4.Fixation of Tissues

2013 Elsevier Ltd

Introduction

Appropriate fixation of tissues for histological exam-ination is extremely important. Without attention to this process, the range of tests performed in a modern histopathology laboratory will be rendered ineffective and practically useless. The concept of fixation of biological tissues in order to understand biological function and structure has led to the development of many types of fixatives over the last century. The mechanisms and principles by which specific fixatives act to harden and preserve tissues and prevent the loss of specific molecules fall into broad categories. These include the covalent addi-tion of reactive groups and of cross-links, dehydra-tion, and the effects of acids, salt formation, and heat, along with combinations of these actions. Compound fixatives may function via several of these mechanisms.Although each fixative has advantages, they all

have many disadvantages. These include molecular loss from fixed tissues, swelling or shrinkage of tissues during the process, variations in the quality of histochemical and immunohistochemical stain-ing, the ability to perform biochemical analysis accu-rately, and varying capabilities to maintain the structures of cellular organelles. One of the major problems with fixation using formaldehyde has been the loss of antigen immunorecognition due to that type of fixation combined with processing the tissue to paraffin wax (Eltoum et al. 2001a, 2001b). However, from a clinical perspective the advent of heat-induced epitope retrieval methods, insti-gated in the early 1990s, have overcome many of these limitations (Shi et al. 1991). Similarly, the anal-ysis of mRNA and DNA from formalin-fixed,

paraffin-embedded tissue has been problematic (Grizzle et al. 2001; Jewell et al. 2002; Steg et al. 2006; Lykidis et al. 2007). All widely used fixatives are selected by compromise; their good aspects are bal-anced against less desirable features. This chapter discusses the basics of fixation, the advantages and disadvantages of specific fixatives, and provides some of the formulae for specific fixatives currently used in pathology, histology, and anatomy.The major objective of fixation in pathology is to

maintain clear and consistent morphological fea-tures (Eltoum et al. 2001a, 2001b; Grizzle et al. 2001). The development of specific fixatives usually has been empirical, and much of the understanding of the mechanisms of fixation has been based upon information obtained from leather tanning and vaccine production. In order to visualize the micro-anatomy of a tissue, its stained sections must main-tain the original microscopic relationships among cells, cellular components (e.g. the cytoplasm and nuclei), and extracellular material with little disrup-tion of the organization of the tissue, and must maintain the tissues local chemical composition. Many tissue components are soluble in aqueous acid or other liquid environments, and a reliable view of the microanatomy and microenvironment of these tissues requires that the soluble components are not lost during fixation and tissue processing. Minimiz-ing the loss of cellular components, including pro-teins, peptides, mRNA, DNA, and lipids, prevents the destruction of macromolecular structures such as cytoplasmic membranes, smooth endoplasmic reticulum, rough endoplasmic reticulum, nuclear membranes, lysosomes, and mitochondria. Each fixative, combined with the tissue processing protocol, maintains some molecular and macromo-lecular aspects of the tissue better than other fixative/

Fixation of tissuesAnthony Rhodes

4

4 Fixationof tissues70

fixative of choice for most pathologists has been 10% neutral buffered formalin (Grizzle et al. 2001).The most important characteristic of a fixative is

to support high quality and consistent staining with H&E, both initially and after storage of the paraffin blocks for at least a decade. The fixative must have the ability to prevent short- and long-term destruc-tion of the micro-architecture of the tissue by stop-ping the activity of catabolic enzymes and hence autolysis, minimizing the diffusion of soluble mol-ecules from their original locations. Another impor-tant characteristic of a good fixative, which helps maintain tissue and cellular integrity, is the inanima-tion of infectious agents, which helps maintain tissue and cellular integrity. It is also important to have good toxicological and flammability profiles that permit the safe use of the fixative (Grizzle & Fredenburgh 2005). The advent of new biological methods, increased understanding of the human genome, and the need to rapidly evaluate the biology of disease processes, means that fixatives should also permit the recovery of macromolecules includ-ing proteins, mRNA, and DNA without extensive biochemical modifications from fixed and paraffin-embedded tissues.Other important characteristics of an ideal fixative

include being useful for a wide variety of tissues, including fatty, lymphoid, and neural tissues. It should preserve small and large specimens and support histochemical, immunohistochemical, in situ hybridization and other specialized procedures. It should penetrate and fix tissues rapidly, have a shelf life of at least one year, and be compatible with modern automated tissue processors. The fixative should be readily disposable or recyclable and support long-term tissue storage giving excellent microtomy of paraffin blocks, and should be cost- effective (Dapson 1993).

Types of fixation

Fixation of tissues can be accomplished by physical and/or chemical methods. Physical methods such as heating, microwaving, and freeze-drying are inde-pendent processes and not used commonly in the

processing combinations. For example, if soluble components are lost from the cytoplasm of cells, the color of the cytoplasm on hematoxylin and eosin (H&E) staining will be reduced or modified and aspects of the appearance of the microanatomy of the tissue, e.g. mitochondria, will be lost or damaged. Similarly, immunohistochemical evaluations of structure and function may be reduced or lost.Almost any method of fixation induces shrinkage/

swelling, hardening of tissues and color variations in various histochemical stains (Sheehan & Hrap-chak 1980; Horobin 1982; Fox et al. 1985; Carson 1990; Kiernan 1999; OLeary & Mason 2004). Various methods of fixation always produce some artifacts in the appearance of tissue on staining; however, for diagnostic pathology it is important that such arti-facts are consistent.The fixative acts by minimizing the loss or enzy-

matic destruction of cellular and extracellular mol-ecules, maintaining macromolecular structures and protecting tissues from destruction by microorgan-isms. This results in one view of a dynamically changing, viable tissue (Grizzle et al. 2001). A fixa-tive should also prevent the subsequent breakdown of the tissue or molecular features by enzymatic activity and/or by microorganisms during long-term storage, because diagnostic/therapeutic tissues removed from patients are important resources which may be re-analyzed in the future.A fixative not only interacts initially with the

tissue in its aqueous environment but also, subse-quently, the unreacted fixative and the chemical modifications induced by the fixative continue to react. Fixation interacts with all phases of processing and staining from dehydration to staining of tissue sections using histochemical, enzymatic or immuno-histochemical stains (Eltoum et al. 2001b; Rait et al. 2004). A stained tissue section produced after specific fixation combined with tissue processing produces a compromise in the picture that is formed of one or more features of the original living tissue. To date, a universal or ideal fixative has not been identified. Fixatives are therefore selected based on their ability to produce a final product needed to demonstrate a specific feature of a specific tissue (Grizzle et al. 2001). In diagnostic pathology, the

Physicalmethodsoffixation 71

cutting, the yolk and egg white can be identified separately. Each component is less soluble in water after heat fixation than the same component of a fresh egg. Picking up a frozen section on a warm microscope slide both attaches the section to the slide and partially fixes it by heat and dehydration. Even though adequate morphology could be obtained by boiling tissue in normal saline, in histo-pathology heat is primarily used to accelerate other forms of fixation as well as the steps of tissue processing.

Microwave fixation

Microwave heating speeds fixation and can reduce times for fixation of some gross specimens and his-tological sections from more than 12 hours to less than 20 minutes (Anonymous 2001; Kok & Boon 2003; Leong 2005). Microwaving tissue in formalin results in the production of large amounts of danger-ous vapors, so in the absence of a hood for fixation, or a microwave processing system designed to handle these vapors, this may cause safety prob-lems. Recently, commercial glyoxal-based fixatives which do not form vapors when heated at 55C have been introduced as an efficient method of micro-wave fixation.

