The stages involved in the process of staining

The
role of special stains in the investigation of granulomatous lung disease

 

Introduction

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Lungs, lymph
nodes, tissues and skin are some of the common sites where granulomas can be
found. In order to exclude an
infection, special stains are usually ordered by pathologists. In granulomatous
lung disease, the most used special stains are The Ziehl-Neelsen(ZN), Grocott
methenamine silver (GMS) and the periodic acid–Schiff stain (PAS) can be used too. Haematoxylin
and eosin (H&E) stain is a routine stain carried out preliminary to the
special stains (Ohshimo et al., 2017). There
are five stages involved in the process of staining and these include fixation,
processing, embedding, sectioning and staining (Alturkistani,
Tashkandi and Mohammedsaleh, 2015).

A wide range
of infectious and non-infectious disorders which are characterised by the
formation of granulomas can be defined as granulomatous lung disease. There are
various outcomes and clinical manifestations in this disease due to the wide
spectrum of pathologies (Ohshimo
et al., 2017). A Granuloma can
be defined as a cluster of inflammatory cells, Langhans giant cells, activated microphages (epithelioid histiocytes) and
lymphocytes (Mukhopadhyay and Gal, 2017. Elongated
nuclei and ill-defined cell borders are observed in epithelioid histiocytes
while ordinary histiocytes have cell borders which are well defined, oval or
kidney bean shaped (Ohshimo et al., 2017).
 Hypersensitivity responses or tenacity
of products which are non-degradable can cause granulomas. Different processes
are involved and these include macrophage activity, antigen or invading
organism, irritant, T cell response, overactivity of B cells and biological
mediators (Zumla and
James, 1996).

Multinucleated
giant cells are formed when macrophages or monocytes fuse, macrophages can be
further modified to epithelioid cells (Zumla and James, 1996). Fibronectin and
factors such as insulin-like growth factor, platelet-derived growth factor, TNF-?
and transforming growth factor-? are some of the cytokines and enzymes involved
in the granuloma active site (Zumla and James, 1996). An increased expression
of major histocompatibility (MHC) class II molecules has been observed in
activated macrophages and CD4+ T lymphocytes. Antigen-presenting cells bearing
MHC class II molecules present the T-helper cells with protein peptides and
thereafter the T-cell induces IL-l on the macrophage. The formation/ beginning
of the granuloma is then promoted by the chemotactic factors that follow. The
expression of MHC class II molecules is increased by IFN-?, and an
IgG fraction that is crystallisable is carried by activated macrophage
receptors. This helps in phagocytosis (Zumla and James, 1996).

This process
results in an epithelioid granuloma which can then advance toward fibrosis. Therefore,
in the development of granulomas, CD4+ T helper (Th1 and Th2) lymphocytes are
essential (Zumla and James, 1996). Th1 -type cells take part in hypersensitivity
responses which are delayed and are known to produce IFN-?, IL-2 and
TNF -?. On the other hand, cytokines IL-4, IL-5, IL-6, IL-9, IL-I0, and IL-13
are produced by the Th2-type cells and they help in development of eosinophilia
and B-cells (Zumla and James, 1996). Infectious granulomas appear to have a
relationship with the Th1 and Th2- type cells. Thus, there are many
immunoregulatory mechanisms involved in the formation of granulomas of
different infections (Zumla and James, 1996). In Infectious lung disease, the
most commonly found organisms are fungi and mycobacteria. For non-infectious
lung disease refer to the flow chart below.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: The differential diagnosis of
granulomatous lung disease. Adapted from (Mukhopadhyay
and Gal, 2017).

 

 

A

B

 C

 

D

E

F

Figure 2: Pictures
obtained after staining of lung tissue in the laboratory. A -shows the perls’
stain, B shows the Masson trichrome stain, C shows the haematoxylin and eosin
stain, D shows the Van Gieson stain, E shows the PAS stain and lastly F shows
the toluidine stain. A x10 magnification was used.

Table 1: Routine
and special stains carried out in the laboratory to stain different tissues.

Stain name
 

What does it stain

Why is this useful?

H%E

Nuclei
(purple) and cytoplasm (pink)

General
morphology, cell identification.

Perls’

Carbohydrates
and carbohydrate macromolecules (magenta colour)

Detection
of iron in biopsy specimens

PAS

Carbohydrates
and carbohydrate macromolecules (magenta colour)

Demonstration
of carbohydrates in tissue sections. Diagnosis e.g. glycogen storage diseases
and fungal infection

Van Gieson

Nuclei
(blue), collagen (red), cytoplasm and erythrocytes (yellow). It can also be
combined with elastic stain (blue/black)

Demonstrates
collagen

Toluidine blue

Acidic
components (different shades of blue).
Mast cells (purple/red)

Can be used
in observation of abnormal presence of mastocytes. Also, to highlight mucins
and cartilage.

