274 14 Amyloid

1. Primary amyloid occurring spontaneously in protein is a tetramer and a vital pathological event
the absence of an apparent predisposing illness. appears to be release of the monomer. The pathogen-
It affects organs and tissues such as heart, esis of amyloid has been recently reviewed (Sipe
muscle, skin, and tongue. 2005).
2. Secondary amyloid occurring in patients with Amyloidosis is considered to belong to the cate-
chronic infective disease, such as syphilis and gory of conformational diseases, because the patho-
TB. Later, inflammatory diseases such as logical protein aggregation is due to the reduced
rheumatoid arthritis were also included in stability and a strong propensity to acquire more
this group. than one conformation. It is thought that such a
3. Tumor-associated amyloid. grouping helps to provide an understanding of
4. Myeloma-associated amyloid. the etiology or episodic onset of these diseases, and
opens the prospect for common approaches to thera-
As more and more amyloid-forming proteins were
peutic stratagems in the same way that recognition
identified, this classification became obsolete and
of bacteria as the causative agents of many infections
Husby et al. (1990) proposed guidelines for a better
allowed the idea of antibiotics being useful in all
classification based on the identity of the amyloid
such conditions, or of steroid therapy being of
fibril protein, which was adopted by the World
potential use for all inflammatory conditions (Carrell
Health Organization-International Union of Immu-
& Lomas 1997; Carrell & Gooptu 1998). In this
nunological Societies (WHO-IUIS) and forms the
context it is interesting to note the development of
currently accepted classification of amyloid.
‘designer’ peptides that bind to Aβ and to prion
Today there are about 25 different unrelated
protein, preventing, and even reversing, the confor-
amyloid-forming proteins (Table 14.1). Amyloidosis
mational change responsible for the respective
nomenclature uses the letter A to designate amyloid
disease processes (Soto et al. 2000). This concept is
followed by an abbreviation of the name of the fibril
becoming increasingly accepted, and it is becoming
protein.
evident that amyloid is but one, albeit definable,
subgroup within a larger group of misfolded or
altered protein deposits which are associated with
Pathogenesis human disease (Table 14.2).

The process that causes proteins to become involved

in amyloid formation, converting them from normal Amyloidosis
functioning proteins into inert amyloid deposits is
the focus of much research. There is little to connect In AL amyloidosis, previously known as ‘primary
the different types of proteins involved (Merlini amyloidosis’, abnormal proteins, monoclonal light
2003). In certain amyloid types, only a limited chains, are produced by plasma cells or B-cells and
portion of the amyloid protein precursor forms the form amyloid deposits in the tissues. These can be
fibril – as is the case in Alzheimer’s disease; several either of kappa (κ) or lambda (λ) isotypes. Systemic
amyloid proteins are rich in β-pleated sheet confor- AL amyloidosis is the most common form of clinical
mation in their native form; prion protein contains amyloid disease in developed countries and causes
no β-pleated sheet and it is the formation of β-pleated the most fatalities. In systemic amyloidosis, deposits
sheets de novo that is the pathological event of the can be present in any or all of the viscera, connective
prion diseases. In some amyloidosis, the whole pre- tissue and blood vessels walls, although intracere-
cursor protein may be involved, or there may be bral amyloid deposits are never found (Pepys 2006).
proteolysis of the precursor protein with liberation AL amyloidosis can also be localized when it is
of a smaller amyloidogenic fragment as with AβPP. restricted to a particular organ or tissue, and usually
In transthyretin (TTR) amyloidosis, the circulating has a benign prognosis. In the skin, the deposits
 Amyloidosis 275

Table 14.1 Unrelated amyloid-forming proteins

Dominant tissues
Abbreviation Protein precursor affected Amyloid type
‡
AA Serum amyloid A protein Spleen, gut Reactive systemic AA amyloidosis
AL (κ & λ) ‡
Immunoglobulin light chain Any tissue except the Systemic AL amyloidosis or
brain parenchyma localized AL amyloidosis
AH‡ Immunoglobulin heavy chain Renal Systemic AL amyloidosis or
localized AL amyloidosis
ATTR‡ Variant transthyretin Peripheral and/or Familial amyloid polyneuropathy
(prealbumin) autonomic nerves, (FAP), familial amyloid
cardiac, gastro-intestinal cardiomyopathy (FAC)
STTR‡ Wild-type transthyretin Cardiac Senile TTR
(prealbumin)
AapoAI‡ Variant apolipoprotein AI Kidney, liver Hereditary systemic amyloidodsis
AapoAII‡ Apolipoprotein AII Kidney, liver Hereditary
‡
AapoAIV Apolipoprotein AIV Kidney, liver Sporadic aging amyloidosis
‡
AGel Gelsolin Kidney Hereditary
ACys‡ Cystatin C Brain Hereditary
AβPP Amyloid β-protein precursor Brain Alzheimer’s disease, Down’s
syndrome, cerebral amyloid
angiopathy
Aβ2M‡ β2-Microglobulin Kidney, prostate Dialysis-associated amyloid,
corpora amyleacea
APrP Prion protein Brain CJD, prion disease
ACal Calcitonin Thyroid Medullary carcinoma of thyroid-
associated disease
AANF Atrial natriuretic factor Heart Senile cardiac amyloid
AIAPP Amylin Pancreas Type II diabetes amyloid,
insulinoma
AFib‡ Fibrinogen A α-chain Kidney Hereditary
ALys‡ Lysozyme Kidney Hereditary
APro Prolactin Pituitary Aging amyloid
AIns Insulin Skin Injection sites
ABri ABriPP Brain Familial dementia (British)
ADan A Dan PP Brain Familial dementia (Danish)
AMed Lactadherin Arteries Aging amyloid in arteries
ALac Lactoferrin Cornea Corneal amyloidosis
AKer Kerato-epitheliun Eyes Familial corneal amyloidosis
‡
ALect2 Leukocyte common antigen 2 Kidney, liver LECT 2 amyloidosis
‡
One of the 14 forms of amyloid proteins that can give rise to systemic disease.
 276 14 Amyloid

Table 14.2 Protein conformation diseases

Conditions Affected proteins Associated diseases
Amyloidosis 25 known in humans – see Table
14.1
Serpinopathies α1-Antitrypsin α1-Antitrypsin storage disease
neuroserpin
Hemoglobinopathies Hemoglobulin Sickle cell anemia
Drug and aging induced inclusion body
hemolysis
Lewy body diseases α-Synuclein Parkinson’s disease
Neuronal inclusion bodies Tau Alzheimer’s disease
Pick’s disease
Progressive supranuclear palsy
Superoxide dismutase Motor neuron disease, AML
Ferritin Familial neurodegenerative disorder
Hirano bodies Actin Alzheimer’s disease
Polyglutamine repeats Huntingtin Huntington’s disease
Ataxin Spinocerebellar ataxias
Androgen receptor Spinomuscular atrophies
Prion diseases Prion protein Creutzfeldt-Jacob disease (CJD)
Variant CJD
Gerstmann-Straussler-Scheinker
Kuru
Fatal familial insomnia
Japanese CAA

cause benign lumps and can be excised or left liver and is an acute phase protein which is synthe-
untreated, but localized amyloid deposits are also sized at increased levels in patients with diseases
common in the bladder and in pulmonary tissue, such as rheumatoid arthritis, TB, Crohn’s disease,
often causing obstruction which can lead to familial Mediterranean fever (FMF) and other
complications. hereditary periodic fevers. Chronically high levels
There are five possible types of heavy chain amy- of SAA are a prerequisite for development of AA
loidosis (AH); G, A, D, E and M – these are rare but amyloidosis.
not unknown. Hereditary systemic amyloidosis is a rare disorder
AA amyloidosis, previously known as ‘secondary that is difficult to treat and often fatal. This group of
amyloidosis’, is an occasional complication of disorders occurs in small clusters around the world
chronic infection and inflammatory conditions, and has an autosomal dominant pattern of inheri-
characterized by an acute phase response in which tance. Its most common cause is a mutation in the
production of serum amyloid A protein (SAA) is TTR (prealbumin) gene, which affects about 10,000
increased. SAA is an apolipoprotein produced in the individuals worldwide. Over 100 mutations, most
 Diagnosis 277

of which are amyloidogenic, are known in TTR. though this is not universal in all islets or in all
The major features of hereditary TTR amyloidosis patients with type 2 diabetes. As well as this, in
include severe and ultimately fatal peripheral diabetic patients amyloid can also be found at the
and/or autonomic neuropathy (familial amyloid site of insulin injection as a result of the injected
polyneuropathy, FAP); cardiac involvement is also insulin adopting a fibillary conformation in vitro
common. Other mutations associated with the dis- (when subjected to certain physical or chemical
order are those in the genes encoding apolipopro- stimuli such as heat or acidity), causing a localized
teins AI and AII, fibrinogen A α-chain, gelsolin, cutaneous lump (Lonsdale-Eccles et al. 2009).
lysozyme, cystatin C and β-protein. In all these It is also frequently cited that transmissible spon-
forms the variant protein is deposited as amyloid giform encephalopathy (TSE) is an example of amy-
fibrils predominantly in the abdominal viscera, loidosis, although in fact amyloid has never been
although cardiac and nerve involvement can occur. determined histopathologically in brains of patients
All amyloidogenic mutations are dominant, but with the disease, nor in cows with bovine spongi-
can display both variable penetrance and expressiv- form encephalopathy (BSE).
ity. Thus there may be marked differences in age of There are other protein misfolding diseases that
onset, amyloid deposition and clinical presentation, are not amyloid or amyloidosis; these diseases
not only between families but also within families produce abnormal aggregates of proteins, for
with the same mutation. In contrast to AA amyloi- example Lewy bodies in Parkinson’s disease and
dosis, where the concentration of the amyloid fibril polyglutamine repeats present in Huntington’s
protein SAA is raised but of normal structure, AL disease. However, it is unclear whether the removal
and hereditary amyloidosis are associated with of these aggregates alters the disease process.
abnormal protein which is inclined to refold as a Another protein deposition disease which is often
β-pleated sheet, resulting in amyloidosis. confused with amyloid is light chain deposition
LECT2 amyloidosis was discovered while study- disease (LCDD) which has a similar histological
ing proteins with leukocyte chemotactic activity appearance. However LCDD deposits lack the affin-
(Yamagoe 1996). The pathogenesis of LECT2 remains ity for Congo red stain and do not produce the char-
to be understood, but there is no evidence that LECT2 acteristic green birefringence of amyloid under
is an inherited condition though the first cases cross-polarized light. Under EM, LCDD deposits are
reported were Hispanic patients (Larsen et al. 2010). granular, which aides the distinction (Gibbs et al.
2011).

Other diseases in which amyloid occurs

Diagnosis
As outlined by Pepys in 2006, amyloid is a histologi-
cal feature of Alzheimer’s disease and type 2 diabe- There is an increased awareness of amyloidosis
tes mellitus but, unlike systemic amyloidosis, it is nowadays and more patients are being recognized.
not known whether the amyloid causes these dis- However some patients are still overlooked. The
eases. In Alzheimer’s there is an abundance of intra- diagnosis requires presence of amyloid in a tissue,
cerebral amyloid deposits composed of β-protein, and the gold standard technique is Congo red histol-
though there is poor correlation between the quan- ogy although EM may aid diagnosis. Biopsies are
tity of amyloid and the cognitive impairment. usually taken to investigate organ dysfunction, for
However, mutations that result in abundant deposi- example of the kidneys in nephrotic patients or of
tion of β-protein as amyloid may result in early- sural nerves in familial polyneuropathies. Amyloid
onset Alzheimer’s disease. is present in up to 90% of rectal and/or subcutane-
In patients with type 2 diabetes, amyloid is fre- ous fat biopsies in systemic AA or AL amyloidosis;
quently found in the pancreatic islets of Langerhans, so much so that rectal biopsies or fine needle
 278 14 Amyloid

aspirates of subcutaneous tissue used to be the main It is vitally important to discriminate between AL,
method of screening (Westermark & Senkvist 1973; hereditary amyloidosis and AA amyloidosis, as their
Pepys 1992). Nowadays techniques have improved treatments are entirely different. AL treatment is
greatly such that cardiac biopsies, after a suggestive aimed at ablating the B-cell clone responsible for the
echocardiogram, are becoming more popular. It amyloidogenic free light chain production using
must be noted that a negative biopsy does not cytotoxic drugs. Hereditary amyloidosis treatment
exclude the possibility of amyloidosis due to sample sometimes involves organ transplantation. Therapy
selection and site. In rectal biopsies, amyloid is for patients with AA amyloidosis involves measures
usually found in the walls of submucosal vessels, so to reduce SAA production by treating the cause of
if the full thickness of the muscularis is not obtained the underlying inflammation.
the deposits will go undetected. The tools available today to differentiate the
The use of SAP scintigraphy allows in vivo amyloid type include direct assessment of the fibril
diagnosis as well as the monitoring of progression type by immunohistochemistry, proteomics and,
and regression of the amyloid deposits with occasionally, fibril sequencing. Indirect, but very
treatment (Hawkins et al. 1993; Hawkins 1994). helpful, investigations include searches for mono-
Unfortunately SAP scintigraphy is unable to clonal immunoglobulins using conventional electro-
visualize amyloid within cardiac tissue because phoresis and immunoassays of serum and urine, the
the heart is a moving organ. The bone scanning serum free light chain assay, assessment of the
method DPD scintigraphy (99mTc-3,3-diphosphono- hepatic acute phase response by measuring SAA
1,2-propanodicarboxylic acid) was serendipitously and CRP and, where indicated, genetic sequencing
discovered to have high affinity for cardiac TTR of genes known to be associated with hereditary
amyloid, and is currently under evaluation at the amyloidosis or the periodic fever syndromes. All
National Amyloidosis Centre as a method for visual- these techniques are frequently employed for a
izing cardiac involvement. patient with amyloidosis.

Differentiation between different Demonstration

amyloid types
In hematoxylin and eosin (H&E) stained sections
With the recognition that different proteins form amyloid appears as an amorphous, eosinophilic,
amyloid and are associated with different clinical extracellular, faintly refractive substance that some-
syndromes came the need to identify particular fibril times displays green birefringence under polarized
types histologically. Furthermore, treatment of amy- light. However, it should be noted that collagen also
loidosis is entirely type-specific; hence the correct has this appearance under polarized light in a H&E-
identification of the fibril type is indispensable in stained section. Amyloid can also be weakly bire-
clinical practice. fringent using a powerful light source when stained
Methods of section pretreatment using trypsin or with periodic acid-Schiff. Whilst large deposits
potassium permanganate before Congo red staining of amyloid can be observed with a H&E-stained
were devised (Wright et al. 1976, 1977). After such section, the small deposits, for example in vessels in
pretreatment some amyloids lose their affinity for rectal or bone marrow samples, may be missed.
Congo red, most notably AA amyloid, whereas AL Dyes used for the demonstration of amyloid are
amyloid is resistant. These methods were always compounds developed by the textile industry. This
equivocal in practice and have been rendered obso- includes Congo red, which was developed as
lete by the use of immunohistochemistry and other the first direct cotton dye in 1884 and has been
techniques to identify the particular amyloid fibril ‘re-invented’ many times in the search for a differ-
protein specifically and reliably. ential method for the detection of amyloid (Puchtler
 Demonstration 279

et al. 1964). As in all histopathological methods, its subsequent green birefringence when viewed
they are often performed on tissues that have been by polarized light has become the gold standard.
formalin fixed and processed in to paraffin wax. Although it was many years before the exact stain-
Sometimes the samples can be left standing in fixa- ing mechanism was understood, it is now well
tive for long periods of time, which may make the established that staining of amyloid by Congo red is
staining less sensitive and intense. Control sections due to hydrogen bonding between the Congo red
must be used in all staining methods, and in the dye and the β-pleated sheet in a highly orientated
demonstration of amyloid they should be cut as they linear and parallel manner on the amyloid fibrils
are needed as they can lose their reactivity if stored (Glenner et al. 1980). Any tissue component that
for long periods. binds Congo red in a linear way also exhibits green
birefringence in polarized light. As well as amyloid,
Congo red dense collagen fibers can also bind Congo red dye
in this fashion, often meaning that formalin-fixed
The molecular formula for Congo red is tissue gives false positives. By using an alkaline
C32H22N6Na2O6S2 (Fig. 14.3). Congo red method this phenomenon is reduced
Congo red is a symmetrical sulfonated azo dye (Puchtler et al. 1962). However Romhányi (1971)
containing a hydrophobic center with two phenyl used 1% aqueous Congo red and claimed that if the
groups bound by a diphenyl bond to give a linear tissue sections were mounted in gum arabic this
molecule that is largely hydrophobic (Turnell & problem is overcome. Bély et al. (2006) adapted
Finch 1992). Congo red is also a fluorescent dye Romhányi’s original method, using a long deparaf-
(Puchtler 1965), although is not specific for amyloid. finization step of up to 5 days, together with a longer
Two factors are important to the Congo red-amyloid incubation in Congo red. This technique has shown
reaction: the linearity of the dye molecule and that the amyloid has a stronger affinity to Congo red
the β-pleated sheet configuration. If the spatial con- and therefore can be seen as more sensitive and
figuration of either is altered, even though the chem- selective. Many different tissue structures will also
ical groupings are left intact, the reaction fails. stain with 1% aqueous Congo red, and so it must be
Furthermore, the Congo red-mediated positive bire- used under strict controlled conditions using known
fringence of amyloid implies that the dye molecules amyloid-positive sections in conjunction.
are arranged in a parallel fashion (Romhányi 1971). The specificity of Congo red staining of amyloid
Recent work confirms the long-held belief that the can also be increased by using an alcoholic method
Congo red molecule intercalates between two combined with high ion strength and high pH.
protein moieties at the interface between two adja- Puchtler et al. (1962) combines all these aspects,
cent antiparallel β-pleated sheets by disrupting the giving a superior method to demonstrate amyloid
hydrogen bonds that are responsible for maintain- and green birefringence under polarized light.
ing the β-sheet polymer, yet allowing maintenance Recent comparison of several Congo red staining
of the integrity of the structure by the formation of methods made during a run of the UK NEQUAS
new hydrogen bonds between protein and dye histology external quality control scheme found that
(Carter & Chou 1998). Highman’s method gave the highest scores. At the
Since its introduction on tissue sections by Benn­ referral center quoted above, they get a number of
hold (1992), Congo red staining for amyloid and biopsies that are either false positive or false nega-
tive. Consequently, they recommend the Putchler
method since the lack of a differentiation step means
there is less intervention by the operator. It should
be noted that false positives and false negatives can
be caused by other factors even in this technique; for
Figure 14.3 Chemical structure of Congo red dye. example, in very thin tissue sections.
 280 14 Amyloid

Highman’s Congo red technique (Highman 1946) Working solutions

This simple method has found wide application. The To 100 ml of stock solution A add 1 ml of 1%
solutions are relatively stable and the method affords aqueous sodium hydroxide and filter.
a high degree of selectivity in practiced hands. To 100 ml of stock solution B add 1 ml of 1%
aqueous sodium hydroxide and filter.
Fixation
Not critical; formal saline gives satisfactory results. Method
1. Sections to water, removing pigment where
Solutions
necessary.
Immerse in alkaline sodium chloride solution for 20
2. Stain nuclei in alum hematoxylin, differentiate, and
minutes.
blue.
0.5% Congo red in 50% alcohol
3. Transfer directly to the alkaline Congo red solution
0.2% potassium hydroxide in 80% alcohol
for 20 minutes.
Method 4. Rinse briefly in alcohol, clear, and mount.
1. Sections to water, removing pigment where
Results (Fig. 14.4)
necessary.
Amyloid, elastic fibers, eosinophil granules red
2. Stain in Congo red solution, 5 minutes.
Nuclei blue
3. Differentiate with the alcoholic potassium hydroxide
solution, 3–10 seconds. Note
4. Wash in water, stain nuclei in alum hematoxylin, Although the stock solutions are stable for a couple of
differentiate, and blue. months, the working solutions do not keep and
5. Dehydrate, clear, and mount. should be used within 20 minutes of preparation.

Results
Amyloid, elastic fibers, eosinophil granules red Congo red technique (Stokes 1976)
Nuclei blue In this method there is no differentiation step as
Congo red is applied in an alkaline alcoholic solution.
Note
Harris’s hematoxylin was originally used as a
Differentiation in Step 3 can be arrested in water and counterstain but any alum hematoxylin will suffice.
resumed if necessary. Over-differentiation can occur.
Fixation
Not critical.
Preparation of solutions
Alkaline Congo red technique (Puchtler et al. 1962) Staining solution
The method obviates the need for a differentiation Dissolve 0.5 g potassium hydroxide in 50 ml distilled
step by the inclusion of a high concentration of water, add 200 ml absolute alcohol and add Congo
sodium chloride. This reduces background red until saturated (about 3 g). Stand overnight before
electrochemical staining whilst enhancing hydrogen use and discard after 3 months.
bonding of Congo red to amyloid, resulting in a
Method
progressive and highly selective technique. The
1. Sections to water, removing pigment where
solutions should be freshly made.
necessary.
Fixation 2. Stain in filtered Congo red solution, 25 minutes.
Not critical. 3. Wash in distilled water, then running tap water,
Stock solutions 5 minutes
Stock solution A 4. Counterstain nuclei in Harris’s hematoxylin, 1 minute.
Saturated sodium chloride in 80% ethanol. 5. Blue, differentiate, hematoxylin if necessary, blue.
Stock solution B 6. Dehydrate, clear, and mount.
Saturated Congo red in 80% ethanol saturated with Results
sodium chloride. Amyloid, elastic tissue, eosinophil granules red
1% aqueous sodium hydroxide. Nuclei blue
 Metachromatic techniques for amyloid 281

2. Stain nuclei in alum hematoxylin, differentiate, and

blue.
3. Rinse in water and then 70% ethanol.
4. Treat with Sirius red solution for 1 hour.
5. Wash in tap water for 10 minutes.
6. Dehydrate, clear, and mount.
Results
Amyloid, elastic, eosinophil, and Paneth cell red
granules
Nuclei blue
Note
The staining solution is liable to precipitation,
especially if excess 20% sodium chloride is added
Figure 14.4 A Congo red stained section of kidney demonstrating during preparation.
amyloid deposition.

Sirius red
Metachromatic techniques for amyloid
Sirius red F3B (direct red 80), is similar to Congo red
and has been proposed to give a more intense stain-
As we move into the next generation of technology
ing reaction which is valuable for photographic pur-
in the modern histopathology laboratory, metachro-
poses. Sirius red stained amyloid, like Congo red,
matic techniques are very rarely used for the iden­
also gives green birefringence with polarized light.
tification of tissue components and will only be
However, as described by Brigger (1975), Sirius red
mentioned briefly.
does not display fluorescence.

Methyl violet
Sirius red technique (Llewellyn 1970)
Llewellyn’s method is a modification of Sweat’s and Methyl violet was the first synthetic dye used for
uses the cotton dye Sirius red F3B – not to be the demonstration of amyloid (Cornil 1875), but
confused with Sirius red 4B, which does not stain the rationale of the staining reaction remains unex-
amyloid. Although this method is considerably simpler plained. It was thought that the mucopolysaccha-
than the original technique, the staining solution does
ride content of amyloid was responsible for the
not keep well and tends to precipitate out.
reaction, though this is now thought to be unlikely.
Fixation The staining reaction of amyloid produces a red/
Not critical. purple coloration. This coloration is also seen by
Preparation of solution other tissue components, especially mucins found
Dissolve 0.5 g Sirius red F3B in 45 ml distilled in rectal biopsies, so is not selective for amyloid.
water. Add 50 ml absolute alcohol and 1 ml 1% Methyl violet is a mixture of tetra-, penta-, and
sodium hydroxide. Stirring the solution vigorously, hexa-parasaniline, so the red/purple coloration of
slowly add just sufficient 20% sodium chloride (above amyloid is probably a polychromatic reaction
4 ml) to produce a fine precipitate when viewed
(Windrum & Kramer 1957). Methyl stained sections
against strong backlighting. Leave to stand overnight
and filter.
have to be mounted using an aqueous mountant,
since dehydration destroys the staining reaction.
Method The amyloid from some primary amyloidosis may
1. Sections to water, removing pigment where fail to give a positive reaction. Due to the low sensi-
necessary.
tivity and lack of specificity, this method is not now
 282 14 Amyloid

recommended for amyloid detection and diagnosis This property is observed in crystals, and is widely
(Westermark et al. 1999). used in mineralogy as a qualitative test for the iden-
tification of various minerals. Birefringence is exhi­
Crystal violet method (Hucker & Conn 1928) bited when an optically active substance has two
This method uses ammonium oxalate, which accen- differing refractive indices (RI), so that two rays of
tuates the polychromatic effect. Formic acid can also light vibrating in perpendicular planes will travel at
be used as an accentuator of the polychromic effect different velocities through the substance, produc-
(Fernando 1961). ing a faster and a slower ray. The substance is said
to show positive birefringence if the plane of vibra-
Methyl green (Bancroft 1963) tion of the slow ray (greater RI) is parallel to the
length of the fiber or crystal, or negative if the plane
This method is slightly more selective, in that tissue
of vibration of the slow ray is perpendicular to the
sections are stained with methyl violet and then
length of the fiber. Normally birefringent substances
differentiated and counterstained simultaneously
appear colorless (white) against a dark background
with methyl green, which replaces the former dye in
when viewed through crossed polarizing filters, but
tissue components other than amyloid.
where the thickness of the crystal or fiber is uniform,
interference colors may be produced when the two
Polarizing microscopy rays are reunited in the analyzer. Such colors are
characteristic of, and can thus be used to identify,
As discussed earlier, when viewed using polarized the substance being viewed. Practical details of
light and an analyzing polarizing filter, Congo polarizing microscopy and birefringence are given
redstained amyloid exhibits a characteristic bright in Chapter 3.
green birefringence, usually termed ‘apple-green Dichroism is due to light-absorbing differences
birefringence’ or ‘anomalous colors’ (Fig. 14.5). This along different planes of an asymmetrical substance.
property is shared by other β-pleated sheet proteins The spatial arrangement of light-absorbing bands
and appears to be specific to that conformation. is such that either light of a definite wavelength
Birefringence and dichroism are optical properties is selectively absorbed, or a change in intensity of
of anisotropic substances, i.e., substances that have white light occurs when light passes through the
different physical properties in different directions. substance in certain planes. The dichroism of Congo
red stained amyloid is that of the dye molecule,
whose linear molecules are bound by hydrogen
bonds along the parallel folds in the β-pleated sheet
protein, producing different light-absorbing charac-
teristics along certain planes of the fibril (Wolman
1965, 1971). The different amounts of absorption of
light may be seen as different colors or different
intensity of a color (Born & Wolf 1999).
Amyloid in unstained sections can be seen to be
weakly birefringent when using a strong light source;
weak birefringence may also often be seen following
methyl violet, toluidine blue, and eosin staining.
Such birefringence is usually faint, unreliable and
non-specific, and is therefore of little diagnostic use.
Figure 14.5 Same field as Figure 14.4 viewed by polarized light By contrast, the bright apple-green birefringence of
(crossed polarizer and analyzer). The characteristic ‘green amyloid following Congo red staining is easily visu-
birefringence’ is clearly seen against the dark background. alized, highly selective, and thought by many to be
 Acquired fluorescence methods 283

the most reliable diagnostic characteristic of amyloid property can be used to detect small amyloid depos-
in current use (Missmahl & Hartwig 1953; Cohen its. The fluorescence should not be considered as
1967; Pearse 1968). This green birefringence, first specific for amyloid as is the apple-green birefrin-
noticed by Divery and Florkin (1927), is an intrinsic gence seen with polarizing microscopy (Puchtler &
property of the amyloid fibril-Congo red complex. Sweat 1965; Westermark et al. 1999).
The thickness of the section is critical; 8–10 µm is
ideal (Wolman & Bubis 1965). Sections which are too
thin may show faint red colors, while yellow bire- Acquired fluorescence methods
fringent colors can be seen if the section is too thick,
if there is strain birefringence in the optical system, The ability of amyloid to fluoresce following treat-
or if the direction of the fibrils is oblique to the direc- ment with fluorochromic dyes was discovered by
tion of the polarizer. Chiari (1947), although little use was made of this
The use of a good microscope of high optical property until Vassar and Culling (1959) recom-
quality with color-corrected optics is essential to mended the basic fluorochrome dye thioflavine T.
visualize faint birefringence. A revolving stage and The method has the advantage of not requiring
subdued background lighting are also advisable. microscopic differentiation and, save for staining of
The strongest possible light source should be used. renal tubular myeloma casts and mast cell granules,
Many laboratory microscopes do not have strong specificity for amyloid was claimed.
enough light sources, and sections should preferen- Thioflavine T staining has enjoyed considerable
tially be viewed using a modern photomicroscope, popularity as a screening method for amyloid, as the
most of which are equipped both with essentially intensity of fluorescence allows good visualization
perfect optics and with powerful lamps. Only by of minimal deposits. It has become evident that the
using such a setup can the smallest of amyloid specificity originally claimed for the method was
deposits be appreciated. overstated and that many other tissue components,
Apple-green birefringence is also given by certain including fibrinoid, arteriolar hyaline, keratin, intes-
other filamentous structures, most notably the tinal muciphages, Paneth cells, and zymogen gran-
neurofibrillary tangles characteristic of Alzheimer’s ules, also have an affinity for the dye. The addition
disease and certain other degenerative brain of 0.4 M magnesium chloride to a 0.1% thioflavine
diseases, as well as the intracellular inclusions seen T solution at pH 5.7 is claimed to improve selectivity
in adrenal cortical cells (Eriksson & Westermark by competitive ionic inhibition (Mowry & Scott
1990). Whilst these structures fulfill many of the 1967). Similar results are obtained by using thiofla-
characteristics of amyloid, they are not currently vine T at pH 1.4, favoring the reaction of the blue
considered so to be, although the matter remains fluorescing dye component responsible for fluores-
under debate (Westermark et al. 2005). Green bire- cence of amyloid (Burns et al. 1967). The mechanism
fringence is also given by cellulose and chitin, both of binding of thioflavine T to amyloid is not known
of which avidly bind Congo red. They are easily but in vitro studies of binding to purified amyloid
distinguished from amyloid on morphological fibrils and synthetic amyloids show that the dye
grounds. Occasionally other structures may appear interacts with the quaternary structure of the
to give green birefringence: most commonly, dense β-pleated sheet rather than with protein moieties,
collagen. This can usually be distinguished from and so the binding is not dependent on any amino
amyloid by its whiter color, and the difference can acid sequence (LeVine 1995).
be emphasized by the use of sections cut at the rec- A related fluorochrome, thioflavine S, has been
ommended thickness. widely used for the demonstration of amyloid
As mentioned previously, Congo red is a fluo­ (Schwartz 1970). However, it is considered to be
rescent dye, and provided that sections have non-specific and is not recommended (Puchtler et al.
been mounted in a non-fluorescent mountant this 1985).
 284 14 Amyloid

Thioflavine T method (Vassar & Culling 1959) Miscellaneous methods

Fixation
Not critical. Many dyes, notably alcian blue and toluidine blue,
Preparation of solution commonly used for the identification of mucopoly-
1% aqueous thioflavine T. saccharides, have been used on amyloid-containing
tissue sections to substantiate histochemically the
Method
mucopolysaccharides frequently found in bioche­
1. Sections to water, removing pigment where
necessary.
mical assays of amyloid tissue extracts. In most
2. Treat with alum hematoxylin solution, 2 minutes.
instances the results have been disappointing.
3. Wash in water and stain in thioflavine T solution, 3 Uptake of these dyes is poor and variable. Differing
minutes. electrolyte concentrations (Mowry & Scott 1967) and
4. Rinse in water and differentiate excess partial pepsin digestion (Windrum & Kramer 1957)
fluorochrome from background in 1% acetic acid, may enhance alcian blue uptake and toluidine blue
20 minutes. metachromasia, respectively, but staining is never
5. Wash well in water, dehydrate, clear, and mount in strong and interpretation difficult. The variable peri-
a non-fluorescent mountant. odic acid Schiff’s staining of amyloid, which is often
Results intense, was thought to indicate the presence of car-
Using a UV light source (mercury vapor lamp), UG1 bohydrate within amyloid fibrils, but this has been
Exciter filter, BG38 red suppression filter, and K430 disproved. The glycoprotein AP component is now
barrier filter: amyloid, elastic tissue, etc. – silver-blue thought to be the origin of this positivity. Alcian
fluorescence. blue borax, with a celestine blue hemalum and van
Using blue light fluorescence quartz-iodine or Gieson counterstain, elegantly demonstrates some
mercury vapor lamp with BG12 exciter filter and K530 amyloid, but the method is not specific (Lendrum
barrier filter: amyloid, elastic tissue, etc. – yellow
et al. 1972).
fluorescence.
There are several silver impregnation methods that
Notes have been used for the demonstration of amyloid,
a. Step 2 quenches nuclear autofluorescence. such as that of King (1948). There are several silver
b. The mountant must be non-fluorescent such as methods used on central nervous system tissue
glycerine-saline (1 : 9 parts) or DPX. Avoid Canada
for the detection of amyloid-containing plaques
balsam, which autofluoresces.
(and neurofibrillary tangles) in Alzheimer’s disease.
c. Thioflavine T deteriorates, especially if kept in
There have been several reviews of these methods
sunlight, as do the stained sections on prolonged
storage. (Lamy et al. 1989; Wisniewski et al. 1989; Wilcock
et al. 1990; Vallet et al. 1992), and the methenamine
silver method of Haga is given in Chapter 17 on the
nervous system.
pH 1.4 Thioflavine T (Burns et al. 1967)
Acid pH increases the selectivity by favoring the
fluorochromic fraction binding to amyloid while
depressing non-amyloid fluorochrome staining. Fibril extraction
Method
Fibril extraction is a tool that utilizes small amounts
As above but use a freshly prepared solution of 0.5%
thioflavine T in 0.1 M hydrochloric acid.
of unfixed tissue to identify the amyloid fibril type
and is especially useful when no other material is
Results available. Briefly, small amounts of unfixed tissue
Amyloid, Paneth cells, and oxyntic cells – silver-blue are washed in a series of buffers and homogenized
or yellow fluorescence according to filters used.
in saline, centrifuged and washed several times,
 Immunohistochemistry for amyloid 285

leaving a suspension and a pellet of fibrils. These are AP is found in variable proportion of all amyloid
then subjected to drying in layers for Congo red fibrils. It is a non-fibrillar component that does not
staining and immunohistochemistry, the optical have a β-pleated conformation, so antisera raised
density of the suspension is measured and SDS- against AP can be used to demonstrate amyloid.
PAGE, immunoblotting and SAP electroimmunoas- However, SAP is also found naturally in basement
says are carried out. Polypeptide analysis of the membranes and other tissue components and so
fibril isolates by N-terminal amino acid sequencing will also bind antiserum raised against AP, and
is also performed when necessary (Tennent 1999). this is not a definitive marker. In early attempts
using immunohistochemistry to identify all the fibril
types, various antigen retrieval methods were used
Immunohistochemistry for amyloid with varied success. Nowadays retrieval methods
are commonplace in most laboratories and, since
Immunohistochemistry is a widely used method in each antibody differs in the epitope that it recog-
histopathology for identification of tissue disease nizes, it is important to try the whole range of
and can be used to determine the amyloid fibril type available antigen recovery methods for each antise-
in most cases (Fig. 14.6). Due to the distinct protein rum tried. As well as testing all the retrieval methods,
nature of amyloid, antibodies can recognize specific the correct way to evaluate the specificity of immu-
epitopes on amyloid fibrils and on associated com- noreactions is to absorb antiserum with its specific
ponents, like amyloid P (AP) and proteoglycans. antibody (Westermark et al. 1999). Antiserum to all
Some of the amyloid proteins can be masked by fixa- known amyloid-forming protein is commercially
tion of the tissue due to the cross-linking of the available, and most are reliable in identifying the
amino acid side groups which mask the antigenic different fibrils.
site. These authors find that antigen retrieval is of Differentiation between AA and AL amyloid is
little, if of any, use for the detection of amyloid. It is clinically important, and both conditions are likely
thought that the β-pleated conformation of the to occur at one time or another at most hospitals.
amyloid can ‘hide’ some antigenic sites and for iden- Identification of the polyneuropathy-related amyloid
tification of amyloid of transthyretin (TTR) type, types, ATTR, AApoAI and AGel, as well as other
oxidation and high molarity guanidine treatment hereditary types such as ALys, and AFib, may be
(Costa et al. 1986) are needed. reasonably expected to be done only at specialist
centers. The context or clinical presentation of the
patient and disease should be considered when
trying to type the amyloid by immunohistochemis-
try so that informed choices regarding sites of tissue
biopsies and use of relevant antisera can be made.
In AL amyloidosis, about 70% of kappa and
lambda types are identifiable, leaving 30% of ‘prob-
able’ AL type, regardless of methods or retrieval
recoveries used. This is due to the amyloid fibrils
being formed from the variable light chain fragment
of the immunoglobulin molecule. Patients with AL
amyloidosis may have very high concentrations of
free light chains in the serum, which may cause high
background staining with kappa and lambda anti-
sera, making interpretation difficult (Pepys 1992).
Figure 14.6 Immunoperoxidase-DAB stained section of the same It is exceptionally rare, although not completely
field as Figure 14.4 using antiserum to LECT2 antibody. unknown, for a patient to have two types of amyloid.
 286 14 Amyloid

