amercare isolator manual
LINK 1 ENTER SITE >>> Download PDF
LINK 2 ENTER SITE >>> Download PDF
File Name:amercare isolator manual.pdf
Size: 1838 KB
Type: PDF, ePub, eBook
Category: Book
Uploaded: 25 May 2019, 18:39 PM
Rating: 4.6/5 from 684 votes.
Status: AVAILABLE
Last checked: 13 Minutes ago!
In order to read or download amercare isolator manual ebook, you need to create a FREE account.
eBook includes PDF, ePub and Kindle version
✔ Register a free 1 month Trial Account.
✔ Download as many books as you like (Personal use)
✔ Cancel the membership at any time if not satisfied.
✔ Join Over 80000 Happy Readers
amercare isolator manualThe low height and roller base frame make it easy to clean the whole external surface of the isolator. The alarm unit also records and logs significant alarm occurances for future action. Based in Thame in Oxfordshire since 1987, Amercare have an unparalleled reputation and international customer base. Find out more or switch them off if you prefer. However, by continuing to use the site without changing settings, you are agreeing to our use of cookies. Generators are shielded with 60mm interlocking lead. The low height and roller base frame make it easy to clean the whole external surface of the isolator. Typical applications include: Manufactured from materials which are highly resistant to cleaning and sanitising agents used in the Pharmacy. Based in Thame in Oxfordshire since 1987, Amercare have an unparalleled reputation and international customer base. Find out more or switch them off if you prefer. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus.http://activecaribe.com/userfiles/kawasaki-ninja-zx9r-service-manual.xml
- Tags:
- amercare isolator manual, amercare isolator manual, amercare isolator manual, amercare isolator manual transmission, amercare isolator manual transfer switch, amercare isolator manual pdf, amercare isolator manual electrical, amercare isolator manual.
The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations. Layout of the enclosed workstations. (A) Schematic illustrating the layout of the enclosed workstations showing the workflow from the entry of oocytes into the system until the embryos are replaced in the uterus. The incubators act as pass-through hatches linking adjacent workstations. (B) Schematic diagram showing the layout of 2 enclosed workstations connected by the double-doored incubation chambers. (C) Image showing the main features of an enclosed workstation with a built-in stereomicroscope and incubators. There are separate hatches to allow introduction of consumables and removal of waste. (D) Image shows the control panel for setting the temperature of the incubator, workstation and hotplate and the CO 2 in the incubator and workstation. The increase in the proportion of embryos developing to the blastocyst stage coincides with the switch from the open to the enclosed workstations. The two dark bars represent the intermediate period when the cohorts of 20 consisted of embryos cultured in the open and enclosed systems. (D) The proportions of blastocysts graded as early (black), expanded (grey) and hatched (white) on day 6. A significantly higher proportion of blastocysts had undergone hatching by day 6 after culture in the enclosed system compared to the open system. There was a corresponding reduction in the proportion of early blastocysts in the enclosed system (P,0.05). (E) Nuclear counts were performed to compare the total number of cells contained in blastocysts cultured to day 6 or 7 in the open system or enclosed system. The cell count of embryos cultured in the enclosed system was significantly higher on day 6 (128.3638.6 vs 92.1643.7; P,0.05) and day 7 (167.7669.7 vs 106.7648.8; P,0.http://tripura-infoway.com/tempimg/kawasaki-oem-manuals.xml01) compared with those cultured in the open system. There are separate hatches to allow introduction of consumables and removal of waste. (D) Image shows the control panel for setting the temperature of the incubator, workstation and hotplate and the CO2 in the incubator and workstation. Time point zero indicates the first measurement taken immediately after placing the dish on the viewing area. The temperature was then measured at 3-minute intervals. In each case 3 independent experiments were conducted. (B) A pre-equilibrated organ culture dish was removed from the incubator and placed on the viewing area of either the enclosed workstation (purple) or Class II cabinet (blue). There was a gradual increase in pH of the media in the Class II cabinet over the time course of the experiment. A Novel Isolator-Based System Promotes Viability of Human Embryos during Laboratory Proc essing.pdf Content available from CC BY 4.0: pone.0031010.pdf A Novel Isolator-Based System Promotes Viability of Human Embryos during Laboratory Processing.pdf Available via license: CC BY 4.0 Content may be subject to copyright. