Potential alternative to Formaldehyde Fixatives  - A Study

 

Ashika Rachael Samuel, Dr. Gheena

Department of General Pathology, Saveetha Dental College and Hospitals, Chennai

*Corresponding Author E-mail:

 

ABSTRACT:

Aim : the aim of this study is to find a potential alternative that can be used in place of the regular fixatives . Materials and method : in this study , we will use sodium nitrite as a fixative. 8 samples of the tissue overlying the extracted teeth will be used. Background : fixation is the foundation step behind the study of pathology and essentially exists to prevent the autolysis and degradation of the tissue and tissue components such that they can be observed both anatomically and microscopically following sectioning. A number of fixatives exist, either having being in use for decades, or in the case of formaldehyde over a century, while others have only been created in the last 10 years. The broad objective of tissue fixation is to preserve cells and tissue components in a “life-like state” and to do this in such a way as to allow for the preparation of thin, stained sections. Sodium  nitrite has been found to inhibit growth of disease-causing microorganisms; give taste and color to the meat; and inhibit lipid oxidation that leads to rancidity. Reason : an innovative method  to detect the effectiveness  of a new salt like sodium nitrite as a fixative in place of the usual toxic formalin.

 

KEYWORDS :


 

INTRODUCTION:

Histology is a microscopic study in which tissue samples from animals and humans are studied. The main purpose of histology is due to its ability to contribute accurate information in forensic sciences, autopsy, diagnosis and education. [1] The histological staining procedure includes five steps, fixation, processing, embedding, sectioning and staining. [2] Among these steps, the first step Fixation aims at preserving the tissue structure and cell integration from degradation. This occurs due to the cross linking proteins of the fixatives. [3] The most common fixative used is Formalin.[1] It has been noticed  that  formalin may be oxidised in the purse me of oxygen leading to the formation of Formic acid . This may interfere while fixing the blood rich tissue specimens and form "formalin pigment ".[4,5]

 

The other known fixatives are neutral buffered formalin  (NBF), paraffin-formalin ,Bouin fixative etc. [6,7 ] However the major drawback noticed is that , these fixatives lead to the denaturation of the proteins in the sample tissue rendering them dysfunctional . [3] Thus here arises the need to identify an alternative fixative that can perform similar function to formalin but eliminate its drawback and produce high efficiency results. It's has been found that sodium nitrite can serve for this purpose. Nitrites have commonly been used in curing of meat. This curing will prevent the meat from becoming rancid. [8] It also protects food against botulinum toxin and preserves its form. [9] As nitrates have the capacity to preserve the form and structure of the food, its application in the field of histology has been studied. Thus sodium nitrate has been experimented with to understand its fixative strength.

 

MATERIALS AND METHOD:

For the study, sodium nitrite powder was mixed in 100 ml of distilled water in varying concentrations ranging from (40g, 50g, 60g and 80g) and stored in uricol containers. These concentration values had been chosen following the pilot study of sodium nitrite solution with meat chunks kept under observation for 48 hours . The concentrations chosen were 10 g, 30g, 45g ,60 g and 80 g. From these concentrations it was seen that the container with 10 g concentration showed the least the fixation . The meat appeared very soft . It was observed that the container with the maximum concentration (80g) showed excellent fixation property . The specimen appeared normal in consistency and had not much change in its structure . Thus choosing this concentration , the study has been carried out with a few other experimental values to attain maximum accuracy in the result . The tissue samples were collected from the extracted teeth. The size of each tissue was approximately 5*5 mm. One sample had been stored in the standard fixative solution - formalin .

 

Fig 1.1 Pilot study with meat chunk in 45 g concentration of sodium nitrite in distilled water

 

RESULTS:

From the study we proved that sodium nitrite has good fixative properties, particularly the 40 and 60 g concentrations. The effectiveness of the fixative also depends on the size of the tissue to be fixed.

