This document discusses the need for standardization of pre-analytic methods for blood-based biomarker studies of Alzheimer's disease. It notes that inconsistent pre-analytic protocols across studies have led to inconsistent findings and failure to replicate results. It reviews existing guidelines from sources like CLSI on optimal procedures for blood collection and processing. These include recommendations around needle size, order of draw, centrifugation speed and time, and maximum processing time. The document promotes efforts by the STAR-B group to establish standard pre-analytic protocols by reviewing ongoing study methods and available guidelines.

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