Q&A

Due to the principle of the test, the G-test requires a high level of blood collection tubes, which are not only sterile, but also pyrogen-free and enzyme-free.

Reasons for failure of blood collection tubes

(1) Bacterial or fungal contamination or the presence of pyrogens (i.e., endotoxins);

(2) Interference from certain ions or other components of the procoagulant.

The optimal time from specimen collection to testing is no more than 2 hours, and samples can be stored at room temperature for up to 6 hours after serum separation.

Serum specimens should be kept refrigerated at 2-8°C for no more than 24 hours. Long-term storage must be below -20°C, preferably -70°C. Avoid repeated freezing and thawing of specimens. Refrigerated transportation is required. For users who conduct 1-2 sessions per week, serum samples should be transferred to pyrogen-free transfer bottles or pyrogen-free EP tubes for storage. Uncentrifuged specimens should not be stored. Serum should not be repeatedly frozen and thawed, as this will change the serum material, especially the proteins, and affect the test results.

G test kit components, after opening and dissolving at room temperature, the excess main agent should be labeled and frozen in time, and then the unfrozen main agent should be used within 15 min; the standard and quality control products should not be left for more than 1 hour after re-dissolution. (In practice, it is found that the shorter the main agent is left at room temperature, the more favorable it is to its preservation stability.)

If the rate values of standard, QC and sample are all low, it may be due to the failure of the main agent or the low temperature of the enzyme marker; if only the rate values of QC and sample are low, it may be due to the uneven mixing of the treatment solution. the instructions in the G test require shaking for 1min, and the QC, standard and treatment solution should be fully mixed in accordance with the instructions strictly.

(1) Contamination of the main agent;

(2) Failure of the main agent (the case of incomplete failure), which may be caused by insufficient freezing temperature.

(3) After adding the main agent, the plate is not put into the enzyme marker for testing in time, then the program should be selected and the plate should be retrieved before adding the main agent so that the test can be carried out as soon as possible;

(4) After adding the main agent, the plate is put into the zymograph, but it can not be started immediately because the temperature of the zymograph has not risen to the required temperature, then the temperature should be observed in advance to see whether the temperature meets the requirements, and if it does not meet the requirements, it should raise the set temperature, adjust back to 37 degrees after reaching the temperature, and click start directly instead of clicking into the plate when starting the detection (or incubation).

(1) The pipette is not allowed to fill the sample volume.

Solution: Calibrate regularly and redo with a different pipette.

(2) It is not a pyrogen-free tip.

Solution: Use a non-thermal tip to redo the job.

(3) The tip is not inserted tightly or leaks, thus the aspiration and spiking are not allowed.

Solution: Use normal non-thermal original gun head to redo the verification. Pay attention to the liquid level of the gun head to avoid using the problem gun head to add samples again.

(1) Whether the specimen has hemolysis, jaundice or lipemia problem.

(2) Once again, it is necessary to observe whether the linearity of the batch of tests and the measured value of the quality control product meet the requirements of the kit and whether the average rate value of the corresponding value of the concentration of each standard product in the preparation of the standard curve is 1.5-2 times the relationship between a, b, c, d and e.

(3) If the user carries out indoor quality control, it is also necessary to see whether the quality control results of the day out of control. If out of control, according to the out of control processing procedures to solve.

(4) First rule out the existence of factors that cause false positives or false negatives.

(5) Understand the clinical background of the patient sampling, such as the duration of the disease, whether the use of antifungal drug therapy.

(6) Whether the temperature of the instrument, the accuracy of sample addition, and the mixing of standards are sufficiently homogeneous.

(7) Find the manufacturer's technical staff to solve whether it is caused by the reagent kit.

(1) Choose a good product and do the performance verification or comparison before use;

(2) Operate in strict accordance with the operating procedures (SOP) specified in the instructions;

(3) Strictly follow the instructions to ensure that the test meets quality standards;

(4) Establish an indoor quality control system and carry out inter-laboratory comparison when available;

(5) Excluding clear false-positive and false-negative factors.

(6) Using vacuum blood collection tubes validated and qualified by manufacturers to ensure qualified specimens.

(7) Serial testing, with serial positives highly suggestive of fungal infection;

(8) The appearance or disappearance of G cannot be used as an indication to start or stop the use of antifungal drugs.

(9) It must be analyzed in combination with clinical, culture, pathology, and other serological tests such as GM test and MN test;

(10) Close communication with the clinic.

(1) Enzyme marker: must be able to use 450nm wavelength for detection (preferably dual wavelength 450nm and 620nm or 630nm).

(2) High-speed centrifuge: it must be able to reach a centrifugal force of 10,000g or more, preferably at 4 degrees.

