INTRODUCTION

Tuberculosis (TB) is caused by bacteria (Mycobacterium tuberculosis) that most often affect
the lungs. Tuberculosis is curable and preventable.TB is spread from person to person
through the air. When people with lung TB cough, sneeze or spit, they propel the TB germs
into the air. A person needs to inhale only a few of these germs to become infected.About
one-quarter of the world's population has latent TB, which means people have been infected
by TB bacteria but are not (yet) ill with the disease and cannot transmit the disease.People
infected with TB bacteria have a 5–15% lifetime risk of falling ill with TB. However, persons
with compromised immune systems, such as people living with HIV, malnutrition or
diabetes, have a much higher risk of falling ill.When a person develops active TB disease, the
symptoms (such as cough, fever, night sweats, or weight loss) may be mild for many months.
This can lead to delays in seeking care, and results in transmission of the bacteria to others.
People with active TB can infect 10–15 other people through close contact over the course of
a year. Without proper treatment, 45% of HIV-negative people with TB on average and
nearly all HIV-positive people with TB will die (WHO 2017).
Mycobacterium avium complex (MAC) is a life-threatening bacterialinfection. The disease
affects people with AIDS who have a severely suppressed immune system and are not taking
anti-HIV drugs or medication to prevent MAC. However, thanks to effective anti-HIV drugs,
MAC has become relatively rare. People with HIV whose CD4 counts are below 50 are at
risk of developing MAC. Symptoms include fever, weight loss, sweats/chills, diarrhoea,
cramping, fatigue, weakness and anaemia (low red blood cell count). Antibiotics are used to
prevent and treat the illness(Koenig, 2010).

M. bovis is most commonly found in cattle and other animals. In people, M. bovis causes TB
disease that can affect the lungs, lymph nodes, and other parts of the body. However, as with
M. tuberculosis, not everyone infected with M. bovis becomes sick. People who are infected
but not sick have what is called latent TB infection (LTBI). People who have LTBI do not
feel sick, do not have any symptoms, and cannot spread TB to others. However, some people
with LTBI go on to get TB disease (CDC, 2011).
EPIDEMIOLOGY
According to the World Health Organization (WHO), about 8.6 million cases (8.3–9.0
million) were estimated to have occurred in 2012, approximately 2.9 of whom were in
women. Most cases are estimated to be in Asia and Africa (58% and 27% respectively), with
the highest incidence in India (range 2.0–2.4 million) and China (0.9 −1.1 million), together
accounting for 38% of the total number of cases.The global TB incidence rate slowly
declined from 1997 to 2001, with an increase in 2001 (due to the rising number of cases
among HIV-infected patients in Africa). Subsequently, a 1.3% per year average reduction
rate has been observed since 2002, reaching 2.2% between 2010 and 2011. The absolute
number of cases is also currently decreasing, though this declining trend only began in 2006.
Based on these findings, the Millennium Development Goal 6 Target for tuberculosis (i.e. “to
halt and begin to reverse the incidence”) has already been achieved(Giorgia et al., 2014)

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In Nigeria, despite the National TB and Leprosy Control Programme (NTBLCP) reporting 94
604 cases in 2012, this number only represents 51% of the cases estimated to have occurred
in the country for that year. Totals of 70 858 and 94 604 cases were reported in 2008 and
2012, showing a 38% increase over 5 years. Changes in the number of cases were uneven,
ranging from a 28.6% decrease to 353% increase from the 2008 baseline. The proportion of
smear-positive cases ranged from 34.8% to 86.2% in 2008 and from 40.3% to 78.1% in
2012(Joshua et al., 2015)

PATHOGENESIS
The primary culprit with tuberculosis is Mycobacterium tuberculosis (MTB), although
humans are sometimes susceptible to Mycobacterium bovis (M.bovis). According to the
World Health Organization, nearly one third of the world has an asymptomatic or latent
tuberculosis infection. In some cases this is harmless, but the ability of tuberculosis to survive
treatment and spread makes diagnosing and understanding infections vital to combatting its
lethality and global prevalence (Rebar, 2014).

Tuberculosis is a highly infectious disease, and is primarily transmitted via inhalation of

aerosol droplets expelled by infected hosts. The infectious dose is 1-200 bacilli, but each
aerosol droplet can contain 1-400 bacilli, making contact without infection nearly impossible.
In addition to its airborne infection, MTB is adept at using the natural defences of the body
for its own advantage (Rebar, 2014).

