White Paper Automation Of X MAP Technology Based Multiplex Assays

: Pdf White-Paper-Automation-Of-Xmap-Technology-Based-Multiplex-Assays White-Paper-Automation-of-xMAP-Technology-Based-Multiplex-Assays 09 2017 clpmag uploads wp-content

Open the PDF directly: View PDF .
Page Count: 12

Whitepaper

Introduction

Automation provides a number of benets to clinical, industrial,

and research laboratories, including elimination of human errors,

improvement in consistency, minimization of contamination, in-

creased throughput, decreased costs, and reduced hands-on time.

At the same time, multiplex assays oer the benets of more data

per sample, faster reaction times, reduced sample consumption,

enhanced scalability, and a number of additional benets due to

elimination of repetitive steps.

Integrating laboratory automation with multiplex assays pro-

vides signicant eciencies to workow processes, especially

when a large number of samples are being analyzed, or when a

highly multiplexed assay is being developed. Automation can be

partially or fully implemented. For example, a low test volume lab

may automate only the washing steps of an assay, whereas a core

facility or high volume testing lab may want to automate all assay

steps—including incubation, microplate washing, thermocycling,

and microplate shaking—into a single, walkaway system. Multiple

automation criteria should be considered for each lab, such as plate

format, speed of liquid handling, deck capacities, and system and

software upgradability for future considerations.

Luminex® xMAP® Technology is designed for multiplex assays

in clinical, industrial, and life science research labs that require

fast and repeated assays of multiple targets. e platform allows

multiplexing of up to 500 unique assays within a single sample.

High sample throughput is needed in diagnostics reference labs or

during clinical trials. High throughput is also needed during drug

screening stages, although multiplexing is also an advantage dur-

ing such processes. A clinical reference lab may test thousands of

samples per day in low to moderate multiplex, while higher mul-

tiplexing may be needed in a research lab testing dozens of tar-

gets per sample. Flexibility to customize assay steps for both the

throughput and multiplexing needs of each individual lab is a dis-

tinct benet of xMAP Technology.

Microsphere or bead-based arrays oer advantages such as re-

duction of sample consumption and hands-on time, optimizing

eciency during assay development and performance. Having

the capability for both protein and nucleic based applications on

a single platform is another advantage for labs with limited bench

space for multiple equipment footprints. Unlike traditional immu-

noassays or nucleic acid arrays, xMAP bead-based assays occur

entirely in a liquid medium without having to interact with solid-

phase substrates. is makes them ideal for rapid reaction times

and liquid handling automation.

As evidenced by the more than 27,000 publications to date,

xMAP Technology has become the most widely used multiplex-

ing technology. Concerns such as uniform dispensing of beads,

well-to-well carryover, and minimization of bead loss during

transfers are easily mitigated with a robust automation protocol.

e aim of this white paper is to provide examples of automation

solutions and how these have been implemented into labs using

xMAP Technology.

Semi-Automated Liquid Handling Workstations

Multiplex assays oer a number of advantages over singleplex as-

says, but require a number of upfront preparation steps before a

fully automated protocol can be implemented. Liquid handling

robots not only have the advantage of improving output eciency

during sample processing, but also during disconnected prepa-

ratory steps. ose individual processes can each be carried out

equally well by appropriate semi-automated equipment that ts

quickly and easily into an existing routine. xMAP Technology itself

is a compilation of various stages of work, including bead coupling,

coupling conrmation, plate preparation, reaction incubation, and

washing steps, all of which can be partially or fully automated.

Standardizing an assay to microplate size (96- or 384-well for-

mat) is an important rst step in reducing both reaction volume

and total assay costs. High throughput single or multichannel pi-

pette dispensers, currently available on the market, are designed

to dispense very precisely measured quantities of any suspension

into dierent microplate formats. e core elements of these ro-

botic systems are a liquid handling arm that is moveable along the

x- and y-axes, a pipetting head mounted on the forefront of the arm

moveable in the z-direction, and pipetting channels that transfer

the liquids from tubes, reservoirs, and plates into the pipetting tips.

Automation of xMAP® Technology-Based

Multiplex Assays

Tomasz Zborowski, M.Sc

2

Automated Liquid Dispensing Systems

Automated dispensers range from single-channel instruments

(one volume at a time) to multichannel dispensers capable of dis-

pensing up to 384 aliquots simultaneously (Table 1). Software-

controlled sample dilution and multiple plate preparations enable

scale up of xMAP protocols for routine operation with greater

reproducibility than manual procedures. Liquid handling robots

may be complemented with innovative options such as tip loading

for serial 96/384-well dilutions (including 96/384 interchangeable

pipetting heads), plate heating and cooling, orbital shaking, and

barcode reading. Some automated dispensers are easily operated

under a laminar ow hood without requiring connection to a com-

puter (e.g. VIAFLO 96/384 from Integra Biosciences Corp.). ese

single and multichannel dispensers can be easy implemented into

a workow for a series of sample dilution steps and a multitude of

plate preparations, prior to the addition of coupled microspheres to

an xMAP Technology assay.

