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1

GENERAL FAQS

3

ASSAY FAQS

7

SOFTWARE FAQS

13

MAINTAINING THE LUMINEX

17

OUR PARTNERS

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This collection of Frequently Asked Questions and tips is not an official document by Luminex Corp. It derives from several sources and our experiences with assays on the Luminex Platform over the years. All hints are to be tried on own risk. In STarStation software most of the mentioned functions are in different locations of the software menus. Nevertheless the functionality is usually similar.

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23 For a complete listing of available product please refer to Biomedica’s MASTERPIECES Catalog for xMAP products.

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22

OUR PARTNERS

GENERAL FAQS

Additional support materials are available on the web pages of our partner companies:

What kind of dynamic range can be achieved with the Luminex 100/200?

APPLIED CYTOMETRIE SYSTEMS / STARSTATION www.appliedcytometry.com/starsupport/

The dynamic range of the Luminex 100/200 can be three to four orders of magnitude depending upon reagent quality and target density

How sensitive is the Luminex 100/200?

LUMINEX

Reagents, reagents, reagents...from the aspect of both immunoassays and DNA applications the system will be only as sensitive as your reagents. The instrument can detect as few as 100 fluorescent phyco-erythrin molecules per microsphere and can report signals as high as 32,767 median fluorescent intensity.

www.luminexcorp.com

How much time is required to read a 96-well plate using the XY-Platform?

MILLIPORE / UPSTATE www.beadlyte.com

The time required to analyze a 96-well plate is approximately 30 minutes to one hour. Read time is dependent upon the total sample volume, the read volume, and the microsphere concentration.

I do not have Flowcytometer experience! Is such expertise needed? No.

ONE LAMBDA www.onelambda.com

PROGEN / MULTIMETRIX

R&D SYSTEMS

What are the indications of successful startup of the Luminex 100/200 and XY Platform? Turn on the Luminex 100/200 and XY Platform and watch for the following indications that the instruments are responding correctly: • Look for the blue lights. One blue light should appear above the Luminex 100/200 sample probe. The other is a small dot in front of the door on the XY Platform. • Listen for the compressor in the Luminex 100/200. It is a low rumbling sound that should come from the front of the instrument soon after it is powered on. Note: The air compressor in the Luminex 200 runs more quietly than that of the Luminex 100. The compressor should still be audible, but at a much lower level. • Place your hand behind the Luminex 100/200 to feel air coming out of the rear fan. • Observe movement of the syringe inside the front middle door of the Luminex 100/200 shortly after the instrument is powered on. Expect a higher pitched mechanical noise as the syringe plunger moves down and then up.

www.rndsystems.com

How do I drain my Luminex Sheath Delivery System? 1. 2.

ZEUS SCIENTIFIC

Click Prime in the Luminex Software (IS Software 2.x) to pressurize the system. Leave the green air tubing connected between the Luminex 100 and the Sheath Delivery System. 3. On the Sheath Delivery System front panel, disconnect the blue tubing from the intake labeled Sheath Out and disconnect the white tubing from the intake labeled Sheath In. 4. On the Sheath Delivery System front panel, insert the white tubing into the intake labeled Sheath Out and insert the blue tubing into the intake labeled Sheath In. 5. Power the unit off/on. 6. Hit the Prime button on the Sheath Delivery System front panel. 7. Sheath fluid will pump from the Sheath Delivery System reservoir into the 20L Sheath Box. Note: To refill the Sheath Delivery System, reconnect the tubing according to color coding and hit the Prime button.

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21 How do I switch from the Sheath Delivery System to Bottles?

What does the Backflush command do?

Steps to revert back to sheath bottles from Sheath Delivery System: 1. Power off and disconnect the SDS from the Luminex 100/200. 2. Turn the black regulator screw inside the centre door of the Luminex 100/200 one full turn counter-clockwise. 3. Attach the sheath and waste bottle containers. Make sure there is sheath fluid in the bottle (to the fill line) and the sheath cap is tightened. 4. Prime the system. 5. As the system is priming, adjust the black regulator screw inside the center door of the Luminex 100/200. Turn clockwise to increase or counterclockwise to decrease, until the sheath pressure value is within +/- 0.1 psi of the normal level of your instrument. Note: Please visually monitor the pressure gauge on the Run Batch tab. Make sure pressure is centered within green tolerance range. 6. When the prime function is complete, loosen the sheath bottle cap to release pressure. 7. Tighten sheath cap and prime the system once again. 8. Make sure that the sheath and air pressure values increase to the normal level once again.

This command flushes sheath fluid from the sheath fluid container through the fluidics system in the opposite direction of sample flow. Backflush the system to perform these tasks: ·to remove obstructions from the cuvette. ·if fluid does not flow through the waste tubing during prime cycles or during sample acquisition. ·if fluid drips from the probe during priming and forms puddles of fluid on the plate. You don’t need to supply solution in a tray. Back flushing takes about 7 seconds.

Where are syringe seal, air filter and sheath filter located? Syringe and sheath filter are front access – behind the front flaps. The air intake filter is located on the right hand rear top side of the instrument. Be careful not to lose the tubing when exchanging the filter

What Clog troubleshooting procedure does Luminex recommend? 1. 2. 3. 4. 5.