After infection, the host either develops a primary infection immediately, or no initial
infection occurs and the disease remains latent within the body. Upon inhalation, tuberculosis
bacteria travel to the lungs and end up in the aveoli, where they are recognized in an
immunocompetent host as foreign and are attacked by the body's macrophages. These
macrophages attempt to engulf the bacteria and dissemble them, normally a part of the
process of a body defeating disease. However in the case of tuberculosis, it is exactly what
the disease wants. Not all of the bacteria cells will be destroyed no matter how excellent the
host’s immune system is, and the survivors infect and hijack macrophages feeding on them
while increasing the bacteria population. Once macrophages are infected, they either kill the
bacteria inside them or the bacteria multiply until they burst the macrophage, leading to
further infection and extracellular bacilli (increasing the likelihood of infecting others via
aerosol transmission). The infected areas gradually transform into granuloma, a wall of
macrophages intended to contain the infection (Rebar, 2014).

This also allows the MTB to continue growing and overwhelm the cells it has infected until
they die. Over time the centres of these granulomas necrotize, leading to the mixture of blood
and sputum in the lungs tuberculosis is known for. This is where the disease's progress
diverges depending on the individual. For the vast majority of cases, these necrotized lesions
heal with some amount of scarring and calcification. The disease is not gone, and these
asymptomatic cases can remain latent for years and even decades. Less than 10% of these
latent tuberculosis cases become full blown secondary infections, but those account for nearly
80% of active tuberculosis cases. Almost all transmission occurs from latent tuberculosis re-
emerging and causing rapid growth of extracellular bacteria (the kind most likely to be

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transmitted through inhalation of aerosol droplets). Because the disease can lie dormant for so
long, seemingly healthy people can easily infect their friends, neighbours, and anyone they
have contact with before they can be treated or isolated. While tuberculosis is not unique
among diseases for lingering in the body and re-emerging (malaria is another notable
example), this characteristic does contribute to its prevalence and lethality (Rebar, 2014).

In the case that the host does not contain the initial infection, or in the case of re-emergence
after a period of lying dormant and latent in the body, tuberculosis infection and symptoms
proceed similarly. The host's immune system attempts to contain the growing infected areas
by killing off tissue around them to contain the spread and deploying T-cells. This leads to
inflammation and some of the other symptoms common to tuberculosis. During primary
progression of the disease, the infection grows by using the very cells the body attempts to
combat it with, leading to steadily growing areas infested with bacteria, also known as
tubercles. These tubercles can break off and travel through the bloodstream, leading to extra
pulmonary tuberculosis or infection. In a small percentage of hosts the tubercles liquefy,
creating bacteria-rich environments and thickening the host's sputum. The lungs fill with
growing populations of free floating bacteria as well as clusters of bacteria in the linings of
the lungs (Rebar, 2014).

Figure1: Picture explaining pathogenesis of tuberculosis.

(CDC, 2000)

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PATHOPHYSIOLOGY
Once inhaled, the infectious droplets settle throughout the airways. The majority of the bacilli
are trapped in the upper parts of the airways where the mucus-secreting goblet cells exist. The
mucus produced catches foreign substances, and the cilia on the surface of the cells
constantly beat the mucus and its entrapped particles upward for removal. This system
provides the body with an initial physical defence that prevents infection in most persons
exposed to tuberculosis. Bacteria in droplets that bypass the mucociliary system and reach the
alveoli are quickly surrounded and engulfed by alveolar macrophages, the most abundant
immune effector cells present in alveolar spaces (Nancy, 2009).

These macrophages, the next line of host defence, are part of the innate immune system and
provide an opportunity for the body to destroy the invading mycobacteria and prevent
infection. Macrophages are readily available phagocytic cells that combat many pathogens
without requiring previous exposure to the pathogens. Several mechanisms and macrophage
receptors are involved in uptake of the mycobacteria. The mycobacterial lipoarabinomannan
is a key ligand for a macrophage receptor. The complement system also plays a role in the
phagocytosis of the bacteria. The complement protein C3 binds to the cell wall and enhances
recognition of the mycobacteria by macrophages. Opsonization by C3 is rapid, even in the air
spaces of a host with no previous exposure to M tuberculosis.The subsequent phagocytosis by
macrophages initiates a cascade of events that results in either successful control of the
infection, followed by latent tuberculosis, or progression to active disease, called primary
progressive tuberculosis(Nancy, 2009).

The outcome is essentially determined by the quality of the host defences and the balance that
occurs between host defences and the invading mycobacteria. After being ingested by
macrophages, the mycobacteria continue to multiply slowly, with bacterial cell division
occurring every 25 to 32 hours.Regardless of whether the infection becomes controlled or
progresses, initial development involves production of proteolytic enzymes and cytokines by
macrophages in anattempt to degrade the bacteria.Released cytokines attract T lymphocytes
to the site, the cells thatconstitute cell-mediated immunity. Macrophages then present
mycobacterial antigens on their surface to the T cells. This initial immuneprocess continues
for 2 to 12 weeks;the microorganisms continue to growuntil they reach sufficient numbersto
fully elicit the cell-mediatedimmune response, which can bedetected by a skin test.For
persons with intact cellmediatedimmunity, the next defensivestep is formation of
granulomasaround the M tuberculosis organisms (Nancy, 2009).

