C cooling = irreversible damage to meiotic spindle

Temperature
Wang W.H, Meng L., Hacket R.J., Odenburg R., Keefe 2001 Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy Hum. Reprod, 16: 2374-2378 (Polscope) Depolarisation of spindle, oocytes are more sensitive then embryos IVF: 37.0 0.5C

Temperature
Tempeature decrease in a dish out of the incubator
39 36 33 30 27 24 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 in LAF not in LAF

- 0.5C/min

2004/23/EC
A less stringent environment than specified in point 3 may be acceptable where: or, where it is demonstrated that the mode and route of application of the tissue or cell to the recipient implies a significantly lower risk of transmitting bacterial or fungal infection to the recipient than with cell and tissue transplantation; or, where it is not technically possible to carry out the required process in a Grade A environment (for example, due to requirements for specific equipment in the processing area that is not fully compatible with Grade A) Placing inverted microscopes to perform ICSI on in a LAF can be incompatible with adequate IVF procedures

2004/23/EC
In point 4 (a), (b), (c) and (d), an environment must be specified. It must be demonstrated and documented that the chosen environment achieves the quality and safety required, at least taking into account the intended purpose, mode of application and immune status of the recipient. Appropriate garments and equipment for personal protection and hygiene must be provided in each relevant department of the tissue establishment along with written hygiene and gowning instructions Interpretation Falling under the exceptions is no free guide to lack of quality!! We will have to gather evidence that we way we are working is leading to good IVF results without jeopardising quality and safety

Introduction - summary
The EU requirements default requirments on clean air are very demanding (GMP pharmaceutical industry) Exceptions to these most stringent conditions may apply to IVF But, even then, action has to be taken to ensure clean air in IVF labs

Outline
1. 2. Introduction Defining air quality Cleanroom classifications Practical aspects 3. 4. Validation of air quality Where are we today

Technical requirements for processing, preservation, storage and distribution

EU Guide to Good Manufacturing Practice-Manufacture of Sterile Medicinal Products 2003/94/EC
http://pharmacos.eudra.org/F2/eudralex/vol4/home.htm
A B C D

Air quality in practical terms

Grade A = LAF Background B,C,D These are different levels of cleanliness of air in which LAFs are operating and personnel is working It is generally accepted that a LAF can only achieve Grade A if the incoming air is of grade B

Classification according to EU GGMP Pharmaceutical Clean Room

In order to reach the B, C and D air grades, the number of air changes should be related to the size of the room and the equipment and personnel present in the room The air system should be provided with appropriate filters such as HEPA for grades A,B and C EU Guide to Good Manufacturing Practice-Manufacture of Sterile Medicinal Products

Cleanroom ISO 14644-1 classification for the main particle sizes

A B-C

Microbial contamination

Outline
1. 2. Introduction Defining air quality Cleanroom classifications Practical aspects 3. 4. Validation of air quality Where are we today

Class A

IVF: Class D

TD2: LAF cabinet (grade A) in grade D background

GMP General Requirements Clean areas Changing rooms Dress code Laminar flows Filtered air

GMP Clean areas

Design Avoid unnecessary entry Clean areas Smooth, impervious, unbroken surfaces Permit cleaning No uncleanable recesses, ledges, cupboards, equipment No sliding doors Ceilings Pipes and cucts Sinks and drains

GMP Changing rooms

Changing rooms Designed as airlocks Flushed with filtered air Separate for entry and exit desirable Hand washing facilities Interlocking system Visual and/or audible warning system

GMP dress code

Grade B background High level cleanroom Bunny suits, mask, gloves Two levels of changing rooms, from street to C, from C to B Hermetically sealed pass-troughs for specimens Grade C background Trouser suit covering wrists and neck, hair and baird covered Changing room Hermetically sealed pass-troughs for specimens Grade D background General protective suit, hair and baird covered Standard of currently better-constructed modern IVF laboratories

Bunny suits

Bunny suits

Trouser suits

The suit was the uniform of the liberated woman

GMP air filtering

Air supply: (HVAC) Generation and supply of filtered air under positive pressure Airflow patterns Failure of air supply Pressure differential monitored and recorded

