|01. Layout of the Lab|
|02. Lab Design|
|03. Oocyte & Embryos in Lab|
|04. Air Quality|
|05. Essential Equipments|
|06. DOs & DONTs|
|07. Experts’ Tips|
|08. Lab Guidelines & Organization|
Action plan for setting up an IVF facility:
IVF lab design should be done based on the expected patient flow and future expansion plans. The budget might vary depending on the quality of equipments that you wish to purchase and patient load expected.
The site of lab is very important as the outcome of IVF depend on lab quality along with other major contributing factors. A pollution free area is ideal. Make sure that the IVF lab area is away from patient traffic locations in the hospital. If the IVF centre is in down town areas keep the IVF lab region to the 3rd or upper floors. The ground floor invite more air pollution.
Design should be done according to the expected expansion plans. Take advice from the experts (not one). Do not solely depend on any vendors for lab design.
There should be minimum requirements for any IVF lab that should be maintained.
Air quality in the IVF laboratory /OT area are of utmost importance. Pollution status of the air in the vicinity of the IVF clinic also should be taken into consideration. Based on these, more air handling units with HEPA/activated carbon filters should be installed with adequate positive pressure.
Although robust data are not available, assisted conception is unlikely to be any less prone to adverse incidents; indeed there have been several high-profile cases which have drawn attention to this problem. Because of the nature of the work undertaken in assisted conception, there is the potential to affect not only future generations but also many patients simultaneously because of storage of biological material. It is therefore important to implement strategies to reduce the likelihood of patient safety incidents. Established methodologies exist for the reactive (root cause analysis) and the proactive assessment of risk (failure mode effects analysis). Furthermore, establishing the detail of a process and its context through process mapping is an important prerequisite for understanding its risk. The knowledge gained through these enquiries enables the implementation of an effective risk management programme Efficient actions against the growing work-related illness are necessary in order to achieve a sustainable development and create a good prerequisite for growth. An organizational culture, which facilitates integration of new methods into the continuous improvement work, must be created.
The success and improvement in outcome depend not only in the lab equipments and quality disposables. The staff training/educational level etc are utmost important.
Proper installation of IVF equipments, proper after-sales service and proper calibration and continuous updates on the new equipments etc are to be taken care.
This is an area which the centre management has to put their active thoughts.
Enough culture disposables, proper culture media procurements from reliable companies, maintenance of standard operating procedures as manuals are essential.
The embryology laboratory should have adequate space to follow good laboratory practice. More specifically:
- The construction of the laboratory should ensure aseptic and optimal handling of gametes and pre-embryos during all phases of the treatment.
- The location of storage areas and equipment such as incubators, centrifuges and cryo equipment should be logically planned for efficiency and safety within each working area.
- Separate office space should be provided for administrative work, such as record keeping and data entry.
- A general wet area in which washing of equipment, sterilization, etc., is performed, should be separate from the embryo laboratory. Moreover, if fixatives are applied, these analyses should be performed in a separate room in a fume-hood.
When commissioning the laboratory, thought should be given to the most recent developments in equipment and facilities. Bench height, adjustable chairs, microscope eye height, efficient use of space and surfaces, sufficient air-condition and the amount of daylight, all contribute to a working environment that minimizes distraction and fatigue. Consideration should also be given to local health and safety requirements.
3.2. Laboratory equipment
- The laboratory equipment used should be adequate for laboratory work and easy to clean and disinfect.
- Critical items of equipment, including incubators and frozen embryo storage facilities, should be appropriately alarmed and monitored.
- All embryo laboratories should have an automatic emergency generator backup in the event of power failure.
- A minimum number of two incubators is recommended. Gas cylinders should be placed outside or in a separate room with an automatic backup system.
- Incubators should be frequently cleaned and sterilized. Nitrogen tanks should be cleaned and sanitized at least every year.
The purpose of the protective measures is also to ensure aseptic conditions for gamete and embryos. The procedures should deal with, but not be limited to, the following:
- Use of laboratory clothing.
- Use of non-toxic (non-powdered) gloves and masks.
- Use of eye and face protection if cryogenic materials are handled.
- Use of vertical laminar-flow benches.
- Use of mechanical pipetting devices.
- Use of fume-hood in case of fixatives.
- Disinfection and sterilization of potentially infected equipment.