When there is staining of amyloid deposits with two tissue sample is then digested with the proteolytic
amyloid fibril antisera by immunohistochemistry, enzyme trypsin. This generates a complex mixture of
we would suggest that proteomics should be under- peptides arising from each of the proteins in the
taken to determine the fibril type. tissue. The mixture is chromatographed on a reverse
The demonstration of prion-derived amyloid phase high-performance liquid chromatography
deposits, APrP, is difficult and most cases are referred (HPLC) column to separate the tryptic peptides,
to the Creutzfeldt-Jakob Disease Surveillance Unit, which are then analyzed directly by electrospray
who have vigorous immunohistochemical criteria mass spectrometry (MS). MS generates information
and obtain consistently good results (Bell et al. about the molecular mass and sequence for each of
1997). It is important to follow these criteria so as to the peptides in the mixture. These MS-derived data
avoid false-positive interference from normal prion are then collated and compared against a database of
protein, and it should be noted that the proteins all proteins using a search engine such as MASCOT,
within all amyloid deposits differ from the normal which identifies the protein(s) present in tissue by
precursor proteins largely by alteration of conforma- using a probability-based algorithm. This LCMD-
tion rather than any antigenically determinable proteomics approach can not only identify the class
character. of proteins in tissues, allowing us to type the amyloid
(e.g. TTR or AL), but also can identify novel amy-
loidogenic proteins (cf. Lec-2) as well as protein vari-
Laser microdissection-proteomics: ants found in TTR such as M30V or V122I.
a new tool for typing amyloid Although LCMD-proteomics requires a substan-
tial investment both in terms of equipment and staff,
Proteomics is a high sensitivity, mass spectrometric it is rapidly becoming the method of choice for
method for identifying proteins. Laser capture micro- typing amyloid tissue and for the identification of
dissection (LCMD) allows one to precisely remove an novel amyloidogenic proteins. Within 10 years, this
area of interest from a tissue section. This can now be approach will be common in leading specialist
coupled with a proteomics approach to offer histolo- centers for the typing of amyloid fibrils.
gists a powerful tool for identifying proteins in both
freshly prepared and formalin-fixed, paraffin embed-
ded tissues. Although slides specifically made for Evaluation of methods
LCMD can be obtained, it is also possible to extract
material from previously cut unstained sections The ‘gold standard’ for amyloid demonstration
(archived as a matter of routine), which allows work remains the use of Congo red staining with ‘apple
to be carried out retrospectively to type amyloid. green’ birefringence. The preferred method for par-
This approach was pioneered at the Mayo Clinic, affin-embedded material remains that of Puchtler
USA, and is now being developed at a number et al. (1962). Thioflavine T and other fluorescent
of centers, including the National Amyloid Centre dyes may offer an alternative to Congo red, but they
in London. A simplified laser microdissection- may be less selective, as other hyaline and fibrinoid
proteomics procedure is outlined below. fibers also give positive results (Cooper 1969).
A tissue sample is mounted onto a slide, dewaxed Positive immunohistochemistry with antisera to
and stained with Congo red. The amyloid is visual- amyloid P component may be useful when used in
ized by its characteristic, apple-green birefringence conjunction with Congo red staining, though it must
and with the use of the fluorescent properties of the be remembered that elastin and collagen fibers will
Congo red dye and using a fluorescent laser capture also stain positively. It is of value to identify the type
microscope. Congo red positive material is excised of amyloid present using immunohistochemistry, as
and collected into a 0.5 ml tube, where it is reduced the success of treatment may well depend on such
and carbamidomethylated to protect cysteines. The identification.
 The future 287

Bodin, K., Ellmerich, S., Kahan, M.C., et al., 2010.

The future Antibodies to human serum amyloid P
component eliminate visceral amyloid deposits.
New treatments are constantly being investigated Nature 468, 760–767.
for the treatment of amyloidosis. At the National
Born, M., Wolf, E., 1999. Principles of optics.
Amyloidosis Centre a bis-d-proline compound,
Electomagnetic theory of propagation interference
CPHPC, which depletes circulating SAP, when com-
and diffraction of light, seventh ed. Cambridge
bined with a treatment using antibodies to human
University Press, Cambridge, pp. 12, 49, 95–103,
SAP, triggers a potent, complement-dependent,
820–823, 843.
macrophage-derived giant cell reaction that swiftly
removes massive murine visceral amyloid deposits Brigger, D., Muckle, T.J., 1975. Comparison of Sirius
without adverse effect (Bodin 2010). The unprece- red and Congo red as a stain for amyloid in
dented capacity of this novel combined therapy to animal tissues. Journal of Histochemistry and
eliminate amyloid deposits may be applicable to all Cytochemistry 23, 84.
forms of systemic and localized amylodoidosis in Burns, J., Pennock, C.A., Stoward, P.J., 1967. The
the future. specificity of the staining of amyloid deposits with
LCMD-proteomics is proving to be a valuable thioflavine T. Journal of Pathology and
technique and is becoming more popular in leading Bacteriology 94, 337–344.
specialist laboratories and could possibly be the Carrell, R.W., Gooptu, B., 1998. Conformational
technique of the future. changes and disease – serpins, prions and
Alzheimer’s. Current Opinion in Structural
Acknowledgments Biology 8 (6), 799–809.

We thank Dr Julian Gillmore for his advice and com- Carrell, R.W., Lomas, D.A., 1997. Conformational
ments. Special thanks to our friends and colleagues at disease. Lancet 350 (9071), 134–138.
the National Amyloidosis Centre for their support Carter, D.B., Chou, K.C., 1998. A model for
and encouragement. structure-dependent binding of Congo red to
Alzheimer beta-amyloid fibrils. Neurobiology of
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 Microorganisms
Jeanine H. Bartlett
15
geographic boundaries to infection, exposing
Introduction weaknesses in host defenses, and in
knowledge. Some agents, such as Ebola, have
We have all heard the expression, ‘The world is been around for many years but the first
getting smaller’. Nowhere is that statement truer human outbreaks were not recorded until 1976.
than in the world of microorganisms. Microorgan- Previous outbreaks would flare up and then
isms (also called microbes) are organisms which burn themselves out, undetected and confined,
share the property of being only individually seen before deforestation and the like altered this
by microscopy. Most do not normally cause disease state.
in humans, existing in a state of commensalism,
• Immunodeficiency states occurring either as
where there is little or no benefit to the person, or
part of a natural disease, such as acquired
mutualism, where there is some benefit to both
immune deficiency syndrome (AIDS), or as an
parties. Pathogens are agents that cause disease.
iatrogenic disease. As treatment becomes more
These fall into five main groups:
aggressive, depression of the host’s immunity
• Viruses often occurs, enabling organisms of low
• Bacteria virulence to become life-threatening, and
• Fungi allows latent infections, accrued throughout
• Protozoa life, to reactivate and spread unchecked.
• Helminths • Emerging, re-emerging, and antibiotic-resistant
With the advent of new and more powerful organisms such as the tubercle bacillus and
antibiotics, improved environmental hygiene, and staphylococcus are a constant concern.
advances in microbiological techniques it was • Adaptive mutation occurring in
widely expected that the need for diagnosis of microorganisms, which allows them to jump
infectious agents in tissue would diminish in impor- species barriers and exploit new physical
tance. This assumption underestimated the infinite environments, thus evading host defenses, and
capacity of infectious agents for genomic variation, resisting agents of treatment.
enabling them to exploit new opportunities to • Bioterrorism has become an increasing concern.
spread infections that are created when host defenses The world’s public health systems and primary
become compromised. The following are currently healthcare providers must be prepared to
the most important factors influencing the presenta- address varied biological agents, including
tion of infectious diseases: pathogens that are rarely seen in developed
• The increased mobility of the world’s countries. High-priority agents include
population through tourism, immigration, and organisms that pose a risk to national security
international commerce has distorted natural because they:
© 2013 Elsevier Ltd
 292 15 Microorganisms

• Can be easily disseminated or transmitted Safety

from person to person
• Cause high mortality, with potential for a Most infectious agents are rendered harmless by
major public health impact direct exposure to formal saline. Standard fixation
• Might cause public panic and social procedures should be sufficient to kill microorgan-
disruption, and require special action for isms, one exception being material from patients
public health preparedness. with Creutzfeldt-Jakob disease (CJD). It has been
The following are listed by the Centers for shown that well-fixed tissue, paraffin-processed
Disease Control and Prevention (CDC) in the blocks, and stained slides from CJD remain infec-
United States as high-risk biological agents: tious when introduced into susceptible animals.
Treatment of fixed tissue or slides in 96% formic acid
• Anthrax
for 1 hour followed by copious washing inactivates
• Smallpox
this infectious agent without adversely affecting
• Botulism section quality (Brown et al. 1990). Laboratory safety
• Tularemia protocols should cover infection containment in all
• Viral hemorrhagic fever (various). laboratory areas and the mortuary, or necropsy area,
These factors, acting singly or together, provide an where handling unfixed material is unavoidable.
ever-changing picture of infectious disease where When available, unfixed tissue samples should be
clinical presentation may involve multiple patho- sent for microbiological culture as this offers the best
logical processes, unfamiliar organisms, and modi- chance for rapid and specific identification of etio-
fication of the host response by a diminished immune logical agents, even when heavy bacterial contami-
status. nation may have occurred.

Size General principles of detection

and identification
The term ‘microorganism’ has been interpreted lib-
erally in this chapter. Space limitation precludes a
The diagnosis of illness from infectious disease starts
comprehensive approach to the subject, and the
with clinical presentation of the patient, and in most
reader is referred to additional texts such as that of
cases a diagnosis is made without a tissue sample
von Lichtenberg (1991) for greater depth. The organ-
being taken. Specimens submitted to the laboratory
isms in Table 15.1 are discussed, and techniques for
range from autopsy specimens, where material
their demonstration are described.
maybe plentiful and sampling error presents little
problem, to cytology samples where cellular material
is often scarce and lesions may easily be missed. A
Table 15.1 Size of organisms
full clinical history is always important, especially
Organisms Size details of the patient’s ethnic origin, immune status,
Viruses 20–300 nm any recent history of foreign travel, and current
Mycoplasmas 125–350 nm medication.
The macroscopic appearance of tissue, such as
Chlamydia 200–1000 nm
abscesses and pus formation, cavitations, hyperkera-
Rickettsia 300–1200 nm tosis, demyelination, pseudo-membrane or fibrin
Bacteria 1–14 µm formation, focal necrosis, and granulomas can
Fungi 2–200 µm provide evidence of infection. These appearances are
Protozoa 1–50 µm often non-specific but occasionally in hydatid cyst
disease or some helminth infestations the appear-
Metazoans 3–10 mm
ances are diagnostic. The microscopic appearance of
 General principles of detection and identification 293

routine stains at low-power magnification often utilize (strept)avidin-biotin technologies. These are
reveals indirect evidence of the presence of infection, based on the high affinity that (strept)avidin (Strep-
such as neutrophil or lymphocytic infiltrates, granu- tomyces avidinii) and avidin (chicken egg) have for
loma formation, micro-abscesses, eosinophilic aggre- biotin. Both possess four binding sites for biotin, but
gates, Charcot-Leyden crystals and caseous necrosis. due to the molecular orientation of the binding sites
Some of these appearances may be sufficiently reli- fewer than four molecules of biotin will actually
able to provide an initial, or provisional, diagnosis bind. The basic sequence of reagent application con-
and allow treatment to be started even if the precise sists of primary antibody, biotinylated secondary
nature of the suspect organism is never identified, antibody, followed by either the preformed (strept)
particularly in the case of tuberculosis. avidin-biotin enzyme complex of the avidin-biotin
At the cellular level, the presence of giant cells, complex (ABC) technique or by the enzyme-labeled
(such as Warthin-Finkeldy or Langhans’ type) may streptavidin. Both conclude with the substrate solu-
indicate measles or tuberculosis. Other cellular tion. Horseradish peroxidase and alkaline phospha-
changes include intra-cytoplasmic edema of koilo- tases are the most commonly used enzyme labels
cytes, acantholysis, spongiform degeneration of (see later chapters).
brain, margination of chromatin, syncytial nuclear
appearance, ‘ground-glass’ changes in the nucleus
or cytoplasm, or inclusion bodies, and can indicate In situ hybridization, the polymerase
infectious etiology. At some stage in these processes, chain reaction
suspect organisms may be visualized. A well-
performed hematoxylin and eosin (H&E) method In situ hybridization (ISH) has even greater
will stain many organisms. Papanicolaou stain and potential for microorganism detection. The use of
Romanowsky stains, such as Giemsa, will also stain single-stranded nucleic acid probes offers even
many organisms together with their cellular envi- greater possibilities by identifying latent viral
ronment. Other infectious agents are poorly visual- genomic footprints in cells, which may have rele-
ized by routine stains and require special techniques vance to extending our knowledge of disease.
to demonstrate their presence. This may be due to Acquired immunodeficiency syndrome (AIDS) and
the small size of the organism, as in the case of human immunodeficiency virus (HIV) are good
viruses, where electron microscopy is needed. Alter- examples. The polymerase chain reaction (PCR) can
natively, the organism may be hydrophobic, or also be a very useful technique to obtain diagnoses
weakly charged, as with mycobacteria, spirochetes, of microbial infections from autopsy tissues and sur-
and cryptococci, in which case the use of specific gical specimens. While fresh/frozen tissues provide
histochemical methods is required for their detec- the best-quality nucleic acids for analysis, DNA
tion. When organisms are few in number, fluoro- and RNA extracted from formalin-fixed, paraffin-
chromes may be used to increase microscopic embedded (FFPE) tissues can be used quite success-
sensitivity of a technique. Finally, there are two tech- fully in both PCR and reverse-transcriptase PCR
niques that offer the possibility of specific identifica- (Tatti et al. 2006; Bhatnagar et al. 2007; Guarner et al.
tion of microorganisms that extend to the appropriate 2007; Shieh et al. 2009). Since formalin cross-links
strain level. proteins and nucleic acids, resulting in significant
degradation, it is critical to design PCR assays tar-
Immunohistochemistry geting small amplicons, typically 500 base pairs or
fewer in length (Srinivasan et al. 2002). To this end,
Immunohistochemistry is now a routine and invalu- it is essential to begin processing of specimens as
able procedure in the histopathology lab for the quickly as possible, ensuring that a 10% concentra-
detection of many microorganisms. There are many tion of formalin is used for fixation, and making
commercially available antibodies for viral, bacte- certain that fixation times are kept to no longer than
rial, and parasitic organisms. Most methods today 48 hours (von Ahlfen et al. 2007; Chung et al. 2008).
 294 15 Microorganisms

Table 15.2 A simplified classification of important bacteria

Gram-positive bacteria Gram-negative bacteria
Cocci Bacilli Cocci Bacilli Coccobacilli
Staphylococcus sp. Bacillus Neisseria Escherichia Brucella
Clostridium Klebsiella Bordetella
Streptococcus sp. Corynebacterium Salmonella Haemophilus
(inc. Pneumococcus) Mycobacteria (weak+) Shigella
Lactobacillus (commensal) Proteus
Listeria Pseudomonas
Vibrio
Pasteurella

Furthermore, the use of real-time PCR technology, pyogenic bacteria to the Gram stain, together with
which often requires small amplicons for successful their morphological appearance (i.e. cocci or bacilli)
detection of products, is ideally suited for use with provides the basis for a simple historical classifica-
nucleic acid extracts from FFPE tissues (Denison et tion (Table 15.2).
al. 2011). (Thanks are due to Amy M. Denison, PhD,
for her assistance with this information.)
Use of control sections
While modern advances in technique are important, The use of known positive control sections with all
emphasis is also placed upon the ability of the special stain methods for demonstrating microor-
microscopist to interpret suspicious signs from a ganisms is essential. Results are unsafe in the absence
good H&E stain. The growing number of patients of positive controls, and should not be considered
whose immune status is compromised, and who can valid. The control section should be appropriate,
mount only a minimal or inappropriate response to where possible, for the suspected organism. A
infection, further complicates the picture, justifying pneumocystis containing control, for instance,
speculative use of special stains such as those for should be used for demonstrating Pneumocystis jir-
mycobacteria and fungi on tissue from AIDS oveci (previously called carinii). A Gram control
patients. It should be remembered that, for a variety should contain both Gram-positive and Gram-
of reasons, negative results for the identification of negative organisms. Post-mortem tissues have pre-
an infectious agent do not exclude its presence. In viously been a good source of control material,
particular, administration of antibiotics to the patient although medico-legal issues have now limited this
before a biopsy is often the reason for failure to in some countries. Alternatively, a suspension of
detect a microorganism in tissue. Gram-positive and Gram-negative organisms can be
injected into the thigh muscle of a rat shortly before
Detection and identification of bacteria it is sacrificed for some other purpose. Gram-positive
and Gram-negative organisms can also be harvested
When bacteria are present in large numbers, in an from microbiological plates, suspended in 10%
abscess or in vegetation on a heart valve, they neutral buffered formalin (NBF), centrifuged, and
appear as blue-gray granular masses with an H&E small amounts mixed with minced normal kidney,
stain. However, organisms are often invisible or then chemically processed along with other tissue
obscured by cellular debris. The reaction of blocks (Swisher & Nicholson 1989).
 The Gram stain 295

The Gram stain Solutions

Crystal violet solution (commercially available)
Crystal violet, 10% alcoholic 2 ml
In spite of more than a century having passed since
Distilled water 18 ml
Gram described his technique in 1884, its chemical Ammonium oxalate, 1% 80 ml
rationale is still obscure. It is probably due to a
Mix and store; always filter before use.
mixture of factors, the most important being
Modified Gram’s iodine commercially available, or
increased thickness, chemical composition, and the
Iodine 2g
functional integrity of cell walls of Gram-positive
Potassium iodide 4g
bacteria. When these bacteria die, they become Distilled water 400 ml
Gram negative. The following procedure is only
Dissolve potassium iodide in a small amount of the
suitable for the demonstration of bacteria in smears distilled water, add iodine and dissolve; add
of pus and sputum. It may be of value to the pathol- remainder of distilled water.
ogist in the necropsy room where a quick technique Ethyl alcohol-acetone solution
such as this may enable rapid identification of the Ethyl alcohol, absolute 50 ml
organism causing a lung abscess, wound infection, Acetone 50 ml
septicemic abscess or meningitis. 0.5% basic fuchsin solution (stock) commercially
available, or
Basic fuchsin or pararosaniline 0.5 g
Gram method for bacteria in smears (Gram 1884) Distilled water 100 ml
Method Dissolve with aid of heat and a magnetic stirrer.
1. Fix dry film by passing it three times through a Basic fuchsin solution (working)
flame or placing on a heat block. Basic fuchsin solution (stock) 10 ml
2. Stain for 15 seconds in 1% crystal violet or methyl Distilled water 40 ml
violet, and then pour off excess. Picric acid-acetone
3. Flood for 30 seconds with Lugol’s iodine, pour off Picric acid 0.1 g
excess. Acetone 100 ml
4. Flood with acetone for not more than 2–5 seconds,
Note
wash with water immediately.
With concerns over the explosiveness of dry picric
5. Alternatively decolorize with alcohol until no more
acid in the lab, it is recommended that you purchase
stain comes out. Wash with water.
the picric acid-acetone solution pre-made. It is
6. Counterstain for 20 seconds with dilute carbol
available through most histology suppliers.
fuchsin, or freshly filtered neutral red for
1–2 minutes. Acetone-xylene solution
7. Wash with water and carefully blot section until it is Acetone 50 ml
dry. Xylene 50 ml

Results Staining method

Gram-positive organisms blue-black 1. Deparaffinize and rehydrate through graded
Gram-negative organisms red alcohols to distilled water.
2. Stain with filtered crystal violet solution, 1 minute.
3. Rinse well in distilled water.
4. Iodine solution, 1 minute.
Modified Brown-Brenn method for Gram-positive and 5. Rinse in distilled water, blot slide but NOT the
Gram-negative bacteria in paraffin sections (Churukian & tissue section.
Schenk 1982)
6. Decolorize by dipping in alcohol-acetone solution
Sections until the blue color stops running. (One to two
Formalin-fixed, 4–5 µm, paraffin-embedded sections. dips only!)
 296 15 Microorganisms

7. Counterstain in working basic fuchsin for 6. Differentiate in preheated acetic alcohol until no
1 minute. Be sure to agitate the slides well in the more color washes out (2% acetic acid in
basic fuchsin before starting the timer. absolute alcohol, preheated to 56°C). This may
8. Rinse in distilled water and blot slide but not take 15–20 minutes; the section should be light
section. brown or straw colored.
9. Dip in acetone, one dip. 7. Rinse briefly in distilled water.
10. Dip in picric acid-acetone until the sections have 8. Stain in Twort’s, 5 minutes.
a yellowish-pink color. 9. Wash in distilled water.
11. Dip several times in acetone-xylene solution. At 10. Rinse in acetic alcohol until no more red runs out
this point, check the control for proper of the section; this takes only a few seconds.
differentiation. (Go back to picric acid-acetone if 11. Rinse in fresh absolute alcohol, clear, and mount.
you need more differentiation.)
Results
12. Clear in xylene and mount.
Gram-positive organisms blue-black
Results Gram-negative organisms pink-red
Gram-positive organisms, fibrin, some fungi, blue Nuclei red
Paneth cell granules, keratohyalin, and Red blood cells and most cytoplasmic green
keratin structures
Gram-negative organisms red Elastic fibers black
Nuclei red
Other tissue elements yellow
Be sure you do not allow the tissue sections to dry at
any point in the staining process. If this occurs after Techniques for mycobacteria
treatment with iodine, decolorization will be difficult
and uneven. These organisms are difficult to demonstrate by the
Gram technique as they possess a capsule contain-
ing a long-chain fatty acid (mycolic acid) that makes
them hydrophobic. The fatty capsule influences the
Gram-Twort stain (Twort 1924; Ollett 1947) penetration and resistance to removal of the stain by
Sections acid and alcohol (acid- and alcohol-fastness), and is
Formalin fixed, paraffin. variably robust between the various species that
make up this group. Phenolic acid, and frequently
Solutions
heat, are used to reduce surface tension and increase
Crystal violet solution (see previous method) porosity, thus forcing dyes to penetrate this capsule.
Gram’s iodine (see previous solution) The speed with which the primary dye is removed
Twort’s stain by differentiation with acid alcohol is proportional
1% neutral red in ethanol 9 ml
to the extent of the fatty coat. The avoidance of
0.2% fast green in ethanol 1 ml
Distilled water 30 ml
defatting agents, or solvents, such as alcohol and
xylene, in methods for Mycobacterium leprae, is an
Mix immediately before use.
attempt to conserve this fragile fatty capsule.
Method Mycobacteria are PAS positive due to the carbo-
1. Deparaffinize and rehydrate through graded hydrate content of their cell walls. However, this
alcohols to distilled water. positivity is evident only when large concentrations
2. Stain in crystal violet solution, 3 minutes. of the microorganisms are present. When these
3. Rinse in gently running tap water.
organisms die, they lose their fatty capsule and con-
4. Treat with Gram’s iodine, 3 minutes.
sequently their carbol fuchsin positivity. The carbo-
5. Rinse in tap water, blot dry, and complete drying
hydrate can still be demonstrated by Grocott’s
in a warm place.
methenamine silver reaction, which may prove
 Techniques for mycobacteria 297

useful when acid-fast procedures fail, particularly

Results
if the patient is already receiving therapy for
Mycobacteria, hair shafts, Russell bodies, red
tuberculosis. Splendore-Hoeppli immunoglobulins
A possible source of acid-fast contamination may around actinomyces, and some fungal
be found growing in viscous material sometimes organisms
lining water taps and any rubber tubing connected Background pale blue
to them. These organisms are acid- and alcohol-fast Notes
but are usually easily identified as contaminants by a. The blue counterstain may be patchy if extensive
their appearance as clumps, or floaters, above the caseation is present. Care should be taken to
microscopic focal plane of the section. avoid over-counterstaining as scant organisms can
easily be obscured.
b. Decalcification using strong acids can destroy
acid-fastness; formic acid is recommended.
c. Victoria blue can be substituted for carbol fuchsin
Ziehl-Neelsen (ZN) stain for Mycobacterium bacilli and picric acid for the counterstain if color
(Kinyoun 1915) blindness causes a recognition problem.
Sections
Formalin or fixative other than Carnoy’s, paraffin.
Solutions
Fluorescent method for Mycobacterium bacilli
(Kuper & May 1960)
Carbol fuchsin commercially available, or
Basic fuchsin 0.5 g Sections
Absolute alcohol 5 ml Formalin fixed, paraffin.
5% aqueous phenol 100 ml Solution
Mix well and filter before use. Auramine O 1.5 g
Acid alcohol Rhodamine B 0.75 g
Hydrochloric acid 10 ml Glycerol 75 ml
70% alcohol 1000 ml Phenol crystals (liquefied at 50°C) 10 ml
Methylene blue solution (stock) commercially Distilled water 50 ml
available, or
Method
Methylene blue 1.4 g
1. Deparaffinize (1 part groundnut oil and 2 parts
95% alcohol 100 ml
xylene for M. leprae).
Methylene blue solution (working) 2. Pour on preheated (60°C), filtered staining solution,
Methylene blue (stock) 10 ml 10 minutes.
Tap water 90 ml 3. Wash in tap water.
Method 4. Differentiate in 0.5% hydrochloric acid in alcohol
1. Deparaffinize and rehydrate through graded for M. tuberculosis, or 0.5% aqueous hydrochloric
alcohols to distilled water. acid for M. leprae.
2. Carbol fuchsin solution, 30 minutes. 5. Wash in tap water, 2 minutes.
3. Wash well in tap water. 6. Eliminate background fluorescence in 0.5%
4. Differentiate in acid alcohol until solutions are pale potassium permanganate, 2 minutes.
pink. (This usually only takes 2–5 dips.) 7. Wash in tap water and blot dry.
5. Wash in tap water for 8 minutes, then dip in 8. Dehydrate (not for M. leprae), clear, and mount in
distilled water. a fluorescence-free mountant.
6. Counterstain in working methylene blue solution Results
until sections are pale blue. Mycobacteria golden yellow (using blue light
7. Rinse in tap water, then dip in distilled water. fluorescence below 530 nm)
8. Dehydrate, clear, and mount. Background dark green
 298 15 Microorganisms

Notes 7. Wash in warm, running tap water for 5 minutes.

The advantage of increased sensitivity of this 8. Counterstain in working methylene blue, one
technique is offset by the inconvenience of setting up quick dip. (Sections should be pale blue.)
the fluorescence microscope. Preparations fade over 9. Wash in warm, running tap water for 5 minutes.
time, as a result of their exposure to UV light. 10. Blot sections and dry in 50–55°C oven for 5
minutes.
11. Once dry, one quick dip in xylene.
Modified Fite method for M. leprae and Nocardia 12. Mount with permanent mountant.
Fixation
Results See Figure 15.1
10% neutral buffered formalin (NBF).
Acid-fast bacilli including M. leprae bright red
Sections Nuclei and other tissue elements pale blue
Paraffin sections at 4–5 µm. Quality control/notes
Solutions Be careful not to overstain with methylene blue and
Carbol fuchsin solution commercially available, or do not allow sections to dry between carbol fuchsin
0.5 g basic fuchsin dissolved in 5 ml of absolute and acid alcohol.
alcohol; add 100 ml of 5% aqueous phenol. Mix well
and filter before use. Filter before each use with #1
filter paper.
Cresyl violet acetate method for Helicobacter sp.
5% sulfuric acid in 25% alcohol
Sections
25% ethanol 95 ml
Formalin fixed, paraffin.
Sulfuric acid, concentrated 5 ml
Methylene blue (stock) commercially Method
available, or 1. Deparaffinize and rehydrate through graded
Methylene blue 1.4 g alcohols to distilled water.
95% alcohol 100 ml 2. Filter 0.1% cresyl violet acetate onto slide or into
Coplin jar, 5 minutes.
Methylene blue, working 3. Rinse in distilled water.
Stock methylene blue 5 ml 4. Blot, dehydrate rapidly in alcohol, clear, and mount.
Tap water 45 ml
Xylene-peanut oil 1 part oil: 2 parts xylene

Method
1. Deparaffinize in two changes of xylene-peanut oil,
6 minutes each.
2. Drain slides vertically on paper towel and wash
in warm, running tap water for 3 minutes. (The
residual oil preserves the sections and helps
accentuate the acid fastness of the bacilli.)
3. Stain in carbol fuchsin at room temperature for 25
minutes. (Solution may be poured back into bottle
and reused).
4. Wash in warm, running tap water for 3 minutes.
5. Drain excess water from slides vertically on paper
towel.
6. Decolorize with 5% sulfuric acid in 25% alcohol,
Figure 15.1 The modified Fite’s procedure is necessary to
two changes of 90 seconds each. (Sections
demonstrate Mycobacterium leprae due to the organism’s fragile,
should be pale pink.)
fatty capsule.
 Techniques for mycobacteria 299

Results Notes
Helicobacter and nuclei blue-violet The greatest problem with this method is overstaining
Background shades of blue-violet or irregularity of staining, with Malachite green. It is
valuable in demonstrating the Legionella bacillus in
Notes
postmortem lung smears.
This simple method allows for good differentiation of
Helicobacter sp. from other organisms.

