Instituto

Politécnico de Lisboa
Escola Superior de Tecnologia da Saúde de Lisboa
Licenciatura em Anatomia Patológica, Citológica e Tanatológica
(Degree in Pathology, Cytological and thanatological)

Formol Líquid versus Formol Gel:

Comparison of Quality of fixation in immunocytochemistry

Students:
Ana Cristina Bastos Santos
Joana Filipa Louro Francisco Antunes Filipe

Course:
Research in pathology

Head Teacher:
Amadeu Borges Ferro

Project advisor:
Master Amadeu Borges Ferro

Lisbon, Setembro de 2012

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1. Description

1.1- Objetives

The investigators set out to evaluate if any statistically significant differences could exist
between 10% buffered formalin liquid (FL) and formol gel (FG), during the stage of fixation.
With this purpose, an analysis statistics descriptive was performed to all the samples analyzed.
From the application of descriptive statistics provided by the software used (SPSS v.20), values
for the average and standard deviation of the two groups of samples under study- samples fixed
in FL and samples fixed with FG-were obtained.
The group of fragments fixed in FL had an average score of 95,36 ( on 100) with a standard
deviation of 6,651 against the group fixed on FG that had an average score of 96,06 (on 100)
with a standard deviation of 5,800 (figure 1).

Average standard deviation

Figure 1: Average and standard deviation of FL and FG

From the analysis of the figures above, we can conclude that the results were slightly better
when fixation was done with FG, than FL. Such conclusion can done not only by the average
value but also by the lower value obtained in standard deviation which portrays the existence of
a greater uniformity of the results when using formol gel as fixative.
However, although slightly better for the FG, the results were very similar, the difference
between them being less than one percent.

1.2- T test – Influence of fixative on quality

In order to ascertain whether the results obtained allow or not to reject the Null Hypothesis (H0)
defined, "There are no statistically significant differences in quality of fixation of fragments were
fixed in 10% formalin Liquid Buffered compared with those fixed in 10% formalin Gel" the T test
was performed for independent samples. Applying the above test a value of p or p-value, is
obtained and it allows, or not, rejecting the set H0.

For the study in question and from the global analysis of all samples, the p-value obtained
exceeds the value of α (.066> .05) (Table 1). Thus, the relationship between both values does
not allow the rejection of H0 defined by what we can say that there are no statistically
significant differences between groups of samples fixed with different fixative solutions

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Table 1 – Output SPSS: Teste T

These results may be further supported by analysis of the results of direct microscopic
observation of the blades in the study – picture 1

Picture 1 - Immunostained with anti-vimentin sérum : FL (left ) and FG (right) – 400x.

The left picture corresponds to a section of a fragment fixed in formalin for 24h in FL, while the
image on the right is a fragment fixed in formaldehyde Gel (FG) for an equal period of time.
Both sections are positive for immunostaining with anti-Vimentin serum.
By direct observation of images we can see that there is no difference in any parameter
evaluated: immunostaining intensity, amount of labeled structures, immunostaining nonspecific,
contrast staining intensity and preservation of morphology.

1.3 - Oneway test ANOVA – Fixation length

Subsequently, in order to verify if the duration of fixation could influence the overall score of
histological sections whose fragments were set with each of the fasteners, the test was
conducted with ANOVA Oneway, whereby was obtained value p-value . For both fasteners, p-
value assumed values above α, specifically, fixed to the fragments in FL p = 0.274 (Table 2),
the FG set at p = 0.344 (Table 3).

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 1.4 - Post Hoc de Tuckey

Even in the study of the influence of the duration of fixation quality of immunostaining, and to
enable multiple comparison of averages (length fixation - 12h, 24h and 48h) a poc hoc test was
performed, more particularly the Tukey .
With respect to the FL, we can see that the value p assumes values between 0.337 and 1.000
(Table 4). In the case of FG, p can assume values between 0.317 and 0.852 (Table 5).