Examples of Commercially Available Single and Multichannel

Pipette Dispensers for Semi-automated Workflows

Table 1a. Single semi-automated pipette dispensers

Company Product Name Plate Format

BioTek®MultiFlo™ Microplate

Dispenser 96-384

Formulatrix Mantis 96-384

Hudson Robotics SOLO™96-384

Thermo Scientific Multidrop™96-384

Table 1b. Multichannel semi-automated pipette dispensers

Company Product Name Plate Format

Agilent Technologies

Agilent™ Bravo™

Automated Liquid

Handling Station

96-384

Apricot Designs i-Pipette/Pro 96-384

Aurora Biomed VERSA 10/110/1100 96-384

BioTek®MicroFill™ Microplate

Dispenser 24-96-384

CyBio Cybi®-FeliX 96-384

Formulatrix Tempest 96-384

Gilson®Quad Z-215 Liquid

Handler 96-384

Hudson Robotics Micro10x™96-384

INTEGRA Biosciences VIAFLO 96/384 96-384

Rainin Liquidator™ Manual

Pipetting System 96-384

Labcyte™Echo® Liquid Handler 96-384

Thermo Scientific

Thermo Scientific™

Versette™ automated

liquid handler

96-384

TOMTEC®Quadra Tower™96-384

Automated dispensers have become more widely adopted as

they incorporate advanced, multifunction robotic features. It is

common practice to continue all washing steps on multichannel

dispensers equipped with an integrated plate vacuum ltration

station and/or magnetic bead separation deck (e.g. Agilent™ Bravo™

Automated Liquid Handling Platform, Agilent Technologies).

Depending on the types of the microspheres used in the multiplex-

ing protocol (MicroPlex® or MagPlex®), laboratories can now per-

form various stages of xMAP assays under one partially automat-

ed, unattended workow, minimizing human-induced variability.

Some liquid handlers are fundamentally simple dispensers,

but also have a built-in robotic functionality that allows multiple

microplates to be moved automatically (e.g., ermo Scientic™

Versette™ automated liquid handler/ermo Scientic®; Precision™

XS/BioTek®, Quadra4®/TOMTEC®). A robotic arm can shuttle 96-

and 384-well plates to and from a plate stacker, which can be con-

gured to hold dierent numbers of microplates. ese multiplate

stacker systems are an ideal solution for robust walkaway capabili-

ties, providing an extra microplate workspace and increased labo-

ratory eciency (Figure 1).

A

C

E

B

D

F

Figure 1

A: Agilent™ Bravo™ Automated Liquid Handling Platform.

B: i –Pipette. C: VIAFLO (Integra Bioscience Corp). D: Liquidator™ 96 (Rainin®)

Advanced robotic liquid handlers with hotels and plate transport functionality.

E: Thermo Scientific™ Versette™ automated liquid handle. F: Quadra4™ (Tomtec®).

A: © Agilent Technologies, Inc. 02/2015. Reproduced with Permission, Courtesy of Agilent

Technologies, Inc.

B: i-Pippette is manufactured by Apricot Designs Inc®. For more information, contact them

at info@apricotdesigns.com.

E: Thermo Scientific and Versette are trademarks of Thermo Fisher Scientific. Photograph

used by permission from Thermo Fisher Scientific

3

Plate Washing Systems

Instruments dedicated to a single function, such as buer dispens-

ing, serial dilution, or plate preparation, provide an easy way to in-

corporate automation for multiplex microsphere-based screening

protocols. Patented xMAP Technology uses microspheres as the

solid phase for a binding reaction with dierent types of antigens,

antibodies, or oligonucleotides. Compared to ELISA, coupling of

capture molecules to xMAP microspheres provides greater surface

area and improved distribution of capture molecules throughout

the clinical sample. Although the assay itself is carried on the sur-

face of the microsphere rather than on the surface of the micro-

plate (as with ELISA), microplate washing is used to remove sample

matrix and unbound materials from the microsphere suspensions

between incubations to ensure accurate and reliable data.

Plate washing can have a substantial impact on immunoassay

results; therefore, standardizing this process can improve a meth-

od’s performance across multiple batches. Usually several washing

steps are required during an assay run. xMAP polystyrene micro-

spheres (MicroPlex® with a diameter of 5.6μm) are typically sepa-

rated from the rest of the sample by vacuum-based bead ltration.

Likewise, LumAvidin® Microspheres are not magnetic and require

vacuum ltration, such as MultiScreen® Filter Plates (Millipore®,

Cat. no. MABV N12) used with a vacuum pump system manifold,

such as the MultiScreen™ Resist Vacuum Manifold from Millipore

(MAVM0960R) dedicated for 96- and 384-well plate formats. e

high throughput compatible MultiScreen® HTS lter plates, also

available from Millipore, have been specically designed for use

with automated equipment. e plate dimensions are standard-

ized to meet ANSI/SBS 2004 1-4 standard compliance for multiwell

plates and are fully integrated with automated gripper arms and

further ltration to waste. Rigid sidewalls with moving grippers

can also provide ample surfaces for bar codes and plate readers. All

of these features are important and should be taken into consider-

ation before the scaling up of any assay.

Automated vacuum ltration systems (96/384-well microplate

washers) process either individual rows or full assay microplates

much more quickly than manual ltration procedures using sepa-

rate vacuum manifolds. Unfortunately, sticky and undiluted bio-

logical uids (serum, plasma, saliva, etc.) might easily clog lter

plates, and even a single clogged well can cause failure and sample

loss for a complete microtiter plate. Leaving the plate on the vac-

uum apparatus too long between wash steps and the addition of

subsequent reagents may allow the plate to dry, and microspheres

may adhere to the lter bottom wells or clump.

Magnetic Separation

In order to keep costs low while achieving the highest possible

throughput, microplate washers may use the principle of magnetic

separation as an alternative to vacuum ltration and plate cen-

trifugation. Second generation xMAP microspheres (superpara-

magnetic MagPlex with a diameter of 6.5μm) contain magnetic

particles, allowing easier separation of the microspheres from the

reaction sample and easy automation of the washing steps. ese

semi-automated devices come in the form of strip washers, full

plate washers, and combination washer-dispensers (Figure 2).