Toluidine blue in Sorenson’s buffer for Helicobacter

Sections
Formalin fixed, paraffin.
Gimenez method for Helicobacter pylori (Gimenez 1964;
McMullen et al. 1987) Solutions
Sections Toluidine blue in pH 6.8 phosphate buffer
Formalin fixed, paraffin. Sorenson’s phosphate buffer, pH 6.8 50 ml
1% aqueous toluidine blue 1 ml
Solutions
Buffer solution (phosphate buffer at pH 7.5, or
Method
0.1 M)
1. Deparaffinize and rehydrate through graded
0.1 M sodium dihydrogen orthophosphate 3.5 ml
alcohols to distilled water.
0.1 M disodium hydrogen orthophosphate 15.5 ml
2. Stain in buffered toluidine blue, 20 minutes.
Stock carbol fuchsin
3. Wash well in distilled water.
Commercial cold acid-fast bacilli stain, 1g
4. Dehydrate, clear, and mount.
or basic fuchsin
Absolute alcohol 10 ml Results
5% aqueous phenol 10 ml Helicobacter dark blue against a variably
Filter before use. blue background
Working carbol fuchsin
Phosphate buffer 10 ml
Stock carbol fuchsin 4 ml Warthin-Starry method for spirochetes (Warthin & Starry
1920)
Filter before use.
Sections
Malachite green
Formalin fixed, paraffin.
Malachite green 0.8 g
Distilled water 100 ml Solutions
Method Acetate buffer, pH 3.6
1. Deparaffinize and rehydrate through graded Sodium acetate 4.1 g
alcohols to distilled water. Acetic acid 6.25 ml
2. Stain in working carbol fuchsin solution, 2 minutes. Distilled water 500 ml
3. Wash well in tap water. 1% silver nitrate in pH 3.6 acetate buffer
4. Stain in malachite green, 15–20 seconds. Developer
5. Wash thoroughly in distilled water. Dissolve 3 g of hydroquinone in 10 ml pH 3.6 buffer,
6. Repeat steps 4 and 5 until section is blue-green to and mix 1 ml of this solution and 15 ml of warmed 5%
the naked eye. Scotch glue or gelatin; keep at 40°C. Take 3 ml of 2%
7. Blot sections dry, and complete drying in air. silver nitrate in pH 3.6 buffer solution and keep at
55°C. Mix these two solutions immediately before use.
8. Clear and mount.
Results Method
Helicobacter red-magenta 1. Deparaffinize and rehydrate through graded
Background blue-green alcohols to distilled water.
 300 15 Microorganisms

2. Celloidinize in 0.5% celloidin, drain, and harden in 2% hydroquinone

distilled water, 1 minute. Hydroquinone 1g
3. Impregnate in preheated 55–60°C silver solution Distilled water 25 ml
(b), 90–105 minutes. Make fresh solution for each use.
4. Prepare and preheat developer in a water bath.
Reducing solution
5. Treat with developer (solution c) for 31/2 minutes at
Mix 10 ml of 2.5% gum mastic, 25 ml of 2.0%
55°C. Sections should be golden-brown at this
hydroquinone, and 5 ml absolute alcohol. Make just
point.
prior to use and filter with #4 filter paper; add 2.5 ml
6. Remove from developer and rinse in tap water for
of 0.04% silver nitrate. Do not filter this solution. When
several minutes at 55–60°C, then in buffer at room
the gum mastic is added, the solution will take on a
temperature.
milky appearance.
7. Tone in 0.2% gold chloride.
8. Dehydrate, clear, and mount. Method
1. Deparaffinize and rehydrate through graded
Results
alcohols to distilled water.
Spirochetes black 2. Sensitize sections in 1% aqueous uranyl nitrate
Background golden-yellow
at room temperature, and place in microwave
Notes oven until solution is just at boiling point, approx.
It is wise to take a few slides through at various 20–30 seconds; do not boil. Alternatively, place
incubation times to ensure optimum impregnation. in preheated 1% uranyl nitrate at 60°C in a
water bath for 15 minutes, or in microwave
oven and bring to boiling point – do not boil;
2% zinc sulfate in 3.7% formalin may be
substituted.
Modified Steiner for filamentous and non-filamentous 3. Rinse in distilled water at room temperature until
bacteria (Steiner & Steiner 1944; Modified Swisher 1987) uranyl nitrate residue is eliminated.
Sections 4. Place in 1% silver nitrate at room temperature and
microwave until boiling point is just reached. Do
Formalin fixed, paraffin.
not boil. Remove from oven, loosely cover jar,
Solutions and allow to stand in hot silver nitrate,
1.0% uranyl nitrate commercially available, or 6–7 minutes; alternatively, preheat silver nitrate for
Uranyl nitrate 1g 20–30 minutes in a 60°C water bath, add slides,
Distilled water 100 ml and allow to impregnate for 11/2 hours.
1% silver nitrate 5. Rinse in three changes of distilled water.
Silver nitrate 1g 6. Dehydrate in two changes, each of 95% alcohol
Distilled water 100 ml and absolute alcohol.
7. Treat with 2.5% gum mastic, 5 minutes.
Make fresh each time and filter with #1 or #2 filter
paper before use. 8. Allow to air dry, 5 minutes.
9. Rinse in two changes of distilled water. Slides
0.04% silver nitrate
may stand here while reducing solution is being
Silver nitrate 0.04 g
prepared.
Distilled water 100 ml
10. Reduce in preheated reducing solution at 45°C in
Refrigerate and use for only 1 month. a water bath for 10–25 minutes, or until sections
2.5% gum mastic commercially available, or have developed satisfactorily with black
Gum mastic 2.5 g microorganisms against a light yellow
Absolute alcohol 100 ml background. Avoid intensely stained
Allow to dissolve for 24 hours, then filter until clear background.
yellow before use. Refrigerate unused portion for 11. Rinse in distilled water to stop reaction.
reuse. 12. Dehydrate, clear, and mount.
 Some important bacteria 301

occur. Staphylococci tend to form clusters. Multi-

resistance to antibiotics is sometimes encountered.
Neisseria meningitidis (meningococcus) is a common
cause of meningitis, and may produce a fulminating
septicemia. Organisms can be seen in histological
sections of meningococcal meningitis, but are diffi-
cult to identify because they are usually within neu-
trophil cytoplasm.
Neisseria gonorrhoeae (gonococcus) is the cause of
gonorrhea. Organisms may be seen within poly-
morphs in sections of cervix, endometrium, or Fal-
lopian tubes in cases of gonorrhea but, again, are
difficult to find. Members of the Neisseria family are
Figure 15.2 Syphilis Treponema pallidum, bacilli (arrowed), seen generally difficult to see in histological sections,
with the modified Steiner technique. The resistance to coloration is although easily detectable in smears of fresh pus or
shared by Helicobacter, spirochetes, and Legionella. cerebrospinal fluid (CSF), characteristically in pairs.
They are easier to detect using the Gram-Twort
Results See Figure 15.2 method.
Spirochetes, cat-scratch organisms, dark brown- Lactobacillus acidophilus (Döderlein’s bacillus) is a
Donovan bodies, non-filamentous black normal inhabitant of the human vagina and is seen
bacteria of L. pneumophila in cervical smears taken in the secretory phase of
Background bright yellow the cycle.
to golden
Corynebacterium vaginale is a short Gram-negative
yellow
bacillus which may cause cervicitis, and is present
Notes in about 6% of women of childbearing age. It may
Bring all solutions to room temperature before using. be seen in cervical smears where it accumulates as
All glassware making contact with silver nitrate should blue-stained masses on the surface of squamous
be chemically cleaned. Avoid the use of metal forceps
cells stained by Papanicolaou’s method, with these
in silver solutions. When doing a bacterial screen,
Gram controls should be run along with diagnostic cells being known as ‘clue cells’.
slides. As spirochetes take longer to develop, Gram Helicobacter pylori is frequently seen in gastric
controls should be used in addition to spirochete biopsies. A spiral vibrio organism is heavily impli-
controls. When Gram controls have a yellow cated as the organism causing many cases of chronic
appearance, remove them to distilled water, and gastritis. It is seen as small, weakly hematoxyphilic
check on microscope for microorganisms. Return to organisms (usually in clumps) in the lumina of
silver solution if they are not ready, and repeat,
gastric glands, often adherent to the luminal surface
realizing that spirochetes will take longer. Most
solutions can be made in large quantities and kept in
of the epithelial cells. With practice, these can be
the refrigerator. identified from an H&E stain. However, Warthin-
Starry, Steiner, Gimenez, toluidine blue, or cresyl
violet acetate methods demonstrate them more
Some important bacteria clearly. A commercial specific antiserum has recently
become available for their demonstration.
Staphylococcus aureus is perhaps the most important Clostridium difficile causes pseudomembranous
pathogen of this group. It causes boils, wound and colitis, an inflammation of the large bowel. This
burn infections, and a form of cavitating pneumonia arises following the administration of broad-
in children and adults. Septicemic states and the spectrum antibiotics; the balance of the normal
formation of multiple scattered abscesses sometimes anaerobic gut microflora is disturbed, allowing the
 302 15 Microorganisms

organism to proliferate unchecked. C. difficile is dif- bacterium may be difficult to stain except with the
ficult to stain but the ‘volcano lesions’ of purulent Dieterle and modified Steiner silver stains, and spe-
necrosis are a good indicator. cific antiserum.
Listeria monocytogenes is the cause of a rare form of Treponema pallidum is the organism causing syphi-
meningitis and may cause septicemia in humans. lis, and is infrequently seen in biopsy specimens as
Focal necrosis with macrophages that contain tiny the primary lesion or ‘chancre’ is diagnosed clini-
intracellular rods arranged in a ‘Chinese letter’ for- cally. The spirochete is quite obvious using dark-
mation, and staining variably with the Gram stain, ground microscopy, as an 8–13 µm corkscrew shaped
are the hallmark of this disease. microorganism that often kinks in the center. Diet-
Mycobacterium tuberculosis remains a significant erle, Warthin-Starry, or modified Steiner methods
pathogen in developed countries where the familiar may demonstrate the organism. In addition, specific
caseating granulomatous lesion and its associated antiserum is also available.
1–2 µm, blunt-ended, acid- and alcohol-fast bacilli Leptospira interrogans is the organism causing lep-
can still be seen. In Africa and other countries, this tospirosis or Weil’s disease. It is a disease character-
organism has developed an opportunistic relation- ized by spirochetes, and is spread in the urine of rats
ship with AIDS, where it is a major cause of death. and dogs, causing fever, profound jaundice, and
Mycobacterium avium/intracellulare are representa- sometimes death. Spirochetes can be seen in the
tives of a group of intracellular opportunistic acute stages of the disease where they appear in
mycobacteria that are frequently present in the later Warthin-Starry and modified Steiner techniques as
stages of immunosuppression, particularly that tightly wound 13 µm microorganisms with curled
associated with AIDS. They frequently persist in ends resembling a shepherd’s crook.
spite of treatment, and are often lethal. The lesions Intestinal spirochetosis appears as a massive infesta-
produced are non-caseating and consist of collec- tion on the luminal border of the colon by spirochete
tions of vacuolated macrophages that often contain Brachyspira aalborgi (Tomkins et al. 1986). It measures
vast numbers of organisms. On occasion, there is 2–6 µm long, is tightly coiled, and arranged perpen-
little evidence of a cellular reaction on an H&E- dicularly to the luminal surface of the gut, giving it
stained section, and the organism is detected only a fuzzy hematoxyphilic coat in an H&E stain. There
by routinely performing an acid-fast stain, such as is no cellular response to the presence of this spiro-
the ZN, on all tissue from AIDS patients. This group chete. It is seen well with the Warthin-Starry and the
also includes M. kansasii. modified Steiner techniques.
Mycobacterium leprae is an obligate intracellular, Cat-scratch disease presents as a self-limiting, local,
neurotrophic mycobacterium that attacks and single lymphadenopathy appearing about 2 weeks
destroys nerves, especially in the skin. The tissue after a cat scratch or bite. Histologically the node
reaction to leprosy depends on the immune status shows focal necrosis or micro-abscesses. Two Gram-
of the host. It can be minimal with a few macro- negative bacteria (Afipia felis and Bartonella henselae)
phages packed with crescentic, pointed, intracyto- have been implicated. Because of the timing or mat-
plasmic bacilli (lepromatous leprosy), or may contain uration factor of the bacterium, it is difficult to dem-
scanty organisms and show florid granulomatous onstrate on paraffin sections, but the modified
response (tuberculoid leprosy). M. leprae is only Steiner and the Warthin-Starry methods are valuable
acid-fast and can often be demonstrated with a stan- techniques for demonstrating this organism.
dard ZN technique.
Legionella pneumophila was first identified in 1977
as the cause of a sporadic type of pneumonia of high Fungal infections
mortality. The small Gram-positive coccobacillus is
generally spread in aerosols from stagnant water Fungi are widespread in nature, and humans are
reservoirs, usually in air-conditioning units. The regularly exposed to the spores from many species,
 Fungal infections 303

yet the most commonly encountered diseases are the paraffin sections. These antibodies have not found
superficial mycoses that affect the subcutaneous or widespread use, however, on fixed tissue where
horny layers of the skin or hair shafts, and cause identification still relies primarily on traditional
conditions such as athlete’s foot or ringworm. These staining methods.
dermatophytic fungi belong to the Microsporum and An H&E stain, a Grocott methenamine (hexamine)-
Trichophyton groups and may appear as yeasts or silver (GMS), a mounted unstained section to look
mycelial forms within the keratin. They are seen for pigmentation, and a good color atlas (Chandler
fairly well in the H&E stain, but are demonstrated et al. 1980) when experience fails permit most fungal
well with the Grocott and PAS stains. As with other infections to be identified to levels sufficient for
infections, the increase in the number of patients diagnoses. However, there is no substitute for micro-
with diminished or compromised immune systems biological culture.
has increased the incidence of systemic mycoses,
allowing opportunistic attacks by fungi, often of low
virulence, but sometimes resulting in death.
When fungi grow in tissue they may display prim- Grocott methenamine (hexamine)-silver for fungi and
Pneumocystis spp. organisms (Gomori 1946; Grocott 1955;
itive asexual (imperfect) forms that appear as either
Swisher & Chandler 1982)
spherical yeast or spore forms. Some may produce
Sections
vegetative growth that appears as tubular hyphae
Formalin fixed, paraffin.
that may be septate and branching; these features
are important morphologically for identifying dif- Solutions
ferent types of fungi. A mass of interwoven hyphae 4% chromic acid commercially available, or
is called a fungal mycelium. Only rarely, when the Chromic acid 4.0 g
fungus reaches an open cavity, the body surface, or Distilled water 100 ml
a luminal surface such as the bronchus, are the 1% sodium bisulfite
spore-forming fruiting bodies called sporangia, or Sodium bisulfite 1g
conidia, produced. Distilled water 100 ml
5% sodium thiosulfate
Sodium thiosulfate 5.0 g
Distilled water 100 ml
Identification of fungi
(A) 0.21% silver nitrate (stock)
Some fungi may elicit a range of host reactions from Silver nitrate 2.1 g
exudative, necrotizing, to granulomatous; other Distilled water 1000 ml
fungi produce little cellular response to indicate Refrigerate for up to 3 months.
their presence. Fortunately, most fungi are relatively (B) Methenamine-sodium borate solution (stock)
large and their cell walls are rich in polysaccharides, Methenamine 27 g
which can be converted by oxidation to dialdehydes Sodium borate decahydrate (borax) 3.8 g
and thus detected with Schiff’s reagent or hexamine- Distilled water 1000 ml
silver solutions. Fungi are often weakly hematoxy- Refrigerate for up to 3 months.
philic and can be suspected on H&E stains. Some Methenamine-silver sodium borate solution
fungi, such as sporothrix, may be surrounded by a (working)
stellate, strongly eosinophilic, refractile Splendore- Equal parts of solutions A and B. Make fresh each
Hoeppli precipitates of host immunoglobulin and time and filter before use.
degraded eosinophils. 0.2% light green (stock)
Fluorochrome-labeled specific antibodies to many Light green 0.2 g
fungi are available, and are in use in mycology labo- Distilled water 100 ml
Glacial acetic acid 0.2 ml
ratories for the identification of fungi on fresh and
 304 15 Microorganisms

Light green (working) digested prior to incubation. A water bath may be

Stock light green 10 ml used effectively to insure an even incubation
Distilled water 50 ml temperature.
Prepare working solution fresh before each use. b. Borax insures an alkaline pH.
c. Sodium bisulfite removes excess chromic acid.
Method
d. Some workers prefer a light H&E counterstain. This
1. Deparaffinize and rehydrate through graded
is especially useful when a consulting case is sent
alcohols to distilled water.
with only one slide, providing morphological detail
2. Oxidize in 4% aqueous chromic acid (chromium for the pathologist.
trioxide), 30 minutes.
e. Solutions A and B need to be made and stored in
3. Wash briefly in distilled water. chemically clean glassware (20% nitric acid), as
4. Dip briefly in 1% sodium bisulfite. does the working solution. This includes graduates
5. Wash well in distilled water and Coplin jars. Do not use metal forceps.
6. Place in preheated (56–60°C water bath) working f. Allow all refrigerated solutions to reach room
silver solution for 15–20 minutes. Check control temperature before using.
after 15 minutes. If section is ‘paper bag brown’
then rinse in distilled water and check under
microscope. If it is not ready, dip again in distilled McManus’ PAS method for glycogen and fungal
water and return to silver. Elastin should not be cell walls
black. Check every 2 minutes from that point Fixation
onwards. (See Note a.) 10% NBF.
7. Rinse well in distilled water.
Sections
8. Tone in 0.1% gold chloride, 5 seconds. Rinse in
distilled water. 3–5-µm paraffin sections.
9. Place in 5% sodium thiosulfate, 5 seconds. Solutions
10. Rinse well in running tap water. Schiff’s reagent, also commercially available
11. Counterstain in working light green solution until a 0.5% periodic acid solution
medium green (usually 5–15 seconds). Periodic acid 0.5 g
12. Dehydrate, clear, and mount. Distilled water 100 ml

Results 0.2% light green (stock)

Light green 0.2 g
Fungi, pneumocystis, melanin black
Distilled water 100 ml
Hyphae and yeast-form cells sharply delineated in
Glacial acetic acid 0.2 ml
black of fungi
Mucins and glycogen taupe to dark gray/ This is the same stock solution used in the GMS.
brown Light green (working)
Background pale green Stock light green 10 ml
Distilled water 50 ml
Notes
a. Incubation time is variable and depends on the Make fresh before each use.
type and duration of fixation, and organism being Method
demonstrated. Impregnation is controlled 1. Deparaffinize and hydrate slides to distilled water.
microscopically until fungi are dark brown.
2. Oxidize in periodic acid solution for 5 minutes.
Background is colorless at this point. Over-
3. Rinse in distilled water.
incubation produces intense staining of elastin and
fungi that may obscure fine internal detail of the 4. Place in Schiff’s reagent for 15 minutes.
hyphal septa. This detail is essential for critical 5. Wash in running tap water for 10 minutes to allow
identification, and is best seen on under- pink color to develop.
impregnated sections. To avoid excess glycogen 6. Counterstain for a few seconds in working light
impregnation in liver sections, section may be green solution.
 A selection of the more important fungi and actinomycetes 305

vaginal moniliasis, the skin and nails, and may be

7. Dehydrate in 95% alcohol, absolute alcohol, and
clear in xylene. found in heart-valve vegetations. It is seen as both
8. Mount in resin-based mountant. ovoid budding yeast-form cells of 3–4 µm, and more
commonly as slender 3–5 µm, sparsely septate, non-
Results
branching hyphae and pseudo-hyphae. While diffi-
Fungal cell walls and glycogen magenta to red
cult to see on H&E, this organism is strongly Gram
Background pale green
positive, and is obvious with the Grocott and PAS
Quality control/notes techniques.
A solution of 5% aqueous sodium hypochlorite Aspergillus fumigatus is a soil saprophyte and a
reduces overstaining by Schiff’s.
commensal in the bronchial tree. It may infect
old lung cavities (Fig. 15.3) or become systemic in
immunosuppressed patients. The fungus has broad,
A selection of the more important fungi 3–6 µm, parallel-sided, septate hyphae showing
and actinomycetes dichotomous (45 degrees) branching. It may be asso-
ciated with Splendore-Hoeppli protein and some-
Actinomyces israelii is a colonial bacterium which can times forms fungal balls within tissue. This fungus
be found as a commensal in the mouth and tonsillar may be seen in an H&E stain and is demonstrated
crypts. It can cause a chronic suppurative infection, well with a PAS or Grocott. When it grows exposed
actinomycosis, which is characterized by multiple to air, the conidophoric fruiting body may be seen
abscesses drained by sinus tracts. Actinomycotic as Aspergillus niger, a black species that can cause
abscesses can be found in liver, appendix, lung, and infection of the ear.
neck. The individual organisms are Gram-positive, Zygomycosis is an infrequently seen disease caused
hematoxyphilic, non-acid-fast, branching filaments by a group of hyphated fungi belonging mainly to
1 µm in diameter. They become coated in ‘clubs’ of the genera Mucor and Rhizopus. They have thin-
Splendore-Hoeppli protein when the organism is walled hyphae (infrequently septate) with non-
invasive. These clubs are eosinophilic, acid-fast, parallel sides, ranging from 3 to 25 µm in diameter,
1–15 µm wide, and up to 100 µm long, and stain branch irregularly, and often show empty bulbous
polyclonally for immunoglobulins. This arrange- hyphal swelling. Grocott and PAS are the staining
ment of a clump of actinomyces or fungal hyphae, methods of choice (Figs 15.4 and 15.5).
which measures 30–3000 µm, surrounded by eosino-
philic protein, is called a ‘sulfur’ granule and is an
important identification marker for certain fungal
groups. These granules may be macroscopically
visible and their yellow color is an important diag-
nostic aid.
Nocardia asteroides is another actinomycete. It is
filamentous and may be visible in an H&E stain, but
is Grocott positive and variably acid-fast using the
modified ZN for leprosy. However, it is difficult to
demonstrate even with the acid-fast bacillus. Its
pathology is similar to that of actinomycosis, but its
organisms are generally more disseminated than
those of actinomycosis.
Figure 15.3 A strong hematoxylin (Ehrlich’s and eosin stain) will
Candida albicans is a common fungus, but with show the fine detail of many infectious agents. The hyphal
immunosuppression can become systemic. It infects structure identifies this as Aspergillus which was colonizing an old
the mouth as thrush, the esophagus, the vagina as tuberculosis cavity in the lung.
 306 15 Microorganisms

Figure 15.4 Rhizomucor spp. (a cosmopolitan, filamentous Figure 15.6 Grocott’s methenamine-silver stains a wide variety of
fungus) is well demonstrated by this PAS stain with light green infectious agents. Here seen with light green counterstain is the
counterstain. method of choice for Histoplasma capsulatum, a dimorphic
endemic fungus.

an H&E stain. The PAS and Grocott procedures

demonstrate these cells well. Infection is found in
the lungs and in the brain within the parenchyma or
in the leptomeninges. Often, these patients are
immunosuppressed.
Histoplasma capsulatum is another soil-dwelling
yeast that can cause a systemic infection in humans
called histoplasmosis. It is especially common along
the southern border of the United States, and where
there are large bird populations. The organism is
usually seen within the cytoplasm of macrophages
that appear stuffed with small, regular, 2–5 µm
Figure 15.5 Immunohistochemistry is being increasingly applied yeast-form cells that have a thin halo around them
to the demonstration of microorganisms using labeled specific
in H&E and Giemsa stains. Langhans’ giant cells
antibody. This figure demonstrates Zygomycetes, a fast-growing
forming non-caseating granulomas may be present.
fungus, with fast red chromogen.
PAS and Grocott stains demonstrate this fungus well
(Fig. 15.6).
Cryptococcus neoformans exists solely in yeast-form Pneumocystis jiroveci. There is still some debate
cells, is variable in diameter (2–20 µm) with ovoid, over the taxonomy of this organism, although recent
elliptical, and crescentic forms frequently seen. analysis of its ribosomal RNA has placed it nearer
There is an extensive mucopolysaccharide coat to a fungal than a protozoan classification (Edman
around the yeasts that is mostly dissolved during et al. 1988). It came to prominence as a pathogen
processing, but, when present, appears as a halo following immunosuppressive therapies associated
around the organism and is visible with special with renal transplants in the 1960s, and has become
stains such as Mayer’s or Southgate’s mucicarmine a life-threatening complication of AIDS. It most fre-
procedures. Yeasts may be free form or within quently causes pneumonia, where the lung alveoli
the cytoplasm of giant cells, staining faintly with are progressively filled with amphophilic, foamy
 The detection and identification of viruses 307

plugs of parasites and cellular debris. It is found

Macchiavello’s stain for rickettsia and viral inclusions,
rarely in other sites such as intestines and lymph modified (Culling 1974)
nodes. The cysts are invisible in an H&E stain, and
Sections
can barely be seen in a Papanicolaou stain, as they
Formalin fixed, paraffin.
appear refractile when the microscope condenser is
racked down. Specific immunohistology is available Method
to use; otherwise, Grocott methenamine-silver is 1. Deparaffinize and rehydrate through graded
alcohols to distilled water.
recommended.
2. Stain in 0.25% basic fuchsin, 30 minutes.
Only electron microscopy or an H&E stain on a
3. Differentiate in 0.5% citric acid, 3 seconds.
resin-embedded thin section will show their internal
4. Wash in tap water, 2 minutes.
structure. The cysts are 4–6 µm in diameter and
5. Counterstain in 1% methylene blue, 15–30
contain 5–8 dot-like intracystic bodies. The cysts
seconds.
rupture and collapse, liberating the trophozoites
6. Rinse in tap water.
which can be seen as small hematoxyphilic dots in
7. Dehydrate, clear, and mount.
a good H&E and Giemsa stain; these attach to the
alveolar epithelium by surface philopodia. Results
Rickettsia and some viral inclusions red
Background blue

The demonstration of rickettsia

The detection and identification
Rickettsial organisms, such as those causing Q fever, of viruses
Rocky Mountain spotted fever, or typhus, rarely
need to be demonstrated in tissue sections. They can While the cytopathic effects of viruses can often be
sometimes be seen with a Giemsa stain, or by using seen in a good H&E stain, and may be characteristic
the Macchiavello technique which also demon- of a single viral group, the individual viral particles
strates some viral inclusion bodies (Fig. 15.7). are too small to be seen with the light microscope,
thus requiring the electron microscope to reveal
their structure. This may allow a rapid and accurate
diagnosis in viral infections; an outline of the value
of electron microscopy in the diagnosis of viral
lesions is given in Chapter 22. Some viruses aggre-
gate within cells to produce viral inclusion bodies,
which may be intranuclear, intracytoplasmic, or
both. These inclusion bodies may be acidophilic and
usually intranuclear, or can be basophilic and cyto-
plasmic. Most special staining methods are modified
trichromes, using contrasting acid and basic dyes to
exploit these differences in charges on the inclusion
body and the host cell. These methods include
Mann’s methyl blue-eosin stain for the Negri bodies
of rabies, Macchiavello’s method, and more recently
the elegant Lendrum’s phloxine-tartrazine stain.
Figure 15.7 Immunohistochemical method demonstrating Rocky Unfortunately, the need for optical differentiation
Mountain spotted fever in kidney. It is caused by the bacterium in these methods increases the chance of technical
Rickettsia rickettsii, which is carried by ticks. error.
 308 15 Microorganisms

The introduction of commercially available mono-

Results
clonal immunohistology to viruses, which are either
Viral inclusions bright red
class or species specific, has revolutionized the Red blood cells variably orange-red
tissue detection of viruses. Hepatitis B virus is a Nuclei blue-gray
good example of the diagnostic value of this tech- Background yellow
nique where the surface antigen (also known as HBs
Notes
or Australia antigen) and the core antigen (HBc)
All tissue is stained red with phloxine, which is then
can be specifically detected immunohistochemically, differentiated by displacement with the counterstain
providing clinically important information about tartrazine. The red color is first removed from muscle,
the stage of this disease. More recently, nucleic acid then other connective tissues. Paneth cells, Russell
hybridization probes have become available and can bodies, and keratin can be almost as dye retentive as
be used to detect genomically inserted viral nucleic viral inclusions, and can occasionally be a source of
confusion.
acid in situ, in cells and tissues that are frozen or
formalin fixed. It should be remembered, however,
that the detection of microorganisms using nucleic
acid probes, unlike specific biotinylated antiserum,
does not necessarily mean active disease. Shikata’s orcein method for hepatitis B surface antigen
(modified Shikata et al. 1974)
Sections
Formalin fixed, paraffin.
Phloxine-tartrazine technique for viral inclusions Solutions
(Lendrum 1947)
Acid permanganate
Sections 0.25% potassium permanganate 95 ml
Formalin fixed, paraffin. 3% aqueous sulfuric acid 5 ml
Solutions Orcein
Phloxine Orcein (synthetic) 1g
Phloxine 0.5 g 70% alcohol 100 ml
Calcium chloride 0.5 g Concentrated hydrochloric acid (gives 1 ml
Distilled water 100 ml a pH of 1–2)
Tartrazine Saturated tartrazine in cellosolve (2-ethoxyethanol).
A saturated solution of tartrazine in 2-ethoxyethanol, Method
or cellosolve. 1. Deparaffinize and rehydrate through graded
alcohols to distilled water.
Method 2. Treat with acid permanganate solution, 5 minutes.
1. Deparaffinize and rehydrate through graded 3. Bleach until colorless with 1.5% aqueous oxalic
alcohols to distilled water. acid, 30 seconds.
2. Stain nuclei in alum hematoxylin (Carazzi’s or 4. Wash in distilled water, 5 minutes, then in 70%
Harris’s), 10 minutes. alcohol.
3. Wash in running tap water, 5 minutes. 5. Stain in orcein solution at room temperature, 4
4. Stain in phloxine solution, 20 minutes. hours, or in a Coplin jar of 37°C preheated orcein,
5. Rinse in tap water and blot dry. 90 minutes.
6. Controlling with the microscope, stain in tartrazine 6. Rinse in distilled alcohol and examine
until only the viral inclusions remain strongly red, microscopically to determine desired staining
5–10 minutes on average. intensity.
7. Rinse in 95% alcohol. 7. Rinse in cellosolve, stain in tartrazine, 2 minutes.
8. Dehydrate, clear, and mount. 8. Rinse in cellosolve, clear, and mount.
 Viral infections 309

the target organ and damage varies with the viral

Results
strain, ranging from massive acute necrosis to
Hepatitis B infected cells, elastic and brown-black
some mucins chronic ‘piecemeal necrosis’ of liver cells, leading to
Background yellow cirrhosis. An eosinophilic ‘ground glass’ appearance
is seen in the cytoplasm of some hepatocytes, due to
Notes
dilated smooth endoplasmic reticulum that contains
The success of this method largely depends on the
tubular HB surface antigen. It is this component that
particular batch of orcein used, and on freshly
prepared solutions. This method relies on can be demonstrated using Shikata’s orcein method,
permanganate oxidizing of sulfur-containing proteins or by specific immunohistochemistry.
to sulfonate residues that react with orcein. Results Herpes viruses are usually acquired subclinically
compare well with those obtained using labeled during early life and enter a latent phase, to be reac-
antibodies, but the selectivity is inferior. tivated during times of immunological stress. These
viruses cause blistering or ulceration of the skin and
mucous membranes, but can cause systemic dis-
Viral infections eases, including encephalitis, in immu­nosuppressed
or malnourished individuals. The cytopathic effects
Whilst not exhaustive, this brief summary reflects of the herpes virus are well seen in Tzanck smears
some viruses that are encountered in surgical and of blister fluid, and include the margination of chro-
post-mortem histopathology (Table 15.3). matin along nuclear membranes, Cowdry type A
Viral hepatitis. To date, five hepatitis viruses have (‘owl’s eye’) inclusion bodies, and syncytial or
been reported, hepatitis viruses (HV) A, B, C, D, and ‘grape-like’ nuclei within giant cells. Cytomegalovirus
E, that show great biological diversity, and three of (CMV) is sometimes seen as a systemic opportunis-
which are incompletely characterized. The liver is tic infection in AIDS patients. It is seen in the

Table 15.3 Viral infections seen in histopathology

Virus Family Genome Disease
Measles Paramyxo SS RNA Measles
Varicella-zoster Herpes DS DNA Chickenpox, shingles
Herpes simplex Herpes DS DNA Cold sores, genital herpes
Cytomegalovirus (CMV) Herpes DS DNA Cytomegalic inclusion disease
Epstein-Barr virus Herpes DS DNA Glandular fever, African
Burkitt’s lymphoma
Human T-cell leukemia virus (HTLV-1) Retro SS RNA Adult T-cell leukemia
Human immunodeficiency virus (HIV) Retro SS RNA AIDS
Human papilloma virus (HPV) Papova DS DNA Human wart viruses
JC virus Papova DS DNA Progressive multifocal
leukoencephalopathy
Poliovirus Picorna SS DNA Poliomyelitis
Molluscum virus Pox DS DNA Molluscum contagiosum
Lyssavirus Rhabdo SS RNA Rabies
DS = double-stranded; SS = single-stranded.
 310 15 Microorganisms

endothelial cells, forming prominent intranuclear from AIDS patients. It produces a distinctive neuro-
inclusions that spill into the cytoplasm where they pathological lesion in AIDS encephalitis consisting
form granular hematoxyphilic clusters. The CMV of microglial nodules, or stars, containing collections
virus causes obvious cytomegaly in the cells it of giant cells, microglia, and astrocytes. Synthetic
infects. All herpes viruses have an identical electron nucleic acid probes have been prepared to HIV
microscopic appearance of spherical, 120 nm, genomes.
membrane-coated particles. Influenza virus (flu) is a contagious respiratory
Papilloma viruses are a family of about 50 wart illness caused by influenza viruses (Fig. 15.8). It can
viruses that cause raised verrucous or papilloma- cause mild to severe illness, and at times can lead to
tous skin warts, or flat condylomatous genital warts. death. According to the Centers for Disease Control
Cytologically, evidence of hyperkeratosis may be (CDC), every year in the United States, on average,
present together with koilocytosis (irregular nuclear 5–20% of the population suffers from the flu, more
enlargement and cytoplasmic vacuolation forming than 200,000 people are hospitalized from flu com-
perinuclear halos). Skin verrucas are associated with plications and about 36,000 people die from flu.
HPV 1–4 strains, genital condylomas with HPV 6, More recently, concern about the influenza A H5N1
11, 16, and 18, and cervical cancer with HPV 16 and strain of bird flu has emerged. Some people, such
18. These uncoated viruses measure 55 nm, are as older people, young children, and people with
mainly intranuclear, and can be detected using elec- certain health conditions, are at high risk for serious
tron microscopy, or immunoperoxidase and gene flu complications.
probes on paraffin sections. SARS (severe acute respiratory syndrome) is a
JC virus is a papovavirus that causes progressive viral respiratory illness caused by a coronavirus
multifocal leukoencephalopathy, a demyelinating called SARS-associated coronavirus (SARS-CoV)
disease, in AIDS and other immunosuppressed (Fig. 15.9). SARS was first reported in Asia in
patients. Intranuclear hematoxyphilic inclusions February 2003. Over the next few months, the
may be seen within swollen oligodendrocytes. illness spread to more than two dozen countries
Molluscum virus produces a contagious wart in in North America, South America, Europe, and
children and young adults called molluscum conta- Asia before the SARS global outbreak of 2003
giosum. Large eosinophilic, intracytoplasmic inclu- was contained.
sion bodies can be seen in maturing keratinocytes
on routine H&E sections, and are seen well with
phloxine-tartrazine. The large 1 µm viral particles
have a typical pox virus structure: brick-shaped
with a superimposed figure-of-eight nucleic acid
sequence.
Rabies virus. This neurotrophic rhabdovirus forms
intracytoplasmic eosinophilic inclusions best seen
in the axonal hillocks of hippocampal neurons of
the brain. Macchiavello, phloxine-tartrazine,
Mann’s methyl blue-eosin, or PAS stains are recom-
mended. Note: given the pathogenicity of these
agents, if suspected, the case should be passed to a
relevant diagnostic center rather than a routine
laboratory.
Human immunodeficiency virus (HIV) consists of at
least two retrovirus strains. The virus is best seen in Figure 15.8 Immunohistochemical method demonstrating Flu A
cultured lymphocytes and is rarely seen in tissues virus infected cells in the bronchus.
 The demonstration of protozoa and other organisms 311

Association of Neuropathologists. Visit their website

(http://www.cjdsurveillance.com) for details on
how to submit specimens for testing; they perform
these tests at no charge for laboratories in the USA.
In addition, both CDC and the World Health Orga-
nization (WHO) also offer guidelines regarding the
handling of suspected and known cases of prion
disease. Visit http://www.cdc.gov and search for
CJD for a fact sheet and other relevant information.
WHO offers a manual in pdf form for downloading.
It gives information about what to do should you
find yourself with a suspected or known positive
case in your lab: http://who.int/bloodproducts/
Figure 15.9 Immunohistochemical method demonstrating the TSE-manual2003.pdf. Remember that these types
previously unrecognized SARS-associated coronavirus which is of cases should never knowingly be handled in
responsible for severe acute respiratory syndrome (SARS). a routine histology lab. Contact your local health
department for additional guidelines.