The post hoc test results thereby strengthening the results obtained in Test ANOVA Oneway we
discussed before, since from this one might say too, that the length of fixation does not
influence the final quality of immunoreactivity.

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 In order to facilitate visualization of the results described above, we shown an image where you
can see two compiled photographs that portray different fragments fixed in both FL and FG in
the three time intervals in the study - 12h, 24h and 48h.
As mentioned, the six pictures presented correspond to different histological sections that only
have in common the fact that they are positive with anti-Ki67 immunostaining.
In the photographs presented on the left, and numbered from one to three, it is possible to
observe microscopic images of three sections corresponding to three distinct blocks of
fragments fixed in FL. The only difference resides in the duration of fixation: 12h for the first
picture, 24h and 48h for 2 to 3.
The photos shown on the right in the image correspond to three sections from three distinct
fragments fixed in FG. Also in this case only differ in the duration of fixation: from 4 to 6, 12h,
24h and 48h.
Thus, despite the above numbered variants we don’t observed statistically significant differences
in the three groups presented (12h, 24h and 48h) in which concern the quality of immunostaining.

Picture 2: Comparison of quality of the immunostaining with primary serum anti-Ki 67 in placenta fragments fixed
with FL (left) and FG (right) with different fixation time: 1 and 3-12 hours; 2 and 4-24 hours; 3 and 6-48h- 400 x

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1.5 –T test – Serums

In order to better understand if the type of fixatives in study, FL or FG, has influence on the
quality and capacity of action of each of the six sera studied, the t test was performed (Table 6).
From the analysis of Table 6 we can easily verify the existence of two distinct situations: for the
serum anti-CD3 and anti-CD34, p-value <α (0.025 and 0.00 respectively), witch means that in
this case the null hypothesis defined can be rejected.
In other words, it can be stated that the quality of immunostained with each of the two antisera
is influenced by the type of fixative used.

Processing data available was even possible to calculate both the mean and standard deviation
for each serum (Table 7).

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 1.5.1 Anti-Pan Cytokeratin

For the serum anti-pan cytokeratin, the results were quite similar. The Global Score average of
fixed samples in FL was 98.95 as opposed to 99.28 for samples fixed in FG. Also with respect to
the standard deviation, the values were very close: 2.004 to 1.656 for FG and FL (Figure 2).
The data provided by the statistical tests are easily confirmed by microscopic analysis of
histological sections. In image 3 you can see, on the left, a section set in FL and on the right,
one set at FG. Both images are the corresponding sections fixed for 12h and positive for serum
anti-pan cytokeratin, AE1/AE3 clones. As can be seen, there are no apparent differences in
staining intensity particular, the amount of labeled structures or in non-specific immunostaining.

Average standard deviation

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 1.5.2 Anti-CD3

The difference found in the averages for immunostaining with anti-CD3 between fixed tissues
with FL and FG is 2.47 percentage points, and was the highest average calculated for the second
fastener. In more detail, the mean Global Scores to FL samples was 85.58 whereas the mean
for samples fixed on FG was 88.05. In case of standard deviation, the values were 7.580 and
7.017 respectively, these being the highest values obtained throughout the study (Figure 3).
Standard deviation values so high reflect the great variability of results observed.

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Although the data obtained from the statistical analysis, the existence of such variability is
extremely difficult to verify when the observation of only small areas of the sections studied, as
can be noted in the image analysis 4.

1.5.3 Anti-CD20

The quality of immunostained with anti-CD20 serum is lower than the immunoblot with serum
anti-pan cytokeratin, clone AE1/AE3. Although, for serum concerned, there is greater uniformity
of values between fixed samples FL and FG are variations significantly lower than those obtained
for serum mentioned above.
When applied to fragments FL fixed in the quality of immunostaining was scored with score
Global average of 93.11 and a standard deviation of 5.810. Moreover, fragments set in FG, the
average value obtained was 93.89 with a standard deviation of 5.391 (Figure 4).