Each washing device has to be specically pre-programmed for

any xMAP-based assay. ere are specic requirements for han-

dling protein versus genomic applications that are dependent on

the type of assay chemistry used (e.g., washing with use of dispos-

able tips due to contamination risk). erefore, washing protocols

must be optimized for bead separation method (magnetic or vacu-

um ltration), combination of washer, magnetic carrier, and plate

type used. Automation serves to simplify and speed processing,

but also tends to improve bead recovery and precision leading to

better assay performance.1

e 3rd Edition xMAP Cookbook (Appendix C) provides an

example of programming instructions for BioTek® ELx405 and

ELx406 microplate washer (magnet P/N 7103016, Dexter LifeSep®

96F technology) for round bottom 96-well plate and MagPlex

beads. e wash programs described below were optimized for

Tecan washers to achieve optimal bead recovery for multiplex

xMAP assays in 96-well plate format with low residual volume per

well (Tecan HydroFlex™ washer with Smart-2 MBS magnetic plate

carrier and Hydrospeed™ washer with Smart-2 MBS carrier with

96HT wash head; recommended plate: Greiner Bio-one 96-well

low bottom thickness of the wells of 0.3mm only, Cat. no. 655096)

(Tecan, personal communication).

BioTek achieved high throughput for handling multiple plate

batches by simple integration of a microplate washer with the

BioStack™ Microplate Stacker (Figure 3). e sensitive gripper arm

with rotational wrist exchanges plates between storage stacks and

an attached microplate washer in less than ten seconds. e robot

allows hands-free processing of up to 50 plates. An ecient alter-

native for a large screening laboratory might be the PerkinElmer

Twister II Microplate Handler for 96-well or 384-well capacity with

the Luminex FLEXMAP 3D® high throughput analyzer, which can

handle deep well and low prole microplates.

A

B

Figure 2

A (from left to right): ELx50™ Microplate Strip Washer (low throughput),

ELx405™ Microplate Washer (high throughput), EL406™ Microplate Washer

Dispenser (high throughput) (Tecan).

B (from left to right): HydroFlex™ (low throughput), HydroSpeed™ (high throughput)

(Tecan).

4

Table 2. Optimized Tecan programs for HydroFlex™ and HydroSpeed™ washers

Reference HydroFlex™ with Smart-2 MBS Magnetic Plate Carrier HydroSpeed™ with Smart-2 MBS Carrier and 96HT Wash Head

Wash Program: Parameters Parameters

96-well μ-clear plate Low bottom thickness Low bottom thickness

Plate Definition File: [GRE96fb_magbeads] [MAG_GRE96ft]

Name: MAG MAG_96

Cycle 1: # of cycles: 1 # of cycles: 1

Soak (bead settling) Time: 90 sec Time: 90 sec

Aspirate Mode: normal

(one asp. point/well)

• z-pos: custom 6mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 8mm/s

Mode: normal

(one asp. point/well)

• z-pos: custom 6mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 2mm/s

Dispense z-pos.: overflow

• disp. volume: 200μl/well

• channel: 1

• disp. rate: 300μl/sec

z-pos.: overflow

• disp. volume: 200μl/well

• channel: 1

• disp. rate: 350μl/sec

Cycle 2: # of cycles: 1 # of cycles: 1

Soak Time: 60 sec Time: 60 sec

Aspirate Mode: normal

(one asp. point/well)

• z-pos: custom 6mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 8mm/s

Mode: normal

(one asp. point/well)

• z-pos: custom 6mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 2mm/s

Dispense z-pos.: overflow

• disp. volume: 200μl/well

• channel: 1

• disp. rate: 300μl/sec

z-pos.: overflow

• disp. volume: 200μl/well

• channel: 1

• disp. rate: 350μl/sec

Cycle 3: # of cycles: 1 # of cycles: 1

Soak Time: 60 sec Time: 60 sec

Aspirate Mode: normal

(one asp. point / well)

• z-pos: cust. 5.5mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 5mm/s

Mode: normal

(one asp. point / well)

• z-pos: cust. 5.5mm

• Asp. time: 1 sec

• Aspirate rate: 1

• Head speed: 1mm/s

AB

Figure 3

A: EL406™ Washer Dispenser integrated with Bio-Stak™ Microplate Stacker. (BioTek)

B: PerkinElmer (formerly Caliper) Twister II Microplate Handler. (©Q3-2016 PerkinElmer, Inc. All rights reserved. Printed with permission)

5

Several devices such as the KiNEDx robot arm, Caliper Twister

II robot (PAA Inc.), or Orbitor™ RS Microplate Mover (ermo

Scientic) can simplify the automation of xMAP instruments.

ese automated robots are able to move microplates rapidly in

and out of the xMAP instrument and between microplate devices

such as liquid handlers, dispensers, or washers located on the labo-

ratory bench (Figure 4). eir extensive vertical reach allows mul-

tiple stacked or high density instruments to be loaded in a small

footprint, and a bidirectional telescoping arm provides superior

reach with unlimited base rotations (360°).

Coupling Microspheres with a Semi-automated Device

xMAP microspheres can be covalently coupled with dierent

reagents for specic bioassays such as antigens, antibodies, or

A A

B

B

C

Figure 4

A. Orbitor™ Microplate Mover attached to KingFisher Flex (Thermo Scientific,

Orbitor, and KingFisher are trademarks of Thermo Fisher Scientific. Photographs

used by permission from Thermo Fisher Scientific.)

B. KiNEDx robot (PAA Inc.) arm integrated with Luminex® 200™ C. Caliper Twister II

robot (PAA Inc.) loading microplate into Luminex® 200™ system. (Photographs used

by permission from Peak Analysis and Automation Ltd.)