Prion disease The demonstration of protozoa and

other organisms
To date, more than eight transmissible neurodegen-
erative diseases have been described affecting the The identification of protozoa is most often made on
central nervous system (CNS). The diseases caused morphological appearance using H&E and, particu-
by prions include Creutzfeldt-Jakob disease (CJD) larly, Giemsa stains. The availability of antisera
and variant CJD (vCJD), Germann-Straussler-Shien- against organisms such as entamoeba, toxoplasma,
ker disease, fatal familial insomnia, and kuru in and leishmania has made diagnosis much easier in
humans, bovine spongiform encephalopathy (BSE, difficult cases (Fig. 15.10a and b).
also known as ‘mad cow disease’), scrapie (in goats
and sheep), and chronic wasting disease (CWD) (in Giemsa stain for parasites
mule deer and elk). Prions are not microbes in the Sections
usual sense because they are not alive, but the illness Fixative is not critical, but B5 or Zenker’s is preferred;
they cause can be transmitted from one animal to thin (3 µm) paraffin sections. (If Zenker’s is not used,
another. All usually produce a characteristic spongi- post-mordant in Zenker’s in a 60°C oven for 1 hour
form change, neuronal death, and astrocytosis in before staining.)
affected brains. The infectious agent is a prion, a Solutions
small peptide, free of nucleic acid and part of a Giemsa stock (commercially available) or
normal transmembrane glycoprotein which is not, Giemsa stain powder 4g
strictly speaking, a virus. Antibodies have been pre- Glycerol 250 ml
pared from prion protein that strongly mark accu- Methanol 250 ml
mulated abnormal protein in these diseases (Lantos Dissolve powder in glycerol at 60°C with regular
shaking. Add methanol, shake the mixture, and allow
1992).
to stand for 7 days. Filter before use.
The CJD Surveillance Center in the USA is an
invaluable source for monitoring and testing human Working Giemsa for parasites
prion disease in the United States. The Center is Giemsa stock 4 ml
Acetate buffered distilled water, pH 6.8 96 ml
supported by the CDC and by the American
 312 15 Microorganisms

Method Protozoa
1. Deparaffinize and rehydrate through graded
alcohols to water. Entamoeba histolytica, the organism causing amebic
2. Rinse in pH 6.8 buffered distilled water. colitis or dysentery, can be found in ulcers that occur
3. Stain in working Giemsa, overnight. in infected colon and in amebic liver abscesses. The
4. Rinse in distilled water. trophozoite (adult form) measures 15–50 µm, con-
5. Rinse in 0.5% aqueous acetic acid until section is tains a small nucleus, and has a foamy cytoplasm
pink. containing ingested red cells and white cell debris.
6. Wash in tap water. They may be seen in granulation tissue within ulcers
7. Blot until almost dry. on routine H&E staining, or in the luminal mucus
8. Dehydrate rapidly through alcohols, clear, and overlying normal-appearing mucosa. They are PAS
mount. positive; brief counterstaining in 1% aqueous metanil
Results yellow emphasizes the ingested red cells.
Protozoa and some other dark blue Toxoplasma gondii, a commonly encountered
microorganisms or­ganism that is spread in cat litter, causes an
Background pink-pale blue acute lymphadenopathy which is often subclinical.
Nuclei blue Affected nodes show non-specific changes and no
organisms. In AIDS and other immunosuppressed
patients this protozoon causes systemic diseases,
including meningoencephalitis where encysted
bradyzoites and free tachyzoites can be seen in
necrotic brain tissue. Cysts also occur in other tissues
such as cardiac muscle, and measure up to 40 µm
with tachyzoites 4–6 µm, which can be seen on H&E.
A Giemsa stain can also be used, but the use of
labeled specific antiserum is recommended.
Leishmania tropica is transmitted by sandfly bite
and causes a chronic inflammatory disease of
the skin sometimes called cutaneous leishmaniasis.
The injected parasite forms (2 µm), or amastigotes,
a are found in large numbers within the cytoplasm of
multiple swollen histiocytes that congregate in early
lesions in the dermis. A related organism, L. don-
ovani, causes a systemic visceral infection, kala azar,
in which the organisms are seen within histiocytes
in spleen, lymph nodes, liver, and bone marrow. The
organisms are hematoxyphilic and can be empha-
sized with a Giemsa stain.
Giardia duodenalis (lamblia) is a flagellate protozoon
that is ingested in cyst form from drinking water
with fecal contamination; the trophozoites migrate
to the duodenum where they may cause severe diar-
b rhea and malabsorption. These organisms can been
Figure 15.10 (a) H&E and (b) immunohistochemical methods seen on an H&E stain, where they appear as eosino-
demonstrating the single-celled parasite Toxoplasma gondii in philic, sickle-shaped flakes with indistinct nuclei
heart. resting on intestinal mucosa that may show little
 Worms 313

evidence of inflammation. When seen in a fresh Acknowledgments

Giemsa-stained duodenal aspirate, they appear kite-
shaped, 11–18 µm in size, binucleate, and have faint The author would like to acknowledge Amy M.
terminal flagella. Denison for assistance with the PCR section in this
Trichomonas vaginalis is a similar flagellate chapter.
protozoon most frequently seen in a Papanicolaou
stain. Inflammatory cells and mildly dysplastic
squamous cells often accompany this parasite as it References
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 Bone
Diane L. Sterchi
16
cavities of long bones, vertebrae, and centers of flat
Introduction bones. It is a mesh of bone strands each about 1 mm
thick. Although it looks less solid than cortical bone,
Bone is considered the most important supporting this arrangement of trabeculae, particularly in the
tissue in the body. It is composed of cells, organic femoral head and vertebrae where it forms an almost
extracellular matrix and inorganic salts. Bone tissue ideal weight-bearing structure, is very strong.
is mineralized in layers which provide great The major components of bone are mineral, cells
strength and flexibility to the skeletal system. It and an organic extracellular matrix of collagen fibers
varies in formation, depending on its function, and ground substance. These are dynamic compo-
across the body. These functional/formation differ- nents as the processes of cell replacement, repair and
ences are also based on the proportion of the differ- remodeling of bone, and the erosion and reforma-
ent inorganic and organic processes incorporated tion of collagen and mineral occur continually
or produced in the formation of a bone. The most throughout adult life.
common mineral in bone is hydroxyapatite which
consists of collagen, proteins and carbonate ions.
The main bulk of bone is approximately 70% Bone collagen
mineral and 30% organic components by weight.
Bone cells, as opposed to marrow cells, are rela- The collagen found in bone differs from other col-
tively sparse. This chapter will review bone mor- lagen in the body in that it becomes mineralized and
phology and its organic and inorganic components, is laid down in bands or lamellae roughly parallel
as well as considering methods on preparing sec- to one another. The collagen fibers within each
tions of bone for analysis that can be used in clini- lamella tend to lie next each other but at an angle to
cal and research histology laboratories. the fibers in adjacent lamellae. A cement of proteo-
glycan ground substance outlining these fibers is
seen in sections only at the cement lines. The orga-
nization of collagen lamellae is responsible for the
Normal bone distinctive micro-anatomical patterns of bone which
are easily seen with polarized light microscopy
Two types of bone can be recognized macroscopi- (Fig. 16.1).
cally in the adult human skeleton. They are cortical The simplest pattern occurs on the periosteal and
or compact bone and trabecular, cancellous, or endosteal surfaces of compact bone as circumferen-
spongy bone. Compact bone is the solid, hard, and tial lamellae, and in trabecular or non-Haversian
immensely strong bone that forms the shafts of long bone where lamellae are roughly parallel to the
bones (e.g. femur and tibia) and exterior surfaces of surface. Cortical bone is composed of Haversian
the flat bones (e.g. ribs and skull). Trabecular bone systems or osteons in which concentric lamellae sur-
is found in the diaphysis, epiphysis, and marrow round channels (Volkmann’s canals) containing one
© 2013 Elsevier Ltd
 318 16 Bone

Unmineralized collagen or osteoid forms a border,

or seam, on surfaces of newly formed bone, and
after osteoid is deposited there is a lag before it
becomes mineralized. Osteoid is normally around
15 µm thick, covering only a small proportion of the
surfaces. In some diseases, such as rickets or osteo-
malacia, it is much thicker and widespread.
On inactive surfaces the osteoid is very thin,
difficult to see, and is completely absent where
resorption is taking place. This process is called
remodeling and consists of resorption and deposi-
tion taking place in equilibrium so that the volume
and shape of bones stays more or less constant. In
later life, remodeling slows down, and deposition
Figure 16.1 Lamellar and non-lamellar (woven) bone of normal
may not keep up with resorption, causing increased
rib from child aged 2 12 years. Celloidin section; polarized light.
bone porosity and brittleness, and, in extreme cases,
the disease osteoporosis.

Bone mineral
The main mineral content of bone is calcium and
phosphate combined with hydroxyl ions to form
hydroxyapatite crystals. The mineral is approxi-
mately 38% calcium and thought to be deposited as
amorphous calcium phosphate in the initial miner-
alization phase. This transforms to hydroxyapatite
by addition of hydroxyl ions to form a crystal lattice
into which carbonate, citrate, and fluoride ions as
Figure 16.2 Haversian systems (osteons, Volkmann’s canals, and well as magnesium, potassium, and strontium can
bone cells) in ground section of undecalcified bone in methyl
be substituted or included. Carbonate is present in
methacrylate, acid etched/surfaced stained with McNeal’s/toluidine
blue stain.
large quantities but probably only in the hydration
shell and on crystal surfaces.
The hydroxyapatite forms needle-like crystals
or more blood vessels. These tubular structures run
about 22 nm in length, resulting in an enormous
longitudinally in the bone and are packed closely
total crystal surface area. Thus, the mineral fulfills
together with irregular interstices filled by the rem-
the obvious function of giving strength and rigidity
nants of older osteons (Fig. 16.2). Cement lines
while approximately 20% remains in the amorphous
outline the boundaries of osteons, and some tra-
form to provide a readily available buffer for main-
becular and circumferential lamellae.
taining total body chemical equilibrium (e.g. pH and
Another collagen fiber arrangement forms non-
enzyme system regulation).
lamellar or woven bone and is found in immature
bone and some pathological conditions. This colla-
gen is not deposited in the lamellae but in thick, Bone cells
short, randomly oriented bundles. When viewed
with polarized light, these appear as coarse fibers There are three types of bone cell other than the
resembling a woven fabric. marrow cells belonging to the hemopoietic system.
 Normal bone 319

Osteoblasts
Osteoblasts are fully differentiated to carry out the
primary function of bone formation by producing
and laying down osteoid. They are seen on surfaces
of actively forming bone as plump cells with
basophilic cytoplasm and eccentric nuclei distal to
the bone surface. The cytoplasm is basophilic due
to ribonucleic acid, and before becoming fully dif-
ferentiated frequently contains glycogen. Acid phos-
phatase is found in osteoblasts and the surrounding
tissues, but decreases at onset of calcification. Upon
completion of bone formation, most osteoblasts
revert to a quiescent (small undifferentiated cell)
state and reside among the heterogeneous cell
population.

Osteocytes
In one respect osteoblasts do represent immature
cells, as some are trapped in the osteoid matrix they
lay down, and become mature osteocytes residing in
tiny spaces or lacunae within the bone. Lacunae are
connected to each other and to the vascular spaces
by canaliculi – tiny channels into which osteocyte
processes project for the purpose of passing fluids
and dissolved substances necessary for cell metabo- Figure 16.3 Schmorl’s picro-thionin stain showing bone
lism (Fig. 16.3). canaliculi. Celloidin section of normal femoral shaft.

Osteoclasts
These are the cells responsible for bone resorption
or erosion. Osteoclasts are large multinucleated
(giant) cells whose cytoplasm contains numerous
mitochondria and alkaline phosphatase. They occur
in small clusters or singly on bone surfaces undergo-
ing resorption and are often seen in the depressions
(Howship’s lacunae) they are actively creating by
erosion. These surfaces have an irregular outline and
lack osteoid (Fig. 16.4). The direction of resorption
is random, with no relationship to lamellar struc-
ture. Osteoclasts respond to altered mechanical
stresses on the skeleton and to growth, and their
activity contributes to remodeling. They also
respond to hormones that can either stimulate or
inhibit their activity. When resorption halts, the Figure 16.4 Osteoclasts lining trabecular bone. Undecalcified rat
process of bone formation resumes (osteoid, knee in GMA microtomed at 2 µm. Hematoxylin and eosin stain.
 320 16 Bone

mineralization, etc.). Cement lines will occur at the new bony shaft by cartilage growth or epiphyseal
junction between old and newly formed bone. plates capable of interstitial growth. Once bone has
ossified, it can only grow by apposition. Remodeling
is continuous. In some places deposition exceeds
Development and growth
resorption, whereas in others this is reversed and
results in the characteristic shape of the bone. When
Bone develops in two different ways according to
bone is fully grown, the three ossification sites unite
the site and shape of the bone. It begins early in the
and the cartilage growth plates disappear.
embryo and is not complete until approximately 15
years of age.
Intramembranous ossification Techniques for analyzing bone
This occurs in flat bones, e.g. skull, sternum, pelvic
Techniques for the demonstration of bone and its
bones. A fibrous membrane first develops at the
components are possibly more varied and difficult
bone formation site in which mesenchymal cells
than for any other tissue. They include:
differentiate into osteoblasts that begin the bone
formation process by laying down osteoid. This • Decalcified bone for frozen, paraffin, and
starts in small islets that gradually unite to become transmission electron microscopy (TEM).
trabeculated and finally form an external layer of • Mineralized bone for frozen, plastic
compact bone. (microtomed or sawn/ground sections),
transmission or scanning EM samples.
Endochondral ossification The technique chosen for examination of bone is
influenced by the initial clinical diagnosis, case
This occurs in long bones and major parts of the
urgency, and the extent of investigation required.
skeleton. This type of bone development begins
Specimens arriving in the laboratory can vary in
with differentiation of mesenchymal cells at sites
size from a needle biopsy a few millimeters long
where bone will be formed, but this bone is laid
to whole appendages, e.g. amputation. Mineralized
down in a cartilage model that resembles the final
sections are used for microradiographic and
shape of the bone. This cartilage model becomes
histomorphometric studies as well as polarized and
covered with a connective tissue sheath or perichon-
fluorescent light microscopy.
drium and grows by both apposition and intersti-
tially. Appositional growth or the laying down of
more cartilage begins towards the exterior and is Biopsies
mainly responsible for increased diameter; intersti-
tial growth is by cells dividing within the model and Biopsies are used for diagnosis of several diseases
mainly occurs towards the extremities, resulting in such as cancers, hemopoietic disorders, and infec-
increased length. tions. These specimens are usually small enough to
In the central part of the model, cells continue to treat much like soft tissue, except that a bone biopsy
differentiate, cartilage begins to calcify, blood vessels usually needs decalcification. This is particularly
invade, and the cartilage is broken up into strands. neccesary if it contains a piece of the cortical bone in
Ossification, by differentiation of perichondrial cells order to produce paraffin sections. A bone marrow
into osteoblasts, begins around the exterior of the biopsy is usually removed with a Jamshidi needle
primary ossification site at the center of the model. for diagnosis of metabolic bone disease. Metabolic
Osteoblasts invade the strands of calcified cartilage bone diseases are diagnosed using trabecular bone
and deposit osteoid that soon becomes calcified. This (Byers & Smith 1967) taken from the iliac crest which
process continues and secondary ossification sites is an accessible bone site representative of skeletal
appear at each end of the model, separated from the bone as a whole.
 Techniques for analyzing bone 321

Sections that are requisite to assess the relation- the trimming and prevents autolysis of the outer
ship between mineralized and unmineralized bone layers of the specimen. The mortuary/morgue area
(osteoid) are best processed and embedded into a has a dual advantage for both limb storage and sub-
plastic, such as methyl methacrylate (MMA), glycol- sequent sample preparation on an available autopsy
methacrylate or a epon-like plastic. The preferred table. Whenever possible, specimen radiography
method used for metabolic bone disease research of large bone specimens helps select the lesion/
and diagnosis is MMA plastic. Plastic is not a diseased area for trimming to a smaller sample size
common embedding medium in clinical laboratories for processing.
but is useful in research and clinical laboratories that
are connected with research. It is also possible to
produce frozen sections from an undecalcified bone
Resection/replacement specimens
biopsy. There are silver stains that demonstrate bone
Benign or low-grade malignant tumors and arthritic
and osteoid in a decalcified, paraffin-embedded
femoral heads resemble large biopsy specimens, fre-
bone section (Tripp & McKay 1972) but many
quently have an established diagnosis and are often
researchers choose the MMA-embedded section.
considered less urgent. In femoral head or knee
It is not practical to bisect (half for paraffin/
replacement surgeries, the bone specimen removed
half for plastics) an iliac crest trephine biopsy if
from the patient is usually received in the laboratory
both paraffin and plastic embedding methods are
whole. Either prior to or after some fixation, a wedge-
employed in the laboratory. Metabolic bone disease
shaped sample is cut from the whole specimen using
laboratories usually prefer a whole trephine bone
a Stryker bone saw or a heavy-duty X-ACTO knife.
core for plastic embedding. Needle biopsies should
This wedge-shaped sample is placed back into fixa-
remain whole for paraffin or plastic methods.
tive for 24–48 hours and then decalcified, processed
and sectioned for pathology evaluation.
Amputation specimens
Large amputation specimens are usually taken Fixation
as a result of tumor, chronic osteomyelitis, and gan-
grene. These specimens are often delivered to the Unless immediate diagnosis is needed using cryo-
laboratory immediately after removal. They often microtomy, all bone specimens must be totally fixed
are not in any sealed container and without fixative, before subjecting them to any decalcification and
and must be dealt with as soon as possible (either processing procedures. Complete fixation helps
in the mortuary or laboratory). The majority of the protect bone and surrounding soft tissue from the
limb is usually discarded or saved/fixed (if requested damaging effects of acid decalcification. Ten percent
by patient) and the area or lesion with actual or neutral buffered formalin (NBF) is suitable for both
suspected involvement in the disease process is paraffin and non-tetracycline labeled bone. It should
retained for final evaluation. Skin, excess muscle, be noted that fixation proceeds faster by reducing
and connective tissue should be cut away from the the size of the bone, opening the bone, and removing
lesion if possible. Excess bone or a joint disarticu- excess skin and soft tissue surrounding the lesion.
lated above and below the lesion should be per- Large specimens can be bisected or reduced in size
formed so that fixation is adequate. The relevant by sawing into multiple slabs, and immersed into
portions should be immersed into a large volume of fixative immediately, or no longer than 48 hours
fixative to insure complete fixation. If it is not pos- after initial fixation. Once cut into smaller pieces, the
sible to inspect the specimen for several hours after samples should be placed into fresh fixative.
receipt, it should be refrigerated at 4°C or it can be For MMA embedding, 10% NBF is generally used
placed in fixative as a whole and kept at 4°C. Placing for fixation. Alcoholic formalin or 70% ethanol fixa-
it in fixative prior to trimming helps in managing tion is the fixative of choice for tetracycline-labeled
 322 16 Bone

bone. Alcohol-based fixatives are not recommended sample will drag through the blade. The first cut is
for bone destined for acid decalcification as alcohol made through the mid-plane, then approximately
can slow or prevent decalcification. Fixatives con- 3–5 mm thick slabs are cut parallel to the first cut. A
taining chloroform (Carnoy’s) and mercury (B5, saw guide plate or wood block held against the first
Zenker’s, Susa), including substitutes of them, cut edge ensures an even slice. It is safer for workers
should be avoided for specimens to be radiographed to hold thinner bones between two wood blocks that
since those fixatives tend to make bone radio-opaque will not ruin blades. Sawing should be at a slow
and unsuitable for specimen interpretation. even rate to match speed of blade cutting into the
bone. Pushing the bone produces uneven cuts, and
may jam or break a blade.
Sawing Bone slabs should be fixed for an additional 24–48
hours, especially if they appear pinkish-red or par-
Good saws are an essential piece of equipment in a tially fixed. After sawing, any bone dust or debris
bone histology laboratory. Other than surgical saws, adhering to slab surfaces can be cleaned away using
there are a range of hobby shop or handyman’s a slow stream of water and a wet paper towel to
bench saws that are designed to cut through stones, brush off the debris. Care must be taken not to push
plastic, and some thin metals. These saws cut debris into marrow spaces or to wash slabs exces-
through cortical bone slowly with cuts no deeper sively before the bone is totally fixed.
than 7.5 cm. Dry saws may need slight modifica-
tions to prevent blade slippage when cutting wet,
fatty bone. Water-cooled saws prevent heat damage Fine-detail specimen radiography
to bone due to high-speed sawing, and are capable
of full-length cuts through long bones and append- Radiographs of bone slabs, blocks, or fragments are
ages, e.g. femur and tibia. Buehler Isomet Low useful for four main purposes:
Speed Saws (Buehler Ltd, USA) are used for trim-
1. To examine the nature and extent of a lesion.
ming specimens, plus cutting bones embedded in
2. To provide a diagram of a lesion prior to block
plastics. This type of saw has a thin diamond-
selection for processing.
impregnated blade with a water-cooling bath and is
3. To check progress of decalcification, i.e.
ideal for bisecting biopsies (only when required)
decalcification endpoint test.
and larger bones (depending on the bone specimen
diameter). It can make precise, debris-free cuts 4. To confirm the presence of foreign materials,
through 8 mm thick bone cores, other cortical or e.g. prosthetic devices, metal or glass fragments
trabecular and MMA-embedded bone specimens. implanted by trauma.
Scalpel blades, fretted wire or jewelers’ saws have Thin bone slices give sharper-image radiographs
been used to cut biopsies with damaging results. than a whole specimen or clinical radiographs.
These cutting devices can crush or fracture fine tra- When using film and not digital images, the use of
beculae, creating ‘fracture artifact’, and force bone ‘soft’ X-rays (of low kV) and high-contrast X-ray film
fragments into marrow spaces, spoiling the bone provides finer detail and clarity (Fornasier 1975).
histology. Many non-specialist departments of pathology
Suitable blade specifications on small saws are use their in-house radiology department. However,
0.5 cm width and 12 to 16 teeth per inch (tpi), making there is the ‘Faxitron’ (Faxitron Inc., USA) cabinet
finer, cleaner cuts than a larger saw blade 1.25 cm X-ray system which can be used for specimen X-rays
wide with 6 tpi. Blades in specifications needed are of bone in clinical and research settings. It comes in
available from tool companies. a free-standing and a tabletop unit. The energy
Soft tissues and dense connective tissue, e.g. output is 10–110 kV with 3 mA tube current. In addi-
tendons, should be removed before sawing or the tion to the manual exposure capability, the unit
 Techniques for analyzing bone 323

should be equipped with its automatic exposure least mineralization in order to provide the quickest
timer with 5-second to 60-minute setting at 1-second possible diagnosis. These pieces can be fixed, rapidly
intervals. The cabinet has adjustable shelf levels for decalcified, and processed to meet urgent clinical
film-to-source distances of 31–61 mm and is fully requirements.
lead lined to shield the operator from X-rays. A The ideal thickness of larger bone pieces is 3–5 mm.
special door interlock safety device automatically If bone slabs are too thick, both decalcification and
turns off the X-ray beam if the door is opened during processing are prolonged while overly thin bone
operation. This is an instrument that uses X-ray film slabs (less than 2 mm) tend to become brittle and
(Kodak X-OMAT 2, Ready Pak; Kodak Ltd) but bend during processing and embedding which may
newer units produce digital and real-time images. cause the tissue to pop out of the paraffin block
When using a Faxitron for decalcification checks, a during sectioning. The dense collagen matrix tends
bone slab should be first radiographed using the to prevent adequate paraffin wax penetration and
automatic exposure timer, and then exposure time, thin pieces are not held firmly in the softer paraffin
kV, and mA is recorded. This eliminates guesswork embedding media during microtomy.
for a first exposure and provides the correct expo- An addition, an aid in area selection of a specimen
sure time and kV for a repeat radiograph or for is radiography when available, a diagram or ‘map’
subsequent manual exposures of adjacent bone slabs of the lesion can be made from the X-ray to locate
of the same thickness. area of interest for processing.
An example of manual exposure requirements is
as follows:
• bone slab 3–5 mm thick on polyethylene sheet Decalcification
(moisture barrier)
In order to obtain satisfactory paraffin sections of
• Kodak X-OMAT 2, Ready Pak (Kodak Ltd)
bone, inorganic calcium must be removed from the
• film-to-source distance (FTSD), 50 cm (lowest
organic collagen matrix, calcified cartilage, and sur-
shelf level)
rounding tissues. This is called decalcification and is
• settings, 30 kV (3 mA) carried out by chemical agents, either acids to form
• exposure time approximately 1 minute. soluble calcium salts, or chelating agents that bind
Exposure time is dependent on specimen thick- to calcium ions. Even after decalcification, the dense
ness (thicker slabs require longer exposures), film-to- collagen of cortical bone is remarkably tough and
source distance (longer distance requires more time), tends to harden more after paraffin processing.
and type of film used. Large, whole bones, e.g. prox- Occasionally, small foci of calcifications in paraffin-
imal end of femur with metal prosthesis, can be embedded or frozen tissues can be sectioned without
radiographed using X-OMAT 2, FTSD (upper shelf), much noticeable damage to the knife or disruption
longer exposure time (approx. 8 minutes), and of surrounding tissue. After hematoxylin staining,
higher kV (70 kV). Soft tissues, cartilage, and tumor these foci usually appear cracked and as dark purple
are more easily seen in underexposed radiographs, granular masses with lighter purple halos.
useful for evaluating surrounding soft tissue The choice of decalcifier is influenced by four
involvement by a bone tumor, e.g. osteosarcoma. inter-dependent factors: urgency of the case, degree
The X-ray film can be developed quickly in a of mineralization, extent of the investigation, and
radiology department and viewed without delay. staining techniques required.
Any acid, however well buffered, has some damag-
ing effects on tissue stain-aridity. This problem
Area selection for embedding
increases with acidity of solutions, i.e. lower pH, and
In urgent cases of suspected tumor or infection, an length of decalcification period. Consequently, the
attempt should be made to select a sample with the rapid decalcifiers are more likely to adversely affect
 324 16 Bone

any subsequent staining, especially if not fixed com- rapid or slow, and give decalcification instructions
pletely. This is most noticeable in cell nuclei with the and warnings against prolonged use. Rapid propri-
failure of nuclear chromatin to take up hematoxylin etary solutions usually contain hydrochloric acid
and other basic dyes as readily as soft tissues never (HCl), whereas slow proprietary mixtures contain
exposed to acid solutions. The staining using acid buffered formic acid or formalin/formic acid. A
dyes is also less affected, but eosin (an acid dye) can study (Callis & Sterchi 1998) found that dilution of
stain tissue a deep, unpleasant, brick red without the a proprietary HCl solution was not deleterious for
preferred three differential shades. These effects effective decalcification or staining, and this is an
on H&E staining can be reduced by doing the decal- option if a strong mixture is considered too concen-
cification endpoint test, post-decalcification acid trated. Chelating reagents such as EDTA mixtures
removal, and adjustment of the stain procedure. are also available pre-mixed. Although proprietary
mixtures have no obvious advantages over solutions
Decalcifying agents prepared in laboratories, their use is increasingly
popular in busy laboratories because they are reli-
As noted previously there are two major types of able, time and cost-effective while addressing some
decalcifying agent, i.e. acids and chelating agents, safety issues by eliminating handling and storage of
although Gray (1954) lists over 50 different mixtures. concentrated acids.
Many of these mixtures were developed for special
purposes with one used as a fixing and dehydrating Strong inorganic acids, e.g. nitric, hydrochloric
agent. Other mixtures contain reagents, e.g. buffer
salts, chromic acid, formalin, or ethanol, intended to These may be used as simple aqueous solutions with
counteract the undesirable swelling effects that acids recommended concentrations of 5–10%. They decal-
have on tissues. Many popular mixtures used today cify rapidly, cause tissue swelling, and can seriously
are from the original formulae developed many years damage tissue stainability if used longer than
ago (Evans & Krajian 1930; Kristensen 1948; Clayden 24–48 hours. Old nitric acid is particularly damag-
1952). For most practical purposes, today’s laborato- ing, and should be replaced with fresh stock. Strong
ries seem to prefer simpler solutions for routine acids, however, tend to be more damaging to tissue
work. Provided the bone is totally fixed and treated antigens for immunohistochemical staining, and
with a decalcifier suitable for removal of the amount enzymes may be totally lost.
of mineral present, the simple mixtures work as well Strong acids are used for needle and small biopsy
or better than more complex mixtures. specimens to permit rapid diagnosis within 24 hours
or less. They can be used for large or heavily miner-
Acid decalcifiers alized cortical bone specimens with decalcification
progress carefully monitored by a decalcification
Acid decalcifiers can be divided into two groups: endpoint test (Callis & Sterchi 1998). The following
strong (inorganic) and weak (organic) acids. As is a list of strong acid decalcifying solutions. The
Brain (1966) suggested, many laboratories keep an formulae and preparations are available in Bancroft
acid from each group available for either rapid diag- & Gamble, sixth ed. (2008).
nostic or slower, routine work.
1. Aqueous nitric acid, 5–10% (Clayden 1952)
2. Perenyi’s fluid (Perenyi 1882)
Proprietary decalcifiers
3. Formalin-nitric acid (use inside a fume hood)
The components in proprietary decalcifying solu-
tions are often trade secrets. Manufacturers provide
Weak, organic acids, e.g. formic, acetic, picric
Material Safety Data Sheets (MSDS) that frequently
indicate the type and concentration of acid. Their Of these, formic is the only weak acid used exten-
product data sheets usually indicate if a solution is sively as a primary decalcifier. Acetic and picric
 Techniques for analyzing bone 325

acids cause tissue swelling and are not used alone not bind to calcium below pH 3 and is faster at pH
as decalcifiers but are found as components in Car- 7–7.4; even though pH 8 and above gives optimal
noy’s, Bouin’s, and Zenker’s fixatives. These fixa- binding, the higher pH may damage alkali-sensitive
tives will act as incidental, although weak, protein linkages (Callis & Sterchi 1998). EDTA binds
decalcifiers and could be used in urgent cases with to ionized calcium on the outside of the apatite
only minimal calcification. Formic acid solutions crystal and as this layer becomes depleted more
can be aqueous (5–10%), buffered or combined calcium ions reform from within; the crystal becomes
with formalin. The formalin–10% formic acid progressively smaller during decalcification. This is
mixture simultaneously fixes and decalcifies, and is a very slow process that does not damage tissues or
recommended for very small bone pieces or needle their stainability. When time permits, EDTA is an
biopsies. However, it is still advisable to have com- excellent bone decalcifier for enzyme staining, and
plete fixation before any acid decalcifier is used. electron microscopy. Enzymes require specific pH
The salts, sodium formate (Kristensen 1948) or conditions in order to maintain activity, and EDTA
sodium citrate (Evans & Krajian 1930), are added to solutions can be adjusted to a specific pH for enzyme
formic acid solutions making ‘acidic’ buffers. Buff- staining. EDTA does inactivate alkaline phosphatase
ering is used to counteract the injurious effects of but activity can be restored by addition of magne-
the acid. However, in addition to low 4–5% formic sium chloride.
acid concentration, increased time is needed for EDTA and EDTA disodium salt (10%) or EDTA
complete decalcification. Formic acid is gentler and tetrasodium salt (14%) are approaching saturation
slower than HCl or nitric acids, and is suitable for and can be simple aqueous or buffered solutions at
most routine surgical specimens, particularly when a neutral pH of 7–7.4, or added to formalin. EDTA
immunohistochemical staining is needed. Formic tetrasodium solution is alkaline, and the pH should
acid can still damage tissue, antigens, and enzyme be adjusted to 7.4 using concentrated acetic acid. The
staining, and should be endpoint tested. Decalcifi- time required to totally decalcify dense cortical bone
cation is usually complete in 1–10 days, depending may be 6–8 weeks or longer, although small bone
on the size, type of bone, and acid concentration. spicules may be decalcified in less than a week. For-
Dense cortical or large bones have been effectively mulae and preparations are available in Bancroft &
decalcified with 15% aqueous formic acid and Gamble, sixth ed. (2008).
a 4% hydrochloric acid–4% formic acid mixture 1. Formalin-EDTA (Hillemann & Lee 1953)
(Callis & Sterchi 1998). The following is a list of 2. EDTA (aqueous), pH 7.0–7.4
weak acid decalcifying solutions. The formulae and
preparations are available in Bancroft & Gamble,
Factors influencing the rate of decalcification
sixth ed. (2008).
Several factors influence the rate of decalcification,
1. Aqueous formic acid
and there are ways to speed up or slow down this
2. Formic acid-formalin (after Gooding & Stewart process. The concentration and volume of the active
1932) reagent, including the temperature at which the
3. Buffered formic acid (Evans & Krajian 1930) reaction takes place, are important at all times. Other
factors that contribute to how fast bone decalcifies
Chelating agents are the age of the patient, type of bone, size of speci-
men, and solution agitation. Mature cortical bone
The chelating agent generally used for decalcifica- decalcifies slower than immature, developing corti-
tion is ethylenediaminetetraacetic acid (EDTA). cal or trabecular bone. Another factor of mature
Although EDTA is nominally ‘acidic’, it does not act bone is that the marrow may contain more adipose
like inorganic or organic acids but binds metallic cells than a young bone. This requires diligent atten-
ions, notably calcium and magnesium. EDTA will tion to make sure specimens stay immersed in
 326 16 Bone

