PCR Cycler Validation - Extended Information

Thermal cycling (PCR) and other temperature related amplification techniques have become standard technology in diagnostics applications and biotechnological research. The results of these amplification techniques have substantial impact on our society. This website provides information concerning the thermal behavior of thermal cyclers, as well as the impact on the generated results. This information contributes significantly to the apprehension of our mutual obligation to ensure that the usage of thermal equipment is accurate, uniform and frequently verified or calibrated, if necessary.

The use of biological positive, negative and threshold controls is insufficient to guarantee reliable PCR results. Validation of cyclers should not be limited to laboratories that comply with ISO, GLP, GMP, FDA, Ster-Lab, Bell-test and other norms and regulations, but is also recommended for all cyclers and their users in the market place. Cycler validation is required for accreditation, and is also recommended by several Associations. Furthermore it soon will be incorporated into the European regulations.

It is obvious that PCR Cyclers should perform as accurately as possible, the displayed temperatures should be comparable to the actual absolute temperatures, and the temperature uniformity (spread) within a block should be acceptably low. If this is not the case, one would at least like to know what these temperature deviations are. This way, you take into account these deviations when developing a protocol, positioning samples in the block or interpreting results.

Thermal cyclers are much more complicated in their design then most users of the instrument can imagine. In general, the design of thermal cyclers can be divided into two sections. One section takes cares of the programming, the power regulations, sensor processing etc. The other section can be categorized as the block design. The block itself is a construction of several parts: the actual reaction block, the sensors, peltier modules, heaters, sealings, as well as a heat sink. These individual layers are constructed on top of each other and they form a “sandwich” construction. Dishes and springs are used to balance the sandwich shrinking and extension, caused by the wide temperature range of operation, 0-100°C generally. All the mentioned components may differ in quality, and wear in time, thus influencing the temperature output of the cycler greatly. Local differences in heat can cause so-called hot and cold spots in a reaction block.

In modern temperature performance testing, on e.g. thermal cyclers, we can distinguish two ways of measuring. The most commonly used technique is static measurements. This means that the thermal device is programmed to a fixed temperature for an extended period of time and measurements take place, on different locations within the device, several seconds or minutes after the device indicates it has reached the desired set point. This is a rough method to determine the accuracy and uniformity of a device and is most suitable for static thermal devices like incubators and ovens. These static measurements are often carried out with single sensor test equipment and their accuracy and repeatability relies greatly on the operator and the used test equipment.

As a consequence of this development, the static measurement method (as described above) is no longer an adequate tool for performance and calibration tests on thermal cyclers. CYCLERtest developed an exciting set of tools for these demanding temperature test applications. One of the main features of this setup is its capability to perform dynamic multichannel temperature measurements with very high accuracy thermistors. This means that up to 16 or 48 sensors take measurements simultaneously during the complete protocol run, on 16 different positions, with an adjustable sample rate (typically 2 measurements/sec/channel). The information gathered by this system is not only valuable to determine the uniformity and accuracy of a thermal cycler, but it also gives more significant information about other parameters, like sample block temperature overshoots, undershoots, temperature drift, ramping speeds, cycle to cycle behavior, block to block behavior, uniformity errors caused by different block loads, and actual cycle times. With this information it is also much easier to perform cycler to cycler protocol transitions.

An underestimated side effect of thermal cycler behavior is the overshoot of cycler block temperature during cycling. In the best scenario, this effect is reproducible, but often it’s not. It occurs on cyclers with limited control capabilities, but also variations in heat transfer capabilities of different sample block materials are also a factor in this. Differences in block loads are not detected by single sensor controlled machines, and only a few multi-sensor equipped machines are capable in detecting different block loads. Some cases show massive overshoots of up to 4 to 6°C for seconds.