These nodular-typelesions form from an accumulationof activated T lymphocytes

andmacrophages, which creates a micro environment that limits replicationand the spread of
the mycobacteria.This environment destroys macrophages and produces earlysolid necrosis at
the centre of thelesion; however, the bacilli are ableto adapt to survive.In fact,
M.tuberculosisorganisms can changetheir phenotypic expression, such asprotein regulation,
to enhance survival. By 2 or 3 weeks, the necroticenvironment resembles soft cheese,often
referred to caseous necrosis,and is characterized by low oxygenlevels, low pH, and limited
nutrients.This condition restricts furthergrowth and establishes latency.Lesions in persons
with an adequateimmune system generally undergo fibrosis and calcification,

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successfullycontrolling the infection sothat the bacilli are contained in thedormant, healed
lesions (Nancy, 2009).

Lesionsin persons with less effective immunesystems progress to primary

progressivetuberculosis.For less immunocompetent persons,granuloma formation is
initiatedyet ultimately is unsuccessfulin containing the bacilli. The necrotictissue undergoes
liquefaction, andthe fibrous wall loses structuralintegrity. The semiliquid necroticmaterial can
then drain into abronchus or nearby blood vessel,leaving an air-filled cavity at theoriginal
site. In patients infected with M tuberculosis, droplets can be coughed up from the bronchus
and infect other persons. If discharge into a vessel occurs, occurrence of extrapulmonary
tuberculosis is likely. Bacilli can also drain into the lymphatic system and collect in the
tracheobronchial lymph nodes of the affected lung, where the organisms can form new
caseous granulomas(Nancy, 2009).

MODE OF TRANSMISSION
Mycobacterium tuberculosis is spread by small airborne droplets, called droplet nuclei,
generated by the coughing, sneezing, talking, or singing of a person with pulmonary or
laryngeal tuberculosis. These minuscule droplets can remain airborne for minutes to hours
after expectoration. The number of bacilli in the droplets, the virulence of the bacilli,
exposure of the bacilli to UV light, degree of ventilation, and occasions for aerosolization all
influence transmission. Introduction of M tuberculosis into the lungs leads to infection of the
respiratory system; however, the organisms can spread to other organs, such as the
lymphatics, pleura, bones/joints, or meninges, and cause extra-pulmonary tuberculosis
(Nancy, 2009).

Tuberculosis (TB) is transmitted from an infected person to a susceptible person in airborne

particles, called droplet nuclei. These are 1–5 microns in diameter. These infectious droplet
nuclei are tiny water droplets with the bacteria that are released when persons who have
pulmonary or laryngeal tuberculosis cough, sneeze, laugh, shout etc. These tiny droplet nuclei
remain suspended in the air for up to several hours. Tuberculosis bacteria, (Mycobacterium
tuberculosis) however are transmitted through the air, not by surface contact. This means
touching cannot spread the infection unless it is breathed in (Ananya, 2017).

Transmission occurs when a person inhales droplet nuclei containing tuberculosis bacteria.
These droplet nuclei travels via mouth or nasal passages and move into the upper respiratory
tract. Thereafter they reach the bronchi and ultimately to the lungs and the alveoli (Ananya,
2017)

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Figure 2: Picture showing how droplet nuclei spread in air.
TB is spread from person to person through the air. The dots in the air represent droplet
nuclei containing tubercle bacilli (CDC, 2000).
SIGNS AND SYMPTOMS

Pulmonary General: Pulmonary and Extrapulmonary

Extrapulmonary
• Coughing • Chills • The symptoms depend on
• Coughing up sputum or • Fever part of body affected by
blood • Night sweats tuberculosis (TB) disease
• Pain in the chest when • Loss of appetite • TB of the spine may cause
breathing or coughing • Weight loss pain in the back.
•Weakness or easy • TB of the kidney may cause
fatigability blood in the urine.
• Malaise (a feeling of • Meningeal TB may cause
general discomfort or illness) headaches or psychiatric
symptoms.
• Lymphatic TB may cause
swollen and tender lymph
nodes, often at the base of the
neck.
• Pleural
•Laryngealcause hoarseness
(CDC 2000)

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LABORATORY DIAGNOSIS
Multiplication of tubercle bacilli in any site of the human body causes a specific type of
inflammation, with formation of a characteristic granuloma. Anatomic pathology involves
examining tissue for suspect TB. Tissue samples may be obtained either by biopsy from a
patient or at autopsy (Nadia and Donald, 2005).

Histology consists of macroscopic examination of lesions that suggest the presence of

tuberculosis if there is access to all or a large part of the affected organ (lymph node or
kidney), and microscopic examination of a sample (Nadia and Donald, 2005).