decalcification solution. Of all the above factors, the may become completely macerated almost as soon
effectiveness of agitation is still open to debate. as they are decalcified.
The optimal temperature for acid decalcification
has not been determined, although Smith (1962)
Concentration of decalcifying agent
suggested 25°C as the standard temperature, but in
Generally, more concentrated acid solutions decal- practice a room temperature (RT) range of 18–30°C
cify bone more rapidly but are more harmful to the is acceptable. Conversely, lower temperature
tissue. This is particularly true of aqueous acid solu- decreases reaction rates and Wallington (1972) sug-
tions, as various additives, e.g. alcohol or buffers, gested that tissues not completely decalcified at the
which protect tissues, may slow down the decalcifi- end of a working week could be left in acid at 4°C
cation rate. Remembering that 1 N and 1 M solu- over a weekend. This practice may result in ‘over-
tions of HCl, nitric, or formic acid are equivalent, decalcification’ of tissues, even with formic acid. A
Brain (1966) found that 4 M formic acid decalcified better recommendation is to interrupt decalcifica-
twice as fast as a 1 M solution without harming tion by briefly rinsing acid off bone, immersing it in
tissue staining, and felt it was advantageous to use NBF, removing from fixative, rinsing off the fixative,
the concentrated formic acid mixture. With combi- and resuming decalcification on the next working
nation fixative-acid decalcifying solutions, the decal- day. Microwave, sonication, and electrolytic methods
cification rate cannot exceed the fixation rate or the produce heat, and must be carefully monitored to
acid will damage or macerate the tissue before fixa- prevent excessive temperatures that damage tissue
tion is complete. Consequently, decalcifying mix- (Callis & Sterchi 1998).
tures should be compromises that balance the Increased temperature also accelerates EDTA
desirable effects (e.g. speed) with the undesirable decalcification without the risk of maceration.
effects (e.g. maceration, impaired staining). However, it may not be acceptable for preservation
In all cases, total depletion of an acid or chelator of heat-sensitive antigens, enzymes, or electron
by their reaction with calcium must be avoided. This microscopy work. Brain (1966) saw no objection to
is accomplished by using a large volume of fluid decalcifying with EDTA at 60°C if the bone was
compared with the volume of tissue (20 : 1 is recom- well fixed.
mended), and by changing the fluid several times
during the decalcification process. Brain, however,
Agitation
pointed out that if a sufficiently large volume of
fluid is used (100 ml per g of tissue) it is not neces- The effect of agitation on decalcification remains
sary to renew the decalcifying agent because deple- controversial even though it is generally accepted
tion is less apparent in a larger volume. Small that mechanical agitation influences fluid exchange
number and similar-sized specimens in one con- within as well as around tissues with other reagents.
tainer is preferred. Therefore, it would be a logical assumption that agi-
Ideally, acid solutions should be endpoint tested tation speeds up decalcification, and studies have
and changed daily to ensure the decalcifying agent is been done which attempt to confirm this theory.
renewed and that tissues are not left in acids too long Russell (1963) used a tissue processor motor rotating
or overexposed to acids (i.e. ‘over-decalcification’). at one revolution per minute and reported the decal-
cification period was reduced from 5 days to 1 day.
Others, including Clayden (1952), Brain (1966), and
Temperature
Drury and Wallington (1980), repeated or performed
Increased temperature accelerates many chemical similar experiments and failed to find any time
reactions, including decalcification, but it also reduction. The sonication method vigorously agi-
increases the damaging effects which acids have on tates both specimen and fluid, and one study noted
tissue so that, at 60°C, the bone, soft tissues, and cells cellular debris found on the floor of a container after
 Techniques for analyzing bone 327

sonication could possibly be important tissue shaken changed often at first, since the reaction with calcium
from the specimen (Callis & Sterchi 1998). Gentle is initially quicker and then slows down as the
fluid agitation is achieved by low-speed rotation, calcium in the tissue is depleted. Minimally calcified
rocking, stirring, or bubbling air into the solution. tissues and needle biopsies decalcified by a strong
Even though findings from various studies are unre- acid may be tested only once. It is wise practice for
solved, agitation is a matter of preference and not a laboratory that performs a high number of bone
harmful as long as tissue components remain intact. biopsy specimens to establish a time range (that
depends on the amount of cortical bone present) for
Suspension ideal decalcification time for bone biopsies. Once the
process is established it would eliminate guessing,
The decalcifying fluid should be able to make contact testing, and handling of those delicate samples.
with all surfaces of a specimen and flat bone slabs Biopsy decalcification is fairly consistent on multiple-
should not touch each other or the bottom of a con- sized biopsies within an hour or two of acid expo-
tainer, as this is enough to prevent good fluid access sure. Wrapping a bone needle biopsy in tissue paper
between the flat surfaces. Bone samples can be sepa- and leaving it wrapped until embedding prevents
rated and suspended in the fluid with a thread or any loss of cells or tissue during the washing or
placed inside cloth bags tied with thread or prefer- decalcification process. Sponges sometimes pull
ably in a cassette. The cassette will provide identifi- cells from a sample when washing and decalcifying.
cation without having to prepare tags for bag Often these may be urgent cases where a shorter
suspension. Some workers have cleverly devised decalcification time is allowed, but the sample must
perforated plastic platforms to raise samples above be carefully treated and incomplete decalcification
a container bottom to permit fluid access to samples. is still possible. If tissue is still slightly under-
decalcified after paraffin embedment and section-
ing, surface decalcification can be done. Problem
Completion of decalcification
blocks should be identified as such so that proper
Ideally, bone should be taken from the acid solution treatment is given should further microtomy be
as soon as all calcium has been removed from it. It requested.
is still possible that the outer parts of a sample will
be overexposed to the acid, although these parts
Decalcification endpoint test
usually stain no differently from inner portions,
which are the last to be decalcified. Tissues decalci- There are several methods for testing the completion
fied in acids for long time periods or in high acid of decalcification, with two considered to be the most
concentrations are more likely to show the effects of reliable. These are specimen radiography, using an
over-decalcification, whether or not all mineral has X-ray unit and the chemical method to test acids and
been removed. EDTA solutions. Another method first used to test
Consequently, it is important for a laboratory to nitric acid is a weight loss, weight gain procedure
control a decalcification procedure by using a decal- that provides relatively good, quick results with
cification endpoint test to know when calcium all acids and EDTA (Mawhinney et al. 1984;
removal is complete and, if incomplete, renew the Sanderson et al. 1995). Although still used, ‘physical’
decalcifying agent. If laboratories do not perform tests are considered inaccurate and damaging to
endpoint testing, it is recommended they should do tissues. Probing, ‘needling’, slicing, bending, or
so. When using formic, HCl or nitric acids, daily squeezing tissue can create artifacts, e.g. needle
testing is recommended unless near the endpoint; tracks, disrupt soft tumor from bone, or cause false-
then test every 3–5 hours when possible. With EDTA, positive microfractures of fine trabeculae, a potential
weekly tests are sufficient unless solution changes misdiagnosis. The ‘bubble’ test is subjective and
are more frequent. It is recommended that EDTA be dependent on worker interpretation.
 328 16 Bone

Methods for chemical testing of acid decalcifying ‘Bubble’ test. Acids react with calcium carbonate
fluids detect the presence of calcium released from in bone to produce carbon dioxide, seen as a layer
bone. When no calcium is found or the result is of bubbles on the bone surface. The bubbles disperse
negative, decalcification is said to be complete and with agitation or shaking but reform, becoming
may entail using one extra change of decalcifier after smaller as less calcium carbonate is reduced. As an
actual completion. EDTA can be chemically end- endpoint test, a bubble test is subjective and unreli-
point tested by acidifying the used solution; this able, but can be used as a guide to check the progress
forces EDTA to release calcium for precipitation by of decalcification, i.e. tiny bubbles indicate less
ammonium oxalate (Rosen 1981). calcium present.
Radiography. This is the most sensitive test for
detecting calcium in bone or tissue calcification. The
method is the same as specimen radiography using
a FAXITRON with a manual exposure setting of
Calcium oxalate test (Clayden 1952) approximately 1 minute, 30 kV, and Kodak X-OMAT
This method involves the detection of calcium in X-ray film on the bottom shelf. It is possible to
acid solutions by precipitation of insoluble calcium expose several specimens at the same time. The
hydroxide or calcium oxalate, but is unsuitable for method is to rinse acid from the sample, carefully
solutions containing over 10% acid even though these place identified bones on waterproof polyethylene
could be diluted and result in a less sensitive test. sheet on top of the X-ray film, expose according to
Solutions directions, and leave bones in place until the film is
Ammonium hydroxide, concentrated. developed and examined for calcifications. Bones
Saturated aqueous ammonium oxalate. with irregular shapes and variable thickness can
occasionally mislead workers on interpretation of
Method
results. This problem is resolved by comparing the
1. Take 5 ml of used decalcifying fluid, add a piece
test radiograph to the pre-decalcification specimen
of litmus paper or use a pH meter with magnetic
stirrer. radiograph and correlate suspected calcified areas
2. Add ammonium hydroxide drop by drop, shaking with specimen variations. Areas of mineralization
after each drop, until litmus indicates solution is are easily identified, although tiny calcifications are
neutral (pH 7). best viewed using a hand-held magnifier. Metal dust
3. Add 5 ml of saturated ammonium oxalate and particles from saw blades are radio-opaque, sharply
shake well. delineated fragments that never change in size.
4. Allow solution to stand for 30 minutes. These are unaffected by decalcification, appearing as
Result gray specks on the bone surface and can be easily
If a white precipitate (calcium hydroxide) forms removed. Spicules of metal, metallic paint, or glass
immediately after adding the ammonium hydroxide, forced deep into tissue by a traumatic injury are also
a large quantity of calcium is present, making it sharply delineated but cannot be removed without
unnecessary to proceed further to step 3 which would damaging the tissue. Radiography only indicates
also be positive. Testing can be stopped and a
the presence of deeper foreign objects, and care
change to fresh decalcifying solution made at this
must be taken during microtomy to not damage
point. If step 2 is negative or clear after adding
ammonium hydroxide, then proceed to step 3 to add the knife (Fig. 16.5).
ammonium oxalate. If precipitation occurs after
adding the ammonium oxalate, less calcium is
Treatment following decalcification
present. When a smaller amount of calcium is
present, it takes longer to form a precipitate in the Acids can be removed from tissues or neutralized
fluid, so, if the fluid remains clear after 30 minutes, chemically after decalcification is complete. Chemi-
it is safe to assume decalcification is complete.
cal neutralization is accomplished by immersing
 Techniques for analyzing bone 329

a: Before decalcification b: Complete decalcification

Figure 16.5 An example of a FAXITRON X-ray (a) before decalcification (b) after decalcification is completed. The bright white in the
sample is calcium. As the calcium is removed, the bone becomes opaque.

decalcified bone into either saturated lithium car- higher percentages of sucrose may prevent the tissue
bonate solution or 5–10% aqueous sodium bicarbon- from fully freezing or freezing unevenly, causing
ate solution for several hours. Many laboratories soft spots in the tissue which will create thick-thin
simply rinse the specimens with running tap water appearing sections or the tissue will fall out due to
for a period of time. Culling (1974) recommended improper freezing.
washing in two changes of 70% alcohol for 12–18 Tissues decalcified in EDTA solutions should not
hours before continuing with dehydration in pro- be placed directly into 70% alcohol, as this causes
cessing, a way to avoid contamination of dehydra- residual EDTA to precipitate in the alcohol and
tion solvents even though the dehydration process within the tissue. The precipitate does not appear to
would remove the acid along with the water. affect tissue staining since EDTA is washed out
Adequate water rinsing can generally be done in during these procedures, but may be noticeable
30 minutes for small samples and larger bones in 1–4 during microtomy or storage when a crystalline
hours in order to not delay processing. Samples crust forms on the block surface. A water rinse after
needing immediate processing, e.g. needle biopsies, decalcification or overnight storage in formal saline,
can be blotted or quickly rinsed to remove acid from NBF, or PBS should prevent this.
surfaces before proceeding to the first dehydrating
fluid. It is important to avoid contaminating the first
Surface decalcification
dehydrating fluid with acids, and washing bones
even for a short time is good practice particularly Surface decalcification is needed when partially
with large bone slabs. decalcified bone or unsuspected mineral deposits in
Acid-decalcified tissues for cryomicrotomy must soft tissue are found during paraffin sectioning. This
be thoroughly washed in water or stored in formal technique is done to prevent knife damage and
saline containing minimal amounts of sucrose (3– torn tissue sections. After finding a calcification, the
10%), or PBS with 3–10% sucrose, at 4°C before exposed tissue surface in a paraffin block is placed
freezing. This helps avoid any residual acid in the face down in 1% HCl, 10% formic, or a proprietary
tissue from corroding the metal knife. Caution: acid solution for 15–60 minutes, rinsed with water
 330 16 Bone

to remove corrosive acids, and re-sectioned. The melting paraffin from bone and going back through
acid removes a few micrometers of calcium from two changes of xylene, two changes of 100% alcohol
the tissue surface, permitting only a few sections to remove residual water, and then reprocessing
to be cut after careful block re-orientation in the back into paraffin. The later is not recommended
microtome to avoid wasting this thin surface and is not always reliable, especially if the tissue was
decalcified layer. not well fixed.
Modern embedding methods using metal molds
with plastic tissue cassettes have all but eliminated
Processing decalcified bone the necessity to mount the paraffin-embedded
tissues on wood, hard rubber blocks, or metal
In today’s laboratories, automated computerized chucks. A labeled cassette contains the tissue
processors with vacuum and pressure options have throughout processing and, after embedding, the
improved the efficiency and quality of tissue plastic back of a block fits into a microtome cassette
processing, particularly bone. Solvents used for clamp. Macro-cassette systems including larger cas-
dehydration (ethanol, isopropanol, reagent and pro- settes, molds, and a special block holder are avail-
prietary alcohol mixtures) and clearing (xylene, able for sledge microtomes. Specimen size is the
xylene substitutes) will work well for bone and soft limiting factor for embedding with cassettes, and
tissue processing. Paraffin waxes developed in with a little creativeness the oversized bone can be
recent years have been improved by the addition of embedded in a paraffin-filled metal pan or similar
plastic polymers and other chemicals that allow container, a warm hardwood block placed directly
better wax penetration and sectioning. Decalcified on top of bone, with all allowed to harden in place.
bone sectioning is made easier after infiltration and This results in a hard back, embedded directly in the
embedding in a harder paraffin wax. Often bone block, which can be clamped tightly in the micro-
infiltrated with routine paraffin for all tissues can be tome, avoiding holding onto softer paraffin, which
embedded in harder paraffin to give firmer support can crack under excessive clamping pressure.
of bone during sectioning. Small bone and needle
biopsies containing little cortical bone can be pro-
cessed with soft tissues. Microtomes and knives
Oversized, thick bone slabs require an extended
processing schedule in order to obtain adequate Bone biopsies and smaller primarily cancellous bone
dehydration, clearing, and paraffin infiltration. blocks can be cut on any properly maintained micro-
Some laboratories specialized in orthopedic work tome. Many newer microtomes are more powerful,
find it advantageous to dedicate one processor to heavier, and automated, making them capable of
extended processing schedules for bone and not sectioning both paraffin and plastic bone blocks.
interfere with routine daily soft tissue processing. Oversized and exceptionally hard, dense bone
With an enclosed automatic processor, time in each samples too difficult to cut on a smaller microtome
dehydrating, clearing solvent, and paraffin may are easier to section on a large sledge or heavy duty
vary from 2 to 4 hours, with larger bone slabs motorized sliding microtome (Polycut, Leica, USA).
needing the longest time. If a bone sample has been There is a wide choice of good microtome blades
endpoint tested for completed decalcification, but including heavy ‘c’ profile steel and the popular dis-
still appears chalky, mushy, and crumbles out of the posable blades. The disposable knives are conve-
block during sectioning, then dehydration, clearing, nient, extremely sharp, single-use blades capable
or paraffin infiltration may be incomplete. Blocks of sectioning properly decalcified and processed
can be melted down and re-infiltrated with paraffin paraffin-embedded bones. Newer microtomes come
for up to 8 hours to see if this improves sectioning. equipped with disposable blade holders, or dispos-
Another possibility is reversing processing by able blade holder inserts can be purchased for older
 Processing decalcified bone 331

model microtomes. High-profile disposable blades near the top of a block or angled in a way to avoid
are slightly thicker and wider than the low-profile compression of the softer cartilage and paraffin into
blades, and tend to ‘chatter’ or vibrate less when the denser bone, creating wrinkles. Generally, hard
cutting denser bones. Heavier steel knives range in tissues cut more easily if cooled by a melting ice block
size from 16 to 18 cm for small microtomes and from to allow water penetration into the tissue surface.
200 to 300 cm for base sledge microtomes with spe- Extensive soaking causes visible tissue swelling
cially designed blades for the Polycut. Because steel away from the block face and, even though the tissue
knives need frequent sharpening, an automatic knife cuts more easily, the sections fall apart on the water
sharpener is a cost-effective, time-saving device bath. A flat ice block made with water-filled polyeth-
when these knives are used routinely. An automatic ylene storage bags keeps blocks dry during cooling,
knife sharpener is a rare find but a multiple plate or paraffin bone blocks can be cooled in a −20°C
type one can also sharpen tungsten carbide knives freezer for a short time. Larger blocks can be cut at
which are used for undecalcified bone cryotomy and room temperature as long as the room is not overly
plastic embedded tissues. Sharpening a tungsten warm or humid. If using a tape method to obtain a
carbide knife frequently can be expensive, and a difficult section, the blocks must be at room tempera-
knife sharpener can save time and money. Unlike ture and dry so that the tape adheres to the block.
steel knives, tungsten carbide knives need to be An optimal thickness for bone sections is the same
reconditioned after multiple sharpenings. as that for soft tissues, 4–5 µm, and cut routinely
from adequately processed blocks. Bone marrow
biopsies should be cut at 2–3 µm for marrow cell
Microtomy identification, and sliding microtome sections may
vary from approximately 5 to 8 µm.
Small bone samples and biopsies usually section
well with knife angles set for routine soft tissue
microtomy. Generally, disposable blades work well Flattening and adhesion
at the manufacturer’s recommended angle settings
for their high- or low-profile blades. Slight adjust- Bone sections adhere to slides well when glass sur-
ment of a knife angle can be attempted if dense corti- faces are coated with some type of adhesive. Slides
cal bone sectioning is not working with the routine come in all types of coating with different levels of
soft tissue knife angle, and can be increased or tissue-adhering properties. All work well but a
decreased at a microtomist’s discretion. Knives must strong charge or coating is preferred when working
be changed frequently, sometimes after cutting one with bone. The most common is a silanized Plus
ribbon or a few sections of cortical bone. When sec- Charge® (Erie Scientific, NH) or poly-L-lysine-coated
tioning any bone sample, a sharp knife is necessary (PLL) slides. If only plain, uncoated slides are avail-
in order to get flat, uncompressed, wrinkle-free sec- able there is a simple coating method one can
tions, along with patience and good microtomy use. Wash slides in soap and water, rinse soap off
skills by the operator. completely, then dip in a gelatin and potassium
Longitudinal sections of cortical bone may section dichromate ‘subbing’ solution, air dry, and store
better when the knife cuts ‘along the grain’ or the slides in a clean dry box until needed (Drury &
length of the bone oriented at right angles to the Wallington 1980). When sectioning numerous
knife. A bone of somewhat rectangular shape can be bone blocks, 10 ml of the chrome subbing solution
embedded or oriented in a block holder so that a can be added to a 2-liter water bath or simply add
smaller corner of sample is cut first with the wider a few gelatin granules to the water as it is heating.
area cut last. This helps reduce knife vibration and If some sections are persistently non-adherent, a
potential gouging of bone out of the paraffin block. solution containing amylopectin, a starch (Steedman
When cartilage is present, it should be located 1960), or a high molecular weight 225 bloom gelatin
 332 16 Bone

in the chrome subbing mixture may be more an important aspect in disease diagnosis, and is
successful. Gelatin should be used sparingly or an used frequently on decalcified bone tumors, bone
excess coating is stained by hematoxylin giving marrow, and cartilage. Most special stains used with
an unsightly blue background underneath and bone sections are available pre-mixed and are more
around sections. convenient to use than mixing your own. However,
While floating on water, cartilage and bone sec- it is better to see the original ingredients/formulae
tions can expand more than the paraffin or other in references to understand the mechanism and to
tissue components, and small folds may form as the help identify proper staining.
sections dry. When this occurs, the water bath
temperature should be lowered to 10–15°C below
Hematoxylin and eosin (H&E)
the paraffin melting point. Flattening a section by
mounting a section in excess water on the slide, then When staining sections of properly decalcified tissue
holding the slide against a hot plate to melt the wax no modifications to the standard H&E techniques
and evaporate the water must be used with caution. are required. There are several ways to counteract
Bone sections may ‘explode’ apart, displacing corti- weak nuclear staining damaged by acids and make
cal bone from trabecular bone and ruining the gross the hematoxylin stain darker. Freshly prepared
morphology. Reducing the surface tension of water hematoxylin, particularly mixtures that lose strength
by floating a section on RT 10% ethanol, picking up over time, e.g. Harris’s, Gill II and III, often stain
the section on a slide, then immediately but slowly darker than solutions near an expiration date. Pref-
lowering the section into a warm water bath allows erence in hematoxylin is using a progressive hema-
a section to flatten gently. If cartilage curling is a toxylin since it does not require differentiation
problem, drying sections flat at 37°C overnight or steps that a regressive hematoxylin does. Restora-
longer may solve this problem. Most bone sections tion of basophilic staining can be attempted by
flatten and dry without problems or special treat- immersing a hydrated section in 4–5% aqueous
ment provided the tissue has been properly pro- sodium bicarbonate for 10 minutes to 2 hours,
cessed and sectioned with a sharp knife. rinsing well with water, and staining with hema-
toxylin. If using a regressive hematoxylin, the acid
differentiation step can either be shortened to one or
Staining methods for decalcified two fast dips in 0.5% acid alcohol or this step can be
bone sections eliminated entirely. When using a progressive hema-
toxylin, blueing solutions should be mild bases, e.g.
Most routine soft tissue staining methods can be Scott’s tap water substitute or saturated lithium
used without modification for staining decalcified carbonate, to avoid bone section loss caused by
bone sections. Acid decalcification, particularly ammonia water blueing. If poor hematoxylin stain-
when prolonged or used with a heat producing ing is a persistent problem, it is recommended that
method, e.g. microwave, sonication, or electrolytic, the decalcification method be evaluated and appro-
can adversely affect the H&E and some special priate changes made to avoid damage to staining.
stains. When the temperature exceeds 37°C during Alcoholic eosin solutions, 0.5–1%, often stain bone
decalcification, Giemsa staining may be too pink and surrounding tissues overly red, and staining
and the historical Feulgen stain for DNA will be time can be shortened from 1 minute to 30 seconds,
negative because of excessive protein hydrolysis. or even 10–20 rapid dips. Another excellent counter-
Staining is successful after EDTA treatment, but the stain for differential staining of bone components is
slower decalcification rate usually rules it out in eosin Y-phloxine B.
favor of faster acid methods. H&E is still the primary In general, most hematoxylin solutions work well
stain used for most final diagnoses with the aid of for staining bone, including mercury-free Harris’s,
special stains. Immunohistochemical staining is now Ehrlich’s, Mayer’s, Cole’s, Gill II or III, and many
 Processing decalcified bone 333

a b
Figure 16.6 (a) Mayer’s hematoxylin and eosin. (b) Ehrlich’s hematoxylin and eosin. Epiphyseal growth plate from proximal femur of an
11-week-old rat.

proprietary mixtures. Some workers prefer Ehrlich’s immature fibers a very pale orange compared to the
hematoxylin to a more specific nuclear stain, e.g. deeper red mature fibers. A trichrome stain (i.e. Mas-
Mayer’s (Fig. 16.6) for its ability to stain articular son’s) remains a standard, popular method to dem-
and growth plate cartilages a deeper blue to purple onstrate collagen fibers in contrast to bone, cells, and
in contrast to the pink collagen and other tissues. In other soft tissues. The immature collagen fibers stain
general, a hematoxylin solution can stain decalcified distinctly but are a paler blue or green compared
bone to show cement lines in Paget’s diseased bone, with darker-stained mature fibers. Trichrome-
new bone, and rapidly formed or remodeled bone stained adult or mature bone often shows areas of
provided the hematoxylin stains darkly enough. A blue or green staining with some bright red areas
good H&E stain can stain all cells and bone compo- that frequently have no relationship to bone struc-
nents including osteoid as long as care is taken to ture. Osteoid is usually stained with the aniline blue
adjust the staining procedure to achieve optimal or light green fiber stains.
results. Polarized light microscopy may be more useful for
positive identification of collagen than these stains
since the finest fibers do not show distinct colors
Collagen stains
with routine light microscopy. Sirius red, which is
Collagen stains can be used to demonstrate mature commonly used for amyloid, is an excellent stain to
and finer immature fibers in certain tumors and a visualize collagen fibers under polarized light. A
fracture callus. van Gieson (VG) picro-fuchsin stains toluidine blue stain demonstrates collagen fibers.
 334 16 Bone

Cartilage and acid mucopolysaccharides differentiated primary tumor. The PAS reaction is
not affected by decalcification but prolonged treat-
Cartilage can be stained to demonstrate mucopoly- ment with strong acids should be avoided. Reticulin
saccharides using various metachromatic staining staining, the silver impregnation of reticulin fibers,
methods or the azure method by Hughesdon (1949), helps in diagnosis of bone tumors, tumor metastasis
recommended for its selectivity and stability. to bone, and myelofibrosis. Reticulin staining is not
The critical electrolyte concentration method of affected by decalcifying agents although the ammo-
Scott and Dorling (1965) provides a more precise niacal solutions can cause a section to release from
identification of acid mucopolysaccharides in carti- the slide, necessitating the use of a stronger section
lage. They used 0.05% 8GX alcian blue in pH 5.8 adhesive.
acetate buffer containing 0.4–0.5 M magnesium
chloride to stain these strongly sulfated mucopoly-
saccharides blue. Another method useful for Bone canaliculi
showing articular cartilage degradation of ground
Osteocytes and their lacunae are large enough to be
substances in arthritic and other diseases is safranin
easily identified in most preparations, including
O-fast green (Rosenberg 1971), which stains the car-
H&E-stained paraffin sections of decalcified bone.
tilage varying shades of red. Toluidine blue O,
Fine canaliculi radiating from lacunae are not easily
0.1–1% aqueous solution, is also commonly used to
seen in H&E-stained sections, but are well demon-
stain NBF-fixed cartilage (Fig. 16.7). Workers should
strated with a modified Holmes’ silver impregnation
be aware that EDTA, as well as some fixatives and
method on buffered, formic acid-decalcified, paraffin-
acid decalcifiers, extract proteoglycans and can
embedded bone sections (Taylor et al. 1993).
result in weak cartilage staining by safranin O and
The major problem in demonstrating canaliculi is
possible false-negative quantitative results (Callis &
the attempt to show spaces too fine for identification
Sterchi 1998).
by routine staining of surrounding bone, so it is
The positive red periodic acid-Schiff (PAS) reac-
necessary to fill the spaces with a substance that
tion demonstrates mucopolysaccharides in new
appears dark against a lighter or unstained back-
bone, calcifying cartilage, and glycogen in some
ground. The simple ‘air injection’ method (Gatenby
early osteoblasts. PAS assists in diagnosis of some
& Painter 1934) is done with undecalcified ground
mucinous metastatic tumors and primary tumors
sections dried, and mounted in hot, melted Canada
with glycogen with a diastase digestion of glycogen
balsam to trap air inside the canaliculi. These look
to help make more precise identification of a poorly
like black threads against the unstained bone and
the balsam but, if sections are too thin or the balsam
too fluid, the air will be displaced.
The picro-thionin method (Schmorl 1934) depends
on deposition of a thionin precipitate within the
lacunae and canaliculi (Fig. 16.3), although frozen or,
historically, celloidin sections were recommended.
Drury and Wallington (1980) indicated there is less
channel shrinkage in such sections as compared to
paraffin sections and the dye precipitate could
penetrate readily. When frozen sections are not rou-
tinely used, one of the staining methods for decalci-
fied bone paraffin sections developed by either
Taylor et al. (1993) or Tornero et al. (1991) could
Figure 16.7 Toluidine blue-stained articular cartilage and bone. provide workers with better options to fit into
Formic acid-decalcified rat femur. Paraffin section. routine paraffin work. Tornero’s group used a
 Processing decalcified bone 335

microwave picro-thionin procedure and felt it gave

7. Dehydrate rapidly, clear in xylene, and mount in
more accurate, uniform staining of canaliculi as permanent mounting media.
compared to Schmorl’s method.
Results
A later reference by Schmorl recommended
Lacunae and canaliculi dark brown-black
aqueous 0.125% thionin and noted an alkaline solu-
Bone matrix yellow or brownish-yellow
tion accelerated and intensified staining by adding Cells red
one or two drops of concentrated ammonia to
Notes
approximately 10 ml staining solution just before
a. Agitate sections gently during steps 3–6. This is
use. Culling (1974) stated the pH of the thionin solu-
particularly important in step 6, and change 70%
tion was critical, with successful staining results alcohol frequently.
dependent on the amount of ammonia added, and b. If bone matrix is decolorized during differentiation
recommended one drop of ammonia in 100 ml solu- (step 6), restore yellow color by returning the
tion. Drury and Wallington (1980) indicated that section to the picric acid solution for a few
thionin dye batches vary considerably, making it seconds before proceeding with dehydration.
necessary to adjust the ammonia content of staining
solutions made from different lots in order to obtain
Schmorl’s modified method with 0.125% thionin
suitable staining, and that other dyes may be used
solution replaced the picric acid with either phos-
with good results, notably azure A.
photungstic or phosphomolybdic acid and was pre-
ferred when staining children’s bones. Culling
Schmorl’s picro-thionin method (Schmorl 1934) (1974) used the modified method and recommended
Fixation extended time in the alkaline thionin solution, then
Any fixative, but avoid mercuric chloride. a few seconds’ treatment in the acid, followed by
fixation of the dye with dilute ammonia. This
Decalcification
method results in blue-black canaliculi and lacunae
Any decalcifying solution.
on a sky-blue background.
Sections
Frozen or celloidin freshly cut.
Immunohistochemistry (IHC)
Solutions
Stock solution Diagnostic immunohistochemical staining is fre-
0.25 % aqueous thionin. quently done on decalcified bone sections embed-
Working solution ded in paraffin (e.g. bone marrow biopsies, tumors,
0.125% thionin: filter 50 ml stock solution and dilute cartilage) and uses the same staining methods and
with 50 ml distilled water. Add 1 or 2 drops of materials as for soft tissue immunohistochemistry.
concentrated ammonia immediately before use.
Care must be taken to fix bone specimens properly
Saturated aqueous picric acid and decalcify with the least damaging agent in the
Method shortest time possible in order to protect antigens
1. Wash sections in distilled water, 10 minutes. from damaging effects of acids. Immunostaining is
2. Stain in thionin solution, 5–20 minutes or longer. possible on 2 µm thick methyl methacrylate sections
3. Wash in distilled water. after complete removal of the plastic with warm
4. Immerse sections in picric acid solution, xylene and a pressure cooker antigen retrieval
30–60 seconds. method (Hand & Church 1998). Glycol methacrylate
5. Wash in distilled water. (GMA) cannot be removed and may inhibit ade-
6. Differentiate in 70% alcohol until the bluish-green quate antibody or immunoglobulin penetration to
clouds of stain cease to form, 5–10 minutes or the antigenic sites, so it is suggested that the bone
longer.
be stained using DAB chromagen after fixation and
 336 16 Bone

before GMA embedding. Counterstains work well subsequent staining. Plastic sections can be either
after microtomy when processed in this manner and transferred to a slide or stained directly on the tape.
it does not diminish or wash out the chromagen. A special tape transfer system is also available for
bone frozen sections.