Freeze-drying and freeze substitution

Freeze-drying is a useful technique for studying soluble materials and small molecules; tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a vacuum chamber at 40C. The tissue can be post-fixed with formalde-hyde vapor. In substitution, specimens are immersed in fixatives at 40C, such as acetone or alcohol, which slowly remove water through dissolution of ice crystals, and the proteins are not denatured; bringing the temperature gradually to 4C will com-plete the fixation process (Pearse 1980). These methods of fixation are used primarily in the research environment and are rarely used in the clinical labo-ratory setting.

routine practice of medical or veterinary pathology, anatomy, and histology, except for the use of dry heat fixation of microorganisms prior to Gram stain-ing. Most methods of fixation used in processing of tissue for histopathological diagnoses rely on chemi-cal fixation carried out by liquid fixatives. Reproduc-ibility over time of the microscopic appearances of tissues after H&E staining is the prime requirement of the fixatives used for diagnostic pathology. Methods of fixation used in research protocols may be more varied, including fixation using vapors and fixation of whole animals by perfusing the animals vascular system with a fixative (Eltoum et al. 2001a, 2001b).Several chemicals or their combinations can act as

good fixatives, and accomplish many of the stated goals of fixation. Some fixatives add covalent reac-tive groups which may induce cross-links between proteins, individual protein moieties, within nucleic acids, and between nucleic acids and proteins (Horobin 1982; Eltoum et al. 2001a, 2001b; Rait et al. 2004, 2005). The best examples of such cross-linking fixatives are formaldehyde and glutaraldehyde. Another approach to fixation is the use of agents that remove free water from tissues and hence precipi-tate and coagulate proteins; examples of these dehy-drants include ethanol, methanol, and acetone. These agents denature proteins by breaking the hydrophobic bonds which are responsible for the tertiary structure of proteins. Other fixatives, such as acetic acid, trichloroacetic acid, mercuric chloride, and zinc acetate, act by denaturing proteins and nucleic acids through changes in pH or via salt for-mation. Some fixatives are mixtures of reagents and are referred to as compound fixatives, e.g. alcoholic formalin acts to fix tissues by adding covalent hydroxymethyl groups and cross-links as well as by coagulation and dehydration.

Physical methods of fixation

Heat fixation

The simplest form of fixation is heat. Boiling or poaching an egg precipitates the proteins and, on


water removed, the structure of the protein may become partially reversed, with hydrophobic groups moving to the outside surface of the protein. Once the tertiary structure of a soluble protein has been modified, the rate of reversal to a more ordered soluble state is slow and most proteins after coagula-tion remain insoluble even if returned to an aqueous environment.Disruption of the tertiary structure of proteins, i.e.

denaturation, changes their physical properties, potentially causing insolubility and loss of function. Even though most proteins become less soluble in organic environments, up to 13% of protein may be lost, for example with acetone fixation (Horobin 1982). Factors that influence the solubility of macro-molecules include:

1. Temperature, pressure, and pH.2. Ionic strength of the solute.3. The salting-in constant, which expresses the

contribution of the electrostatic interactions.4. The salting-in and salting-out interactions.5. The type(s) of denaturing reagent(s)

(Herskovits et al. 1970; Horobin 1982; Papanikolau & Kokkinidis 1997; Bhakuni 1998).

Alcohol denatures protein differently, depending on the choice and concentration of alcohol, the pres-ence of organic and non-organic substances, and the pH and temperature of fixation. For example, ethanol denatures proteins > phenols > water and polyhydric alcohols > monocarboxylic acids > dicar-boxylic acids (Bhakuni 1998).

Other types of coagulant fixative

Acidic coagulants such as picric acid and trichloro-acetic acid change the charges on the ionizable side chains, e.g. (NH2 NH3+) and (COO COOH), of proteins and disrupt electrostatic and hydrogen bonding. These acids also may insert a lipophilic anion into a hydrophilic region and hence disrupt the tertiary structures of proteins (Horobin 1982). Acetic acid coagulates nucleic acids but does not fix or precipitate proteins; it is therefore added to other fixatives to prevent the loss of nucleic acids.

Chemical fixation

Chemical fixation utilizes organic or non-organic solutions to maintain adequate morphological pres-ervation. Chemical fixatives can be considered as members of three major categories: coagulant, cross-linking, and compound fixatives (Baker, 1958).

Coagulant fixatives

Both organic and non-organic solutions may coagu-late proteins, making them insoluble. Cellular archi-tecture is maintained primarily by lipoproteins and by fibrous proteins such as collagen; coagulating such proteins maintains tissue histomorphology at the light microscopic level. Unfortunately, because coagulant fixatives result in cytoplasmic flocculation as well as poor preservation of mitochondria and secretory granules, such fixatives are not useful in ultrastructural analysis.

Dehydrant coagulant fixatives

The most commonly used coagulating fixatives are alcohols (e.g. ethanol, methanol) and acetone. Meth-anol is closer to the structure of water than ethanol. Ethanol therefore competes more strongly than methanol in the interaction with hydrophobic areas of molecules; thus, coagulant fixation begins at a concentration of 5060% for ethanol but requires a concentration of 80% or more for methanol (Lillie & Fullmer 1976). Removal and replacement of free water from tissue by any of these agents has several potential effects on proteins within the tissue. Water molecules surround hydrophobic areas of proteins and, by repulsion, force hydrophobic chemical groups into closer contact with each other and hence stabilize hydrophobic bonding. By removing water, the opposite principle weakens hydrophobic bonding. Similarly, molecules of water participate in hydrogen bonding in hydrophilic areas of proteins; so removal of water destabilizes this hydrogen bonding. Together, these changes act to disrupt the tertiary structure of proteins. In addition, with the

Chemicalfixation 73

Trichloroacetic acid (Cl3CCOOH) can penetrate hydrophobic domains of proteins and the anion pro-duced (CCOO) reacts with charged amine groups. This interaction precipitates proteins and extracts nucleic acids. Picric acid or trinitrophenol slightly dissolves in water to form a weak acid solution (pH 2.0). In reactions, it forms salts with basic groups of proteins, causing the proteins to coagulate. If the solution is neutralized, precipitated protein may redissolve. Picric acid fixation produces brighter staining, but the low pH solutions of picric acid may cause hydrolysis and loss of nucleic acids

Non-coagulant cross-linking fixatives

Several chemicals were selected as fixatives second-ary to their potential actions of forming cross-links within and between proteins and nucleic acids as well as between nucleic acids and proteins. Cross-linking may not be a major mechanism at current short times of fixation, and therefore covalent addi-tive fixatives may be a better name for this group. Examples include formaldehyde, glutaraldehyde, and other aldehydes, e.g. chloral hydrate and glyoxal, metal salts such as mercuric and zinc chlo-ride, and other metallic compounds such as osmium tetroxide. Aldehyde groups are chemically and bio-logically reactive and are responsible for many his-tochemical reactions, e.g. free aldehyde groups may be responsible for argentaffin reactions (Papaniko-lau & Kokkinidis 1997).

Formaldehyde fixation

Formaldehyde in its 10% neutral buffered form (NBF) is the most common fixative used in diagnos-tic pathology. Pure formaldehyde is a vapor that, when completely dissolved in water, forms a solu-tion containing 3740% formaldehyde; this aqueous solution is known as formalin. The usual 10% for-malin used in fixation of tissues is a 10% solution of formalin; i.e., it contains about 4% weight to volume of formaldehyde. The reactions of formalde-hyde with macromolecules are numerous and complex. Fraenkel-Conrat and his colleagues, using

simple chemistry, meticulously identified most of the reactions of formaldehyde with amino acids and proteins (French & Edsall 1945; Fraenkel-Conrat & Olcott 1948a, 1948b; Fraenkel-Conrat & Mecham 1949). In an aqueous solution formaldehyde forms methylene hydrate, a methylene glycol as the first step in fixation (Singer 1962).

H C O H O HOCH OH2 2 2= +

Methylene hydrate reacts with several side chains of proteins to form reactive hydroxymethyl side groups (CH2OH). If relatively short fixation times are used with 10% neutral buffered formalin (hours to days), the formation of hydroxymethyl side chains is probably the primary and characteristic reaction. The formation of actual cross-links may be relatively rare at the currently used relatively short times of fixation.Formaldehyde also reacts with nuclear proteins

and nucleic acids (Kok & Boon 2003; Leong 2005). It penetrates between nucleic acids and proteins and stabilizes the nucleic acid-protein shell, and it also modifies nucleotides by reacting with free amino groups, as it does with proteins. In naked and free DNA, the cross-linking reactions are believed to start at adenine-thymidine (AT)-rich regions and cross-linking increases with increasing temperature (McGhee & von Hippel 1975a, 1975b, 1977a, 1977b). Formaldehyde reacts with C=C and SH bonds in unsaturated lipids, but does not interact with carbohydrates (French & Edsall 1945; Hayat 1981).The side chains of peptides or proteins that are

most reactive with methylene hydrate, and hence have the highest affinity for formaldehyde, include lysine, cysteine, histidine, arginine, tyrosine, and reactive hydroxyl groups of serine and threonine (see Table 4.1) (Means & Feeney 1995).Gustavson (1956) reported that one of the most

important cross-links in over-fixation, i.e. in tanning, is that between lysine and the amide group of the protein backbone. Due to the shorter fixation times of current diagnostic pathological and biologi-cal applications, cross-linking reactions with the protein backbone are unlikely to occur (French & Edsall 1945; Fraenkel-Conrat et al. 1945, 1947;


