Health: Clinical Diagnosis and Management

Histopathology or cellular pathology is a division of pathology that deals with the microscopic analyses of diseased cells in a tissue section. Therefore, histopathology is the study of fine microscopic details, anatomical changes and anomalies in the experimental tissue samples as a result of diseases (1).

The person who deals with histopathology known as pathologist or histopathologist. Histopathologists are medical professionals who possess a broad knowledge of pathological and clinical aspects of diseases. Many diseases or disorders look very alike, thus, being competent to distinguish small dissimilarities is an important task for the histopathologist. Histopathologists are at the front position of research into various prevailing diseases such as cancer (2).

The key use of histopathology is in medical science where it involves the examination of biopsy samples and autopsy. Detailed autopsy inspection involves measuring clinical, macroscopic, and radiology aspects, looking for primary pathologies, determining how they transmit to the terminal occurrence, looking for original causative aspects to that pathology, and comparing with the experimental medical traits. In the circumstance of tumour pathology, only macroscopic study is hardly adequate (3). Nowadays, in the age of genetics, paraffinized tissue may be beneficial for DNA archiving. Some pathological conditions may provide unclear macroscopic forms (perforated gastric ulcers, tumour mimics such as intestinal TB and pulmonary fungal infections). Admitted by many histopathologists, mainly cardiovascular disorders provide little additional information histologically and assessment is basically for eliminating contributing elements (e.g. Arteritis) (2 and 3). 

Histopathological specimens can be prepared by the following 3 methods:

1. Whole mount: Whole mounts are preparation of entire organism. E.g. fungi and parasite. These preparations must not be more than 0.2-0.5 mm in width (3).
2. Section: The mainstream of histopathological experiments are done by tissue sections cut in 3 to 5 mm. thick sections. These sections can be stained and mounted permanently for histopathological studies (3).
3. Smear: These preparations are suitable for blood samples, bone marrow and other bodily fluids like aesthetic or pleural fluid. Smears are also prepared by pressing a grease free slide in contact with the damp surface of tissues or crushing soft tissues between two slides (3).

Five Problems Encountered During Practical which could have led to Poor Section Qualities:

Scratch or Tears across Part of the Section

Cause: Dust, carbon, suture or calcium etc. in wax or tissue.

Remedy: Examination of block below a magnifying glass. If dust in wax is found re-embed. Removing of suture from the tissue can be done with scalpel. If calcium is found, decalcifying the block should be done (4).

Section displays thick and thin horizontal appearances

Cause: Loose block, loose knife, blunt edged knife, very firm tissues.

Remedy: Tightening of block and knife, sharpening of knife and softening of tissue or embedding in harden wax (4).

No ribbon formation

Cause: Block is not parallel to ribbon, tissue may be incompletely infiltrated or contaminated with water or IPA, paraffin may be too hard etc. or knife tilted too much.

Remedy: correction of the alignment and tilt, re-embed (4).

Sections are folded or compressed

Cause: Blunt spot on the knife, border of the block is not parallel to the knife, soft region in the wax due to existence of clearing agent (4).

Remedy: Trim edges, align block along the knife, re-infiltration of tissues and re-embed.

Tissue is mummified or dried-out

Cause: Mechanical failure of the tissue processing machine.

Remedy: Placing the dried specimen into the following rehydrating solution for 18-24 hours-

Distilled water- 70.0 ml

Sodium Carbonate: 1.0 gm

Absolute ethyl alcohol: 30.0 ml (4)

Tissue-based diagnosis and research are highly based on the routine H&E staining and special stains. These stains colours transparent tissue sections, thus, allow histopathologists and researchers to examine tissue morphology, cell structure, microorganisms such as bacteria or fungus and prevalence or presence of particular cell types under microscope (5 and 6).

Routine H&E refers to Hematoxylin and Eosin staining which stains the nucleus blue and cytoplasm pink. This is called routine staining because H&E staining is used regularly in histopathological research laboratories as it offers the pathologists a very detailed sight of the tissue. Often H&E staining gives sufficient information for a disease diagnosis based on tissue organization, any nuclear changes, abnormalities or presence of particular indicators (5).

Special stains refers to any stain other than H&E. There are hundreds of special stains but few are used in clinical histopathology when H&E staining does not provide adequate information. Some commonly used special stains are Perls’ Prussian Blue Iron, Masson's Trichrome, Periodic Acid Schiff, Modified GMS Silver stain, Alcian Blue and Ziehl Neelsen (6).

Formalin fixed deposits are form from haemoglobin by the action of formaldehyde at acidic pH. It can easily be fixed with simple formalin solution like formal-saline. As solutions age, formic acid is created from the formaldehyde and drops the pH. This sequentially causes deposition of crystals of formalin pigment or acid formaldehyde haematin. Formation of formalin pigment can be effectively stopped by using 10% neutral buffer formalin (NBF) (7).

These deposits are found both extracellular and intracellular spaces in tissues after fixation. Formalin deposition occurs in nearly all types of tissue after a long period but forms much faster and prevalent in blood samples (7).

In H&E staining, Hematoxylin is a basic dye whereas Eosin is an acidic dye. Basic dyes react with acidic or anionic (basophilic) components of cell such as DNA, RNA and nucleoproteins. Acidic dyes like Eosin react with basic or cationic components of cell. Proteins and other components of cytoplasm, cell wall and extracellular fibres are basic in nature (acidophilic), thus, they react with Eosin and forms pink coloration (5).

Mordants are substances that causes certain staining reactions to take place by forming an anchor between the stain and the tissue. Mordants facilitate the binding reaction of stain. This anchoring is referred as lake. Without lake, most dyes are not capable of binding to and stain the tissue. E.g. Potassium alum and Ammonium (mordants) for Hematoxylin which makes acidic Hematoxylin basic in nature (8). 

References

1. Allen DC, Cameron IR, editors. Histopathology specimens: clinical, pathological and laboratory aspects. Springer Science & Business Media; 2012 Aug 14.
2. Flynn C, James J, Maxwell P, McQuaid S, Ervine A, Catherwood M, Loughrey MB, McGibben D, Somerville J, McManus DT, Gray M. Integrating molecular diagnostics into histopathology training: the Belfast model. Journal of clinical pathology. 2014 Feb 3: jclinpath-2014.
3. McPherson RA, Pincus MR. Henry's clinical diagnosis and management by laboratory methods. Elsevier Health Sciences; 2016 Mar 31.
4. Culling, C.F.A., 2013.Book of histopathological and histochemical techniques: including museum techniques. Butterworth-Heinemann.
5. Bancroft JD, Layton C. The hematoxylins and eosin. Bancroft's Theory and Practice of Histological Techniques, Expert Consult: Online and Print, 7: Bancroft's Theory and Practice of Histological Techniques. 2013:173.
6. Carson FL. Histotechnology. American Society of Clinic; 2015.
7. Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. Journal of Histotechnology. 2013 Jul 18.
8. Gersh IS. Fixation and staining. The cell “(J. Brachet and AE Mirsky, eds.) p. 2014 May 10:21-66.

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