Remove the sample probe and sonicate the smaller end for 2-3 minutes. Flush distilled water through the probe with a syringe from the narrow end out through the larger end. Replace sample probe and verify sample probe height. Run 3 Backflush, 3 Drain, 2 Alcohol Flush, and 3 Washes with distilled water. Attempt to calibrate by using 4-5 drops/well. Calibration reagents should not be diluted. Events/sec should reach at least 250 and calibration should pass.

What are the Order Codes for Maintenance items? What procedures should I follow if Calibration and/or Controls fail in the Luminex IS Software? 1. 2. 3. 4. 5.

Verify that the correct Lot numbers and Target Values are selected. Vortex the calibration vials. Verify that the calibration microspheres have not been diluted. Verify that at least five drops of calibration microspheres were used. Verify that xMAP Sheath Fluid or other Luminex approved sheath fluid is being used. 6. Ensure that the Gortex seal in the waste container cap is not wet and that the waste container cap is vented. 7. Verify that the correct wells have been selected if using the XY Platform. 8. Remove the sample probe and sonicate the narrow end for 2-3 minutes. 9. Using a syringe, flush the sample probe with distilled water from the narrow end out through the larger end. 10. Readjust the sample probe height upon replacement in the arm. 11. Complete 3 Backflush, 3 Drain, 2 Alcohol Flush and 3 Washes with water. 12. Repeat calibration. Verify that the pressure readings are between 6 and 9 psi and the Events/Second rate reaches at least 250. If it continues to fail, contact Technical Support.

Can I purchase a second Sample Probe – to have it ready in cases of troubleshooting? Yes, the order code is: LMNX-CN-0007-01 Probe for XYP

LMNXCN-0028-01/1 LMNXCN-0001-01 LMNXCN-0014-01/1

Sheath Filter without Quick Disconnect Air Intake Filter Syringe Seal

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5 1. 2. 3.

Turn on the Luminex 100 analyzer, XY Platform, and PC. Wait for the Luminex 100 IS software to start. Remove the clear plastic shield that covers the sample probe area. In a 96-well microtiter plate where overall height is no more than 0.75 inches, place the appropriate alignment tool in the plate. - For a standard plate with flat-bottomed wells, stack two of the larger (5.08 mm diameter) alignment disks together and place into a well. - For a half volume plate with flat-bottomed wells, stack two of the smaller (3.35 mm diameter) alignment disks together and place them into a well. - For a filter bottom plate, stack three of the larger flat discs together into a well. - For a plate with conical wells, place one alignment sphere into a well. - For round-bottom wells, stack two small disks in a well. - Note: Alignment discs and spheres can be placed in any well as long as the well is designated in the software. 4. Click Eject/Retract to eject the XY Platform. Place the 96-well microtiter plate on the XY Platform with position A1 in the top left corner and click Eject/Retract to retract the plate. 5. Use the 3/32 hexagonal allen wrench to loosen the height adjustment locking screw. 6. Click the Maintenance tab. knurled tubing 7. Use the drop down menu on the bottom, right corner of the screen to define the connector well being used for adjusting the vertical probe height. Click Sample Probe Down to lower the sample probe. height 8. Using the thumb wheel, lower the probe until it just touches the top of the adjustment thumb wheel locking alignment discs or sphere. screw 9. Use a 3/32 hexagonal allen wrench to tighten the height adjustment locking screw. Do not overtighten. 10. Click Sample Probe Up to raise the sample probe. 11. Replace the clear plastic shield that covers the sample probe area.

What procedures should I follow if the Luminex IS Software reports a sample acquisition failure? • • • •

•

Protect the microspheres from light during storage and incubation. Vortex or pipette the samples up and down to verify that the microspheres in the sample are fully resuspended. Verify that the sample probe height has been adjusted properly. If you can calibrate and run system controls successfully, but cannot run samples, then the assay may need to be evaluated. If samples consist of concentrated biological fluids, a dilution may be required. We recommend that concentrated biological fluids, such as serum or plasma be diluted at the concentration given in the assay description or 1:5. In commercial assay systems follow the package insert! If you cannot calibrate, see “Procedure for Calibration/Control Failure in the Luminex 100 IS Software

Incomplete run under Luminex 2.3 •

•

The IS 2.3 software allows you to complete an incomplete batch- when you finish that batch it would generate the output file. You can also export the portion that you have run (export batch) and that should generate an output file. File -> Open Incomplete Batch opens a dialog that displays a list of batches that are incomplete (crash, Cancel All or marked for deletion).

How to run a maintenance command in the middle of the batch: 1. 2. 3. 4. 5. 6.

Select pause Wait until the current command is complete & the status has changed from Pausing to Paused Click Cancel All Run your maintenance command or maintenance batch Click Open Incomplete Batch and select your original batch Click the Start button to continue where the batch left off

What Procedures should I follow for long term Storage of my Luminex instrument? Additional helpful hints for vertically aligning the sample probe follow: •

Hold the door of the XY Platform open as you perform the height adjustment. The goal is to adjust the probe to exactly the point where it is resting on top of the alignment tools but not pressing down too hard on the 96-well plate. After tightening the height adjustment screw, click the “Sample Probe Up/Down” button a few extra times. As the probe raises and lowers, watch through the XYP door to make sure the tray does not bounce up or plunge down.

•

If the gold wheel on the sample arm is difficult to turn, instead, grip the brown knurled tubing connector above the sample arm with your thumb and forefinger and push the probe up and down.

