troubleshooting problems with lateral flow assays

Troubleshooting Problems with Lateral Flow Assays

Michael A. Mansfield

OEM Diagnostics Group

EMD Millipore

Bedford, MA

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Complexity of a Lateral Flow Test

BIOLOGICAL CHEMICAL

PHYSICAL ENGINEERING

TROUBLESHOOTING | 25 JUNE 2015

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The Design Goal

Develop processes that result in a foolproof product.


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Complaints

A defect is reported.

Product is returned.

Defective product is analyzed.

The failure mode is identified.

Corrective action is implemented.

FAST!

Often not so fast!

What is a typical sequence of events?


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Why does the description of the failure not suggest a root cause?

The reported failure is an endpoint observation, often from a QC

technician or end user.

– Low sensitivity

– False negative

– False positive

– High background

– Irregular or slow flow

There are many possible causes for “typical” failures.


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What are the possible causes of “low sensitivity?”

Membrane flow too fast

Inactive capture reagent

Insufficient capture reagent

Insufficient conjugate in test

Inactive conjugate

Inactive analyte

pH or salts incorrect

System flowing too fast

Not enough conjugate reaching the

test line

Not enough sample reaching the test

line

Each of these possibilities

has material, design, and

process elements.


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Where do you start troubleshooting?

Confirm the reported failure.

– Is the failure described adequately and correctly?

– What is the frequency of the failure?

– Can the failure be reproduced?

– Does the confirmation process reveal any other problems?

Are other problems observed that are consistent with the

described failure?


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How do you go about distinguishing the possible root causes?

Basic Rule: Check the obvious and simple

things first.


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#1 - Investigate the basics.

Materials history

Process variability

Unusual observations during production

This is an obvious place to start and may provide a clue where to

look first.


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#2 - Start with the finished device.

Take the device apart before starting lengthy experimental

investigations.

Inspect the strips.

– Consistency of assembly

– Contamination

– Physical damage

A housing and tapes can hide a lot of problems.


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#3 - Substitute components that are known to work.

If a material is thought to be the problem, substitute a lot from a

previously released batch of devices.

If a chemical or biological component is suspect, substitute a lot

from a previously released batch of devices.

This assumes:

– There are retains of the key components.

– There are no stability issues.


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Absorbent pad

#4a - Employ the half-stick assay to speed the investigation.

Easier to troubleshoot problems with the

membrane as well as biological and

chemical components

Eliminates variation from conjugate

pads, sample pads, housings, and

associated assembly processes

Many variables can be checked quickly.Membrane

T

C


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#4b - Increase the complexity as needed.

Add sample padAdd conjugate pad

CONJUGATE PAD

SAMPLE PAD


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What corrective actions are needed?

Consider the nature of the manufacturing operation.

Automated Processes

- Machinery

- Engineering controls

- Process variables

- Material consistency

- Operator training

Manual Processes

- Large number of employees

- Consistency of training

- Consistency of execution

day to day

person to person


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What corrective actions are needed?

Vendors

– Improve consistency

– Additional (relevant) specifications

Manufacturing and QC

– Institute relevant incoming QC

– Refine processes

– Enhanced training

– Are the specifications too stringent?

End user

– Improve operating instructions


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Troubleshooting Examples

TROUBLESHOOTING | 14 MAY 2013

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EXAMPLE #1 - The problem

The strips run inconsistently, and sensitivity

is variable. Strips made with membrane

from the same roll don’t work the same. In

some cases, the flow is so slow that the strip

never finishes running. All of our chemistries

are optimized.


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EXAMPLE #1 - What did the analysis show?

Absorbent Pad

Membrane

Conjugate Pad

Sample Pad

Absorbent Pad

Membrane

Conjugate Pad

Sample Pad

Absorbent Pad

Membrane

Conjugate Pad

Sample Pad

Top View of

Master Cards


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Absorbent Pad

Membrane

Conjugate Pad

Sample Pad

Angled View toward Conjugate Pad


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The conjugate pad was not consistently overlapping or contacting

the membrane.

The cards were being assembled by hand.

In final devices, variations in overlaps were obscured by the

housing.

