Simoa HD-1 Analyzer Troubleshooting Guide. USER Sep 2014 Software Version 1.3 Beta

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Simoa HD-1 Analyzer

Troubleshooting Guide

USER-104-28

17 Sep 2014

Software Version 1.3 Beta

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Customer Support

Customer support is available 8 AM to 8 PM, EDT.

Phone: 1-877-786-8749 Email: [email protected]

Contact Us

Quanterix Corporation 113 Hartwell Avenue Lexington, MA 02421 Phone: 617-301-9400 email: [email protected]

The Quanterix products referenced in this document are for research use only and are

not for diagnostic or therapeutic procedures.

Simoa and Quanterix are registered trademarks of Quanterix Corporation.

Amicon is a registered trademark of Merck KGaA, Darmstadt, Germany. ProClin is a registered trademark of Rohm and Haas Company. EZ-Link is a trademark of Thermo Scientific. HulaMixer is a trademark of Life Technologies Corporation. NanoDrop is a trademark of Thermo Fisher Scientific.

This product is protected by US and Foreign patents and patent filings.

Quanterix Corp. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Quanterix Corp.

The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.

Quanterix Corp. makes no representations that this document is complete, accurate, or error-free and assumes no responsibility and will not be liable for any errors, omissions, damage, or loss that might result from any use of this document, even if the information in the document is followed properly.

This document is not part of any sales contract between Quanterix Corp. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.

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Instrument Troubleshooting

Problem Suggested Action

Need to diagnose root causes of instrument problems. Create a support package and then power-cycle the instrument. The support package will help Quanterix™ determine root causes and implement corrective action.

1 Touch Start at the left of task bar.

2 Touch the Search Box to access the keyboard. 3 In the Search Programs and Files field, enter

support package. At the top of the Program List,

you should see this program name: Create Support

Package.

4 Touch Create Support Package. The file package will build and zip onto the desktop. The zipped file has the current date as its name.

5 Turn the instrument off, then on again. You do not obtain results for the first 8 samples in an

assay run. The Simoa system expects the first 10 cuvettes to beloaded into the instrument before a run begins. If the cuvettes are not loaded, no beads are added to the first 8 samples.

Be sure to run the Prepare for Run maintenance task before you begin a run. This task loads 10 cuvettes into the instrument.

Bead fill starts low and rises as run continues. Beads were not resuspended according to the instructions in the assay package insert before they were loaded into the instrument.

Be sure to follow the package insert instructions regarding preparation of the bead reagent. Note that the Simoa system displays a reminder message regarding the need to resuspend the beads. Homebrew results data issues. Confirm instrument function and data reliability by

running a PSA assay and examining the results. The PSA calibration is very stable.

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USER-104-28 09-Sep-2014 Page 5

Problem Suggested Action

In Resources Tab: Number of Discs Available is not

correct. “System detects no disc on the disc carrier.” If both drawers are open when discs are scanned, thenumber of discs will not be accepted by the software. Only one drawer can be open when scanning the disc barcode.

First sample flagged and has no data. Prior run was not in multiples of 24 (total calibration and samples). To ensure processing of the first sample, always program calibrators and samples in multiples of 24 (24, 48, 72, etc.).

Problems arising from non-Quanterix beads. Only Quanterix beads have been tested and are supported.

Run is blocked by inadequate resources. Resources

required are twice the expected amount. While setting up the run, the plate rack was insertedtwice. To clear, return to the Setup Run tab, remove the sample plate, and set up the run again.

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USER-104-28 09-Sep-2014 Page 7

Assay Troubleshooting

Problem Causes and Suggested Actions

Positive signal in negative control. Reagents or samples are contaminated. Use fresh reagents and pipette carefully.

The detector antibody is detecting the coating antibody. Check the background of coating antibody and detection antibodies.

Excess antibody is causing nonspecific binding. Reduce the amount of antibody.

High background across entire plate. Conjugation time is too long.

Use the recommended conjugation time. There is a problem with the substrate solution.

Use fresh substrate solution and incubate substrate in the dark. Laboratory glassware introduced contaminants.

Ensure that reagents are fresh and prepared in clean glassware. Antibody loss during buffer exchange. The buffer exchange step was performed incorrectly.

Make sure that you have collected the entire volume of concentrated antibody. Rinse the filter membrane and collect the rinsate.

The antibody is incompatible with the buffer exchange method. Try a different buffer exchange method, such as dialysis. Try a buffer with a different pH.

You used an incorrect baseline buffer instead of the antibody buffer.

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Low signal. Target protein is not expressed or is expressed at a low level in the sample used.

Increase the amount of sample used. Ensure that you are using a positive control within the detection range of the assay.

Insufficient antibody.

Check that you are using the recommended amount of antibody. You may need to increase the antibody concentration to

optimize results.

The capture beads are not performing adequately.

Prepare the bead concentrate again, making sure to carefully follow the instructions in the Homebrew bead coating protocol in the Homebrew Assay Development Guide (USER-181-01). The detector is not biotinylated.

Prepare the detector concentrate again. Carefully follow the instructions in the Homebrew detector biotinylation protocol in the Homebrew Assay Development Guide (USER-181-01). Check the biotin/antibody ratio by biotin quantification assay (Pierce, 28005).

Increase the molar excess ratio for biotinylation.

Substitute other biotinylation reagents in the Homebrew detector biotinylation protocol to produce more efficient biotinylation.

The detector and/or SBG concentration is too low. Increase the detector and/or SBG concentration. Reagents are not fresh or not at the correct pH. Ensure that reagents are fresh and prepared correctly. Aggregated beads. One of the conjugation parameters is incorrect.

Verify that you are using the correct amount of beads, EDAC concentration, and antibody concentration.

Reduce the antibody concentration (e.g., 1 mg/mL). Verify the pH in the conjugation reaction.

Visually confirm that the rotation speed during incubation is high enough to prevent beads from settling. Increase the speed if necessary.

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USER-104-28 09-Sep-2014 Page 9

The antibody concentration is too high. The concentration was measured incorrectly.

Repeat the measurement, making sure that you use correct technique and the proper measuring equipment. Make sure that you are subtracting the background correctly and using the correct antibody extinction coefficient.

The buffer exchange was incomplete. Repeat the buffer exchange.

Poor bead conjugation efficiency. One of the antibody buffer components is hampering coating (for example, Tris).

Try using a buffer with different components.

Repeat the buffer exchange multiple times to remove the component that is interfering with coating.

Antibody concentration is too low. Increase the antibody concentration. Low yield of detector antibody. Detector antibody was lost during filtering.

Carefully follow the Homebrew detector biotinylation protocol in the Homebrew Assay Development Guide (USER-181-01) and collect the entire volume, including from rinsate.

Over-biotinylation is causing precipitation. Reduce the molar excess of biotin reagent. Poor quality calibration curve. Calibrators are not properly prepared.

Prepare the calibrators again.

One or more reagents are performing incorrectly.

Make sure that all reagents have been correctly prepared and diluted to the correct concentration.

Make sure that all reagent bottles are filled and loaded properly. Check reagent quality and use substitutes for any reagents that are not performing adequately.

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Low-level calibrators have been contaminated by high-level calibrators.

Prepare the calibrators again using proper technique. A high-level calibrator is saturated.

Shift the calibrator range lower. Low-level calibrators are flat. Shift the calibrator range higher.

Simoa HD-1 Analyzer Troubleshooting Guide. USER Sep 2014 Software Version 1.3 Beta