PROCEDURES FOR IMMUNE ASSESSMENT
(FALL 1997)
Overview
I) Recruitment Call;
If R agrees to biomedical assessment, screen for allergies to influenza Vaccine eggs or contact lens solution.
If R doesn't want to do biomedical assessment, recruit for interview Only.
2) Forward R's name to nurse for scheduling.
3) Nurse schedules time for vaccination and calls in to office
4) On day of appointment call to confirm visit and check fur febrility.
5) At R's home
IMPORTANT; Consent forms must be signed the first time one of our staff meets with respondent, the nurse or interviewer must know who has first contact!!
a) Verify that R is NOT febrile
b) Have R sign consent form
c) Collect PREvaccination blood sample (see Blood Protocol)
d) Administer vaccine (see Vaccination Protocol)
e) Schedule tentative time 1st POST-vaccination blood draw (approximately 5 weeks post vaccination, can be 1 week either side)
6) At end of day return to the office;
a) Return leftover vaccine to cold room
b) Finish processing blood samples
c) Report that vaccination was completed and leave consent form in Gayle's mail slot
7) POST-vaccination blood draws
a) 1st draw is 4-6 weeks post vaccination (targetted at 5 weeks post)
-Phlebotomist confirm appointment or reschedule and report to office
-Collect sample (see Blood Protocol)
-Finish processing sample at the office
-Remind R that we will be in touch to collect and sample in about 6 months
-Report blood draw completed to office
b) 2nd draw is approximately 6 months post vaccination (follow procedures at 7a above)
Supplies
The following supplies are needed for BLOOD DRAWS:
- Penrose tourniquets
- alcohol
- needles (21 and 22 gauge) for vacutainers
- butterflies (needle for hard draws)
- red top vacutainer tubes (2 per draw) ganze
- bleach
-band-aid strips
- holders for vacutainer needles
- red disposable containers for needles
-biohazard bags
-Latex gloves (medium and large)
- disposable absorbant pad
- test tube racks
- test tube labels for vacutainer tubes
-box that holds 81 specimens
-cooler and coldpack (-20 degrees)
- closed end pipettes
-cryo vials (5 per draw)
1) Organize supplies and label tubes: Labels should include ID#, Date, and time of blood draw (e.g. PRE, POST1, or POST2)
2) At R's home or blood draw site
a) Collect 20 ml (2 - 10ml tubes) of blood via standard venipuncture of the antecubital vein using asceptic technique.
b) Put blood samples in test tube rack til clotted, then place in cooler for transport back to office.
c) of needle in portable sharps disposal container and put other disposable materials into red biohazard bags.
3) Prepping blood samples for analysis at lab (room 2635 MSC)
a) Label 5 cryo-vial using same format as for vacutainer tubes (see #1 above)
b) Centrifuge blood for 10-15 minutes minutes at 3 (when using lab centrifuge),
c) If clot is adherent use wooden stick/swab to "rim" clot
c) Using a closed-end Pipette draw off 2 ml aliquots of sera per sample into each cryo-vial (sera from the 2 vacutainers for a single individual can be mixed).
d) 2 cryo vials of sera, for each respondent, will be stored separately Place vials into appropriate storage boxes, mark location on grid map and freeze- Samples will be stored according to timing of draw (e.g. all pre-samples will be stored together' all postl samples etc.).
e) Place all disposable materials in red biohazard bags.
Supplies
Vaccination
- alcohol
- gauze
- band-aid spots
- gloves
- Influenza vaccine in syringe with needle for single use
- Hepatitis A vaccine in syringe with needle for single use Emergency Kit ( Nurse should always carry 2 ER kits) Diphenhydramine 50 mg/ml Epinephrine 1:1000 ratio lmg/ml
1 - 26 gauge (5/8 inch) sub-q needle
3 - lcc Tuberculin syringes
1 - 25 gauge (5/8 inch) needle
1 - 23 gauge (1 1/2 inch) needle
1 - 19 gauge (1 1/2 inch) needle
Alcohol pads
Emergency contact info- Dr. X, MD, PhD
Flu Vaccine Procedure
1) Organize supplies
2) At R's home or vaccination site:
a) Check for febrility
b) Vaccination procedure
-Instruct R to relax their arm by placing it in their lap.
