- Proper sample collection is critical to achieving valid results. If in doubt, please contact the core.
- Depending upon the specific protocol, a volume of whole blood is usually collected in an EDTA coated tube and centrifuged for 15 minutes at 4°C.
- Use no more than 10 IU heparin per ml of blood collected. An excess can result in falsely high values in some assays.
- Samples with gross hemolysis or lipemia may yield false results.
- Plasma is preferred over serum because it is cleaner, easier to work with, and therefore gives better results.
- The sample should be aliquoted into an appropriately labeled tube (use a separate tube for each assay). For canine and human use 5 ml 12 x75 mm (e.g. Sarstedt tube #55.526) with push caps. For mouse & Luminex use 0.5 ml 30 x 7.8 mm (e.g. Sarstedt tube #72.699) microfuge tubes.
- See below for tissue prep and saliva collection procedures.
A preservative must be added to the following samples (instructions for preparing these solutions are at the bottom of this page):
- Active Ghrelin: Pefabloc SC (or HCL/PMSF)
- Catecholamines: EGTA-glutathione (if not added during sampling see FAQ)
- C-peptide: Trasylol (aprotinin)
- GLP-1: DPP-IV inhibitor
- Glucagon: Trasylol (aprotinin)
- PYY-Active (3-36): Trasylol (aprotinin) & DPP-IV inhibitor
- PYY-Total: Trasylol (aprotinin)
- Each tube should be uniquely labeled (e.g. Date, Sample ID, Investigator's name/initials, and type of assay requested).
- Sample tubes are placed in a robot for pipetting purposes. Do not use long, bulky labels which will prevent the tubes from fitting correctly into the rack. Please make your labeling as simple as possible.
- Patient labels can be denoted with a date and number. We do not need patient names. When samples are identified by name only, additional time is required to prepare the work list.
- Samples in tube boxes should be organized in the order to be assayed. Please include the iLab email submission confirmation with the samples.
- Samples should be stored at -20 to -70°C (catecholamines at -70°C). To avoid multiple freeze/thaw cycles samples must be aliquoted into separate tubes for each assay (e.g. aliquot enough plasma in one tube for insulin, another tube for glucagon, etc).
Active Ghrelin Pefabloc SC (AEBSF) solution (or HCL/PMSF as an alternative)
- The active form of ghrelin is very unstable and labile in serum/plasma due to the nature of the octanoyl group on serine-3. Samples should be processed as quickly as possible and kept on ice to retard the breakdown of active ghrelin.
- We recommend adding to Pefabloc SC (AEBSF) - Sigma Cat # 76307 to the blood upon collection.
- Care must be taken when using heparin as an anticoagulant, since excess will provide falsely high values. EDTA is recommended - use no more than 10 IU heparin per ml of blood collected.
- Specimens can be stored at 4ºC if they will be tested within 4 hours. For longer storage, specimens should be aliquot and stored at -20ºC or below. Multiple freeze/thaw cycles should be avoided.
- Avoid using samples with gross hemolysis or lipemia.
Catecholamine EGTA-Glutathione solution (if not added during sampling see FAQ)
- 4.5 g EGTA (for 50 ml solution)
- 3.0 g Glutathione (for 50 ml solution)
- The solution must be titrated with NaOH to pH of 6.0 -7.4
- Use two beakers to prepare:
- Beaker #1 - Measure EGTA and Glutathione and place in 50 ml beaker
- Beaker #2 - in a small 15 ml glass or plastic beaker, measure 5 ml of 10N NaOH solution and fill to 15 ml with di-H2O
- Beaker #1 - Bring volume of solution to 30 ml with Di-H2O (solution will be very cloudy) - place a stir bar in the solution and place on magnetic stirrer
- Place pH probe in beaker # 1 and stir solution slowly while titrating.
- Add NaOH solution from beaker #2 with a dropper while stirring. pH to 6.0-7.4
- Use a funnel to pour in to a 50 ml calibrated flask and bring up to 50 ml volume with Di-H2O.
- Pour solution into dark glass bottle and keep in refrigerator. (Must be kept in a dark place, so do not leave out on counter too long).
