• 1. ADXBLADDER Samples
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  • 1. How much urine does the test require?

    Greater than 10mL and up to 50 mL of urine is required to run the ADXBLADDER test. The patient should always be asked to provide the whole urine void, then an appropriate portion of that void can be aliquoted for use in the ADXBLADDER assay. Ensure the sample is well mixed before aliquoting.

    More urine increases the potential sample available for testing.

    For example:

    50mL urine 500μL 4 x 100μL samples
    25mL urine 250μL 2 x 100μL samples
    10mL urine 100μL 1 x 100μL samples

    We have found that showing the patient a collection container with an example volume of 50mL of coloured water significantly increases the number of patient samples greater than 50mL.

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  • 2. The sample is greater than 50mL, what do I do?

    Between 10 and 50 mL of urine is required to run the ADXBLADDER test. If you have a greater volume than 50ml, this may be decanted into separate tubes and processed as additional patient samples, or simply discarded.

    It is important to ensure the whole urine sample has been thoroughly mixed to prevent premature sedimentation prior to decanting into the centrifuge tubes, which could result in inconsistencies between samples.

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ADXBLADDER Sample Preparation

  • 1. ADXBLADDER Sample Preparation
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  • 1. I can’t see a cell sediment pellet in the centrifuge tube when I spin down the urine sample

    Not all samples will create a pellet that is easy to see with the naked eye in a conical centrifuge tube, and this is not unusual in our experience. In these cases extra care should be taken when discarding the urine supernatant to ensure any cells in the tube are not lost. Please remember that the inability to see a pellet with the naked eye does not necessarily indicate the absence of any cells.

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  • 2. The lysate is not homogenous/difficult to pipette, what do I do?

    Some samples may be viscous after lysing or there may not be enough liquid in the prepared lysate to test. There are a number of ways to deal with these:

    • 1. Centrifuge the lysate for a maximum of 5 minutes at 9500g and test the supernatant
    • 2. Use a larger pipette tip to pipette the viscous liquid
    • 3. Add more lysis buffer (not greater than the volume of lysis buffer initially added) and vortex
      to mix – the results for these samples should include the calculation to account for the extra dilution step and should be interpreted with caution, especially if the results fall near to the analytical cut-off.
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  • 3. How long can I store urine samples before processing?

    Urine must be processed within 48 hours of collection.
    Samples may be stored at room temperature for up to 8 hours and then at 2 - 8°C for up to 48 hours prior to processing. Do not freeze urine before processing.

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  • 1. ADXBLADDER Protocol
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  • 1. Can I automate ADXBLADDER?

    ADXBLADDER has been developed as a manual ELISA, but like other manual ELISAs it may be validated on open automated platforms by the user. Application notes are available for some platforms – contact your Arquer Diagnostics distributor for assistance. Each lab should validate ADXBLADDER on their system.

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  • 2. Do I have to run Positive and Negative Controls every time I run the ADXBLADDER assay?

    Yes, the Positive and Negative Controls are used to calculate the results and the validity of the assay.

    Patient sample results cannot be determined positive/negative without the positive control to normalise the data. The negative control ensures there are no inherent issues with the plate, such as high background, which could give falsely elevated results.

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  • 3. Can I pool samples?

    Pooling of samples has the potential to dilute any positive samples to such a degree that they are no longer positive, especially those that contain small amounts of MCM5 positive cells in the urine. Arquer Diagnostics DO NOT support pooling of samples.

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  • 4. Should I wait until I have enough samples to fill the microplate?

    No; although ADXBLADDER requires that you run the Positive Control and Negative Control (lysis buffer) each time you run the test and therefore it is most efficient to run as many tests as possible. The ADXBLADDER kit has sufficient reagents to run up to 18 wells of positive control.
    We appreciate that it is not always desirable to wait so the microplate is presented as 12 x 8 microwell strips that can be run independently.

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  • 5. Should I test in duplicate?

    Arquer Diagnostics test samples and controls in duplicate and take a mean of the reading in order to control for pipetting errors.

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  • 6. Can I manually wash the ADXBLADDER assay?