Corrective Actions:

Increased training of assembly personnel

Higher level of visual inspection during and after assembly

process

Further investigation revealed:


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Example #2 - Misalignment of Pad Materials

Strips developed in R&D

– Excellent sensitivity & specificity

– Highly reproducible

Transferred into manufacturing

– Automation equipment designed in house

Major problems encountered

– Manufacturing using the same lots of materials

– Strip performance very inconsistent

– Product unable to meet QC specifications


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EXAMPLE #2 - Misalignment of Pad Materials


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EXAMPLE #2 - Materials Crushed During Assembly


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EXAMPLE #2 - Inconsistent Contact


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EXAMPLE #2 - What happened?

A mechanical engineer was told to design and build an assembly

machine.

He wasn’t told about the criticality of alignment and the

compressibility of the materials.

When these requirements were explained, the engineer added

guides to maintain alignment and reduced the compression to

prevent crushing.


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The Challenges of Cutting

The cutting stroke has to be strong enough to cut through the

thickest material – the backing card – cleanly and consistently.

The cutting stroke has to be gentle enough that there is no

damage to the most fragile materials – the membrane and glass

fiber pad.

The cutting stroke must not disturb the position of the materials.


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Damage from Cutting

Nicked Edges

Stray Fibers


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Example #3 – Poor Test Strip Performance

Flow of the sample was not uniform.

Hydrophobic spots appeared on the membrane when strips were

run.

Variations in sensitivity and specificity – Some membrane lots

were OK while others were not.

“We need a membrane that performs more consistently.”


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Strips were removed from the cassette to observe the entire flow

stream.

TEST

LINE

FLOW

AIR LOCK

TIME


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Damage from Cutting

NORMAL DAMAGED

TOP LAMINATE

MEMBRANE

POLYESTER

ADHESIVE CARD

EDGE VIEW

DAMAGED(cross section)

channel



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Example #3 – Corrective Actions

The cutting machine needed to be re-evaluated for:

– Frequency of cleaning

– Frequency of routine maintenance

The adhesive materials needed to be reassessed.

Then, they could rationally look at other problems.


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EXAMPLE #4 – Delamination

Delamination is separation of the top laminate from the strip.

TOP LAMINATE

MEMBRANE

POLYESTER FILM

ADHESIVE CARD

Adhesive not adhering

to membrane

Membrane not adhering

to film

???


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The manufacturer received a new lot of membrane.

Some slits from the new lot showed varying levels of delamination

of the cover tape from the membrane.

The artifact was difficult to control by working with the cutting

fixture.


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ABSORBENT PADCONJUGATE PAD MEMBRANE

INT

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Side View of Strips


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Using a properly maintained cutter, very thin

strips can be produced.

Delamination – Not Always Width Dependent

3 mm

1 mm


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Assembly into Cassettes

The cassette must be compatible with the dimensions

of the strip.

If the cassette is merely a holder for the test strip, it

must not interfere with the function and flow of the test

strip.


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EXAMPLE #5 – Assembly Problems

Indentations Cracks Leakage

Finished devices were not meeting specification for sensitivity and

specificity.

Cassettes were taken apart after running strips.


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EXAMPLE #6 – Abnormal Sample Flow


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EXAMPLE #6 – Abnormal Sample Flow

Damage to the membrane from placement into the housing was

having a large negative impact on sample flow.

Changing the physical design of the strip and/or housing was

required to produce a uniform flow front along the entire length of

the strip.


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Example #7 – False positives

2 min

10 min

T C


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Example #7 – False positivesSide View of Conjugate Pad Region

Initial

After 12 min


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Example #7 – False positives

Summary of test strip artifacts

– Colloidal gold is the wrong color.

– False positive signal develops as soon as the gold particles

pass the test line.

– Colloidal gold flows in streaks.

– Release of colloidal gold is very slow.

Approaches to fixing the artifacts

– First, redevelop the colloidal gold conjugates.

– Then, address the other issues with test strip performance.


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Conclusions

Effective manufacture of lateral flow test strips is a combination of

biological, chemical, physical, and engineering issues.

All aspects of manufacture must be considered when attempting to

minimize variation in the final product.

Problems that are seen on the membrane can be caused by

design and assembly issues affecting other parts of the test.


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To learn more about our products and services, visit

http://www.emdmillipore.com/diagnostics


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