Author: Joe Grove – some of the collection protocol may be out of date.
Protocol for collection of patient blood and isolation of IgG.
Collection of patient blood:
3 consent forms (see attached) are required per patient, 1 for patient, 1 for
doctor and 1 for researcher. Consult patient records and identify HCV +ve
(HIV –ve) patients. Approach the consultant due to see the patient and ask
them to gain consent and organise blood to be taken, aim to collect 20ml in
red top vacutainers.
Handling of HCV infected blood:
All isolations to be carried out in tissue culture lamina No. 4.
Protective gear (lab-coat, disposable plastic apron, gloves) must be worn
while handling blood samples.
Place all equipment/samples on a plastic tray (easier to clean in case of
spillage). Only use disposable Pasteur pipettes, do NOT use needles or other
sharp materials, or pipettes that are also used for routine cell culture work.
Pipettes and all other plasticware (including vacutainers) contaminated with
blood MUST be soaked in 10% Trigene solution for 24hrs prior to disposal in
a sealable waste pot.
Discard all material into yellow sharps bin (including closed vacutainers and
paper towels); bin to be incinerated after use (do NOT leave in the hood for
other people to use, even it is only half full!).
Clean hood with 10% Trigene and 70% Ethanol after use.
Immediately clean up spillages with paper towel and 10%Trigene.
Do NOT pool any (clotted) blood from vacutainers into falcon tubes!
Isolation of serum
Collect in Red Top Vacutainers (+clot activator)
Spin vacutainers 10 min at 3000 rpm (brake 4) at 4-6 C (in sealed buckets!)
Carefully take off supernatant using a disposable Pasteur pipette and transfer
to Falcon tubes, make sure not to disturb the red blood cells (if you
accidentally mix serum/plasma and red blood cells, re-spin supernatant for 5
min and transfer to fresh falcon tube)
Heat inactivate sera by incubation at 56C for 1 hour. Remove other objects
and glass thermometer from water bath prior to doing so and clean water bath
with 70% ethanol after use.
Separate sera in to 2.5 ml aliquots and store in locked -80. – tubes labelled
with “biohazard” sticker and stored in re-sealable plastic bag.
IgG isolation from patient sera.
Using designated HCV+ve or HCV-ve 4ml protein G column stored in cold
room in IgG isolation box.
1. Filter samples, to do this; dilute 1/4 in PBS, spin @ 2000rpm for
10mins and 0.2uM syringe filter in to fresh tube.
2. Wash column with 10 mls sterile PBS
3. Pass sample through column to capture IgG.
4. Wash column with 10mls PBS.
5. Elute IgG with 12mls 0.1M Glycine (pH2.7) discarding the first 2ml,
collecting the remainder.
6. pH test the elute and neutralise with 1M tris pH 9.0 (~650ul/10ml)
7. Wash column with 10mls PBS.
8. Pass 10mls PBS 0.1% NaN3 through column before storage.
9. Dialyse IgG over night against PBS.
10. 0.2uM filter sterilise IgG.
11. Quantify protein concentration of a 1/100 dilution of IgG using a
spectrophotometer @ 280nm, use following formula.
(X/1.35) X 100 = [IgG] (mg/ml)
Any equipment that has come into contact with patient sera must be soaked in
10% Trigene for 24hrs and then placed into a yellow sharps bin which must
be disposed of immediately, as above.
CONSENT FORM
Study Title: The Immune System in Chronic Liver Diseases
1. I confirm that I have read and understand the information sheet dated
20th August 2007 for the above study.
2. I understand that my blood donation for this research is voluntary and
that I am free to withdraw participation at any time without my medical
care or legal rights being affected.
3. I understand that isolated blood will be stored for a limited time until the
experiments are competed.
4. I agree for 25ml of blood (about 2 full table spoons) to be taken for this
study.
5. I agree to take part in the above study.
Name of patient
Date
Signature
Name of person taking Consent
(if different from Principal Investigator)
Date
Signature
Principal Investigator
Date
Signature
1 copy for patient, 1 for researcher, 1 to be kept with hospital notes
Information sheet 3 (for patients with chronic liver disease) 20 August 07
The Immune System in Chronic Liver Diseases
Please keep this information sheet for future reference
1. Introduction to the Research.
This study is being organised and funded by the Liver Unit in Birmingham. We
are interested in how your white blood cells which as part of the immune system
have evolved to fight infections might also be involved in the development of liver
diseases.
2. What is the research study about?
We believe that the immune system plays an important role in the normal liver by
fighting bacteria and viruses to prevent them from infecting the liver. However,
under some circumstances white blood cells can be misdirected to damage
normal tissues leading to chronic liver damage. In our studies we will isolate
white cells from blood taken form your vein. We can then grow these cells in the
laboratory and study how they might lead to liver damage. For some experiments
it is necessary to store the isolated cells and/or small pieces of tissue for
subsequent experiments.
3. Why have I been chosen?
Because you are chronically infected with Hepatitis C virus.
4. What will I have to do?
If you agree to take part in our research study we would like to take a blood
sample from you (about 2 tablespoons of blood, 25 ml). This blood sample is
taken in addition to the routine samples taken but it can be done when you are
having blood removed for your routine tests. The removal of this small amount of
blood will not in any way effect your condition as your body can rapidly replace
the cells removed.
5. What are the benefits?
There is no direct benefit to you from taking part although the information we
gain may help us to understand liver diseases and develop new treatments in the
future.
6. What are the risks?
There are no additional risks for you.
7. What are the alternatives?
This study does not involve any change in treatment and you are free to not take
part in it.
8. What if I do not want to take part?
Your participation in the study is entirely voluntary and will not affect your
present or subsequent care in any way.
If you decline to be involved or
subsequently withdraw from the study this will also have no effect on your
present or future treatment.
9. What happens to the information?
Your participation in this study will be confidential.
No personal information
which could identify you will leave the hospital. Any samples that are stored will
not be labeled with you personal information.
10. Are there any restrictions on what I might eat or do?
There are no restrictions on what you can eat or do apart from those that relate
to your routine treatment.
11. Confidentiality agreement.
Your participation in this study will be confidential.
identify you will leave the hospital.
No information which could
12. Who else is taking part?
Other patients who are chronically infected with Hepatitis C.
13. What if something goes wrong?
The study does not involve any change in your treatment so there are no risks for
you.
14. What happens at the end of the research study?
The results of this research will be kept and published so that other research
groups can benefit from the results gained. Cells and tissue samples that are not
longer needed will be destroyed.
15. Is my doctor being paid for including me in the study?
No, neither your doctor nor the hospital receives any money.
16. What if I have more questions or do not understand something?
If you have any questions please ask your doctor, or contact the Liver Research
Laboratories using the number below. Should you wish to see the overall results
of the study, or the publication, which may result, please ask your study doctor.
17. What happens now if I decide to take part?
If you decide to take part, we will ask you to take more blood than we need to
treat you (about 2 tablespoons full) and will use the blood for our research.
18. What happens if I change my mind during the research study?
If you change your mind at any time, do let us know and we will destroy any
samples we have already taken.
19. This study has been reviewed by the South Birmingham Local
Research Ethics Committee.
20. Contact name and number
If you need any further information this can be obtained from Professor David
Adams or Dr. Mathis Heydtmann who can be contacted via the hospital
switchboard (0121-472-1311).