Download Chapter 32 Troubleshooting Procedure for ArtisanLink Special

Automating Special Stains using Artisan™ and ArtisanLink Special Staining System
Chapter 32 Troubleshooting Procedure for ArtisanLink
Special Staining System
Debra Cobb, HT, MBA
The special stains that are available for the ArtisanLink Special
Suggested Reading
Staining System include:
White PI (2007). World Congress of Pathology Informatics. Brisbane,
Australia.
Acid-Fast Bacteria (AFB) Stain/
Alcian Blue Stain pH 2.5
Ziehl-Neelsen Stain
Automation in histology is here to stay. With the shortage of
Troubleshooting Issues
experienced histotechnologists, an increased workload, and
When troubleshooting ArtisanLink Special Staining System, the
demands for faster turnaround times, laboratories are looking for
ways to produce consistent, quality-stained slides with less staffing
histotechnologist will not only be required to rely on his/her knowledge
of histology procedures and special stains chemistry, but also on his/
Alcian Blue/PAS Stain
Gram Stain
in the most efficient time.
Iron Stain
Alcian Blue/PAS
The arrival of automated instruments has forced histotechnologists
ArtisanLink software. Because the instrument applies reagents as
to familiarize themselves with hardware, software and chemistry in
well as heats and incubates according to the instructions given by the
order to adequately troubleshoot automated platforms. Understanding
user, it is important that the user programs the instrument properly
(PAS-M) Stain/
the differences and the similarities between manual and automated
taking into consideration that all other processes such as grossing,
Mucicarmine Stain
special staining is half the battle when it comes to troubleshooting.
sectioning, fixation, etc are optimized before the sample is placed
Hematoxylin Stain
Jones’ Basement Membrane
Congo Red Stain
her computer skills in order to optimize staining protocols using the
on the instrument. In other words, how to optimize individual slides
Elastic Stain
PAS-Green Stain
Periodic Acid-Schiff (PAS) Stain
Giemsa Stain
Grocott’s Methenamine Silver
Reticulin/No Counterstain
Every histotechnologist understands that to achieve quality staining
results performed manually the following basic factors should be in
place in the laboratory:
(GMS) Stain
Gomori’s Trichrome Stain
Back to Basics
The most common staining issues on ArtisanLink are:
Staining artifacts
 Background staining
Masson’s Trichrome (TRI) Stain
1.Proper grossing, infiltration of tissue with fixative, and dehydration
steps.
Gomori’s Green Trichrome Stain
based on prestaining processes can only be defined by a user.
Warthin Starry Stain
Feulgen Stain
2.A certain tissue thickness recommended for a particular procedure.
For instance, it is recommended to cut tissue for staining with
Congo Red into a thickness of 8 µm and tissue for staining with
Jones’ Basement Membrane Stain is recommended to be cut
Conclusion
into a thickness of 2 µm.
In conclusion, the ArtisanLink Special Staining System provides
3.Drying slides at the right temperature because drying slides at
the flexibility of manual staining coupled with the precision and
temperatures above 62° C can cause damage to the tissue leading
consistency of automation. Unlike the manual method, there is no
to a suboptimal staining.
mixing or monitoring of time dependent reaction steps. Furthermore,
The same basic factors are required for staining slides on an
chemical hazards from mixing of reagents are eliminated and the
automated instrument such as ArtisanLink. However, two more
variability of special stains results is greatly reduced. Last, but not
factors should be taken into consideration:
least, the instrument allows the histotechnologist to perform other
tasks while the slides are stained. The new ArtisanLink Special
Staining System also allows one instrument to communicate with two
more instruments with specialized software.
Inconsistent staining
Staining Artifacts
Problem:
(1)Tissue slides stored for long periods of time (months to years).
(2)Dust particles settling on slides, especially, those that are charged.
(3)Dispensing of reagents from dirty bulk liquid containers (Fig. 1).
Solution:
(1) For slides that may be dusty, it is recommended to try the following
procedure after the deparaffinization and hydration steps:
Place the slides in running tap water for 2-3 minutes.
(a)The user must follow the procedures and instructions as provided
by the instrument manufacturer.
(b)The user must follow a daily maintenance procedure in addition to
the biannual or annual preventive maintenance procedure.
