CASE STUDY

Xylene-Free Tissue Processing

Xylene-Free Tissue Processing:Good Results and Less Exposure
for Staff and Environment
Pathology North Hesse, Kassel, Germany
Pathology North Hesse Laboratory
Pathology North Hesse, in total, has a staff of 42 with anadditional, five medical specialists and five assistant physicians.
In the histology laboratory, reporting to laboratory heads
Karin Flecke and Karoline Janas, there are twenty permanent
medical technical assistants working full- or part-time, to process
the incoming specimens quickly and reliably for diagnosis.

ProfilePathology North Hesse is a private institute for pathology,
founded 2007 in Kassel, Germany with Prof. Dr. med.
Josef Rüschoff as medical director. It is a consolidation andexpansion of two independent pathology laboratories. Later,a branch laboratory in Eisenach, Thuringia, was added, inorder to properly serve this region. The management of thePathology North Hesse is in the hands of four associatepartners – all medical specialists in pathology.
The service area of North Hesse Pathology includes hospitals
and medical practices in Kassel and the surrounding region.
There are synergies with the discovery life science
company, Targos, located in the same building, which
specializes in the development of biomarkers andservices in molecular pathology.

Technical equipment supporting the laboratory work includes
seven tissue processors, three tissue embedding stations
and four sectioning stations. Start of work is 6:00 a.m. – thefirst specimens are sectioned starting 6:30 a.m., continuing
until noon. The team’s goal is to process the tissue quickly,
to enable a fast diagnosis and return of results. Under normalconditions, the specimens of the previous day are reported
back around 1:00 p.m.
In the histology section, the workload varies between 300 to
400 specimen per day. In 2021, about 227,000 blocks wereprocessed, resulting in about 358,000 slides.
Alongside the histology laboratory, there is a smaller cytology
laboratory that handles about 30 samples a day, as well asa laboratory for IHC (Immunohistochemistry) samples. For
immediate sectioning, two cryostats are available.

Health Risks in Histology
In pathology, classification of tumors and definition of diseases
comes from the analysis of tissue specimens. With the rightdiagnosis it is possible to develop a treatment for patients, and,in the best case, to initiate the healing of the patient. This is theaim of the pathologist, supported by the technical staff in thelaboratory.
However, it should not be forgotten that the job of a medical
technical assistant is one of the most unhealthy professions1.
In processing tissue to be permanent and stable, hazardous
fixation fluids and solvents are used. Therefore, the commitment
to health of the patients can risk the health of laboratory
personnel.
During the normal process, tissue is processed in four steps:

Since the health and life of patients depend on properdiagnoses, a solid quality management system is very importantfor Pathology North Hesse. Since 2013, the laboratory has
been accredited by the DAkkS (Deutsche Akkreditierungsstelle
GmbH; German accreditation body) following DIN EN ISO/IEC
17020 (conformity assessment). These accreditations require
quality assurance and competency on highest levels. The
Institute participates in internal as well at external audits andinterlaboratory comparisons.
In relation to the work in the laboratory, this means – thelaboratories work very precisely, cassettes are put intoalphanumeric order before the start of the tissue processing, inthe order of their laboratory entry, and different colored cassettes
help to identify the different tissues types at one glimpse. Thepre-processing arrangement does not add much time earlyin the work cycle – yet helps to save time in the end, since theworkflow can proceed directly after tissue processing without
any additional sorting steps. The arrangement of the cassettes
also helps avoid mistakes.

•First, the tissue is fixed, to stop degradation processes
and to make the tissue stable. For this step, formalin/
formaldehyde is used in most of the pathology laboratories.

•After fixation the tissue is getting dehydrated – most oftenwith the help of alcohol.

•The third step, degreasing and clearing is done with xylene.

•The last step, embedding into paraffin or other media,
stabilizes the tissue and enables sectioning.

All those steps may include contact to formaldehyde andexposure to organic solvents and are, therefore, potentiallyhazardous for the user.
In the last few years, several methods have been developed tominimize the health risks in the laboratory. Technique can help
here – e.g. a good ventilation system removes solvent vapors
directly at their origin. Simple steps, like the usage of containers
with lids preventing the fixative from evaporating at the workstation, reduce the amount of solvent and formalin vapor in theatmosphere of the laboratory.
At the instrument level, there have been a number of technicalenhancements improving safety: There are tissue processors
on the market with built-in protection for users. The Excelsior™
ES, AS and Revos™ tissue processors from Epredia, forexample, have downdraft ventilation included in the instrument. It

automatically starts when the instrument is opened for loading orunloading of the samples. That way, the hazardous formalin andorganic solvent vapors are removed in a manner that prevents
the user contact. Those vapors are then routed through a variety
of filters neutralizing the formalin and binding the xylene.

substitutes. For user safety, xylene should be banned from use
in laboratories wherever possible. For example, in most of thetissue processing procedures, xylene can be substituted by theless-harmful isopropanol.