Figure 5

A. Inverse magnetic particle processing

B. KingFisher Flex—front view with see-through lid and plates. KingFisher is a

trademark of Thermo Fisher Scientific. (Photographs used by permission from

Thermo Fisher Scientific.)

oligonucleotides. Although the coupling protocol typically involves

some level of manual work, automation of the bead coupling pro-

cess can improve lot to lot variation (CV%). In addition to protocols

listed in the literature, the xMAP Cookbook provides the general

guidelines for the coupling of biomolecules to microspheres. An ef-

cient, semi-automated protocol for bead coupling uses MagPlex

Microspheres on the KingFisher™ Flex instrument (ermo

Fisher Scientic), originally designed for nucleic acid isolation

and purication but adapted for the multiplexing needs of xMAP

Technolog y.2 e device features an innovative inverse magnetic

separation technique, which eliminates the aspiration and other

liquid handling steps that can lead to waste or contamination in

the assay. e KingFisher coupling protocol results in almost 100%

bead recovery.

In principle, the KingFisher approach involves a mechanical

arm composed of a row of disposable plastic sleeves that is low-

ered into the plate wells. e sleeves move like a plunger slowly up

and down, mixing the liquid reagents and the suspended magnetic

beads. After the mixing step, a second arm composed of a row of

magnets slides into the sleeves to collect all beads (along with any

coupled molecules) and transfers them to new solution (Figure 5).

By adapting a standard 96-well format plate for experimen-

tal throughput needs, individual assay stages in the KingFisher

Flex pipeline can be automated independent of each other by

the end user. e following procedure describes how to perform

protein coupling to MagPlex beads in 96-well format plate. Using

the KingFisher Flex as a semi-automatic device enables auto-

mated activation and conjugation steps (up to 1mL when using

ermo Scientic Deep Well 96 Plates) with ecient magnetic

bead transfer.2

6

Materials for automated protein coupling:

• Carboxylated MagPlex microspheres (Luminex Corp., Austin,

TX, USA) Local distributors are listed on the Luminex web page:

www.luminexcorp.com

• Activation buer (100mM Na2HPO4 + 0.005% (v/v) Triton X-100),

pH 6.2

• Sulfo-NHS (Pierce, Cat. No. 24510 or 24520 - no weigh format)

• EDC (Pierce, Cat. No. 77149)

• Coupling buer (50mM MES + 0.005% (v/v) Triton X-100), pH 5.0

• Storing buer (PBS + 1% (w/v) BSA) containing 0.05% NaN3)

• Antibody/protein to be coupled (any suitable source)

• KingFisher plate 96-well ermo (ermo Fisher Scientic, Cat.

No. 97002540)

• KingFisher Flex (ermo Fisher Scientic)

Method:

1. Sonicate and vortex the selected MagPlex beads stocks thor-

oughly for at least 10 sec and transfer 300μL (1.25 x107 beads) of

each bead stock solution to the respective wells in the 96-well

plate (KingFisher plate 96-well ermo).

2. Magnetic beads of the rst plate are transferred by the

KingFisher Flex particle handler into second plate for washing

with 250μL activation buer (100mM Na2HPO4 + 0.005% (v/v)

Triton X-100).

3. For carboxyl group activation, MagPlex beads are moved into

wells containing 150μL activation buer with 5mg/mL EDC

and 5mg/mL sulfo-NHS. Activation time: 20 min with slow

agitation at room temperature.

Table 3. Sandwich immunoassay protocol for magnetic bead handler

The key settings, such as incubation time, position, speed of motion, strength of shaking movements, and number of washing steps can be programmed accordingly.

Step Plate content Volume µl

Mixing parameters

Time Temp Speed

1. Transfer Magnetic bead array 50

2. Analyte capture—6

cycles Sample 100 10 min, pause 2min RT Very slow

3. Wash + transfer Wash buer 100 20 sec RT Slow

4. Wash + transfer Wash buer 100 20 sec RT Slow

5. Detection antibody—5

cycles Detection antibody mix 100 10 min, pause 2 min RT Very slow

6. Wash + transfer Wash buer 100 20 sec RT Slow

7. Wash + transfer Wash buer 100 20 sec RT Slow

8. Detection reagent—4

cycles SAPE solution 100 10 min, pause 2 min RT Very slow

9. Wash + transfer Wash buer 100 20 sec RT Slow

10. Wash + transfer Wash buer 100 20 sec RT Slow

11. Bead release to assay

plate Assay buer 100 20 sec RT Slow

Adapted from 2 Poetz O, Henzler T, Hartmann M, Kazmaier C, Templin MF, Herget T, Joos TO. Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling. Mol Cell Proteomics 2010

9(11):2474–2481.

4. Activated magnetic beads are transferred by magnetic particle

handler to wash plates (two times wash with 250μL coupling

buer (50 mM MES + 0.005% (v/v) Triton X-100).

5. It is important to determine optimal amounts of antigens or

antibodies in separate preliminary experiments. Antigens or

antibodies diluted to an applicable protein concentration in

coupling buer and distributed on a 96-well assay plate, are

incubated with the activated beads and agitated for 2h at

room temperature.

6. Coupled beads are transferred by KingFisher Flex for further

washing with 250μL wash buer and resuspended in 200μL

blocking/storage buer (PBS + 1% (w/v) BSA) containing 0.05%

NaN3 and stored in the dark at 2–8°C until further use.

Following coupling conrmation, the microsphere population will

be pooled by KingFisher Flex to form an array on a 96-well format

plate. To perform a multiplex sandwich immunoassay, all assay

reagents including wash buer, detection antibody mixture, strep-

tavidin-R-phycoerythrin (SAPE), and prediluted samples can be

placed simultaneously on the KingFisher Flex for further magnetic

particle processing. It takes approximately 3–4 hours to process an

assay. Standard settings for the xMAP instrument (96-well/2500

beads per well, sample volume 100μL, minimum bead count per

region 100) enables plate reading within 30 minutes (FLEXMAP

3D®). An example of a multiplexed sandwich immunoassay proto-

col using KingFisher Flex can be found in Table 3.

e use of a magnetic particle handler such as the KingFisher

Flex device enables the quantitative transfer of magnetic beads

from the sample well into the wells containing washing solutions

or other assay reagents (e.g., activation buer, capture/detection

7

Figure 6

A. Custom MagPlex microspheres in MTP tubes (Luminex Photograph) B. A per-

manently bonded, unique 2D barcode is laser-etched onto the base of every storage

tube to securely identify and track the content of every tube (Luminex Photograph)

C. 2D barcode tube reader (e.g. Thermo Scientific VisionMate 2D barcode)

(Photograph used by permission from Thermo Fisher Scientific.)

