Vacuum sealing and cooling as methods to preserve surgical specimens
Thomas Kristensen1, Birte Engvad1, Ole Nielsen1, Torsten Pless2, Steen Walter3 and Martin Bak1
2
1
Department of Pathology, Odense University Hospital, Odense, Denmark
Department of Surgical Gastroenterology, Odense University Hospital, Odense, Denmark
3
Department of Urology, Odense University Hospital, Odense, Denmark
Introduction
Recently, a vacuum based refrigeration system for preservation of surgical specimens has been proposed as a logistically feasible and safe alternative to formalin
fixation at the surgical theatre.
However, the system combines vacuum sealing and cooling, which is known to
delay degradation processes in surgical specimens, and the extent to which vacuum
sealing alone has an effect on tissue preservation remains to be tested.
Materials and methods
We compared the effect of vacuum sealing to the effect of i) cooling, ii) cooling
in addition to vacuum sealing and iii) no treatment, in up to five different organs
(Figure 1).
The evaluation of tissue preservation was based on integrity of up to 15 different
epitopes using IHC analysis, morphology using histological examination and RNA
integrity using real-time quantitative polymerase chain reaction (PCR) analysis. We
modified the scoring system described by the NordiQC to assess histology as well as
IHC staining quality (Figure 2-3).
Figure 1. A total of 70 experimental samples and six reference samples were
included for each of the five organ types included in the study. The six reference
samples were collected at the experimental time = 0 h. Experimental samples
were collected after 1, 2, 4, 8, 20, 44 or 92 h. RT: Room Temperature. Samples
labelled “IM” were formalin fixed for approximately 48 h before further processing and paraffin embedding and analyzed with respect to IHC and morphological
endpoints. Samples labelled “R” were snap frozen and stored at -80°C until
subsequent RNA extraction and analysis of RNA integrity. Samples labelled “R”
were not analyzed with respect to tissue morphology or epitope integrity.
Results
A time-dependent decrease in the integrity of cellular morphology was observed
in all four experimental treatment groups during the experimental period of 92 h.
Morphological integrity was reduced at a higher rate in samples stored at room
temperature (RT) compared to samples stored at 4°C, independently of whether
the samples were vacuum sealed or not (Figure 4A).
When examining the IHC analysis individually for each antibody, the mean of all IHC
integrity scores within a tissue type or the mean of all IHC integrity scores in all tissues, the same overall result was observed; IHC epitope integrity was reduced in a
time dependent manner consistent with that observed for cellular morphology, the
quality was reduced at a higher rate in samples stored at RT compared to 4°C, and
no tissue preserving effect of vacuum sealing was detected (Figure 4B).
No detectable reduction in integrity of ABL1, B2M or GUS mRNA was observed in
any of the four treatments in spleen and kidney. In liver, however, only samples
from the two 4°C treatments showed the pattern with little or no RNA degradation
over time. In contrast, samples from both RT treatments produced similar results
with detectable degradation being evident after 20 h (Figure 5).
Figure 3. IHC staining quality scores illustrated using E-cadherin antibody (clone
HECD-1) in four formalin fixed paraffin embedded kidney samples representing
the score values 1 – 4. [A] Optimal IHC staining quality (score value 4), vacuum
sealing and cooling at 4°C, 1 h. [B] Good IHC staining quality (score value 3),
vacuum sealing and cooling at 4°C, 44 h. [C] Borderline IHC staining quality (score
value 2) showing weak staining, vacuum sealing and room temperature, 44 h.
[D] Poor IHC staining quality (score value 1) showing hardly any staining, vacuum
sealing and room temperature, 92 h. x 100 for all panels.
Figure 5. [A] Integrity of ABL mRNA in spleen presented as the qRT-PCR Ct
value as a function of time in the six treatment groups; room temperature
with vacuum (――), room temperature without vacuum (――), 4°C with
vacuum (- -  - -), 4°C without vacuum (- -  - -), FineFIX fixation (······) and
formalin fixation (······). cDNA from a fixed amount of total RNA was used for
all samples. Each data point represents the mean of qRT-PCR triplicate analysis.
Experimental samples were collected at the time points 1, 2, 4, 8, 20, 44 and 92
h. Experimental time = 0 h is depicted as 0.1 h. Samples producing no amplification are represented by Ct = 41. [B] Integrity of GUS mRNA in spleen. Results
highly similar to those observed in spleen were observed in kidney (data not
shown) [C] Integrity of ABL mRNA in liver. [D] Integrity of GUS mRNA in liver.
Within each organ type, results highly similar to those observed for ABL and GUS
were observed for B2M (data not shown).
Conclusion
Figure 2. Morphological integrity scores illustrated in four formalin fixed paraffin
embedded liver samples representing the score values 1-4. [A] Optimal morphological integrity (score value 4), vacuum sealing and cooling at 4°C, 1 h. [B]
Good morphological integrity (score value 3) showing slight nuclear shrinkage,
vacuum sealing and cooling at 4°C, 20 h. [C] Borderline morphological integrity
(score value 2) showing marked nuclear shrinkage, vacuum sealing and cooling
at 4°C, 92 h. [D] Poor morphological integrity (score value 1) showing loss of cell
definition and extensive cell lysis, vacuum sealing and room temperature, 92 h.
x 100 for all panels.
Figure 4. [A] Morphological integrity scores as a function of time in the four
treatment groups; room temperature with vacuum (――), room temperature
without vacuum (――), 4°C with vacuum (- -  - -) and 4°C without vacuum
(- -  - -). Each data point represents the mean of the score values in the five
tissues. [B] IHC staining quality scores as a function of time in the four treatment
groups. Each data point represents the mean of all score values from all antibodies in all five tissues. Quality score value 4 corresponds to optimal, 3 corresponds
to good, 2 corresponds to borderline and 1 corresponds to poor morphological
integrity or IHC staining quality. Experimental time = 0 h is depicted as 0.1 h in
both panels.
[email protected]
No tissue preserving effect of vacuum sealing was
observed for any of the endpoints included in the
present study. Instead, storage at 4°C was demonstrated to preserve tissue to a higher degree than
storage at RT, independently of whether the tissue
was subjected to vacuum sealing or not.
We conclude that vacuum sealing is not an alternative to cooling on ice. If used, the vacuum sealing
system should be combined with cooling as a
logistically convenient method to transport surgical
specimens between surgical theatres and pathology
departments.
Acknowledgments
The study was supported financially by Axlab,
Vedbæk, Denmark. The TissueSafe instrument was
provided by Milestone, Bergamo, Italy.