- Use of disposable material; after usage, it must be discarded immediately in the proper waste containers. Potential infectious materials must be disposed of in a manner that protects laboratory workers and maintenance, service, and housekeeping staff from exposure to infectious materials in the course of their work.
- Needles and other sharps should be handled with extreme caution and discarded in special containers. If possible, glassware should be omitted in the laboratory, otherwise the Pasteur pipettes and broken glassware should be discarded in special containers.
Composition of the media:
The IVF media formulation more or less simulates the physiological fluids. In vivo, the oocyte is fertilized in the ampulla of fallopian tube, and then makes its way as a pre-embryo to the womb. The pre-embryo arrives there at the stage of morula. This is 4-5 days after ovulation. The faloopian environment where growth of pre-embryo happens is dynamic micro environment with endothelial cells continuosly responding to changing hormonal milieu. In near future such a system could be replicated in vitro by “ microfluidic embryo culture systems. The pH level of the IVF medium is usually about 7.4
Embryos are on a continuum of development from the time of initiation of oocyte growth in the primordial follicle through implantation and fetal development.
The buffer system most commonly used is bicarbonate buffer under 5-6% carbon dioxide gas (CO2) in air. In the natural system the buffer consists of 24 mM HCO3- with CO2 which is found in the lungs at a partial pressure of about 40mm Hg. This partial pressure corresponds to 5%. The CO2 in the liquid phase is in equilibrium with that in the gas phase which is achieved only gradually. It is therefore necessary to place the IVF culture medium in the CO2 incubator well in advance (with cap opened). In practice an incubation period of 16 hours seems to be necessary.
The osmolarity of IVf cultue media chosen is 285 mOsm per kilograms water, corresponding to blood plasma. With OCC (oocyte cumulus comples) or embryo culture in open culture systems there is a high chance of increase in osmolarity in the media as a function of time and humidity in the chamber. Hence, oocytes and spermatozoa are cultured in incubators with high humidity of above 80%.
Another possibility to counteract evaporation is to use mineral oil in order to close off the drops of medium from the environment . It is however necessary to equilibrate the oil beforehand at high humidity with 5% CO2 . Also during the inspection of the oocytes for pronuclei, which most laboratories will carry out under normal atmospheric conditions, less evaporation will occur when using mineral oil. Apart from evaporation the dissolved CO2 will also not escape quickly from the medium. This inhibitory effect of the oil on the diffusion of carbon dioxide gas can also be a disadvantage . If due to a protracted period outside the incubator CO2 still escapes, then it will take longer to return to equilibrium after being put back in the incubator , than it would without the oil. Another additional advantage is that the oil acts as a barrier to dust particles and any micro-organisms from the atmosphere.
A controlled work environment is of high priority due to the complexity of the process. This includes hygienic conditions to minimize microbial contamination, a warmed work surface for the sustenance of biological matter and provisions for the use of microscopes. There is still debate over the use of laminar air flow hood for oocyte/embryo manipulations. The advantage of using such systems is to avoid any contamination for your culture media. The disadvantage lies in the desiccation or temperature change in embryo loaded media droplets by air flow. A closed system integrated with a good stereo zoom microscope with gas-control and temperature control is more physiological.
As well as ensuring a good pure medium , the gas phase in the CO2 incubator should also comply with high standards . Higher standards are required for the maintenance of cylinders for medical gases than those set for technical gases.
Most incubators will be used for a mixture of carbon dioxide and air. There are also laboratories which want a different atmosphere in the incubator. The oxygen content in particular is then often set lower. As already mentioned, IVF media are calculated on a carbon dioxide content in the incubator of 5%.
Co2 incubators are easily contaminated with bacterial growth or moulds due to the high humidity. Regular cleaning is therefore necessary. For this it is best to use cleaning agents specially suited to tissue culture. Alcohol should be used with moderation due to the negative effects that alcohol has on oocytes and early embryos. Most of te infections in culture systems happens due to seminal bacterial infections which are resistant to common antibiotics. A bacterial culture and sensitivity is a must for all males before IVF procedure. Check the swim-up or density gradient based sperm preparation under your inverted microscope in ICSI workstation at 200/400 magnification to make sure that there is no bugs in your insemination drop.