Table 4.1

Actionofm

ajorsingleorcom

bina

tionfix

atives

Categ

oryof

fixative

Deh

ydrants

Aldeh

yde

cross-lin

kers

Com

bina

tion

mercu

ric

chloride

with

form

alde

hyde

oracetic

acid

Osm

ium

tetroxide

Picricacidplus

form

alinand

ac

eticacid

Com

bina

tionalcoho

ls

plusformalin

Exam

ples of

catego

ryEtha

nol

Metha

nol

Acetone

Form

alde

hyde

Glutaraldeh

yde

Zen

kers

B5Po

st-fixatio

n

after

glutaralde

hyde

Bouins

Alcoh

olic formalin

Effect on proteins

Precipita

tes

with

out chem

ical

additio

n

Cross-linkers: add

s active hydroxym

ethyl

grou

ps to am

ines,

amides, some reactive

alcoho

ls, an

d sulfydryl

grou

ps; cross-links

amine/am

ide or

sulfydryl side chains

of proteins

Add

itive plus

coag

ulation

Add

itive cross-

links; some

extractio

n

some

destruction

Add

itive and

non

- ad

ditive coag

ulan

t some extractio

n

Add

itive plus

precipita

tion

mRNA/DNA

Slight

Slow

ly cross-links;

slightly extracts

Coa

gulatio

nSlight

extractio

nNo actio

nSlight

Lipids

Extensive

extractio

nNo actio

nNo actio

nMad

e insoluble

by cross-links

with

dou

ble

bond

s

No actio

nExtensive extractio

n

Carbo

hydrates

No actio

nNon

e on

pure

carboh

ydrates;

cross-linking

of

glycop

roteins

No actio

nSlight

oxidation

No actio

nNo actio

n

Qua

lity of H

&E

staining

Satisfactory

Goo

dGoo

dPo

orGoo

dGoo

d

Chemicalfixation 75Categ

oryof

fixative

Deh

ydrants

Aldeh

yde

cross-lin

kers

Com

bina

tion

mercu

ric

chloride

with

form

alde

hyde

oracetic

acid

Osm

ium

tetroxide

Picricacidplus

form

alinand

ac

eticacid

Com

bina

tionalcoho

ls

plusformalin

Effect on

ultrastructure

(organ

elles)

Destroys

ultrastructure,

includ

ing

mito

chon

dria,

proteins,

coag

ulates

Goo

d (N

BF) to

excellent preservation

with

glutaraldeh

yde;

adequa

te to go

od in

Carson-Millon

igs

Poor

preservatio

nUsed for

visualization of

mem

bran

es

Poor ten

ds to

destroy

mem

bran

es

Poor

Usual

form

ulation

701

00% solution

or in

com

bina

tion

with

other types

of fixative

Form

alde

hyde

(37

%)

10

% V/V aqu

eous

solutio

n bu

ffered with

ph

osph

ates to pH

7.2

7.4.

Glutaraldeh

yde 2%

bu

ffered to pH 7.4

Mercuric

chlorid

e combine

d either with

acetic acid

plus

dichromate or

with

form

alde

hyde

plus acetate

1% solution

buffe

red to

pH 7.4

Aqu

eous picric

acid, form

alin,

glacial a

cetic acid

10% formalde

hyde

(37%

) with

90%

etha

nol

Impo

rtan

t varia

bles/issues

Time, spe

cimen

thickness

shou

ld be used

on

ly for small o

r thin spe

cimen

s

Time, tem

perature,

pH, concen

tration/

specim

en thickne

ss

Toxic

Extrem

ely toxic

Mito

chon

dria and

integrity of nu

clea

r mem

bran

e de

stroyed; not

approp

riate for

some stains;

morda

nt

Time, spe

cimen

dimen

sion

s. N

ote

good

fixative for

rena

l tissues

Special u

ses

Preserves sm

all

non-lipid

molecules such as

glycog

en; preserves

enzymatic activity

Gen

eral all-roun

d fixative; best for

ultrastructure if

used

with

osm

ium

tetroxide

post-fixatio

n

Excellent for

hematop

oietic

tissues

Ultrastructural

visualization of

mem

bran

es;

lipids on

frozen

sections

Morda

nt for

conn

ective tissue

stains (trichrom

e)

Goo

d ge

neral

fixative; goo

d for

specific

immun

ohistochem

i- cal rea

ctions and

go

od to de

tect

lymph

nod

es in

fatty

tissue; rem

oves fats

from

tissue


dialysis (Fraenkel-Conrat et al. 1945, 1947; Fraenkel-Conrat & Olcott 1948a, 1948b; Fraenkel-Conrat & Mecham 1949).The principal type of cross-link in short-term fixa-

tion is thought to be between the hydroxymethyl group on a lysine side chain and arginine (through secondary amino groups), asparagine, glutamine (through secondary amide groups), or tyrosine (through hydroxyl group) (Tome et al. 1990). For example, a lysine methyl hydroxyl amine group can react with an arginine group to form a lysineCH2arginine cross-link; similarly, a tyrosine methyl hydroxyl amine group can bind with a cysteine group to form a tyrosineCH2cysteine cross-link. Each of these cross-links between macromolecules has a different degree of stability, which can be mod-ified by the temperature, pH, and type of the envi-ronment surrounding and permeating the tissue (Eltoum et al. 2001b). The time to saturation of human and animal tissues with active groups by formalin is about 24 hours, but cross-linking may continue for many weeks (Helander 1994).When formaldehyde dissolves in an unbuffered

aqueous solution, it forms an acid solution (pH 5.05.5) because 510% of commercially available form-aldehyde is formic acid. Acid formalin may react more slowly with proteins than NBF because amine groups become charged (e.g. N+H3). In solution, this requires a much lower pH than 5.5. However, the requirement for a lower pH to produce N+H3 groups may not be equivalent to that required in peptides. Acid formalin also preserves immuno-recognition much better than NBF (Arnold et al. 1996), and indeed the success of Taylor in the early days of immunocytochemistry to demonstrate immunoglobulins in paraffin-processed tissue sec-tions, most probably relied on the fixation of the tissues in acid formalin (Taylor et al. 1974). The dis-advantage of using acid formalin for fixation is the formation of a brown-black pigment with degraded hemoglobulin. This heme-related pigment, which forms in tissue, is usually not a great problem unless patients have a blood abnormality (e.g. sickle cell disease, malaria).Formaldehyde primarily preserves peptide-

protein bonds and the general structure of cellular

Fraenkel-Conrat & Olcott 1948a, 1948b; Fraenkel-Conrat & Mecham 1949; Gustavson 1956).

Reversibility of formaldehyde-macromolecular reactions

The reactive groups may combine with hydrogen groups or with each other, forming methylene bridges. If the formalin is washed away, reactive groups may rapidly return to their original states, but any bridging that has already occurred may remain.Washing for 24 hours removes about half of reac-

tive groups, and 4 weeks of washing removes up to 90% (Helander 1994). This suggests that actual cross-linking is a relatively slow process, so, in the rapid fixation used in diagnostic pathology, most fixation with formaldehyde prior to tissue processing stops with the formation of reactive hydroxymethyl groups.For long-term storage in formalin, the reactive

groups may be oxidized to the more stable groups (e.g. acids NHCOOH) which are not easily removed by washing in water or alcohol. Thus, fol-lowing fixation, returning the specimen to water or alcohol further reduces the fixation of the specimen, because the reactive groups produced by the initial reaction with formalin may reverse and be removed. Although it was initially thought that cross-linking was most important in the fixation of tissue for bio-logical uses (based on the limited number of cross-links over short periods of fixation), it is likely that formation of these hydroxymethyl groups actually denatures macromolecules and renders them insol-uble. As these washing experiments have not been reproduced, the actual mechanisms and their impor-tance to fixation by formaldehyde are uncertain. As well as simple washing under running water, over-fixation of tissue may be partially corrected by soaking the tissue in concentrated ammonia plus 20% chloral hydrate (Lhotka & Ferreira 1949). Fraenkel-Conrat and his colleagues frequently noted that the addition and condensation reactions of formaldehyde with amino acids and proteins were unstable and could be reversed easily by dilution or

Chemicalfixation 77

phospholipids (Hayat 1981). At room temperature glutaraldehyde does not cross-link nucleic acids in the absence of nucleohistones but it may react with nucleic acids at or above 45C (Hayat 1981).