Histology is an aid to diagnosis when bacteriological techniques cannot be applied. It is

especially useful for extrapulmonary tuberculosis. It is helpful to consider histological
examination and bacteriological techniques as complementary (Nadia and Donald, 2005).

TYPES SAMPLES
1. ASPIRATION OF THE LYMPH NODES
Affected peripheral lymph nodes, particularly cervical nodes, can be aspirated.
Aspirationshould be performed at the upper pole of the node (Nadia and Donald, 2005).

2. BIOPSY OF THE SEROUS MEMBRANES

Effusions of the serous membranes can be aspirated. However, the effusions are much less
useful for diagnosis than histology or, even better, culture of the biopsy specimen (Nadia and
Donald, 2005).

3. TISSUE BIOPSY
WITHOUT SURGERY
 THE SEROUS MEMBRANES: Biopsy of the pleura (with an Abrams or Castelain
needle) and the pericardium is performed closed. As a result the fragments are not
always sampled from the site of the lesions. In contrast, when biopsy of the
peritoneum is performed during laparoscopy, samples can be taken directly from a
suspect lesion. Whatever serous membrane is affected, several fragments should be
sampled during a single biopsy.
 THE SKIN: Skin biopsy.
 THE REPRODUCTIVE ORGANS: Biopsy of the endometrium by curettage.
 DIFFERENT ORGANS AFTER ENDOSCOPY: Bronchial biopsy during
bronchoscopy, pleural biopsy using thoracoscopy, gastric biopsy during endoscopy, or
liver biopsy during laparoscopy. As these biopsies are performed under direct
observation, fragments of suspect lesions can be sampled using biopsy forceps (Nadia
and Donald, 2005).

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DURING SURGERY
A surgical intervention can be performed to confirm the diagnosis by sampling of a deep or
superficial lymph node, a bone fragment or part of an organ. During the surgical intervention
a sample can sometimes be examined immediately in order to decide on the next step (Nadia
and Donald, 2005).

POST MORTEM
After death from an unknown cause, tissue samples taken at autopsy can be analysed (Nadia
and Donald, 2005).

 MACROSCOPIC ASPECT
MORPHOLOGY
Primary tuberculosis almost always begins in the lungs. Typically, the inhaled bacilli implant
in the distal airspaces of the lower part of the upper lobe or the upper part of the lower lobe,
usually close to the pleura. As sensitization develops, a 1- to 1.5-cm area of gray-white
inflammation with consolidation emerges, known as the Ghon focus. In most cases, the center
of this focus undergoes caseous necrosis. Tubercle bacilli, either free or within phagocytes,
drain to the regional nodes, which also often caseate. This combination of parenchymal lung
lesion and nodal involvement is referred to as the Ghon complex. During the first few weeks
there is also lymphatic and hematogenous dissemination to other parts of the body. In
approximately 95% of cases, development of cell-mediated immunity controls the infection.
Hence, the Ghon complex undergoes progressive fibrosis, often followed by radiologically
detectable calcification (Ranke complex), and despite seeding of other organs, no lesions
develop (Kumar et al., 2010).

Histologically, sites of active involvement are marked by a characteristic granulomatous

inflammatory reaction that forms both caseating and noncaseating tubercles. Individual
tubercles are microscopic; it is only when multiple granulomas coalesce that they become
macroscopically visible. The granulomas are usually enclosed within a fibroblastic rim
punctuated by lymphocytes. Multinucleate giant cells are present in the granulomas.
Immunocompromised people do not form the characteristic granulomas (Kumar et al.,,
2010).

VARIOUS TYPES OF MACROSCOPIC LESIONS ARE SYMPTOMATIC OF

TUBERCULOSIS. CERTAIN LESIONS CAN BE OBSERVED ON CLINICAL
EXAMINATION OF A PATIENT:
ULCERATIONS on the surface of the skin or the mucous membranes are irregularly
draining sinuses with raised edges, containing necrotizing granuloma (Nadia and Donald,
2005).

FISTULAS AND SINUSES form in the absence of natural drainage (adenitis, cold abscess).
Other lesions can be observed on endoscopy (laparoscopy, fibroscopy, thoracoscopy,
coelioscopy) (Nadia and Donald, 2005).

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ISOLATED NODULES present as disseminated whitish or yellowish granuloma. These
granuloma can be of different sizes: from miliary granulation of less than 1 mm diameter to
tuberculoma which can reach up to 20 mm in diameter (Nadia and Donald, 2005).

DIFFUSE LESIONS may be gelatinous, and are grey or yellow in colour. On examination
of a sample excised during surgery or autopsy any of these lesions can be observed.
Dissection of the sample (lung, kidney) can sometimes reveal tuberculous cavities, which
present in the form of cavities filled or covered with caseating granuloma. These types of
lesions are most characteristic of tuberculosis (Nadia and Donald, 2005).

