Working Group on Biological Measures - Summary of Findings

Biological Measures Working Group:

Biochemical markers of Alzheimer's disease:  Standardization and validation

Mony de Leon NYU and Pankaj Mehta IBR

 Acquisition and Storage of Samples

1. Sampling protocols should be uniform across collaborating sites. This includes time of day, and fasted state. Samples should be efficiently handled, placed on ice or centrifuged promptly. Records will be kept documenting these steps.

2. Proper collection into tubes with preservatives (e.g. EDTA) or whole blood for DNA extraction should be specified and tubes suitable for long-term storage (polypropylene) need to be considered. Vendors should be specified.

3. Size of the aliquots for storage is important and should reflect the needs of the individual lab assays. Ideally these would be standard.

4. Attention should be given to a system of labeling and tracking the samples collected, stored, and distributed.

Assay Standards

1. Standard ELISA assay protocols (with assured antibody uniformity) should be detailed. Discuss analytic recovery of the protocols.

2. Other assay techniques e.g. HPLC, mass spectrometry etc also need to be detailed along with supporting quality control procedures including equipment calibration.

3. Explicit information is required regarding the source and history of standards, eg pooled CSF or synthetics.

4. Information on the effect of freeze-thaw cycles is important and should be considered for any assay where this is relevant.

5. The accuracy and confidentiality of patient and lab data must be assured.

6. Entry procedures and checking of errors must be detailed.

7. Intra-assay and inter-assay reliability indices should be provided and procedures for handling deviations specified.

8. Clinical (CLIA certified) labs should be used to get cell counts and albumin levels to insure that CSF samples are not contaminated by blood.

9. It should be stated if samples will be assayed individually or in batch.

10. Indicate the throughput of the individual labs.

11. Specify if more than one lab will provide values for a specific analyte.

General Collection Procedures of body fluids:  Blood, CSF, Urine

Blood: For the collection of plasma, use purple top tubes with EDTA as the anticoagulant.  To collect serum use red top tubes without any anticoagulant.  Aliquot plasma or serum in polypropylene tubes (not glass tubes), and store at -80 C.

CSF: To avoid getting values that depend on the gradient effect, it may be necessary to analyze a specified portion of the CSF.  For example, serial sampling of CSF in five portions, and test each portion to rule out the gradient effect.  CSF chemistry will determine if the spinal tape is traumatic.  Aliquot CSF and store at -80 C.

Urine: Collect 24-hour urine, if possible.  The second choice is to collect the first void in the morning.  Urinalyses, rule out urinary tract infection and express the data in terms of creatinine levels. 

Store urine at –80 C .

STANDARD CLINICAL LABORATORY ASSESSMENTS

Analyzed by CLIA-certified clinical laboratory

To be performed annually, using age- and gender-specific reference ranges

POSSIBLE ADDITIONAL SCREENING  LABORATORY ASSESSMENTS

Screen Only

Hepatitis C Surface Antigen

Hepatitis B Surface Antigen

HIV

Urine drug screen (alcohol, cocaine, narcotics, benzodiazepines, THC, barbiturates, amphetamines)

Working group findings on specifications for samples for future (unspecified) analytes

Matrix: Blood--serum fraction and whole cell fraction

Anticoagulant/Preservative: None (coagulated blood) (red top tube)

Volume: 10 ml (2 X 5ml tubes)

Cost: (From Fisher catalog) $28.50/100 5ml tubes

Method: Allow blood to clot for 30 min.  Remove clot, and freeze at -80 o C (whole cell fraction).  (Could aliquot whole cell fraction into 200 ml samples, if desired, by diluting with an equal volume of PBS/EDTA). Spin supernatant for 10 min at 3,500 g .  Remove supernatant, aliquot into 200 ml samples, and freeze at -80 o C (serum fraction).  Discard pellet.

Matrix: Blood--plasma fraction and whole cell fraction

Anticoagulant/Preservative: Na-EDTA (lavender top tube), or Na-citrate (light blue top tube), or heparin-Na (green top tube).

Volume: 10 ml (2 X 4.5 or 5ml tubes)

Cost: (From Fisher catalog) $30.43/100 (lavender top), $32.01/100 (light blue top), $46.20/100 (green top)

Method: Spin for 10 min at 3,500 g .  Remove supernatant, aliquot into 200 ml samples, and freeze at -80 o C (plasma fraction).  Freeze pellet at -80 o C (whole cell fraction).  (Could aliquot whole cell fraction into 200 ml samples, if desired, by diluting with an equal volume of PBS/EDTA).

Matrix: CSF

Anticoagulant/Preservative: None

Volume: 10-15ml

Cost: Baxter LP tray costs $28.00/tap (catalog price)

Method: Keep sample cold and process as soon as possible.  Spin for 10 min at 3,500 g .  Aliquot supernatant as 0.5 or 1.0 ml samples, and freeze at -80 o C.  Pellet could be frozen for white cells, if desired.