Osmium tetroxide fixation

Osmium tetroxide (OsO4), a toxic solid, is soluble in water as well as non-polar solvents and can react with hydrophilic and hydrophobic sites including the side chains of proteins, potentially causing cross-linking (Hopwood et al. 1990). The reactive sites include sulfydryl, disulfide, phenolic, hydroxyl, car-boxyl, amide, and heterocyclic groups. Osmium tetroxide is known to interact with nucleic acids, specifically with the 2,3-glycol moiety in terminal ribose groups and the 5,6 double bonds of thymine residues. Nuclei fixed in OsO4 and dehydrated with alcohol may show prominent clumping of DNA. This unacceptable artifact can be prevented by pre-fixation with potassium permanganate (KMnO4), post-fixation with uranyl acetate, or by adding calcium ions and tryptophan during fixation (Hayat 1981). The reaction of OsO4 with carbohydrates is uncertain (Hayat 1981). Large proportions of pro-teins and carbohydrates are lost from tissues during osmium fixation; some of this may be due to the superficial limited penetration of OsO4 (i.e.


procedure, followed by bleaching of the section in sodium hypochlorite solution (Hypo). However, this is not effective on mercuric chloride fixed tissues which have been stored for a number of years as paraffin blocks. In these tissues, retrospective analy-sis by immunohistochemistry and molecular tech-niques becomes unreliable due to the formation of much larger aggregates of mercuric pigment which cannot be removed subsequently by Lugols iodine. The chemistry of fixation using mercuric chloride is not well understood. It is, however, known that mer-curic chloride reacts with ammonium salts, amines, amides, amino acids, and sulfydryl groups, and hardens tissues. It is especially reactive with cyste-ine, forming a dimercaptide (Hopwood 2002) and acidifying the solution:

sulfydryl 2 R S H HgCl

R S Hg H Cl

( ) +( ) + ++

2

2 2 2

If only one cysteine is present, a reactive group of RSHgCl is likely.Mercury-based fixatives are toxic and should be

handled with care. They should not be allowed to come into contact with metal, and should be dis-solved in distilled water to prevent the precipitation of mercury salts. Mercury-containing chemicals are an environmental disposal problem. These fixatives penetrate slowly, so specimens must be thin, and mercury and acid formaldehyde hematein pigments may deposit in tissue after fixation. Mercury fixa-tives (Hopwood 1973) are no longer used routinely except by some laboratories for fixing hematopoietic tissues (especially B5). A potential replacement for mercuric chloride is zinc sulfate. Special formula-tions of zinc sulfate in formaldehyde replacing mer-curic chloride in B5 may give better nuclear detail than formaldehyde alone and improve tissue pene-tration (Carson 1990).

Special fixatives

Dichromate and chromic acid fixation

Chromium trioxide dissolves in water to produce an acidic solution of chromic acid, with a pH of 0.85.

osmium dioxide (+4 valence state) is facilitated by a reaction with solutions of ethanol.In addition to its use as a secondary fixative for

electron microscope examinations, OsO4 can also be used to stain lipids in frozen sections. Osmium tetroxide fixation causes tissue swelling which is reversed during dehydration steps. Swelling can also be minimized by adding calcium or sodium chloride to osmium-containing fixatives (Hayat 1981).

Cross-linking fixatives for electron microscopy

Cell organelles such as cytoplasmic and nuclear membranes, mitochondria, membrane-bound secre-tory granules, and smooth and rough endoplasmic reticulum need to be preserved carefully for electron microscopy. The lipids in these structures are extracted by many fixatives with dehydrants (e.g. alcohols). Therefore for ultrastructural examination it is important to use a fixative that does not solubi-lize lipids. The preferred fixatives are a strong cross-linking fixative such as glutaraldehyde, a combination of glutaraldehyde and formaldehyde, or Carsons modified Millonigs, followed by post-fixation in an agent that further stabilizes as well as emphasizes membranes such as OsO4.

Mercuric chloride

Historically, mercuric chloride was greatly favored for its qualities of enhancing the staining properties of tissues, particularly for trichrome stains. However, it is now rarely used in the clinical laboratory due to the health and safety issues involved with the use of a mercury-containing fixative, and also due to the reduced reliance on special stains. A further major disadvantage of mercuric chloride fixation is the inevitable formation of deposits of intensely black precipitates of mercuric pigment in the tissues. This subsequently gives them inferior value for immuno-histochemical and molecular studies. In recently fixed tissues, these precipitates can be readily removed by a Lugols iodine step in the staining

Compoundfixatives 79

compounds included the commercially available HOPE (HEPES-glutamic acid buffer mediated Organic Solvent Protection Effect) fixative and the reversible cross-linker dithio-bis[succinimidyl pro-pionate] (DSP) for immunocytochemistry and expression profiling, in addition to zinc-based fixa-tives. They concluded that a novel zinc formation (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and NBF for DNA, RNA and antigen perseveration. DNA and RNA fragments up to 2.4kb and 361bp in length, respectively, were detected by PCR, reverse tran-scriptase PCR and real-time PCR in the Z7 fixed tissues, in addition to allowing for protein analysis using 2D electrophoresis. Nucleic acids and protein were found to be stable over a period of 614 months. Moreover, the fixative is less toxic than formaldehyde formulations. Whilst this fixative appears to show great promise, it should be borne in mind that fixation in NBF will also allow the extraction of similarly sized fragments of DNA and RNA for analysis by PCR-based technologies, within this time frame.

Metallic ions as a fixative supplement

Several metallic ions have been used as aids in fixa-tion, including Hg2+, Pb2+, Co2+, Cu2+, Cd2+, [UO2]2+, [PtCl6]2+, and Zn2+. Mercury, lead, and zinc are used most commonly in current fixatives, e.g. zinc-containing formaldehyde is suggested to be a better fixative for immunohistochemistry than formalde-hyde alone. This does however depend upon the pH of the formaldehyde, as well as the zinc formalde-hyde (Arnold et al. 1996; Eltoum et al. 2001a).

Compound fixatives

Pathologists use formaldehyde-based fixatives to ensure reproducible histomorphometric patterns. Other agents may be added to formaldehyde to produce specific effects that are not possible with formaldehyde alone. The dehydrant ethanol, for

Chromic acid is a powerful oxidizing agent which produces aldehyde from the 1, 2-diglycol residues of polysaccharides. These aldehydes can react in histo-chemical stains (PAS and argentaffin/argyrophil) and should increase the background of immunohis-tochemical staining (Grizzle 1996a).Actual chromic salts (i.e. chromium ions in +3

valence state) may destroy animal tissues (Kiernan 1999) but chromium ions in their +6 state coagulate proteins and nucleic acids. The fixation and harden-ing reactions are not understood completely but probably involve the oxidation of proteins, which varies in strength depending upon the pH of the fixative, plus interaction of the reduced chromate ions directly in cross-linking proteins (Pearse & Stoward 1980). Chromium ions specifically interact with the carboxyl and hydroxyl side chains of pro-teins. Chromic acid also interacts with disulfide bridges and attacks lipophilic residues such as tyro-sine and methionine (Horobin 1982). Fixatives con-taining chromate at a pH of 3.55.0 make proteins insoluble without coagulation. Chromate is reported to make unsaturated but not saturated lipids insol-uble upon prolonged (>48 hours) fixation and hence mitochondria are well preserved by dichromate fixatives.Dichromate-containing fixatives have primarily

been used to prepare neuroendocrine tissues for staining, especially normal adrenal medulla and related tumors (e.g. phaeochromocytomas). However, reliance on the chromaffin reaction used to identify chromaffin granules following dichromate fixation has greatly diminished, being replaced by immuno-histochemistry to a range of neuroendocrine markers, to include neuron-specific enolase, chromagranin A, and synaptophysin (Grizzle 1996a, 1996b).

