There's inconsistent terminology and methodology when measuring individual differences in circadian timing, preference, and behavior. This protocol combines gold standard techniques for categorizing circadian phenotypes and conducting diurnal performance testing. Well, the main advantage of this protocol is that we can accurately assess circadian phenotypes by looking at both circadian phase and sleep behavior, and this can be done with individuals in their home environment.
Demonstrating the participant process will be Cheryl Isherwood from the University of Surrey. Invite participants who meet the inclusion criteria to attend an initial meeting. And obtain written informed consent from all participants interested in taking part in the experiment.
Ask the participants to complete the Munich ChronoType Questionnaire, which assesses individual differences in sleep, wake variables and light exposure on work and free days. For physiological sample collection, pre-label seven milliliter polypropylene collection tubes with the participant ID number, morning or evening, and then individual sampling number. Different colored labels can also be used for morning and evening sampling tubes to help distinguish between samples.
Prepare a sample collection record sheet for both morning and evening protocols to allow the participants to timestamp the samples after they are taken, including a participant ID number, date for seasonal information, and location for calculating the photo period. It is critical that universal time is used to ensure there are no issues with a.m. and p.m.
time codes. Demonstrate how to collect the saliva samples, informing the participants that it is essential to acquire the morning samples immediately upon waking before getting out of bed, and that the evening samples must be acquired in dim light, preferably, red light. Researchers should measure this, if possible.
Instruct the participant to collect the samples on a free day when they are able to go to bed and wake up at preferred times, without the need for an alarm. Give each participant a wrist activity monitor, or actigraph, to be worn for at least two weeks to gather rest and activity patterns, and light data throughout the study period. Set each actigraph to collect actigraphy data for rest and activity analyses, setting the parameters according to what is required.
Instruct the participants how to use the actigraphs, ensuring that they wear it on their non-dominant wrist, and preventing sleeves from covering the device to allow light data to be gathered. In combination with actigraphy, and to facilitate sleep, wake analysis derived from the actigraphic data, give each participant a sleep diary to be completed on a daily basis. For a cortisol awakening response morning sample collection, have the participants collect the samples upon waking while still in bed into the appropriately labeled sample tubes, every 15 minutes for the first hour, and evaluation every 30 minutes for the next one to two hours.
When all of the samples have been collected, the participants should store their samples in the freezer at minus 20 degrees Celsius until their collection by the research team. For dim light melatonin onset, the participant should sit indoors under dim light, and collect saliva samples in the appropriate vial every 30 minutes from three to four hours before habitual bedtime until one to two hours after habitual bedtime. The participant should also record the time each sample is taken.
Where possible, researchers should measure the light conditions in order to monitor intensity and spectral composition. For the evening sampling, it is essential that participants understand the importance of remaining seated indoors under dim light conditions. Participants may use the toilet, or consume a non-caffeinated drink between samples, as long as they are seated again for 15 minutes before the next sample is due to be collected.
Ensure that all other rooms have the same light conditions, so that the participant remains in dim light, preferably red light, for the duration of the sampling period. If food is consumed between the samples, the participants must wash out their mouths with water 15 minute prior to the collection of the next sample. Once all of the samples have been collected, the participants should store their samples in a freezer at minus 20 degrees Celsius until their collection by the research team.
The diurnal performance testing can be conducted at home or in the laboratory, and should be arranged according to the study hypothesis based on the number of time points being investigated at specific clock times. Upon retrieval of the saliva samples and sleep diary, extract the daily bedtime and wake up times from the sleep diaries, and enter these data into the manufacturer software to obtain actigraphic variables relevant to the study. To determine the melatonin and cortisol concentration in the participants'saliva samples at each time point, perform a radio immunoassay with each participant's samples according to standard protocols.
Calculate the individual DLMOs as the time points at which melatonin concentrations exceed two standard deviations of the three baseline initial saliva collections. Calculate the cortisol peak as the time of the highest cortisol concentration recorded during the morning cortisol awakening response. A variable should be allocated as zero if it is in the early circadian phenotype category, one if it is in the intermediate circadian phenotype category, and two if it is in the late circadian phenotype category.
Calculate a total circadian phenotype score from the five variables collected. Based on this score, sub-categories can also be determined as indicated in the table. All of the participants categorized as early circadian phenotypes had a score between zero to one.
And all of the late circadian phenotype participants had scores between eight to 10. To confirm the results of this representative experiment, the group averages were compared for each circadian phenotype variable. Other sleep variables, including the sleep duration, efficiency, and latency, did not differ significantly between the groups.
The corrected mid-sleep on free days was significantly correlated with the dim light melatonin onset, peak time of cortisol awakening response, sleep onset, and wake up time. These data indicated that the different circadian phenotype groups demonstrated clear differences in their sleep onset, offset times, as well as in their physiological variables. Significant diurnal variations were observed at the whole group level for the Karolinska Sleepiness Scale and Psychomotor Vigilance Task.
When each group was analyzed separately, subjective sleepiness showed significant diurnal variations, with ECPs reporting high sleepiness in the evening, and LCPs'highest sleepiness in the morning. For Psychomotor Vigilance performance, significant diurnal variations were found in LCPs, but not in ECPs. It's important to explain each step of the protocol clearly and to make sure that the participant understands during the setup interview to maximize study compliance.
Once the participants are categorized into circadian phenotypes, the performance testing element can be amended, changing timings, scaled up by increasing time points, or changed, by using different tasks, depending on the study aims. This protocol can be used in any study requiring an accurate and thorough assessment of circadian phenotype as a screening tool for inclusion and exclusion criteria, and also to control for circadian phenotype in time sensitive research. This approach provides a detailed method for combining gold standard biological phase markers with objective and subjective sleep patterns to determine individual circadian phenotypes.