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. Moreover, blastocysts produced in the isolator- based system contained significantly more cells and their development was accelerated. Citation: Hyslop L, Prathalingam N, Nowak L, Fenwick J, Harbottle S, et al. (2012) A Novel Isolator-Based System Promotes Viability of Human Embryos during Laboratory Processing. Funding: Funding received from the Medical Research Council (G0601157 and GO400232), One North East, and Rege Ner8. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manus cript. Newcastle Fertility Centre receive a percentage of the royalties. There are no products in development to declare. The product is currently marketed under Vitrosafe.http://eco-region31.ru/boss-flanger-bf-3-manual It is likely that exposure of oocytes and embryos to unphysiological conditions is an important contributory factor. Laboratory processing during IVF typically involves the use of open-fronted microbiological (Class II) safety cabinets and stand- alone incubation chambers. Manipulations requiring removal of gametes and embryos from the incubator, therefore require exposure to ambient laboratory conditions which may result in deviations from physiological temperature and pH. The conventional practice is to culture human gametes and embryos in bicarbonate-buffered culture media under mineral oil in a CO 2 enriched incubator with either ambient (21) or reduced (5) O 2. Thus, exposure to ambient conditions can result in an upward drift in the pH of the culture medium.To reduce the impact of environmental stresses due to physicochemical instability and exposure to chemical and microbial contaminants, we aimed to develop a means of protecting oocytes and embryos from exposure to ambient conditions throughout the IVF process. This approach enabled us to provide a controlled and monitored environment from the time oocytes are harvested from the ovary until embryos are transferred to the uterus. Analysis of clinical and laboratory data before and after the installation of the enclosed workstations indicates that protection from ambient conditions promotes increased cell proliferation and viability of human embryos. Methods Measurement of temperature and pH Measurements of the pH and temperature of culture medium were obtained in 3 independent experiments using a calibrated pH meter (Jenway, UK) and thermocouple (Digitron, UK). Record- ings were taken from tissue culture dishes placed on the microscope viewing area and containing 500 m l of pre-equilibrated bicarbonate buffered G1 (Vitrolife Ltd) culture medium overlaid with 700 m l of mineral oil.https://www.mizuingenieria.com/images/91-polaris-250-service-manual.pdf In the case of the open system, the dishes of media were pre-equilibrated in a BB6220 Heraeus CO 2 incubator (Thermo Scientific). The enclosed system measurements were conducted following pre-equilibration in the custom built incubators (Vitrosafe Systems Ltd). In vitro fertilization and culture of human embryos All culture media were purchased from Vitrolife (Gothenburg, Sweden). Oocyte retrieval was conducted using MOPS-buffered (G-MOPS) medium in the open-fronted system and bicarbonate- buffered (G-IVF) medium in the enclosed system. In both systems, oocytes for intra-cytoplasmic sperm injection (ICSI) were prepared by enzymatic removal of surrounding cumulus cells using hyaluronidase (Hyase, Vitrolife) in G-MOPS medium. Sperm injection was also preformed in G-MOPS medium. Injected oocytes were transferred to organ culture dishes containing bicarbonate-buffered G1 medium under mineral oil as described above. The bicarbonate-buffered medium G-Fert or G-IVF was used for fertilization by conventional IVF. The best quality embryos were selected for replacement based on morphological appearance and cell number on day 2 or day 3 after oocyte retrieval. Remaining embryos were cryopreserved, donated to research, or discarded. All procedures in the enclosed system were conducted in heated air, which except for cumulus cell removal and sperm injection, was enriched with 7 CO 2. Following detection of a copulatory plug, the mice were sacrificed and one oviduct was transferred to an open-fronted cabinet and the other oviduct to an enclosed isolator-based system. Pronuclear stage zygotes with surrounding cumulus cells were transferred to a hyaluronidase-containing medium (Hyase, Vitrolife) in either G-MOPS for samples processed in the open-fronted system, or in G1 embryo culture medium for samples in an enclosed research workstation. Following dissociation of the cumulus cells the zygotes were transferred to G1 