•

Using a spare plate identical to the one used for sample acquisition, cut off the side of row 1. Hold the door of the XYP open as you perform the height adjustment. You will be better able to see the probe hitting the top of the alignment tools in A1 with the side of the plate cut off.

What does the Drain command do? The cuvette in the system can be drained temporarily and refilled in preparation for running. Draining the system helps to remove debris from the bottom of the cuvette. Use drain only for troubleshooting purposes. When draining, you do not need to supply solution. Draining takes approximately 2 minutes and should be followed by a alcohol wash with 70% isopropanol. Any fluid that drains from the system (typically 125ml) drains to the reservoir as the default.

1. 2. 3. 4. 5. 6. 7.

Run a sanitize with household bleach Run a sanitize with distilled water Run 4 washes with distilled water Remove the sample probe from the instrument Use a syringe to flush with distilled water from narrow to the larger end Place it back to the sample arm Wrap sample probe end with parafilm (LX200)

Can bead carry over - from one well to the next - cause false results? Yes. This is easily and automatically corrected by setting instrument parameters to count 50-100 bead events per bead set in a multiplex assay and having results reported as the "median". This will omit out-lying beads with MFI values that will falsely skew the mean. Templates for diagnostic assays are prepared by the manufacturer and account already for all these factors, thus being optimized for best possible result and easy of use.

Why is median the most used statistic for analysis in the IS software? Although the results are not published, Luminex collaborated with a group of statisticians to investigate the sampling distributions of the microsphere-based results. From actual assay results (immunoassays - sandwich capture and competitive formats and nucleic acid hybridizations), samples were drawn and then used in simulations to determine confidence intervals, etc. for various levels of reporter signal. The study showed that the sampling distributions tend to be somewhat skewed - not unexpected since we are dealing with biological assays - with some assay formats being more so than others. The skew appears to come from beads that are "outliers" in the assay, i.e. either very high reporter signal or very low reporter signal. This could be

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19 due to a number of reasons including heterogeneous bead populations - variation in the coupling efficiencies, inadequate suspension or mixing of beads in the reaction, sample carryover (stray beads from another well carried over in the fluidics or an the sample probe), etc. As test statistics go, the mean is much more sensitive to outliers than the median. With the skewed distributions that we have observed and simulated from the bead populations, the median is the more logical choice. lt gives a better measure of central tendency, is less susceptible to the impact of outliers, and is a useful barrier to inter-sample carryover when considering patient test results. Although the median statistic is used for analysis in the Luminex 100 IS Software, many other statistics are may be exported as raw, uninterpolated data via the Output.csv file (Microsoft Excel compatible). Other statistics include: mean, trimmed mean, peak, trimmed peak, %CV, trimmed %CV, standard deviation, trimmed standard deviation, trimmed median, count, trimmed count.

Weekly Preventative Maintenance

Definitions for Statistics in Luminex Software:

•

Luminex statement: one calibration is good for up to 4 weeks.

•

Some labs calibrate every day.

•

Some labs have established routine to calibrate once a week.

• •

Count - Gated events, if gates are set Median - The middle reporter (RP1) value in a list of ascending ordered values

•

Mean - Average of all reporter (RP1) values in the list

• •

Standard Deviation - Reporter (RP1) standard deviation from the mean %CV - Coefficient of variation or relative dispersion (= Standard Deviation/Mean x 100)

• Peak - Most frequent measure in a histogrammed distribution (similar to Mode) Trimmed Statistics: • Trimmed - All trimmed statistics remove the lower and upper five percent of the extreme reporter (RP1) values, then used for Count, Mean, Standard Deviation, %CV, or Peak

•

Remember to dismount/mount Probe/Needle 1x week and sonicate the narrow end with deionised water for min.10 minutes! Prolongation will not harm the needle.

•

Use a syringe to flush the probe with distilled water from narrow end out through the larger end.

•

Replace the sample probe and readjust the height for the plates you will be using.

•

Run 3 Backflushes, 3 Drains, 2 Alcohol Flushes and 3 washes with distilled water.

How often shall I calibrate my Luminex instrument! The recommendations differ from source to source. It will be responsibility of the laboratory to establish the calibration routine according to their needs. Here are some examples:

In any case there are several additional circumstances forcing you to run a calibration (refer to the manual). The most common ones are: •

exceeding the temperature range (+- 3°C) indicated in the “Run Batch” window.

• •

The instrument has remained idle for a long period of time. Sample acquisition is problematic.

•

Calibration Checkpoints

•

Trimmed Mean - Average reporter (RP1) value for trimmed microsphere population •Trimmed Standard Deviation - Reporter (RP1) standard deviation from the mean of the trimmed microsphere population

1. 2. 3.

•

Trimmed %CV - Coefficient of variation of trimmed microsphere population

I have not received the Quality Certificate Document with the target

•

Trimmed Peak - Most frequent measure in a histogrammed distribution of the trimmed microsphere population (similar to Mode)

values of my Calibrators and Controls. Where can I get it?

Trimmed Count - Number of microspheres in the region for which the reporter (RP1) values fall within the nth and the (100-nth) percentile where n = 5%. All Trimmed statistics are calculated off of the Trimmed Count microsphere population.