Masson Trichrome

Nuclei
(blue), erythrocytes, muscle, cytoplasm and keratin (Bright-red)
Collagen
(blue or green)

Differentiation
between smooth muscle and collagen in tumours. Also show increase of collagen
in diseases

 

Infectious
lung diseases

Before the
use of special stains, it is important to begin with the haematoxylin-eosin
(H&E) stain (Myers and Tazelaar, 2008). Reason being that after
critical scrutiny most fungi can be identified using the H&E stain, though
mycobacteria are not visible using this stain. Fine-drawn differences in
morphology between organisms can be seen using the H&E stain and the
organisms can be placed in the context of the associated tissue reaction. The Histoplasma is exempt from this rule due
to its visibility being impossible on this stain (Mukhopadhyay and Gal, 2017).

Normally for fungi, the histochemical stains used are Groccot’s
methenamine silver stains (GMS) while the Ziehl–Neelsen (ZN) stain is used for mycobacteria (Mukhopadhyay and
Gal, 2017). Although for fungi, the periodic acid–Schiff
stain (PAS) can also be performed, it is of less preference in comparison
to the GMS stain. This is because there is less contrast between fungi and
background debri when the periodic acid–Schiff stain is used (Mukhopadhyay and
Gal, 2017). Furthermore, the auramine/auramine-rhodamine fluorescence technique
can be used as an alternative to the ZN stain in mycobacteria. This is because
the technique is more sensitive than acid-fast stains and has shown to be
equivalent to culture (Mukhopadhyay and Gal, 2017). The
auramine rhodamine fluorescence has sensitivity of 80% meanwhile the ZN
stain has a sensitivity of 60%. However, specificity in the auramine rhodamine
fluorescence is low (84%) when compared to the ZN stain (98%) (Ohshimo et al.,
2017). It is
therefore advised that after the H stained sections have been examined,
the GMS stain be used before the ZN stain as it is more time consuming (Mukhopadhyay and Gal, 2017). Most organisms can be picked out
at low magnification of x20 objective or x10 ocular except the Histoplasma which can require using a
x40 objective. Additionally, more time can be spent examining the ZN-stained
sections if a negative result is shown with GMS and since mycobacteria are
difficult to find and are few, it is important to use a x40 objective (Mukhopadhyay and
Gal, 2017).

Figure 3: Comparison
of the most common fungal causes of lung granulomas and some stains. From (Mukhopadhyay and Gal, 2017).

Furthermore, because GMS also stains dust particles, mucin and elastic
tissue, it can be misleading in identification of microorganisms. Although
pollen and dust particles lack budding forms related to fungal yeasts,
characteristic shapes and are small (size), they do appear like fungal yeast
and this could cause certain challenges. 
In every case it is important to check standard tissue controls because
if a GMS stain is poorly performed it may result in a false negative result. Histoplasma yeasts
may stain weakly with GMS in necrotizing granulomas and this may result in them
being overlooked (Mukhopadhyay and Gal, 2017).

To add on, due to the use of xylene in routine processing in the ZN
stain, mycobacteria are difficult to find and are a few, thus is it important
to spend some time at high magnification e.g. x40 objective and x10 0cular
lens. To detect organisms that appear only on certain planes, fine focus should
constantly be adjusted or a high-power oil immersion objective can be used. Occasionally,
organisms can be found within the granulomatous rim or periphery of the
necrosis despite being common in the necrosis centre. Tuberculous and
nontuberculous can then be differentiated after mycobacteria are identified (Majeed
and Bukhari, 2011). Unfortunately for speciation, there is not much reliability
in the morphologic appearance of mycobacteria. It’s mostly based on
microbiologic culture-made smears instead of histologic material that is
paraffin-embedded or formalin-fixed. Only nontuberculous mycobacteria have been
included in histologic studies that claim particular morphologic features for
specific mycobacterial species. Features such as size (can however be
associated with certain species), cording and beading can hinder accuracy and
speciation of mycobacteria even in smears made from microbiologic culture
material.

                            

Figure 4: Infectious necrotizing granulomas. A
shows tuberculosis, B is the same as A and the arrow shows single acid-fast
bacterium. C shows nontuberculous mycobacterial infection.  Isolation of
mycobacterium intracellular was done in cultures. Very similar to
tuberculosis, necrotizing granuloma with abundant necrosis observed. On the
bottom right, there are epithelioid histiocytes. D, Same case as C, Arrows show
mycobacteria and there is identical morphology as of organism noted in B. Histoplasmoma
is shown in E, despite the granuloma being larger, it is identical to
granulomas A, C and F. The last picture is the same case as E.  Histoplasma yeasts
(H%E, original magnifications ×100 – A, C, and E). For B and D – ZN stain with
magnification of ×1600 and for F, GMS stain with magnification ×400 was used.
From (Mukhopadhyay and Gal, 2017).

