Histopathology and Haematology: Dealing with Liver Specimens and Staining Methods

This article discusses the laboratory procedures for dealing with liver specimens in histopathology and haematology, including tissue fixation, tissue crossing, tissue processing, embedding tissue in paraffin, microtomy, and staining methods. Specific tests that can be performed to assist in diagnosis, such as Perl's iron staining, D/PAS, and reticulin staining, are also covered. The article concludes with a provisional diagnosis of hemochromatosis and a differential diagnosis of liver cirrhosis.

SCMED 3034 Histopathology and Haematology Scenario
Questions:
1. How laboratory would deal with the liver specimen?
Tissue fixation
This is the first step that is done on a specimen after the piece of the tissue has been removed.
It can be from the liver, heart or spleen. Tissue fixation is achieved by putting the tissue
removed in 10% neutral buffered formalin. This will help prevent tissue autolysis which is
degeneration of chains of protein molecules immediately after the tissue is removed.
Formalin does this by crosslinking the chains of protein molecule ends which are loose thus
controlling more decomposition from happening (Carriel, Campos, Aneiros-Fernández, &
Kiernan, 2017).
Tissue crossing
This is the thorough inspection of tissue for diseases then measured and described. Any
information concerning the tissue is written down or typed. The tissue biopsy thereafter is
kept safe in plastic tissue cassette that is inserted in a path of formalin (DeWitt.et.al 2015).
Tissue processing
According to Bulte.er.al (2018), tissue processing moves the tissue through several chemical
reagents which include; formalin, 70%, 80%, 96%, and 100% alcohol, liquid paraffin and
xylene. Formalin function to prevent tissue from decomposing. The four deferent alcohol

grades make the tissue dehydrated slowly and alcohol is removed from the tissue by xylene
after dehydration. Xylene is replaced by liquid paraffin then it enters the tissue permanently.
Embedding tissue in paraffin
The tissue from plastic cassette is placed in a mold after being removed. Liquid paraffin is
then filled with it and the cassette part with hospital number of the patient is placed on top of
the mold to form a paraffin block after cooling (Arni, Caiezel, Weder and Hillinger, 2017).
Microtomy
This is cutting of thin biopsy tissue sections and placing them on a glass slide. The paraffin
block is placed in a block holder in an instrument called a microtome passing across a blade.
The block advances forward by three or four micrometers each time it passes the blade. This
is equivalent one tissue cell thickness. This results in a biopsy tissue section and paraffin in a
long ribbon. The ribbon is then picked and placed in a warm water bath allowing wrinkles to
smooth out. A glass slide is then slipped under the section and lifted from the water hence
microtomy (Ferry.et.al 2015).
Staining
A 60 degree Celsius oven is used to dry the tissue and then made to pass a series of chemical
reagents. The biopsy tissue is passed through xylene to remove alcohol and paraffin alcohol
to remove xylene. The tissue is then rehydrated in preparation for staining. Hematoxylin and
eosin (H and E) are the two reagents used for staining. Hematoxylin stain the nucleus black

from dark blue and area surrounding the nucleus is stained by eosin light pink and the tissue
that is outside the cell dark pink to red (Katzka.et.al 2015).
2. Specific tests that could be performed within the histology laboratory to assist in the
diagnosis and reasons for choosing them.
Perl’s iron staining
This is a appropriate method in displaying iron in the tissues. The section of the biopsy
tissue is treated with hydrochloric acid that is dilute in order for ferric irons to be released
from binding proteins. These irons react with potassium Ferro cyanide to produce an
insoluble compound which is blue. This is called the Prussian blue reaction. Iron is store
d as ferritin or hemosiderin in the liver. Ferritin is a common form of iron storage and its
capacity is about 4500 iron (III) ions per protein molecule. Since the Perl’s method stain
iron (III) ions, then it is an important test in measuring iron stores (Sun.et.al 2015).
D/PAS (Periodic Acid-Schiff)
According to Wang and Hasnain (2017), periodic Acid-Schiff is used together with
diastase enzyme an enzyme that digests glycogen. This method can also be used for
periportal liver staining of the AAT polymer inclusions which are found in alpha-1
antitrypsin deficiency disease. This method is used to detect polysaccharides like
glycogen and mucosubstances such as mucins, glycolipids and glycoproteins.
Adenocarcinomas secrete mucin. Since liver is the most common site of tumor
metastasis, then this method of staining is important. When the liver is damaged, iron
storage and metabolism is affected and therefore iron overload can result.
Reticulin stain

This method is used to visualize reticular fibers and it is used mostly in liver
histopathology. Under normal circumstances, a fine-lace-like reticular fiber secreted by
fibroblasts support the cellular components of the liver. In disease conditions this support
is altered and may either increase or decrease. This depends on the type of disease
condition (Goodman 2018).
3. Expected results from the staining methods.
Perl’s staining results
This is also called Prussian blue reaction. In this method Ferro cyanide combines any
ferric ion in the tissue resulting in bright blue pigment called the ferric Ferro cyanide or
Prussian blue. The final results are iron stains blue, the nuclei stain red and the
background stains pink.
Periodic acid-schiff stain
On a periodic Acid-Schiff, alpha antitrypsin globules, diastase resistant stain, with a
characteristic magenta color, are found at the lobule periphery.
Reticulin stain
In diseases like liver cirrhosis there is an increase in number of reticular fibers. Their
gauge is also increased. In iron overload, there is presence of hepatocellular granules
when using this method.
4. Additional non-histologic test recommended to the referring clinician.
Serum transferrin saturation- this is a test that used to determine the amount of iron
bound to a protein named transferrin which carries iron in the blood. Transferrin
saturation value is considered high if it is more than 45% (Yeap, 2015).

Testing for gene mutations- this is testing for DNA mutations in the HFE gene (Hollerer,
Bachmann and Muckenthaler, 2017).
5. One provisional diagnosis for the study case and one differential diagnosis (possible
alternative).
The possible provisional diagnosis is hemochromatosis (Porto et.al, 2016). This is a
metabolic disorder in which the body loads too much iron. It is a genetic disorder due to
gene mutation and if left untreated can be fatal to the individual. The most common
symptoms of this disease condition are; abdominal pain, lack of energy, memory fog,
heart flutter s, loss of sexual drive, and irregular heartbeat. This is the most possible
diagnosis as the old female lady has most of these signs.
Alternative diagnosis is liver cirrhosis (Huguet et.al, 2018).

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