under mineral oil for onward culture until the 8- cell stage when embryos were transferred to G2 medium. Nuclei were visualized using an epi-fluorescence microscope (Nikon TE2000) fitted with an Apotome (Zeiss, Germany) or by confocal microscopy (Zeiss LSM 510 META). Nuclear counts were performed using Metamorph or Zeiss LSM 510 imaging software. We determined the accuracy of cell counting by different operators and imaging systems by performing a correlation to determine whether there were differences between counts (Fig. Research using human embryos was approved by the Newcastle and North Tyneside Research Ethics Committee and licensed by Human Fertilisation and Embryology Authority (HFEA). Written informed consent was obtained from all embryo donors. Comparison of clinical data During the period when the new isolator equipment was being fitted and validated, our conventional equipment was transferred to temporary facilities so that the clinical service could continue.Statistics Logistic regression models were used to investigate factors associated with positive pregnancy outcomes, including group, age, and time trends. Chi-squared or 2 sample Student t-tests were used to test for significant differences. We applied moving average analysis of groups of 20 embryos to obtain trend information on blastocyst development. A correlation was carried out to determine whether there were differences between blastocyst nuclear counts performed by different operators. The chain of workstations was configured to link adjacent patient treatment rooms in which oocyte retrievals and embryo transfers are conducted (Fig. 1A). To avoid removing embryos from the enclosed system, we designed double-doored incubation chambers, which can be accessed from adjacent workstations (Fig. 1B). This enabled us to maintain oocytes and embryos within the enclosed environment from the time of oocyte harvest until transfer of embryos to the uterus. The air supply to the critical workarea, and to the incubators was passed and recirculated through HEPA filters to remove particulates, and through activated carbon filters to remove VOCs. A major advantage of this system is that the supply air to work area and to the incubation chambers can be heated and enriched with CO 2 providing environmental control during manipulation as well as during incubation (Fig. 1D). In addition to the standard temperature, humidity and CO 2 controls, the incubators are fitted with O 2 sensors to facilitate culture in reduced O 2. All environmental sensors are linked to an alarm and recording system. Thus, each workstation was designed to provide a self- contained controlled physiological environment for manipulation and incubation of gametes and embryos. Given the technical complexity of IVF and ICSI procedures, ergonomic considerations were paramount. Two prototype workstations were manufactured and tested extensively prior to manufacture of the complete interlinked system. Following initial trials the design was modified to improve operator access to the incubator compart- ments. After a period of approximately 2 weeks in which operators became accustomed to working in the enclosed environment, it was found to be entirely feasible to perform all procedures, including intracytoplasmic sperm injection (ICSI) and preimplan- tation genetic diagnosis (PGD), within the new system. The manufacture and installation of the system was controlled via an agreed validation master plan. Installation of the complete system was followed by a period of extensive testing for compliance with design specifications, and validation by an independent contractor. Stability of environmental conditions Our primary aim in developing the enclosed chain of workstations was to prevent fluctuations in temperature and pH during open manipulation of oocytes and embryos. We therefore compared stability of temperature and pH in the open and enclosed systems using conditions designed to simulate open manipulations of embryos and gametes in our laboratory. This is most likely due to the overlay of mineral oil causing a reduced rate of CO 2 loss from the culture medium. However, following replacement in the incuba- tor, the pH of the culture medium did not begin to drop for 15 minutes and failed to return to 7.31 during the 90 minute period of measurement (Fig. 2C). Thus, using a conventional system, the increase in pH although modest, remains elevated for a prolonged period following replacement in a CO 2 -controlled incubator. Carbon filters were included in the design to protect against VOCs. Using a VOC meter with a detection limit of 0.1 ppm, we were unable to detect VOCs within the workstations or incubators. Background lab readings were also below the detection limit of the