1.1. |
Aerosol Management System (AMS) |
| 1.1.1. |
While sorting viable infectious material (infected cells) under high pressure the following guidelines must be followed for proper aerosol containment. All sort operators in this section must be trained and certified by the VRC flow core facility prior to any cell sorting operations. |
| 1.1.2. |
The AMS must be on and functioning according to the manufacturer guidelines. Appendix A shows the aerosol flow and the locations of the vacuum gauge and monitor. The vacuum monitor should be set to 20% and the vacuum gauge must read between 1.0 and 1.5 inches of HoH. If it is outside of this range, check to make sure that the filter is seated correctly. If gauge does not come into range replace HEPA filter unit and tubing. |
| 1.1.3. |
The sheath waste tank must contain enough hypochlorite to provide a final concentration of 10% when filled (1L beach to a final 10L waste collected). If full empty before starting cell sorting procedures. |
| 1.1.4. |
The Accudrop camera system must be functioning normally according to the manufacturer guidelines. This camera system is used to monitor the sort stream and alerts the operator to potential increased aerosols. In this situation the operator can correct the sort stream and reduce aerosol contamination. The FACSAria is equipped with a Sweet Spot, which is used during all sorting operations. This device can detect stream drifts and possible clogs and automatically shutdown the stream. |
1.2. |
Measurement of Containment and Tolerances |
| 1.2.1. |
The AMS must be tested under simulated worse case failure mode. In this mode the instrument is set to 70 psi and 50,000 particles/second, with the waste catcher blocked to create large aerosol. This is done by covering waste catcher with a cut off piece of sample tubing (Appendix B). |
| 1.2.2. |
Add glass slide to the AeroTech concentrator and place directly on top of sort chamber (Appendix C and Appendix D). Adjust the pressure to the AeroTech concentrator to 55 LPM (liters per minute). Close door to deflection plates but do not install tube holders. The main sort chamber should also be closed and the AeroTech device should be placed directly on top of this chamber door. |
| 1.2.3. |
Turn AMS on (20%) and check for proper vacuum function (1.0-1.5 inches of HOH). |
| 1.2.4. |
Place Glo-Germ particles onto the sample station and adjust either the particle concentration or the flow rate to achieve a particle rate of 50,000 particles per second. Note: When creating aerosols, which could contain Glo-Germ particles, it is recommended the operator wear a 0.2µm particle mask. |
| 1.2.5. |
Begin acquiring Glo-Germ particles and allow collection for 5 minutes. (see Appendix E, Figure A). |
| 1.2.6. |
Turn off vacuum to AeroTech concentrator and remove slide from inside. Put in a fresh slide and continue collecting aerosol with the AMS turned off for another 5 minutes (positive control). Stop sample acquisition and remove waste catcher plastic sleeve. |
| 1.2.7. |
Examine glass slides for bright green fluorescence using a fluorescent microscope equipped with a FITC filter (520-640nm, see Appendix E, Figure B). Glo-Germ particles will form a pattern as directed by the holes found in the plate cover (see Appendix F). Therefore, when present, the location of the particles can be found in this pattern.
|
| 1.2.8. |
Scan the entire slide on 10x and count all Glo-Germ particles. The positive control slide can be used as a reference to distinguish between fluorescent debris and actual Glo-Germ particles. Record all data (Appendix G). |
| 1.2.9. |
Acceptable Tolerance: No Glo-Germ particles detected after 5 minutes of active air sampling in front of sort chamber door with no tube holder in place and the AMS turned on. The positive control slide must contain greater than 100 particles after 5 minutes of active air sampling with the AMS turned off and no tube holder in place. |
1.3. |
Infectious sort procedure. |
| 1.3.1. |
The flow cytometer must pass all tolerances of aerosol containment as described in this procedure. If these tolerances are not met, infectious cell sorting is not permitted. |
| 1.3.2. |
The operator must wear personnel protection as outlined in section 4.0. If the operator is not protected as described in this section, infectious cell sorting is not permitted. |
| 1.3.3 |
A warning sign must be posted on the outside of the flow cytometer lab (see Appendix H) and the room is limited to two individuals during the sort procedure.
