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Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(3):103-105
Original Article
Importance of pH of Fixatives Used for Fixation of
Histopathology Specimens – An Un-Recognized Issue
Usman Hassan, Muhammed Kashif Baig, Sajid Mushtaq
Department of Pathology, Shaukat Khanum Memorial Cancer Hospital & Research Centre, Lahore
Abstract sensitive techniques are being introduced in histopathology
Objective: Adequate fixation of the surgical specimens which have very strong impact on diagnosis. So, a good
is very important to ensure optimal histopathology, fixation is not only necessary for light microscopic
immunohistochemistry and genetic tests results. To examination but also for more advanced and sensitive
achieve these results, it is very important to use good techniques like special stains, immunohistochemistry and
fixatives with pH maintained at 7. We measured pH
genetic studies like polymerase chain reaction and in-situ
values of the histopathology specimens received from hybridization.3,4
different parts of country to determine whether fixative There are many fixatives commercially available and each
being used is appropriate or not. has its own advantages and disadvantages. An ideal fixative
Material and Methods: It was a descriptive cross should have maximum advantages and least number of
sectional study carried out at Armed Forces Institute of disadvantages. One such fixative is formaldehyde. It is
Pathology, Rawalpindi, Independent Medical College widely used in our operation theatres and histopathology
and Mian Muhammad trust hospital Faisalabad on one departments for transport and fixation of surgical
thousand consecutive samples received for 1,4
specimens. It is suitable for most of the tests carried out at
histopathology reporting. To assess the pH, fixative of histopathology department like light microscopy, special
the sample was taken out in the kidney tray and pH stains, immunohistochemistry and genetic studies.
meter was dipped in it and value of pH noted. There are many important factors involved in fixation like
Results: The specimen pH was recorded. The mean hydrogen ion concentration (pH), depth of penetration,
value came out to 6.1 ranging from 1.3 - 8.8. The osmolarity, concentration, duration and temperature. Among
maximum number of readings fell between 6.1 -7.0 (n these factors, the one which is very important and easily
530 cases). The second highest group was 5.1- 6.0 which measurable is hydrogen ion concentration (pH). pH values
comprised of 269 cases. About 366 specimens have pH of different fixatives vary. In general pH is usually adjusted
5
less than 6 and 104 specimens above 7, whereas for ideal to the physiological range by use of a suitable buffer.
fixation, the ideal recommended pH is 7. Satisfactory fixation occurs between pH 7 and 8. Outside
Conclusion: Unfortunately, most of the specimens this range there are changes which are detrimental to the
received did not have ideal pH. It is very important to ultra-structure of tissue. After being surgically removed, pH
educate medical and paramedical staff, who deal with within the tissue cells is known to decrease, due to anoxia.
the surgical specimens, about the importance of pH and As many normal tertiary and quaternary structures of most
also how to ensure proper fixation of the specimens. This proteins are stable over a limited pH range about neutrality
will help in achieving ideal histopathology results. i.e. pH of 7, many buffer systems are available to decrease
Key Words: Histopathology, Fixatives, pH this acidic environment and to preserve cellular proteins.
The most commonly used in our laboratories is phosphate
buffer.6,7
Introduction Although there are many issues which need to be addressed
In histopathology, tissues are fixed before they are examined regarding specimen handling but the one which is the most
microscopically. Fixation is the foundation for the basic, easily controllable and of utmost importance is
8,9,10
subsequent stages in the preparation of sections through to adequate hydrogen ion concentration of the fixative.
From our experience we have noted that many times
Address for Correspondence: morphological details are compromised due to use of poor
Dr Usman Hassan fixative in operation theatre. Whenever the pH of these
E mail:drusmanhassan256@gmail.com fixatives is retrospectively checked it is not up to the mark.
th th Most of the time it is towards the acidic range. The rationale
Received: Oct.12 2015 Accepted:Dec. 7 2015 of this study is to determine the frequency of pH values of
1,2
the making of diagnosis. Nowadays, more and more
103
Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(3):103-105
fixatives used to fix surgical specimens received at AFIP good results. A good fixation helps in preparing good
Rawalpindi, Independent Medical College and Mian quality slides, carrying out proper immunohistochemical
Muhammad Trust Hospital Faisalabad from different stains and cytogenetic studies which can give us best results.
medical centers of our country. This is especially important in morphologically difficult
lesions in which immunohistochemical and genetics studies
Material and Methods have to be done. This study was carried out to find out the
It was a descriptive cross sectional study carried out at pH of fixative used to fix histopathological specimens which
Armed Forces Institute of Pathology, Rawalpindi, were received from different parts of country. The pH is an
important factor to determine fixative ability to fix the
Independence Medical College and Mian Muhammad Trust specimens properly.5,6,7 Unfortunately the lack of education
Hospital Faisalabad on one thousand consecutive samples regarding fixation of specimens in doctors results in poor
received for histopathology reporting. Request forms of fixation of specimens. This badly effects proper processing
these samples were retrieved and department identification of the tissue and also subsequent ancillary techniques. It is
numbers and name of referring hospitals were noted. evident from the results of this study that 366 specimens had
Hydrogen ion concentrations (pH) of fixatives used for pH less than 6 and 104 specimens above 7, whereas for ideal
fixation of surgical specimens were measured with the help fixation, the ideal recommended pH is 7.
of an instrument called pH meter. To assess the pH, fixative This study enlightens the importance of the fixative pH. All
of the sample was taken out in the kidney tray and pH meter doctors and paramedical staff related to surgical specimens
was dipped in it and value of pH noted. Data was entered in need to know the importance of proper pH. It can be easily
SPSS version 17. Mean, median and mode were calculated determined from the results of this study that most submitted
for the pH values. specimens have pH either below or above the recommended
pH. This means we do not know how to make proper
Results fixative. This can have serious implications on the biopsy
The specimen pH was recorded. The minimum pH was 1.3 results. This should be made a permanent part of curriculum
and maximum pH was 8.8. The mean value came out to 6.1. of medical students and staff to educate them how to process
The mean, median and mode were 6.18, 6.3 and 6.4 a surgical specimen once surgery is done. We cannot deny
respectively. The maximum number of readings fell between the fact that once histopathology specimens are poorly fixed,
6.1 -7.0 (n = 530 cases). The second highest group was 5.1- we cannot revert back the changes which had already been
6.0 which comprised of 269 cases. About 366 specimens occurred in specimen due to poor or bad fixative pH. This
have pH less than 6 and 104 specimens above 7, whereas for can be controlled at first sight by allowing surgical
ideal fixation, the ideal recommended pH is 7 (Table 1). specimens to fix in proper pH fixative. This will further
affect the interpretation of immunohistochemistry,
8-12
Table1: Frequency of specimens with different pH cytogenetic and PCR studies.
pH groups Number of specimens
1.0-2.0 1 Conclusion
The poor fixation due to inappropriate pH can affect the
2.1-3.0 4
processing, staining including special staining and
immunohistochemistry, cytogenetic studies and also their
3.0-4.0 8
interpretation which can lead to wrong diagnosis and
4.1-5.0 84
treatment of patient. In order to overcome all these
5.1-6.0 269
problems, medical and paramedical staff involved in dealing
6.1-7.0 530
with surgical specimens should be properly educated
7.1-8.0 103
regarding the handling and fixation of surgical pathology
8.1-9.0 01 13,14
specimens.
Discussion
Fixation is a chemical process by which biological Conflict of interest
tissues are preserved from decay and prevented from This study has no conflict of interest declared by any author.
autolysis and putrefaction. Fixation prevents biochemical References
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