Histopathology is a stimulating and demanding science with a distinguishing advantage of remaining one of the principal diagnostic tools whose valuable contribution for predicting biological behaviour of diseases and controlling patient management is phenomenal.
It is a medical science with a distinct history, evolution, techniques, sampling methods, processing, interpretation and diagnostic fallibility. The clinician samples tissues and send it to the laboratory for histopathological processing in a standard concentration of fixative. Through biopsy interpretation and assessment of surgical resections and autopsies, the Histopathologists is able to differentiate between normal and abnormal tissues.
The professional input of the medical histologist (histotechnologist/scientist) is necessary to make the tissue visible for microscopic studies.
The Clinician and Histopathologists must adopt the following critical thinking process to make a diagnosis:
(1) Knowledge: to have a sound working knowledge of symptoms and diseases
(2) Comprehension: to understand the tissue and organ systems that may be involved both in normal and diseased conditions
(3) Application: to be able to identify and name pathological processes that may occur
(4) Analysis: to have the ability to discriminate one pathological process from another
(5) Synthesis: to decide the most likely causes from epidemiological data
(6) Evaluation: to decide on likely diagnosis.
How did histopathology evolve as a medical science and diagnostic tool? How and where are samples collected? What are the important substances demonstrated in the histopathology laboratory? What is the role of autopsy in histopathology diagnoses? How are diseases diagnosed and interpreted? What are the issues involved in the diagnostic fallibility of histopathology? These are the focus of this paper which shall be elucidated under the following subheadings:
• History of histopathology
• Histopathology Sampling methods
• Important Substances and Structures Demonstrated
• The Role Of Autopsy
• Diagnosis of Diseases
• Biopsy Interpretation
• Diagnostic fallibility
History Of Histopathology
Several discoveries led to the emergence of histopathology as a medical specialty. It evolved from combined advancement in anatomy, physiology and pathology. Johannes Muller (1801-1858) takes the credit for the birth of histopathology. He was the first to use the microscope to reveal the nature and structural characteristics of cancer in 1838.
As scientists recorded a breakthrough in the use of microscopes to study tissue elements, efforts began to be made to improve on the quality of materials studied. Several chemical agents were used to preserve tissues until formalin became a universally acceptable fixative. It was discovered by Blum in 1893.
Initially, freehand sections were prepared using razors. Later, instruments were designed to hold specimens so that thinner slices could be cut. The use of the microtome for animal tissues occurred in 1848. The Cambridge rocker (1885) became widely used in pathology laboratories for its ability to section serial sections. Unfortunately however, it could not section tough tissues and large blocks. The introduction of the sledge microtome helped to overcome this limitation. The rotary microtome soon came to fore for sectioning tissue blocks. Motorized microtomes are now available in modern laboratories to reduce carpal tunnel syndrome caused by repetitive motion.
The paraffin wax method was developed slowly over a period of time by several researchers to solve the problem involved in cutting thin sections. Fixed, dehydrated and cleared tissues were infiltrated with molten paraffin wax which provide firm support for tissues and make sectioning easier.
With time, there arose justified need for dyes to make differential colouring of tissues possible for microscopic studies. Early researchers used naturally occurring dyes such as madder, indigo and carmine. Leeuwenhoek had used saffron from crocus to study muscle but hematoxylin was first successfully used by Wilhelm von Waldeyer in 1863. The hematoxylin and eosin method was introduced in 1875 by Wissowzky. The periodic acid Schiff method was developed by McManus in 1946. Silver nitrate preparations were used in histopathology and dated back to the mid 1880’s.
By 1960, automatic stainers started appearing in histopathology laboratories. Productivity was enhanced and time saved.
Welch was the first to use a frozen section to diagnose breast cancer during surgery in 1891. Before then, Francois Raspail (1794-1878) was regarded as the ‘Founder of Histochemistry’. The tissue is frozen and the fluid in it freezes and then provides support for the cutting of sections.
As things progressed, tissue processors were introduced to shorten the time required for histopathological processing of tissues. The first automated tissue processor was made in Germany in 1909.
The field of enzyme histochemistry was created in 1939 when Gomori and Takmatsu developed methods for demonstrating the enzyme alkaline phosphatase in frozen sections. This is widely applied in the diagnosis of muscle biopsies in neuromuscular disorders, some enzyme deficiency disorders and bone marrow biopsies.