-Using the influenza vaccine syringe, insert the needle straight into the deltoid muscle centered between the shoulder and the axillae and the posterior and anterior of the arm. Repeat with the Hepatitis A vaccine in an area 2 cm distal.
-Monitor the R for at least 15 minutes after vaccination to assess for allergic reaction
Note: The vaccine can be kept at room temperature, but efforts should be made to keep it stored in cooler.
In Case of Emergency- Shortness of breath, dizziness, loss of consciousness
1) Lay subject down, elevate legs above heart
2)Epinephrine 1:1000 (1 mg/ml)
Subcutaneous or intramuscular
0.2 ml, wait 15 min, if not improved -->
0.3 ml, wait 15 min, if not improved -->
0.5 ml, if not improved --> call ambulance (911)
Maximum= 1mg = 1ml
3) If shortness of breath, resolves then give Benadryl, 50mg per oral one dose and then non-emergency transportation to physician or ER for further evaluation.
NOTE: In the event the subjects ate reluctant to receive their vaccines, they should be told that UW studies show that only 1% of subjects experience a fulll range of symptoms following their shot. That range of symptoms includes fever, chills, etc. Moreover, the flu shot given in our study is a relatively new version, and thus is quite pure. That means that the odds are infinitesimal that the flu shot will actually make the subject "sick." Of course, roughly 70% of subjects experience some tenderness or soreness at the site of the influenza vaccine, but the experience of being unable to lift one's arm, etc. is very very rare. There are almost no side effects from the Hepatitis A vaccine. Both shots are given in one arm to allow sleeping on the other side.
Overview
1) Call R to schedules time for vaccination, record date and time of appointment on Vaccination/Blood Draw Appointment Sheet. Schedule of appointments for a given week must be turned into office by Friday of the previous week. This information is critical for coordinating lab personnel responsible for processing blood once it is received in the office.
NOTE: when scheduling appointments keep in mind that the sample has to be returned to the lab for processing so that it can be frozen within 24 hours.
2) Approximately 24 hours prior to appointment call to confirm visit and check for febrility.
3) At R's home:
IMPORTANT; Consent forms must be signed the first time one of our staff meets with respondent.
a) Verify that R is NOT febrile
b) Have R sign appropriate consent form (if you are the first person to have contact with subject)
c) Collect PRE-vaccination blood sample (see Blood Protocol)
d) Administer influenza and hepatitis A vaccines (see Vaccination Protocol)
e) Schedule tentative times for the POST-vaccination blood draws according to the following schedule:
Post Draw 1 - 13-16 days post vaccination (approximately 2 weeks)
Post Draw 2 - 28-34 days post vaccination (at least 4 weeks)
f) If R desires, complete and initial Vaccination confirmation form.
4) IMPORTANT: BLOOD SAMPLES MUST BE RETURNED TO THE LAB SO THEY CAN BE PROCESSED AND FROZEN AT -70 DEGREES WITHIN 24 HOURS OF DRAW.
At end of a trip completed within 24 hours of drawing first blood sample, return to the office and;
a) Return leftover vaccine to cold room
b) Put blood samples on the Life Histories counter in the lab.
c) Report that vaccination was completed and leave consent form in Gayle's mail slot
along with date and time of follow up blood draws
If your trip involves an overnight stay and you will not be able to return the blood samples to the lab within 24 hours of being drawn, YOU MUST NOTIFY GAYLE OR THE LAB STAFF IN ADVANCE, and then Fed-Ex the blood using the kit and directions provided.
7) POST-vaccination blood draws
-Confirm appointment or reschedule and verify/report to office
-Collect sample (see Blood Protocol)
-Return blood sample to lab (see above).
-After 2nd followup remind R that we will be in touch to collect and sample in about 6 months
-Report blood draw completed to office
Supplies
The following supplies are needed for BLOOD DRAWS:
- Penrose tourniquets
- alcohol
- needles (21 and 22 gauge) for vacutainers
- butterflies (needle for hard draws)
- large vacutainer tubes with blue speckled caps (2 per draw) gauze
- bleach
-band-aid strips
- holders for vacutainer needles
- red disposable containers for needles
-biohazard bags
-Latex gloves (medium and large)
- disposable absorbant pad
- test tube racks
- test tube labels for vacutainer tubes
-box that holds 81 specimens
-cooler and coldpack (-20 degrees)
- closed end pipettes
1) Organize supplies and label tubes: Labels should include ID#, Date, and time of blood
draw (e.g. PRE, POST1, POST2, or POST3)
2) At R's home or blood draw site
a) Collect 16 ml (2 - 8ml tubes) of blood via standard venipuncture of the antecubital vein using asceptic technique.
b) Put blood samples in test tube rack in box for transport back to office.
c) Put needle in portable sharps disposal container and put other disposable materials into red biohazard bags.