Can x-MAP kits be used in a FlowCytometer? No! In purchasing products utilizing x-MAP technology, the customer is accepting an end-user license agreement that states that Luminex ® microspheres contained within are for use only with Luminex® instrumentation systems. All 100 bead sets are automatically assigned to discrete non-overlapping positions within the Luminex ® bead map. User manipulation or judgment is not required for "gating" bead sets or quantifying reporter fluorochromes. Reporter fluorescence is displayed as a numerical value in MFI units (Mean Fluorescence Intensity).

Can I use an empty Sheath Fluid container as waste tank?

Sample Probe Height adjustment must be correct Air & Sheath Pressure should be in “green” during priming. Usually in the 6-9 psi range Events/second averages 250 or more

Go to the Luminex Web-Page. In the Support/FAQ section you can download all the available lots of Calibrators and Controls. http://www.luminexcorp.com/support/calibration/index.html

Do I really have to manually fill the form with calibrator values for a new lot? For Luminex IS 2.x software: yes – unless you know another lab operating the same lots. In this case you can export/import the lot data in the form For STarStation Software: no. Import CAL and CON target Values via ACS website: http://www.appliedcytometry.com/starsupport/technotes/104.html

Very good idea!

How do I adjust the vertical alignment of the Luminex 100 sample probe using the Luminex 100 IS Software? (“needle adjustment”) Sample probe height remains a critical factor in efficient sample acquisition when using the XY Platform with the Luminex 100. Remember that the sample probe should be re-adjusted anytime the instrument is moved, the probe is removed and replaced for cleaning purposes, the type of plate you are running changes, or any time sample acquisition is slow or sporadic. Note: Be sure the plate you are using is completely flat - warped plates can lead to incorrect probe height adjustment. To adjust the Sample Probe vertical height:

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7 Athena Daily Startup Template You will need 8 empty wells of a blank microplate. 1. 2. 3. 4. 5. 6.

Run "Athena Daily Startup" Drag-drop the first well of assay to the start location Fill Reservoir with 70% Ethanol Fill 8 wells (1 Strip) with 250 µl Sheath Fluid Eject - put in the plate - Retract Press "Start"

Note: Remember to remove the 70% Ethanol solution from the Reservoir and fill with Sheath Fluid! Athena Daily Shutdown Template You will need 8 empty wells of a blank microplate. 1. 2. 3. 4. 5. 6. 7.

Run "Athena Daily Shutdown" Drag-drop the first well of assay to the start location Select "Save and Load" --> "Finish" Fill Reservoir with Household bleach (Hypochlorite: 15-20 %) or 1M NaOH Fill 8 wells (1 Strip) with 250 µl deionised water each Eject - put in the plate - Retract Press "Start"

ASSAY FAQS

What steps should I follow to prevent leaks from filter bottom plates? When washing samples in a filter plate with wash buffer, buffer will sometimes adhere to the bottom of the plate under one or more wells. The fluid on the bottom of the plate can draw the fluid from within the samples down through the filters causing a leak. If this occurs, wipe or blot the bottom of the filter plate with absorbent material after completing a wash cycle on the vacuum manifold. In addition to blotting the plates, make sure your sample probe height is adjusted correctly for the filter plate wells. If the sample probe is too low, it may puncture the filters and cause leaking.

Bead handling: helpful hints • •

Note: Remember to remove the Fluid from the Reservoir!

Instrument Calibration Template

Do not expose the beads to light. Use a foil to cover vial or dark vials when you have to prepare dilutions. Transferring beads out of low volume vials: - vortex - pulse-spin in mini-centrifuge or flick manually - mix with pipette tip before transferring

You will need 8 empty wells of a blank microplate.

•

1.

Run the "Athena Daily Startup" Remember to remove the 70% Ethanol solution from the Reservoir and fill with Sheath Fluid! 2. Run "Instrument Calibration" 3. Drag-drop the first well of assay to the start location (this example – A1) 4. Select "Save and Load" --> "Finish" 5. Mix the Calibrators and Controls very well (vortex 30 sec max speed) 6. A1 -> 3 drops Cal-1 7. B1 -> 3 drops Cal-2 8. C1 -> 3 drops Con-1 9. D1 -> 3 drops Con-2 10. Eject - put in the plate - Retract 11. Press "Start"

•

•

Do not puncture the filter when pipetting.

Note: Remember to wash 4x with sheath fluid after any calibration to prevent carryover for the following assay!

•

When washing do not over dry your filter – you may not be able to resuspend your beads again. The filter starts to dry from the center, which is visible. Do not handle filter plates on surfaces covered with absorbent material!!

STarStation Startup and Instrument Calibration Script STarStation scripts and the STarStation QC-Plate enables very easy daily setup. This function allows for automatic warm-Up , cleaning, removal of air bubbles and calibration of the system – all in one.

Mix, mix, mix: vortexing and mixing is important to obtain a good suspension. As a rule of thumbs: you hardly can “over-vortex” a bead suspension. When adding beads to a plate: either use your tip to mix in the vial, or put the plate on a shaker. Alternatively you can try to vortex the whole plate by using a standard lab vortex, holding the plate to the side or on top.

Do I have to sonicate my beads? •

Usually bead suspensions are not sonicated but vortexed strongly.

•

In some assays however (like Athena) you have to sonicate the beads before handling them. Follow the instructions. Be sure not to sonicate too long (usually up to 60sec is enough-depending on the ultrasonic device). Doing so may harm the beads!

Filter Plate handling: helpful hints

• •

Make sure that you removed all drops after washing (by blotting and/or wiping) before you proceed with your protocol.

