Techniques for Diagnosis of Specimen

Question 1

Describe how your laboratory would deal with this specimen?

When the liver core specimen in formalin arrive at the Histology laboratory lab, the first thing that the staff would do is the specimen accessioning, which the specimen is being accessioning y labelled it with number in order to identify each of the specimen for each of the patient (Edward C., 2013). Since the specimen arrived already been fixated with formalin in order to prevent it from decaying and also at the same time try to preserve the cells and tissues to be in a “life-like” state (Rolls Geoffey, 2011).

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After fixation, the next step would be grossing or known as “cut-up”, in which in this case the liver specimen need to be dissect into small pieces in order to be fit into the cassettes. After the specimens being put in the cassettes, it is then being load onto the tissue processor for processing through the wax. Tissue processors are used for preparation of paraffin section (Rolls Geoffey, 2011). It will allow the specimen to be in a molten paraffin wax state after a sequence of different solvents infiltrated in the specimen (Rolls Geoffey, 2011).

Embedding is the next step where after processing, the specimen is being placed in an embedding center, placed in wax filled molds after it being removed from the cassettes (Rolls Geoffey, 2011). This is the stage where the liver specimen is being carefully oriented because in order to see the abnormal area to be visible under the microscope and thus determine the plane thoroughly (Rolls Geoffey, 2011). Using the appropriate block, the specimen is filled with wax and let it solidify for a few minutes (Rolls Geoffey, 2011). A stable base has been provided for clamping in the microtome when the cassette is now filled with wax and forming as part of the block (Rolls Geoffey, 2011).

Now, section cutting process can be preceded. Section cutting is been done by using the instrument called microtome by using an extremely fine steel blade (Hubscher, S., 2008). To get only a single layer of cells that is makes up the section, the paraffin sections are usually being cut at a thickness of 3 – 5 µm (Hubscher, S., 2008). Since sectioning making it in a form of ribbons since they will stick together edge to edge making it easier to picked up onto the microscopic slides on the floated sections on the surface of warm water in a flotation bath in order to flatten them (Hubscher, S., 2008). The specimen are ready for staining after a thoroughly drying (Hubscher, S., 2008).

Question 2

Which specific test could be performed within your histology laboratory to assist in the diagnosis? Why did you choose these?

Based on the received liver core specimen, it shows that the total iron body content is quite high which about 20gm. Thus, the most specific test to assist in the diagnosis of the iron concentration is Perl’s Prussian blue test (PPB). Perl’s Prussian blue stain is the major histochemical stain that is used to detect as well as identified the ferric iron (Fe³⁺) in particular tissues ^[4]. By the principle of hydrolysis of mineral acid, this ferric ion then is being released from the deposition of tissues, which are protein bound. Based on chemistry’s concept itself, iron in ferric state wills formed ferric ferrocyanide since it is reacted with hydrochloric acid. Thus, this makes the insoluble blue compound visible ^[5].

There is no colored product by ferrous ions themselves, thus their reaction cannot be seen. Within this technique, usually red in color will represents a nuclei in that particular normal cell, yellow in color for erythrocytes, deep blue stained for ferric salts and lastly, stained as blue or black for the asbestos bodies. The stain is composed of aqueous hydrochloric acid, aqueous potassium ferrocyanide and a neutral red stain^[6].

Measuring 10ml of hydrochloric acid, which is about 2% in concentration, starts the staining method. Then, 2% of potassium ferrocyanide was added and ensure that the mixture mixed thoroughly by shaking hem well. Next, placed the slides on the rack for staining step and carefully filtered the solution onto the slides and leave it with that solution for about 15 minutes. Later, carefully removed the excess solution from the slides by rinsed it using the distilled water. The step continued by filtered 1% of neutral red onto the slides and leaves it for about 5 minutes. The purposed of this step is to let the slides for having the capable time so that it will be able to attach to the dye completely ^[7]. About 5 minutes later, thoroughly rinsed the slides by using distilled water to remove any excess dye, which has not been attached to the cells components. The washing step should not be decreased below 5 minutes as thorough washing is required to prevent a heavy dye precipitate resulting from the neutral red counterstain ^[8]. The slides are then are blotted by using filter papers. Quickly, the slides were rinsed in 70% of industrial methylated spirit and at the same time agitating the slide by making a slosh up and down. Then, placed the slides in absolute industrial methylated spirit (100%) and further agitated for another one to two minutes.