Fixatives for DNA, RNA, and protein analysis

Lykidis et al. (2007) conducted a comprehensive analysis of 25 fixative compounds, many reputed to provide improved preservation of DNA and RNA and proteins in tissues for immuno-cytochemical analysis, whilst at the same time ensuring optimal morphological preservation. The


authors to suggest that unbuffered formalin is a better fixative than NBF with respect to immuno-recognition of many antigens (Arnold et al. 1996; Eltoum et al. 2001b). This no doubt aided the detec-tion of antigens before the early 1990s, prior to the advent of heat-induced epitope retrieval methods in immunocytochemistry. However, minimal delay in effectively fixing very labile antigens, such as the estrogen receptor, is vital in the immunohistochemi-cal testing for a range of clinically important prognostic and predictive biomarkers. Whilst form-aldehyde fixation remains the recommended method for optimal preservation of morphological features, proteins and nucleic acids in a clinical environment, the most reliable way of achieving optimal formalin fixation is through its buffering at pH 7.27.4 (i.e. neutral buffered formalin).At the acidic pH of unbuffered formaldehyde,

hemoglobin metabolic products are chemically modified to form a brown-black, insoluble, crystal-line, birefringent pigment. The pigment forms at a pH of less than 5.7, and the extent of its formation increases in the pH range of 3.0 to 5.0. Formalin pigment is recognized easily and should not affect diagnoses except in patients with large amounts of hemoglobin breakdown products secondary to hematopoietic diseases. The pigment is removed easily with an alcoholic solution of picric acid. To avoid the formation of formalin pigment, neutral buffered formalin is used as the preferred formaldehyde-based fixative.Acetic acids and other acids work mainly through

lowering pH and disrupting the tertiary structure of proteins. Buffers are used to maintain optimum pH. The choice of specific buffer depends on the type of fixative and analyte. Commonly used buffers are phosphate, cacodylate, bicarbonate, Tris, and acetate. It is necessary to use low salt-buffered formalin in the new complex tissue processors in order to keep the machine clean, and reduce problems in its operation.

Duration of fixation and size of specimens

The factors that govern diffusion of a fixative into tissue were investigated by Medawar (1941). He

example, can be added to formaldehyde to produce alcoholic formalin. This combination preserves mol-ecules such as glycogen and results in less shrinkage and hardening than pure dehydrants.Compound fixatives are useful for specific tissues,

e.g. alcoholic formalin for fixation of some fatty tissues, such as breast, in which preservation of the lipid is not important. In addition, fixation of gross specimens in alcoholic formalin may aid in identify-ing lymph nodes embedded in fat. Some combined fixatives including alcoholic formalin are good at preserving antigen immunorecognition, but non-specific staining or background staining in immunohistochemical procedures can be increased. Unreacted aldehyde groups in glutaraldehyde-formaldehyde fixation for example may increase background staining, and alcoholic formalin may cause non-specific staining of myelinated nerves (Grizzle et al. 1995, 1997, 1998a, 1998b; Arnold et al. 1996; Grizzle 1996b).

Factors affecting the quality of fixation

Buffers and pH

The effect of pH on fixation with formaldehyde may be profound depending upon the applications to which the tissues will be exposed. In a strongly acidic environment, the primary amine target groups (NH2) attract hydrogen ions (NH+3) and become unreactive to the hydrated formaldehyde (methy-lene hydrate or methylene glycol), and carboxyl groups (COO) lose their charges (COOH). This may affect the structure of proteins. Similarly the hydroxyl groups of alcohols (OH) including serine and threonine may become less reactive in a strongly acidic environment. The extent of formation of reac-tive hydroxymethyl groups and cross-linking is reduced in unbuffered 4% formaldehyde (Means & Feeney 1995), which is slightly acidic (French & Edsall 1945), because the major methylene cross-links are between lysine and the free amino group on side chains. The decrease in the effectiveness of formaldehyde fixation and hence cross-linking in such a slightly acid environment has led some

Factorsaffectingthequalityoffixation 81

laboratories, thin specimens may be fixed in NBF for only 56 hours including the short time of fixation in tissue processors. The extent of formation of cross-links during such rapid NBF fixation is uncertain. Consequently, the formation of hydroxy-methyl groups may predominate, as opposed to more resilient cross-linking. It has been suggested that rapid fixation is acceptable as long as histochemical staining remains adequate; and that immunohistochemistry and other molecular tech-niques are probably enhanced by shorter times of fixation using an aldehyde (e.g. formaldehyde)-based fixation. However, recent studies investigat-ing the time taken to adequately fix clinical cases of breast cancer tissue for subsequent immunohisto-chemical detection of estrogen receptors illustrate that this practice can be detrimental to the optimal preservation of important antigens and should be avoided. Goldstein et al. (2003) found that 68 hours was the minimum time required to adequately fix breast tissue for immunohistochemical testing of estrogen receptors, regardless of the size and type of specimen. Consequently, the current guidelines for estrogen receptor and progesterone receptor testing produced by the American Society of Clinical Oncol-ogy (ASCO) and College of American Pathologists (CAP) recommend this minimal fixation time in neutral buffered formalin for all clinical breast cancer specimens (Hammond et al. 2010).

Temperature of fixation

The diffusion of molecules increases with rising tem-perature due to their more rapid movement and vibration; i.e. the rate of penetration of a tissue by formaldehyde is faster at higher temperatures. Microwaves therefore have been used to speed formaldehyde fixation by both increasing the tem-perature and molecular movements. Increased vapor levels, however, are a safety problem (Grizzle & Fredenburgh 2001, 2005). Most chemical reactions occur more rapidly at higher temperatures and therefore formaldehyde reacts more rapidly with proteins (Hopwood 1985). Closed tissue processors have their processing retort directly above the paraf-fin holding stations which are held at 6065C,

found that the depth (d) reached by a fixative is directly proportional to the square root of duration of fixation (t) and expressed this relation as:

d k t=

The constant (k) is the coefficient of diffusability, which is specific to each fixative. Examples are 0.79 for 10% formaldehyde, 1.0 for 100% ethanol, and 1.33 for 3% potassium dichromate (Hopwood 1969). Thus, for most fixatives, the time of fixation is approximately equal to the square of the distance which the fixative must penetrate. Most fixatives, such as NBF, will penetrate tissue to the depth of approximately 1 mm in one hour; hence for a 10 mm sphere, the fixative will not penetrate to the center until (5)2 or 25 hours of fixation. It is important to note that the components of a compound fixative will penetrate the tissue at different rates, so that these aspects of the fixative will be best manifest in thin specimens.Gross specimens should not rest on the bottom of

a container of fixative: they should be separated from the bottom by wadded fixative-soaked paper or cloth, so allowing penetration of fixative or pro-cessing fluids from all directions. In addition, unfixed gross specimens which are to be cut and stored in fixative prior to processing should not be thicker than 0.5cm. When surgical specimens are to be processed to paraffin blocks, the time of penetra-tion by fixative is more critical. Specific issues related to the processing of tissues have been reviewed by Grizzle et al. (2001) and Jones et al. (2001).Fixation proceeds slowly and the period between

the formation of reactive hydroxymethyl groups and the formation of a significant number of cross-links is unknown. Ninety percent of reactive groups can be removed by 4 weeks of washing (Helander 1994), confirming that cross-linking is not a rapid process and may require weeks for completion of potential bonds.Proteins inactivate fixatives, especially those in

blood or bloody fluids. Bloody gross specimens should therefore be washed with saline prior to being put into fixative. The fixative volume should be at least 10 times the volume of the tissue speci-men for optimal, rapid fixation. Currently in some


making the retort slightly warmer than room temperature.

Concentration of fixative

Effectiveness and solubility primarily determine the appropriate concentration of fixatives. Concentra-tions of formalin above 10% tend to cause increased hardening and shrinkage (Fox et al. 1985). In addi-tion, higher concentrations result in formalin being present in its polymeric form, which can be depos-ited as white precipitate, as opposed to its mono-meric form HO(H2CO)H, which at 4% provides for greatest solubility (Baker 1958). Ethanol concentra-tions below 70% do not remove free water from tissues efficiently.

Osmolality of fixatives and ionic composition

The osmolality of the buffer and fixative is impor-tant; hypertonic and hypotonic solutions lead to shrinkage and swelling, respectively. The best mor-phological results are obtained with solutions that are slightly hypertonic (400450 mOsm), though the osmolality for 10% NBF is about 1500 mOsm. Simi-larly, various ions (Na+, K+, Ca2+, Mg2+) can affect cell shape and structure regardless of the osmotic effect. The ionic composition of fluids should be as isotonic as possible to the tissues.

Additives

The addition of electrolytes and non-electrolytes to fixatives improves the morphology of the fixed tissue. These additives include calcium chloride, potassium thiocyanate, ammonium sulfate, and potassium dihydrogen phosphate. The electrolytes may react either directly with proteins causing dena-turation, or independently with the fixatives and cellular constituents (Hayat 1981). The choice of electrolytes to be added to fixatives used on a tissue processor may vary. Fixatives buffered with electro-lytes such as phosphates may cause problems with some processors due to precipitation of the salts. The

addition of non-electrolyte substances such as sucrose, dextran, and detergent has also been reported to improve fixation (Hayat 1981).