•

During washing adjust the power of your vacuum to ensure smooth removal of liquids. Typically this is done within +-10 seconds. o To strong vacuum may cause reduced ability to resuspend the beads o To weak vacuum may prevent liquid removal in some vials – especially in assays with high serum concentration or overnight incubation procedures

•

If the vacuum is not strong enough because you just use part of the filter plate, you can cover the unused part of the plate with a plate seal to increase the pressure.

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17 • • •

After washing wipe or blot the bottom of the filter plate with a paper towel or other absorbent material to ensure removal of drops. Visually check that! Vortex the plates after reagent addition or for bead resuspension either utilizing a plate shaker (preferred) or a standard vortex . During incubation: cover the plate with a seal or at least with the plastic cover that came with the plate. Protect the plate from light!!

MAINTAINING THE

How should I prepare my serum samples? High concentrations of serum may block the filters in your filter bottom plate or may cause clogging during the data acquisition on the Luminex instrument. When running assays with high serum concentrations (like cytokine assays) or even undiluted serum (like Labscreen assays) it is a good idea to clear your serum samples as good as possible before you start processing them. There are basically 2 ways to do so: •

use a commercial filter system to filter your samples

•

spin down your samples in a centrifuge for 10min at 10.000rpm

What kind of plates can I use to dilute serum samples? •

Follow the assay protocol.

• •

Usually you can use any preferred method that you have established in your lab. Some assays come with dilution plates – but you can use any other preferred blank low binding plate as well.

•

A very comfortable method is to use dilution racks’ with disposable tubes (this example is produced by “Greiner”). This enables you to operate 8-channel pipettes and higher volmes – by that reducing your workload greatly.

Can I refreeze the beads after thawing once? No! Some kits (like LABScreen kits) are delivered and stored at -20°C or below. After thawing once the beads should be stored at 4°C and be used within 3 month.

Can I measure intra-cellular Cytokines on my Luminex? Cytosolic cytokines that have not been secreted can be measured by lysing cells or tissue and analysing the cell lysate. The Luminex® instrument is fully optimized to run up to 100 parameters simultaneously and is not designed to scan or sort individual cells.

What biological samples can be used with x-MAP kits? This depends mainly on the assay design and the kit protocol of the manufacturer. We know of customers using kits for: cell culture supernatants, serum, plasma, tears, blood spots, lavage fluid, urine, tissue lysates, cell lysats, DNA, RNA, amplicons …. In addition keep in mind that many research panels are available for several species like mouse, rat…

Can I run multiple test systems on the same plate? Yes. On the Luminex-side of the Software you can combine assays with identical incubation schemes easily to a “multi-batch”. In diagnostic test systems utilizing special softwares, the results are usually treated as if run separately. So you do have the comfort of processing several test systems in parallel!

How long does a test take to run? There is no general answer to this. Depending on the assay system and the number of parameters used in the test system the time saving can be enormous. The following example demonstrates for the AtheNA ANA Test System: it saves time, labour and cost compared to the IFA and ELISA. A time comparison is shown below.

Maintenance Schedule Though neither difficult or time consuming, maintenance is extremely critical to proper operation of the instrument. Maintenance must be performed, at a minimum, each morning and each afternoon that the machine is used. This process is not labor intensive, involving only a few mouse clicks on the part of the user. In addition to daily maintenance, other types of maintenance (e.g., calibration, cleaning) are necessary. Maintenance Schedules are responsibility of the laboratory and should be established according to recommendations of the manual, frequency of use and the individual service contract with Luminex Corp. Here is an example for how to schedule maintenance tasks:

Daily

Daily Startup, Calibrate, wash between assays, shut down

Routine tasks

Wash between plates, check sheath fluid and waste bottle

Weekly

Visual inspection, clean sample probe (sonicate), flush the system

Monthly

Run calibrations and controls, clean exterior surfaces.

Every 6 month

Replace syringe seal, clean ventilation filter

Yearly

Replace sheath filter and air intake filter

For official Luminex recommendations: please refer to the manual. For detailed procedures: please refer to the manual. For all order codes for spare parts: please refer to the manual.

Is there a Maintance Log Sheet available that I could use? Yes. The Luminex manual supplies you with a log that you could use in the “Maintenance and Cleaning” Section. Alternatively you may love to use the summary sheet of all maintenance requirements which can be printed simply by pressing a button on the main screen of AtheNA Software (if installed) – if not refer to the Instrument manual).

Daily Maintenance Templates This is not an official recommendation by Luminex. It shows how to use the maintenance templates installed on some Luminex instruments. If you do not use these templates follow the instructions of your manual.

Though neither difficult or time consuming, maintenance is extremely critical to proper operation of the instrument. Maintenance must be performed, at a minimum, each morning and each afternoon that the machine is used. This process is not labor intensive, involving only a few mouse clicks on the part of the user. In addition to daily maintenance, other types of maintenance (e.g., calibration, cleaning) are necessary. A summary sheet of all maintenance requirements can be printed simply by pressing a button on the main screen of AtheNA Software (if installed – if not refer to the Instrument manual).

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9 I want to run samples in Duplicate. Where is the according function? There is no “duplicate” button to tick – or any similar function. Generally the Luminex IS Software assigns Samples and Standards with identical Sample IDs/Names as Replicates. Thus it is very easy to create any number of replicates – just by naming the standards or samples identically. The Analysis Module will calculate average and other statistics based on identical Sample or Standard IDs.