Supplies
Vaccination
- alcohol
- gauze
- band-aid spots
- gloves
- Influenza vaccine in syringe with needle for single use
- Hepatitis A vaccine in syringe with needle for single use
Emergency Kit ( Nurse should always carry 2 ER kits)
Diphenhydramine (Benadryl) 25 mg/ml
Epinephrine 1:1000 ratio lmg/ml
1 - 26 gauge (5/8 inch) sub-q needle
3 - lcc Tuberculin syringes
1 - 25 gauge (5/8 inch) needle
1 - 23 gauge (1 1/2 inch) needle
1 - 19 gauge (1 1/2 inch) needle
Alcohol pads
Emergency contact info- Daniel Muller, MD, PhD
Influenza/Hepatitis A Vaccine Procedure
1) Organize supplies
2) At R's home or vaccination site:
a) Check for febrility
b) Vaccination procedure
-Instruct R to relax their arm by placing it in their lap.
-Using the influenza vaccine syringe, insert the needle straight into the deltoid muscle centered between the shoulder and the axillae and the posterior and anterior of the arm. Repeat with the Hepatitis A vaccine in an area 2 cm distal or proximal.
-Monitor the R for at least 15 minutes after vaccination to assess for allergic reaction
Note: The vaccine can be kept at room temperature, but efforts should be made to keep it stored in cooler.
In Case of Emergency- Shortness of breath, dizziness, loss of consciousness
1) Lay subject down, elevate legs above heart
2)Epinephrine 1:1000 (1 mg/ml)
Subcutaneous or intramuscular
0.2 ml, wait 15 min, if not improved -->
0.3 ml, wait 15 min, if not improved -->
0.5 ml, if not improved --> call ambulance (911)
Maximum= 1mg = 1ml
3) If shortness of breath, resolves then give Benadryl, 50mg per oral one dose and then non-emergency transportation to physician or ER for further evaluation.
NOTE: In the event the subjects ate reluctant to receive their vaccines, they should be told that UW studies show that only 1% of subjects experience a fulll range of symptoms following their shot. That range of symptoms includes fever, chills, etc. Moreover, the flu shot given in our study is a relatively new version, and thus is quite pure. That means that the odds are infinitesimal that the flu shot will actually make the subject "sick." Of course, roughly 70% of subjects experience some tenderness or soreness at the site of the influenza vaccine, but the experience of being unable to lift one's arm, etc. is very very rare. There are almost no side effects from the Hepatitis A vaccine. Both shots are given in one arm to allow sleeping on the other side.
Processing and Freezing Human CPT Blood Samples:
Supplies:
15 ml Conical Tubes (2625, TC supplies)
2 ml Cryovials (2619, at nurse's workbench)
Sterile PBS (2625, refrigerator, cold room)
RPMI Freezing Media (2625, refrigerator)
Freezing Cooler (2619, -70 freezer)
5 & 10 ml Sterile Pipettes (2625, TC supplies)
Bleach (2625, under sink)
Gloves (2619, nurse's workbench, 2625, near centrifuge)
Solutions:
Freezing Media:
50 ml RPMI-1640 (cold room)
1.7 ml P/S (10,000U/10mg) (2619, in blue -20 freezer)
1.7 ml L-Glut (200mM) (2619, in blue -20 freezer)
16 ml FCS (20% final conc) (2619, in blue -20 freezer)
12 ml DMSO (15% final conc) (2625, near laminar flow hood)
PBS:
0.21 gm KH2PO4 (2619, located on chemical shelf)
9.0 gm NaCl (2619, located on chemical shelf)
0.726 gm Na2HPO4.7H2O (2619, located on chemical shelf)
H20 to 1 Liter (pH 7.2), autoclaved
CPT Procedure (black & blue speckled tubes):
Be sure to wear gloves during this procedure!! Put all used pipettes in the orange biohazard bag, and all glass CPT tubes in the red sharps container.