 

DISCUSSION:

The primary objective of fixation is to prevent all the degenerative processes that occur once the tissue is deprived of the blood supply . The tissue ingestion that occurs due to the release of micro organisms due to rupture of outer membrane leads to autolysis. This phenomenon is greatly reduced by the properties of fixation. Furthermore,  the other steps in histochemical procedure where the tissue is subjected to various chemicals and waxes need to be protected against any form of damage. Fixation aids in this aspect and protects the tissue . [10,11] Despite all the advantages , it is also noted that the fixative can cause some preliminary changes to the tissue like shrinkage, swelling , hardening etc when subjected to an aqueous environment. Even during processing , there may be some morphological changes in the non aqueous environment .[11]

 

There are two main types of Fixation - chemical and physical means. Physical fixation includes heating, microwaving and cryo- preservation. Heat fixation is now replaced by micro wave techniques. Cryo - preservation refers to freeze drying and it is not applicable to the diagnostic tissue specimens. [11] The chemical methods include perfusion and immersion techniques. In general, a fixative is a single agent dissolved in a particular solvent or it may be due to the combination of one or more agents . [12]

 

The most widely accepted fixatives are formalin . It is prepared by diluting one part of formalin in nine parts of water as a buffer. It is accepted that washing of the tissue specimens with formalin can reverse some reactions however the cross links will still remain the same. [13]  Another main advantage of formalin is its ability to preserve the peptide in the cellular proteins. [14] The formalin also reacts with lysine , tyrosine, threonine, serine, glutamine and others to form a cross links . These cross links are responsible for maintenance of the tissue in it normal physiological state. [15]

 

Though formalin serves to be so advantageous, it is highly hazardous to man. It's entry into the body through the eye, skin or respiratory tract can be very harmful to man. It may cause irritation, corrosion or allergy to man. Many studies have proved formalin to be carcinogenic to man kind. Common cancers caused are nasopharyngeal cancer, sinonasal cancer, myeloidleu-kaemia. [14] Some undesired effects are also seen with the fixation. The formalin may undergo oxidation reaction and form Formic acid resulting in fall in pH. This Formic acid will react with the haemoglobin and form a new compound, acid formaldehyde haemoglobin. This is a brown black granular pigment which gets deposited on the blood rich tissues. It is often misinterpreted as a micro organism or pigment. [16]  

 

Thus due to the above reasons, alternatives to formalin have come into use. Mostly used alternatives are gluteraldehyde and genipin. Gluteraldehyde posses equivalent functions as formalin. The cross linking of gluteraldehyde are highly irreversible. [15] The gluteraldehyde slowly degrades to glutaricacid  which enables the conversion of a cyclic compound to an oligomeric compound When this fixative is used for electron microscopy as a primary fixator, a secondary fixation is done with osmium tetroxide.[17] Genipin is a naturallyoccuring fixative. It acts with the amino acids and converts it into a blue compound . It is believed that the use of this fixative on tissue builds its resistance collagenase degradation. However, it's action on nucliec acid is yet to be proven .[18]

 


In this  study , the soft  tissue samples that were collected from the tooth extraction cases and were fixed using sodium nitrite for 48 hours. The samples were carefully observed for any kind of physical change.  The tissues were just a little soft in nature . After fixation was complete , the tissues sample underwent processing for 2 and half hours in the various dehydrating agents that aimed  at washing the samples and removing the water content .


 

Fig 1.2 fixation in the sodium nitrite

 

The samples were then placed in  the embedding cassettes and put into the liquid paraffin till it solidified .After 24 hours they were removed from the casettes and and checked for the physical conformation . All tissue samples appeared in the same form and there was no deformation.

  Fig 1.3 Tissue sample after processing

 


The tissues were then placed into the blocks and hot wax was poured over it to obtain the blocks for the next step called sectioning. The blocks were obtained and the sectioning was successfully done . The sectioned components were now eligible for staining and slide preparation .