Several types of macroscopic lesion are generally present on a single excised sample.
Nevertheless, however clear the diagnosis seems to be, the examination must be completed
by microscopic examination of tissue segments after specific staining (Nadia and Donald,
2005).

 MICROSCOPIC ASPECTS
The involvement of an organ by tuberculosis is associated with an inflammatory reaction at
the affected site. The inflammation occurs in three successive stages that can be simultaneous
as acute, subacute and chronic and that have different histological aspects (Nadia and Donald,
2005).

A granuloma is a clump of cells that forms when the immune system tries to fight off a
harmful substance but cannot remove it from the body.A necrotizing granuloma is an area of
inflammation in which tissue has died. Necrotizing means dying or decaying (Nadia and
Donald, 2005).

THE ACUTE PHASE

Infection by the tubercle bacillus first leads to a rapid, nonspecific inflammatory reaction
manifested by exudative lesions that are not particularly specific to tuberculosis. The focus of
the inflammation is the site of a sero-fibrous exudate with numerous macrophages in the
centre. At this stage the bacillus can be observed at the centre of this site of inflammation
(Nadia and Donald, 2005).

THE SUBACUTE PHASE

Lysis of the bacilli liberates the phospholipids from their capsule, provoking a specific tissue
reaction and the formation of follicles, “Koëster follicles”. Two kinds of follicular lesions can
be observed:
THE EPITHELIOID GIANT CELL FOLLICLE
A rounded focus containing:
NUMEROUS EPITHELIOID CELLS: these are monocytes with an egg-shaped centre,
abundant cytoplasm and indistinct cytoplasmic edges. Several Langhans giant cells, generally
situated at the centre of the follicle. These are large cells with abundant cytoplasm, indistinct
edges and multiple centres arranged in the shape of a crown or a horseshoe. Langhans cells

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are caused by the fusion of epithelioid cells. Epithelioid cells and Langhans cells are created
from the metamorphosis of monocytes under the action of lymphokinesand a peripheral
crown of lymphocytes.
This follicle does not contain necrosis and is not specific to tuberculosis. It is common to
“granulomata”: tuberculous leprosy, sarcoidosis and connective tissue diseases.
THE NECROTIZING GRANULOMA: the epithelioid giant cell follicle presents with
central caseating necrosis. This lesion is very specific to tuberculosis. Caseating necrosis is a
fine-grained, homogeneous, eosinophilic necrosis(Nadia and Donald, 2005).

THE CHRONIC PHASE

THE FIBROUS FOLLICLE: the tuberculous follicle gradually develops into a fibrous
follicle. Collagenous fibres invade the tuberculous focus, which is enclosed in a fibrous shell
with fibroblasts and lymphocytes, forming a fibro-caseating follicle that is then transformed
into a fully fibrous follicle. This follicle can become entirely calcified.
Isolated or clustered follicles of varying types and sizes can be observed. There are usually a
number of visible lesions at the different acute, subacute or chronic stages (Nadia and
Donald, 2005).

METHODS
CYTOLOGICAL TECHNIQUES
 MATERIAL SAMPLED BY BIOPSY OF TISSUE SPECIMENS
After biopsy of tissues such as lymph nodes, a smear is made by spreading the sample on a
slide.The smear is then air-dried and stained with May-Graunwald-Giemsa.
 ASPIRATED FLUID
Part of the aspirated fluid is taken into a test tube, it is centrifuged at 2000 rpm. A smear
is madeof the centrifuged sediment, which is then stained. In general, smear
examinationsof aspirated fluid have a lower diagnostic yield than smears of tissue(Nadia
and Donald, 2005).

HISTOLOGICAL TECHNIQUES
Whatever type of specimen is available, the following steps should be followed to prepare it
forhistological examination:
PREPARE THE SAMPLE FOR EXAMINATION:
1. Obtaining a fresh specimen
Fresh tissue specimens will come from various sources. It should be noted that they can very
easily be damaged during removal from patient or experimental animal. It is important that
they are handled carefully and appropriately fixed as soon as possible after dissection. Ideally
fixation should take place at the site of removal, perhaps in the operating theatre, or, if this is
not possible, immediately following transport to the laboratory (Geoffrey, 2018).