A

C

B

antibody mixture, SAPE reporter solution). e device does not re-

quire vacuum ltration or advanced liquid transfer steps, which

are often the basis for fully automated workstations. Such worksta-

tions (e.g., Tecan or Hamilton) trade these more exible options to

ensure high throughput.

KingFisher Flex is also well-suited to run commercially avail-

able bead-based assay kits, delivering a highly practical laboratory

solution for a more robust workow. A number of laboratories have

published protocols and results for such approaches. Using this

instrument, Poetz et al2 used three WideScreen® proling panels

(EMD Millipore) to analyze multiple receptor tyrosine kinases and

their degree of phosphorylation within the same sample. Setting up

a workow that employs magnetic bead arrays and a KingFisher

Flex processor, Heubach et al3 demonstrated a faster and more

cost-ecient approach for generating bead assays with up to 500

dierent peptides for further protein-peptide interaction studies.

Several studies have shown that a semi-automated ermo Fisher

concept adopted for dierent xMAP protocols might be used for

ecient screening of multiple analytes.4,5,6

To simplify the complete semi-automated workow, routine

procedures such as pipetting immunoassay reagents into multiple

plates can easily be performed with multichannel pipette dispens-

ers to reduce handling errors. KingFisher Flex can accommodate

several 96-well microtiter plates in the same amount of time lled

with individual buers for washing and incubation, but some

steps still require human intervention (e.g. nal transfer of 96-well

plates into an xMAP instrument). Luminex produces custom for-

mat MagPlex microspheres (RUO) in MTP tubes that also can be

used for automated bead coupling, using all kinds of multichannel

liquid handling options that help to simplify laboratory workow

(Figure 6).

Fully Automated Immunoassay Platforms

Ideally, a fully automated workow for xMAP-based assays would

perform hands-free steps such as bead coupling, sample pre-pro-

cessing (plate preparation with required dilution), washing (mul-

tiple steps, including bead separation), plate transferring (for incu-

bation or thermal cycling), and loading into the xMAP instrument.

At this point, there are few instruments or kit manufacturers that

have developed fully automated solutions dedicated for a large va-

riety of tasks and suited to protein/genomic applications and spe-

cic market segments.

Tecan, a Swiss company specializing in the development,

Figure 7

Top: Luminex® 200™ integrated with fully automated Freedom EVO liquid handling

workstation. (Tecan)

Bottom from left to right: FLEXMAP 3D with swivel base and Tecan washer

located under xMAP instrument with rotation for ease of maintenance; Freedom

EVO robust solution for MAGPIX (Tecan).

8

Figure 8: Magnetic carriers for the HydroSpeed plate washer.

Left, top to bottom: 384-well (MBS 384 carrier), 96-well two magnets (smart-2 MBS

96) and 96-well one magnet (MBS 96 carrier) for optimized magnetic bead washing.

Right: smart-2 MBS carrier for automated washing with Tecan's HydroFlex™.

production, and distribution of fully automated workow solu-

tions, conducts dierent assay chemistries with advanced robot-

ics.As shown in Figure 7, all xMAP instruments (Luminex 100/200,

MAGPIX, and FLEXMAP 3D) can be integrated with the Tecan

Freedom EVO® liquid handling platform, which has been actively

adopted by many molecular diagnostic, genomic, proteomic, and

drug discovery laboratories.7 e Freedom EVO® series oers four

dierent worktable capacities (width: 75, 100, 150, and 200 cm),

each with similar tip conguration (1, 2, 4, 8, 96, and 384) for the

100μL–5000μL volume range. Tecan created a user-friendly au-

tomated workstation for performing all pipetting/liquid transfer

tasks (from both reservoirs and 1.5 mL centrifuge tubes into all

wells), plate movements, separation of microspheres from assay

suspensions (vacuum ltration or magnetic bead separation), and

mixing steps. e highly eective shaker (Te-Shake™) handles stan-

dard 96–384 microplates, deep-well plates, and PCR tubes, with

the added option of sample heating if necessary. For the robotic

aspect any plate which corresponds to the Society of Biomolecular

Screening (SBS) standards can be used with Freedom EVO for

plate logistics and carriers. Common plate brands integrated with

the EVO device are: Greiner®, Nunc®, Corning®, Macherey-Nagel®,

and Eppendorf®.

Due to an automation module available in the Luminex

xPonent® software, Tecan drivers enable communication between

the xMAP instrument and Freedom EVO, which allows the robotic

manipulator arm to transfer the assay plate to the xMAP instru-

ment. e typical, fully automated process includes the pipetting

steps for all reagents, PosID™ barcode tube scanning, an incuba-

tor tower for dark refrigeration/incubation of up to six plates (5°C

to 60°C), and plate storage (hotel option). e Freedom EVOware®

software controls for any errors during the performance, also pro-

viding automatic xMAP system maintenance, mid-run calibration

routines, and dynamic assay scheduling for parallel processing of

multiple plates.

Two types of washers can be integrated into a Freedom EVO®

device: HydroSpeed™or HydroFlex™. HydroSpeed is an advanced

microplate washer optimized for washing cells or xMAP beads

Figure 9

A. Deck layout design from JANUS ® CS Autoplex Workstation On.-board gripper

moves plate into integrated Luminex® 200™ analyzer.