Immunohistochemisty methods were introduced into histopathology laboratories when Taylor and Burns developed a working method for IHC suitable for formalin-fixed paraffin-embedded tissues in 1974. Prior to the time, researchers had used 3,-3 diaminobenzindine (DAB) to create a stable colored compound at the site of the protein in the tissue. The peroxidase antiperoxidase method, which inserts a secondary antibody, that attaches to the primary antibody and a peroxidase antiperoxidase complex was developed by Sternberger et al. This occurred in 1970. Today, IHC methods have revolutionized cancer diagnosis in the histopathology laboratory.
Laboratory Information Systems started arriving in clinical laboratories in the mid-1970’s but workable packages for surgical pathology became available later.
Histopathology Sampling Methods
For an accurate histological diagnosis, a representative sample from the area of investigation is a must. The methods and techniques applied for collecting these samples are categorized into surgical and imaging and are performed outside the laboratory. Sound knowledge about them is a necessity because they are an integral part of the diagnostic process.
Tissue samples from a living body is called biopsy while samples from a dead body is called autopsy.
Biopsies are invasive techniques which carry element of risks. These risks include:
(1) Infection or bleeding from biopsy site
(2) Contamination from biopsy instrument
(3) Perforation of tissues or organs
(4) Dissemination of tumours
(5) Local paralysis
(6) Pneumothorax and sepsis
(7) For brain biopsies, it is stroke, seizure or death
For any biopsy to be carried out therefore, there must be a genuine clinical indication. Tissue biopsy sampling methods are highlighted as follows:
Advanced Breast Biopsy Instrumentation (ABBI)
This is also known as large-core breast biopsy-a surgical technique that involves removing breast lesions under image guidance. The patient is made to lie face down on a prone biopsy table and a stereotactic mammography imaging with computers is used to pinpoint the exact location of a breast mass based on X-rays taken from two different angles. Up to 20mm of breast tissue may be taken and the procedure allows for the removal of the entire lesion in one nonfragmented piece while minimizing the amount of healthy tissue taken.
Aspiration Biopsy Cytology (ABC)
This procedure plays an important role in the preoperative diagnosis of soft-tissue tumours. The process involves the extraction of cells from tissue mass found in areas such as breast, liver, thyroid and lymph node. This method uses a thin, hollow needle and syringe to aspirate cells from masses that could be palpated through the skin. For deep lesions, X-ray or ultrasound guidance is necessary.
Brush Biopsy Cytology
This method is used for sampling ureter, mouth, bronchus, biliary tract and esophagus for the purpose of detecting cancer cells. For sampling the ureter, a cystoscope, guide wire, uteroscope, nylon or steel brush and biopsy forceps are employed.
This is also known as computed axial tomography (CAT scanning). It combines the use of a computer and X-rays. It is particularly useful for the diagnosis of lung disease, locating and imaging tumours and for facilitating needle biopsies.
This technique is used to diagnose cervical cancer in patients that have had abnormal cervical smears or biopsies. It is an extensive form of cervical biopsy in which a cone-shaped sample of tissue is removed from the inner surface of the cervix.
This sampling method uses a large-bore needle to aspirate cellular materials through insertion from organs such as kidney, liver, breast and prostate. The procedure is performed in much the same way as fine needle aspirations but often require local anesthetic.
Crosby Capsule Biopsy
This technique is used for obtaining small bowel biopsies for the investigation of malabsorption states such as gluten enteropathy. The patient is made to swallow a capsule attached to a thin tube. X-ray is taken to observe when the capsule is at the exact site. Negative pressure is then created in the tube and a small portion of the bowel mucosa is sucked into the capsule. A small cutting device in the capsule is activated and a biopsy of the mucosa taken.
This technique involve scrapping of tissues from cavities or growths using a curette, a scoop or spoon- shaped surgical instrument. It is a popular method for uterine samples.
Endoscopy is the examination and inspection of the interior of body organs, joints, or cavities through an endoscope and may be taken through a natural body orifice or a small surgical incision. The endoscope is a device that uses fiberoptica and powerful lens systems to provide lighting and visualization of the interior of tissues. Therefore, endoscopy is a general term that refers to biopsies taken using endoscopes. It include the following:
1. Gastroscopy-Endoscopic biopsies taken from the stomach
2. Colonoscopy or Sigmoidoscopy-Endoscopic biopsies taken from the Colon
3. Cystoscopy-Endoscopic biopsies taken from the bladder
4. Bronchoscopy-Endoscopic biopsies taken from the lungs
5. Arthroscopy-Endoscopic biopsies taken from the joints
6. Colposcopy-Endoscopic biopsies taken from the cervix
7. Laparoscopy-Endoscopic biopsies taken from the abdominal cavities.
Endoscopic Retrograde Cholangiopancreatography(ERCP)
This is an X-ray examination of the pancreatic and bile ducts and is used in the diagnostic assessment of patients with suspected pancreaticobiliary disease.