Selecting or avoiding specific fixatives

The choice of a fixative is a compromise, balancing their beneficial and detrimental effects. Kiernan (1999) originally produced a table of the actions of fixatives; this was later modified and published by Eltoum et al. (2001b), and Table 4.1 is a further modi-fication of the latter.However, specific fixatives are unsuitable for most

uses and should be avoided. The main problem with fixatives used in histological staining is the loss by solution/extraction of molecules that are targets of specific histochemical methods. Typically, some mol-ecules are soluble in aqueous fixatives (e.g. glyco-gen), while others are soluble in organic-based fixatives (e.g. lipids). Some fixatives may chemically modify targets of histochemical staining and thus affect the quality of special stains (e.g. glutaralde-hyde for silver stains); this includes modification of staining secondary to changes in pH induced by fixa-tion. A good discussion of the effects of fixation on histochemistry is by Sheehan and Hrapchak (1980).The table of Sheehan and Hrapchak (1980) modi-

fied by (Eltoum et al. 2001b) has been changed so that harmful methods of fixation could be identified rapidly. Table 4.2 of this chapter is a further modifi-cation of the table.

Fixation for selected individual tissues

Eyes

The globe must be firmly fixed in order to cut good sections for embedding. Eyes may be fixed in NBF, usually for about 48 hours; to speed fixation one or two small windows can be cut into the globe (avoid the retina and iris) after 24 hours. After gross descrip-tion, the anterior (iris) and posterior (e.g. optic nerve) are removed with a new, sharp razor blade and the components of the globe are fixed for an

Fixationforselectedindividualtissues 83

Table 4.2 Incompatiblestainsandfixatives

Targetofspecialstain Typeofspecialstain Fixativebestavoided Preferredfixative

Amebas Bests carmine Alcohol or alcoholic formalin

Aqueous fixative

Cholesterol and cholesterol esters

Schultzs methodDigitonin

10% NBF (frozen section) 10% NBF (frozen section)

Bouins; Zenkers Bouins; Zenkers

Chromaffin granules Ferric ferricyanide reduction test

Orths; Mllers

Gomori-Burtner methenamine silverPeriodic acid-Schiff (PAS)

Orths; Mllers

Mallorys aniline blue collagen stain

10% NBF; Bouins; Heidenhains mercuric chloride

Dichromate and alcohol bases

Connective tissue Wilders reticulum 10% NBF; Zenkers; Hellys Bouins

No picric acid fixativesMassons trichrome NBF tissues must be

post-fixed with (Bouins)Mallorys analine blue collagen stain

Zenkers All except preferred

Copper Mallorys stain Alcohol-based fixatives Formalin

Degenerating myelin Marchis method Orths for 48 hours;10% NBF

All except preferred

DNA/RNA Feulgen Ethanol Bouins, strong acids

Elastic fibers Gomoris aldehyde fuchsin

10% NBF No chromates

Fats/lipids Nile blue sulfate Formal calcium All except preferredOsmic acid (frozen section)

10% NBF All except preferred

Oil red O (frozen section) 10% NBF Zenkers; HellysSudan black B (frozen section)

10% NBF Zenkers; Hellys

Fibrin Mallorys phosphotungstic acid hematoxylin

Zenkers Bouins

Weigerts stain for fibrin Absolute ethanol; Carnoys alcoholic formalin

Bouins

Glycogen Bauer-Feulgen Carnoys or Gendres Aqueous fixativePAS Acid alcoholic formalin Aqueous fixativeBests carmine Absolute alcohol; Carnoys Aqueous fixative

Glycoproteins Mller-Mowry colloidal iron

Alcoholic formalin Carnoys Chromates

Hemoglobin Lepehnes (frozen section) Short time in 10% NBF ZenkersDunn-Thompson 10% NBF Bouins, Zenkers, Hellys


Table 4.2 (continued)

Targetofspecialstain Typeofspecialstain Fixativebestavoided Preferredfixative

Hepatitis B surface antigen

Orcein No chromatesAldehyde fuchsin No chromates

Iron Mallorys stain Alcohol-based fixatives Formalin

Juxtaglomerular cells of kidney

Bowies stain Hellys All except preferred

Melanin pigments DOPA oxidase Fresh frozen or formalin All except preferred

Mitochondria Carson-Millonigs Dehydrants, ethanol methanol, acetone

Mucoproteins PAS Glutaraldehyde

Neuroendocrine granules

Rapid argyrophil Fontana-Masson

10% NBF Ethanol, methanol, acetone

Pancreas , , & cells

Trichrome-PAS 10% NBF or Hellys Zenkers, Bouins Alcohol based

Paneth cell granules Phloxine tartrazine 10% NBF Acid

Peripheral nerve elements

Bielschowskis for neurofibrils and axis cylinders

36 weeks in 10% NBF All except preferred

Bodians for myelinated and non-myelinated nerve fibers

9 parts ethanol, 1 part formalin


Nonidezs for neurofibrils and axis cylinders

100 ml 50% ethanol plus 25 g chloral hydrate


Rio-Hortega for neutrofibrils 10% NBF All except preferredImmunohistochemistry biotin-streptavidin

Formal zinc Alcoholic formalin

Phospholipids Smith-Dietrich (frozen section)

Formal calcium All except preferred

Bakers acid hematin (frozen section)

10% NBF All except preferred

Pituitary cells Congo red for cells 10% NBFGomoris aldehyde fuchsin for cells

Bouins NBF requires mordant

Silver stains Fontana-Masson-Grimelius Glutaraldehyde

Spirochetes Giemsa Bouins; Zenkers BouinsGrams technique Zenkers BouinsLevaditi ZenkersWarthin-Starry 10% NBF All except preferred

Uric acid crystals Gomoris methenamine silver for urate

Absolute ethanol All except preferred

Gomoris chrome alum hematoxylin-phloxine

Bouins Avoid chromates

Usefulformulaeforfixatives 85

additional 48 hours, or more, in buffered formalde-hyde, before being processed. Embedding may be in celloidin or paraffin. Perfusion fixation of the eye is recommended for studies of the canal of Schlemm and/or the aqueous outflow pathways.

Brain

The problem of fixing a whole brain is to render it firm enough to investigate the neuroanatomy and to produce sections to show histopathology and to respond to immunochemistry if required. Conven-tionally this fixation takes at least 2 weeks. Adickes et al. (1997) proposed a perfusion technique which allows all of the above to be accomplished and the report issued in 56 days. This method depends on the perfusion of the brain via the middle cerebral arteries. Fixatives may also be enhanced by the use of microwave technology (Anonymous 2001; Kok & Boon 2003; Leong 2005). Alcoholic formalin should not be used for fixation if immunohistochemistry is to be performed using biotin-avidin (streptavidin) methods (Grizzle et al., unpublished data).

Breast

Clinical samples should be fixed in 10% NBF for between a minimum of 68 hours and a maximum of 72 hours, and should be sliced at 5mm intervals after appropriate gross inspection and margins des-ignation. Time from tissue acquisition to fixation should be as short as possible in order to prevent lysis of clinically important biomarkers, such as estrogen receptors, progesterone receptors and the human epidermal growth factor receptor-2 (HER2). They should be placed in a sufficient volume of NBF to allow adequate tissue penetration. If the tumor specimen has come from a remote geographical location, it should be bisected through the tumor on removal and sent to the laboratory immersed in a sufficient volume of NBF (Hammond et al., 2010).

Lungs

Lung biopsies are typically fixed in NBF. The lungs from autopsies may be inflated by and fixed in NBF via the trachea or major bronchi, and in our experi-ence these lungs can be cut within 2 hours. Gross

sections are fixed overnight and sections to be pro-cessed and cut the next day.

Lymphoid tissue

Special care should be taken with all lymphoid tissue, as many organisms (e.g. Mycobacterium tuber-culosis and viruses) may sequester themselves in the lymphoid reticular system. The lymphoid tissue is usually sliced and a representative sample of fresh tissue taken for special studies (e.g. flow cytometry or molecular analysis). The rest of the lymph node is fixed in NBF, though some laboratories fix part of the tissue in B5 or zinc.

Testis

Biopsies of the testes are fixed routinely in NBF.