1. Invert CPT tubes 8-10 times before spinning.
2. Balance centrifuge buckets and CPT tubes, using 15 ml conical tubes for balance.
3. Spin at 2700 rpm, room temperature, 20 minutes. Spin without the brake on (light off).
4. Inspect the tubes to see if the gradient has formed properly. (It is possible to respin if the gradient has not formed)
5. In a Laminar Flow hood remove the stopper from each of the two tubes for a subject.
6. Using a 5 ml pipette carefully remove the plasma (be sure not to disturb the lymphocyte layer) from both tubes and distribute in 5 cryovials total.
7. Label cryovials with the subject #, date, draw # (i.e. pre, post I, post II), and plasma.
8. Carefully wash the cells out of the CPT tube using about 5 mls PBS for each tube.
9. Transfer the PBS/Cells from both CPT tubes to a 15 ml conical tube (be sure tubes are labeled with subject ID#'s) .
10. Spin the cells down at 1700 rpm, room temperature, 15 minutes, with the brake on (light on).
11. Pour off the supernant into a waste container containing bleach, thump cells to resuspend, and add 10 mls PBS to tube.
12. Respin as in #10.
13. Pour off the supernant, thump cells, and resuspend the cell pellet in 5 mls of Freezing Media.
14. Aliquot the cells in 5 cryovial, 1ml each. Label tubes as before, except label these tubes cells.
15. Freeze plasma in -70o freezer in the appropriate tower and box, record location in GREEN notebook. The plasma is being stored in Tower K Box 7. Continue storing samples in boxes 8, 9 etc as needed.
16. Place cells in cryofreezer and store at -70o overnight.
17. Transfer cells to Liquid Nitrogen, and record cell location in BLACK notebook. These cells are being stored in Tower 4 Box 4. Continue storing in boxes 5, 6, etc as needed.
18. Pour the bleach waste solution down the drain, and flush with plenty of water.
(NOTE: Accuspin System Histopaque tubes can be stored at room temperature or refrigerated, if the tubes have been refrigerated they MUST be brought to room temperature before using, protected from light)
1. Check that the 'frit' (disk) is above the liquid. If it is not, centrifuge at 2100 RPM for 30 seconds.
2. Label Tubes with subjects ID#.
3. In a Laminar Flow hood, remove the blue stoppers from each of the 4 tubes for each subject and freely pour 15-30 ml fresh blood finto the upper chamber of each prefilled Accuspin System tube.
4. Centrifuge at 2100 RPM for 10 minutes at 18-26 degrees C (room temperature).
5. Inspect the tubes to see if the gradient has formed properly, (It is possible to respin if the gradient has not formed) .
6. Using a 5 ml pipette carefully remove the plasma, being careful not to disturb the lymphocyte layer (see attached diagram of separated layers) and distribute in 5 cryovials total.
If there is excess plasma, remove to within 0.5 cm of the opaque interface containing mononuclear cells.
7. Label cryovials with the subject #, date, draw # (i.e. pre, post I, post II), and plasma.
8. Transfer cells to a clean 15 ml conical tube and add 5 ml PBS.
10. Spin the cells down at 1700 rpm, room temperature, 15 minutes, with the brake on (light on).
11. Pour off the supernant into a waste container containing bleach, thump cells to resuspend, and add 10 mls PBS to tube.
12. Respin as in #10.
13. Pour off the supernant, thump cells, and resuspend the cell pellet in 5 mls of Freezing Media.
14. Aliquot the cells in 5 cryovial, 1ml each. Label tubes as before, except label these tubes cells.
15. Freeze plasma in -70o freezer in the appropriate tower and box, record location in GREEN notebook. The plasma is being stored in Tower K Box 7. Continue storing samples in boxes 8, 9 etc as needed.
16. Place cells in cryofreezer and store at -70o overnight.
17. Transfer cells to Liquid Nitrogen, and record cell location in BLACK notebook. These cells are being stored in Tower 4 Box 4. Continue storing in boxes 5, 6, etc as needed.
18. Pour the bleach waste solution down the drain, and flush with plenty of water.