This is a surgical procedure performed on a lesion that is small enough to be removed easily. General or local anesthesia is used. Marking of excision biopsy margins with sutures or metal clips by the surgeon is necessary to aid future biopsy in case of incomplete incision.
Fine Needle Aspiration Cytology (FNAC)
This procedure involves the insertion of a needle into the mass and negative pressure created in the syringe. Cellular material is drawn as a result of the negative pressure. The needle is moved back and forth to aspirate enough material for diagnosis. This procedure prevents the patient from having an open surgical biopsy. It does not require anesthetic and discomfort is usually minimal. Bleeding is the most common complication. Infection is rare.
This technique samples a large, sometimes inaccessible mass that cannot be removed easily. The surgeon cuts into the mass and removes a sample that is then used to establish a definitive diagnosis before the continuation of major surgery.
Otherwise known as touch preparations. Smears are prepared by pressing the cut surface of freshly dissected samples e.g. lymph node onto the surface of a microscope slide. The result is a thin layer of cells that may be stained and examined microscopically.
Liquid Based Cytology
This is an automated alternative to the conventional Pap smear. A sample fromm the cervix is collected using a plastic brush device that is detached after specimen collection and placed into a vial of transport medium.
In the laboratory, the transport media vials are vortex mixed and the cell suspension passed through a density gradient centrifugation process to remove mucus and blood cells. The cell pellet is then re-suspended and a thin-layer sample transferred to a microscope slide that can be stained and examined microscopically.
Loop Electrosurgical Excision Procedure (LEEP)
This procedure is used to treat cervical dysplasia, a pre-cancerous change of the cervical epithelium that can be identified on cervical smear examination. The method uses a thin wire loop electrode attached to an electrosurgical generator that transmits a painless electrical current to the loop. As the loop comes in contact with the cervix, the tissue is rapidly heated, causing the cells to separate. The loop quickly cuts a margin around the affected cervical tissue and removes sufficient tissue for definitive treatment and pathologic evaluation. The LEEP specimen is larger that a colposcopic sample and similar in size to a cone biopsy.
A small, sharp, hollow tube called punch is placed over a lesion after a local anesthetic. The tool is then pushed down and slowly rotated to cut out a circular piece of skin. When the punch is removed, the circular skin sample if lifted up with a forceps or a needle and the skin is cut away. The technique is used for skin generally. However, it is also used for cervix after an abnormal cervical smear.
Used for obtaining skin samples that affect only the top layers of the skin i.e. dermis and epidermis with a razor blade or scalpel. A deep shave may be necessary to evaluate pigmented moles or other skin tumours.
Most applicable to Pap smear. The surface of the cervix is scraped with a spatula-shaped instrument t obtain the sample of cells. The cells obtained are carefully smeared onto a glass slide, stained and examined microscopically for any abnormality.
This is a specialized radiological technique used to evaluate masses that are either too small to be palpated directly through the skin or are located in an inaccessible part of the body such as the brain. The most common use of this technique is a stereotactic breast biopsy where a special computer is used to guide a needle to an abnormality seen on mammography.
Trephine biopsies of the bone marrow are usually performed on the posterior iliac crest and should be carried out when clinically indicated. The trephine needle removes a sample of bone marrow of at least 1.6cm that is used to prepare biopsies, smears, or films for the diagnosis, staging and progress of disorders involving the blood cells such as myelomas, lymphomas and leukemias.
Important Substances And Structures Demonstrated By Histopathology Staining Methods.
A brief highlight of substances and structures demonstrated to aid histopathology diagnosis is vital to elucidate the pivotal role this clinical specialty play in medical diagnosis. Staining techniques applied for the substances shall also be mentioned without delving into the details. The disease conditions where they are useful will also be mentioned.
The following substances and structures are worth of attention for this work:
(5) Elastic Fibers
(6) Collagen Fibers
(7) Reticulin Fibers
(8) Amyloid Fibrils
(9) Endocrine Glands
(12) RNA and DNA
These are a large group of substances which exist in normal and pathological tissues. They were previously classified based on their structures, site in the body and staining reactions. Recently however, Kiernan classified them broadly as polysaccharides, proteoglycans and glycoproteins.
The Periodic Acid Schiff method is the most popular stain for carbohydrates. It was developed by McManus in 1946 and mostly used to demonstrate the presence of glycogen in tissue sections. Glycogen is normally found in the liver, endocervix and fresh muscle but pathologically present in adenocarcinomas, mesotheliomas, seminomas and in clear cell carcinomas.