 


Scoring

Ease cutting

Block stability and storage

General appearance

Morphological appearance of nuclei

Morphological appearance of cytoplasm of epithelial cells

Morphological appearance of connective tissue

Artefacts

0

Very difficult

Very poor

Distortion of the general architecture , improper orientation and staining

Not clearly visible

Hazy and distorted

Not clearly distinguished , hazy

Artefacts are numerous

1

Difficult

Poor

Orientation is proper with definition of a few structures and improper staining

Distorted appearance

Clearly  visible and large number of vacuoles . Improper staining

Semblance of connective  tissue observed , improper staining

Artefacts restricted to those elicited due to fixation of tissue processing . Improper staining

2

Very easy ( too soft )

Medium

Structures are visible in proper orientation , margins not distinct , improper staining

Distinct in appearance , nucleoli is not visible

Distinct , less number of vacuoles . Staining not uniform

Collagen fibres are distinct in few areas and hazy in others . Improper staining

Less artefacts

3

Normal

Good

All structures are distinct in proper orientation to each other and exhibit proper staining .

Distinct nuclear memebrane and nucleoli

Clearly seen, distinct , homogeneous, no vacuoles , uniform staining

Individual Collagen fibres can be made out distinctly with a distinct nucleus . Staining is proper .

No artefacts

 

Fig 1.4 Block preparation before sectioning

 


This was the scoring criteria for the various slide prepartions.

 

In the last step , using heamatoxylin  and eosin stain , the tissue was stained and viewed under a microscope . The  scoring was done based on a few criteria and it was graded .


 

Fig 1.5 Histopatholoigical slide formalin (40X ),80g(10X), 60g (40X),50g (40X) and 40g (40X) respectively

 


The slides demonstrated excellent staining and most of  e cellular components were clearly visible .

Concentration

Ease Of cutting

Block stability on storage

General appearance

Morphology of nuclei

Morphology of cytoplasm

Morphology of connective tissue

Artefacts

40 g

3

Good

2

3

2

0

2

50 g

2

Good

0

0

0

0

0

60 g

3

Good

2

2

2

2

2

80 g

2

Good

0

1

0

0

0

Formalin

3

Good

3

2

3

3

3


The grading was done and it was noticed that the concentration of 40 g and 60g showed the best fixation properties. In comparing these two, the most suitable fixative concentration found was 40g. Thus its proved from the study that sodium nitrite can be used as an alternative fixative. Care should be taken that the size of  the tissue used must be a little larger in order to attain maximum success rate with this compound .

 

CONCLUSION:

The need for discovering a potential alternative for formaldehyde was highly essential due to its hazardous properties to man. And it was proven in this study, the experimental sodium nitrite solution could replace formaldehyde with a proper concentration. This compound has no harmful affects to man and also exhibits most of the properties as a standard fixative. Thus, we can opt for sodium nitrite as an alternative fixative .

 

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5.       Walker JF: Formaldehyde, 3rd ed. Am Chem Soc Monograph Series. Reinhold, New York, 1964.

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7.       Iyiola, S.,  and Avwioro,  O.  G.  (2011). Alum  haematoxylin  stain for  the demonstration of  nuclear  and  extra  nuclear substances.  Journal  of  Pharmacy  and  Clinical  Sciences.

8.       Bryan, N., and Ivy, J., (2015). Inorganic nitrite and nitrate: evidence to support consideration as dietary nutrients. Nutrition Research, 35, 643-654.

9.       Nigam, P. K., and Nigam, A. (2010). Botulinum Toxin. Indian Journal of Dermatology, 55(1), 8–14.

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15.     NTP. Report on carcinogens, Twelfth Edition. National Toxicology Program, USA Department of Health and Human Services, 2011; http://ntp.niehs.nih.gov/ntp/roc/twelfth/roc12.pdf November 7, 2011

16.     Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. J Histotechnol 2001;24;173 -190.

17.     Rolls GO, Farmer NJ, Hall JB. Artefacts in histological and cytological preparations. In Woods A and Ellis R eds. Laboratory histopathology. New York: Churchill Livingstone, 1994;5.3-1 - 5.3-29.

18.     Bozzola JJ, Russell LD. Electron microscopy: principles and techniques for biologists. Boston: Jones and Bartlett, 1992

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Received on 26.06.2016          Modified on 08.08.2016

Accepted on 10.08.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(11):1872-1876

DOI: 10.5958/0974-360X.2016.00383.8