Muscle biopsies

Biopsies of muscle are received fresh. A portion is separated for enzyme histochemistry. The tissue for routine histological assessment is fixed in NBF and embedded so the fibers of the specimens are viewed in cross-section and longitudinally. After processing this is stained with H&E, a trichrome stain, and Congo red if amyloid is suspected.

Renal biopsies

Renal core biopsies should be subdivided into three and each piece should contain adequate numbers of glomeruli. Each portion is then preserved, depend-ing upon the method to be used for analysis:

NBF for routine histology Buffered glutaraldehyde (pH 7.3) for

ultrastructural analysis Snap frozen in isopentane and liquid nitrogen

for immunofluorescence examination.

Useful formulae for fixatives

Gray (1954) lists over 600 formulations for various fixatives. The following is a list of the fixatives and formulae most commonly used by biomedical scien-tists and histotechnologists. Many of these formulae


Formal (10% formalin), saline

Tap water 900 mlFormaldehyde (37%) 100 mlSodium chloride 9 g

Formal (10% formalin), zinc, unbuffered

Tap water 900 mlFormaldehyde (37%) 100 mlSodium chloride 4.5 gZinc chloride or (zinc sulfate) 1.6 g (or 3.6 g)

Zinc formalin is reported to be an excellent fixative for immunohistochemistry.

Formalin, buffered saline

Tap water 900 mlFormaldehyde (37%) 100 mlSodium chloride 9 gSodium phosphate, dibasic 12 g

Formalin, buffered zinc

10% neutral buffered formalin 1000 mlZinc chloride 1.6 g

Mercuric fixatives

A problem with fixation in mercury solutions is that several types of pigment may combine with the mercury. These pigments are removed from sections by using iodine treatment followed by sodium thiosulfate.

Zenkers solution

Distilled water 250 mlMercuric chloride 12.5 gPotassium dichromate 6.3 gSodium sulfate 2.5 g

Just before use add 5 ml of glacial acetic acid to 95 ml of above solution. This is a good fixative for bloody (congested) specimens and trichrome stains.

Hellys solution

Distilled water 250 mlMercuric chloride 12.5 g

are based on those presented in standard textbooks of histochemistry (Sheehan & Hrapchak 1980; Carson 1990; Kiernan 1999). They vary slightly from text to text, but these variations are unlikely to cause problems.For routine histology, 10% neutral buffered forma-

lin (NBF) is frequently used for initial fixation and for the first station on tissue processors. NBF is com-posed of a 10% solution of phosphate buffered form-aldehyde. Formaldehyde is commercially supplied as a 3740% solution and in the following formulae is referred to as 37% formaldehyde.

Neutral buffered 10% formalin

Tap water 900 mlFormalin (37% formaldehyde solution) 100 mlSodium phosphate, monobasic, monohydrate

4 g

Sodium phosphate, dibasic, anhydrous 6.5 g

The pH should be 7.27.4There are other formulations of NBF and related

fixatives. NBF purchased from commercial compa-nies may vary widely in its aldehyde content, and commercial companies may add material such as methanol (Fox et al. 1985) or other agents to stabilize NBF preparations.

Carsons modified Millonigs phosphate buffered formalin

Formaldehyde (3740%) 10 mlTap water 90 mlSodium phosphate, monobasic 1.86 gSodium hydroxide 0.42 g

Deionized water can be used if tap water is hard and/or contains solids. The pH should be 7.27.4. This formula is reported to be better for ultrastruc-tural preservation than NBF.Sometimes the term formal is used to refer to

10% formalin or 37% formaldehyde.

Formal (10% formalin), calcium acetate

Tap water 900 mlFormaldehyde (37%) 100 mlCalcium acetate 20 g

This is a good fixative for preservation of lipids.

Usefulformulaeforfixatives 87

Potassium dichromate 6.3 gSodium sulfate 2.5 g

Just before use add 5 ml of 37% formaldehyde to 95 ml of above solution. It is excellent for bone marrow extramedullary hematopoiesis and interca-lated discs.

Schaudinns solution

Distilled water 50 mlMercuric chloride 3.5 gAbsolute ethanol 25 ml

Ohlmachers solution

Absolute ethanol 32 mlChloroform 6 mlGlacial acetic acid 2 mlMercuric chloride 8 g

This fixative penetrates rapidly.

Carnoy-Lebrun solution

Absolute ethanol 15 mlChloroform 15 mlGlacial acetic acid 15 mlMercuric chloride 8 g

This fixative penetrates rapidly.

B5 fixative

Stock solution:Mercuric chloride 12 gSodium acetate 2.5 gDistilled water 200 ml

Add 2 ml of formaldehyde (37%) to 20 ml of stock solution just before use.Frequently used for bone marrow, lymph nodes,

spleen, and other hematopoietic tissues.

Dichromate fixatives

There is a variation among the names attributed to the formulae of dichromate fixatives but not in the formulae themselves. Time of fixation (24 hours) is critical for dichromate fixatives. Tissue should be washed after fixation and transferred to 70% ethanol. Failure to wash the tissue after fixation may

cause pigments to be precipitated. Extensive shrink-age occurs when tissues are processed to paraffin blocks.

Millers or Mllers solution

Potassium dichromate 2.5 gSodium sulfate 1 gDistilled water 100 ml

Mllers or Regauds solution

Potassium dichromate 3 gDistilled water 80 ml

At time of use add 20 ml of formaldehyde (37%).

Orths solution

Potassium dichromate 2.5 gSodium sulfate 1 gDistilled water 100 ml

At time of use add 10 ml of formaldehyde (37%).

Lead fixatives

See special fixatives.

Picric acid fixatives

Many picric acid fixatives require a saturated aqueous solution of picric acid. Aqueous picric acid 2.1% will produce a saturated solution and 5% picric acid a saturated solution in absolute ethanol.

Bouins solution

Saturated aqueous solution of picric acid 1500 mlFormaldehyde (37%) 500 mlGlacial acetic acid 100 ml

Bouins solution is an excellent general fixative for connective tissue stains. The yellow color can be removed with 70% ethanol, lithium carbonate, or another acid dye, separately or during the staining sequence. Bouins solution destroys membranes; therefore intact nuclei cannot be recovered from Bouins fixed tissue and there may be extensive shrinkage of larger specimens.


Clarkes solution

Absolute ethanol 60 mlGlacial acetic acid 20 ml

This solution produces good general histological results for H&E stains. It has the advantage of pre-serving nucleic acids while lipids are extracted. A short fixation is recommended and tissues are trans-ferred to 95% ethanol following fixation.

Carnoys fixative

Acetic acid 10 mlAbsolute ethanol 60 mlChloroform 30 ml

Carnoys fixative is useful for RNA stains, e.g. methyl green pyronine, and for glycogen preserva-tion. It shrinks and hardens tissues and hemolyzes red blood cells. It may destroy the staining of acid-fast bacilli. It is useful in cytology to clear heavily blood-stained specimens.

Methacarn

Acetic acid 10 ml100% methanol 60 mlChloroform 30 ml

Causes less hardening and less shrinkage than Car-noys, but with the same pattern of staining.

Dehydrant cross-linking fixatives

Compound fixatives with both dehydrant and cross-linking actions include alcohol-formalin mixtures.Alcohol-formalin fixation or post-fixation can be advantageous in large specimens with extensive fat. Lymph nodes can be detected much more easily in specimens with alcohol-formalin fixation due to the extraction of lipids and to texture differences com-pared with tissues fixed in NBF. The preparation of alcohol-formaldehyde solutions is complex, espe-cially buffered forms of this compound fixative. It is probably best to purchase commercial preparations of buffered alcohol-formaldehyde. For use in post-fixation (e.g. after 10% NBF), Carson (1990) recom-mends the following formula:

Hollandes solution

Distilled water 1000 mlFormaldehyde (37%) 100 mlAcetic acid 15 mlPicric acid 40 gCopper acetate 25 g

A useful fixative for gastrointestinal biopsies and endocrine tissue; specimens are washed before exposure to NBF.

Dehydrant fixatives

Dehydrant fixatives act to remove free and bound water, causing a change to the tertiary structure of proteins so that they precipitate, leaving the nucleic acids relatively unchanged. Ultrastructure is destroyed by any of these four dehydrants due to the extraction of lipids, and each may cause exces-sive shrinking of tissue components after more than 34 hours of fixation. Each of these fixatives can be modified by adding other chemicals to produce spe-cific effects.