HEPA filtration was developed by the Atomic Energy Commission during World War II to remove radioactive particles from the air in manufacturing plants A HEPA filter must remove at least 99.97% of all airborne particles by particle count at a size of 0.3 microns In hospital operating rooms, burn centers, laboratories and manufacturers of critical products like computer chips, where particle and bacteria free air is mandatory, HEPA filtration systems are used to remove bacteria and other airborne contaminants

Ventilation

LAFs
1) Laminar flow hoods for maintaining a clean to sterile work environment Some laminar flow hoods blow a curtain of air across the work surface carrying possible contaminants (including microorganisms) away from the work surface and out into the laboratory These hoods actually can increase your chances of becoming contaminated with microorganisms and should not be used for containment

LAFs
2) biosafety cabinets used to keep the specimen and the operator safe There are 3 classes of biosafety cabinets, I, II and III Class I safety cabinets are rare and do not protect the work area much

LAFs
Class II biological safety cabinets provide personnel, environmental and product protection This is the type of hood that most IVF units require Air flow is drawn around the operator into the front grille of the cabinet, which provides personnel protection In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet Because cabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or ducted out of the building (Type B BSC)

LAFs
Class III The Class III biological safety cabinet was designed for work with biosafety level 4 microbiological agents, and provides maximum protection to the environment and the worker. It is a gas-tight enclosure with a non-opening view window Access for passage of materials into the cabinet is through a dunk tank (that is accessible through the cabinet floor) or double-door pass-through box (such as an autoclave) that can be decontaminated between uses. Reversing that process allows for safe removal of materials from the Class III biosafety cabinet. Both supply and exhaust air are HEPA filtered

LAFs
Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (usually about 0.5 inches of water pressure)

LAFs
Hoods must be properly maintained and tested. Also, how you set up your work area in the hood may increase or decrease your chances of cross-contamination. Because of the large bulky microscopes that we use in IVF and that often must protrude out of the hood into the laboratory for us to use, it is very difficult to use a Class II hood efficiently (the microscope will interrupt the vertical flow of air across the front of the hood)

Class I

Class II

Class I - II

Class III

IVF lab design

HEPA 14 ISO 7 >10 Pa B in operation C at rest

Outline
1. 2. Introduction Defining air quality Cleanroom classifications Practical aspects 3. 4. Validation of air quality Where are we today

Validation

ISO 15189, 5.3.2 Equipment shall be shown (upon installation and in routine use) to be capable of achieving the performance required and shall comply with specifications relevant to the examinations concerned

Validation
Validation or qualification in case of equipments or environments means establishing documented evidence that provides assurance that a specific process, equipment or environment will consistently produce a product meeting its predetermined specifications and quality attributes

Validation of air quality

1. Document all procedures attaining by far and large to air quality 2. Measure, monitor, maintain Particle count measurements CFU measurement Monitoring of pressure, air changes, verification of ventilation systems VOC measurement Maintenance certificates 3. Show benchmark results (fertilisation, pregnancy results)

Measure, Monitor and Maintain

Particles: Particle counter (laser based) Microbiological Settle plates or other Hygiene monitor Monitor air ventilation system Maintenance of LAFs and air ventilation systems On regular basis VOC: not mentioned in EU Directive but important to IVF Get professional help where needed

Particle Counter
Measures in seconds Proof of Air Quality Easy to use Particles from 0.3 micron to 5 micron Sizes and Counts particles Data output

Microbiological control

Hygiene Monitor
Detects Bacteria Collects and Measures ATP Proof of Hygiene standards Results in 10 seconds

Swab a 10x10 area

Required testing (ISO14644-1)

VOC: origine
Traffic - pollution Paintings glue Packing materials isolation Plastics Machines Laboratory equipment Compressed gasses Staff Alcohol anesthetics

Cohen et al.Hum. Reprod 1997

Cohen et al.Hum. Reprod 1997

Nunc dishes

Cohen et al. Hum. Reprod 1997

Effects of VOC Cohen et al., Hum Reprod vol. 12, 1997

Effects of VOC Cohen et al., Hum Reprod vol. 12, 1997

VOC : the practice

Elimination of possible sources No alcohol Anesthetic gasses Measure VOC concentrations Capturing - Cryo concentration - Gas Chromatography (GC) - Mass Spectroscopy (MS) Adsorbent tubes (aldehydes) - High performance liquid chromatography (HPLC) ACS badge Eco sensor VOC meter Filtration Active charcoal absorption Oxydation (Potassium permanganate) Photo-Catalytic Oxidation

