1.1 Basic concepts
1.1.1 Quality Control (QC)
Quality control is a management process that monitors the entire process, eliminates errors, prevents changes, and maintains the standardization status. This process is carried out through a feedback loop.
1) determine the object of control;
2) Specify the standard (expected value) of the control object;
3) Develop or select control methods and means;
3) Measuring actual data;
4) Compare or compare the difference between the actual data and the expected value and explain the reason for this difference. When the predetermined error range is exceeded, the alarm signal is sent and the feedback channel is interrupted.
5) Take action to resolve the difference. The means of restoring the original state (the original standard state) works.
Quality control is mainly carried out using quality control charts. A quality control chart is a graph that compares the performance data of a test with the calculated expected "control limit." This performance data is selected in chronological order when it is normally performed according to the procedure, and its purpose is to detect the "traceable" cause of variation during the inspection process. The cause of the error of "retrievable" refers to other reasons than random errors. The "control limit" is calculated by statistics, and we will introduce it in detail later (see
1.3. Indoor quality control procedures).
1.1.2 Errors Experimental errors are classified into three types: systematic errors, random errors, and gross errors.
Systematic error refers to the error that a series of measurement results have the same tendency as the true value or the target value. It has obvious regularity and can be repeated under certain conditions. It can be prevented and corrected by quality control.
Random error, also known as accidental error, is an accidental, unanticipated error that is difficult to avoid and correct. The distribution of random errors in the inspection work conforms to the normal distribution law.
Negligence error is an artificial liability error. Work can be avoided by strengthening laboratory management and quality control.
1.1.3 In the normal distribution and standard deviation ELISA test, when the same sample is tested for more than 20 times, it will be found that the data (the absorbance of the measurement results) are distributed on both sides of the mean, and most of them are concentrated near the mean. If the measured value is plotted on the abscissa and the frequency of occurrence is plotted on the ordinate, a bell-shaped graph can be drawn. As shown in Figure 5-1, the top of the clock is the mean value, and the other values ​​are symmetrically distributed with the mean value. This is the normal distribution.
In other words, when ELSIA detects the same sample for a certain number of times, the data within ±1SD of the mean (X) accounts for 68% of the data of the group, and the data distributed within the range of X±2SD accounts for the total. 95% of the time,
The data distributed in the X±3SD range accounts for 99% of the total. When we require the test results to be acceptable within the X±2SD range, 95% of the data will be eligible.
1.1.4 True value The value measured by the exact, optimal decisive method is called the true value. The true value is generally undetectable. The value measured by a reliable and decisive method, called the target value, usually uses the target value to represent the true value.
1.1.5 accuracy (accuracy)
It refers to the degree to which the measurement result is close to the true value (or target value). Accuracy cannot be expressed in numbers and is often measured in terms of inaccuracy. The degree of deviation between the measurement result and the target value is called the deviation, which indicates the inaccuracy of the test.
Absolute deviation = mean of test - true value (or target value)
Relative deviation = absolute deviation true value 或 (or target value) × 100%
1.1.6 Precision (Precision)
It refers to the degree of closeness between each measurement result and the average value when the measurement is repeated for the same sample, that is, the degree of coincidence between the repeated measurement values.
1.1.7 Standards
1. International Standards The fixed value materials determined by the WHO or the corresponding organization and determined by affirmative, recognized and accurate physical or chemical methods.
2. International biological activity standards are materials based on biological responses to international active units calibrated by WHO or corresponding tissues.
3. Reference standard serum The legal materials produced by the National Standardization Organization according to international standardization. Can be used to identify instrument and identification method accuracy.
1.1.8 clinical decision leuel
When a certain analyte reaches a certain level, the clinician must take medical measures. This concentration of the analyte is called a clinically decisive level.
1.2 Quality Control Serum quality control serum is the serum of the existing target value. One or several parts are added to each routine test, and the results are used to understand the situation of this test. If the result of the quality control serum test can control the error within a certain range, it means that the test does not have an impermissible error. If an abnormal result exceeding the allowable error range occurs, indicating that the test is unqualified, the cause should be sought, and after correction, the sample to be tested is re-examined. Therefore, quality control serum plays an important role in quality control work.
1.2.1 Use of quality control serum The hepatitis B marker substance control serum prepared by the Clinical Laboratory of the Ministry of Health can be kept at -20 °C for half a year.
When the frozen state is melted, it should be mixed first, and the unused portion can be stored at 4 ° C for 5 days. It is not advisable to repeat ice melting or self-packing. The quality control serum required for the indoor quality control work of a certain test is generally prepared in 3-6 months. Homemade undetermined quality control serum, before a batch of quality control serum will be used up, you need to prepare the next batch of quality control serum. Quality control serum requires stable performance, the potency is unchanged in the long term, and its physical and chemical properties should be similar to the patient sample, so that it can effectively monitor.
