This information is intended for (potential) CYCLERtest distributors and MTAS users and provides general and technical information about the MTAS (Mobile Temperature Acquisition System). This system is used for performing thermal performance and temperature quality validations on thermal equipment, especially thermal cyclers used for the PCR® amplification process.

Reaction and process parameters in PCR® amplifications are not the only criteria that affect the quality of the results. Most PCR® protocols use three temperatures in the process, (92°C-96°C) denaturation step, (37°C-60°C) annealing step and (70°C-74°C) extension step. This process is performed on a so-called thermal cycler that achieves the temperature cycling. The instruments for this application need to have excellent qualities in:

• Reliability - performing temperature cycling without interruption and failures
• Robustness and durability
• Temperature Accuracy - the average block temperature must be within a specified range of the set temperature
• Temperature Uniformity - the well to well difference (variation) must not exceed certain limits
• Reproducibility - consistence in achieving the target temperatures at the same time in each cycle

For verification of the temperature quality of PCR® instruments sample blocks, certain methods are available. Of course a simple mercury thermometer can be used for these specific temperature measurements, although this type of sensor is not suitable for accurate temperature measurements due to its robustness and inaccuracy. Other commercially available "hand-held" temperature measurement instruments may have a higher accuracy, but the used sensors do not match this special application. Besides that, it is only possible to measure static temperatures on certain block locations sequentially, while the PCR® process is a dynamic process. With "hand-held" instruments, measurements are performed manually.

Because there was no system available for accurate dynamic and multi-channel temperature validations on thermal cyclers, GENO-tronics developed a temperature measurement system specially for this application, called MTAS. The system consist of special designed hardware and temperature probes for performing very accurate and dynamic multi-channel temperature measurements. Very important for performing correct, accurate and reliable temperature measurements is the temperature sensor. The sensors used with the MTAS (see next paragraph) are precisely designed and manufactured with the same cone angle and shape as the sample well. This means that for every sample block type there is a specific designed temperature probe. High thermal conductive cement with special parameters is used for molding the sensor in the exact shape. The bare sensors are not commercially available and are specially manufactured for suiting the highly sophisticated MTAS applications. The sensors have a high accuracy and low drift and variation over time. Important parameters and specifications of the measurement system are the accuracy and the repeatability of the system. It is common that verifying the quality of a certain instrument, the accuracy and resolution of the measurement system used has to be minimal three times better then the instrument to be verified. Also the repeatability, the reproducibility and drift of the complete measurement system are very important parameters. The MTAS is designed to match these recommended parameters (see the paragraph "specifications" for the technical details)

As mentioned in the previous paragraph, GENO-tronics and CYCLERtest both located in The Netherlands, have developed a high sophisticated temperature measurement and validation system for capturing and analyzing the temperature performance of thermal cycling equipment, especially instruments used for PCR(r) reactions, cycle sequencing instruments, incubators etc. The system has unique features and capabilities and is suitable for temperature validation of all kind and brands of instruments in the market e.g. thermal cyclers manufactured by MJ Research Inc, Applied Biosystems, Eppendorf, Biometra, MWG , Hybaid etc. The system has been calibrated against the ITS-90 temperature standard and verified by an independent calibration laboratory (KEMA), which is accredited by the "Dutch Council of Accreditation". A great advantage of the MTAS the transportability of the system and offers "on-site" testing and "in field" validations. For the customer this is very convenient, because it is not necessary to send the instrument to a test laboratory for the validation. MTAS test results are analyzed and reported in an automated process and do not need and allow many manual operations. CYCLERtest's policy is that only trained and certified technician are allowed to do temperature validations with the MTAS . Due to this policy, CYCLERtest educates and certifies engineers for operation of the system and perform correct and reliable temperature measurements.

Every temperature measurement, regardless which type of thermal cycling instrument is tested, is performed using a standardized and prescribed temperature protocol. This enables comparison of the results of every measurement performed, whether the tested instrument model is identical or different. This standard protocol is designed to support all relevant temperatures related to instrument specifications and de PCR(r) process. Data and results from the measurements are collected in a central database and the instrument specific "field " performance are calculated . The "field" performance can be different from the cycler manufacturer specifications.

A unique feature of the MTAS is the option to determine the quality of a temperature gradient of a thermal cycler. Since the MTAS is multi-channel the temperature probes can be located at strategic locations in the sample block to measure the gradient. The measurement is also performed dynamically. See the illustration on the next page for a picture of a typical measured gradient with the MTAS. For more background information please study the "Thermal Performance and Validation" brochure of CYCLERtest BV, the Netherlands.