Preparation of mineralized bone Block impregnation for

osteoid demonstration
Sections demonstrating bone mineral and its rela-
tionship to the unmineralized components of bone This method is used solely for the negative demon-
must be prepared by methods that do not interfere stration of osteoid in which the calcium in mineral-
with the mineral substance, i.e. undecalcified bone ized bone is replaced by silver before decalcification
sections. Mineralized bone must be cut with tung- and paraffin sectioning. Block impregnation tech-
sten carbide-tipped knives and needs special, hard niques suffer from inherent defects of peripheral or
support to avoid cracked or crumbling tissue sec- over-impregnation near the surface with an incom-
tions. Paraffin is too soft and fails to match the plete reaction deeper in the tissues. As long as these
hardness of bone or provide strong, solid support artifacts are recognized in a finished preparation, the
needed to prevent the fragmented mineralized osteoid seams counterstained reds are clearly delin-
sections. eated next to the blackened mineralized bone. The
Acrylic resins and plastics are now widely used deeper, centrally located trabeculae are pale with
and the preferred embedding media for undecalci- only the outlines of lacunae and some blackened
fied bone and their use has revolutionized how this canaliculi. The advantage of this method is that
bone is examined. Frozen sections provide some good-quality paraffin sections are easily prepared.
support of cancellous bone, but the bone itself tends
to look damaged and somewhat fragmented even
though a diagnosis could be made from the soft
Silver staining of bone prior to decalcification
tissue components.
(Tripp & Mackay 1972)
Fixation
Adhesive tape methods 99% ethyl alcohol.
Tissue
Adhesive tape methods or tape transfer methods 1–2 mm thick bone pieces.
have been used to maintain the intact sections of
Solutions
undecalcified double-embedded bone sections
2% aqueous silver nitrate
during microtomy. Two methods, one for undecal-
Reducer
cifed bone embedded in MMA (Hardt 1986) and the
Sodium hypophosphite 5g
other for decalcified, paraffin-embedded bone 0.1 M sodium hydroxide 0.2 ml
(Eurell & Sterchi 1994), are used for sectioning dif- Distilled water 100 ml
ficult blocks. Clear adhesive packaging tape is rolled
5% aqueous sodium thiosulfate (anhydrous)
onto the trimmed block face and the cut section
Decalcifier
sticks to the tape during and after sectioning. The 10% aqueous formic acid
tape-section combination is then attached to an
van Gieson’s picro-fuchsin
adhesive coated slide and either dried on a hot
Method
plate or clamped firmly between sheets of polyeth-
1. Wash in several changes of distilled water, 4 hours.
ylene or wood inside a 60°C oven overnight. During
2. Place in 2% silver nitrate, 48 hours in complete
the staining process, the tape releases in xylene,
darkness.
leaving the section ‘transferred’ onto the slide for
 Preparation of mineralized bone 337

diseased bone with moderate to severe osteomala-

3. Rinse in three changes of distilled water, 15–20
seconds each. cia, can be rapidly diagnosed on an H&E stained
4. Wash in running tap water, 4 hours. frozen section. Other stains can be done on bone
5. Place in reducer, 48 hours. frozen sections, including a modified Romanowsky
6. Wash in running tap water, 1 hour. method for patterns in bone remodeling and carti-
7. Place in sodium thiosulfate solution, 24 hours. lage development (Dodds & Gowen 1994), enzyme
8. Wash in running tap water, 1 hour. and immunohistochemical methods. Unstained
9. Decalcify in 10% formic acid. sections can be examined with polarized light to
10. Process to paraffin wax, cut, and mount. see woven and lamellar patterns in bone.
11. Dewax and bring sections to water. Laboratories not using MMA embedding tech-
12. Stain with van Gieson’s stain, 2 minutes. niques may find bone cryotomy a valuable addi-
13. Dehydrate, clear, and mount. tion to their facility. Frozen sections permit rapid
diagnosis on some bone diseases. Rapid or ‘snap’
Results
freezing bone samples in liquid nitrogen-cooled
Edges of mineralized bone black
Bone brown to yellow
2-methylbutane (isopentane) must be used care-
Osteoid red fully as some bones can shatter in the extremely
cold (−120°C) temperature. A dry ice/isopentane
Notes
bath (−70°C) snap freezes bone coated with 4%
a. For cellular detail, an adjacent NBF-fixed, non-
aqueous polyvinyl alcohol (PVA, water soluble,
impregnated block should be processed.
124,000 MW) or embedded in optimum cutting
b. Radiographic decalcification endpoint test cannot
be used with the radio-opaque silver deposits. temperature compound (OCT), gently and without
c. Nitric and hydrochloric acid decalcifiers may attack shattering. Hexane can be substituted for isopen-
the silver deposits. tane (Dodds & Gowen, 1994). In brief, the tech-
d. NBF-fixed bone may be used provided nique is:
formaldehyde is completely removed by distilled
water washes before impregnation.

1. Mount bone on cork or embed in a cryomold with

OCT.
Frozen sections
2. Snap freeze carefully in ‘syrupy’ (thawing)
isopentane cooled by liquid nitrogen (−120°C) or
Using a modern cryostat, patience, a slow steady
with dry ice/isopentane (−70°C).
cutting speed, and a tungsten carbide-tipped steel
3. Place bone in cryostat at −30 to −35°C. Remount
knife, frozen sections from trephine and needle
frozen bone onto a metal chuck with OCT to
biopsies of cortical and trabecular bone can be cut provide maximum stability during sectioning.
with ease and minimal section damage. A knife 4. Cut section at 5–7 µm, pick up section on slide,
with a tungsten carbide edge is much harder than a and fix with fixative of choice. Post-fixation in 95%
steel edge and cuts calcified bone without frag- alcohol, 5 minutes, removes fat.
menting a section or damage to the edge. For 5. Stain in Harris, Gill II or Gill III for 1 minute or
demonstration of bone marrow cells, tumor, and longer or desired intensity.
calcified bone components, the hematoxylin stains 6. Rinse with water or a blueing reagent to ‘blue’
cell nuclei and mineralized bone intensely blue section; avoid ammonia water.
and eosin stains osteoid and other soft tissues 7. 1% alcoholic eosin approximately 10–30 seconds
or desired intensity.
shades of red. Some bone with metabolic diseases,
8. Dehydrate, clear, and mount in permanent
such as Paget’s, renal osteodystrophy, and hyper-
mounting medium.
parathyroidism showing advanced changes or
 338 16 Bone

bone specimens require extended time in dehydra-

Notes
tion, clearing, and MMA infiltration compared to
a. Formalin-fixed biopsies can be rinsed, immersed in
5–20% sucrose for 1–8 hours at 4°C to replace decalcified bone paraffin processing.
water before freezing and improve sectioning, Although some workers ‘wash’ the polymeriza-
being known as cryoprotection. Higher tion inhibitor from the monomer (Difford 1974),
concentrations of sucrose for cryoprotection may many now use an unwashed monomer method suit-
delay freezing and cause uneven freezing. able for microtomed or sawn ground sections.
Optimization and practice should be used to Enclosed automatic processors should not be used
determine the amount of sucrose that is used.
with toxic MMA monomers, although bone labora-
b. Fresh frozen sections can be fixed, rinsed, and
tories use these processors for alcohol and xylene
then decalcified in 10% EDTA before
immunostaining. steps but finish MMA infiltration steps with hand
c. Enzyme staining can be done on fixed or unfixed processing. Approximate time per change in sol-
sections. vents for all processing steps depends on bone size:
d. A special tape transfer system (Cryojane; Leica, small, 24–30 hours; medium (3–5 mm), 48 hours or
Inc.) is available for cryomicrotomy of undecalcified more; large, 2–4 days.
bone and other difficult tissues, keeping sections Procedures for MMA processing and embedding
intact and adhered to special polymer-coated developed by Sterchi (1996) are similar to those
slides (Schiller 1999). in Chapter 8. Sterchi used two changes each of
e. Any hematoxylin can be used with staining 95% and 100% ethanol: xylene or (1 : 1) MMA
intensity optimized for worker preference.
monomer/100% ethanol. MMA infiltration times are
the same as processing times. Sterchi’s infiltration
mixtures vary from those in Chapter 8 by using
Plastic embedding 100 ml monomer and adding to: Mixture #1, 1 g
benzoyl peroxide (BPO) only; Mixture #2, 5 ml dibu-
Mineralized bone sections are best studied when tylphthlate 1 g BPO, 15 g poly methyl methacrylate
the embedding medium matches bone hardness to powder (996,000 MW; Aldrich, MO); Mixture #3
permit intact sections necessary to examine bone (same as #2) but 1.5 g BPO. Embed in fresh Mixture
density or defects in mineralized components in #3, polymerize at RT or in a 37°C water bath (small
relation to the bone cells, cartilage, osteoid, and bone), cure blocks for 4–6 hours at 60°C, then freeze
other soft tissues. Synthetic resins for both EM blocks for 2 hours to remove polypropylene
(Epon) and light microscopy plastics (GMA, MMA) containers.
work well for these purposes. Epoxy EM resins are
suitable only for ultra-thin sectioning of tiny bone
Sectioning methacrylate-embedded bone
pieces; their hydrophobicity causes poor stain pen-
etration into tissues. Bone embedded in MMA can be sectioned by either
Methacrylates were used for specimen whole- microtomy (1–20 µm) or sawing and grinding for
mount displays in museums before use as support thick sections (150 µm to 1 mm). Thick ground sec-
media for sections. Although Woodruff and Norris tions are necessary for microradiography, bone con-
(1955) favored an n-butyl/ethyl methacrylate taining metal or other implant material, and
mixture, methyl methacrylate (MMA) is now the extremely large bones that cannot be sectioned. An
preferred plastic for undecalcified bone work. MMA ultra-miller on a large sliding microtome (Polycut E;
mixed with polyethylene glycol (Boellaard & von Leica, USA) can precisely mill a perfectly flat 15–
Hirsch 1959) or dibutylphthlate is softer and more 20 µm section. Ultra-milling and grinding wastes
elastic. Glycol methacrylate, a softer plastic than bone and must be used carefully. Thin bone sections
MMA, used with bone biopsies results in ‘laddered’ cut more easily with a motorized microtome
bone sections cut with glass knives. Mineralized designed for plastic work.
 Preparation of mineralized bone 339

Sawing Grinding and polishing

Hand sawing and grinding can be done by clamping Motorized metallurgical grinder/polishers are ideal
a block in a vice, cutting a slab with a fretted wire for final finishing of MMA-embedded bone wafers
saw, and then grinding the slab to produce a section to a required thickness for microscopic examination
thin enough for staining and microscopic examina- and microradiography. These machines have vari-
tion. Hand preparations result in thick, uneven able speed adjustments, rotating base plates to hold
slices with deep scratches requiring extra grinding self-adhesive grinding papers or polishing cloths,
to obtain a smooth section. This is time consuming running water for removing debris, and even have
and wastes more specimen even though adequate special adapters to hold a specimen during the
sections are possible with practice and careful grinding process. Grinding papers remove scratches
sawing/grinding techniques. with progressively coarser to finer grit papers (360,
Saws designed for cutting metallurgy samples are 400, 600 grits), followed by polishing with an
recommended for obtaining precise, flat, thin sec- aqueous 1 µm alumina for a mirror-smooth surface.
tions. A Micro-Grinding System (EXAKT Technolo- Residual fine scratches produce unsightly patterns
gies, Oklahoma, USA) specifically developed for in microradiographs and surface stained sections. A
bone work by Donath (1988a, 1988b) is complete with dial caliper can be used to check section thickness
plastic embedding media, plastic slides, special saws, throughout the grinding process. Hand grinding is
and a grinder/polisher. Low- or high-speed metal- inexpensive, simple, and with practice will produce
lurgical saws (Isomet, Buehler Ltd, IL, USA) cut adequate sections but automated grinders have the
easily through bone blocks with or without metal advantages of speed and producing multiple and
implants using diamond-impregnated cut-off blades, flatter sections. It is not recommended to grind
and produce sections in need of less grinding. These unembedded cortical bone between abrasive glass
machines cut wafers down to 100 µm thick and, by plates (historical method) without support of the
resetting the micrometer, accurate serial sectioning is plastic. It may fracture the bone; grind away fine
possible. These saws are water cooled to disperse trabeculae, cells and soft tissues without complete
heat produced by cutting while continuously clean- removal of scratches.
ing the blade and block face for debris-free sections. Microtome sections of undecalcified bone
The blade thickness (kerf) increases with larger
blade diameters and each cut with a blade wastes Automated microtomes (Leica 2165, Leica USA, or
only as much tissue as its own thickness (kerf loss). Olympus Cut 4060E, Triangle Biomedical Systems,
It is advisable to use a smaller-diameter blade with NC) and a D profile tungsten carbide-tipped steel
the thinnest kerf whenever possible. Larger blocks knife section, both MMA and GMA. Tungsten
generally need sturdier, large-diameter blades for carbide knives help avoid the ‘laddered’ bone sec-
proper clearance through a block and no flexing of tions seen after cutting with glass knives. Large
blade while cutting even though the kerf loss will be blocks need to be sectioned on a larger, sliding
greater. motorized microtome (Leica Polycut E or S) with
Milling cutters and precision cutting machines special tungsten carbide knives.
(Malvern Microslice, UK) can cut sections to approx-
Staining methyl methacrylate-embedded bone
imately 150 µm. Sawn sections are ground and pol-
ished to produce the final thickness and remove Methacrylate bone sections can be stained in two
scratches while ultra-milled sections need no further ways depending on the type of section available.
grinding or polishing. The MMA-embedded slab Sections, 4–8 µm thick, attached to an adhesive-
section is glued to white or clear plastic slides with coated glass slide can be stained after either soften-
cyano-acrylate glue to endure the stress of grinding ing or removing the plastic. When section loss is a
and polishing. problem, sections can be stained by ‘free-floating’ in
 340 16 Bone

a dish or attached to adhesive tape. Ultra-milled or staining can be done by mildly decalcifying (acid
ground sections, 20–200 µm thick, glued to plastic ‘etching’) the exposed bone surface with 1% formic
slides, can be ‘surface’ stained, a unique, effective acid for 1 minute to remove a few micrometers of
method for examination of mineralized bone and calcium from the section to permit better dye pene-
its components. tration into the bone, thereby enhancing staining of
Unstained thin sections examined with polarized bone. MMA is very hydrophobic and only certain
light demonstrate collagen patterns within the bone. low molecular weight dyes actually penetrate
There are many staining methods for MMA bone MMA for suitable staining of softer tissue compo-
sections, including methods necessary to distinguish nents with the aid of heat or alkaline stain solutions
the osteoid from the mineralized components. Micro- (pH 7–9). Some methods used for surface staining
radiographic evaluation can be done on 100 µm thick include methylene blue-basic fuchsin, potassium
sections followed by surface staining of the same permanganate-oxidized methylene blue (Sander-
section for microscopic examination and correlation son’s Rapid Bone Stain, Surgipath, USA), modified
of two methods. Paraffin staining methods often do MacNeal’s tetrachrome (Schenk et al. 1984), and
not stain MMA sections well, and must be adapted 0.75% toluidine blue in phosphate buffer, pH 7–8
for these sections by longer staining times, or plastic (Eurell & Sterchi 1994). These methods stain calcified
removal (‘deplasticize’) or softening with solvents bone and its components distinctly, i.e. osteoid, cal-
(‘etching’) to permit stain penetration. cification fronts, lacunae, canaliculi, osteons, osteo-
Stains for calcium are of prime importance and clasts, osteoblasts, marrow cells, collagen, and other
include hematoxylin, solochrome cyanin, and the soft tissues, various shades of dark to light blue or
von Kossa silver nitrate methods. Trichrome stains blue-green, and cartilage shades of deep purple to
will distinguish calcified bone stained with some violet. Basic fuchsin, light green, and van Gieson’s
hematoxylin and the fiber stain from the osteoid are used as counterstains with these methods.
stained with the cytoplasmic stain. Thus, a Masson’s
trichrome stains bone blue and osteoid red. A modi-
Mounting after staining
fication of this method by Goldner (1937) results in
clear, brilliantly detailed staining of osteoclasts, Deplasticized stained sections on glass slides are
osteoblasts, fibroblasts, and cells from marrow or mounted in the same way as paraffin sections using
tumors. Some workers feel the modified MacNeal’s alcohol dehydration, xylene clearing, and mounting
tetrachrome and Movat’s pentachrome methods with a synthetic mounting medium. Free-floating
give superior staining results for differentiating sections tend to wrinkle and, while in clearing agent,
osteoid from mineralized bone (Schenk et al. 1984). can be flattened with a brush or rolled between
Various toluidine blue methods with pH ranges of pieces of smooth filter paper, then mounted with
7–9 are routinely used to specifically stain trabecular synthetic resin with a weight placed on top of the
mineralization fronts, cartilage, and other bone cover glass to keep the section flat until the medium
components. dries. Strong clamping devices maintain flat sections
Detection of tetracycline fluorescence in bone can briefly but cause the mounting media to retract
be done on either unstained sections or sections during storage.
stained with Villanueva’s mineralized bone stain Surface-stained sections in MMA cannot be dehy-
(Sanderson 1997). drated, cleared, or mounted. Methyl methacrylate is
Surface staining methods for 20–200 µm thick pol- softened by alcohols, and is soluble in xylene and
ished bone MMA sections mounted on white plastic other mounting media solvents. Their use results in
slides are helpful for examining oversized bone sec- ugly cracking of plastic in and around a section.
tions or sections containing metal implants. This Surface-stained sections are usually not perma-
staining technique provides surprisingly excellent nently coverslipped. To examine surface-stained
cellular detail, and is commonly used to study the sections, place a cover glass on top of the dry section
interface of the bone with a metal implant. Surface or a drop of immersion oil to aid the resolution of
 Preparation of mineralized bone 341

the section under the scope. Examine with the

Buffer solution A 94.7 ml 47.35 ml 23.67 ml
brightest light setting on the microscope. If using the Buffer solution B 5.3 ml 2.65 ml 1.33 ml
immersion oil, wipe the oil off after removing the Distilled water 100.0 ml 50.0 ml 25.0 ml
temporary coverslip. The slide will feel oily but do 200.0 ml 100.0 ml 50.0 ml
not use any solvents to clean the oil off since it will
*Check the molecular weight of the dry chemical to
not alter the stain after long-term storage whereas refigure the molarity of the solution when making new
the solvent will. stock solution.

Working toluidine blue O solution

Hematoxylin and eosin for MMA-embedded tissue
Phosphate buffer, pH 8.0 40 ml
These methods are useful for diagnosis of suspected 1% toluidine blue stock 3 ml
osteomalacia and distinguish mineralized bone from Working basic fuchsin solution
osteoid, with nuclei and other soft tissues stained Phosphate buffer, pH 8.0 40 ml
similarly to decalcified bone paraffin sections. 1% basic fuchsin stock 1 ml
1% formic acid**
Formic acid (88%) 1 ml
Distilled water 99 ml
Hematoxylin and eosin-like stain (Eurell & Sterchi, 1994)
**Or use premixed formic decalcifying solution.
Modified toluidine blue/basic fuchsin (TB/BF) for MMA
sections (Fig. 16.8) Method
1. Place slides into 1% formic acid, 30 seconds with
Solutions slight agitation.
1% stock toluidine blue O 2. Rinse slides thoroughly in running tap water.
Toluidine blue O 1.0 g 3. Blot slide with gauze or paper towel to dry.
Distilled water 100 ml
4. Mix working toluidine blue staining solution.
1% stock basic fuchsin 5. Preheat toluidine blue staining solution to
Basic fuchsin 1.0 g 57–60°C in microwave.
Distilled water 100 ml 6. Place slides into preheated toluidine blue, cover
Phosphate buffer solution, pH 8.0 and let stain for 5 minutes.
Buffer solution A: 0.2 M disodium hydrogen 7. Rinse in running tap water.
orthophosphate (28.39*g 8. Blot slides to drain off excess water.
Na2HPO4 in 1 L H2O) 9. Mix working basic fuchsin staining solution.
Buffer solution B: 0.2 M sodium dihydrogen
10. Preheat basic fuchsin staining solution to 57°C in
orthophosphate (27.60*g
microwave.
NaH2PO4 in 1 L H2O)
11. Place slides into preheated basic fuchsin, cover
and let stain for 3–5 minutes. depending on the
staining intensity preferred.
12. Rinse in running tap water.
13. Dry completely, then coverslip.
Note: Do not let slides dry completely during any
step of staining procedure except before
coverslipping.
Results
Calcified bone Pink to purple
Osteoid Dark pink
Cell cytoplasm Pink
Nuclei Blue to blue purple
Cartilage Violet

Figure 16.8 Example of the hematoxylin and eosin-like stain.

A 4 µm section of rabbit TMJ embedded in methyl metacrylate.
 342 16 Bone

Hematoxylin and eosin (Wallington 1972)

Reagents
Cole’s hematoxylin
1% aqueous eosin
Method
1. Deplasticize with xylene and hydrate sections to
distilled water.
2. Stain in freshly filtered Cole’s hematoxylin,
60 minutes with occasional agitation.
3. Wash well in alkaline tap water.
4. Stain in eosin solution, 30 minutes.
5. Wash in tap water.
6. Dehydrate, clear, and mount.
Results
Osteoid pink
Calcified bone purple brown
Nuclei blue

Solochrome cyanine
The solochrome cyanine stain differentiates osteoid
from newly laid down bone and older bone (Fig.
16.9). The following method gives stronger sharper
staining compared to 1% solochrome cyanine R in Figure 16.9 Solochrome cyanine method showing osteoid
2% acetic acid (Matrajt & Hioco 1966) but the proce- and mineralized bone. Undecalcified section of methacrylate-
dures are essentially the same. embedded iliac crest biopsy from a patient with osteomalacia.

Solochrome cyanine (Hyman & Poulding 1961) Results

Mineralized bone light blue
Solution
Calcification front dark blue
Solochrome cyanine R 1g
Osteoid light red-orange
Concentrated sulfuric acid 2.5 ml
Wide osteoid and orange light red-orange with
Mix well until dye incorporates into the resulting
bands pale blue
‘sludge’. Add 500 ml of 0.5% aqueous iron alum
Nuclei blue
(ferric ammonium sulfate).
Mix and filter.
Staining for bone mineral
Method
1. Deplasticize with xylene and hydrate sections to The classic von Kossa (1901) silver method is used
distilled water. to stain the mineral component (calcium phosphate)
2. Stain in solochrome cyanine solution, 60 minutes. in bone, and is a negative stain for osteoid with the
3. Using a microscope, differentiate in warm (30°C) calcium component blackened by silver deposition.
alkaline tap water until mineralized areas appear Osteoid is counterstained red by either the van
blue and other areas light red. Over-differentiation
Gieson’s or safranin O (Figs 16.10 and 16.11). This
causes all parts to become blue.
can also be used as a ground section surface stain
4. Dehydrate, clear, and mount.
but without the acid ‘etching’ removal of calcium.
 Preparation of mineralized bone 343

Von Kossa method (modified Von Kossa 1901)

Solutions
1% aqueous silver nitrate
2.5% sodium thiosulfate
1% safranin O or van Gieson’s picro-fuchsin
Method
1. Deplasticize with xylene, and hydrate sections to
distilled water.
2. Place in silver nitrate solution, expose to strong
light for 10–60 minutes and watch the mineralized
bone turn dark brown to black, indicating a
completed reaction.
3. Wash in three changes of distilled water.
4. Treat with sodium thiosulfate, 5 minutes.
5. Wash well in distilled water.
6. Counterstain as desired.
7. Dehydrate, clear, and mount.
Results
Mineralized bone black
Osteoid red
Notes
1. Long-wavelength UV light from sunlight or a quartz
halogen microscope lamp is preferable to a tungsten
filament light bulb, and accelerates the reaction.
Figure 16.10 Von Kossa’s silver deposition method giving a
2. van Gieson’s picro-fuchsin counterstaining may
negative demonstration of osteoid, counterstained with safranin O.
interfere with birefringence of osteoid.
Undecalcified section of methacrylate-embedded iliac crest biopsy
from a patient with osteomalacia. Ground section.
Goldner’s trichrome method
This staining technique can be more valuable than the
von Kossa method in investigations of metabolic
diseases, e.g. Paget’s, renal osteodystrophy, and
hyperparathyroidism, because of excellent staining of
cells. Osteoblast and osteoclast activity is easily
assessed, an important factor for both diagnosis and
evaluating the effects of treatment in these disorders
by repeated bone biopsies. An additional advantage
is that metastatic tumor cells in bone marrow are
easily identified.
Solutions
Weigert’s iron hematoxylin
Ponceau-fuchsin-azophloxin stock solutions
Ponceau de xylidine solution
Ponceau de xylidine 0.75 g
Figure 16.11 Surface-stained canine bone. Rapid bone stain
Acid fuchsin 0.25 g
counterstained with van Gieson’s. Bone (red), osteocytes and
Acetic acid 1 ml
giant osteoclasts (blue). Undecalcified methyl methacrylate-
Mix, and add to distilled water 100 ml
embedded ground section.
 344 16 Bone

Azophloxin solution Demonstration of aluminum

Azophloxin 0.5 g Patients receiving hemodialysis for chronic renal
Acetic acid 0.6 ml
failure may deposit aluminum at the mineralization
Mix, and add to distilled water 100 ml
sites in bone, producing an osteomalacia-like pattern.
Final working stain solution
Aluminum can be demonstrated by either the alu-
Ponceau-fuchsin solution 5–10 ml
minon or solochrome azurine method, with the
Azophloxin 2 ml
0.2% acetic acid solution 88 ml latter considered the most reliable. Two newer
methods eliminated staining of free-floating sections
Light green solution
(see method below) and used sections attached to
Light green 1g
Acetic acid 1 ml glass slides. One is a modified acidic solochrome
Mix, and add to distilled water 500 ml azurine method for GMA-embedded bone sections
(Huffer et al. 1996), and the other is an aluminon
Phosphomolybdic acid-orange G solution
Phosphomolybdic acid 3g method for the study of uremic bone fixed with NBF
Orange G 2g instead of absolute ethanol and embedded in MMA
Dissolve in 500 ml of distilled water, and add a crystal (Maloney et al. 1982).
of thymol.
Method
1. Deplasticize with xylene and hydrate sections to Solochrome azurine method for aluminum in bone
water. biopsies (modified from Denton et al. 1984)
2. Immerse sections in alkaline alcohol solution Sections
(90 ml of 80% ethanol and 10 ml of 25% Undecalcified bone embedded in MMA.
ammonia), 1 hour.
Solutions
3. Rinse in water, 15 minutes.
Stock solution
4. Stain in Weigert’s hematoxylin, 1 hour. 1% aqueous solochrome azurine (CI 143830). Stable
5. Rinse in tap water, 10 minutes. for a long period of time.
6. Rinse in distilled water, 5 minutes.
Working solution
7. Stain in final Ponceau-fuchsin-azophloxin solution, Adjust pH of 1% solochrome azurine stock solution to
5 minutes. pH 5 with 25% acetic acid. This forms an important
8. Rinse in 1% acetic acid, 15 seconds. precipitate needed for staining; do not filter this
9. Stain in phosphomolybdic acid-orange G solution, solution. Prepare working solution immediately before
20 minutes. use and discard after use.
10. Rinse in 1% acetic acid, 15 seconds.
Method
11. Stain with light green, 5 minutes.
1. Free-floating MMA sections to distilled water. Use a
12. Rinse in three changes of 1% acetic acid. small Petri dish to contain all solutions for
13. Rinse in distilled water, blot dry, and mount. free-floating sections throughout the procedure.
Results 2. Stain sections in working stain solution (pH 5.0) at
Mineralized bone green RT for 18 hours or overnight.
Osteoid orange-red 3. Wash gently in distilled water for 20–30 seconds.
Nuclei blue-gray 4. Counterstain with 1% neutral red.
Cartilage purple 5. Wash in distilled water.
6. Blot dry and leave in drying oven overnight.
7. Mount in synthetic mounting medium.
Results
Aluminum dark blue-purple
Nuclei and background shades of red
 Preparation of mineralized bone 345

Aluminon method (after Irwin 1955) Microradiography

Fixation
Microradiographs are high-resolution, fine-detail
Fix bone in absolute ethanol.
contact X-rays of thinner ground sections for micro-
Sections scopic examination and evaluation of bone mineral
MMA, free-floating sections. density and distribution. The denser, highly miner-
Solutions alized areas appear almost white as fewer X-rays
Buffer solution penetrate them, and less dense, non-mineralized
5 M ammonium chloride 60 ml areas show shades of yellowish-gray grading to
5 M ammonium acetate 60 ml black against the black background of the exposed
6 M HCl 10 ml film (Fig. 16.12).
Mix, and check pH (should be approx. pH 5.2). The requirements for microradiography, even
Working stain solution though similar to those for fine-detail specimen
Aurinetricarboxylic acid (‘aluminon’) 2g radiography, are even more stringent: a fairly pow-
Buffer solution, as above 100 ml erful, controllable source of ‘soft’ X-rays (low kV),
Dissolve aluminon in a few milliliters of buffer, and even overall section thickness, and high-resolution
bring final volume to 100 ml with remaining buffer.
Heat to 60°C. Filter immediately before use.
Differentiating solution
Buffer solution, as above 50 ml
1.6 M ammonium carbonate 22 ml
Check pH; should be approx. 7.2.
Method
1. Bring sections to water.
2. Stain for 5–10 minutes at 60°C; use freshly filtered
stain, preheated to 60°C (see Notes a and b).
3. Rinse in distilled water.
4. Differentiating solution for 3–5 seconds.
5. Wash in distilled water.
6. Counterstain in 1% aqueous methylene blue for
1 minute.
7. Rinse in distilled water.
8. Dehydrate in alcohols and mount in synthetic resin
medium.
Results
Sites of aluminum bright red
Background blue
Notes
a. Sections embedded in MMA and other acrylics,
e.g. LR White, may detach from glass slides in this
60°C solution.
b. Free-floating MMA sections often wrinkle in the
60°C solution. Multiple sections and careful
handling are recommended.
Figure 16.12 Microradiograph of normal femoral shaft showing
different densities of mineralization. The lightest areas indicate the
heaviest mineral deposition.
 346 16 Bone

film or photographic plates are necessary for this

After exposure, the mammography film or plates
procedure. must be developed exactly according to manufacturer
Mineralized bone sections embedded in MMA are instructions, using solutions and temperatures
preferred and must be perfectly flat, measure specified for these products, then dried. The dried
approximately 70–130 µm thick, and be free of film is cut to fit on a larger glass slide mounted under
debris and scratches. Thicker sections create blurred a larger cover glass and secured at the edges by
images and thinner sections transmit too many cellophane tape. An exposed area on a photographic
plate is mounted under a cover glass with Eukitt’s or
X-rays. Sections must be in tight, close contact with
an equivalent medium. Examination should be made
the film or plate and developed under controlled using a 10x objective on light microscope in a dark
conditions for comparative work. room.
Microradiography has been done with X-ray
crystallography units, and, although not called
microradiography, even greater resolution work is Fluorescent labeling
accomplished with the BSEM or backscatter electron
imaging scanning electron microscope (Bloebaum Tetracycline antibiotics form fluorescent complexes
et al. 1990). The Faxitron X-ray unit produces satis- with calcium at the sites of bone mineral deposition
factory results, and Dunn et al. (1974) described a (Milch et al. 1957, 1958) and provide an effective in
standard procedure with this unit using 20 kV, vivo tracer of bone formation in these areas. The
20 cm film to source distance (FTSD) for 75 minutes drug localizes rapidly in newly mineralized sites of
with 5 × 5 cm glass Kodak High Resolution Plates, bone or teeth and appears as a bright fluorescent line
Type 1A. Unfortunately, photographic plates and under UV light. Two or more doses administered to
films suitable for microradiography are frequently patients at known intervals provide a method for
discontinued. Workers should locate replacement estimating the rate of bone remodeling (Frost 1983a).
film with a resolving power of 2000 lines/mm or The measurable distances between parallel uptake
higher and high-contrast, fine-grain emulsions, lines indicate the amount of bone deposited at each
properties that help reduce long exposure times time interval between doses (Fig. 16.13). In order to
(Boivin & Baud 1984). Some workers are finding retain tetracycline labeling, mineralized bone is
success using mammography film (Kodak MIN R fixed in 70% ethanol or alcoholic formalin, embed-
2000; Kodak, Rochester, NY) at higher kV, longer ded in MMA, sectioned, mounted unstained and
FTSD, and short exposure time (L. Jenkins, personal
communication). Using the example below as a
guideline, workers should be able to optimize set-
tings needed for any new film or photographic
plates.

An example of microradiography using mammography

film in Faxitron (L. Jenkins, personal communication, 2000)
Bone section, approximately 100 µm thick, pressed
tightly against film inside a vacuum cassette.
A Kodak MIN R 2000 mammography film.
Film-to-source distance (FTSD) 50 cm (lowest shelf
level).
Tube, 55 kV. Figure 16.13 Tetracyline-labeled cortical bone from a young
horse. Two doses were given at 3 weeks. Specimen was
Tube current, 3 mA.
alcohol-fixed, undecalcified, methacrylate-embedded section
Exposure time is approximately 5 seconds.
illuminated with 480 nm wavelength UV light.
 Morphometry of bone 347

viewed with a UV light microscope at approximately bone work are being used to reduce the time needed
360–480 nm wavelength. After UV light evaluation, for measurements and calculations of final results.
sections can be stained with toluidine blue for A standardized, generally universal system of
further microscopic examination. nomenclature, symbols, and units for bone histo-
morphometry was summarized by Parfitt (1988)
and it is recommended that workers be familiar
Morphometry of bone with and use this system. This is a terminology list
of primary measurements for volume, surface, thick-
The general principles and methods of morphome- ness, mineralization rate, formation rate, and so on.
try are outlined in Chapter 23 of this book. This Basic measurements are confined to trabecular bone
section very briefly deals with some application of and are:
techniques for evaluating bone disorders, particu- (a) trabecular bone volume and surface
larly in metabolic bone disease (MBD).
(b) eroded (resorption) surface
Normal bone containing trabeculae undergoes
constant remodeling or resorption by the osteoclasts (c) osteoid surface
and formation by osteoblasts. In disease, remodeling (d) mineralized surface
can be disturbed and cause either too much or too (e) osteoid thickness
little of one of these dynamic processes to occur in (f) wall thickness (of new bone layers at formation
the bone. site)
Assessment of relative amounts of bone trabecular
(g) mineral apposition rate (calcification rate)
tissue, osteoid, resorption and deposition (apposi-
(Recker 1983)
tion) can be made by a subjective microscopic exam-
ination of the section. This is adequate where Calculations are made from collected data (Parfitt
changes are obvious, but subtle alterations may et al. 1987), and results correlated to the various
require accurate measurements to detect an abnor- diseases. Chapter 23 explains how area measure-
mality. In metabolic bone disease, tetracycline is ments are numerically equated with volume mea-
administered at specific times prior to biopsy to help surements. The derivation of some of these values is
determine the amount of active mineralization in shown below:
bone (Fig. 16.12), followed by an iliac crest biopsy
and subsequent MMA embedment. Briefly, bone Bone volume (%)
histomorphometry can be used for detection and
assessment of disease severity or effects of treat-
area of osteoid
ments of MBD, e.g. post-menopausal osteoporosis. Osteoid volume (%) =
area of trabeculae and
Recker (1990) listed eight metabolic bone diseases marrow space
with tentative indications for bone biopsy, although
this list is anticipated to expand with acquisition of Osteoid surface (%)
more knowledge and treatment of MBD. length of trabecular surface
Histomorphometric analysis can be done with covered by osteoid
=
manual, semi-automated, or automated methods. A total length of trabecular surface
manual method could include a standard micro-
osteoid volume
scope with eyepiece reticules, digitized tablets Osteoid index (%) =
osteoid surface
(image display), image storage, and a computer for
data storage and output. Increasingly, modern auto- Resorption surface (% )
mated computerized image analysis systems with a length of trabecular surface
video camera, a screen with screen grid for area occupied by lacunae
=
counting, and software designed specifically for total length of trabecular surface
 348 16 Bone

There are many other derived parameters used to tissue without destruction of the sample. MicroCT
describe bone dynamics described by Frost (1983b). works on the same principles as clinical CT. A radia-
Histomorphometric values for normal males and tion source releases X-rays through a sample; the
females with relation to age, and values for the X-rays that make it through the sample are collected
various diseases compared to age and sex-matched on the detector. In many MicroCT units the sample
normal controls have been published, and are useful rotates while projection images are collected at
as reference guidelines (Melsen et al. 1983). It is various angles. Generally up to a thousand projec-
important to be aware of pitfalls in techniques for tions are taken greater than 190 degrees around the
bone morphometry; these are well discussed in sample. Reconstruction then takes the planar projec-
Recker’s book (1983). An example of a problem is tions and compiles them into a three-dimensional
with serial biopsy evaluation of a severe disease, for image. This 3D image can then be manipulated in
example Paget’s, where known variations occur all three orthogonal planes. Changing the distance
from site to site and even within the same bone. It of the sample from the X-ray source and detector or
is important that each laboratory establish its own the number of projections can influence the resolu-
set of normal values, along with careful standard tion of the scan (Hsieh 2003).
operating procedures (SOPs) for sample prepara- Different tissue types block or ‘attenuate’ X-ray
tion, staining techniques, and microscopy used for radiation at varying degrees. This attenuation prop-
bone morphometry. If standardized stains or magni- erty of tissue of differing densities is what creates
fication are not used, measurements made using dif- the contrast in the image. Bone has a higher density
ferent stains can result in different values for the than tissue, hence its ease of visualization with CT.
same biopsy. A standardized magnification must be With a 3D image one can perform geometric analysis
used to estimate surface values, otherwise higher and quantify trabecular size, number thickness,
values are produced with increased magnification as spacing and connectivity, along with bone mineral
finer surface convolutions are resolved. Detailed density and content (Abe et al. 2000). The use of
discussions of bone morphometry can be found in contrast agents can allow for ‘virtual histology’ of
Bone Histomorphometry: Techniques and Interpretation tissue (Johnson et al. 2006) (Fig. 16.14).
(Recker 1983); Proceedings of the International Work-
shop on Bone Histomorphometry (Jee & Parfitt 1980);
and a useful review by Revell (1986).
Acknowledgments

Micro computed tomography (CT) The author would like to thank the following for
their contributions to this chapter: Gayle Callis, for
Micro computed tomography (aka MicroCT) is a writing the previous version; and Chris Bull, for his
tool that allows the micron analysis of bone and expertise in CT and providing Figure 16.14.
 Morphometry of bone 349

Figure 16.14 Example of a specimen CT scan showing different dimensional measurement capabilities.