Finally, always placed the slides mount in a DPX-type mountant since other mounting media results in fading of the stain. Again, the slides were agitated for about 2 minutes to ensure that there was no gas trapped in the slide ^[9]. Then, covered the slides with cover slips by placing a mountant upon the cover-slip which is to cover the section on the slide and the slide removed from DPX and then was gently touched on the side of the slide with the section to the cover slip.

Question 3

What are the expected results from the staining methods you have chosen?

The pattern of iron deposition can be obtain with the help of iron staining.⁴ The iron stain shows the features of the possible cause of excess iron in the body. ⁴ The degree of the iron deposition and many grading methods exist to grade the extent of deposition in the liver by using iron stain.⁴

The main organs that store excess of iron is the liver. Iron stored in cell in soluble compound is called ferritin while insoluble form is called hemosiderin. Only hemosiderin that can be seen using H&E stain while the ferritin cannot be seen. The hemosiderin appears as coarse golden brown refractile granules. On the Perl's Prussian Blue stain, the ferritin appear as a fain bluish blush and the hemosiderin appears a deep blue in color.⁴

Figure 1 The liver core specimen^1,2

This is the result obtain from the patient’s liver core specimen. It can be seen in the Figure 1 of large blue granules mark. This is how the presence of iron in the cell. The nuclei pigment appears red and the iron pigment appears deep blue in color with the Prussian blue stain. This means there is excess of iron stored in the liver of this patient.³

For a normal human being, excess of iron is stored in the year. Increase of the ferric iron stores can be identified as Hemochromatosis. The excess iron in body can lead to increase iron store in the liver. The iron is stored in the intracellular compartment of the liver.²

Question 4

What additional non-histological tests would you recommended to the following clinican?

For the additional non-histological tests, serum ferritin blood test is recommended to be used by clinician in order to detect the content of iron in patient’s body (Adams P, 2008). It is an enzyme-linked assay that can be performed on blood sample from nonfasting patient (Adams P, 2008). 70% of the total iron store in patient’s body can be found in her haemoglobin while the 20% of the total iron stored as ferritin (Hicks R, 2013). The increase or decrease of ferritin level will indicate the changing of iron level in her body (Hicks R, 2013). The normal serum ferritin level is not more that 200ng/ML in women and normal iron level in human body is about 5 gram (Hicks R, 2013).

Firstly, by referring to the Figure 2, the serum ferritin blood test will be started by drawing the blood from a vein of the patient (KidsHealth , 2014). The surface of patient’s skin will be cleaned with antiseptic and the tourniquet (elastic band) is placed around the upper arm to cause blood swell in the targeted vein (KidsHealth , 2014). Then, a needle will be inserted into the targeted vein causing the blood to withdraw from the vein and collected in a syringe (KidsHealth , 2014). When the procedure is completed, the elastic band is removed from the patient’s body part (KidsHealth , 2014). When the blood has been collected in the syringe, the needle will be removed and the targeted area will be covered with cotton in purpose to stop the bleeding (Hicks R, 2013).

Question 5

Provide one provisional diagnosis for the case study and one differential diagnosis (possible alternative).

A provisional diagnosis is basically the first diagnosis or the "working diagnosis" that is made by a medical professional and it is usually not clarify as the final diagnosis^[4]. This type of diagnosis is generally assign when the presenting problems meet some of the criteria for a disorder, but more information required for the accurate diagnosis and it might be modified as the patient's care continues and more details of diagnosis presented^[4]. Besides, the provisional diagnosis might change depending on the patient's ongoing condition, his or her response to any treatments that is offered, and also the patient's level of comfort with the new diagnosis.