Also the microscopic examination of histological sections studied denoted the absence of
significant differences in the quality of immunostained by anti-CD20. Two of these sections are

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represented, in part, on the image 5, where one can observe on the left part of a section fixed
in FL and the right, a section fixed in FG

1.5.4 Anti-CD34

For the serum anti-CD34, the difference between the average score for Global fixed tissues FL
and FG is more pronounced. In the first case, the average global score obtained was 97.10 with
a standard deviation of 3.956. Regarding fixed in FG, the average score was 99.03 with a
standard deviation of 2.392 (Figure 5).
Microscopically, this difference can be detected by analyzing the image 6. In this case, when
observing the section fixed on FL, shown at left, is easily proved that besides the lowest amount
of labeled structures compared to when the section shown on the right, also the specific staining
intensity is lower.

1.5.5 Anti-Ki 67

The results obtained by the anti-Ki67 immunostaining were satisfactory, whereas, in this case,
there is a contradiction until now the trend noted since the average value is set at higher for
fragments FL. More specifically, the mean Global Scores for FL is 98.79 as opposed to 98.01

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obtained for the FG. In case of deviation, the value obtained for the FL was 2.928 while for the
FG was 3.198 (Figure 6).

The image 7, presented below, is related to the microscopic aspect of the two sections fixed for
12 hours and subjected to immunoblotting with anti Ki-67: the image on the left refers to
section fixed in FL in fixed and right fixed in the FG.

1.5.6 Anti-Vimentina

Anti-Vimentin Serum follows the trend set out for serum anti-Ki67. Also in this case the average
values obtained were higher for fragments fixed in FL, 98.62 to 98.09 as opposed to FG. The
values of standard deviation were 2.800 and 3.103 for fixed fragments in FL and FG,
respectively (Figure 7).

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Microscopically it can be confirmed that homogeneity results both in the intensity of the specific
marking, as the amount of labeled structures, staining intensity and contrast marking
nonspecific. Pictured 8 is possible to observe two distinct areas relating to sections fixed in FL,
left, and FG, right by 12 hours.

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2. Final conclusion
According to the results obtained and presented in the previous chapter, and although
the average Global Scores for Formaldehyde Gel (96.06) present slightly higher than
the average Global Scores for Liquid 10% buffered formalin (95.36) it can be stated
that there are no statistically significant differences in quality of attachment compared
the two fasteners study, 10% Liquid Buffered Formaldehyde and Formaldehyde Gel
10%. This holds true both in the type of fixative used as the fixation time concerns.
Thus, the results obtained confirm those presented in previous studies, where one can
read that "there are overlapping both in the macroscopic results, both in morphology
and tissue preservation, there are slight variations were not significant."
However, this study also evaluated the influence of fixation quality of immunoreactivity.
With respect to this parameter, it was found that each serum reacts differently in
certain tissues fixed with fixative. However, despite the above, only two sera
immunocytochemical studies, anti CD3 and anti-CD34, there is the existence of
significant differences.

Thus, taking into consideration all the results presented above, it can be concluded
that, and answering the question posed at the beginning of starting work,
Formaldehyde Gel 10% is able to match the quality of fixation afforded by Liquid
Formaldehyde 10% buffered.

Besides the above, and assuming the producer announced that the emission of toxic
gases is lower in formaldehyde gel, it can be stated that this fixation is presented as
the alternative to formaldehyde fixation on routine laboratories of Pathology, data also
confirmed by studies directed by both LaFrinieri Galang and as for Souls and
colleagues.

Thus, it is also concluded that the overall goal is the researchers proposed, contributing
to the decrease in exposure of Pathology Technicians toxic gases released by Liquid
10% Buffered Formalin. This last fact proves extremely important because the toxic
gases released by liquid Formol be the main risk factor chemical that are subject to
these professionals.

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