1.2.2 Threshold quality control Serum control serum and undetermined values. If only one quality control serum is used, it is generally set at the boundary between the normal value and the abnormal value, and the qualitative measurement is at a weak positive level, which is called the critical value. The definition of the critical value of hepatitis B markers should be based on clinical requirements to provide a unified standard for weakly positive judgments.
Threshold quality control serum can be used as a positive control in the kit and a third control other than the negative control. It can sensitively reflect the detection level of the kit and ensure that the specimen with weak positive reaction is not missed.
1.2.3 Preparation of quality control serum Each laboratory can select the quality control serum provided by the clinical center according to its own conditions, or prepare the quality control serum used in this room according to the following methods (taking hepatitis B quality control serum as an example).
1) Collect fresh serum that is free of hemolysis, jaundice, and no bacterial contamination.
2) Heat at 56 ° C for 10 hours to live.
3) Remove the precipitate by centrifugation or filtration.
4) Dilute the collected serum to the desired concentration with 10% calf serum or normal human serum (PBS buffer). It is better to dilute with normal human serum because its composition is closer to the test specimen.
5) Filtering and sterilizing. Pack the small ampules in one-time use, seal them, and store them at 20 °C. Do not freeze and thaw repeatedly.
The level at which the test substance is required to be detected is often considered to be the level at which the quality control serum should be selected. If there are other requirements for this test, the required concentration of the control should be added.
6) Calibration content. The mean value of the >±2SD data was deleted as the target value for 20-30 measurement results and compared with the known fixed value serum.
1.3 Indoor quality control procedures Clinical test results, there may be no error between each time or every day. Determine the allowable error range, which is not caused by clinical misdiagnosis and missed diagnosis. The following steps are used to determine the quality control range.
1) Measurement error under optimal conditions.
2) Errors of known values ​​of serum under routine test conditions.
3) Errors in serum of unknown values ​​under routine test conditions.
4) Requirements for clinical application. An allowable error range should be determined for any test, and the premise is to meet clinical requirements. If the allowable error is set too small, there is no clinical significance, but it takes a lot of manpower, material resources and time to comply with the rule. Conversely, if the allowable error is set too large, the monitoring system will not be aware of the clinically required error of detection and lose the meaning of quality control.
1.3.1 Determination of the optimal value of quality control serum (OCV) under the optimal conditions. Under the best conditions of the laboratory (including operators, reagents, instruments, etc.), the quality control serum is detected 20-30 times. The measured result is calculated, and the mean and standard deviation (SD) of the set of data are obtained to indicate the best working quality of the laboratory.
The determination of OCV in the HBsAg ELISA assay is now illustrated. The quality control serum used is critical serum,
The HBsAg concentration was 5 ng/ml. In the laboratory, select the technician with the best quality and the most skilled operation to carry out serious and special measurement. Select the best kit. Before the test, carefully correct, adjust, debug and use the constant box and sampler. The sample is sucked, etc., which is tested under the best and most ideal conditions. Negative controls and positive controls were simultaneously measured in addition to the quality control serum. And two measurements were made to obtain two absorbance values ​​(A values), and X was obtained. 20 times in a row, find 20
X, ie X1...X20. From these 20 data, X and SD of OCV are obtained.
1.3.2 Determination of the known value of the quality of the serum (routine conditions variance-known value, referred to as RCVK) under normal conditions.
The technician who performs the routine test puts the quality control serum in the routine test sample under the condition of routine test, and performs 20 tests, and the result is calculated by the same OCV method. It is generally accepted that the SD of the RCV is acceptable within twice the SD of the OCV.
If it is too large, you should find the reason to make it close to the SD value of the OCV. After the laboratory conditions are improved (for example, correcting the sampler, correcting the washing operation, adjusting the incubation temperature, etc.), the RCVK measurement is repeated. If the SD value of the RCVK is smaller, indicating that the OCV is not measured under optimal conditions, the OCV should be retested. Under normal conditions, RCVK must be better than
OCV is big. The quality control is used to control the conditions so that the RCVK data is as close as possible to the OCV value. The RCVK data reflects the quality of the laboratory's daily work, used as a quality control chart, controls the results of the indoor test, the results of the daily test, and whether the report can be issued.
1.3.3 Under normal conditions, the determination of the unknown value of the serum variability (routine conditions variancl-unknown value RCVU) is sometimes used to avoid subjectivity, and then RCVU determination.
The measurement procedure is the same as RCVK, but the operator of the test does not know the value of the quality control serum, or performs routine tests under the condition that the operator does not know which is the quality control blood serum to exclude the subjectivity of the operator. This is not illustrated here.