AtheNA ANA Test System

ELISA

Total assay time – 2 hours, 15 minutes

Total assay time – 10 hours•

Hands-on time – 15 minutes

Hands-on time – 1 hour, 15 minutes

Can I run SNP assays on the Luminex? pr-ag 09.03.2006IgGBorr Mylab 0574|Melanie Who Thursday Mar 09 2006

Yes. Single Nucleotide Polymorphisms (SNPs) are specific mutations within a DNA sequence. SNPs induce variations between individuals such as signalling pre-disposed risk of disease, adverse reaction to drugs and response to drug treatment. At least10 million individual SNPs exist within the human genome. The key to design SNP assays on the Luminex platform are the FlexMAP Beads: FlexMAP beads are 100 Luminex beads with 100 unique anti-tag DNA regions attached to their surface. These antitags allow specific SNP sequences to be determined in assay procedures such as the Allele-Specific Primer Extension (ASPE) assay. Our partner ACS is also the only company in Europe selling the FlexMAP beads at present.

A1,Leerwert B1,Negativ C1,Positiv D1,6B10352712

The Web based Tag-It™ Oligo Design Software application can be used to prepare orders for tagged gene specific oligonucleotides (GSOs) and their respective FlexMAP™ beads. https://tagit.luminexcorp.com/tagit/welcome.jsp The significance of the anti-tag is as follows:

E1,6B10352731-1:100

•

Each bead number has a specific anti-tag.

• •

DNA used for a SNP assay has a primer designed to compliment the specific antitag on the chosen bead. A specific anti-tag primer is designed for each SNP being analysed. During the assay, biotinylated dNTPs are used.

•

When the beads and the DNA strands are mixed together, the DNA strands will hybridise with each specific anti-tag sequence contained on each bead.

•

When the beads are acquired on the Luminex machine, they are identified into their specific region on the bead map (red leaser), and then the biotinylated SNP sequence coupled to SA-PE is determined by the green laser (see principles of assay).

Note: STarBase, the SNP analysis module for STarStation software is available as an additional product. This software makes SNP easy with several advanced functions. For detailed information please contact your local Biomedica representative. • Results are displayed graphically as allelic scatter plots •

Genotype calls are automatically made from preset templates

•

Thresholds can be set to refine your results with questionable samples

• •

Perform up to 100 simultaneous DNA and RNA assays from a single sample Visualise, analyse and quantify genotyping experiments with ease

I acquire my data right this moment. Which are the control beads in my assay to get a first impression? •

•

In Athena assays the control beads are labelled “NS-Cntrl” if you watch in the “Diagnostics” tab window or in “Acquisition details”. There will be no practical use of this information because the assays utilizes a sophisticated flexible intra-well calibration system. You cannot compare any of the values on the level of raw data! In Multimetrix assays you will find a bead labelled “CR1” as a positive control bead.

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15 •

•

In LABType/Class I assays Bead#13 is control for Exon 2, Bead#32 is control for Exon 3 Bead#35 runs as negative control bead In LABType/Class II assays Bead#34 runs as positive control bead Bead#35 runs as negative control bead

What are the rules to call a positive LABScreen PRA result valid? • •

First (=highest) result (raw data) must be >500 Second highest (raw data) must be >300

•

For all LABScreen assays: PC/NC Ration must be >2

PCR/LT: Do I need to use loading buffer to gel my amplicon? No.

Do not be irritated: Luminex sells the original and therefore fully compatible version of the instrument in several OEM versions. Most of these are identical in configuration and therefore fully compatible with other softwares. There are just few “closed” versions of Luminex instruments on the market.

Will my LABtype/LABScreen/AtheNA/Multimetrix/Plexus Assay run on STarStation Software? YES. STarStation Software is the best Software solution on Luminex platform when it comes to optimizing your workflow in a pure research environment. Features like the multi user login,optimized assay handling, one-buttonstartup, automatic generation of standard curves and the ability to reanalyse you data easily are ideally suited to industrial and clinical research centers. As any Starstation instrument comes with additional original Luminex IS Software preinstalled you will stay perfectly compatible with any other software like HLAVisual and many others.

How shall I change settings in Luminex IS 2.3 for different analysis

PCR/LT: How long should I run my gel?

software’s running on my Luminex PC?

The speed depends on the voltage. The higher the voltage - the shorter the time. Typical times would be around 15min at 200V for a regular gel. Running a minigel lasts typically 3-5min.

The main concern here are the settings for the output data in Luminex IS software. The menu is located in Tools/Options/Data Export (see picture #1) • HLA Visual: everything in “Additional Export Stats” and “Additional Batch Information” should be tagged (see picture #2). In addition “Copy to Output.csv too..” should be tagged and in “Export location Label Style” you should tag “Both…” • AtheNA: everything in “Additional Export Stats” and “Additional Batch Information” should be untagged. • PrAG: everything in “Additional Export Stats” and “Additional Batch Information” should be untagged. • STarStation: STarStation Software does not interfere with Luminex IS 2.3 settings at all. • OpenOffice: feel free to decide what data you want in your output.csv file. Whatever you have tagged you will get.