|
| 1.3.4. |
Turn on and verify that the AMS is working correctly. This device must have a vacuum pressure of 1.0-1.5 SCFM. If this tolerance is not met, infectious cell sorting is not permitted. |
| 1.3.5. |
Close all barriers around the sort chamber as described in section 5.2. If this is not done, infectious cell sorting is not permitted. |
| 1.3.6. |
While under a laminar flow hood, all samples for sorting must be filtered through a 100um mesh prior to sorting. This reduces the potential for clogging and decreases the risk of creating aerosols. |
| 1.3.7. |
Remove sample from the laminar flow hood and place onto the sample station. Start sort and monitor the sort performance using the Accudrop camera. If during the sort the stream is deflected (due in part to a clogged flow cell tip), the sort is designed to stop automatically and block the sort tubes. The sort will not restart until the operator has cleared the clog. Use the following procedure to remove a clog from the Cytometer. |
|
1.3.7.1. Remove the sample from the sample chamber. |
|
1.3.7.2. Turn stream off and then on again to see if drop delay and stream returns to normal pattern. If clog is not fixed select the nozzle flush procedure and use Coulter Clenz. |
|
1.3.7.3. If the nozzle can not be cleared, remove and sonicated for 2-5 minutes. Clean nozzle with 10% beta-dyne for 2 minutes before placing in sonicator. |
|
1.3.7.4. Sorting can be resumed after the flow cell tip is cleared. |
| 1.3.8. |
After sort is completed, samples can be removed immediately after sample tube is unloaded from the sample station. |
| 1.3.9 |
When sort is finished, run 10% betadyne through the sample lines for 2 minutes and spray and wipe down the inside and outside of sort chamber with 70% ETOH. |
2.0 |
Personnel Safety Equipment (Primary Barriers) |
2.1 |
Dupuy HEPA filter suit and respiratory system (Appendix I): This system is used before any potentially infectious sort where infectious aerosols maybe formed during this procedure. |
| 2.1.1. |
Connect battery to helmet and turn on to ensure the helmet fan is working correctly and turn OFF. |
| 2.1.2. |
Attach disposable boots. |
| 2.1.3. |
Place helmet inside face shield of suit by attaching the Velcro strips. |
| 2.1.4. |
Check the HEPA filter is located over the helmet motor. |
| 2.1.5. |
Attach battery pack to belt on the inside of the respiratory suit and turn on battery. |
| 2.1.6. |
Pull respiratory suit over the top on head after adjusting the sleeves. |
| 2.1.7. |
Attach gloves and check for proper airflow. The airflow should filter from the top of the helmet and exit out of the bottom. |
2.2. |
Use of gloves and glasses or face shields. |
| 2.2.1. |
Double gloves are worn at all times within the BLS-3 facility. |
| 2.2.2. |
Before leaving the flow cytometry laboratory and before entering the anteroom area, either decontaminate gloves with 70% ethanol or replace with a clean pair of gloves. |
| 2.2.3. |
Face shielding or laboratory glasses are worn if the potential for spills or splashing could occur. |
| 2.2.4. |
Remove and dispose of all protective equipment after entering the anteroom. |
| 2.2.5. |
Remove contaminated gown first followed by the removal of gloves. |
| 2.2.6. |
Wash hands before leaving the anteroom. |
2.3. |
Laboratory Protective Clothing |
| 2.3.1. |
Disposable laboratory gowns are worn at all times in the BSL-3 facility. |
| 2.3.2. |
It is recommended that disposable gowns must be closed in the front and opened in the back when performing procedures, which have the potential of creating spills or splashing. |
| 2.3.3. |
When indicated respiratory suits must be worn such as described in this section especially in procedures, which can produce infectious aerosols. |
| 2.3.4. |
Sleeves and or disposable boots should be worn when potential spills or splashing could occur. |
3.0 |
Laboratory Facilities (Secondary Barriers) |
| 3.0.1. |
Doors to the laboratory are closed at all times. |
| 3.0.2. |
If special procedures are preformed warning signs are posted on the laboratory door identifying the special hazard, special protection and contact person to notify in case of an emergency. |
| 3.0.3. |
The proper use of both class II and class III biological hoods must be used when handling infectious agents (see Appendix J, references; 9,10 and 11). |
| 3.0.4. |
HEPA filters in biological hoods are checked and cleaned annually. |
| 3.0.5. |
Biological hoods must be free of obstructions, which might compromise the airflow. |
4.0. |
Bio-safety References (see Appendix J). |
|
back to top |