    Arquer Diagnostics Ltd. only endorse automated washing of the ADXBLADDER microplate. Any manual washing must be validated by the user.

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  • 7. Is ADXBLADDER affected by other components in the urine?

    Arquer diagnostics have tested the assay for sensitivity to a range of contaminants that can commonly be found in some urine samples such as blood or cellular components. See the product “Instructions for Use” document “12.3 Potentially Interfering Substances” for details.

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Interpreting ADXBLADDER

  • 1. Interpreting ADXBLADDER
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  • 1. The Positive or Negative controls fall outside of the ranges given in the IFU

    Positive Control or Negative Control results that fall outside of the ranges given in the IFU invalidate the assay, in this case the assay should be repeated. If the control results remain outside of the given ranges the assay should be considered void. If issues persist, contact your local Arquer Diagnostics distributor.

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  • 2. My sample is close to the positive/negative cut-off

    The cut-off has been calculated using the data collected from a prospective clinical trial of 856 patients. If the sample returns results near the cut-off, with a valid assay, there is no reason to doubt the validity of these results. As usual, all results should be interpreted in conjunction with patient history.

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  • 3. False Positives – positive results from known bladder cancer negative patients

    Check the “Instructions for Use” document Section - 11. “PERFORMANCE LIMITATIONS”. False positive results may come from a number of potential sources:

    • Proliferative cells in the urine from other sources such as prostate/other genitourinary cancers and prostatitis or damage caused by renal calculi and kidney stones or recent urological investigations.
    • Contamination of test when making up controls – we suggest that controls are prepared in a separate area to those where sample preparation/ELISA testing is conducted. Change gloves between control and specimen preparation
    • Additionally, published literature has shown that up to 30% of bladder cancers are not detected by initial cystoscopy1,2.
      1. 1. Jocham D et al. Photodynamic diagnosis in urology: state-of-the-art. Eur Urol 53(6): 1138-48 (2008)
      2. 2. Zhangqun Ye et al. A comparison of NBI and WLI cystoscopy in detecting non-muscle-invasive bladder cancer: A prospective, randomized and multi-center study. Sci Rep.; 5:10905 (2015)
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  • 4. False Negatives – negative results from known bladder cancer positive patients

    No test is 100% accurate. However, the greatest potential for false negatives is loss of sample cells or whole pellets in specimen preparation. Check the below points to ensure optimal specimen preparation.

    • Urine samples are thoroughly mixed to re-suspend the cells prior to decanting for centrifugation
    • The cell pellet is retained when liquid is decanted after centrifugation. We suggest decanting the liquid back in to the specimen collection container to ensure that if the cell pellet is dislodged from the centrifuge tube it is retained and can be spun down again.
    • Cells are thoroughly suspended in lysis buffer.
    • <5% of total bladder tumours are leiomyosarcomas. These cancers of the mesenchymal
    • layer develop beneath the bladder epithelial layer and therefore do not come into contact with the urine and as such are unlikely to be detected by ADXBLADDER. These cancers are also difficult to diagnose by cystoscopy and usually require additional imaging techniques to diagnose.

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  • 5. The duplicates of a sample have significantly different score

    In samples where the two replicates give clinically discrepant results (i.e. one well is positive, the other negative), the test should be repeated once using the retained sample. If the result remains discrepant then an invalid result is reported - if the retest is concordant then the re-test result is the final result. The sample must be re-tested within 24 hours. Samples should not be subjected to more than one freeze thaw cycle as freeze-thaw effects can lead to reduced capture of the MCM5 protein in the assay. If this cannot be achieved, a fresh sample must be obtained for re-test.

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  • 6. The duplicates of a control have significantly different score

    Any Positive Control or Negative Control duplicates that are significantly different may indicate a procedural issue such as inadequate washing or a pipetting error, and patient results from such assays should not be reported. Check that all equipment is functioning correctly before repeating the assay. If issues persist, contact your local Arquer Diagnostics distributor.

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  • 1. Other
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  • 1. Can I use ADXBLADDDER for Veterinary Applications?

    ADXBLADDER has been developed for human use. However, the MCM5 protein is well conserved across the animal kingdom. Any veterinary applications should be validated by the user.

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