PCR/LT: How can I prevent evaporation in my PCR-plate? By the use of pressure pads suiting your PCR-cycler! These pads are available from One Lambda for several popular cyclers. They are used to enhance the pressure of the lid to hermetically seal the plate.

PCR/LT: Is there any alternative to ice usage after the neutralisation in LABType assays? Yes. You can use cool racks as shown in the picture. These racks are available from several companies (the picture shows “Eppendorf”)

PCR/LT: We have a thermocycler different to PE 9600/9700 in our laboratory. How can we adopt our cycler to One Lambda protocols? All the protocols in One Lambda assays where optimized to the mentioned popular and widespread cyclers. If you have a different one you may try to adjust the ramp speed of your instrument to 1,5°C / sec down and 1,0°C / sec up respectively. In any case you will have to validate your assay.

What are the settings for my centrifuge to spin down the beads during a washcycle in One Lambda “flicking” protocols? Some assay protocols (like LABtype and LABscreen) recommend the “flicking method” for washing bead assays. In this very reliable method you either use a PCR-plate or a conical ELISA plate for your assay. Utilizing a plate compatible swing bucket in your centrifuge enables you to spin down the beads – thus forming a pellet. Plate spinning is usually done at 1300g. Depending on the rotor of your centrifuge the rpm value may vary. Please refer to the manual of your centrifuge to obtain or calculate the correct value. Popular centrifuges are set in the 2500-3200 rpm range. This may be different for your model!!!!

AtheNA and PrAG Software are identical in their setting-needs.

I selected the data file for analysis in PrAG-software – and nothing happens. The window to the right is still empty. May I still validate my results or is my assay lost? Your assay is not lost. Most probably you have selected a wrong Lot number during preparation of the patient list. Follow this procedure to correct the wrong Lot entry in the data file: 1. locate the patient list in the directory specified in the "Options" menu of PrAG-software under "LIS-Path". 2. open the patient list in a text editor. Remark: do not use Open Office or Excel. This may make your file incompatible. 3. the first 4 letters in line 4 refer to the lot selected in the software during setup of the assay. Lot specific information is used by the software to calculate result indexes correctly. 4. Correct the Lot number accordingly ; save the file. 5. Restart your evaluation.

I want to prepare a template with the standards in Duplicate, but I cannot find the according function?

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11 What format do I need to follow to add a patient list in IS?

How can I spin PCR plates in my centrifuge?

Creating external patient lists can be very useful if you run several test panel on the same samples. Here are the steps to follow: • You apply a Patient List to any batch or multi-batch only during batch setup at the Luminex Batch Setup dialog box. • You can create a Patient List text file using Windows Notepad or a text editor. • A patient list must be in a text file format (.txt) to be imported into IS 2.2. The patient list text file must meet the following requirements: - The first line of text in the file must be "LX100IS Patient List" - The second line of text in the file must be "[Accession#, Dilution Factor]" - Any following lines of text should be only in the format, "x,y" Where x=accession ID number for the patient and y=dilution factor (if no dilution factor use "1"). - Patient list entries are case sensitive.

As mentioned before you need to have a compatible swing bucket in the rotor of your centrifuge. In addition you will need a PCR plate adaptor fitting in your centrifuge. Many vendors of centrifuges supply these adaptors (like “Eppendorf”). As a low cost alternative you may try to use standard PCR plate racks as shown in the picture (CLP-Atlas; # 3510.A 96x0,2ml tube rack)

Example: LX100IS Patient List [Accession#, Dilution Factor] Joe, 1 Sue, 1 Leo, 1 Max, 1 Eve, 1

Can I see the beads during processing an assay? Sometimes. The microspheres produced by Luminex Corp. have a diameter of 5,6 µm. As such they are far below visibility by naked eye. They will be visible under microscope or – under certain circumstances - in pellet form. • •

In Filter plate assays generally you will not be able to see them. Neither in Suspension nor on the filter membrane after washing. In some flicking assays (like LABscreen PRA) you may be able to see a tiny bluish pellet after spinning

Where do I find the templates for my assay? • • •

For One Lambda assays: whenever you receive a new lot you download the files from download.onelambda.com For Zeus assays: You receive a CD with every kit. The files are on the CD

Note: If you attempt to load an improperly formatted patient list file through the batch setup dialog, then the command list grid may label the samples in an unexpected manner. The file should always list the patient information per line as: Patient Name, Dilution Factor. If additional numbers are added to the right of the dilution factor, then the grid will concatenate these values into a single dilution factor. For example: (“Bob, 10, 5, 3” would produce Name = “Bob” and Dilution Factor = “1053” instead of “10”).

For Multimetrix/Progen assays: You receive a CD with every kit. The files are on the CD • For Research assays: you have to learn how to create templates. Creating a template is a matter of several minutes. Once you have prepared the template you do not need to do that every time you run an assay. If you have problems obtaining or creating templates – please consult the kit inserts or contact your Biomedica representative to assist you.

Can I run any Assay on any Version of the Luminex machine?

What is the naming convention for New Batches?

No. As routine or diagnostic assays usually utilize very user-friendly software for calculating final results there are minimum requirements to be fulfilled on the instrument side. Here is a general guideline on compatibility of various Luminex Versions and retails. Details may or may not be answered on request on a case by case basis.