Advanced Chemical Sensors

(www.acsbadges.com)

Eco sensor C-21

Permanent monitoring acoustic alarm 80 dB

VOC Meter
VOCs at low levels (0.1ppm) Pin-point the source Equipment and consumables screened Stores data

Filtration - CODA

Air conditioning Laboratory air Gas lines Incubators

Incubator Filters - Prospectively Randomized Study

Photo-Catalytic Oxidation

CO CO2 + H2O NxO

Z-IVF AIRe

Active Carbon Filter + potassium permanganate HEPA filtration removes particles >0,3M Photo-Catalytic Oxidation: converts toxic compounds into CO2 and water Ultraviolet light

Validation of air quality

1. Document all procedures attaining by far and large to air quality 2. Measure, monitor, maintain Particle count measurements CFU measurement Monitoring of pressure, air changes, verification of ventilation systems VOC measurement Maintenance certificates 3. Show benchmark results (fertilisation, pregnancy results)

Measuring quality: benchmarks Mayer J., RBM online, Vol 7, No 6, 2003

Benchmarks
Transfers D3 - D5, feb 2004 - jan 2006 (ICSI)
80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 1 3 5 7 9 11 13 15 17 19 21 23
78,0 76,0 74,0 72,0 70,0 68,0 66,0 64,0 62,0 60,0 58,0 1 3 5 7 9 11 13 15 17 19 21 23

% fertilisation (ICSI) feb 2004 - jan 2006

%ETD3 %ETD5 GeenET

%BV

Embryoquality D5 vs transferrate
90,0

Outcome Feb 2004 - Dec 2005

50,0 45,0 40,0 35,0 30,0 25,0 20,0 15,0 10,0 5,0 0,0 1 3 5 7 9 11 13 15 17 19 21 23

80,0 70,0 60,0

%AD5 %ETD5

50,0 40,0 30,0 20,0 10,0 0,0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

%poshCG/cycle %DELIVERIES/PoshCG

Pregnancies - outcome

Outline
1. 2. Introduction Defining air quality Cleanroom classifications Practical aspects 3. 4. Validation of air quality Where are we today

Where are we today

Directive 2004/23/EC = GMP GMP IVF Very limited evidence in IVF literature on relation between air quality and IVF outcome In view of the EU Directive we have to start to collect evidence

Particles and microbiological

Boone W. Boone W. Control of air quality in an assisted reproductive technology laboratory. technology laboratory. Fertil
Steril. Vol. 71, No. 1, 1999

Journal of Assisted Reproduction and Genetics, 21, 347 348, 2004

Von Wyl S, Bersinger N.A. Air quality in the IVF laboratory results and survey.

Ortu S Contrles particulaires et biologiques de lair lhpital Hygine et infections nosocomiales, Revue Francophone
des Laboratoires, novembre 2005, 376, 51-57

VOC: literature
Cohen J, Gilligan A,Esposito W, Schimmel T and Dale B: Ambient air and its potential effects on conception in in-vitro. vitro. Hum. Reprod Reprod. 12, 1742 - 1749 (1997) Hall J, Gilligan A, Schimmel T, Cecchi M and Cohen J: The origin, effects and control of air pollution in laboratories used for human embryo culture. Hum.
Reprod (suppl): 1998

Mayer J et al. Mayer J et al. Prospective Randomized Analysis of the Impact of an IVF Incubator Air Filtration System on Clinical Pregnancy Rates. 11Th World Congress on In Vitro Fertilization
and Human Reproductive Genetics, 1999 Sydney Australia 1999 Sydney Australia

VOC: literature
Boone W. Boone W. Control of air quality in an assisted reproductive technology laboratory. technology laboratory. Fert &
Steril. Vol. 71, No. 1, 1999

Journal of Assisted Reproduction and Genetics, 21, 347 348, 2004

Von Wyl S, Bersinger N.A. Air quality in the IVF laboratory results and survey.

Pfeffer et al. Mesure des composs organiques volatises (COV) et des aldehydes dans un laboratoire de fcondation in Vitro (FV), CO 01 FFER, 2005

What will tomorrow bring

Make an estimation of your situation Most favourable scenario: you comply Premises of sufficient quality but only LAFS or clothing to be replaced Worst case scenario: whole ventilation system to be changed or to be installed Best option: new cleanroom facilities?