B. Additionally, pipetting station, vacuum filtration for plate washing (option for

magnetic bead separation), blotting and mixing station. (©Q3-2016 PerkinElmer,

Inc. All rights reserved. Printed with permission).

in both 96- and 384-well plates, while HydroFlex is designed for

automated microplate strip washing and vacuum ltration perfor-

mance for 96-well microplate formats only. e HydroSpeed wash-

er provides two standard magnetic carriers (MBS 96- and 384-well

carrier), and one an innovative patent-pending design that uses

two magnets per well for optimized washing results with xMAP

microspheres (Smart-2 MBS 96 carrier), as shown in Figure 8.

is conguration provides a more robust assay performance and

guarantees higher bead recovery (typically ≥90%) in comparison

to polystyrene microspheres ltration. Moreover, HydroFlex wash-

ers with optional magnetic bead separation are required to use

a 96-well at bottom plates, such as Greiner Bio-One, Germany

(Cat. no. 655096).

Software drivers for Research Use Only (RUO) applications with

several third party washers including Embla®, BioTek405™ TS/LS,

Biotek EL406, and Biotek ELx405™, are also available from Tecan.

xMAP platforms (Luminex 100/200, MAGPIX, and FLEXMAP 3D)

are not restricted to a specic manufacturer for kits or reagents,

which gives the end user more exibility in terms of running dier-

ent tests under one deck.

e JANUS® CS Autoplex Workstation from PerkinElmer has

been developed to automate a number of proteomic applica-

tions, such as multiplexed cytokine immunoassays (e.g., Millipore

Human Cytokine LINCOplex™ Kit, cat. no #HCYTO-60K) to im-

prove biomarker screening and to more easily process commercial-

ly available kits (Figure 9). is provides a tremendous advantage

over manual processing and guarantees ecient management

A

B

9

of xMAP bead-based protocols, which are suitable for setting up

multi-batches in large-scale studies.8 e JANUS liquid handling

system and the Luminex 100 analyzer were evaluated by testing

multiplex ow immunoassays for the detection of IgG type-specic

IgG antibodies to Herpes Simplex Virus (HSV) types 1 and 2.9 Intra-

and interassay reproducibility studies showed excellent precision

and resulted in a 50% reduction in turnaround time compared to

routine testing by EIA. Together, both instruments can create a

prospective solution for improving laboratory medicine, and oer-

ing physicians more rapid and accurate clinical information.

Nucleic Acid High Throughput Solutions

ere are dierent requirements for handling protein versus ge-

nomic applications that are dependent on the type of assay chem-

istry used. erefore, each automation platform has to be pro-

grammed for a specic xMAP application and chemistry. Over the

past few years, nucleic acid-based assays have become accepted in

clinical diagnostics owing to their high specicity, low sample re-

quirement, and ease of automation. In addition, recent advances

allow such assays to be congured in xMAP multiplex format, en-

abling eective screening for multiple assay targets.

Luminex xTAG® Technology provides a method for the simulta-

neous detection of 150 dierent nucleotide sequences in a single

reaction. Proprietary sets of 24 base oligonucleotides (also known

as anti-TAG sequences) are covalently coupled to MagPlex beads.

Each MagPlex-TAG bead region’s anti-TAG is complementary to a

specic TAG sequence incorporated into reporter molecules. is

approach has been validated and xTAG Technology has become

an industry standard in genetic disease screening, gene proling,

microbial detection, and characterization of antibiotic resistance

associated with bacterial SNPs.10

Song et al11 utilized xTAG with Allele Specic Primer Extension

(ASPE) chemistry to develop a reliable and cost-eective multi-

plexed genotyping assay for simultaneous detection of 11 muta-

tions in g yrA, gyrB, and parE of S. enterica serovars, Typhi, and

Paratyphi A that result in nalidixic acid resistance (NalR) and/

or decreased susceptibility to uoroquinolones. e assay is ca-

pable of rapidly screening and identifying the underlying genetic

changes in quinolone-resistant isolates and demonstrates that

the xMAP platform is ideal for molecular epidemiological analy-

sis. is assay might be adapted to full automation workstations

and used as a screening tool more easily than techniques such as

sequencing, which are more suited for biomarker discovery. It is

important to note that a potential limitation for such automation

implementation is the requirement of sample preprocessing to ex-

tract and purify the nucleic acid of interest. However, there have

been signicant advances in addressing these limitations over the

past few years, and several semi- or fully-automated systems that

perform rapid nucleic acid extraction are now available. Currently,

the Freedom EVO 150 is capable of extracting DNA from 96 sam-

ples including controls and subsequent PCR set up in less than

3.5 hours.12 High throughput performance of up to three 384-well

PCR plates in less than eight hours is achieved with the dynamic

Figure 10

Luminex® 200™ integrated with MICROLAB® STAR –HLA solution (Microlab is a reg-

istered trademark of Hamilton Company in the U.S. and/or other countries.)

scheduling of the Freedom EVOware software. In order to keep

the complexity and cost low while reaching the highest possible

throughput, the thermocycling, plate sealing, and plate peeling

steps might be performed oine.

A number of commercially available standalone workstations

as well as “homemade” integrated systems have been developed for

nucleic acid preparation during the last few years. Today, fully au-

tomated devices cover critical steps of genomic applications from

nucleic acid extraction (DNA/RNA) to PCR set up with thermocy-

cler integration (e.g., using automated extraction platforms from

QIAGEN, bioMérieux, Roche, Chemagen, Tecan, and Hamilton).

