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).

1. Specific tests that could be performed within the histology laboratory to assist in the diagnosis and reasons for choosing them.

Tissue crossing

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).

1. 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.

1. 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).

Tissue processing

Testing for gene mutations- this is testing for DNA mutations in the HFE gene (Hollerer, Bachmann and Muckenthaler, 2017).

1. 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).  

References

Arni, S., Caviezel, C., Weder, W., & Hillinger, S. (2017). A strategy to overcome the interfering problem of tissue embedding OCT compound in activity based multiplex profiling of tyrosine kinase substrates.

Bulte, J. P., Halilovic, A., Kalkman, S., van Cleef, P. H., van Diest, P. J., Strobbe, L. J., … & Bult, P. (2018). Assessment of HER 2 status in breast cancer biopsies is not affected by accelerated tissue processing. Histopathology.

Carriel, V., Campos, F., Aneiros-Fernández, J., & Kiernan, J. A. (2017). Tissue fixation and processing for the histological identification of lipids. In Histochemistry of Single Molecules (pp. 197-206). Humana Press, New York, NY.

DeWitt, J., Cho, C. M., Lin, J., Al-Haddad, M., Canto, M. I., Salamone, A., … & Khashab, M. A. (2015). Comparison of EUS-guided tissue acquisition using two different 19-gauge core biopsy needles: a multicenter, prospective, randomized, and blinded study. Endoscopy international open, 3(5), E471.

Ferry-Galow, K. V., Lawrence, S. M., Navas, T., Makhlouf, H. R., Butcher, D. O., Gouker, B. A., … & Kummar, S. (2015). Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers.

Goodman, Z. D. (2018). Liver Biopsy Diagnosis of Cirrhosis. In Diagnostic Methods for Cirrhosis and Portal Hypertension (pp. 17-31). Springer, Cham.

Huguet, A., Latournerie, M., Debry, P. H., Jezequel, C., Legros, L., Rayar, M., … & Thibault, R. (2018). The psoas muscle transversal diameter predicts mortality in patients with cirrhosis on a waiting list for liver transplantation: A retrospective cohort study. Nutrition, 51, 73-79.

Katzka, D. A., Geno, D. M., Ravi, A., Smyrk, T. C., Lao-Sirieix, P., Miramedi, A., … & Kryzer, L. A. (2015). Accuracy, safety, and tolerability of tissue collection by Cytosponge vs endoscopy for evaluation of eosinophilic esophagitis. Clinical Gastroenterology and Hepatology, 13(1), 77-83.

Porto, G., Brissot, P., Swinkels, D. W., Zoller, H., Kamarainen, O., Patton, S., … & Keeney, S. (2016). EMQN best practice guidelines for the molecular genetic diagnosis of hereditary hemochromatosis (HH). European Journal of Human Genetics, 24(4), 479.

Sun, H., Walsh, A. J., Lebel, R. M., Blevins, G., Catz, I., Lu, J. Q., … & Wilman, A. H. (2015). Validation of quantitative susceptibility mapping with Perls’ iron staining for subcortical gray matter. Neuroimage, 105, 486-492.

Wang, R., & Hasnain, S. Z. (2017). Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology. PLOS Pathogens.

Yeap, B. B., Divitini, M. L., Gunton, J. E., Olynyk, J. K., Beilby, J. P., McQuillan, B., … & Knuiman, M. W. (2015). Higher ferritin levels, but not serum iron or transferrin saturation, are associated with Type 2 diabetes mellitus in adult men and women free of genetic haemochromatosis. Clinical endocrinology, 82(4), 525-532.

Hollerer, I., Bachmann, A., & Muckenthaler, M. U. (2017). Pathophysiological consequences and benefits of HFE mutations: 20 years of research. haematologica, 102(5), 809-817.