These could be acid mucins, neutral mucins or sialomucins depending on their chemical compositions. Generally, they have the ability to absorb ferric iron from colloidal iron solutions.
Mucins in goblet cells of the G.I.T. is demonstrated by the Best Carmine method. The Hales’ colloidal iron method demonstrates acid mucins (blue), the high iron diamine method identifies sulfated mucins (brown-black). The alcian blue method devised by Steedman in 1950 remains the most versatile method for acid mucins.
Demonstration of mucins are significant in the diagnosis of primary tumours of the lower G.I.T. and some metastases from adenocarcinomas of the lower G.I.T.
Many pigments could be found in the human body under normal and pathological conditions. Pigments develop from fixation process, disease process, some are naturally occurring while others are introduced from outside the body otherwise known as exogenous pigments.
Pigments created by the body’s metabolism are of most interest to pathologists. They include hemosiderin, melanin, chromaffin substance, lipochrome, lipofuscin, hemoglobin and Dubin-Johnson pigment. However, iron pigments and melanin pigments are the most commonly demonstrated pigments in routine histopathological diagnosis.
Perl’s Prussian blue method is used to demonstrate iron pigment and aid in the diagnosis of bone marrow disorders and hemosiderosis of the liver.
Melanin is naturally occurring in the skin and the substantia nigra in the midbrain but pathologically present in melanin –producing neoplasms such as neveu or melanoma. Methods for the demonstration of melanin include Masson Fontana (black) and the Diazo method.
Lipids could be classified as simple lipids, compound lipids and derived lipids. A wide range of stains for lipids (fat) are possible on histological sections. Fat exists normally in some organs of the body and in the skin but indicative of pathological process when found inside the liver, or as a lipoma or liposarcoma.
Common fat stains include osmium tetroxide, Sudan III, Sudan IV, Oil red O, Sudan Black. Less commonly used stains are Bakers acid hematein, Luxol fast blue and Nile blue sulphate methods. Fat stains are useful in the diagnosis of neuronal and other storage disorders.
Elastic tissue is present in the skin, ligaments and the elastic laminae of blood vessels. It is also very abundant in the lung and the wall of the aorta.
The Verhoeff elastic method combined with Van Gieson is the most popular elastic fibers stain used in histopathology laboratories. Orcein is reputed to be the stain of choice for delicate elastic fibers in skin and favoured by dermatopathologists. The Gomori aldehyde fuschsine method introduced in 1950 is a progressive method for elastic fibers.
Collagen forms a coarse extracellular framework or scaffolding; its fibers are the coarse connective tissue fibers. They are doubly refractile and stain red with van Gieson’s stain. It is well demonstrated by the trichrome methods and in polarized light, collagen is brilliantly bi-refringent. In present times, Masson’s trichrome introduced in 1929 is the most popular connective tissue stain. Nuclei is stained blue-black, cytoplasm red, and connective tissue green or blue depending on counter stain chose.
Reticulin is the name given to the chemical entity or substance of which reticular fibers are composed; it is a protein to which fatty acids and polysaccharides are bound.
Reticulin fibers are demonstrated by silver impregnation, the gold method and the periodic acid Schiff technique.
Certain neoplasms produce abundant reticulin which assumes characteristic patterns. These are neoplasms derived from mesodermal elements e.g. rhabdomyosarcomas, hemangiosarcomas and also fibroblastic tumors.
In the liver, reticulin stains are often helpful in early cirrhosis. They are indispensable in the diagnosis of early fibrosis of the marrow and are helpful in differentiating certain kidney lesions.
Amyloid fibers are seen in amyloidosis- a group of diseases resulting in the deposition of insoluble protein in the interstitial spaces of blood vessels and various organs. There are at least five types. Secondary amyloid is usually created in response to chronic inflammatory processes including pulmonary infection, tuberculosis and rheumatoid arthritis as well as some neoplasms.
A popular amyloid stain is Congo red. The carbohydrate component can be demonstrated by the PAS method and iodine. Pretreatment with potassium permanganate will reduce secondary amyloid staining. Amyloid is metachromatic and bi-refringent in polarized light.
Myelin are demonstrated for the study of axonal diseases especially when they become degenerated. Myelin is the material that forms the extended surface membrane of Schwann cells in the peripheral nervous system and of oligodendrocytes in the central nervous system. It forms a multiplayer sheath around the axons of neurons and provides electrical insulation.
Staining methods for its demonstration include Weigert Pal, Kultschitsky and Loyez. The PTAH method will also demonstrate myelin. Modern methods of demonstration are the Luxol fast blue and eriochrome cyanine R (Solochrome cyanin).