VOC meter. This indicates that under normal working conditions, VOCs are not a major concern in our laboratory environment. However, we believe that the carbon filters are important for protecting against the unforeseen events in which VOCs generated in the external environment might enter the laboratory air supply. Furthermore, carbon filtration of the re-circulated air in the enclosed system was deemed necessary to remove any internally generated VOCs.The incubators act as pass-through hatches linking adjacent workstations. ( B ) Schematic diagram showing the layout of 2 enclosed workstations connected by the double-doored incubation chambers. ( C ) Image showing the main features of an enclosed workstation with a built-in stereomicroscope and incubators. There are separate hatches to allow introduction of consumables and removal of waste. ( D ) Image shows the control panel for setting the temperature of the incubator, workstation and hotplate and the CO 2 in the incubator and workstation.Blastocyst formation was determined by the appear- ance of a well defined trophectoderm layer surrounding a fluid filled blastocoel cavity and an inner cell mass (ICM) (Fig. 3A). We found that the overall proportion of embryos developing to the blastocyst stage by day 7 was 30 in the open system compared Figure 2. Comparison of temperature and pH stability between the enclosed workstations and conventional open-fronted cabinets. ( A ) A pre-equilibrated organ culture dish was removed from the incubator and placed on the viewing area of either the enclosed workstation (purple) or Class II hood (blue). In each case 3 independent experiments were conducted. ( B ) A pre-equilibrated organ culture dish was removed from the incubator and placed on the viewing area of either the enclosed workstation (purple) or Class II cabinet (blue). There was a gradual increase in pH of the media in the Class II cabinet over the time course of the experiment.All embryos included in the above comparison were cultured in an atmosphere of 7 CO 2, however, the O 2 tension in the incubation chambers of the enclosed system was 5 compared Figure 3. Comparison of development to the blastocyst stage between the enclosed workstations and conventional open-fronted cabinets. ( A ) Images show examples of early, expanded and hatched human blastocysts indicating the trophectoderm and the ICM. ( B ) Development of embryos to the blastocyst stage was compared between the open and enclosed systems. A significantly higher proportion of embryos developed to the blastocyst stage in the enclosed system (P, 0.05). ( C ) Three point moving average of development to the blastocyst stage in consecutive cohorts of twenty embryos. The two dark bars represent the intermediate period when the cohorts of 20 consisted of embryos cultured in the open and enclosed systems. ( D ) The proportions of blastocysts graded as early (black), expanded (grey) and hatched (white) on day 6. A significantly higher proportion of blastocysts had undergone hatching by day 6 after culture in the enclosed system compared to the open system. There was a corresponding reduction in the proportion of early blastocysts in the enclosed system (P, 0.05). ( E ) Nuclear counts were performed to compare the total number of cells contained in blastocysts cultured to day 6 or 7 in the open system or enclosed system. The cell count of embryos cultured in the enclosed system was significantly higher on day 6 (128.3 6 38.6 vs 92.1 6 43.7; P, 0.05) and day 7 (167.7 6 69.7 vs 106.7 6 48.8; P, 0.01) compared with those cultured in the open system.Analysis of blastocysts in which nuclear staining was performed indicated that those cultured in the enclosed system contained significantly (P, 0.05; Fig. 4B) more cells on Day 6 compared with those cultured in the open system. Taken together these findings indicate that the increased blastocyst formation and cell number of embryos cultured in the enclosed system was not solely a consequence of culturing embryos in reduced O 2 tension. In view of the constraints associated with working with human embryos, we performed a series of controlled experiments to test the effect of the enclosed system on the development of mouse embryos to the blastocyst stage. Comparison of the proportions of embryos developing to the blastocyst stage showed significantly higher development in the enclosed system (P, 0.05; Figure 4C), confirming our findings with human embryos. However, while the mouse blastocyst cell counts were slightly higher in the enclosed system (Fig. 4D), the difference was not significant. This may be due to the fact that the mouse embryos were cultured only from the 1-cell stage and therefore had reduced exposure to ambient conditions compared