Instruments such as Biomek® 4000 (Beckman Coulter), Zephyr®

Molecular Biology Workstation (PerkinElmer) or VERSA™ GENE

(Aurora Biomed Inc.) have been designed to perform nucleic acid

purication or real-time PCR with high precision, throughput,

and accuracy. Precise pipetting of reagents, preparation of master

mix, creation of dilution series, and sample addition guarantees

hands-free preparation of the PCR plate for amplication. Also,

popular chemistries used in genomic applications are avail-

able from companies such as Promega, Applied Biosystems™,

Invitrogen™, and QIAGEN.

IVD Automation

As the IVD market continues to grow globally, it must adapt to new

testing environments, innovative technologies, and new types of

clinical markers. Key assay components, such as buers, antibod-

ies, and detection reagents used in multiplex must be optimized

to work together to produce a sensitive result that is consistent

across all measurement channels and assay parameters. erefore,

it is clear that there is a demand for technologically advanced in-

struments with greater automation that oer reproducible results

independent from the environment in which they are performed.

In February 2010, Hamilton Robotics announced the launch

of solutions for HLA antibody screening and identication and

HLA DNA typing methods based on Gen-Probe’s (now Immucor’s)

10

Figure 11

From left to right: LABXpress™ Pipettor (aspirate/dispense into 96-well reaction

plate, mixing, shaking, centrifuging, reading on xMAP instrument). The arm with

grid automatically transfers plates to the integrated reader and acquired data are

analyzed with HLA Fusion software. (One Lambda photographs used by permission

from Thermo Fisher Scientific.)

LifeCodes HLA reagents (Press release BoxID 324512, Martinsried,

22.02.2010). Filling the market gap for histocompatibility and trans-

plant immunology labs that require a high degree of multiplexing

and throughput, Hamilton nalized workow solutions for blood

banks and long term storage systems (-20°C to 80°C) under one liq-

uid handling deck. Hamilton Robotics developed fully automated

solutions for IVD sample preparation that reduce hands on time,

minimize operational variation, and deliver consistent results. e

Microlab® STAR™ liquid handling workstation is based on patented

Monitored Air Displacement pipetting technology (MAD), where

the risk of contamination of critical assays is reduced to an abso-

lute minimum (Figure 10). e system allows for real-time detec-

tion of pipetting errors when an insucient amount of liquidhas

beenaspirated for a single, multiple, or partialdispense, or due to

introduction of air and blocked tips (TADM—Total Aspirate and

Dispense Monitoring). e combination of MAD pipetting with

compressed CO-RE tip attachment technology meets IVD require-

ments for precision, even in critical situations when the STAR

channels must pipette liquids with extremely low viscosity and

high vapor pressure (e.g., methanol/acetone).Depending on the

complexity of the xMAP application, three unique workstations

are currently available: STARlet, STAR, and STARplus (deck size:

1, 1.5, and 2 meters). Up to 16 independent 1000μL channels can be

combined with any of Hamilton's multiprobe heads (96,

384 or nanopipetting) on a single instrument with a pipette vol-

ume range of 0.5 to 1000μL. is results in a doubling of throughput

by simultaneous preparation of two microplates. e 96 channel

head is able to pipette liquids in range of 1 to 1000 μL and the 384

probe head from 0.5 to 50μL. All of these features are important for

integration of any xMAP instrument with the Microlab® STAR™ de-

vice, especially for the FLEXMAP 3D which can analyze 384 sam-

ples within 1.5 to 2 hours. Similar to other fully automated liquid

handling systems, the Microlab STAR is controlled by advanced

software (Venus One), which allows exible robot arms mounted

on the decks to transfer the plates between individual system

components (e.g., pick a plate from a microplate stacker and place

it into an adjacent xMAP instrument or pass microplates from a

plate stacker to a pressured control vacuum system or to magnetic

washers). Hamilton has integration experience with the follow-

ing washers: Biotek ELx405R, Tecan Power Washer 384, ermo

Wellwash Ascent, MD Embla 96/384, and MD Embla Skan Wash

300/400 (Molecular Devices).

xMAP Technology, together with Hamilton Robotics, provides

an appropriate platform to study the impact of dierent HLA

antibodies isotypes of on transplantation outcomes. e xMAP

screening approach oers high sensitivity, which is comparable to

the high sensitivity of cross-match by ow cytometry.13 An ideal

t for the transplant community is One Lambda’s LABType® SSO

and LABScreen® IVD labeled products; especially LABScan 3D

(Luminex FLEXMAP 3D), which can detect up to 500 HLA speci-

cities in a single test, eliminating the need to run multiple tests.

A sample preparation device called LABXpress™ Pipettor (One

Lambda) can accommodate eight plates under one deck, and guar-

antees hands-free capabilities suited for high-volume solid organ

and bone marrow transplant centers (Figure 11).

Demand for excellent quality and reproducible tests in health-

care continues to be driven by IVD kit providers together with

automation manufacturers. ere are other fully automated plat-

forms that come to the forefront, which are more critically de-

veloped around very specic methodologies. eradiag (formerly

Biomedical Diagnostics) has launched a comprehensive range of

tests based on xMAP Technology for the diagnosis of autoimmune

pathologies, allergies, and early detection of heart failure. e com-

mercially available FIDIS™ kits are processed with a liquid handling

platform called eralis, which lls the laboratory IVD gap for crit-

ical public health tests.

Summary and Conclusions

Multiplex bead-based assays have become established in high-

throughput laboratories, including combinations of multiple semi-

automated workstations to carry out the traditionally manual

parts of xMAP protocols. e immediately obvious benets of la-

bor savings, better quality data, and cost reduction are key drivers

in integrating independent liquid handling devices (liquid dispens-

ers, plate washers, plate handlers, etc.) into small-scale, benchtop

or walkaway automation applications for xMAP Technology.