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Further reading
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Chappard, D., Blouin, S., Libouban, H., et al., 2005. preparation and staining in decalcified bone. In:
Microcomputed tomography of hard tissues and Sheehan, D.C., Hrapchak, B.B. (Eds.), Theory and
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(3), 23–25(AM). pp. 96–98.
 Techniques in neuropathology
J. Robin Highley • Nicky Sullivan
17
• Meninges
Introduction
• Blood vessels

Neuropathology has classically been seen as some- The neuron is an excitable cell that is responsible
thing of a dark art by general histologists because of for processing and transmitting information.
the tradition of using a large number of obscure and Neurons communicate with each other via intercel-
often capricious stains. However, with the ascen- lular interfaces called synapses. At the synapse, an
dancy of molecular pathology, many of the more electrical impulse in the presynaptic neuron causes
unreliable stains are being replaced by immunohis- it to release a chemical transmitter that diffuses
tochemistry. As such, many of the preparations that across a narrow gap to influence the electrical activ-
have been described in previous editions of this ity of the postsynaptic target neuron. Neurons have
volume have been omitted from this chapter as they several components (Fig. 17.1):
are no longer in use. Nonetheless, a number of reli- • The cell body (or ‘soma’), which contains
able and useful tinctorial and metal-based stains various subcellular organelles responsible for
remain in common usage. These are described in the metabolic upkeep of the cell.
this chapter together with some preparations that • The axon, an elongated fibrous process that
are still employed, albeit less frequently. Finally, in transmits electrical impulses away from the
neuropathology as in most other areas of histologi- soma to synapses with either other neurons
cal practice, hematoxylin and eosin (H&E) remains or muscle fibers. This may be a meter or more
the most useful and commonly used stain, as it dem- in length in the case of the lower motor
onstrates most cell types well and with good detail. neurons that reside in the lower (lumbar) spinal
cord and innervate muscles of the lower leg.
• The dendritic tree, a plexus of cell process
The components of the normal responsible for receiving synaptic inputs from
nervous system other neurons.
• The nucleus, which resides in the cell body and
The nervous system can be subdivided into the is the site of storage of the cell’s genetic code
central, peripheral and autonomic nervous systems. in DNA.
This chapter will concern itself principally with the Two naturally occurring pigments may be
central nervous system and secondarily with the observed to accumulate in the brain with age: lipo-
peripheral nervous systems. The principal compo- fuscin and neuromelanin. Both are generally
nents of the central nervous system are: believed to represent cellular waste products. Lipo-
• Neurons fuscin is a yellow-brown, autoflourescent, granular
• Glial cells substance composed of peroxidized protein and
© 2013 Elsevier Ltd
 354 17 Techniques in neuropathology

Oligodendrocyte
Dendrites

Nucleolus Myelin sheath

Nissl bodies

Nucleus

Axon
Cell body

Astrocyte

Blood vessel

Synapse

Figure 17.1 Diagram showing a neuron with its component parts together with other cells of the central nervous system. (Courtesy of Patrick
Elliott of the Medical Illustration Department, Royal Hallamshire Hospital, Sheffield, UK.)

lipids. It is seen in larger neurons, such as the lower oxidative metabolism of catecholamines (Sulzer
motor neurons of the spinal cord and pyramidal et al. 2008).
cells of the hippocampus in the context of Glial cells are the support cells of the nervous
Alzheimer pathology. Large amounts of lipofuscin- system. They are diverse in nature, the principal
like pigment accumulate in the context of inherited types being:
neuronal ceroid lipofuscinoses, of which Batten’s
disease is the most common (Goebel & Wisniewski • Astrocytes, which have a number of functions.
2004). Neuromelanin is most commonly seen in the They maintain the extracellular ion and
cytoplasm of neurons of the substantia nigra neurotransmitter balance. They are involved in
and the locus ceruleus. It is the cause of the macro- repair and scarring responses to brain damage
scopic pigmentation of these structures. Under and also form part of the blood–brain barrier,
the microscope, it is a dark brown, granular mate- which protects the brain from harmful blood-
rial that is believed to be the by-product of borne substances.
 Techniques for staining neurons 355

• Oligodendroglia, which form myelin, a

phospholipid sheath around nerve axons that
enhances the speed of conduction of impulses.
• Ependymal cells, which line the ventricles of
the brain and central canal of the spinal cord.
Neuropil is a term used to denote the feltwork of
neuronal processes in which neuron cell bodies
reside. Central nervous system tissue is classically
subdivided into gray matter, which contains the
majority of neuronal cell bodies and little myelin,
and white matter, which is predominantly formed
of myelinated axons and few neurons.
The meninges form three layers of protective cov-
ering over the brain. The outer layer, beneath the
Figure 17.2 Anterior horn cells in spinal cord. Notice their large
skull, is formed by the dura mater. It is a tough,
size and the prominent nucleolus. Paraffin section, stained with
fibrous membrane. The arachnoid mater is a more toluidine blue. Similar results can be obtained with cresyl fast
delicate, fibrillary covering that lies inside the dura violet.
mater and is more closely adherent to the brain
surface, but it does not invaginate into the surface
infoldings of the brain (or sulci). The pia mater is the
most delicate covering. It is closely apposed to the Motor neurons generally have very coarse
brain surface, following its contours down into the (‘tigroid’) Nissl substance, and regions such as the
depths of sulci. anterior horns of the spinal cord, where these cells
are abundant, are good tissues to use when learning
these stains (Fig. 17.2). For paraffin-embedded sec-
Techniques for staining neurons tions of formalin-fixed tissue, the cresyl fast violet
stain is reliable and relatively straightforward. As
such it is by far the most commonly used Nissl prep-
Tinctorial stains for Nissl substance
aration. Toluidine blue may also be used, whilst Ein-
Hematoxylin and eosin (H&E) preparations demon- arson’s gallocyanin method, being more suited to
strate most important features of neurons. However, alcohol-fixed tissue, is largely unused (Kellett 1963).
Nissl preparations are also popular for examining
the basic architecture of neural tissue and its com-
ponents. These are often combined with the luxol Cresyl fast violet (Nissl) stain for paraffin sections
fast blue myelin stain. Granules of Nissl substance Fixation
are found in the cell body (Fig. 17.1) and correspond Alcohol, Carnoy’s or formalin.
to rough endoplasmic reticulum. They are baso- Sections
philic due to the associated nucleic acid (Palay & Paraffin 7–10 µm or 25 µm (see Note b).
Palade 1955). Many basic dyes (e.g. neutral red,
Preparation of stain
methylene blue, azur, pyronin, thionin, toluidine
Cresyl fast violet 0.5 g
blue and cresyl fast violet) stain Nissl substance.
Distilled water 100 ml
Variation in the stain used, pH and degree of dif-
Differentiation solution
ferentiation allow preparations to label either Nissl
substance alone, or Nissl substance in combination Glacial acetic acid 250 μl
Alcohol 100 ml
with cell nuclei.
 356 17 Techniques in neuropathology

axons. Thus, antibodies to phosphorylated

Method
NF-H mark axons but not cell bodies in normal
1. Dewax sections and bring to water.
nervous system tissues. Antibodies to non-
2. Cover with filtered cresyl fast violet; stain for
10–20 minutes. phosphorylated neurofilament will label
3. Rinse briefly in distilled water. neuronal somata (Trojanowski et al. 1986).
4. Differentiate in 0.25% acetic alcohol until most of Microtubule-associated protein 2 (MAP-2) is a
the stain has been removed (4–8 seconds). protein involved with microtubule assembly
5. Briefly pass through absolute alcohol into xylene and is expressed by neurons in dendrites and
and check microscopically. cell bodies (Maccioni & Cambiazo 1995;
6. Repeat steps 4 and 5 if necessary, giving less Shafit-Zagardo & Kalcheva 1998). It is therefore
differentiation when repeating. often used to as a marker of neuroepithelial
7. Rinse well in xylene and mount in Canada balsam differentiation (Wharton et al. 2002; Blumcke
or DPX. et al. 2004).
Results
2. Cytoplasmic proteins. PGP9.5 and neuron-
Nissl substance purple-dark blue
specific enolase (NSE) are strongly expressed in
Neurons pale purple-blue
Cell nuclei purple blue neurons and can be reliably labeled by
commercially available antisera. Unfortunately,
Notes
they are not specific for neuronal cells, making
a. If only Nissl substance is required to be
interpretation tricky. They are best used in the
demonstrated, the stain is acidified with 0.25%
context of a broad antibody panel (Ghobrial &
acetic acid.
Ross 1986; Wilson et al. 1988).
b. Estimation of cortical neuronal density is made on
25 µm thick sections. 3. Neuronal nuclear proteins. NeuN is a neuron-
c. The cresyl violet method can be used as a specific DNA binding protein, which starts to
counterstain when demonstrating myelin with the be expressed around the time of initiation of
Kluver and Barrera method (see below).
terminal differentiation of the neuron (Mullen
et al. 1992). Antibodies to NeuN therefore label
neuronal nuclei, and neuronal components of
Immunohistochemistry of neurons other tumors (Edgar & Rosenblum 2008).
However, in the context of neuro-oncology, it
The protein targets of antibodies used in immuno-
lacks specificity, being expressed to a variable
histochemical preparations for the demonstration of
degree in a diverse range of primary brain
neuronal elements can be classified into four main
tumors. Therefore, NeuN is best used as part of
groups:
a panel of antibodies in the investigation of
1. Neuronal cytoskeletal proteins. Neurofilaments clear cell primary brain tumors, but is of
(NF) are intermediate filaments specifically limited utility for other tumors (Preusser et al.
expressed by mature neurons. They are 2006).
composed of protein subunits that are classified 4. Proteins associated with neurosecretory
by molecular weight into NF-L, NF-M, and granules. Antisera to these proteins can be
NF-H which may be variably phosphorylated useful to establish neuronal and
(Gotow 2000). Antisera raised against different neuroendocrine differentiation (Koperek et al.
neurofilament proteins in different states of 2004; Takei et al. 2007). Synaptophysin is a
phosphorylation are available. NF-H, in membrane glycoprotein component of
particular, and NF-M, to a lesser extent, are presynaptic neurosecretory vesicles. The cell
normally unphosphorylated in the neuronal body of normal neurons is usually unstained
cell body, but become phosphorylated in the by synaptophysin (Fig. 17.3), resulting in early
 Techniques for staining neurons 357

methods and are rarely performed in time-

constrained modern diagnostic laboratories. Immu-
nohistochemistry has largely replaced the old silver
preparations for the demonstration of axons as it is
reliable and produces adequate results for diagnos-
tic neuropathology with ease. However, for formal
quantitation of axons, many still find that Palmgren’s
method is superior to neurofilament immunohisto-
chemistry (Chance et al. 1999). This technique has
classically been used for staining axons of the
peripheral nervous system. However, it is also an
excellent preparation for staining central nervous
system axons as well. Palmgren’s method uses
potassium nitrate to suppress staining of reticulin. It
Figure 17.3 Large neuron from an area of cortical dysplasia is considered that with the Palmgren method, cresyl
stained with synaptophysin. Note intense staining within the violet preparations and immunohistochemistry for
neuropil and weak cytoplasmic staining.
neurofilament and MAP-2, there is no longer require-
ment for older silver preparations such as that of
Bielschowsky (Bielschowsky 1902) and Marsland
claims that cell body labeling was a feature of
(Marsland et al. 1954).
neoplastic neuronal cells that differentiated
Silver techniques, such as the Palmgren method,
them from native neurons (Miller et al. 1990).
require great care and attention to detail such as
However, it is now evident that a population of
clean glassware and pure distilled water for a suc-
normal neurons also show cell body labeling
cessful outcome. Stock solutions should be well
which detracts from the use of this feature as a
maintained and not more than a few months old (in
diagnostic marker (Quinn 1998). Synaptophysin
some cases less than a week).
is a useful marker of neuroendocrine
differentiation and so also stains cells in
metastatic neuroendocrine tumors
(Wiedenmann et al. 1987). Chromogranin A is a
protein of the dense core matrix of Modified Palmgren’s method for nerve fibers in
neurosecretory granules, and antibodies to it paraffin-embedded material (Palmgren 1948)
can be used to identify cells containing dense Fixation
core vesicles (Nolan et al. 1985). It is therefore Formalin fixed tissue.
used predominantly to elucidate Sections
neuroendocrine differentiation in tumors. Paraffin sections 6–10 µm. Sections should be on
coated slides.
Techniques for staining axons and Preparation of solutions
neuronal processes Acid formalin
Concentrated formaldehyde (40% w/v) 25 ml
The annals of neurohistology describe several Distilled water 75 ml
1% nitric acid 0.2 ml
methods to demonstrate various special structures
of the neuron, including axons (viable and degener- Silver solution
ate), dendrites, synapses, dendritic trees and periph- 30% silver nitrate 25 ml
eral nerve endings. Many of these were block 20% potassium nitrate 25 ml
5% glycine 0.5 ml
impregnation and free-floating frozen section
 358 17 Techniques in neuropathology

Reducer yellow to dark amber. It is important that at the

Pyrogallol 10 g reduction step the slides are gently agitated to ensure
Distilled water 450 ml an even reduction of the tissue; if the sections are not
Absolute ethanol 550 ml dark enough, they can be rinsed in distilled water and
1% nitric acid 2 ml steps 4–6 repeated but with a shorter time in the
Fixing bath silver solution.
5% sodium thiosulfate. The hotter the reducer, the faster the reduction
will take place and it may well be uneven, leading
Method
to suboptimal preparations. Sections can be toned
1. Take sections to distilled water. using gold chloride prior to fixing, which is an optional
2. Treat sections with acid formalin for 5 minutes. step.
3. Wash in three changes of distilled water for The original method used an intensifying step prior
5 minutes. to fixing: this employs aniline. Some have found this
4. Leave in filtered silver solution for 15 minutes at to be of little value. The method was originally
room temperature. designed for use with paraffin sections. However, it
5. Without rinsing, drain the slide and flood the can be applied to cryostat sections which have been
section with reducer that has been heated to pretreated with 20% chloral hydrate overnight prior to
40–45°C. Rock the slide gently and add fresh carrying out the Palmgren method.
reducer. Leave for 1 minutes. A beaker placed on
a hot plate is useful for this stage.
6. Wash in three changes of distilled water. Examine
Examination of axons in peripheral nerve in diag-
microscopically and, if necessary, repeat from
step 4, reducing the time in the silver solution and nostic neuropathology now relies largely on tolu-
decreasing the temperature of the reducer to idine blue-stained semi-thin resin-embedded tissue.
30°C. Capricious techniques such as Eager’s method for
7. Wash in distilled water. detecting degenerating axons are no longer in use
8. Fix in 5% sodium thiosulfate, 5 minutes. (Eager et al. 1971).
9. Wash in tap water. The Golgi preparation and its variants are excel-
10. Dehydrate in alcohol. Clear and mount in DPX. lent for the visualization of the three-dimensional
Result nature of the neuron and its dendritic processes.
Nerve fibers brown or black
However, modern diagnostic neuropathology prac-
tice has no requirement for this. Golgi techniques
Notes are occasionally used in research (e.g. Garey 2010)
In the original method the silver solution contained 5% although new antisera are increasingly allowing
acetic acid rather than 5% glycine.
immunohistochemical indices of these aspects of cell
The only proviso when using glycine is that it morphology. It is suggested that the interested
must be made up fresh prior to use; as it is only
reader consider the Pugh and Rossi modification for
stable for approximately one week. However the
Palmgren silver, once made up, is stable for several use on paraffin-embedded tissue (Pugh & Rossi
weeks. 1993) if a Golgi stain is to be attempted.
The silver incubation time may need to be
increased for tissues which have had a short formalin
fixation time, but as a rule of thumb you should see a Myelin
slight yellow tinge to the tissues when the optimum
time has been reached. The reducer keeps for several Myelin forms an electrically insulating sheath
months.
around axons. It is approximately 80% lipid and 20%
The original method stated that it had to stand for
protein and is formed from sheet-like processes of
24 hours before use, but this is not the case. The
reducer will darken with time, changing from pale
glial cells that are concentrically wrapped multiple
times around the axon. This greatly improves the
 Myelin 359

speed and efficiency of impulse conduction along

Luxol fast blue stain for myelin with cresyl violet
the axon. Myelin is formed by oligodendrocytes in counterstain (Kluver & Barrera 1953)
the central nervous system and Schwann cells in the
Fixation
peripheral nervous system. A single oligodendro-
Formalin.
cyte may myelinate multiple axons in its vicinity,
whereas a single Schwann cell may only myelinate Sections
a single segment of a single axon. Loss of myelin (as Paraffin, 10–15 µm.
is seen in multiple sclerosis in the central nervous
Preparation of solutions
system or Guillain-Barré in the peripheral nervous
Luxol fast blue
system) can be severely debilitating. Luxol fast blue 1g
Modern tinctorial stains for myelin are simple and Methanol (absolute) 1000 ml
reliable and can be performed on formalin-fixed 10% acetic acid 5 ml
paraffin-processed tissue. Many can be combined Mix reagents and filter.
with a Nissl stain to demonstrate neuronal localiza-
Cresyl violet stock solution
tion. Older methods may give more even and con-
Cresyl violet 0.5 g
sistent staining, but are considerably more time
Distilled water 100 ml
consuming and have fallen from use (Weigert 1904;
Loyez 1910; Weil 1928). Both luxol fast blue and Acidified cresyl violet solution
solochrome cyanine preparations are now favored. Add 0.8 ml of 10% acetic acid to 100 ml of stock
cresyl violet solution and filter before use.
Luxol fast blue is a copper phthalocyanine dye
which is employed in myelin staining of paraffin- Method
processed tissue (Kluver & Barrera 1953). This can 1. Take sections on slides to 95% alcohol (not
be combined with cresyl violet or hematoxylin to water).
outline cellular architecture (Fig. 17.4) or with peri- 2. Stain in luxol fast blue solution, 2 hours at 60°C,
odic acid-Schiff (PAS) to demonstrate myelin degra- or 37°C overnight.
dation products in demyelinating disease. It can be 3. Wash in 70% alcohol.
used on central nervous system tissue only. 4. Wash in tap water.
5. Differentiate in 0.1% lithium carbonate solution
until the gray and white matter are distinguished.
This may be more easily controlled by using
0.05% lithium carbonate followed by 70–95%
alcohol instead.
6. Wash in tap water.
7. Check differentiation under the microscope.
Repeat step 5 if necessary.
8. Stain in cresyl violet solution, 10–20 minutes.
9. Drain sections and transfer to 70% alcohol. Avoid
placing the section in water at this stage as the
cresyl violet staining loses some of its intensity.
Gently agitate the sections; the cresyl violet dye
will flood out. The 70% alcohol differentiates the
cresyl violet stain. Optimally, the cresyl violet
should be removed, leaving the cell bodies and
Nissl clearly visible. Do not over-differentiate; the
70% alcohol will take out the cresyl violet and to a
Figure 17.4 Macro section of a human hippocampus certain extent the luxol fast blue. The cresyl violet
demonstrating geographical variation in myelin content. Luxol fast counterstain will deepen the color of the luxol fast
blue.
 360 17 Techniques in neuropathology

blue stained myelin from turquoise to a deep 4. Differentiate in 5% iron alum until all the nuclei are
blue. unstained. Wash frequently in distilled water, and
10. Dehydrate, clear in xylene, and mount in DPX. examine microscopically.
5. Wash in running tap water.
Result
6. Counterstain if desired.
Myelin blue
Cells violet-pink 7. Dehydrate, clear, and mount.
Result
Notes
a. If the section is over-differentiated with lithium Myelin sheaths blue
carbonate/alcohol, the section can be restained Notes
with the luxol fast blue and then differentiated to a. The staining solution keeps well.
obtain the optimum staining result. This may apply
b. Neutral red, Neutral fast red, Piero-Ponceau S, or
to tissues which have very low amounts of myelin
van Gieson can be used for counterstaining.
(e.g. baby/neonatal brains). These tissues can be
very challenging in achieving optimum staining.
Unfortunately, once the cresyl violet counterstain
has been applied the over-differentiation cannot be Immunohistochemistry for S-100 is useful in the
rectified.
diagnosis of tumors derived from Schwann cells
b. Some histologists prefer to differentiate the cresyl
both in the central nervous system and peripherally
violet using 0.25% acetic acid in 100% alcohol.
(Hirose et al. 1986; Winek et al. 1989). Many antisera
c. The use of thick sections is important for the
are used as markers of myelination, the most useful
visualization of myelin tracts.
being myelin basic protein and myelin associated
d. Other counterstains may be used such as neutral
red. This will result in the myelin appearing purple/ glycoprotein (Itoyama et al. 1980a, 1980b; Ludwin &
blue due to a slightly different color balance, and Sternberger 1984; Lindner et al. 2008). However,
is a matter of personal preference. given the reliability and simplicity of the tinctorial
The solochrome cyanine stain is a simple and stains, immunohistochemical myelin makers are
rapid technique for demonstration of myelin in both largely unused in routine diagnostic neuropathology
the central and peripheral nervous systems. and remain the preserve of research laboratories.
Myelin loss may occur in a region of brain
damaged by any of a number of processes such as
trauma, neoplasia, multiple sclerosis or toxic insult.
Page’s solochrome cyanine technique for myelin in It may also occur secondary to the loss of axons
paraffin sections emanating from a lesioned brain region. In modern
Fixation practice, degeneration of myelinated tracts is
Formalin. most commonly demonstrated by showing loss of
Sections normal myelin staining by either luxol fast blue or
Paraffin, 6–10 µm. Cryostat section, 10 µm. solochrome cyanine preparations, or by showing a
microglial reaction using CD68 immunohistochem-
Preparation of solution
istry (e.g., Ince et al. 2008). Historically, the Marchi
Solochrome cyanine RS 0.2 g
technique (Swank & Davenport 1935) and neutral
Distilled water 96 ml
10% iron alum 4 ml lipid stains have been used to detect early and
Concentrated sulfuric acid 0.5 ml late myelin degeneration products, respectively.
However, the Marchi technique requires block stain-
Method
ing or free-floating sections and lipid stains cannot
1. Take sections to water.
be performed on paraffin-embedded material. Given
2. Stain for 10–20 minutes at room temperature.
these issues, the sensitivity of CD68 immunohisto-
3. Wash in running water.
chemistry and the ease of the tinctorial preparations
 The neuroglia 361

for normal myelin (see above), the Marchi and

neutral lipid techniques have been rendered
obsolete.

The neuroglia

The term neuroglia refers to the supporting cells

of the central nervous system and comprises
ependymal cells, astrocytes, oligodendrocytes, and
microglia. As is becoming a recurrent theme, immu-
nohistochemistry is increasingly replacing tinctorial
stains for their identification.

Figure 17.5 Reactive astrocytes in white matter, stained by

Ependymal cells anti-GFAP immunohistochemistry technique with hematoxylin
nuclear counterstain. Fine GFAP-containing processes form a
Ependymal cells are epithelioid and line the ventri- felt-like mat in which the stellate cell bodies are evident.
cles of the brain and the central canal of the spinal
cord. They are easily located with conventional
stains such as H&E and immunohistochemistry for Astrocytes are principally classified into proto-
GFAP, vimentin and S-100. Immunohistochemistry plasmic and fibrous forms. These are similar in func-
for epithelial membrane antigen (Uematsu et al. tion. However, whereas protoplasmic astrocytes
1989; Hasselblatt & Paulus 2003) labels both normal have shorter, thicker, highly branched processes and
and neoplastic ependymal cells, whilst are generally found in the gray matter, fibrous astro-
cytokeratin markers are negative. cytes have longer, thinner, less-branched processes
and usually reside in white matter. Astrocytic reac-
Astrocytes tions in the cerebellum are characterized by Berg-
mann or radial astroglia which have processes that
Astrocytes have multiple, fine processes and (in run radially from the Purkinje cell layer of the cortex
their reactive state) are ‘star-shaped’ (hence the to the pial surface.
name). On standard H&E sections, only the nucleus In response to injury of the brain parenchyma,
of resting astrocytes is distinct, as the cell body astrocytes react by increasing in size with a more
cannot be discerned from background neuropil. prominent, eosinophilic cytoplasm. The nucleus
These nuclei are slightly larger with more open moves from a central to a more eccentric position
granular chromatin than that of the more compact within the cell cytoplasm and processes become
oligodendrocyte. Modern neuropathology relies more prominent. Astrocytic gliosis is a response to
most heavily on GFAP (Fig. 17.5) immunohisto- permanent injury, whereby astrocytes proliferate to
chemistry for demonstration of astrocytes, although fill tissue defects with a fibrous glial scar.
antibodies to S-100, αB-crystallin and glutamine In neuro-oncology, astrocytic differentiation is
synthetase may also be used. Metal-based and tinc- best demonstrated by GFAP immunohistochemistry.
torial methods such as Cajal’s gold sublimate, PTAH GFAP immunoreactivity is also seen in other tumors
(Chan & Lowe 2002) and Holzer (1921) are no longer including ependymoma, oligodendroglial tumors
in use as these are variously more expensive, more and choroid plexus tumors (Eng & Rubinstein 1978;
technically demanding or less specific than their Velasco et al. 1980; Eng 1983; Doglioni et al. 1987).
immunohistochemical equivalents. Astrocytic tumors also label with vimentin and
 362 17 Techniques in neuropathology

S-100, but these are also seen in many other tumor However, these epitopes are not expressed by oli-
types, rendering them of little use for differential godendroglial tumors (Nakagawa et al. 1986).
diagnosis. Astrocytes occasionally show cross-reac- Olig2 is a transcription factor that regulates oligo-
tivity as seen for the pan cytokeratin AE1/AE3 (Cos- dendroglial development and is expressed by the
grove et al. 1989). Therefore, it is expedient to use nuclei of oligodendrocytes and oligodendroglial
other cytokeratin markers such as CAM5.2 or tumors (Yokoo et al. 2004). Sadly, it is not specific
MNF116 to exclude the diagnosis of epithelial cell for oligodendrogliomas as it labels other morpho-
tumors, such as metastatic adenocarcinoma. logically similar tumors (Preusser et al. 2007) and is
The proliferation marker Ki-67 (MIB-1) is often also expressed by astrocytomas (Ligon et al. 2004).
used in the assessment of surgical neuropathology A reliable immunohistochemical marker to distin-
specimens as an aid to grading tumors and to help guish oligodendroglioma from astrocytomas has
differentiate reactive from neoplastic astrocytic pop- not yet been found. Finally, deletion of chromo-
ulations. The latter will tend to have a higher number somes 1p and 19q (most commonly investigated by
of nuclei labeled with this marker. fluorescence in situ hybridization) is a well-
An emerging and potentially more powerful tool recognized molecular feature of oligodendroglio-
for the diagnosis of diffuse oligodendroglial and mas and appears to be associated with a better
astrocytic neoplasms is the use of antibodies to iso- prognosis and response to treatment (Bourne &
citrate dehydrogenase 1 (IDH1) carrying the R132H Schiff 2010).
mutation. This is the most frequent mutation in
diffuse gliomas (Hartmann et al. 2009). There is an Microglia
emerging literature that appears to demonstrate that
antisera to this mutant protein may be used to dif- Unlike other glial cells, microglia are mesodermal in
ferentiate reactive gliosis from grade II and III astro- origin. They are believed to be derived from blood-
cytomas (Camelo-Piragua et al. 2010; Capper et al. derived monocytes that move into the brain during
2010) and oligodendrogliomas from lesions with embryonic development (Kim & de Vellis 2005).
similar morphologic appearances (Capper et al. Microglia serve as the resident innate immune
2011). system and, under certain pathological conditions,
may develop into full-blown macrophages. They are
involved in most, if not all, known forms of CNS
Oligodendrocytes pathology (Graeber & Streit 2010). Microglia are
most commonly subclassified into resting, activated
Oligodendrocytes are glial cells that form the and ‘amoeboid’ forms. Resting microglia are classi-
myelin of the central nervous system and are cally ramified in morphology, whilst activated
present in both gray and white matter. As noted microglia are rod-shaped and amoeboid are (as the
above, a single oligodendrocyte may myelinate name suggests) amoeboid.
axons from multiple neurons. In H&E and cresyl A number of established immunohistochemical
violet preparations, oligodendrocytes have small markers for microglia are available, including CD68
(7 µm) round to oval nuclei with compact chroma- (PGM1), human alveolar macrophage (HAM)-56,
tin. The cytoplasm is indistinct from the surround- class II major histocompatibility complex (MHC;
ing neuropil although oligodendroglial tumors particularly in inflammatory states) and HLA-DR-II
may show artifactual perinuclear halos in paraffin antibodies. Although these do not label other glial
sections. Silver preparations to demonstrate oligo- cells, they do label infiltrating macrophages from the
dendroglia (Penfield 1928; Stern 1932) are rarely circulation. Non-immunohistochemical prepara-
used, now replaced by immunohistochemistry to tions, such as that of Penfield (Penfield 1928) and
myelin basic protein and myelin associated glyco- Weil and Davenport (Stern 1932) are not specific,
protein label oligodendrocyte processes (see above). and they are no longer used.
 Neurodegeneration 363

In the majority of cases, neuropathological assess-

Neurodegeneration ment of neurodegeneration tends to broadly focus
on dementing illnesses and motor degeneration and
Neurodegenerative conditions are largely diseases
there is considerable overlap between the two.
of old age. As the population ages, these conditions
Investigations of dementia tend to uncover one, or
place an increasing burden on health and social
a combination of, three types of pathology, namely:
care systems. This, together with the escalation in
Alzheimer’s disease; vascular dementia (multi-
research into neurodegeneration in recent years, has
infarct dementia); or dementia with Lewy bodies. A
resulted in an increasing workload on neuropathol-
small number of dementia cases show frontotempo-
ogy units.
ral lobar degeneration. The neurodegenerative dis-
Sadly, it is often the case that a definitive diagnosis
eases of the motor system that are diagnosed at
of any neurodegenerative condition cannot be made
autopsy tend to focus on motor neuron disease (also
without autopsy. In most studies, the accuracy of
known as amyotrophic lateral sclerosis) and condi-
clinical diagnosis of the cause of a dementing illness
tions that cause Parkinsonian clinical features. Other
is in the order of 75%. An autopsy does not benefit
neurodegenerative diseases of the motor system
the deceased, but does have wider benefits, namely:
(e.g., Huntington disease, spinocerebellar ataxia and
• Neuropathological autopsies can yield data and Friedreich’s ataxia) tend to be diagnosed by genetic
tissue to assist research. tests.
• Neuropathological autopsies provide The microscopic examination of the brain for
epidemiological data, allowing the prevalence neurodegeneration is often an iterative process,
of different neurodegenerative diseases to be whereby an initial examination is performed using
monitored. fairly standard tinctorial preparations (most favor
• Autopsy findings are often of considerable H&E). After this, more specialist preparations
educational benefit for both senior and junior (usually immunohistochemistry) are performed
clinicians as well as pathologists. (Lowe 1998).
• An increasing number of neurodegenerative Many neurodegenerative diseases are character-
conditions are familial, often with known ized by accumulations (or inclusions) of protein, for
causative mutations. Accurate the majority of which there are now commercially
neuropathological characterization can available antisera. These diseases are thus often clas-
therefore guide genetic counseling. sified by the particular protein that forms the patho-
The pathological characterization of neurode­ logical aggregates that characterize the disorders.
generative disease is a staged process. The first These categories are: the tauopathies (e.g. Alzheim-
step is removal of the brain, sometimes with the er’s disease, Pick’s disease, supranuclear palsy, cor-
spinal cord in addition. These are examined fresh ticobasal degeneration and argyrophilic grain
and samples may be taken and snap-frozen for disease), the synucleinopathies (Parkinson’s disease,
studies that require unfixed tissue. The brain is dementia with Lewy bodies and multiple system
then fixed in formalin, ideally by suspending it by atrophy), prion disorders and TDP-43 proteinopa-
the basilar artery in a large volume of formalin for thies (motor neuron disease, frontotemporal lobar
three or more weeks. Other methods, such as degeneration with TDP-43). These inclusions are
simply allowing the brain to rest on the bottom of detailed in Table 17.1.
the formalin container result in unacceptable dis- Ubiquitin is a small regulatory protein that binds
tortion. Following formalin fixation, the brain is misfolded or other aberrant proteins, and labels
examined macroscopically both intact and after them for destruction by the proteasome. Many
slicing (usually coronally). Appropriate blocks of dementing illnesses are characterized by accumula-
tissue are taken, processed and paraffin embedded tions of ubiquitylated protein; the location and
for microscopy. form of these accumulations can provide valuable
 364 17 Techniques in neuropathology