 Rinse in deionized water.
Place the slides in Artisan™ Wash Solution for 5 minutes before
loading them on ArtisanLink.
(2) For slides that have dispensing artifacts, it is recommended to use
clean bulk liquid containers.
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Troubleshooting Procedure for ArtisanLink Special Staining System
Troubleshooting Procedure for ArtisanLink Special Staining System
Figure 1. Artifacts on slides caused by
Figure 3a. Nuclear Fast Red precipitate.
dirty bulk liquid containers.
Figure 3b. Crystal Violet showing shards.
Reagent packs that are placed on the instrument need to be checked
for any type of build-up of chemical deposits that can be dispensed
on the slide during the staining run. It is recommended to check the
dispenser tip and to mix and prime the packs before daily use (Fig. 2).
It is also important to store the reagent packs tip down to prevent
damage to the dispenser’s internal parts.
Staining artifacts may also arise from reagent dyes. Dyes such as
Nuclear Fast Red or Crystal Violet are known to cause artifacts.
For instance, Nuclear Fast Red is sensitive to cold. If the reagent is
stored in the refrigerator or left out on the loading dock in freezing
weather, there is a risk that the reagent will start to precipitate.
Once this reaction starts, it will continue regardless of correcting
the storage temperature. This precipitate will then be dispensed onto
the slide (Fig. 3a). Crystal Violet is another example of a dye that
has a tendency to leave crystal-like shards on the tissue (Fig. 3b).
Figure 2. Dispenser showing build-up of chemical deposits.
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Troubleshooting Procedure for ArtisanLink Special Staining System
Troubleshooting Procedure for ArtisanLink Special Staining System
Reagents used on ArtisanLink have been validated and optimized for
Background Staining
Inconsistent Staining
 Reagent packs not primed before use. This can cause either a
staining quality as well as for correct storage and usage temperatures.
Background staining is a common problem when using charged
This may be caused due to:
non-dispense of reagent or an incomplete dispense of reagent. It
It is important that the user follows the recommended temperature
slides or when there are additives in the water bath or by heat. When
for storage and usage for each of the staining kits. All ArtisanLink
using a methenamine silver solution, a “mirroring” also know as grey
staining kits have been validated to be used at room temperature.
haze can occur on the slide. “Mirroring” is defined as metallic silver
Deviating from the recommendations may cause an uneven staining,
deposited non-specifically over large areas of the front and back of
shorten the shelf life, and possibly cause a false negative staining.
the glass slide. The silver solution deposits a fine, brown-black,
Reagent storage and usage temperatures can be located on the
granular precipitate over the section and the glass portion of the
reagent packs and in the package inserts.
is important to always prime the packs before daily use to ensure
Inconsistent tissue thicknesses from slide to slide. For instance,
thicker sections will show a strong staining, whereas thinner
sections will show a weak staining (Fig. 5). It is important to keep
to the correct thickness throughout the microtomy sectioning to
obtain a consistent staining.
proper dispensing of reagents.
 Uneven staining. This can be caused by the reagent not spreading
evenly across the tissue section or sections. This is particularly
problematic with reagents containing acids such as acetic acid
or periodic acid. The solution to this problem is to soak the slides
in an Artisan™ Wash Solution for five minutes before loading the
slide (Fig. 4). Microscopically, this type of precipitate is usually not
slides onto the slide carousel. This procedure conditions the slides
found on the same plane as the tissue (1).
for spreading the reagents (Fig. 6).
Figure 5. Inconsistent tissue thicknesses from the same run. Warthin-Starry
Stain.
Cold reagent packs not brought to room temperature before use. All
reagent packs used on ArtisanLink Special Staining System must
be brought to room temperature (22-25°C). Allow 45 minutes for
Figure 6. Periodic Acid-Schiff Diastase (m/D) staining of liver tissue. The uneven
the reagent packs to warm up before placing them on ArtisanLink.
staining is due to amylase not spreading evenly- causing part of the liver section
Using a cold or cool reagent pack may prevent the chemical
to stain undigested glycogen.
reaction from taking place, and will give the impression that the
Figure 4. Examples of “mirroring”/grey haze. The cause of this effect is over-
instrument is producing inconsistent staining results within the
heating of the methenamine solution.
same run.