In addition, large robotic systems require dedicated, highly

trained personnel and have steeper learning curves to become

fully familiar with these devices. Nevertheless, automation holds

the promise of increasing sample throughput and improving data

output. Given the rise of molecular testing, it is also likely that

there will be multiplex testing combinations that include both

traditional immunoassays as well as nucleic acid-based molecular

assays, all of which must be optimized to work together under one

automation deck.

e examples of xMAP-based assays mentioned in this review

have been used to illustrate the various means of multiplexing

presently in practice and to open possibilities of further integra-

tion of multiplexing and laboratory automation. e author does

not attempt to provide a listing of every assay, application, or

11

REFERENCES

1. Paul H, Banks P. Automated workows for Luminex xMAP® assays.

Biotek (Internet). Cited 2015 September. Available from: www.biotek.com.

2. Poetz O, Henzler T, Hartmann M, Kazmaier C, Templin MF, Herget T,

Joos TO. Sequential Multiplex Analyte Capturing for Phosphoprotein

Proling. Mol Cell Proteomics 2010 9(11):2474–2481.

3. Heubach Y, Planatscher H, Sommersdorf C, Maisch D, Maier J, Joos TO,

Templin MF, Poetz O. From spots to beads—PTM-peptide bead arrays

for the characterization of anti-histone antibodies. Proteomics 2013

Mar;13(6):1010-5.

4. Bigalke B, Pötz O, Kremmer E, Geisler T, Seizer P, Puntmann VO,

Phinikaridou A, Chiribiri A, Nagel E, Botnar RM, Joos T, Gawaz M.

Sandwich immunoassay for soluble glycoprotein VI in patients with

symptomatic coronary artery disease. Clin Chem 2011 Jun;57(6):898-904.

5. Planatscher H, Rimmele S, Michel G, Pötz O, Joos T, Schneiderhan-Marra

N. Systematic reference sample generation for multiplexed serological

assays. Sci Rep 2013;3,3259.

6. Luckert K, Götschel F, Sorger PK, Hecht A, Joos TO, Pötz O. Snapshots

of protein dynamics and post-translational modications in one

experiment--beta-catenin and its functions. Mol Cell Proteomics 2011

May;10(5):M110.007377.

7. Allinson JL, Automated immunoassay equipment platforms for analyti-

cal support of pharmaceutical and biopharmaceutical development.

Bioanalysis 2011 Dec;3(24):2803-16.

8. Bourdon D, Tack L. Fully Automated High roughput Biomarker

Screening using xMAP Polystyrene Bead-based Multiplex Cytokine

Detection Assay. Perkin Elmer (Internet). Cited 2015 September. Available

from: www.perkinelmer.com.

9. Binnicker MJ, Jespersen DJ, Harring JA. Evaluation of three multiplex ow

immunoassays compared to an enzyme immunoassay for the detection

and dierentiation of IgG class antibodies to herpes simplex virus types

1 and 2. Clin Vaccine Immunol 2010 Feb;17(2):253–257.

10. Deshpande A, White PS. Multiplexed nucleic acid-based assays for

molecular diagnostics of human disease. Expert Rev Mol Diagn 2012

Jul;12(6):645-59.

11. Song Y, Roumagnac P, Weill FX, Wain J, Dolecek C, Mazzoni CJ, Holt

KE, Achtman M. A multiplex single nucleotide polymorphism typing

assay for detecting mutations that result in decreased uoroquinolone

susceptibility in Salmonella enterica serovars Typhi and Paratyphi A. J

Antimicrob Chemother 2010 Aug;65(8):1631-41.

12. Stangegaard M, Frøslev TG, Frank-Hansen R, Laursen SS, Jørgensen M,

Hansen AJ, Morling N. Automated extraction of DNA and PCR setup us-

ing a Tecan Freedom EVO® liquid handler. Forensic Science International:

Genetics Supplement Series 2 2009;2(1):74-76.

13. Heinemann FM. HLA genotyping and antibody characterization

using the Luminex multiplex technology. Transfus Med Hemother

2009;36(4):273-278.

platform that is currently available, but rather to guide readers to-

ward looking for novel liquid handling techniques for integration

with xMAP instruments. Since not all screening assay chemistries

are compatible with automated platforms, new robotic innovations

will continue to contribute to the development of cost-eective

xMAP-based applications.

Acknowledgements

All images are used at the courtesy of the companies listed in the

references. e instruments are used as examples in this article

and represent those that the author has either used or has learned

of during the course of his career.

12

To learn more, please visit: www.luminexcorp.com

©2016 Luminex Corporation. All rights reserved. Luminex, xMAP, FLEXMAP 3D, MAGPIX, MAGPLEX, and xTAG are trademarks of Luminex Corporation,

registered in the U.S. and other countries. Luminex 100/200 is a trademark of Luminex Corporation. WP1149.0215

Automated Liquid System Providers

Company Site Address

Agilent www.agilent.com

Apricot Designs www.apricotdesigns.com

Aurora Biomed www.aurorabiomed.com

Beckman Coulter www.beckmancoulter.com

BioMicroLab www.biomicrolab.com

Biosero www.bioseroinc.com

BioTek Instruments www.biotek.com

Cetac www.cetac.com

CyBio www.cybio-ag.com

Eppendorf www.eppendorfna.com

Gilson www.gilson.com

Hamilton Robotics www.hamiltonrobotics.com

Integra Biosciences www.integra-biosciences.com

Labnet International www.labnetlink.com

Molecular Devices www.moleculardevices.com

Peak Analysis and Automation www.paa-automation.com

PerkinElmer www.perkinelmer.com

Rainin www.rainin.com

Tecan www.tecan.com

Thermo Fisher Scientific www.thermoscientific.com

Titerek-Berthold www.titertek.com

Tomtec www.tomtec.com

Note:This table provides some examples of automation vendors and does not indicate that these platforms have been tested with xMAP Technology or are compatible with all xMAP

applications. Consult your vendor for more information.