Table 17.1 Inclusion body immunostaining

Inclusion Disease Immunohistochemistry
Neurofibrillary tangle (Fig. 17.6a, b) Alzheimer’s disease Tau protein, ubiquitin/p62
Lewy body (Fig. 17.7a) Parkinson’s disease α-Synuclein, ubiquitin/p62
Cortical Lewy body (Fig. 17.7b) Dementia with Lewy bodies α-Synuclein, ubiquitin/p62
Motor neuron disease/FTLD inclusion Motor neuron disease, some Ubiquitin/p62, TDP-43
(Fig. 17.8) forms of FTLD
Glial cytoplasmic inclusion Multiple system atrophy α-Synuclein, ubiquitin,
p62
Glial cytoplasmic inclusion Progressive supranuclear palsy tau, ubiquitin/p62
Corticobasal degeneration
Pick body Pick’s disease Tau protein, ubiquitin/p62

diagnostic clues. Therefore, immunohistochemistry aggregates of peptides generated by the cleavage of

for ubiquitylated proteins is often a useful early step amyloid precursor protein (a membrane-associated
in neuropathological diagnosis. For this, we favor protein of unknown function) by β- and γ-secretases.
antibodies to p62. This protein binds ubiquitylated Deposits of β-amyloid become surrounded by
proteins and shuttles them to the proteasome dilated and distorted neuronal processes to form
(Wooten et al. 2006). Antibodies to p62 can be used senile plaques. Senile plaques and neurofibrillary
to label pathological, ubiquitylated aggregates of tangles are the histological hallmarks of Alzheimer’s
tau, α-synuclein and TDP-43 (Kuusisto et al. 2008). disease (Figs 17.6 and 17.9).
Further, p62 immunohistochemistry has greater Vascular dementia is an umbrella term for a variety
specificity for pathological aggregates than ubiqui- of conditions characterized either by multiple large
tin immunohistochemistry, leaving non-pathological or small regions of infarction throughout the brain,
features unlabeled. or by smaller numbers of infarcts in functionally
As noted above, autopsies in neurodegeneration important structures such as the thalamus or hip-
principally concern dementia and motor system pocampus (Jellinger 2008). The picture is somewhat
degenerative diseases, many of which overlap. Of complicated by the fact that many cases with a
the dementing illnesses, the vast majority are diag- heavy burden of vascular pathology will addition-
nosed as Alzheimer’s disease, vascular dementia, ally have a coexistent burden of Alzheimer- or Lewy
dementia with Lewy bodies and frontotemporal body-type pathology.
lobar degeneration. However, given the consider- Dementia with Lewy bodies is characterized by neo-
able public health concerns surrounding prion dis- cortical and limbic neuronal cytoplasmic inclusions
eases, cases will also occasionally be assessed for of α-synuclein – the Lewy bodies that give this con-
consideration of this diagnosis. dition the name. Lewy bodies were first described
Alzheimer’s disease is characterized by ubiquity- in Parkinson disease, where they are visible as
lated accumulations of tau and β-amyloid. Hyper- round, intensely eosinophilic, hyaline intracytoplas-
phosphorylated tau forms flame-shaped neuronal mic neuronal inclusions in the substantia nigra and
intracytoplasmic inclusions known as neurofibril- locus ceruleus of the midbrain and brainstem. They
lary tangles and neuritic fibrillary deposits known are composed of ubiquitylated α-synuclein and can
as neuropil threads. β-Amyloid is formed from therefore be detected by immunohistochemistry for
 Neurodegeneration 365

a b
Figure 17.6 (a) Tangles in neurons stained by the Gallyas silver technique. (b) Tangles in neurons stained by immunohistochemistry for
phosphorylated tau (AT8). In the background large numbers of neuropil threads can be seen.

antisera to these proteins or p62 (Lowe et al. 1993; transmissible nature and because of the risk of trans-
Dickson 1999; Goedert 1999; Kuusisto et al. 2008). mission to laboratory staff.
Frontotemporal lobar degeneration (FTLD) is an The normal function of prion protein is unclear,
umbrella term for a number of neuropathological and yet it is a normal constituent of cell membranes
entities, all of which predominantly manifest clini- and most highly expressed in neurons. When mis-
cally as frontotemporal dementia. The different folded, it is capable of causing disease. In humans,
forms of FTLD are classified by the immunoreactiv- most cases are sporadic (largely Creutzfeldt-Jakob
ity of the proteinaceous intracytoplasmic inclusions disease, CJD), approximately 10% of cases are famil-
that characterize them (Cairns et al. 2007; Mackenzie ial and a very few have been caused by medical
et al. 2009, 2010). However, this is a complex and intervention. Prion disease due to the consumption
constantly evolving field and classifications tend to of matter containing misfolded prion proteins is
rapidly become outdated in their finer details. In implicated in variant CJD (vCJD) and (historically)
essence, the majority of cases are classified as tauop- kuru (Johnson 2005).
athies (due to their accumulations of intracytoplas- Clinically, sporadic CJD is characterized by a
mic tau). Of the non-tauopathies, most cases show rapidly progressive multifocal neurological dys-
intracy­toplasmic inclusions of ubiquitylated, phos- function, sleep disturbance, myoclonic jerks, ataxia
phorylated TDP-43 (designated FTLD-TDP), and a and a terminal severe globalized cognitive impair-
few are characterized by inclusions of FUS/TLS or ment. Death occurs after approximately 8 months
neurofilament. A small residuum are characterized (Johnson 2005).
by intracytoplasmic inclusions with immunoreactiv- Pathologically, CJD is characterized by neuronal
ity to p62 and ubiquitin alone (FTLD-UPS), or no loss, gliosis, spongiform change (giving them their
observable reactivity at all (so-called dementia alternative name of spongiform encephalopathies)
lacking distinctive histology). and the deposition of protease-resistant prion
The prion diseases are rare neurodegenerative dis- protein. From a practical perspective, it should be
orders. They are of considerable interest due to the noted that prion protein is a normal constituent of
inherited nature of some forms, the tragic and rapid the nervous system. The pathological form is there-
progression of these conditions, the public health fore detected by first treating sections with a
monitoring of these conditions on account of their protease enzyme to eradicate immunoreactivity to
 366 17 Techniques in neuropathology

a b
Figure 17.7 (a) H&E staining of substantia nigra from a patient with Parkinson’s disease. The brown color is normal neuromelanin. Two
neurons contain Lewy bodies – rounded eosinophilic inclusions with a pale ‘halo’ around them. (b) Immunohistochemistry for α-synuclein
showing Lewy bodies and Lewy neurites in the amygdala of a case of dementia with Lewy bodies.

a b
Figure 17.8 Motor neuron disease (amyotrophic lateral sclerosis). (a) p62 immunohistochemistry showing a skein-like cytoplasmic
inclusion in a lower motor neuron. (b) TDP-43 immunohistochemistry showing two motor neurons with ‘compact’ cytoplasmic inclusions.

normally folded, physiological prion protein. This which are characterized by neuronal intracytoplas-
leaves immunoreactivity only for pathological, mic accumulations of hyperphosphorylated tau,
misfolded, protease-resistant prion protein. and multiple system atrophy, which is character-
Parkinsonism is characterized by tremor, hypoki- ized by glial cytoplasmic inclusions of α-synuclein.
nesia, rigidity, and postural instability. It is most Most cases of motor neuron disease, in common
commonly caused by Parkinson’s disease, which is with FTLD-TDP, are characterized by neuronal and
pathologically characterized by Lewy bodies in glial cytoplasmic accumulations of hyperphosphor-
brainstem and midbrain structures (see above and ylated, ubiquitylated TDP-43 (Fig. 17.8).
Fig. 17.7). Less common causes include progressive Following consideration of the principal forms
supranuclear palsy and corticobasal degeneration, of dementia as described above, it will be apparent
 Neurodegeneration 367

that in order to provide a full diagnostic service a

Gallyas method for Tau pathology (Gallyas 1971)
laboratory should have access to optimized immu-
This method gives excellent staining of neurofibrillary
nohistochemistry for hyperphosphorylated tau, β-
tangles, and the neuritic pathology surrounding
amyloid, α-synuclein, p62 or ubiquitin (ideally the plaques, although the amyloid component itself is
former), TDP-43, neurofilament and protease- unstained. It may also be used for a number of other
resistant prion protein. neurodegenerative diseases (especially argyrophilic
grain disease).
Fixation
Stains for detection of the changes of Formalin-fixed tissues.
Alzheimer’s disease Sections
Paraffin-processed sections, 8 µm thick.
As noted above, the two types of Alzheimer-
Solutions
type pathology are (1) features associated with
hyperphosphorylated tau (principally neurofibril- 1. 5% periodic acid
lary tangles and neuropil threads) and (2) features 2. Alkaline silver iodide solution
Sodium hydroxide 40 g
associated with β-amyloid (principally neuritic
Potassium iodide 100 g
plaques and congophilic amyloid angiopathy). The
Distilled water 500 ml
tinctorial stains and immunohistochemical prepara- 1% silver nitrate 35 ml
tions used in the characterization of Alzheimer-type Dissolve the sodium hydroxide in water, then add
pathology reflect this dichotomy: the potassium iodide and wait until dissolved. Slowly
• Classically, Cross-modified Palmgren (Cross add the silver nitrate and stir vigorously until clear.
1982) and the Gallyas technique (Gallyas 1971) Then add distilled water to give a final volume of
1000 ml.
have been used to demonstrate neurofibrillary
tangles as well as the neuritic pathology 3. 0.5% acetic acid
associated with neuritic plaques. These 4. Developer working solution
have largely been replaced by Add 3 volumes of stock solution II to 10 volumes of
stock solution I. Stir and add 7 volumes of stock
immunohistochemistry for
solution III. Stir and wait to clear.
hyperphosphorylated tau (Fig. 17.6b).
Stock solution I
• β-amyloid has historically been demonstrated
Sodium carbonate (anhydrous) 50 g
by methenamine silver (which will also stain a
Distilled water 1000 ml
minority of tangles; Fig. 17.9a) and thioflavine
Stock solution II (dissolve consecutively)
S. These have been replaced by
Distilled water 1000 ml
immunohistochemistry after formic acid
Ammonium nitrate 2g
pretreatment (e.g. BA4; Fig. 17.9b). Silver nitrate 2g
• Some preparations can be used to detect both Tungstosilicic acid 10 g
forms of pathology, although these tend to be Stock solution III (dissolve consecutively)
less sensitive than those preparations that Distilled water 1000 ml
focus on one pathology. Thus, the modified Ammonium nitrate 2g
Bielschowsky technique underestimates Silver nitrate 2g
the β-amyloid pathology load (Lamy et al. Tungstosilicic acid 10 g
Formaldehyde (conc.) 7.3 ml
1989).
5. 0.1% gold chloride
Guidelines for the dissection and staining of speci-
6. 1% sodium thiosulfate (‘hypo’)
mens in order to accurately characterize Alzheimer-
7. 0.1% nuclear fast red in 2.5% aqueous
type pathology have been laid down (Mirra et al.
aluminum sulfate
1993; Alafuzoff et al. 2008; Alafuzoff et al. 2009).
 368 17 Techniques in neuropathology

a b
Figure 17.9 Methenamine silver (a) and immunohistochemistry for β-amyloid (b), showing senile plaques in cerebral cortex from cases of
Alzheimer’s disease.

Method 10. Rinse in distilled water.

1. Take sections to distilled water. 11. Place in 1% sodium thiosulfate solution for
2. Place in 5% periodic acid for 5 minutes. 5 minutes.
3. Wash in distilled water for 5 minutes twice. 12. Wash in tap water.
4. Place in alkaline silver iodide solution for 13. Counterstain in 0.1% nuclear fast red for
1 minute. 2 minutes.
5. Wash in 0.5% acetic acid for 10 minutes. 14. Wash in tap water.
6. Place in developer solution (prepare immediately 15. Dehydrate, clear, and mount in DPX.
before use) for 5–30 minutes.
7. Wash in 0.5% acetic acid for 3 minutes. Results
8. Wash in distilled water for 5 minutes. Neurofibrillary tangles and plaque neurites black
9. Place in 0.1% gold chloride for 5 minutes. Nuclei red
 Neurodegeneration 369

Methenamine silver method for senile plaques (Yamaguchi Sections

et al. 1990) Paraffin sections, cut at 6–8 µm.
This preparation has largely been superseded by βA4
immunohistochemistry. It stains amyloid plaques well, Solutions
but detects only a subset of neurofibrillary tangles. 20% silver nitrate solution
Silver nitrate 20 g
Fixation Distilled water 100 ml
Formalin fixed.
Developer
Sections
Formalin 20 ml
Paraffin, 8 µm. Distilled water 100 ml
Solutions Concentrated nitric acid 1 drop
Working solution Citric acid 0.5 g
5% hexamine 50 ml
0.2% ammonia washing solution
5% sodium tetraborate 2.5 ml
Ammonia 0.2 ml
5% silver nitrate 2.5 ml
Distilled water 100 ml
Add the reagents in the above order, i.e. silver nitrate
last. ‘Hypo’
1% sodium thiosufate.
10% formalin in tap water
Method Method
1. Take sections to water. 1. Take sections to water.
2. Rinse in distilled water. 2. Place slides in 20% silver nitrate for 20 minutes in
3. Place sections in working solution for 3–4 hours a refrigerator (4oC).
at 60°C. 3. Place slides in distilled water while performing
4. Check microscopic appearance of plaques and step 4 below.
tangles at regular intervals until stained black. 4. To the 20% silver nitrate (used in step 2) add
5. Rinse in distilled water. ammonia, drop by drop, stirring vigorously until
6. Place sections in 10% formalin in tap water for precipitate turns clear. Add two more drops of
5 minutes. ammonia. Return slides to this solution for
7. Rinse in tap water. 15 minutes (4oC) in a fridge.
8. Place sections in 5% sodium thiosulfate for 5. Place slides in 0.2% ammonia. This is a holding
5 minutes. step whilst the working developer solution is
made.
9. Rinse in tap water.
6. To 50 ml of the ammonical silver solution
10. Dehydrate, clear, and mount in DPX.
(from step 4), add 3 drops of the developer
Results solution; place this in a clean Coplin jar. Drain the
Amyloid plaques black slides of the ammonia wash and place in the
Some tangles (rare) black ammonical silver/developer solution. The
Background yellow-brown development time will vary between 2 and 5
minutes approximately, depending on the ambient
temperature. As the development proceeds the
Modified Bielschowsky method for plaques and tangles tissue will turn a golden brown. Check
(Litchfield & Nagy 2001) microscopically until the tangles and plaques are
optimally demonstrated.
This preparation is a reasonable compromise
between plaque and tangle labeling. It can therefore 7. Wash in distilled water.
be used for diagnostic purposes. 8. Fix sections in ‘hypo’ for 5 minutes
9. Wash in distilled water.
Fixation
10. Dehydrate, clear, and mount in DPX.
Formalin fixed.
 370 17 Techniques in neuropathology

samples of tissue from the CNS are coming (or

Result
have already come in many countries, including
Tangles black
Plaques black the UK) to represent the bulk of neuropathological
Background brown practice.
The majority of these specimens are taken for the
Notes
diagnosis and/or treatment of neoplasia. The tissue
a. Some laboratories have found that variable
samples are often small and require very careful
ambient room temperature causes variability and
inconsistency in staining. Performing the silver and handling. Some centers prefer neurosurgical biop-
ammonical silver stages at 4°C overcomes this sies to be placed on a sterile polythene sheet which
problem. If this is not a concern for a particular is folded over the specimen to reduce drying, con-
laboratory, these steps can be performed at room tamination or loss. Other centers prefer samples to
temperature. be placed directly into a clean pot. The biopsies
b. The original method used separate solutions of should be transported rapidly to the neuropathol-
20% silver at steps 1 and 4; however, with
ogy laboratory. If this is not possible, they may be
meticulous technique it is more cost-effective to
placed in formalin fixative. Adequate formalin
use the same silver solution throughout.
should be used (10 times the volume of tissue). On
arrival at the laboratory, portions of tissue may be
sampled for intraoperative diagnosis by smear, snap
Neuropathology laboratory frozen for molecular analysis or intraoperative diag-
specimen handling nosis of cryostat sections, or fixed in glutaraldehyde
for electron microscopy. Following this, the remain-
Molecular neuroscience has flourished in recent der of the specimen is fixed in formalin.
years and neuropathology laboratories find that Poorly handled tissue is a source of frustration
they are only one part of a larger team dedicated to for any neuropathology service. Anyone who has
the diagnosis and characterization of neurological worked in such a laboratory will be well acquainted
disease. Thus, the neuropathology service remains with the small tube into which a large tumor has
central to the handling and storage of tissue as well been stuffed that requires a second ‘neurosurgical’
as the procurement of that tissue (either by biopsy procedure to extract it, piecemeal, poorly fixed and
or autopsy) and must now do this in a manner that terribly disrupted. The hopelessly desiccated frag-
facilitates the work of other disciplines that use the ment of tissue stuck to the bottom of a glass tube
tissue. that has taken several hours to reach the laboratory
The principal histological specimens submitted to is also well known. We could go on, but instead beg
or taken by the neuropathology service are: that histology staff strive to educate their neurosur-
• Neurosurgical biopsies and excision specimens. gical and theater colleagues in the appropriate
• Peripheral nerve biopsies. handling of tissue!
• Muscle biopsies. See Appendix I and previous
edition. Intraoperative diagnosis from smear preparations
• Brain, spinal cord and other specimens taken at and frozen sections
autopsy.
The histological and cytological detail that can be
achieved from smear and frozen section prepara-
Brain and spinal cord biopsies and tions is substantially less than can be achieved from
excision specimens paraffin-based histology. Therefore, diagnoses made
by these techniques can only ever be viewed as an
Given the declining autopsy rate that is seen inter- approximation and subject to review following par-
nationally (Burton & Underwood 2007), surgical affin histology. As there is often only a small amount
 Neuropathology laboratory specimen handling 371

of tissue available, it is possible that performing an such that pressure on the nerve at one point will
intraoperative diagnosis may compromise the ability result in pressure fluctuations that cause disruption
to achieve a definitive diagnosis on paraffin-based at locations away from the site of compression. The
histology as a feature that is crucial for diagnosis fragility of nerve biopsies make it imperative that
may be lost in the tissue used for smear or frozen they should be performed only by specialist staff
section. It is therefore vital that prior to making (usually a neurosurgeon or neurologist) who are
smear or frozen section preparations from small trained and experienced in this procedure. The
biopsies the neuropathology staff are certain that number of practitioners should be minimized so as
this is necessary. to concentrate the expertise in these individuals.
Representative, small pieces of tissue (approxi- Useful technical guidelines for the taking, process-
mately 1 mm in diameter) are dissected from the ing and interpretation of nerve biopsies have been
biopsy and placed at one end of a plain glass micro- published (Sommer et al. 2010).
scope slide. A second glass slide is used to crush the Peripheral nerve biopsy should only be per-
specimen and is then drawn across the slide to formed after consultation between the laboratory
produce a uniform smear. Unlike blood film prepa- and clinical teams. A minimum of 20 mm, and
ration, the two slides are held flat together during preferably 30 mm, of nerve should be taken.
smearing, maintaining a gentle and even pressure. Ideally, a member of the neuropathology technical
Alternatively, the tissue may be lightly touched to a staff should attend the biopsy room in order to
single glass slide, allowing a few cells to adhere to receive the specimen as soon as possible. This
the slide in order to minimize handling artifacts. should be placed gently on a piece of dry card, to
Fresh postmortem tissue can be used to practice this which it will naturally adhere. The nerve should
method and gain familiarity with normal appear- not be put in fixative, sutured, clamped, stapled,
ances. The slides are immediately fixed in acetic pinned or traumatized in any way.
alcohol (Wolman’s solution) and stained with H&E, On arrival at the laboratory, a fresh scalpel blade
aqueous toluidine blue or both. We find that the is used to divide the biopsy into at least three seg-
former gives better cytoplasmic definition and the ments: one for paraffin-based histology, one for
latter more nuclear detail. This technique allows semi-thin preparations and electron microscopy and
rapid sampling of several areas from a biopsy. All one for teased fiber preparations. Further portions
cell types in the CNS are readily identifiable by this may be taken for snap freezing. The 2 mm portions
method (Moss et al. 1997). Certain lesions may be at the ends of the biopsy are usually damaged at
too tough to smear, in which case a frozen section surgical removal and are thus inappropriate for his-
may be used. tology. They may, however, be frozen for molecular
studies. Glutaraldehyde is the preferred fixative for
processing to semi-thin sections and electron micros-
Peripheral nerve biopsies copy. Opinion is divided on the best fixative for
paraffin-based nerve histology, although many
The situations in which peripheral nerve biopsy prefer formalin (10% NBF).
have been shown to provide clinically useful infor- Following glutaraldehyde fixation, the nerve may
mation include the management of infection, inflam- be cut into 1 mm pieces, osmicated and embedded
matory and immune disorders (e.g., vasculitis and (see also Chapters 22 and 8 on electron microscopy
granulomatous diseases), amyloidosis and neopla- and plastic embedded sections, respectively). These
sia (Dyck et al. 2005). blocks may be used for electron microscopy or the
Peripheral nerve tissue is exceptionally delicate preparation of semi-thin (1–3 µm) sections. Semi-
and prone to artifacts due to handling, and it is thin sections of transversely oriented tissue are cut
imperative that this is kept to a minimum. In this and stained with toluidine blue or methylene blue-
context it is worth recalling that myelin is a liquid, azure II-basic fuchsin.
 372 17 Techniques in neuropathology

For paraffin processing, fixed material is pro-

50% ethanol in deionized water
cessed and cut at 6–8 µm. It is helpful to include a
Basic fuchsin, stock solution
transverse and a longitudinal segment for examina- Basic fuchsin 0.5 g
tion. Different centers vary in the stains prepared as 50% ethanol 50 ml
a matter of routine. However, these may include
Basic fuchsin, working solution
H&E, a trichrome stain to assess fibrosis, a myelin Basic fuchsin stock solution 1 ml
stain, an axon stain and an amyloid preparation Deionized water 19 ml
such as Congo red. Many biopsies are performed
Method
for inflammatory disorders such as vasculitis. As
1. Filter methylene blue/azure A solution into Coplin
these can be focal, patchy disorders, serial sections
jar.
through the paraffin block should be performed.
2. Put into a water bath set at 65°C.
It may be necessary to augment this with
3. Stain sections in this solution for 30 minutes.
immunohistochemistry.
4. Rinse well in distilled, filtered water.
Teased fiber preparations allow the examination
5. Do not allow sections to dry.
of the pattern of myelin formation along individual
6. Filter basic fuchsin working solution onto slides
axons (Asbury & Johnson 1978). This can give infor- and stain at room temperature for 4 minutes.
mation on whether there have been past episodes of 7. Rinse well in distilled water.
myelin loss with re-myelination, or whether there is 8. Drain, and allow to dry.
myelin fragmentation due to axonal loss. 9. Mount in DPX.
Result
Myelin blue
Other tissue elements light blue
Methylene blue-azure II-basic fuchsin Collagen pink/red
This method is based on that of Humphrey and Elastin red
Pitman (1974). Notes
Fixation a. If staining for leprosy bacilli in sections, stain in
Glutaraldehyde. filtered working solution of basic fuchsin for a strict
2 minutes only. Follow this by washing well in
Sections distilled water.
Semi-thin resin sections. b. Do not leave sections stained with methylene blue/
Solutions azure A in distilled water before counterstaining as
the blue will wash out and result will be too pale.
0.2 M sodium dihydrogen orthophosphate
dihydrate
NaH2PO4.2H2O 3.121 g Preparation of teased nerve fibers (Asbury & Johnson 1978)
Distilled water 100 ml
Nerve fibers can be processed to glycerine or
0.2 M disodium hydrogen orthophosphate unpolymerized Araldite for teasing. The latter method
Na2HPO4 2.839 g produces a firmer consistency to the individual fibers
Distilled water 100 ml and is easier to work with. The stiffness of the fibers
0.1 M phosphate buffer, pH 6.9 also relates to the amount of osmication and the
Methylene blue/azure A concentration of the osmium used.
Methylene blue 0.39 g Tissue
Azure A 0.06 g Fresh nerve.
Glycerol 30 ml
Methanol 30 ml Fixation
0.1 M phosphate buffer, pH 6.9 90 ml Segment of nerve is fixed in 0.1 M phosphate-
Distilled water 150 ml buffered 3.6% glutaraldehyde, for 4–16 hours.
 Neuropathology laboratory specimen handling 373

to be examined are aligned in parallel across the

Method
slide.
1. Wash twice in phosphate buffer, 15 minutes.
For diagnostic purposes, in order to avoid possi-
2. Under a stereomicroscope using two pairs of fine
forceps, carefully remove the epineurium by only ble sampling errors, at least 100 fibers should be
gripping and pulling the connective tissue. sampled (Dyck et al. 2005). However, in severely
Separate the nerve into individual or small bundles demyelinated cases, it may prove difficult to get
of fascicles. This allows a uniform osmication, enough black fibers. When enough fibers are
disregarding the variation in size of the specimen obtained, use the fine forceps to pick up a small
that one may have received. droplet of partially polymerized resin. Apply a thin
3. Osmicate in 0.1 M phosphate-buffered 2% osmium line of the resin along one of the aligned ends of the
tetroxide, 4 hours.
parallel-arranged fibers (the resin should not touch
4. Wash twice in phosphate buffer, 15 minutes each.
the fibers at this stage). Trim a coverslip to size. Hold
5. Briefly rinse in distilled water and process through
the coverslip at an angle to the surface of the slide
50, 80, and 95% alcohol for 10 minutes each.
and carefully touch the line of resin with the lower
6. Dehydrate in two changes of 100% alcohol,
15 minutes each. edge of the coverslip. Carefully lower the coverslip
7. Process through two changes of propylene oxide, until it almost touches the slide, and let go. Lay the
15 minutes each. slide on a flat surface in a 37°C oven and let the resin
8. Mix in equal parts of propylene oxide and Araldite slowly spread longitudinally along the fibers to fill
CY212 resin for 1 hour. the entire gap between the slide and the coverslip.
9. Mix in unpolymerized Araldite CY212 resin Ring the coverslip with nail varnish. Any trapped
overnight (the specimen can be kept in this resin bubbles should be left undisturbed. With practice,
at 4°C for up to 1 year). this mounting technique will allow the well-aligned,
Notes loosely attached fibers to remain undisturbed and
a. Laboratories which do not have access to araldite without overlapping on the slide.
processing can use a simplified method where,
following fixation, the nerve biopsy is washed in
distilled water (1 hour), osmicated (4 hours to
overnight), washed in distilled water (1 hour),
Muscle biopsies
treated with 60% glycerol overnight, and then
teased as described below. The handling and preparation of muscle biopsies are
b. Any remaining tissue from the protocol outlined in covered in Appendix I and in previous editions.
Note a can be subsequently paraffin processed Biopsy samples of skeletal muscle may be taken
and stained with H&E. either using a biopsy needle or at open operation.
The aim of histology is to provide an undistorted
picture of muscle fiber architecture. A detailed
To tease fibers after preparation, place the pro- evaluation of muscle disease relies heavily on
cessed nerve on a glass slide under a stereo- enzyme histochemistry, which necessitates the use
microscope in a pool of unpolymerized Araldite. of cryostat sections of unfixed skeletal muscle (see
Using fine forceps and sharp needles, remove the Table 17.2).
perineurium from a fascicle. Keep dividing the The practice of performing immunohistochemisty
nerve bundles into halves until single or small for fast and slow myosins is beginning to replace
bundles of two to three fibers (black) can be carefully ATPase enzyme histochemistry as a method for dis-
teased out. When separating a smaller bundle from tinguishing fiber types. Major histocompatibility
a larger one, it is helpful to hold onto the smaller one complex (MHC) class I proteins are overexpressed
and pull the larger one slowly away. Slide the fiber in some inflammatory myopathies, a feature that can
across the slide in a trail of resin onto an adjacent be detected by immunohistochemistry (Dubowitz &
slide. Care should be taken to insure that the fibers Sewry 2007).
 374 17 Techniques in neuropathology

Table 17.2 Staining methods for muscle biopsies

Method Demonstrates
H&E Morphology
Gomori trichrome Inclusion bodies, connective tissue, ragged red fibers and tubular aggregates
PAS ± diastase Glycogen
Oil red O Lipids
ATPase, pH 9.4 Myosin loss and myofiber atrophy of Type 1 and 2 fibers
ATPase, pH 4.6 Type 2B myofibers
ATPase, pH 4.3 Type 2C myofibers
NADH-TR Internal fiber architecture, mitochondria and tubular aggregates
Alkaline phosphatase Regenerating myofibers, autoimmune connective tissue disorders
Acid phosphatase Inflammatory cells; necrotic fibers; enhanced lysosomal enzyme activity
Non-specific esterase Inflammatory cells, necrotic myofibers, enhanced lysosomal enzyme activity
Cytochrome c oxidase Mitochondrial disorders
Succinic dehydrogenase Mitochondrial disorders
Myoadenylate deaminase Enzyme deficiency
Myophosphorylase Type V glycogenosis (McArdle’s disease)
Dystrophy-related Dystrophin 1, 2, and 3 (Figs 17.10a, b), sarcoglycans (α, β, γ, and δ),
immunohistochemical stains dysferlin, merosin, caveolin, emerin, calpain-3, spectrin

a b
Figure 17.10 (a) In a normal muscle, dystrophin is localized beneath the cell membrane of muscle fibers. (b) In Duchenne muscular
dystrophy, this staining pattern is absent.

A small portion of muscle may be fixed for electron it will be stiffened and can be put into the main bulk
microscopy if clinically indicated. The sample may of fixative. Staining of motor end-plates and axons
be fixed lightly stretched, longitudinally in a special may be performed on fresh tissues (Coers 1982). A
clip, or stretched out by pinning and fixed with a few detailed description of muscle histology and biopsy
drops of buffered glutaldehyde. After a few minutes, handling is given by Dubowitz and Sewry (2007).
 Neuropathology laboratory specimen handling 375

Central nervous system tissues taken Most of these problems can be reduced by using
at autopsy a longer processing schedule and the use of coated/
charged slides. Rushing fixation, processing or
After removal and macroscopic examination in the drying slides will lead to suboptimal preparations
autopsy room, the brain should be immersed in a and frustration.
10-liter bucket of formalin. It should be suspended The prion diseases present special methodological
by the basilar artery from a piece of string tied to the problems for autopsy and subsequent tissue han-
two attachments of the bucket handle. This may be dling. The misfolded prion, being the transmissible
done by passing the string under the basilar artery, element of these diseases, is a Group 3 Hazard and
or by hooking the basilar artery to the string with a is extremely resistant to usual decontamination
curtain hook or safety pin. The latter method is best methods. Dissection of a brain where prion disease
avoided as the safety pin tends to rust. The brain is a possibility should be done in an appropriate
should not be allowed to touch the bottom or sides laboratory containment facility using disposable
of the bucket. The brain should then be left to fix for instruments and appropriate protective clothing.
3 or more weeks. The spinal cord may be suspended All contaminated tissues and fluids should be
by the dura mater in a suitably long measuring cyl- contained. Sampled tissues should be treated with
inder with a weight attached to the dura mater at formic acid prior to paraffin embedding (Brown
the lower end, to avoid artifactual contraction of the et al. 1990). Work surfaces and instruments may be
cord during fixation. While some laboratories prefer sterilized in 2M sodium hydroxide for 1 hour. Glass-
the minimization of tissue distortion that this ware can be cleaned in sodium hypochlorite
method provides, others consider this is an unneces- (20,000 ppm). In cases of vCJD, prion protein (an
sarily complicated method and prefer to immerse infective hazard) also resides in lymphoid tissues
the cord in the brain bucket beneath the fixing brain. such as tonsil, lymph node, spleen and bone marrow
If molecular techniques are likely to be required, (Ironside et al. 2000). Detailed protocols for the han-
portions of brain and spinal cord may be snap- dling of tissues that may be affected by prion disease
frozen in liquid nitrogen. have been published (Bell & Ironside 1993; World
Whilst paraffin processing of neurosurgical mate- Health Organization 2004; Department of Health
rial can be carried out using most routine overnight 2011).
schedules, autopsy CNS material requires longer
processing, depending upon block size. This can be Acknowledgments
anything from 48 hours to 5 days. Central nervous
system tissue has a high lipid content due to the Thanks to Sebastian Brandner for providing us with
presence of myelin. This lipid makes it prone to his protocol for methylene blue-azure II-basic fuschin
processing artifact, largely due to poor dehydration. staining and to Patrick Elliott of the Medical Illustra-
For this reason, some laboratories use isopropyl tion Department, Royal Hallamshire Hospital, Shef-
alcohol for the last two changes of alcohol to improve field, for his illustration depicted in Figure 17.1.
tissue dehydration during tissue processing.
Processing artifacts include:
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