1. Sid O’Bryant, Ph.D.
Associate Professor
Internal Medicine, Division of Geriatrics
Sid.O’Bryant@unthsc.edu
Standardization of
Methods for Studies
of Blood Based
Biomarkers of
Alzheimer’s Disease

2. Concerns for Study Design
• 46% of laboratory error from preanalytic phase of testing
• Laboratory testing errors are distributed:
• 7% analytic, 93% pre- & postanalytic
• Lack of standardization of sample collection is a barrier to
the field
• What is the concern?
• Inconsistent findings in the literature
• Failure to replicate findings
• Different protocols across ongoing large-scale studies
• To date, largely a failure to learn from the CSF literature
Becan-McBride 1999, CLSI H3-A6 – Procedures for collection of
diagnostic blood: 6th ed; Plebani 1997; Henriksen et al in press -
BBIG

3. Are pre-analytic variables
important?
• Blood collection devices can impact results and can be major source of preanalytic
error – tube stoppers, stopper lubricants, tube walls, surfactants, clot activators,
needles
• Problem encountered with needles is hemolysis; silicone lubricants can interfere with antigen-
antibody reaction in immunoassays; needle components (chromium, iron, manganese, nickel) can
falsely elevate blood metal levels
• Type of collection tube
• Heparin binds nonspecifically to proteins impacting separation and mass spectrometric detection of
peptides; heparin has lead to falsely low albumin levels; can impact fibrinogen levels
• Order of blood draw
• Can have carryover of tube additives; order of draw is different for microcollection tubes
• Impact of preanalytic processing varies according to the markers
• BNP – standards available – EDTA whole blood or plasma only acceptable choice, etc.
• CD40 ligand – plasma only and preanalytic conditions “are critical”
• CRP – more robust against preanalytic variability
Bowen 2009; Apple et al 2007; Weber 2006;
Aziz 2003

4. STandards for Alzheimer’s Research in
Blood biomarkers (STAR-B)
• Blood-Based Biomarker Interest Group – Professional Interest Area
(PIA) of the Alzheimer’s Association
• 1st meeting in Boston at AAIC
• Wednesday, July 17, 2013 from 1-2pm at the Westin Boston Waterfront
Hotel (425 Summer St, Boston, MA 02210, USA), Otis meeting room
• STAR-B is part of this PIA
• Review of ongoing protocols for pre-analytic processing
• Review of currently available guidelines
• White Paper in Process
• Recent publication accepted from BBBIG in Alz & Dementia (Henriksen,
O’Bryant, Hampel, Trojanowski, Montine, Jeromin et al in press – The
Future of Blood-Based Biomarkers for Alzheimer’s Disease)

5. Cohort Fasting Needle Serum Plasma Centrifuge Speed/
Time
Processing Time Storage Method
(Immediate)
Storage Method
(Long-term)
TARCC No Serum-
separating
tubes (tiger
tops)
BD EDTA tubes
(purple top)
1300xg/
10 min
Room temperature
2 hours; sample
processing started
within 1hr of draw
Frozen on wet ice
if unable to be
frozen
immediately;
placed in -20º or -
80 until sent to
biobank. Aliquoted
at processing
-80 º
ADNI Yes 21G (plain red tops) EDTA tubes 3000rpm/
15 min
2 hours; sample
processing started
within one hour
following blood draw
Frozen on dry ice
for 20 minutes
then shipped
same day; upon
arrival samples
are thawed,
realiquoted and
placed in -80º
-80º
AIBL Yes 21G Sarstedt s-
monovette
serum-gel
(brown top)
a) Sarstedt s-
monovette lithium
heparin (green top)
b) Sarstedt s-
monovette EDTA
tubes (with PGE
added) (purple top)
Serum: 1800xg/
15 min
20 º
Plasma:
Step1: 200g/
10 min
20º
Step2: 800g/
15 min
20º
Total processing
must be completed
within 3.5 hours;
Blood processing
must be started
within 20 minutes of
after blood draw
Frozen
immediately at -
80º
Liquid nitrogen
ADCS EDTA tubes 3000rpm/
10 min
-80º -80º

6. Cohort Fasting Needle Serum Plasma Centrifuge Speed/
Time
Processing Time Storage Method
(Immediate)
Storage Method
(Long-term)
NACC Ideal but not
required
Small gauge
needle
EDTA or Heparin
tubes
Quick freeze with
dry ice, then
placed in -80º
freezer
-80º
HABLE Yes 21G Serum-
separating
tubes (tiger
tops)
BD EDTA tubes
(purple top)
1300xg/
10 min
Room temperature
2 hours; sample
processing started
within 1hr of draw
-80º ; aliquoted at
processing
-80º
DIAN Yes Butterfly Red Top Plain
tubes
EDTA tubes
(lavender top)
2000xg/
15 min
Room temperature
Process serum after
allowing to sit in
room temperature
for 30 min
Flash Freeze on
dry ice at site,
shipped, thawed,
then realiquoted,
re-frozen and
stored at -80º
-80º
Liquid Nitrogen
(N=4)
ACS Yes 22G EDTA tubes 2000xg/
15 min
4º
Within 1-2 hours of
collection
Receive samples
on wet ice, flash
freeze on dry ice
then placed in -80º
freezer/liquid
Nitrogen
-84º
Liquid Nitrogen (for
n=4)
Araclon Yes recommended EDTA tubes 2500xg/
15 min
-80º -80º
King’s
Dementia
Studies
Yes 21G or 23G
(depending on
vein size)
Serum Tube
(gold top)
EDTA tubes (purple
top)
3000rpm/
8 min
Time from blood
draw to samples
freezing kept within
2-3hours
-80º -80º

7. What guidelines are already
available?
• Clinical and Laboratory Standards Institute (CLSI)
• H3-A6 –Procedures for the collection of diagnostic blood
specimens by venipuncture approved standard – 6th ed
• Needle size – 19-23g (21g is standard – Bowen 2009)
• Selecting vein
• Order of blood draw:
1. Blood Culture Tube
2. Coagulation Tube (blue top)
3. Serum w or w/o clot activator or gel (red top)
4. Heparin w or w/o gel separator (green top)
5. EDTA w or w/o separator (lavender top)
6. Glycolytic inhibitor (gray top)

8. What guidelines are already
available?
• Clinical and Laboratory Standards Institute (CLSI)
• H18-A4 –Procedures for the handling and processing of blood specimens for
common laboratory tests – 4th ed
• Serum/plasma should be physically separated from contact with cells
ASAP (<2hrs)
• Serum
• Clotted before centrifugation (30-60m) if patient is not on anticoagulant
therapy
• Relative centrifugal force (RCF; g-force) should be utilized over revolutions
per minute
• Biobank procedures (Table 2)
• Centrifugation at 2000g for 10min
• Do not store aliquots from serum/plasma that have been in contact w
cells for >2hrs

9. Standards / Best Practices
• Review of ongoing protocols and currently available
guidelines can be pulled together as a starting point:
• Fasting
• 21g needle
• 2hrs processing time (started w/in 1hr of collection)
• Order of blood draw
• 2000g centrifuge x 10min
• EDTA plasma