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 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 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)