EBMT ADWP/IWP Consensus Guidelines for Preparation and Storage of Biologic Samples In

APPENDIX

EBMT ADWP/IWP consensus guidelines for preparation and storage of biologic samples in patients with autoimmune diseases treated with stem cell transplantation

Studies with biological samples in patients receiving stem cell transplantation often show contradictory results due to the clinical heterogeneity of the respective autoimmune disease and the large variety in the methods that are used to prepare and store biological samples.With the guidelines provided here, we aim to define “good laboratory practice” in handling of biological samples and to harmonizemethods for preparation and storage of biologic samples.It is emphasized that these guidelines are not strict rules how biological samples must be prepared, rather they are suggestions showing how contributing centers process biological samples in their laboratories. Where possible, laboratories should participate in internal and external quality assurance schemes for flow cytometry, molecular genetics and immunological markers. National and/or EU regulations should be followed in relation to biobanking of cells and tissues.

Contents

1. Collection and storageof serum samples
2. Preparation and storage of peripheral blood mononuclear cells (PBMC)
3. DNA preparation
4. RNA preparation
5. Culture of dermal fibroblasts
6. Collection and processing bronchoalveolar lavage (BAL)
7. Collection and processing cerebrospinal fluid (CSF)

1. Collection and storage of serum samples

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Tobias Alexander and Attilio Bondanza

(1)Perform standard phlebotomy with the system generally used for the preservation of serum in the local clinic. Do not use any anticoagulant.

(2)Preserve at least 10ml peripheral blood from each patient (equivalent to ca. 3-4 ml serum). Store at 4°C until centrifuging.

(3)Within 12 hours, centrifuge tubes at 400g for 10min at room temperature.

(4)Retrievethe clear serum with a 500µlpipette and aliquot it in cryotubes with 250µlserum each.

(5)Label each aliquot with the individual patient code number and date of phlebotomy.

(6)Store immediately at -20° (better -70°, if available) until further use.

1. Preparation and storage of peripheral blood mononuclear cells (PBMCs)

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Tobias Alexander and Attilio Bondanza

(1)Perform standard phlebotomy with the system generally used for blood-cell counts in the clinic. Use heparin or EDTA as anticoagulants. EDTA usually gives higher recovery yields.

(2)Obtainat least 20ml peripheral blood from each patient. Store at room temperature until centrifuging.

(3)Within 12 hours, isolate PBMCs by Ficoll-Hypaque density gradient centrifugation. Wash extensively with saline or PBS containing a source of proteins (bovine serum or albumin).

(4) Retrieve the cells, count and aliquot them in cryovials with complete RPMI medium containing 20% fetal calf serum (FCS) and 10% Dimethyl sulfoxide(DMSO).

(5)Label each aliquot with the individual patient code number and date of phlebotomy. Prepareat least 3 aliquots of 1x106 cells each and at least 3 aliquots of 5x106 cells each.

(6)Store immediately in liquid nitrogen until further use.

1. DNA preparation

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Tobias Alexander and Attilio Bondanza

(1)Perform standard phlebotomy with the system generally used for blood-cell counts in the clinic. Use heparin or EDTA as anticoagulants. EDTA usually gives higher recovery yields.

(2)Obtain at least 20ml peripheral blood from each patient. Store at 4°C until centrifuging.

(3)Within 12 hours, isolate PBMCs by Ficoll-Hypaque density gradient centrifugation. Wash twice with saline or PBS containing a source of proteins (bovine serum or albumin).

(4)Retrieve the cells, count them and make aliquots of 1x106 cells in 500 µl tubes.Aspirate the supernatant and centrifuge the tubes at 1800g for 3minutes.

(5)Label each aliquot with the individual patient code number and date of phlebotomy.

(6)Store immediately the pellets at -70°C until further use.

1. RNA Preparation

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Tobias Alexander and Attilio Bondanza

(1)Perform standard phlebotomy with the system generally used for blood-cell counts in the clinic. Use heparin or EDTA as anticoagulants. EDTA usually gives higher recovery yields.

(2)Obtainat least 20ml peripheral blood from each patient. Store at 4°C until centrifuging.

(3)Within 12 hours, isolate PBMCs by Ficoll-Hypaque density gradient centrifugation. Wash extensively with saline or PBS containing a source of proteins (bovine serum or albumin)

(4)Retrieve the cells, count them and make aliquots of 3x106 cells in 500 µltubes. Aspirate the supernatant, re-suspend in 500µlRNA-Buffer (RLT buffer).

(5)Label each aliquot with the individual patient code number and date of phlebotomy.

(6)Store immediately at -70°C until further use

1. Culture of dermal fibroblasts

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Thomas Daikeler and Dominique Farge

(1)Chop skin biopsy into 1mm pieces and carefully spread the skin fragments on the surface of a sterile 25ml tissue culture flask.