(3) 37℃ constant temperature incubator (or other alternative constant temperature air bath), must be able to set the temperature to 37℃.

(4) Boiling equipment: recommended to use the company to verify the qualified metal bath or induction furnace.

(5) Vortex mixer.

(6) 300μL multi-channel pipette (row of guns) and matching tips: it can reduce the time difference of adding samples from different wells.

(7) 100μL and 1000μL single-channel pipettes and matching tips.

(8) Measuring cylinder for preparing wash solution and reagent bottle for preserving wash solution.

(9) Plate sealing film: users need to prepare a part of spare according to the number of experiments done by each box of reagents.

(10) Centrifuge tube: 1.5ml.

Most of the concentrated washes used in the enzyme immunoassay test are phosphate buffer, but because of the different concentration values between the reagents, it is strictly prohibited to mix the reagents of different manufacturers or projects, but Dynamiker's Antigen and Antibody Concentrated Wash can be used for mixing.

Due to the difficulty of pediatric blood collection, the sample serum is insufficient in the case of pre-treatment can be processed according to 180 μL of serum + 60 μL of treatment solution, such as serum enough should be strictly in accordance with the 300 μL of serum + 100 μL of treatment solution to be processed.

(1) The use of floats that are easily deformed, such as foam, deforms the floats in the water bath, resulting in inadequate pretreatment. The sample should be treated in a full boiling water bath;

(2) The supernatant was not aspirated immediately after treatment, resulting in the supernatant being absorbed by precipitation. The supernatant should be aspirated promptly;

(3) Some centrifuges display 10,000 g, but the actual centrifugal force may not reach 10,000. the centrifugal time should be extended or the centrifugal force should be increased;

(4) When processing individual samples, there may be adhesion and delamination on the wall, and the amount of supernatant can satisfy the test, so care should be taken when aspirating samples to avoid aspirating the precipitate. Increasing the centrifugation time can solve this problem and also increase the sample supernatant. You can also mash the precipitate with the tip of a gun and centrifuge again to obtain a sufficient amount of supernatant.

(1) Inaccurate addition of liquid, make sure the amount of liquid added is accurate;

(2) In the process of plate washing, the operation of plate dumping is not standardized, the pollution caused by the impact, correct the operation;

(3) The enzyme labeling plate is placed at room temperature for a long time, which leads to failure, try to shorten the time of placing at room temperature;

(4) After adding the primary antibody or termination solution, the plate shaking time is too short or no shaking, not fully mixed, should be extended appropriately;

(5) When adding samples, take the wrong reagent bottle and add the wrong reagent, the reagent label should be carefully checked;

(6) Incubation time is not performed as required;

(7) The liquid spilled during the operation, the volume change is obvious or cause pollution between the wells

(8) The plate washing operation is not patted dry, and there is liquid residue in the wells.

Foreign literature reports comparable to invasive Aspergillosis (IA) clinical symptoms an average of 5-8 days earlier than high-resolution CT scans an average of 7.2 days earlier than the start of empirical antifungal therapy an average of 12.5 days earlier

The explanation for this phenomenon is that neutrophils are able to phagocytose and rapidly kill mycelium, which reduces the fungal load in the body and thus reduces the amount of GM released into the bloodstream, and there are mannose receptors on the surface of granulocytes, which can bind GM, both of which lead to a false-negative result in the GM test.

All studies have shown that the GM test has a high specificity, while the sensitivity fluctuates (29%-100%).Mennink-Kersten et al. suggest [25] that there are several reasons for this.

(1) Aspergillus growth and antigen release are related to the nutritional conditions and pH of the surrounding environment. If nutrition is adequate and pH is at 7.5, the fungus can sustain growth and release GM, but when inflammation develops, fungal growth is inhibited and GM release decreases.

(2) Relates to exposure to monoclonal-associated antigenic determinants: Whether the EB-A2 monoclonal antibody recognizes and binds to antigenic surface determinants is an important factor affecting the sensitivity of the method.ELISA testing of bronchoalveolar lavage fluid from a patient with a confirmed Aspergillus infection yielded a positive result. However, serial testing of serum antigens resulted in negative results. The investigators concluded that the furanogalactan antigen was definitely present in the blood and was not detected because the GM antigen binds to something in the blood that obscures the antigenic determinants that bind to the EB-A2 monoclonal antibody.

(3) Host's antifungal treatment: whether or not antifungal treatment is carried out before the test is an important factor related to the sensitivity of the experiment.Marr et al [26] analyzed 986 sera from 67 patients and found that the sensitivity of the EI ISA method inside patients who did not receive antifungal treatment could reach 80% , whereas the sensitivity in patients who had been treated with antifungal treatment was only 20%. Therefore, article 10 of the 2016 IDSA guidelines for the diagnosis and treatment of Aspergillosis [27] suggests that routine screening of blood for GM is not recommended in patients receiving antifungal therapy or prophylaxis, but bronchoscopic samples from such patients can be tested for GM (highly recommended; high level of evidence)

Yes, after incubating outside the device at room temperature for 20 minutes, tap rapid test, read a test in about 10s, and you can test several consecutively.