1.3.4 Quality Control Chart Through the above three steps, the indoor quality control chart can be started, and the quality control block diagram is based on RCVK and SD. The quality control chart can be used to monitor the results of each test. When another batch of kits and another batch of quality control serum are not replaced,
The quality control chart can be continued.
The S/CO value of the quality control serum is below the range of -2SD, which is an "alarm". The cause should be searched and the cause identified should be recorded on the quality control chart.
In the ELISA test, the error tolerance range of various test items has to be concluded in practice. The above is just an example of the quality control method, not a conclusion. 2SD is a generally accepted allowable error limit. When one batch of quality control serum is placed in each batch, one more than 2SD should be used as an "alarm", and the second time beyond 2SD is "out of control". When the quality control process occurs, when there is out of control, when there is a loss of control, the cause should be found, usually caused by the failure of the kit or the quality control serum. Replace the kit or replace the quality control serum to find out the cause and correct it and retest. If the test results are still not up to the required or the cause is not found, the OCV test should be repeated. If the result of the OCV test is still good, there is a problem with the normal operation.
It is generally considered that: 1 exceeds 3SD at one time; 2 exceeds 2SD for two consecutive times; 33-5 times continuously within 2SD of one side;
45 to 7 consecutive sides of the horizontal axis are out of control.
In the third and fourth cases, it is often difficult to detect records alone, but this loss of control can be clearly found on the quality control chart.
1.3.5 Statistical calculation method--"immediate" quality control The quality control method introduced above is basically the same as the quality control method of clinical chemical measurement, but ELISA has its particularity.
The most appropriate quality control method has yet to be established. Some laboratories do not perform daily ELISA testing, and ELSIA
The kit has a short expiration date, and it is difficult to continuously measure 20 times with a batch of kits. Using the "immediate method" quality control statistical method, only the continuous measurement 3 times, the third test result can be quality control.
The specific calculation method for the establishment of the "immediate method" is as follows:
1) First arrange the measured values ​​from small to large
2) Calculate X and SD.
3) Calculate the SDI upper limit and the SDI lower limit.
4) Compare the SDI upper limit, the SDI lower limit value, and the number in the SDI value. When the upper limit of SDI and the lower limit of SDI are <n2SD, it means that it is within the control range, and can continue to measure downwards, and continue to repeat the above calculations; when the upper limit of SDI and the lower limit of SDI are between n2SD and n2SD, The value is in the "alarm" state in the range of 2SD~3SD. When the SDI upper limit and the lower SDI value have a value of >n2SD, the value is out of the 3SD range and is "out of control". The values ​​in the "alarm" and "out of control" states should be discarded and the quality control serum and patient samples re-measured. It is only the value of this control that is lost. The other measured values ​​can still be used.
Immediate statistical control method for quality control of ELISA.
When the detected value exceeds 20 times, it is no longer necessary to use the "immediate method" quality control statistical calculation, and can be transferred to the quality control of the conventional quality control chart. The value of the first 20 times and the SD are used as the quality control frame diagram, and the 21st numerical value can be clicked in order.
1.4 inter-room quality assessment (external quality assessment (EQA)
The inter-room quality evaluation referred to as the inter-room quality assessment is a series of methods used by the quality control center to continuously and objectively evaluate the test results of each laboratory, and find the inaccuracy of indoor quality control is not easy to find, understand the laboratory Differences in results and help correct them to make them comparable. The results of each laboratory test were reported to the quality control center, and after statistical analysis, the results were compared. This evaluation does not control the daily test reports issued by each laboratory, but rather a retrospective evaluation. The indoor quality control mainly monitors the precision of the test results, while the inter-room quality control mainly controls the accuracy of the test results and cannot substitute each other. The laboratory participating in the quality assessment should first do indoor quality control.
1.4.1 Method of inter-room quality assessment
1. Investigation of quality control materials This is a common form of inter-room quality assessment at home and abroad. The inter-inspection center of the hepatitis B marker ELISA test is used to regularly issue quality control materials to each laboratory. Each laboratory carries out the inspection on the specified date, and the inspection results are reported to the department's clinical inspection center. The Department of Clinical Inspection conducted statistical analysis and sent the evaluation results back to the laboratories. Through evaluation, each laboratory understands the quality of work in this room, finds gaps, and tries to improve to continuously improve the quality of inspection.
This evaluation method has certain shortcomings, that is, each laboratory often treats the quality control materials specially. When testing, special kits are selected, special technicians are selected for inspection, and some laboratories and the results are modified. This makes the EQA results not reflect the daily work of the lab.
2. Send an observer to the laboratory for reagent investigation. This investigation is not notified in advance, and an observer is temporarily sent to the laboratory to designate a set of specimens for routine evaluation.
This kind of investigation method is easy to find the actual problems existing in the laboratory, and can directly give guidance and help, solve problems and improve the quality of inspection. Such surveys can often use real samples to avoid some of the shortcomings of quality control.
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