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2. Fixation
The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution
(formalin). This will slowly penetrate the tissue causing chemical and physical changes that
will harden and preserve the tissue and protect it against subsequent processing steps.
Generally this will mean that the specimen should fix for between 6 and 24 hours. Most
laboratories will use a fixative step as the first station on their processor.
Following fixation the specimens may require further dissection to select appropriate areas
for examination. Specimens that are to be processed will be placed in suitable labelled
cassettes (small perforated baskets) to segregate them from other specimens.
If it is recent, it has a yellowish white, cheesy texture; on ageing it becomes greyish and
chalky. When caseating material is obtained (from aspiration of an abscess or fistulation of a
lymph node), tuberculosis is the first diagnosis that comes to mind. Sometimes the granuloma
softens, liquefies and drains away, leaving a cavity (Geoffrey, 2018).
3. Dehydration
Because melted paraffin wax is hydrophobic (immiscible with water), most of the water in a
specimen must be removed before it can be infiltrated with wax. This process is commonly
carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing
concentration until pure, water-free alcohol is reached. Ethanol is miscible with water in all
proportions so that the water in the specimen is progressively replaced by the alcohol. A
series of increasing concentrations is used to avoid excessive distortion of the tissue
(Geoffrey, 2018).
4. Clearing
Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it
with wax because wax and ethanol are largely immiscible. We therefore have to use an
intermediate solvent that is fully miscible with both ethanol and paraffin wax. This solvent
will displace the ethanol in the tissue, then this in turn will be displaced by molten paraffin
wax. This stage in the process is called “clearing” and the reagent used is called a “clearing
agent”. The term “clearing” was chosen because many (but not all) clearing agents impart an
optical clarity or transparency to the tissue due to their relatively high refractive index.
Another important role of the clearing agent is to remove a substantial amount of fat from the
tissue which otherwise presents a barrier to wax infiltration (Geoffrey, 2018).
5. Wax infiltration
The tissue can now be infiltrated with a suitable histological wax. Although many different
reagents have been evaluated and used for this purpose over many years, the paraffin wax-
based histological waxes are the most popular. A typical wax is liquid at 60°C and can be
infiltrated into tissue at this temperature then allowed to cool to 20°C where it solidifies to a
consistency that allows sections to be consistently cut (Geoffrey, 2018).
 

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 6. Embedding or blocking out
Now that the specimen is thoroughly infiltrated with wax it must be formed into a “block”
which can be clamped into a microtome for section cutting. This step is carried out using an
“embedding centre” where a mould is filled with molten wax and the specimen placed into it.
The specimen is very carefully orientated in the mould because its placement will determine
the “plane of section”, an important consideration in both diagnostic and research histology.
A cassette is placed on top of the mould, topped up with more wax and the whole thing is
placed on a cold plate to solidify. When this is completed the block with its attached cassette
can be removed from the mould and is ready for microtomy. It should be noted that, if tissue
processing is properly carried out, the wax blocks containing the tissue specimens are very
stable and represent an important source of archival material(Geoffrey, 2018).
 HAEMATOXYLIN AND EOSIN STAINING PROCEDURE
PROCEDURE
Dewax sections in three changes of xylene for 5 minutes each.
Hydrate the sections through descending grades of alcohols each 2 minutes (100%, 90% and
70%).
Wash in running tap water for 2 minutes.
Stain in Harris haematoxylin for 2-5 minutes, Mayer’s haematoxylin for 5-10 minutes or
Ehrlich’s haematoxylin for 15 -20 minutes.
Wash in running tap water for 5 minutes.
Differentiate in 1% acid alcohol (1 quick dip).
Wash in running tap water.
Blue the sections using scott’s tap water for 1 minute or by allowing the slides in running tap
water for another 10 minutes.
Give 10 dips each in ascending grades of alcohol (70%, 90%).
Stain in Eosin for 30 seconds to 2 minutes.
Dehydrate through ascending grades of alcohols each 10 dips (70%, 90%, and 100%).
Clear in 2 changes of xylene 10 dips each.
Mount with DPX.
RESULT
Nuclei: blue, black
Cytoplasm: Pink
Muscle fibres: deep red
RBCs: orange red
Fibrin: deep pink(Suvarna et al., 2013).

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Figure 4: H and E stained slide showing alveoli of from lung tissue
(1) Showing megakaryocytes (thin arrow). A discrete well-formed non-caseating granuloma
(2) Composed of histiocytes, epitheloid cells and Langhan's type multinucleated giant cell
(thick arrow). (Marie, 2012).

 GORDONS AND SWEETS STAINING PROCEDURE

Deparaffinise sections with xylene then take through alcohols to water.
Oxidise in acidified potassium permanganate for 3 minutes
Rinse in distilled water.
Decolourise with 2% oxalic acid for 1 min
Rinse in distilled water.
Mordant in 4% iron alum for 10 minutes
Rinse in distilled water.
Impregnate in ammoniacal silver solution for 11 seconds
Rinse quickly in distilled water.
Immediately reduce with 10% aqueous formalin for 2 minutes
Wash in running tap water for 2 minutes
Tone in 0.2% gold chloride (Not liver cores-see technical point 4) for 2 minutes
Rinse in distilled water.
Fix with 2% aqueous sodium thiosulphate (hypo) for 2 minutes
Wash in water for 2 minutes
Counterstain with neutral red for 2 minutes
Dehydrate, clear and mount.
RESULTS
Reticulin fibres: Black
Nuclei: Red(Roy, 2011).

