Transcript PARTNERS PrEP STUDY

IMMUNOLOGY LABORATORY PBMC
ISOLATION SOP
by Kizza D Martin Ssemambo
Procedure for processing PBMCs for
Tenofovir levels
Materials
• 8ml blue top-CTP with Sodium Citrate (2 tubes
per draw)
• 5oml conical tube
• 2ml cryovial
• Sterile normal (0.9%)
• Ice cold 70% methanol (prepare fresh just prior to
start of pharmacokinetic blood processing
procedure)
Preparation of Ice Cold 70% Methanol
Take 7 parts of methanol and mix with 3 parts
of distilled water. For example,7ml of
methanol and mix with 3ml of distilled water.
Or 70ml of methanol and mix well with 30ml
of distilled water. Store in -70c freezer until
needed
PBMC –Processing and Storing Requirements
1.
2.
3.
Invert the CPT tubes (these should be two 8ml CPT
tubes drawn) gently to mix the anticoagulant
thoroughly. Keep upright at room temperature
until centrifugation remix (invert several times)
CPT tubes before centrifugation . To maximize cell
yield, the time from blood draw to centrifugation
and lysis should be 9 ½ hours or less. Record draw
time on the tracking sheet. (Appendix A).
Spin CPT tubes at room temperature (18-25c)in a
horizontal rotor centrifuge for 25 minutes at 1700
RCF. Record centrifugation start time on the
tracking sheet.(appendix A)
Gently invert the tube, without disturbing the
underlying gel, to suspend the PBMCs in the
plasma and transfer the cells from both CPT tubes
with a pipette to a 50ml conical tube
4. Add sterile isotonic (normal) saline (0.9% to bring total
volume to 30ml.record the exact time saline is added
to cells in conical tubes and the exact volume of the
plasma/saline in the PBMC lysate preparation record
log (appendix A)
5. Remove a 0.5ml aliquot and send for cell counting ( cell
count should be performed within 1 hour of step
5,addding saline). Do not wait for the results of the cell
count, immediately proceed through the next steps.
When the result of the cell count is received, record
total number of cells counted in cells/ml and % viability
manually using hematocytomer and trypan blue or by
an automated method that can give % viability.
6. Centrifuge the conical tube for 15minutes at 00 RFC to
pellet the cells
7. Aspirate as much supernatant as possible
without disturbing the cell pellet. Carefully, as to
not disrupt the cell pellet, invert the tube on a
piece of absorbent tissue to remove as much
plasma/saline as possible from the tube.
8. Add 1.0ml of well-mixed, ice cold 70% methanol
solution to the 50ml conical tube containing the
PBMC pellet. Vortex lightly to lysate the cells and
suspend the PBMC contents. (prepare fresh lysing
solution on each serial PK day) this step should be
completed in 9.5 hours from collection.
9. Completely transfer the contents of the 50ml
conical tube to a 2.0ml conical cryovial using a
pipette.
10. freezing-do one of the following:
• Use a mixture of dry ice and methanol (follow
local chemical safety requirements) to flash
freeze the specimens.
• Use liquid nitrogen to flash freeze the specimens.
• Alternatively, place the specimens directly into
the -70c freezer.
11. Record the exact time the samples are frozen in
the PBMC lysate preparation record log (appendix
A)
Rapid isolation, washing, and extraction of PBMCs
are essential to minimize loss of cells and cellular
contents, including drug contained within.
Plasma-processing and storage
• Invert the EDTA tubes gently to mix the anticoagulant thoroughly.
Keep upright at room temperature until centrifugation. The time
from blood draw from centrifugation should be 60mins or less.
Record the exact time in the plasma preparation record log.
(appendix B)
• Spin EDTA tubes at room temperature (18c-25c) for 10mins at
800RCF. Record the exact time the tubes begin centrifugation in the
plasma preparation record log. (appendix B)
• Remove all plasma from top layer of each tube and aliquot into
1.0ml each into cryovials.
• Store at -70c. These samples will be used for determination of
plasma concentrations. Record the exact time the samples are
frozen in the plasma preparation record log. (appendix B)