Another reason for an uneven staining may be the placement of
the tissue sections on the slide close to the slide clip. This can be
rectified by making sure that the tissue sections are centered on the
To prevent “mirroring” or grey haze, it is important to check the
slide. Placing the sections close to the slide clip causes a “wicking”
temperature of the slide heater and/or the incubation time set in the
effect which prevents complete aspiration of the reagents causing an
software. Using non-charged slides (except for decalcified tissue) can
uneven staining.
also help lessen the amount of background staining when performing
stains with silver nitrate solutions.
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Troubleshooting Procedure for ArtisanLink Special Staining System
Chapter 33 Tips on using the ArtisanLink
Special Staining System
Jamie M. Nowacek, BS, HT(ASCP)CM, QIHC, PMP
Issue
Staining Artifact
Manual Procedure
ArtisanLink Procedure
Store reagents at correct temperature;
Store reagent packs at correct temperature;
(See package insert for specification)
(See package insert for specification)
Filter reagents before use
Mix and prime reagents before use
Use clean slides
Use clean slides
Clean glassware in soap and hot water
Clean bulk liquid containers
Rinse in deionized water and air dry
Check dispenser tip for debri build-up
ArtisanLink Staining System is an automated special stains slide
processor that can be used as a stand-alone system or operated
in a networked configuration using a Local Area Network (LAN) or
a Laboratory Information System (LIS). The system uses special
stains kits and accessory reagents packaged in patented cartridges
that provide precision dispensing of reagents for optimal staining
quality. ArtisanLink utilizes a patented “reaction chamber” and can
handle multiple tasks in a laboratory in a single run. With a broad
menu of special stains, the system can optimize laboratory workflow
through automation dramatically improving productivity. This article
Background Staining
Use acid clean glassware
Use the default protocol for silver stains
Do not use metal forceps
Do not add adhesives to the water bath
Employ proper fixation, dehydration and infiltration
Employ proper fixation, dehydration and infiltration
Increase or decrease staining time by looking
Cut all sections into a thickness of 4 µm, except for Jones’ Basement
at individual slides under the microscope
Membrane Stain for kidney which should be cut into a thickness of 2 µm
provides tips on hardware and software usage as well as some
recommendations regarding the use of slides and reagents to get
Inconsistent Staining
and Congo Red Stain which should be cut into a thickness of 8 µm
the most out of the ArtisanLink Staining System1.
Hardware Tips
The following is a four-point inspection to keep ArtisanLink running
smoothly:
 Waste management: The ArtisanLink Staining System utilizes
For an uneven staining, adjust the volume
Soak the slides in Artisan™ Wash Solution for five minutes
a decrement system to track waste accumulation and capacity
of reagent used to ensure proper spreading
before placing them on the instrument
requirements. A special aspiration system allows the instrument
Figure 1. Waste bottle showing the orange waste gasket.
to separate waste into four categories: metals, alcohol, combined
dyes and water solubles. Depending on the laboratory workload
Conclusion
Reference
volume, 2 L or 4 L bottles can be configured in the software.
The solution to staining artifacts, background staining and incon-
1. Brown RW (Ed.) (2009). Histologic Preparations: Common Problems
and Their Solutions. College of American Pathologists, Northfield IL,
2009.
Upon completion of each session, a waste valve rinse should be
sistent staining for both the manual and the automated procedures
is very similar. The table above shows the comparison.
performed to prevent contamination of the tubing and bottles. The
user should inspect the inner orange waste gasket when replacing
the cap to check that this has not been torn or ripped2 (Fig.1).
ArtisanLink Special Staining System provides the histotechnologist
Keeping software and bottle inventory evenly matched at all times
with a tool to produce the same diagnostic quality stains as found
will prevent overflow and consequential aborted runs. Each time
in a manual staining technique. However, unlike a manual staining,
a bottle is removed, perform the Rinse valves (Fig. 2) from the
ArtisanLink allows the histotechnologist to walk away from the
ArtisanLink bulk fluid screen to enable optimal aspiration.
special staining bench to pursue other tasks.
1
This article provides tips for experienced users. 2A torn or ripped orange waste gasket may be the result of excessive torque applied when screwing/unscrewing
the cap.
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