- Add 20 µl of EGTA-Glutathione per 1 ml of whole blood (dog/human); add 2 µl per 100 µl of whole blood (mouse/rat)
- Mix, Spin, Remove plasma for analysis
- Glutathione and EGTA are purchased from Sigma Aldrich
- Catalog numbers Glutathione G4251 and EGTA E4378
C-peptide / Glucagon Trasylol (aprotinin) solution
- Vanderbilt users can purchase trasylol from the core. Aprotinin concentrate (~200,000 KIU/ml, PentaPharm) can be ordered from Centerchem.
- Dilute with water to 20,000 KIU and of that add 50 µl per ml plasma or blood to give a final concentration of 1000 KIU per ml.
- DPP-IV inhibitors are commercially available
The core does not process tissues but the following protocols can be used:
Catecholamine Tissue Prep
This procedure has been used to measure catecholamines in liver, muscle, kidney, gut, and pancreas.
Grind a small amount of the tissue to a powder with mortars and pestles chilled in liquid nitrogen. (The tissue is freeze-clamped at the time of study and kept at -80 until grinding and processing; best if it is processed the same day or the next day, but not longer than 48 h after study.)
Chill a tube (approx 10-13 ml volume, round bottom) in liquid nitrogen, and then weigh out approx 300 mg tissue into the cold tube, scooping the powdered tissue with a metal spatula chilled in liquid nitrogen. Record exact weight of tissue.
Return cold tube to liquid nitrogen immediately after weighing; don’t ever let the tissue thaw.
Add glutathione/PCA solution (1 ml/100 mg tissue)
Glutathione/PCA solution is 5 mM glutathione in 0.4N perchloric acid
0.384 g reduced glutathione and 8.6 ml 70% PCA; bring up to 250 ml with deionized water
Homogenize well, cap and put on ice (not liq nitrogen) until all samples are done.
Centrifuge, take off clear supernatant (some tissue will float – take off only the clear fluid), put in new tube and freeze at -70°C until analysis.
Amino Acid Tissue Prep
Insitu, or as quickly as possible, collect and weigh tissue (freeze clamp in liquid nitrogen)
Use ice cold 10% SSA (5-Sulfosalicylic acid) 3X volume to powdered tissue (i.e. 1g tissue:3ml SSA) and homogenize using a polytron homogenizer
Centrifuge @3000g for 15 minutes
Decant and save the supernatant for amino acid analysis
We need >50 ul of the supernatant for analysis
We will give you the concentration of the solution expressed in µmol/L. The investigator will measure protein concentration in the pellet (from SSA/ tissue prep) and express the amino acid concentration as µmol/mg
Purine Nucleotide Tissue Prep
Insitu collected and weighed, 100 mg of tissue (free clamp in liquid nitrogen)
add 1 ml cold 0.4 M perchloric acid with 0.5 mM EGTA
homogenize, sit 1 minute on ice, spin cold ~3200 g 5 minutes
decant supernatant-and add 340 ul 0.5M K2CO3, mix, ice 5 minutes
spin cold @3200g 5 minutes, decant (SAVE SUPERNATANT), freeze, analyze
Obtaining high-quality saliva samples can be problematic. The core's salivary cortisol and testosterone assays require a minimum of 400 µl of clean saliva. Care must be taken to ensure that saliva is not diluted with liquids or contaminated with mucous or food debris. Time of sampling should also be considered since cortisol secretion is circadian.
Salimetrics SalivaBio Saliva Collection and Handling Advice
A number of publications suggest that saliva collection methods can affect results:
Tools for drools (2015)
In collaboration with Dr Blythe Corbett and the SENSE lab the Vanderbilt Hormone Assay & Analytical Services Core tested the effect of several collection methods on the results from our assays: passive drool, chewing wax, gum (1 & 3 min), or Salivette (Sarstedt code blue, with synthetic swab). We did not detect a collection effect on cortisol (n=11, 5M/6F healthy adults) but found that testosterone was reduced with Salivette collection (n=5 males). Salivary testosterone was below the level of detection in the adult female subjects. Unpublished results.