1. Ethanol, absolute2. Ethanol, 95%3. Ethanol, 7095%4. Methanol, 100%5. Acetone, 100%

Methanol is useful for touch preparations and smears, especially blood smears. Many alcohol mix-tures may undergo slow reactions among ingredi-ents upon long-term storage; in general most alcohol-based fixatives should be prepared no more than 12 days before use. Acetone fixation should be short (1 hour) at 4C only on small specimens. Acetone produces extensive shrinkage and harden-ing, and results in microscopic distortion. It is used for immunohistochemistry, enzyme studies, and in the detection of rabies. Cold acetone is especially useful to open the membranes of intact cells (e.g. grown on coverslips or microscope slides) to facili-tate entrance of large molecules (e.g. antibodies for immunohistochemical studies). Trade secret ingre-dients stabilize commercial formulations.

Fixationanddecalcifation 89

Glacial acetic acid 5 mlEthyl acetate 25 mlTap water 30 ml

Another alcoholic form of Bouins solution is as follows:

Stock Bouins solution 75 ml95% ethanol 25 ml

This solution is excellent for lymph nodes (24 hours) and for fatty tissue (48 hours).A closely related fixative is:

Rossmans solution

Tap water 10 mlFormaldehyde (37%) 10 mlAbsolute ethanol 80 mlLead nitrate 8 g

Fix for 24 hours at room temperature. This is a good fixative for connective tissue mucins and umbilical cord.

For metabolic bone disease

Phosphate buffer

Tap water 1000 mlNaH2PO4H2O 1.104 gNaHPO4 (anhydrous) 4.675 g

Fixative

Phosphate buffer 900 mlFormaldehyde (37%) 100 ml

Adjust pH to 7.35.

Fixation and decalcifation

Bouins decalcifying solution

Saturated aqueous solution of picric acid (10.5 g per 500 ml)

500 ml

Formaldehyde (37%) 167 mlFormic acid 33 ml

Absolute ethanol 650 mlDistilled water 250 mlFormaldehyde (37%) 100 ml

Carson recommends this formula because she noted that the concentration of ethanol should be less than 70% to prevent the precipitation of phosphates in 10% NBF saturated tissues. For initial fixation the following formulae can be used:

Alcoholic formalin

Ethanol (95%) 895 mlFormaldehyde (37%) 105 ml

Alcohol-formalin-acetic acid fixative

Ethanol (95%) 85 mlFormaldehyde (37%) 10 mlGlacial acetic acid 5 ml

Methanol may be substituted for ethanol with care; similarly, various mixtures of ethanol, acetic acid, and formalin may be used.

Alcoholic Bouins (Gendres solution)

This fixative is similar to Bouins except it is less aqueous and there is better retention in tissues of some carbohydrates (e.g. glycogen). Fixation should be between 4 hours and overnight followed by washing in 70% ethanol, followed by 95% ethanol (several changes). This is the one alcoholic fixative that improves upon aging (Lillie & Fullmer 1976).

Gendres solution

95% ethanol saturated with picric acid (5 g per 100 ml)

800 ml

Formaldehyde (37%) 150 mlGlacial acetic acid 50 mlTo increase the effectiveness of alcoholic Bouins, if there is no time for aging, the following formula has been recommended (Gregory 1980):

Equivalent to aged alcoholic Bouins

Picric acid 0.5 gFormaldehyde (37%) 15 ml95% ethanol 25 ml


Carson, F.L., 1990. Histotechnology: a self-instructional text. American Society of Clinical Pathologists, Chicago, IL.

Dapson, R.W., 1993. Fixation for the 1990s: a review of needs and accomplishments. Biotechnic and Histochemistry 68, 7582.

Eltoum, I.-E., Fredenburgh, J., Grizzle, W.E., 2001a. Advanced concepts in fixation: effects of fixation on immunohistochemistry and histochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues, special methods of fixation. Journal of Histotechnology 24, 201210.

Eltoum, I., Fredenburgh, J., Myers, R.B., Grizzle, W., 2001b. Introduction to the theory and practice of fixation of tissues. Journal of Histotechnology 24, 173190.

Fox, C.H., Johnson, F.B., Whiting, J., Roller, P.P., 1985. Formaldehyde fixation. Journal of Histochemistry and Cytochemistry 33, 845853.

Fraenkel-Conrat, H., Mecham, D.K., 1949. The reaction of formaldehyde with proteins. VII. Demonstration of intermolecular cross-linking by means of osmotic pressure measurements. Journal of Biological Chemistry 177, 477486.

Fraenkel-Conrat, H., Olcott, H.S., 1948a. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. Journal of the American Chemical Society 70, 26732684.

Fraenkel-Conrat, H., Olcott, H.S., 1948b. Reactions of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. Journal of Biological Chemistry 174, 827843.

Fraenkel-Conrat, H., Cooper, M., Olcott, H.S., 1945. The reaction of formaldehyde with proteins. Journal of the American Chemical Society 67, 950954.

Fraenkel-Conrat, H., Brandon, B.A., Olcott, H.S., 1947. The reaction of formaldehyde with proteins. IV. Participation of indole groups. Gramacidin. Journal of Biological Chemistry 168, 99118.

Fixation for fatty tissue

Bouins solution 75 ml95% ethanol 25 ml

May require up to 48 hours for good sections of lipomas or well-differentiated liposarcomas.

Note

This chapter is an introduction to fixation. More detailed and advanced issues related to fixation are included in several other texts/references (Sheehan & Hrapchak 1980; Eltoum et al. 2001a, 2001b; Grizzle et al. 2001). As discussed, various formulae may vary within a few percentages, but most of these formulae produce equivalent results.

References

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Grizzle, W.E., Myers, R.B., Manne, U., Srivastava, S., 1998b. Immunohistochemical evaluation of biomarkers in prostatic and colorectal neoplasia. In: Hanausek, M., Walaszek, Z. (Eds.), John Walkers methods in molecular medicinetumor marker protocols, vol. 14. Humana Press, Totowa, NJ, pp. 143160.

Grizzle, W.E., Stockard, C., Billings, P., 2001. The effects of tissue processing variables other than fixation on histochemical staining and immunohistochemical detection of antigens. Journal of Histotechnology 24, 213219.

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Hopwood, D., 2002. Fixation and fixatives. In: Bancroft, J.D., Gamble, M. (Eds.), Theory and

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4 Fixation of tissuesIntroductionTypes of fixationPhysical methods of fixationHeat fixationMicrowave fixationFreeze-drying and freeze substitution

Chemical fixationCoagulant fixativesDehydrant coagulant fixativesOther types of coagulant fixativeNon-coagulant cross-linking fixativesFormaldehyde fixationReversibility of formaldehyde-macromolecular reactionsGlutaraldehyde fixationOsmium tetroxide fixationCross-linking fixatives for electron microscopyMercuric chloride

Special fixativesDichromate and chromic acid fixationFixatives for DNA, RNA, and protein analysisMetallic ions as a fixative supplement

Compound fixativesFactors affecting the quality of fixationBuffers and pHDuration of fixation and size of specimensTemperature of fixationConcentration of fixativeOsmolality of fixatives and ionic compositionAdditives

Selecting or avoiding specific fixativesFixation for selected individual tissuesEyesBrainBreastLungsLymphoid tissueTestisMuscle biopsiesRenal biopsies

Useful formulae for fixativesNeutral buffered 10% formalinCarsons modified Millonigs phosphate buffered formalinFormal (10% formalin), calcium acetateFormal (10% formalin), salineFormal (10% formalin), zinc, unbufferedFormalin, buffered salineFormalin, buffered zincMercuric fixativesZenkers solutionHellys solutionSchaudinns solutionOhlmachers solutionCarnoy-Lebrun solutionB5 fixative

Dichromate fixativesMillers or Mllers solutionMllers or Regauds solutionOrths solution

Lead fixativesPicric acid fixativesBouins solutionHollandes solution

Dehydrant fixativesClarkes solutionCarnoys fixativeMethacarn

Dehydrant cross-linking fixativesAlcoholic formalinAlcohol-formalin-acetic acid fixativeAlcoholic Bouins (Gendres solution)Gendres solutionEquivalent to aged alcoholic BouinsAnother alcoholic form of Bouins solution is as follows:Rossmans solution

For metabolic bone diseasePhosphate bufferFixative

Fixation and decalcifationBouins decalcifying solution

Fixation for fatty tissueNote

References

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4.Fixation of Tissues

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