with human embryos. Clinical outcome data To determine whether improved development in vitro was matched by increased embryo viability in vivo, we compared clinical outcomes following transfer to the uterus of embryos cultured either in the open or enclosed systems. To control for Figure 4. Effect of the enclosed system and 21 O 2 tension on blastocyst development of embryos. ( A ) Development of human embryos to the blastocyst stage was significantly lower in the open system compared with the enclosed system when incubated in 21 O 2;P, 0.05. ( B ) Nuclear counts were performed to compare the total number of cells contained in human blastocysts cultured to day 6 in the open system or enclosed system in 21 O 2. The cell count of human embryos cultured in the enclosed system in 21 O 2 was significantly higher compared with those cultured in the open system (P, 0.05). ( C ) A significantly higher proportion of mouse embryos developed from the 1 cell to the blastocyst stage in the enclosed system compared with the open system (P, 0.05). Both sets of embryos were cultured in 21 O 2.( D ) Nuclear counts were performed to compare the total number of cells contained in mouse blastocysts cultured in the open system or enclosed system in 21 O 2.We tested this possibility by fitting a single logistic regression model to the data combined over all three groups, taking female age into account. We then tested for trends against time in the residuals between observed and model-fitted clinical pregnancy rate. If there are no time trends then a cumulative sum of residuals will have no systematic pattern. Periods of negative slope indicate periods of poor performance compared with the overall rate, and periods of positive slope indicate the opposite. The results showed consistently worse than overall average performance throughout the periods covered by Groups 1 and 2 and consistently better for Group 3 (Fig. 6). The start of Group 3 coincides precisely with a highly significant (p, 0.001) change from a negative to positive slope. We therefore conclude that the improved treatment outcome was due to the beneficial effects of the enclosed system rather than to a general improvement in the outcome of assisted conception procedures. Discussion Here we describe a novel enclosed isolator-based system designed to minimise the risk of environmental insult during laboratory processing for assisted conception treatment. We find that it is entirely feasible to conduct laboratory assisted conception procedures within this system. Measurements of the physicochem- ical parameters within the critical work area showed that the system is capable of maintaining stable temperature and pH. Analysis of the data before and after the introduction of the enclosed system indicates that it promotes increased development to the blastocyst in vitro. Moreover human embryos developing to the blastocyst stage showed accelerated development and contained significantly more cells than those cultured in the conventional system. Consistent with this we found that following transfer to the uterus, embryos created and cultured in the enclosed system resulted in a significantly higher implantation and clinical pregnancy rate compared with those cultured in the open system. Our primary goal in developing the enclosed system was to minimize exposure of oocytes and embryos to pH and temper- ature fluctuations. In contrast to the enclosed system, pH measurements of bicarbonate-buffered culture medium with an oil overlay showed an upward drift to 7.55 6 0.02 in the open system followed by a prolonged (. 90 min) recovery period upon return to the CO 2 -enriched incubator. However, the impact of this on embryo viability is unclear. Following the introduction of the enclosed system, we dispensed with the use of MOPS-buffered medium for oocyte retrieval. This may have contributed to the beneficial effects of the enclosed system. Conversely, our use of MOPS- buffered medium in ambient CO 2 for sperm injection in the enclosed system, may have contributed to lower magnitude of the increase in implantation rates for ICSI compared with IVF treatment. Our ongoing work seeks to validate bicarbonate buffered-media for sperm injection and to determine whether the outcome of ICSI treatment can be further improved by eliminating MOPS-buffered medium from our ICSI procedures. Table 1. Comparison of patient profiles. Groups 1 and 2 were processed in the open system but in different laboratories. Group 3 was processed in the enclosed system.Our main dataset on human blastocyst development (presented in Fig. 3) was obtained during a period when the enclosed system incubators were set to run at 5 O 2. Subsequent data obtained from human embryos indicate that the improvement