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  ZIEHL-NEELSEN STAINING TECHNIQUE
Dewax tissue section.
Hydrate the sections in descending grades of alcohol.
Flood the sections with Carbolfucshin, heat until steam rises and leave 10 minutes.
Wash in tap water.
Differentiate with 1% acid alcohol until the tissue appears pale pink.
Wash in running tap water for 10 minutes.
Counter stain with methylene blue solution for 1 minute.
Dehydrate the sections in ascending grades of alcohol (70%, 90%, and 100%).
Clear in xylene.
Mount in DPX.
RESULT
Acid fast bacilli: Bright red
Nuclei: blue (Suvarna et al.,, 2013).

Table 1: Reading method for smears stained by Ziehl-Neelsen (immersion lens x 100)
Number of AFB Code
No AFB per 100 immersion fields 0
1–9 AFB per 100 immersion fields exact number of AFB
10–99 AFB per 100 immersion fields +
1–10 AFB per field ++
More than 10 AFB per field +++
(Nadia and Donald, 2005).

AURAMINE
Place the fixed smear on a staining rack and flood slide with rhodamine-auramine for 15
minutes.
Do not let surface dry.
Wash off the stain with distilled water.
Flood slide with fluorescent decolourizer (i.e acid-alcohol) for 2-3 minutes.
Rinse thoroughly with distilled water.
Flood slide with potassium permanganate for 3-4 minutes. Do not allow slide to dry.
Rinse thoroughly with distilled water and air dry.
Examine microscopically under the same light source as used for fluorescent microscopy.

Result and Interpretation:

Positive Test – Acid-fast organisms fluoresce reddish-orange against a dark background.
Negative Test – Non-acid-fast organisms will not fluoresce or may appear a pale yellow,
quite distinct from the bright acid-fast organisms(Tankeshwar, 2015).

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Table 2: Reading method for smears stained by auramine (dry lens x 25)

Number of AFB Code

No AFB on the slide 0
1–10 AFB on the slide doubtful (use Ziehl-Neelsen)
Fewer than 1 AFB per field but more than +
10 on the slide
1–9 AFB per field ++
10–99 AFB per field +++
More than 100 AFB per field ++++

(Nadia and Donald, 2005).

Figure 5: picture showing

1: Mycobacteria tuberculosis on ZiehlNeelsen Stain
2: Mycobacteria tuberculosis on an Auramine–Rhodamine stain.
(Marie, 2012).

 PERLS PRUSSIANS BLUE

Deparaffinise and bring the sections to water.
Treat the sections with freshly prepared acid Ferro cyanide solution for 10-30 minutes.
Wash well in distilled water.
Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red.
Wash rapidly in distilled water.
Dehydrate, clear and mount.

15
RESULT
Ferric ion = blue
Nuclei = red
Background = pink (Suvarna et al.,, 2013).

Figure 6: Perls Prussian blue stained slide showing Intra-alveolar hemosiderin

deposition in lung tissue. (Marie, 2012).

 IMMUNOHISTOCHEMISTRY PROCEDURE
Streptavidin-Biotin method
Three to five microns thick tissue sections were cut from paraffin blocks. These sections were
deparaffinised in fresh xylene (5 minutes) and then rehydrated with decreasing order of
ethanol (5 minutes each). Alcohol was removed by distilled water (3 washings). Sections
were kept in citrate buffer (pH 6.0) and antigen retrieval was followed by microwave heating
(5-10 minutes). Sections were brought to room temperature and washed in tris buffer (pH 7.6)
for three times (5 minutes each). The endogenous peroxidase activity of the tissue was
blocked by incubating the tissue in 3% hydrogen peroxidase for 10 minutes. Again sections
were washed in tris buffer for three times (5 minutes each). Sections were treated with non-
immune goat serum (10 minutes) to block the non-specific binding sites followed by washing
with tris buffer. Excess buffer was drained. Sections were then covered with primary
antibody diluted in tris buffer solution (primary antibody: tris buffer solution = 1:30) and
incubated for overnight in moist chamber at 4-7°C. Next day the sections were brought to
room temperature and rinsed in tris buffer three times (5 minutes each). Link antibody was
added and sections were incubated for 90 minutes and this was again followed by tris
washing. Subsequently Streptavidin peroxidase conjugate was added to the sections for 45
minutes. Finally, the chromogen diluted in the substrate buffer (substrate: chromogen = 50:1)
was added to section and left for 35-45 minutes. Finally, the sections were counterstained

16
with 10% Hematoxylin (2-3 minutes) followed by washing in running tap water, dried and
mounted with DPX mountant. Brown colour reaction products indicated positive
staining(Madhu and Puja, 2006).