(2)Leave the cultures in standard incubator for 14-21 days inculture medium with antibiotic supplements.

(3)Trypsinise and split the cells, after primary culture, the subsequent growth can be in antibiotic free medium.

(4)For most experiments, fibroblasts should be used between second and fifth passage. Alternatively early passage cells can be frozen and used in future experiments.

(5)For freezing of fibroblast cultures, remove media and discard, trypsinise and wash 2-3mls TE, then trypsinise with 7-8ml TE for 5 minutes, add 8 ml 10% FBS and centrifuge, add 1ml freezing media(14mls 10% FBS, 2mls DMSO, 4mls neat FBS) to pellet per cryo-tube. Usually split T75 into 2/3 cryo tubes. 1ml to each tube. Put samples in -80°C freezer before transferring to liquid nitrogen.

1. Collection and processing bronchoalveolar lavage (BAL)

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Thomas Daikeler and Dominique Farge

A)BAL procedure

It is recommended to perform BAL in the right middle lobe or linguala. Another site may be lavaged additionally, based on individual pattern of lung involvement seen in computed tomography scans. For BAL, 200 ml of sterile, pyrogen free saline solution (isotonic 0.9% NaCl) should be installed at room temperature and recovered in 4 aliquots of 50 ml each.

B)Processing BAL samples

(1)Volumes of all aliquots recovered should be measured and fluid recovery (expressed as a percentage of fluid instilled) should be established.

(2)It is recommended not to pool the first aliquot with the fluid recovered subsequently. The last three aliquots should be pooled and mixed thoroughly, and then filtered through sterile gauze to remove mucous particles.

(3)The total cell number should be counted in a small aliquot of the pooled BAL fluid. Former washing procedure may result in a considerable loss of cells. Cells should be separated from the fluid by centrifugation at low speed (500 g – 800 g) for 10 to 15 minutes as soon as possible.

(4)Cytocentrifuge preparations are recommended as a routine procedure to obtain differential cell counts. They may be prepared using pooled BAL fluid or after the cells are resuspended in buffered medium without addition of the serum. For differential cell counts cytospins should be stained with May-Grunwald-Giemsa and at least 500 cells should be counted (using random field counting method). It is recommended that differential cell counts are made every time by two independent technicians.

(5)For BAL supernatant storage,BAL supernatant should be separated from cells as soon as possible and frozen at -80°C. It is recommended to divide the supernatant before freezing into 1.0-2.0 ml aliquots

(6)For BAL cell storage,cell pellets should be resuspended in RPMI-1640 medium with 20% FCS and 10% DMSO at a concentration of 1-10 x 106 cells/ml. If molecular studies are planned DMSO should be avoided. The cells should be rapidly frozen and stored in liquid nitrogen.

1. Collection and processing cerebrospinal fluid (CSF)

Harmonized protocol from Charité (University Medicine, Berlin) and S. Raffaele Hospital Scientific Institute, Milano)

Prepared by Paolo Muraro

A)Acquisition of CSF from lumbar puncture (LP)

(1)Atraumatic spinal needle LP kits should be used to minimize risk of post-LP headache. Draw with a sterile polypropylene syringe, or allow flow under gravity.

(2)Collect 5-ml fractions and place in place in ice bath, with a maximum of 25 mL per time point. Send the first 2 mL for clinical laboratory analysis. It is important to match fractions when comparing across patients, as protein concentration varies depending on the draw volume (Blennow, 1993).

(3)Patients should rest in a recumbent position for 1 h post-LP receive liberal fluid intake, and avoid exertion for 24-48 h to minimize risk of headache.

B)Processing and storage of CSF

(1)Transport CSF on ice and process immediately after collection as significant cell lysis contamination will occur within 1 hour.

(2)Collect CSF samples into low protein binding polypropylene tubes (e.g., Eppendorf brand LoBind® tubes). Avoid polystyrene and glass tubes, which will result in significant protein loss.

(3)Centrifuge CSF at 2,000 g and 4˚C for 10 min. Draw off supernatant and place in new, low binding tube, and document volume of fluid collected. Centrifugation will only remove RBCs, not serum derived proteins. Samples found to contain > 10 RBCs/µL CSF may be inappropriate for proteomic analysis.

(4)Resuspend cell pellet in appropriate preservation agent according to objectives/protocol, e.g. RNALater.

(5)Additives or preservatives may be combined with CSF depending on experimental objectives. Protease and phosphatase inhibitors are recommended.

(6)Snap freeze samples in liquid nitrogen, and store at or below -80˚C to minimize proteolytic breakdown. Avoid repeated freeze and thawing cycles and storage at -20˚C.

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