Technically there is none – but it will be wise to follow certain convention to facilitate easier access to your data. The most common convention is: • Year-Month-Date-Assay System-Lot

•

•

• •

Luminex 1.7 or older (the “old” Luminex): NO. Hands off. Still you may be able to run certain LABType or LABScreen assays from One Lambda. Most research applications will be able to acquire raw data – but there is no software to handle standard curves etc. For analysis you may need additional statistic software or a least a spreadsheet like OOCalc or Excel. Some instruments however may be updated to IS 2.3. Luminex 100/200 IS V2.3 / AtheNA / LABScan / STarStation: YES. This is the best solution when it comes to compatibility. Any standard software will be compatible to this configuration – though there may be some settings that you have to change to make output file formats “like” each other. Any research assay will run with these configurations too. Bioplex Systems: NO. Bioplex Software is not compatible with most other evaluation softwares. Still you may be able to establish certain research assays. Other “closed/modified” versions of Luminex Instruments (not Luminex branded): NO. As long as the Instrument does not run the original Luminex IS Software, you can consider it to be not compatible

The question around compatibility and “openness” of the instruments is mainly: • Does the instrument run the original Luminex 2.3 software? •

As a rule of thumbs: If it does not – hands off.

• For research assays many laboratories use “experiment IDs” instead or with the “Assay System” information. Be aware that you are restricted to Windows file naming convention. Do not use “strange” / local characters.

I run assays from different vendors on my Luminex. How should I set the vertical Probe Alignment? There is no general recommendation. 3 ways how to deal with it: • Set the alignment whenever you change the plate. • •

Use one plate type for reading out all results (e.g Nunc plates and Milipore Filter plates sometimes are ok) Try to find a compromise that fits to all your plate needs. Limiting factor (and as such usually the start for your evaluation) is the assay with the lowest sample volume for reading out the results! In any case you have to test the combinations used in your lab.

In which order does the Luminex read the samples? The system analyzes microtiter plate samples in the following order: vertically, from top-to-bottom within the column and left-to-right after each column. In StarStation software however you are free to use any other preferred plate layout.

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13 How quick shall I read out my assay after labelling? This depends on the assay design mainly. Most xMAP assays are rather stable after labelling/stopping. When you decide not to read your assay immediately: store it in the dark at 4°C and do not forget to vortex/shake the plate before reading. Some assay may require another wash or spin before resuspending to the recommended volume for reading. •

LABScreen assays: store at 4°C up to 24h

• •

LABType assays: storage at 4°C for longer than 4h may reduce signal Athena: read within 1h

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Multimetrix: read within 1 h

•

Research: many research assays can be stored at 4°C up to 24h (or even much longer)

Do I have a second chance to read my assay? Usually Yes. For troubleshooting reasons you may try to read an assay a second time. During first reading the sample volume in the wells was increased due to dilution with sheath fluid. The decreased concentration of microspheres and the dilution obviously will increase the reading time. In any case you should shake (2min) or vortex the plate before rereading.

SOFTWARE FAQS

Luminex IS: How can I import result into MS-Excel on my local PC? Luminex IS data files are saved in CSV (Comma Separated Values) format which is widespread in the world of laboratory IT. On the Luminex instrument usually English number settings are used. For example, the number 1,234.56 has a comma as the digit grouping symbol and a period for the decimal symbol. The number settings for most European regions in Windows 2000 and XP Regional Options will use a period as the digit grouping symbol and a comma for the decimal symbol, i.e., 1.234,56. If you try to import CSV-files on a computer that has the Windows Regional Options set for any region other than English (United States), data exported from Luminex Software in CSV-format may be imported as text rather than as numerical values. The data set will therefore not be recognized by Excel as containing valid numbers, and calculations cannot be performed.

Sometimes you may want to follow this procedure to increase the microsphere concentration again: •

(verify correct sample probe adjustment)

•

vacuum your filter plate or spin/flick your Whatman low volume plate

• •

resuspend with assay buffer or sheath fluid to the volume stated in the assay instructions vortex or shake

•

read plate as usually (accept slower reading time compared to first read)

There are basically 2 ways to deal with that: 1.

The trick to circumvent this problem is to temporarily change the regional settings of your Windows System to English (United States). Then import the CSV-file (usually C:/MyBatches/Output/yourbatch.csv) by dragging it over the Excel Icon on your desktop. Excel will open, load and interpret the CSV as numeric values resulting in a beautiful spread sheet that you can directly use for further calculations etc. Do not forget to save the file (“save as”) in .XLS format and to switch back to your local regional settings. Regional Options may be changed in Windows 2000 and XP as needed. Excel must be closed at the time the Regional Options are set in order for the new settings to take effect in the application. If the application is not closed at the time the settings are changed, the application must be closed and restarted before the settings will become active.

2.

If you have Open Office installed on your Luminex PC you can easily load your file to OO (ok the import message without changing anything) and save it as Excel file. Then move it to your Local PC and load it directly to Excel. This approach has the advantage to have an office package available on the PC and to be able to associate .CSV files in windows directly to Calc which is part of the OO package. To do so you click on any .CSV File and rightclick “properties”. In the “open with” dialog you search for “Calc” of the OO Package and select it. By this you will permanently associate .CSV files to be opened with OO-Calc whenever you directly double-click them.

Note: For STarStation software just directly import your data to Exel by utilizing the Exel-Import module.

Where can I change the Regional Settings on my local PC? Win2000: Start/Settings/Control Panel/Regional Options. Windows XP: Start/Settings/Control Panel/Regional and Language Options