O'bryant alz forum presentation 6.13.13

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O'bryant alz forum presentation 6.13.13

  1. 1. Sid O’Bryant, Ph.D.Associate ProfessorInternal Medicine, Division of GeriatricsSid.O’Bryant@unthsc.eduStandardization ofMethods for Studiesof Blood BasedBiomarkers ofAlzheimer’s Disease
  2. 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 tothe 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 literatureBecan-McBride 1999, CLSI H3-A6 – Procedures for collection ofdiagnostic blood: 6th ed; Plebani 1997; Henriksen et al in press -BBIG
  3. 3. Are pre-analytic variablesimportant?• Blood collection devices can impact results and can be major source of preanalyticerror – 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) canfalsely elevate blood metal levels• Type of collection tube• Heparin binds nonspecifically to proteins impacting separation and mass spectrometric detection ofpeptides; 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 variabilityBowen 2009; Apple et al 2007; Weber 2006;Aziz 2003
  4. 4. STandards for Alzheimer’s Research inBlood 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 WaterfrontHotel (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 – TheFuture of Blood-Based Biomarkers for Alzheimer’s Disease)
  5. 5. Cohort Fasting Needle Serum Plasma Centrifuge Speed/TimeProcessing Time Storage Method(Immediate)Storage Method(Long-term)TARCC No Serum-separatingtubes (tigertops)BD EDTA tubes(purple top)1300xg/10 minRoom temperature2 hours; sampleprocessing startedwithin 1hr of drawFrozen on wet iceif unable to befrozenimmediately;placed in -20º or -80 until sent tobiobank. Aliquotedat processing-80 ºADNI Yes 21G (plain red tops) EDTA tubes 3000rpm/15 min2 hours; sampleprocessing startedwithin one hourfollowing blood drawFrozen on dry icefor 20 minutesthen shippedsame day; uponarrival samplesare thawed,realiquoted andplaced in -80º-80ºAIBL Yes 21G Sarstedt s-monovetteserum-gel(brown top)a) Sarstedt s-monovette lithiumheparin (green top)b) Sarstedt s-monovette EDTAtubes (with PGEadded) (purple top)Serum: 1800xg/15 min20 ºPlasma:Step1: 200g/10 min20ºStep2: 800g/15 min20ºTotal processingmust be completedwithin 3.5 hours;Blood processingmust be startedwithin 20 minutes ofafter blood drawFrozenimmediately at -80ºLiquid nitrogenADCS EDTA tubes 3000rpm/10 min-80º -80º
  6. 6. Cohort Fasting Needle Serum Plasma Centrifuge Speed/TimeProcessing Time Storage Method(Immediate)Storage Method(Long-term)NACC Ideal but notrequiredSmall gaugeneedleEDTA or HeparintubesQuick freeze withdry ice, thenplaced in -80ºfreezer-80ºHABLE Yes 21G Serum-separatingtubes (tigertops)BD EDTA tubes(purple top)1300xg/10 minRoom temperature2 hours; sampleprocessing startedwithin 1hr of draw-80º ; aliquoted atprocessing-80ºDIAN Yes Butterfly Red Top PlaintubesEDTA tubes(lavender top)2000xg/15 minRoom temperatureProcess serum afterallowing to sit inroom temperaturefor 30 minFlash Freeze ondry ice at site,shipped, thawed,then realiquoted,re-frozen andstored at -80º-80ºLiquid Nitrogen(N=4)ACS Yes 22G EDTA tubes 2000xg/15 min4ºWithin 1-2 hours ofcollectionReceive sampleson wet ice, flashfreeze on dry icethen placed in -80ºfreezer/liquidNitrogen-84ºLiquid Nitrogen (forn=4)Araclon Yes recommended EDTA tubes 2500xg/15 min-80º -80ºKing’sDementiaStudiesYes 21G or 23G(depending onvein size)Serum Tube(gold top)EDTA tubes (purpletop)3000rpm/8 minTime from blooddraw to samplesfreezing kept within2-3hours-80º -80º
  7. 7. What guidelines are alreadyavailable?• Clinical and Laboratory Standards Institute (CLSI)• H3-A6 –Procedures for the collection of diagnostic bloodspecimens by venipuncture approved standard – 6th ed• Needle size – 19-23g (21g is standard – Bowen 2009)• Selecting vein• Order of blood draw:1. Blood Culture Tube2. 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. 8. What guidelines are alreadyavailable?• Clinical and Laboratory Standards Institute (CLSI)• H18-A4 –Procedures for the handling and processing of blood specimens forcommon laboratory tests – 4th ed• Serum/plasma should be physically separated from contact with cellsASAP (<2hrs)• Serum• Clotted before centrifugation (30-60m) if patient is not on anticoagulanttherapy• Relative centrifugal force (RCF; g-force) should be utilized over revolutionsper minute• Biobank procedures (Table 2)• Centrifugation at 2000g for 10min• Do not store aliquots from serum/plasma that have been in contact wcells for >2hrs
  9. 9. Standards / Best Practices• Review of ongoing protocols and currently availableguidelines 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

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