 RADIOLOGIC STUDY
Anyone with a cough that lasts for two weeks or more or with unexplained chronic fever
and/or weight loss should be evaluated for TB. Chest X-ray is the primary radiologic
evaluation of suspected or proven pulmonary TB. Radiological presentation of TB may be
variable but in many cases is quite characteristic. Radiology also provides essential
information for management and follow-up of these patients and is extremely valuable for
monitoring complications. Chest X-ray is useful but is not specific for diagnosing pulmonary
TB, and can be normal even when the disease is present. Therefore, it cannot provide a
conclusive independent diagnosis and needs to be followed by sputum testing(Yon, 2015).

 NUCLIEC ACID AMPLIFICATION (NAA)/ POLYMERASE CHAIN

REACTION (PCR)
Newer diagnostic techniques for faster detection of M tuberculosis include nucleic acid
amplification tests. In these tests, molecular biology methods are used to amplify DNA and
RNA, facilitating rapid detection of microorganisms. One method is the polymerase chain
reaction assay, which can be used to differentiate M tuberculosis from other mycobacteria on
the basis of genetic information and provides results within hours. Although the test can
provide rapid confirmation of M tuberculosis in sputum specimens positive for acid fast
bacilli, it has limitations, including high cost, low sensitivity, and low availability(Nancy,
2009).

COMPLICATIONS
 HAEMOPTYSIS can be light, moderate or extensive. Massive haemoptysis, caused
by the eros7ion of an arterial wall, is a rare but dramatic complication that can result
in sudden death (Nadia and Donald, 2005).
 PNEUMOTHORAX, caused by rupture of a cavity into the pleural space, is a
serious complication. Bacilli from the cavity can infect the pleural space, leading to
pyopneumothorax. In the latter event, pleural drainage may be required in addition to
anti-tuberculosis treatment (Nadia and Donald, 2005).
 CONTIGUOUS PLEURISY can accompany active pulmonary tuberculosis (Nadia
and Donald, 2005).

Some complications may occur in patients successfully treated for tuberculosis who
subsequently present with sequelae:
 BRONCHIECTASIS: repeated acute pulmonary infection and haemoptysis are the
most common manifestations. It should not be confused with recurrence of
tuberculosis disease, which must always be confirmed by bacteriological examination
(Nadia and Donald, 2005).

17
  CHRONIC RESPIRATORY FAILURE may occur in patients who have previously
had extensive tuberculosis, in whom large parts of the lung have been destroyed
(Nadia and Donald, 2005).
 PNEUMOTHORAX may result from rupture of a bulla in association with lung
scars; this type of pneumothorax does not lead to infection of the pleura. It is usually
benign, and can usually be relieved within 48 hours with simple medical treatment
(Nadia and Donald, 2005).
 ASPERGILLOMA, due to infection by Aspergillus fulmigatusof a healed cavity,
may present with haemoptysis and often requires surgical excision(Nadia and Donald,
2005).

PROGNOSIS
TB is a severe and often deadly disease without treatment. After 5 years without treatment,
the outcome of smear positive pulmonary TB (PTB) in HIV-negative patients is as follows:
– 50-60% die (case fatality ratio for untreated TB);
– 20-25% are cured (spontaneous cure);
– 20-25% develop chronic smear-positive TB.
With adequate treatment, the case fatality ratio (CFR) often falls to less than 2 to 3% under
optimal conditions.
Similar CFRs are seen with untreated EPTB and smear-negative PTB, with an equivalent fall
in CFR with adequate treatment. Untreated TB in HIV-infected patients (not on
antiretroviral) is almost always fatal. Even on antiretroviral, the CFR is higher than in non-
HIV infected patients(Francis and Michael, 2017).

CONCLUSION
Although bacteriology remains the key examination for confirming the diagnosis of
tuberculosis, histology does play an important role, particularly for confirming the diagnosis
of extra pulmonary forms. Combining histological techniques with bacteriology increases the
yield of histology and can enhance the confirmation of diagnosis of extra pulmonary
tuberculosis.

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RECOMMENDATION

 Care should be taken when handling samples that comes into the laboratory.
 Laboratory personnel should ensure that all the staining techniques mentioned above
and more (if need be) be carried out on sections to enable proper diagnosis.
 Tuberculosis is a curable disease, early detection helps in the treatment and curing of
this infection, proper handling of samples and processing of these samples on time
can help achieve this. Cytological samples when brought to the laboratory should be
given immediate attention (i.e processed on time) to enable early detection of this
bacterium before getting tissue for further diagnosis
 Government on its part can help in organising seminars that will help to enlighten the
public on the need for going for test especially when experiencing any of the
symptoms associated with tuberculosis.

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