Another special and unique MTAS test is a temperature quality test under "load conditions". Load tests simulate realistic conditions that would be experienced in a research and clinical environment. During this test, tubes filled with a certain amount of demi-water (dummy loads) are placed into the sample block to simulate realistic situations. Temperature probes are also placed in the sample block to monitor the block temperature during this test.

MTAS test results are presented in a detailed report, that is generated automatically. MTAS offers three types of reports: "Compact", "Standard" and "Extended" which differ from each other in the presented information. Secondly MTAS offers to include conclusions about the test results and recommendations for the future use of the instrument. The reports are generated in Microsoft Word, which comes standard with the MTAS and has the advantage that the reports can be treated as standard Word documents. An issue is that data in the reports cannot be modified, which is an important issue in GLP/GMP Quality Programs. The layouts of the reports cannot be changed and modified; CYCLERtest's policy is to have uniformity and recognizability in the service. A copy of an example "Standard Report" is available on request.

A "Certificate of Thermal Validation" accompanies every test report. This certificate represents the results of the measured and calculated main parameters of the tested instrument and reflects to "Thermal Performance Classification". Important is that there is no "judgment" made if the instrument performance is good or bad; this is left to the client's/customer's interpretation. Tested instruments are qualified against three different "Thermal Performance Classifications". Class I means that the main parameters as instrument accuracy, homogeneity and ramp speeds are within the manufacturer's specifications. Classification II indicates that the main parameter results are not within the manufacturer specifications but are within the field specification range. All other results are specified in Class III.

The MTAS system consists of the following components:

• MTAS-16 hardware module
• Intelligent CyclerProbe set (available in several models and formats)
  • 16 x 0.2ml: 16 temperature sensors for 0.2ml block format
  • 16 x 0.5ml: 16 temperature sensors for 0.5ml block format
  • 16 x 384 fit: 16 temperature sensors for 384 block format other special models available on request
• MTAS-card
• State-of the-art Notebook with MTAS-16 Acquisition Software and MTAS-DAS
• Analysis Software pre-installed
• Connection cable
• Disposables, cleaning
• Robust, light-weight storage and transportation case

Specifications are subject to change without prior notice

The MTAS (hardware module) is controlled and operated by two separate software applications, 1) the MTAS-16 temperature acquisitions software and 2) MTAS-DAS Data Analysis software.
The data acquisition part captures and processes the temperature data from the hardware module. Each channel is acquired, plotted in real-time to a graph and stored to hard disk. The software indicates the actual temperature of each channel (up to 16) and calculates the average temperature and the temperature spread. The data in the log file is coded and can only be decoded by the Data Analysis Software. The MTAS-Card provides measurements credits; for each measurement a certain amount of credits is decreased from the card. The "Data Analysis Software" analysis the captured data, calculates main instrument parameters as accuracy, spread, ramp speeds etc. and creates a report of the results.
One of the strongest points of the DAS is that for every temperature hold in the test protocol, parameters and data for each individual channel are calculated. The next illustration shows an example of the calculations.

As mentioned earlier, only trained personnel is allowed to operate the MTAS. Therefore CYCLERtest organizes a 1-day training course at their facilities when delivering the MTAS. The training session includes a technical briefing about thermal cyclers, theory of temperature measurements, software training, data interpretation and report writing. Also practical assessments are part of the training. After the course is successfully ended the engineer will receive a "Certificate of MTAS Certified Engineer". If preferred an in-house training session at the distributors location is possible. Please ask for the possibilities.

For interest in the MTAS or more information about CYCLERtest Service we encourage you to contact CYCLERtest BV, The Netherlands.

Accuracy
The average temperature of the sample block, measured in more then two sample well locations simultaneously, compared to a traceable standard of absolute temperature (NIST).

Uniformity
Sample wells in the sample block of the thermal cycler should heat uniformly relative to each other. Uniformity, spread or homogeneity is the highest measured temperature values minus the lowest temperature at the same time

Static Temperature Verification
Measure the temperatures of several locations in the sample block at a static temperature hold sequentially. Measurements are not taken at the same time stamp

Dynamic Temperature Monitoring
Monitoring the temperature continuously on more than 4 locations simultaneously during the temperature changing process.

MTAS is a registered trademark of GENO-tronics BV CyclerProbe, MTAS-Card, are trademarks of GENO-tronics BV All other brand names are (registered) trademarks of the respective holders and manufacturers.

The Polymerase Chain Reaction Process (PCR(r)) is covered by patents owned by Hoffman-Laroche, Inc. (4.683.195, 4.683.202, 4.965.188)