This week's business learning, the Immune Chamber team shared a case of five false positives for hepatitis B. Everyone actively participated, seriously discussed, reached some consensus, and had a deep understanding of the advantages and disadvantages of the enzyme exemption method.
Case after
Patient: Quan Mou, male, 58 years old, from the Department of Rheumatology and Immunology, has been diagnosed with rheumatoid arthritis. On December 9, 2021, blood was drawn for infectious disease screening. Because the patient is a new agricultural medical insurance, according to the regulations, we used the enzyme exemption method to conduct four infectious disease tests on this specimen, and the first results are as follows.
From the above test results, it can be seen that the five results of hepatitis B in the patient are "big three yangs" and are in the active period of hepatitis B. Out of work habits, colleagues in the immune room used the enzyme exemption method for review, and the results were still "big three yangs". Later, the specimen was tested again by electrochemiluminescence. The results were diametrically opposed, with all the indicators being negative.
From the perspective of detection technology, although electrochemiluminescence detection is superior to enzymatic immunity in terms of sensitivity and specificity. However, as a very commonly used detection method, enzyme immunity has considerable accuracy. However, why do the results of the two methods for this patient differ so much? Could it be that the enzyme-free test results are inaccurate?
In order to further verify the results, the immunization chamber colleagues sent the specimens to the reagent manufacturer, who used PEG and high-speed centrifugation methods to process the specimens, and then tested again, and the results were negative. It shows that there is a false positive problem in the detection of this specimen by enzyme exemption method.
After the immunization room reported, colleagues from various professional groups raised questions and discussed.
Question 1: Enzyme exemption method is a traditional infectious disease screening method, the first and first review results are "big three yang", why did you think of using electrochemiluminescence technology to review?
Colleagues in the immune room explained that there are two main reasons for the review of the results.
1. Although it is displayed as "big three yangs", the absorbance values are near the left of the critical value, and such an interval is prone to false yang or false negative results.
2. The patient is active rheumatoid arthritis, and there can be a large number of interfering antibody components in such patients, which is easy to cause false positive results.
Question 2: What influencing factors are likely to cause abnormalities in the results of the enzyme exemption method?
The ELISA procedure includes steps such as reagent and sample preparation, dosing, incubation, washing, color development, and colorimetry. Irregularities at each stage can have an impact on the results of the assay, and there may be endogenous and exogenous factors in the patient specimen that interfere with the enzyme immunoassay.
1. Endogenous factors of the specimen such as nurse blood collection pollution, specimen containing rheumatoid factor (RF), complement, heterophilic antibodies, etc.
2. The setting of the critical value is low ELISA uses the critical value to judge the result, if the OD value is in the detection critical value "gray area", it may obtain false positive/false negative results.
3. When batch testing specimens, if the plate washer is limited, the waiting time will be prolonged, which will then cause the incubation time to be extended, resulting in abnormal results.
Question 3: What is the reason for the abnormal results?
By tracking the patient's test information, the key cause of the false positive result was found, that is, the patient's IgM rheumatoid factor concentration was extremely high, and some autoimmune antibodies were also positive and had considerable titers.
4. How to effectively avoid the abnormal results of the enzyme exemption method caused by the interference factors?
Under the existing conditions in the laboratory, in order to reduce the probability of false positives, the following measures can be taken according to the facts.
1. Formulate strict laboratory operating procedures, but all OD values are low, or special patients such as autoimmune disease patients need to review the results.
2. Do a good job in the pre-treatment of specimens, try to use vacuum blood collection vessels, centrifuge sufficiently (when the weather is cold, the blood specimens are first placed in a 37 ° C water bath for 1 to 2h), no hemolysis, lipids, fibrin filaments.
3. Select recognized, better quality reagents, and different batch size reagents are not mixed.
4. Do a good job in the daily maintenance and regular calibration of instruments and equipment.
5. Set a "gray area" suitable for your own laboratory, and strengthen the review of specimens in the gray area. Other clinical data and laboratory results are also closely combined, follow-up if necessary.
6. Use the self-contained yin and positive standards and suitable weak yang property controls for indoor quality control.
7. When the amount of specimens is too large, it can be operated in batches at the wrong time to ensure that the incubation time is accurate.
8. Laboratories with conditions can carry out neutralization tests.
Inspection to seek truth, test to seek truth, but all accurate inspection, all need rigorous work attitude and rich professional knowledge to support.
Source: Dean Experimental Diagnosis
Editor: Ren Mileage Reviewer: Xiao Ran