In definitive speciation of mycobacteria,
molecular methods such as polymerase chain reaction (PCR) and microbiology
culture are the only useful methods. PCR is useful in determining the species
when a positive result is noted with histologic stain but negative with those
of cultures. Also, it is useful when there was no submission of the biopsied
tissue for culture. Given these points, PCR is more sensitive than the ZN
stain. If PCR is not available or shows negative result, empiric therapy can be
initiated (Mukhopadhyay and Gal, 2017).

Non-infectious
granulomatous Lung disease

Sometimes
even though there is careful examination, organisms are not visible/found
within the granulomas, this is also observed in the necrotizing granulomas.
When presented with such cases re-evaluation of special stains is the most
productive next step. For overlooked infections such as the Histoplasma infection, in several cases
it is essential to then re-examine the GMS-stained sections. If use of a
special stain does not visualise the necrotic portion of the granuloma, it may
be useful to recut the block and the stain can be repeated. Studies carried out
by Goodwin and Snell did show that ten of seventeen histoplasmomas were
diagnosed in a recut after including the necrotic centre (Goodwin and Snell, 1969). However, a
lot of necrotizing granulomas still remain unexplained regardless of all these
steps taken. Suggestions have been made that there could be a possibility of
such cases showing infectious granuloma but unfortunately an inflammatory
reaction may have removed or killed the organism. A descriptive diagnosis is
then recommended and this should also include absence or presence of necrosis
and also if the organism identifiable is absent.  If histologic examination does not yield a
desired result, then results of cultures may be useful. Despite the negative
results in the auramine-rhodamine and ZN stains from the surgically excised
granulomas, mycobacteria mainly nontuberculous still grow in culture.
Ultimately, after all the other options have been exhausted the small portion
of narcotizing granulomas that are unexplained can be categorised as anti-neutrophil
cytoplasmic antibody (ANCA)–related vasculitides or histoplasmosis.

                                  

Figure 5: Non infectious
non-necrotizing granulomas. A shows sarcoidosis, there is distribution of
granulomas along the pleura (top). The three arrows show the interlobular
septum while the bronchovuscular bundles are shown by the arrowhead. There is
no inflammation in the adjacent lung paranchema as it is only localized to the
granulomas. B shows sarcoidosis, this time the non-necrotizing granuloma is
well formed (surronded by concetric fibrosis). C shows hypersensitivity
pneumonitis. Mild thickening of the interstitium is the main abnormality. At
this magnification, no granulomas are visible. D also shows hypersensitivity
pneumonitis, a poorly formed granuloma (loose cluster of histocytes)
characterised by interstitial chronic inflammation. E shows the hot tub lung;
the arrows represent the granulomas that show bronchiole predilection. F also
shows the tub lung in this picture a non-necrotizing granuloma that is well
formed is seen with an airspace. In A, C and E, haematoxylin-eosin was used
with magnifications ×20 and for B, D and F ×200 magnification was used. From (Mukhopadhyay
and Gal, 2017).

Conclusion

To summarise,
histological staining has proven to be very useful in the differential
diagnosis of lung disease. It is important to exclude lung disease because
infection is the common cause of pulmonary granulomas. Granulomatous lung
disease can either be infectious or non-infectious (mentioned on flowchart).
Though necrotizing granulomas are known to be common in infectious lung
disease, it is important to note that sometimes both necrotising and
non-necrotising granulomas can be observed in infectious lung disease.
Furthermore, depending on the immune status of the patient tuberculosis may
show non-necrotising granulomas (Ohshimo et al.,
2017).

After
preliminary information has been acquired after use of the H&E stain,
special stains can be used to visualise or highlight different tissue elements.
In granulomatous lung disease, the most commonly used special stains are the
GMS, ZN and PAS. GMS staining shows the cell walls of both dead and living
fungal organisms. The ZN stain is responsible for staining mycobacteria and it
is known to be less sensitive when compared to the auramine-rhodamine
fluorescence technique. Additionally, the ZN stain is known to stain the
acid-fast bacilli bright red, other tissue (pale blue) and other caseous
material (very pale greyish blue). The PAS stain is useful in visualising the
walls of living fungi) and has already
been mentioned on table 1. Lastly the GMS stain is known to stain fungi black.
However, for speciation of mycobacteria other molecular methods such as PCR or
microbiology culture can be used. All these methods help in the identification
of microorganisms responsible for different infections (Ohshimo et al., 2017).