in blastocyst formation and cell number was maintained when embryos were cultured in a gas phase of 21 O 2 in the enclosed system. Under these conditions we also observed increased mouse blastocyst formation. However, in contrast to human, the mouse blastocysts cell counts were equivalent between the two systems. This may be because the mouse embryos were fertilized in vivo and were therefore subjected to fewer manipulations. Thus, the detrimental effect of the open system was likely reduced compared with human embryos. This implies that events occurring during the first cell cycle are particularly susceptible to effects of in vitro manipulation, and that this has a negative impact on subsequent development. Thus, meaningful evaluation of enclosed culture systems should encompass the entire period of development from insemination onwards. Figure 5. Effect of the enclosed system on IVF and ICSI clinical outcome measures. Embryos from Groups 1 and 2 patients were cultured in the conventional open-fronted system but in different laboratories. Group 3 were conducted in the enclosed system. Cumulative sum of residuals between observed clinical pregnancy rate and that fitted by an overall logistic regression model, plotted against time. During periods of stable performance the plot should have a constant slope: positive when performance is consistently better than average, negative for the opposite. The vertical lines mark the group boundaries. Regions with positive slopes indicate performance consistently better than average, regions with negative slopes indicate the opposite.However, we found that following a two week period of becoming accustomed to working in the isolators, all procedures could be performed with ease. The ergonomic design of the system also took account of the need for all internal surfaces to be accessible for routine cleaning. The system can also be fumigated if required. Among the additional advantages of the enclosed system, we find that the controlled environment is excellent for training inexperienced operators as it protects against potential negative effects of the longer manipulation times. In addition, the separate incubation compartments offer increased protection against cross- contamination between patients, which is especially important in cases of high-risk infections such as Hepatitis B and HIV. Furthermore, by providing a physical barrier between the operator and the critical workarea, the enclosed system offers increased operator protection during treatment of high-risk cases. It also offers the possibility of reduced risk of operator DNA contami- nation during embryo biopsy for pre-implantation diagnosis (PGD). We believe that this is crucial for preventing a build- up of VOCs within the enclosed work area. In conclusion, we have shown that it is possible to perform laboratory processing for IVF and ICSI within an enclosed isolator-based environment and that this is associated with improved embryo quality in vitro and increased implantation following transfer to the uterus. By offering protection against external influences such as chemical pollutants, and protection from temperature and pH fluctuations, the system facilitates consistent and reproducible outcomes in assisted conception treatments. Supporting Information Figure S1 Analysis of the relationship of nuclear counts between operators and imaging systems. Analysis of the relationship between nuclear counts performed by different operators and imaging systems was carried out to determine the accuracy of cell counting. A correlation was carried out to determine whether there were any differences between counts. As there was no difference, the limits of agreement were deduced (Bland and Altman 1999) by calculating 2 times the standard deviation (SD) of the differences between the 2 counts. Scatter diagrams of the difference between the counts (y-axis) and the average of 2 of the counts (x-axis) were plotted to visualize the data. ( A ) Brightfield image of a human blastocyst on left. Image on right shows z-projection of a nuclear stained human blastocyst. Nuclear staining was carried out using Hoescht 33528. The cell number was determined from single planes which have been used here to produce the z-projection. 50 m m scale bar indicated. ( B ) Analysis of the relationship of nuclear counts between operators. The difference between counts from two different researchers plotted against the average of the counts, the dotted lines represent 2 times the SD. ( C ) Analysis of the relationship of nuclear counts between imaging systems. The difference between counts using 2 different imaging systems plotted against the average of the counts, the dotted lines represent 2 6 the SD. Ref: Bland JM, Altman DG.