For this case the 40-year-old post-menopausal woman presents and claimed that she had a long term lethargy, loss of sex drive, abdominal pain for 2 months and discoloration of the skin. Based on the symptoms presented the most probable provisional diagnosis for her is Hemochromatosis as the symptoms presented are quite identical (Table 1). Hemochromatosis is the most common form of iron overload disease, in this cases the total iron body content recorded was 20g, where the normal iron levels for women ranges from 12 to 15.5g of hemoglobin per deciliter of blood^[2]. Primary hemochromatosis or else known as hereditary hemochromatosis, is an inherited disease meanwhile, secondary hemochromatosis is caused by anemia, alcoholism, and other disorders^[1]. The Hemochromatosis causes the body to absorb and store too much iron. Apart, the extra iron builds up in the body's organs such as liver, heart and pancreas can lead to an organ damage^[1].

Table 1 Patient Presented Symptoms and Hemochromatosis Symptoms

There is also another type of diagnosis and it is called a differential diagnosis or possible alternative. The differential diagnosis is the process of comparison among diseases exhibiting similar sign and symptoms^[3]. When a patient has symptoms that are common to more than one disease or condition, the medical professional will make a list of the possibilities, and then eliminate them based on the symptoms that may not fit some of the possibilities^[3]. In this case, when evaluating a patient with suspected hemochromatosis, alcoholic liver disease and multiple transfusions should also be considered^[2].

Alcoholic liver disease

Liver biopsy in the alcoholic disease patients may show a modest increase in iron. In contrary to patients with hemochromatosis, the hepatic iron levels alcoholic disease patients are relatively normal and iron stores are less than 4 g^[2].

Multiple transfusions

Hypertransfusion is performed in patients with sickle cell anemia, beta thalassemia major and also myelodysplastic syndrome^[2]. Such patients may receive as many as 100 units of red blood cells, which may contain as much as 20-25 g of iron, similar to or more than the amount retained in many symptomatic patients with hereditary hemochromatosis^[2].

References

1. Mayoclinic.org. 2014. Hemochromatosis Symptoms - Diseases and Conditions - Mayo Clinic. [online] Available at: http://www.mayoclinic.org/diseases- conditions/hemochromatosis/basics/symptoms/CON-20023606 [Accessed: 26 Jan 2014].
2. Emedicine.medscape.com. 2014. Medscape: Medscape Access. [online] Available at: http://emedicine.medscape.com/article/177216-differential [Accessed: 26 Jan 2014].
3. Torrey, T. 2014. differential diagnosis. [online] Available at: http://patients.about.com/od/glossary/g/diffdiagnosis.htm [Accessed: 26 Jan 2014].
4. Schimelpfening, N. 2014. FAQ: What Is a Provisional Diagnosis Vs. Differential Diagnosis?. [online] Available at: http://depression.about.com/od/diagnosis/f/provisional.htm [Accessed: 26 Jan 2014].
5. Edward C. (2013). Histotechniques. Web Path. Retrieved January 26, 2014, fromhttp://library.med.utah.edu/WebPath/HISTHTML/HISTOTCH/HISTOTCH.html
6. Rolls Geoffey (2011). An Introduction to Specimen Preparation. Leica Biosystem. Retrieved January 26, 2014, from http://www.leicabiosystems.com/pathologyleaders/an- introduction-to-specimen-preparation/
7. Hubscher, S. (2008). Tissue Pathways for Liver Biopsies for the Investigation of Medical Disease and for Focal Lesions.
8. E-med.co.il. 2014. [online] Available at: http://www.e-med.co.il/emed/new/Usersite/Presentations/Liver_Diseases/10.files/slide0014_image0 70.jpg [Accessed: 26 Jan 2014].
9. Library.med.utah.edu. 2014.Hepatic Pathology. [online] Available at: http://library.med.utah.edu/WebPath/LIVEHTML/LIVER018.html [Accessed: 26 Jan 2014].
10. Ukessays.co.uk. 2014.H&e stain and perls prussian blue technique. [online] Available at: http://www.ukessays.co.uk/essays/sciences/h-and-e-stain-and-perls-prussian-blue- technique.php [Accessed: 26 Jan 2014].
11. Rashmil, S. (n.d.). Retrieved from http://www.dako.com/index/knowledgecenter/kc_publications/kc_publications_conne ction/kc_publications_connection14.htm/28829_2010_conn14_special_stains_interpret ation_liver_biopsies_saxena.pdf
12. Adams P (2008) Management Of Elevated Serum Ferritin Levels Journal. US National Library of Medicine, National Institute of Health (2008 May; 4(5):333-334. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093720, on January 27^th 2014.
13. Hicks R (2013) Ferritin blood test. Web MD. Retrieved from http://www.webmd.boots.com/a-to-z-guides/ferritin-blood-test, on January 26^th 2014.
14. KidsHealth (2014) Blood Test: Ferritin (Iron). Retrieved from http://kidshealth.org/parent/system/medical/test_ferritin.html#, on January 25^th 2014.