Only one test can be put in the standard test at one time, so it is more recommended to incubate it outside the device and do the rapid test, the standard test is easy to be contaminated.

If you choose heated centrifugation, you will need EP tubes, centrifuges, water baths or metal baths and other consumables.

There will be some influence, so you need to ensure the temperature and humidity control in the laboratory to ensure the accuracy of the results. The sensitivity of the test cannot be guaranteed when the ambient temperature is below 10℃ or above 40℃ and the relative humidity is above 80%.

1. Pre-treatment: insufficient pre-treatment

2. Incubation time: Incubation time is too long, which will result in weakening of the fluorescence signal of the bound group, leading to low data when taking readings, resulting in false negatives.

3. Incubation temperature: the incubation temperature will affect the binding efficiency.

4. Excessive spiking volume may lead to sample reflux, resulting in abnormal results such as false positives.

There may be two reasons. One is that the G test generally precedes the positive GM test, and there may be a window of time for detection; the other is that the GM concentration in the body is elevated for a short period of time, some of which is transient, and there may be a missed detection time or the GM is at a low value (below the detection limit of the reagent). Therefore, it is recommended to perform dynamic testing in high-risk groups, twice a week, in order to increase the positive detection rate.

Basic research has shown that Aspergillus mainly invades the blood vessel wall, along the vessel wall, and basically does not exist in the blood, so blood culture is negative. The appearance of positive is caused by contamination. However, experts also have different views on this point of view, because some laboratories do culture Aspergillus from the blood of IA patients. Therefore, I personally believe that the key to a positive blood culture for Aspergillus should be combined with the clinic, and then determine whether the result is contamination or meaningful.

Table 4 compares the fungal species detected by the G test and GM test respectively. The G test gives positive results for all fungal diseases except Cryptococcus and Saprophytes (including Trichoderma, Rhizoctonia, etc.), while the GM test is for Aspergillus. However, Penicillium can cross-react with GM antibodies, resulting in a false-positive result on the GM test, and the situation is similar for some Cryptococcus.

If the G test is positive and the GM test is negative, it is possible that

(1) Fungal infections other than Aspergillus, such as Candida.

(2) The presence of a factor causing a false-positive G test in a patient without a fungal infection.

If the G test is negative and the GM test is positive, it is possible that

(1) Patients with granulomatous deficiency, patients with granulocyte phagocytic dysfunction, are unable to release 1,3-β-D-glucan from the fungus.

(2) In the early stage of infection, the growth and secretion of Aspergillus is stronger than the death digestion and cleavage of Aspergillus, and the substance 1,3-β-D-glucan detected by the G-test does not reach the positive level.

IFD is often associated with longer-term use of broad-spectrum antibiotics; therefore, IFD and severe bacterial infections often coexist.Whether the G test or GM test is true positive (i.e., the patient does have a comorbid IFD) or false positive (the patient has only a bacterial infection, not an IFD) depends on a comprehensive clinical analysis. For example, does the patient have a granulocyte deficiency? Has the use of broad-spectrum antibiotics failed? Is there a new lesion on high-resolution CT of the chest on top of the original lesion? Do the lesions have features of varicocele pneumonia (e.g., halo sign, air crescent sign, cavitation, etc.)? Are cultures of blood and other sterile sites positive for fungal cultures or direct microscopic examination for mold findings?

In short, the clinic is the final judge of the results of the G test and GM test, and other laboratory tests for fungi (microscopy, culture, PCR technology) also have very important reference value.

(1) If G test is positive and Mn test is negative:

a. G test is a pan-fungal test, which can detect invasive fungal infections other than Cryptococcus and Trichoderma, if there is G positive and Mn negative, it may be other fungal infections, such as Aspergillus fumigatus.

b. Mn (candida mannan) is a structural component of the cell wall of Candida, but its antigen is thermally unstable and easily degraded.

(2) Negative G test and positive Mn test:

a. Differences in release time and amount of marker release:

First of all, this phenomenon out of line may be related to the release time and amount of release of various test markers, for example, (1-3)-β-D glucan is not distributed on the surface of the fungus, and the glucan can only be released by phagocytosis and processing by phagocytes;

b. Presence of interfering factors that affect the test:

This may be a false negative on the G test or a false positive on the Mn test. For example, taking the antiviral drug acyclovir can cause a false positive Mn test and a negative G test.