Standard Operating Procedures (SOPs) - Guidelines for biological sample collection

CSF samples should be centrifuged at least 30 minutes but maximum 120 minutes after collection.

LUMBER PUNCTURE SOP:

The lumbar puncture should be performed ideally between 8am and 11am.
Local guidelines / routines for antiseptic cleansing and anaesthesia are to be followed.
Position the patient in a reclining or sitting position with back arched.
Use a Spinal Needle Quincke Type Point 0.7 x 75 mm (75 – 90 mm) for the LP.
Insert the needle through the L3/L4 or L4/L5 interspace.
In case of a bleeding at the puncture site. The first 1ml of CSF should be discarded and this should be recorded.
Collect 10 mls of CSF from each subject. Use a POLYPROPYLENE TUBE ONLY for CSF collection (i.e. 15ml polypropylene tube)
Mix the CSF gently by turning the tube up side down for 3-4 times (cap on).
Centrifuge the CSF in the polypropylene tube at 2000g for 10 minutes at +4°C, to eliminate cells and debris
Transfer the centrifuged CSF into another sterile polypropylene tube being careful to avoid disturbing the pellet of cells and debris at the end of the tube.
Aliquot CSF in 1ml aliquots, into sterile 1.5ml PRELABELED POLYPROPYLENE CRYOVIALS with a screw cap.
The patient should be allowed to rest for 30 minutes – 1 hour after the LP procedure – with medical supervision.

In the local biobank freeze:

As the SERUM tubes require a 20-30 minute standing before processing, begin with the PLASMA processing.

Plasma samples should be centrifuged after at least 30 minutes but maximum 120 minutes after blood collection.
Serum samples should be centrifuged after at least 60 minutes but maximum 120 minutes after collection
Centrifuge the sample at 2000G (min. 1800G, max. 2200G), for 10 min, room temperature.
Time between taking blood and freezing must be kept to a minimum (max. 2 hours). In case freezing is planned more than 2 hours after collection store the sample at +4 °C until freezing.

PLASMA : 2x 10ml EDTA TUBES

Collect 20mls of blood in EDTA tubes.
For APOE genotyping, whole blood without centrifuging is required. Transfer 1ml of whole blood from one of the EDTA tubes directly into 1.5ml PRELABELED 1.5ml polypropylene cryovial. Store sample immediately at -80º. Do not snap freeze (ie. do not dip in liquid nitrogen to freeze).
Taking the remaining blood volume in the EDTA tubes and balance in the centrifuge and spin for 5 minutes at 1,380 rcf.
Remove tubes from centrifuge and place on ice without shaking/inverting the tubes.
Using the pipette, draw off the supernatant (upper layer) taking care not to disturb the interface and transfer to a polypropylene tube on ice.
Transfer 1ml aliquots of plasma into PRELABELED 1.5ml polypropylene cryovials.

SERUM: 2 X 10ml SST TUBES

Collect 15mls of blood in SST tubes (with silica plug).
A 5ml sample of the serum will be used for the paired blood test for albumin CSF- to- serum ratio.

Let the serum SST tubes (with silica plug) stand at room temperature for 20-30 minutes to clot.
Balance the tubes in a centrifuge and spin for 5 minutes at 1,380 rcf.
Remove tubes from centrifuge and place on ice without shaking/inverting the tubes.
Using a pipette, draw off the supernatant (upper layer) taking care not to disturb the interface and transfer to a polypropylene tube on ice.
Transfer 1ml aliquots of serum into PRELABELED 1.5ml polypropylene cryovials.
Freeze the cryovials of serum at -80º. Do not snap freeze (ie. do not dip in liquid nitrogen to freeze).
The time taken from blood draw to placing the serum samples in the -80°C freezer should not exceed 35 to 45 minutes. This includes the 20-30 minutes of standing time required for the sample to clot.

In the local biobank freeze:

Serum and plasma at -80°C. Do not snap freeze (ie. do not dip in liquid nitrogen to freeze.
Whole blood for DNA lower or equal to -20°C