Xylene-free working procedures
At Pathology North Hesse, the dehydration of tissues has moved
to a xylene-free procedure wherever possible to protect thelaboratory staff. In 2004, the first Excelsior ES was purchased.
From that point forward, most tissue samples were processed
without xylene usage. Meanwhile, in the histology laboratory,
there are six Excelsior and one Revos tissue processor, of which
five are operated without xylene. Xylene is needed for efficient
clearing only when processing of very fatty tissue like breastand intestine.
In the Eisenach Branch Laboratory, two of the three tissue
processors are running permanently xylene-free – the team has
found the xylene-free procedure is not only safer in handling, butalso results in higher-quality tissue.

Excelsior AS Tissue Processor from Epredia

In the onboard storage area for solvent containers, hazardous
vapors are removed and neutralized with filters as well.
Additionally, the instruments use pre-filled containers which do
not need to be decanted and can be placed directly into theinstrument.

“Among others, gynaecological, urological and dermatologicaltissue samples, treated with alcohol only, are easier to section.
If you dehydrate with xylene, the specimen gets harder andtherefore more difficult to cut”, says Ms. Karoline Janas, one of
the two laboratory heads at Pathology North Hesse.
From time to time, Ms. Janas gets calls from colleagues, whohave issues with the quality of their embedded tissues. “Veryoften, this is just an issue with the dehydration step”, according
to Ms. Janas – and this can easily be fixed by using a xylene-freeprocess for tissue processing.

Beyond instrument features and methods that help users avoid
exposure to hazardous substances, there are ways to avoidhazardous substances completely and use harmless“...tissue samples, treated withalcohol only, are easier to section.
If you dehydrate with xylene, thespecimen gets harder and,therefore, more difficult to cut”
Karoline Jonas, Deputy Head of Entry Laboratory

Protocol for xylene-free embedding
and classic embedding with xylene
Xylene-Free Dehydration
Solvent

Conclusion

Classic Dehydration with Xylene
Time (hrs)Solvent

Time (hrs)

70% Isopropanol

00:30

70% Isopropanol

00:30

80% Isopropanol

00:45

80% Isopropanol

00:45

90% Isopropanol

00:45

90% Isopropanol

00:45

96% Isopropanol

00:45

96% Isopropanol

01:00

100% Isopropanol

00:45

100% Isopropanol

01:00

100% Isopropanol

01:00

100% Isopropanol

01:30

100% Isopropanol

01:00

Xylene00:45

100% Isopropanol

01:30

Xylene01:00

100% Isopropanol

01:30

Xylene

01:15

Paraffin

01:00

Paraffin

00:45

Paraffin

01:15

Paraffin

01:00

Paraffin

01:15

Paraffin

01:00

Total time

12:00

Total time

11:15

The dehydration of the tissue with both protocols runs overnight.
The fixation time is not included in this table – it varies with the type of the tissue.

Protocols used in the laboratory for xylene-free and classic dehydration are very similar.
Both protocols take about the same amount of time to dehydrate the tissue. Since bothprotocols are optimized for an overnight run, time in not a major factor.
In addition to the potentially slower time for tissue processing, higher safety for thelab personnel, and the better sectioning quality – there are two more reasons for thechange to dehydrating tissue just with isopropanol. If the tissues allows: Running onlywith isopropanol saves reagents – per reagent rotation, just one bottle of isopropanolis used, instead of two bottles – isopropanol and xylene. This saves time for changing
of one bottle (and, depending on the source of the solvents, time for decanting). It also
saves money, since the costs for xylene and its disposal can be omitted.
Many institutes with xylene-free dehydration replace xylene with ethyl alcohol. Ethyl
alcohol can also be replaced by isopropanol at each step of the dehydration process.

1

https://www.businessinsider.de/most-unhealthy-jobs-in-america-2017-4?op=1
https://finance.yahoo.com/news/the-15-jobs-that-are-most-damaging-to-yourhealth-155706120.html?guccounter=1

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North Hesse, used with permission. MX43032_US 0822

At Pathology North Hesse in Kassel, theconsumption of xylene is reduced to theminimum. In many cases, during tissue
processing, xylene can be replacedwith isopropanol as the only alcoholfor dehydration. Isopropanol is the only
alcohol where the user can go without theintermedium (xylene).
The resulting tissue blocks often havebetter usability than those processedwith xylene, due to being less hard
and rigid. Those tissue preparations
can be used the same way asclassically dehydrated tissues. They
are also usable for the IHC without any
restriction. “There have never beenissues.”, says laboratory head Ms.
Flecke.
Only when used for very fatty tissue
have the results without usage of xylene
been less satisfying than with classicalprocessing methods.
“We have used this method for fourteenyears”, according to Ms. Janas, “and weonly see benefits. Tissue quality and safety
at work are always our number one priority.”
Pathology North Hesse
info@patho-nordhessen.de