TITLE: Hair cortisol analysis protocol
AUTHORS: Katy    Parker, Matt Bristow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">There has been considerable interest in the measurement of cortisol in hair as a biomarker of stress since work by Davenport et al. (2006). The methodology developed by our laboratory is intended to allow for a scalable method for hair analysis that can be undertaken for large numbers of hair samples and has the minimum number of steps to reduce the chance of error and reduce costs. Our method reduces the number of steps where the sample is transferred to another container as it is kept in the same tube from initial weighing through to end of the methanol incubation. The procedure is also very scalable. The ‘rate limiting’ step for our hair analysis procedure is the grinding of the hair and this has been partially automated using the MP Biological Fast Prep grinder which is highly scalable.  Final quantification of cortisol is performed via an ELISA assay, an assay which is rapid and cost effective.</div><div class = "text-block">References:</div><div class = "text-block"><span>Davenport, M. D., Tiefenbacher, S., Lutz, C. K., Novak, M. a., & Meyer, J. S. (2006). Analysis of endogenous cortisol concentrations in the hair of rhesus macaques. </span><span style = "font-style:italic;">General and Comparative Endocrinology</span><span>, </span><span style = "font-style:italic;">147</span><span>(3), 255–261. https://doi.org/10.1016/j.ygcen.2006.01.005</span></div></div>

[STEPS]
?. [Hair cutting/weighing]
Ensure analytical balance is calibrated.
?. [Hair cutting/weighing]
Place clean beaker on balance.
?. [Hair cutting/weighing]
Scan sample id into spreadsheet.
?. [Hair cutting/weighing]
Place corresponding barcoded 4.5ml sample tube (ThermoFisher Scientific 342800-0045) in beaker and tare.
?. [Hair cutting/weighing]
Create a sample length measure: Measure out required sample length on plastic card, marking length with permanent pen.
?. [Hair cutting/weighing]
Hold scalp end of hair sample on line of sample length measure, pull hair taut and cut at very edge of measure using sharp scissors. Clean scissors with 70% IMS after use.
?. [Hair cutting/weighing]
Transfer hair sample to sample tube using clean tweezers.
?. [Hair cutting/weighing]
Add/remove hair as necessary using tweezers to get as close as possible to 25mg. Record exact weight. For samples less than 25mg transfer whole sample and record weight. Cap sample tube.
?. [Hair sample washing]
Add 2ml isopropanol (Propan-2-ol, >=99.8%, CAS:67-63-0) each tube in batch (batches of approximately 10 samples), replace cap.(Batch size depends on individual speed, ensure samples are not left soaking in isopropanol for extended length of time, aim for no longer than 5 minutes - 10 minutes maximum)
?. [Hair sample washing]
Shake batch of samples on platform shaker for 3 minutes.
?. [Hair sample washing]
Remove isopropanol using pipette and long tips, this must be done carefully to ensure no hair is removed along with isopropanol.
?. [Hair sample washing]
Add another 2ml isopropanol to each tube in batch, replace cap.
?. [Hair sample washing]
Remove isopropanol using pipette and 1250µl tip (Sarstedt 70.1186.100), this must be done carefully to ensure hair is not removed along with isopropanol.
?. [Hair sample washing]
Leave samples uncapped in fume cabinet at room temperature for a minimum of 48 hours to dry completely.
?. [Sample grinding]
Add 3 x ceramic balls (MP Biomedicals 116959050) to each sample and replace cap securely. (We had previously used 5 ceramic balls until testing established that 3 ceramic balls gave equivalent results)
?. [Sample grinding]
Place samples into FastPrep-24 (MP Biomedicals), and grind 6 times at speed 6 m/s for 30 seconds each, allowing 5 minute cool down between grinds.
?. [Extraction into methanol]
Add 2ml methanol (Analytical reagent grade, CAS:67-56-1) to each sample tube. Replace caps tightly.
?. [Extraction into methanol]
Rack samples and position horizontally on platform shaker.
?. [Extraction into methanol]
Shake continuously for 24 hours at room temperature to extract cortisol.
?. [Transfer of clear supernatant]
After incubation, remove samples from platform shaker. Shake sample and tip contents of tube (including hair powder,  methanol and ceramic balls) into the insert of corresponding pre-labelled Salivette tube (Sarstedt 51.1534).
?. [Transfer of clear supernatant]
Centrifuge Salivettes at 1500RCF for 5 minutes.
?. [Transfer of clear supernatant]
Dispose of Salivette inserts containing ceramic balls, replace caps.
?. [Transfer of clear supernatant]
Centrifuge Salivettes (now just containing methanol and hair powder) at 3000 RCF for 15 minutes.
?. [Transfer of clear supernatant]
Transfer 1.4ml clear methanol supernatant to corresponding pre-labelled 2ml tube (VWR 211-0093). Cap up samples as transferred.
?. [Sample dry down]
Uncap samples and load into Scanvac vacuum centrifuge.
?. [Sample dry down]
Run at 1700RPM for 3 hours at 37° to completely dry down Methanol.
?. [Sample dry down]
Replace caps and store at -80°C until day of assay.
?. [Reconstitution]
On day of assay reconstitute samples by adding 150µl Salimetrics assay diluent. Replace caps.
?. [Reconstitution]
Vortex samples for 10 seconds, then invert samples and vortex for a further 10 seconds.
?. [Reconstitution]
Samples should be left to sit for a minimum of 20 minutes after being vortexed.
?. [Reconstitution]
Soon before start of assay, again vortex samples for 10 seconds, then invert samples and vortex for a further 10 seconds.
?. [Reconstitution]
Centrifuge samples at 1200RCF for 2 minutes.
?. [Reconstitution]
The samples can then be assayed according to Salimetrics assay protocol.
?. [Hair sample washing]
Shake batch of samples on platform shaker for 3 minutes.
?. [Hair cutting/weighing]
Clean tweezers with 70% IMS and ensure balance is clean. Repeat from step 4 with remaining samples.