There are incubators which use a layer of water at the bottom. Due to evaporation the humidity in these incubators will be 80% or more . There are also incubators which actively mist water in the recirculation air stream, and can keep the humidity up to 90% by continuously measuring the relative humidity in the incubator. Every time an incubator is fitted with an infrared CO2 indicator, then the reading of the carbonic acid content will not be abnormal. However, if the incubator is equipped with a conductivity indicator for the content of carbonic acid, then the reading depends on the relative humidity and will thus be different from the actual level until the relative humidity in the incubator has increased to the level set.
Calibration of the incubator:
Calibration of the incubator can best be done with an infrared CO2 indicator controlled and calibrated using gas. This calibration gas is available in small cylinders and is always accompanied by an analysis report giving the cylinder number
|Gas Analyzer for Calibration of Incubaors|
|Galaxy CO2 & O2 analyzer||rsbiotech.com/Calibration|
|The Fyrite Gas Analyzer||Bacharach Inc., Pittsburgh, USA|
|Anagas CO2 monitor||wolflabs.co.uk/Anagas_CO2_Monitor.htm|
How to avoid temperature drops while manipulating the oocytes, embryos:
1. Switch on your microscope stage warmers well in advance of the actual procedures.
2. Try to procure best quality stage warmers/test tube warmers.
3. Regular (daily) checking of the temperature by thermometer in a log-sheet.
4. Keep your tissue culture disposables in a 370 C incubator 2-3 hours in prior to the procedure.
5. Warm the ET catheter upto physiological temperature before ET.
6. Make sure all HEPES buffered media were kept in 370C well in advance.
7. Try to minimize the exposure of zygotes while denuding the OCC for fertilization check. If the oocytes are tangled in the spider-web net like situation, try using higher diameter denuding pipettes or try with tuberculin syringe to cut apart the cumulus net. Always do it under equilibrated oil. If you find that the procedure is taking much time bring the dish back to CO2 incubator for an interval.
8. Junior embryologists /trainee embryologists should have ample practice on hand held denuding pipettes before doing the actual cases.
Time of Insemination
Concentration and incubation volume
Pre-implantation development of the embryo
The moment of Embryo transfer:
In vivo, after fertilization the embryo will reach the uterus at the morula-blastocyst stage, which is about 4-5 days after ovulation.
Transfer of embryos on Day 2:
The embryo is in 2-cell to 4-cell stage, but 6-8 cell stage also be observed. Ideal for IVF lab set-ups with minimal or less-optimal culture conditions and one advantage is that the embryo is outside of women’s body for only short period. Ideal ET time for those flying embryologists (free-lancing), and IVF set up in remote areas with less technical supports where there is doubts on the cold-chain maintenance of culture media supply.
Staessen et al (1993) found a difference in the implantation rate between good-quality 4-cell and 2-cell embryos of 21% and 14% respectively.
Transfer of embryos on Day 3:
On day 3 the embryo has developed to the 6-8-cell stage, and even a 10 -12 cell stage does occur. ET on day 3 is preferred by some, as it is not possible to make a right choice of on day 2 when there are more embryos of same quality. Usually a day 3 morning transfer is practiced in most of the centres with surplus embryos transferred to blastocyst media for extrended culture.
Transfer of blastocysts on Day 5:
The first IVF human pregnancy was achieved by blastocyst transfer.At this point in development, the embryo has between 60 and 100 cells distributed in two areas: an outer embryo lining (which will later form the placenta), and an inner mass (which will later become the fetus). During a natural cycle, the embryo develops into the blastocyst stage as it is leaving the fallopian tubes and entering the uterus. An embryo needs to have entered into the blastocyst stage once it arrives in the uterus to ensure proper implantation.
Not all embryos are strong enough to reach the blastocyst stage of development; in fact, approximately 50% of all embryos die soon after the third day of development. By holding transfer off until the blastocyst stage, your embryologist will be better able to ensure that your embryos are healthy and capable of further development. Blastocyst transfer is associated with almost a 50% success rate, compared to the usual 35%.
CO2 incubators allow to closely, reliably and repeatedly mimicking the environmental conditions that sperm, oocytes, blastocysts and developing embryos encounter in-vivo.
A minimum 2 CO2 incubators are essential in any program.
