Glucose Sensor Calibration

We recommend allowing 3 days after implantation for the sensor to stabilize.� The 14-day sensing lifetime of the sensor is assumed to start after this 3 day stabilization.� This time may vary based on the rat type and individual physiology.� The procedures described below should be viewed as guidelines, and some changes may be appropriate for specific experimental paradigms and rat strains.

For calibration of the CGMS biosensor, Pinnacle recommends using Two-Point Calibrations approximately once a week along with daily single point resting glucose measurements.� In general this calibration strategy follows the time course below:

� Days 3, 7, 14 � Two point calibration as described below

� All other days � Single point resting glucose measurement at approximately the same time each day.� For most situations, there will be no loss of accuracy if single point readings are not done every day (i.e., weekends).

In a typical scenario, the sensor will be implanted and telemetry established to ensure a proper run-in, and the animal returned to its cage. �Our standard protocol is to perform the surgery on Friday, so the stabilization period is complete on Monday.� The first two point calibration would then be on Monday with additional 2 point calibrations on each subsequent Friday.

Performing a Two-Point Calibration:

A two point calibration is typically performed by making two separate blood glucose measurements.� One is taken when the animals is in a quiet or resting state. This serves as the baseline measurement and is the same as the single point resting glucose measurement.�� A second blood glucose measurement is then taken following administration of a glucose bolus.� The glucose bolus may be administered orally, IV or IP.� A second blood glucose measurement should be taken after the glucose has peaked and has started its return to baseline (see Figure 1) - approximately 10 to 15 minutes for a bolus dose administered IP.� This timing may change depending on the experimental model or bolus route.� Pinnacle recommends monitoring the biosensor signal closely following bolus administration to assist in determining the proper time-point for the second glucose measurement.

To obtain a valid Two Point Calibration, the difference between the resting glucose concentration and the peak of the bolus response should be greater than 100 mg/dL.� Pinnacle recommends an IP dose of 1 g/kg glucose to achieve this difference, however other doses and route combinations are also valid.� Blood glucose measurements may be obtained through a variety of methods including blood vein catheterization, tail vein bleed or tail tip cutting/bleeding.�

Blood Sampling Technique:

For convenience over the course of the two week experiment and to minimize animal movement or stress while obtaining the blood sample, Pinnacle recommends using a tail tip cleavage to obtain the blood sample.� In this method, the tip of the animal�s tail is cut under anesthesia during the CGMS biosensor implantation procedure.�� The scab covering this wound may then be easily removed for daily blood glucose measurements with minimal perturbation to the animal.

Blood Glucose Reference Measurement:

Pinnacle recommends using the Roche Diagnostics Accu-Chek Nano for blood glucose measurements but there are a number of glucose monitors that will also work well.� We also recommend that all blood glucose reference measurements be made in duplicate, using separate test strips and monitors and be performed in quick succession to ensure consistency.� If the two measurements are highly divergent, a third measurement should be taken and the measurement that is not in agreement discarded.

Entering Calibration Values into Software:

To store the calibration values and types directly into Sirenia CGMS acquisition, press the �Calibration� button to evoke the panel� shown below.

Up to 3 calibration readings can be taken at one time.� If only one is taken, leave the other fields blank.� When the calibration values have been entered, select the type of calibration.� That is, point 1 of a two point, point two of a two point, or a single point, and press �save� to enter the calibration data into the data set being collected.� If activated, the calibration coefficients will automatically be applied to the incoming data, so concentration values can be displayed in real time, and alarms can be tied to change and rate of change in mg/dl as well as nA.�

The values entered, and additional calibration options, are available in CGMS Analysis for detailed post processing of the data if required.