References:

[4] Liver Pathology, In-text: (Google Books, 2014), Bibliography: Google Books. 2014. Liver Pathology. [online] Available at: http://books.google.com.my/books?id=worDZk6fSWwC&pg=PA289&lpg=PA289&dq=why+used+perl's+prussian+blue+to+detect+iron&source=bl&ots=4RfzVoc3O5&sig=BBCSmpM1Cwt6xMxrNBwH8c5hvU4&hl=en&sa=X&ei=AkDmUpLRC8SprAfC_4DYBw&redir_esc=y#v=onepage&q=why used perl's prussian blue to detect iron&f=false [Accessed: 28 Jan 2014].

[5] Perls Prossian Blue Staining Protocol, In-text: (Ihcworld.com, 2014), Bibliography: Ihcworld.com. 2014. Perls Prossian Blue Staining Protocol. [online] Available at: http://www.ihcworld.com/_protocols/special_stains/perls_prussian_blue_ellis.htm [Accessed: 28 Jan 2014].

[6] Theory and Practice of Histological Techniques, In-text: (Google Books, 2014)

Bibliography: Google Books. 2014. Theory and Practice of Histological Techniques. [online] Available at: http://books.google.com.my/books?id=Dhn2KispfdQC&pg=PA235&lpg=PA235&dq=why+Perls+prussian+blue+is+stain+with+1%+neutral+red&source=bl&ots=JyrzjwVyH8&sig=9Qlm0_6T31awaDEuhGSChtQz3mQ&hl=en&sa=X&ei=04LmUsb8POW7iAf9uYGgBw&redir_esc=y#v=onepage&q=why Perls prussian blue is stain with 1% neutral red&f=false [Accessed: 28 Jan 2014].

[7] H&e stain and perls prussian blue technique, In-text: (Ukessays.co.uk, 2014)

Bibliography: Ukessays.co.uk. 2014. H&e stain and perls prussian blue technique. [online] Available at: http://www.ukessays.co.uk/essays/sciences/h-and-e-stain-and-perls-prussian-blue-technique.php#ixzz2rcLX4KTJ [Accessed: 28 Jan 2014].

[8] Perls’ Technique For The Demonstration of Haemosiderin – Method and Tips, In-text: (skinpathonline, 2011), Bibliography: skinpathonline. 2011. Perls’ Technique For The Demonstration of Haemosiderin – Method and Tips. [online] Available at: http://skinpathonline.wordpress.com/2011/06/20/perls’-technique-for-the-demonstration-of-haemosiderin-–-method-and-tips/ [Accessed: 28 Jan 2014].

[9] Perls M. Nachweis von Eisenoxyd in gewissen Pigmenten. Virchov’ s Arch Pat Anat und Phsiol und Klin Med 1867; 39: 42-48. Van Gieson I. Laboratory notes of technical methods for the nervous sistem. New York Med J 1889; 50: 57-60. A.F.I.P.. Laboratory Methods in Histotechnology. Washington D.C. A.F.I.P. 1994. [ Accessed : 28 Jan 2014]