One is exclusively for media equilibration
Another one for Fertilization/embryo culture
Triple gas Incubator:
Research has shown that incubators with an atmosphere comprising CO2, O2, and N2 provide a more natural environment than a plain CO2 incubator, giving better embryo quality and higher success rate.
A good investment if going for Blastocyst culture.
Vertical Laminar Flow hoods:
This HEPA filtered working environment provides clean air for gamete/embryo handling. This could be an integrated laminar flow hood with 2 stereozoom microscopes, a bench top triple gas incubator and an imaging device/monitor with recording facility.
Stereo Zoom Microscopes:
Stereomicroscopes produce three-dimensional, laterally correct and upright images. Their major benefits include large object fields and large working distances. Stereo microscopes, also called dissecting microscopes, are really two compound microscopes which focus on the same point from slightly different angles. This allows the specimen to be viewed in three dimensions. As opposed to compound microscopes, the image is upright and laterally correct (not upside down and backwards). Stereo microscopes are relatively low power compared with compound microscopes,
usually below 100x. They can have a single fixed magnification, several discrete magnifications, or a zoom magnification system. Working distance is much longer than with a typical compound microscope as well, allowing work to be done on the specimen while it is being observed through the microscope (hence the name “dissecting microscope”). Many stereo microscopes are modular in design allowing a variety of stands, eyepieces, objectives, and lighting techniques to be implemented depending on the intended use.
Stereo zoom microscope is used in IVF lab for egg harvesting during ovum pick up, for insemination, embryo changes from media to media, embryo loading to ET catheter, vitrification device etc.
Stereo zoom microscope is usually integrated into the Heated laminar flow table. For a busy IVF lab 2-3 stereo zoom microscopes are essential. One can be dedicated exclusively for vitrification of oocytes/embryos with the base where dishes are kept having no-heating plate.
ICSI work station:
ICSI for male factor infertility is widely practiced in most of the IVF clinics. To perform ICSI (Intracytoplasmic sperm injection), one need an Inverted microscope fitted with a micromanipulator.
Inverted microscopes are available from Nikon, Olympus, Leica, Carl Zeiss etc. The micromanipulators those are available in the market are Narishige, Eppendorf, Research Instruments (RI), Cell Robotics etc.
These are specifically designed for aseptic handling of oocytes and embryos to minimize microbial contamination. Built-in liquid based heating system. Built-in stereomicroscope fitting, transmitted light source, heated plate by electrical heating or water circulation heating. The IVF chambers provide a triple gas incubator kind of environment.
Centrifuge machine with swing-out rotor is ideal fro the sperm preparation in IVF
Bench top Incubator:
This kind of bench top incubators is ideal for existing Laminar flow hood environment in IVF labs
370 C incubator:
These simple incubators can warm up HEPES-buffered media in IVF and can be used to warm-up the disposables
Liquid Nitrogen Cans:
Enough number of Liquid Nitroger dewars are essential for storage of sperm and embryos. One with wide mouth is ideal.
Jason E Swain (2010) the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality, RBM Online, 21, Issue 1, Pages 6-16 (July 2010)
Staffing and Direction:
1.1. The laboratory is directed by an appropriately qualified and experienced scientist or medical doctor according to national rules.
1.2. There are appropriate numbers of staff with the required experience to undertake the workload of thelaboratory.
1.3. All new staff are given a comprehensive orientation and introduction programme.
1.4. Continuing medical education (CME) is recommended for all personnel involved.
1.5. Individual responsibility of each member of staff and line of responsibilities are indicated in a written procedure.
2. Policies and procedures
2.1. All laboratory procedures must include provision for unique patient identification whilst retaining patient confidentiality.
2.2. laboratory results should be reported according to a written procedure. They should be validated, dated and include unique patient identity. Any interpretation of results should be accurate, comprehensive and clinically relevant.
2.3. There is a record of all reagents, calibration and quality control material.
2.4. There is a written, signed and dated protocol for every procedure, written transmission of results and regular maintenance of equipment.
2.5. All the procedures should be gathered in a manual kept in the laboratory and available for consultation.
2.6. A log book should be maintained to permit a regular evaluation of the results.
For further details visit :
Canadian ART Lab Professionals Competency Profiles:
Continue at: http://www.lifeinvitro.com/art-lab-pecs.html
The text above is owned by the site above referred.
Here is only a small part of the article, for more please follow the link