In the process of research and development of reagents, sufficient consideration must be given to stability, which is related to whether all predetermined properties of the product can be achieved during clinical application within the set validity period. The stability of an in vitro diagnostic reagent or measurement system is usually quantified in time. Stability can be quantified by the time at which a certain amount of change in a metrological performance characteristic occurs or the amount of change in a characteristic over a certain period of time. The stability study mainly involves two parts, namely the stability of the reagent and the stability of the applicable sample.
Stability of reagents
1. Real-time stability
The real-time stability test is the period for which the reagent can be stably stored under the specified storage conditions. There are two ways to verify real-time stability. The first is to store the reagents according to the specified storage conditions, take them out for experiments at regular intervals (such as 1 month or 3 months), and compare them with the test results at the beginning of the verification. If the change trend within the expected validity period is within the acceptable range, you can choose to stop the validation until 3 months after the expected validity period or continue the validation until the change trend of the test results exceeds the preset acceptable variation range. If the change trend within the expected validity period exceeds the acceptable range, the validity period of the reagent should be shortened. The second method is suitable for reagents that are sensitive to temperature changes, and the shelf life of the reagents stored at different temperatures can be estimated by referring to the Arrhenius equation.
2. Accelerated stability
Accelerated stability evaluation is a study that examines various physical, chemical and biological properties of in vitro diagnostic reagents by setting extreme conditions such as temperature, humidity, light or vibration, so as to infer the validity period of in vitro diagnostic reagents. Since the main components of most diagnostic reagents are proteins, in accelerated stability studies, increasing the temperature to examine the stability of reagents has become a common method, usually at least 15°in long-term stability studies. C. For example, the stability of a certain reagent (the validity period is set to 1 year. The accelerated stability research method is: put the three batches of kits in a 37°C constant temperature incubator, and take them on the 0th, 3rd, 6th, and 10th days respectively. For the original unopened reagents, the product should be tested with reference to the relevant testing methods in the product technical requirements. All indicators should meet the requirements of the performance indicators in the proposed technical requirements.
3. Transportation stability
Transportation stability examines the stability of the reagents in the actual storage and transportation process, to verify whether the sales, delivery and transportation process will have an adverse effect on the performance of the reagents, and whether the packaging materials and assembly methods of the reagents meet the transportation needs. The verification of transportation stability can be carried out in two ways: simulated transportation and actual shipment. Temperature and sloshing are the two major factors to consider in simulating transportation. The temperature can be adjusted by equipment such as thermostats and refrigerators, and shaking can be achieved by equipment such as shakers. By designing different temperatures and shaking frequencies, the transportation process can be simulated to a certain extent, and even more extreme conditions can be designed to check whether the performance of the reagent can still meet the requirements after extreme transportation conditions. The benefits of simulated transport are strong controllability, high repeatability, and extreme conditions can be designed. The actual shipment can help verify whether the reagents are packaged and shipped and the insulation measures meet the requirements. The design point of such experiments mainly lies in the choice of destination. For example, sending to a city with high temperature in the south in summer and sending to a city with a low temperature in the north in winter are two typical destinations. According to the difference in temperature between the location and the destination, the packing method and the insulation measures taken at the time of delivery can be adjusted, and the verification results can guide the delivery of the reagents after they are on the market.
4. Unpacking stability
If the reagents in non-single-serving packages cannot be used up at one time, if the user cannot restore the package to the original state due to limited conditions, such as vacuum packaging, etc., the unsealing stability of the reagents should be verified. When designing the verification experiment, the user's secondary packaging and storage conditions, as well as the possible storage period, should be considered. For example, the factory packaging is vacuum packaging, and it is packed in a plastic ziplock bag after the first opening. The reagents for 96 people need to be used up in one month. These conditions need to be verified in the stability of the open package. For reagents whose major components are liquid, open bottle stability should be verified. For example, the storage conditions and shelf life of some components using the freeze-drying process after reconstitution. With the increasing degree of mechanization of reagent users, more and more newly developed reagents are matched with automatic or semi-automatic instruments, such as non-single automatic chemiluminescence system, automatic or semi-automatic enzyme-linked immunosorbent assay System, etc. After the reagents are opened and stored on the detection system, the temperature of the reagent storage area of the system may not meet the reagent requirements, and the open storage may also cause the reagents to volatilize, which requires verification of the on-board stability of the reagents.
The stability of the sample
1. Stability under different storage conditions
The analyte in the sample may have different activities under different storage conditions, so the storage conditions and storage period of the applicable samples should also be verified during the reagent development. The stability of some common test indicators was excerpted from "The Use of Anticoagulants in Laboratory Examinations" published by the World Health Organization in 2002. If the detection index is alanine aminotransferase (ALT), its stable time in whole blood at room temperature is 4 days. If it is in the form of serum or plasma, it can be stable for 7 days when stored at -20 °C, and it can be stable when stored at 4-8 °C. 7 days, stable for 3 days when stored at 20-25°C. Another example is triglyceride (TG), which is stable in whole blood at room temperature for 7 days. If it is in the form of serum or plasma, it can be stable for 1 year when stored at -20 °C, and it can be stored at 4 to 8 °C. Stable for 7 days, and stored at 20-25°C for 2 days. The stability verification of the applicable sample does not need to determine the maximum period of stable storage, but only to verify whether the test results of the reagent on the sample will be affected by the change of the storage conditions of the sample and the extension of the storage time during the routine inspection cycle. changes happened.
2. Freeze-thaw stability
If the applicable sample can be stored frozen, i.e. at -20°C or lower, the freeze-thaw stability of the sample needs to be verified. Similarly, the freeze-thaw stability verification of applicable samples does not need to determine the maximum number of repeated freeze-thaw cycles. It only needs to verify whether the test results of the reagents on the samples are required under routine testing requirements, such as 2 to 3 times of freezing and thawing. Will change as the number of freeze-thaw cycles of the sample increases.
3. Additive influence
There are three main types of additives added to samples: anticoagulants, stabilizers and preservatives. If the applicable sample contains two types of serum and plasma, the effect of anticoagulant on the test results of the sample should be verified; if the analyte is stable for a short time under in vitro conditions and a stabilizer needs to be added, the added anticoagulant should be verified. Whether the stabilizer will affect the test results of the samples; if some samples need to be stored for a long time and need to be added with preservatives, it should be verified whether the selected preservatives will affect the test results of the samples.