Demonstration of acid-fast , gram positive and negative organisms is very popular in histopathology laboratories. The demonstration of Mycobacterium tuberculosis is of keen interest to pathologists especially in areas where tuberculosis is endemic.
Robert Koch in1882 was the first to develop a workable method for the demonstration of this organism. Others such as Ehrlich, Franz Ziehl, Rindfleisch and Neelson improved upon the method of staining the organism. The Ziehl Neelsen stain commonly used today is applicable to paraffin sections.
Poor staining results for some acid-fast organisms which contain reduced amount of lipid in their coats. To improve on this, methods that uses oil to remove paraffin wax from sections was devised. The most prominent among them is the Fite Faraco method for Mycobacterium leprae. In advanced laboratories, fluorescent method using auramine-rhodamine is favored for the visualization of sparse acid-fast bacilli.
The Gram stain devised by Christian Gram is the widely used method for differentiating gram-positive and negative organisms. This method and its variations is still widely applicable to paraffin sections. Modifications of the Gram method are the Gram-Twort method, (1924), the Brown and Brenn method (1931) and the Brown and Hopps method (1973).
RNA and DNA
The demonstration of RNA and DNA is vital in studying the cell population of tumours. The Feulgen method for nucleic acids was developed in 1942 and popular in histopathology laboratories. Nucleic acids are hydrolyzed by hydrochloric acid to create aldehydes which are demonstrated by Schiff’s reagent.
The Unna-Pappenheim method developed earlier stains DNA with methyl green and RNA with pyronine Y. Fixation is Carnoy fluid give best results.
The Role Of Autopsy
Certain occurrences reinforce the role of autopsy is histopathological diagnoses. Among such factors are:
(1) Many disorders are unrecognizable before death
(2) Errors occur in biopsy sampling
(3) Discordance between primary clinical diagnosis and that obtained from autopsy has been found to be high especially in malignant tumors.
Comparing histological diagnoses with those obtained from autopsies often improve diagnostic methods and help rectify errors from biopsy sampling.
However, it should be noted that confidence in modern methods of diagnosis such as radiological imaging techniques, computerization, plastination and other audiovisual teaching methods has heralded a worldwide decline in autopsies requests.
Diseases, Diagnoses And Biopsy Interpretation
Diseases are broadly classified into malignant and nonmalignant groupings. Each has its agents and causes. Genetic agents causes chromosomal abnormalities which results in diseases such as Down’s syndrome, achondroplasia and cystic fibrosis.
Diseases such as osteomalacia, burns, liver disease, tuberculosis, influenza, schistosomiasis, hay fever, thyroiditis, schizophrenia are acquired and caused by vit. D deficiency, physical agents, drugs, bacteria, viruses, parasites, autoimmunity, psychogenic factors respectively. They are all nonmalignant diseases.
Malignant diseases could be caused by chemical agents, environmental agents and oncogenic agents. Skin cancer could result from arsenic exposure, mesothelioma could result from asbestos exposure. Other diseases such as leukemia, mouth and throat cancer and cancer of the scrotum could result from exposure to azo dyes, benzene, betel nut and soot respectively.
Environmental factors such as dioxin, radiation, smoking and sunlight could cause lymphoma, thyroid cancer, lung cancer and skin cancer respectively.
Human paillomavirus, Epstein-Bar virus and hepatitis B virus are oncogenic agents that could lead to the following malignant diseases-cervical cancer, Burkitt’s lymphoma and liver cancer respectively.
Diagnosis involves recognizing a particular disease and giving it a name. Diseases are easily recognized from disorders in the structure and malfunction of tissues. The patient’s sample is compared with what is known as normal. Normality is a bell-shaped curve of normal distribution rather than a discrete, single locus.
In making tentative diagnoses, macroscopic appearances are supported by microscopic examination. Benign conditions such as lipomas, fibroid uteri, and dermoid cysts can be readily recognized by macroscopic appearances. So also could carcinomas of the breast, bowel and ovary be readily identified.
In microscopic examination, the pathological process is assigned a name e.g. papilloam, carcinoma and tissue type e.g. squamous and glandular.
In tumor pathology, the degree of differentiation (grading) and depth of invasion (staging) are major prognostic factors.
Grading is usually classified as low grade or high grade. For staging, the TNM system is one of those currently in use. T is the extent of the primary tumor, N indicates the absence or presence and extent of regional lymph node metastases, and M indicates the absence or presence of distant metastases.
While the importance of histopathology in the diagnoses of diseases is not in doubt, it is however honorable to admit that errors do occur. The expression of error is aptly described as diagnostic fallibility. These inaccuracies critically affect patient care and are categorized as oversights or misinterpretations.
Risk management strategies must be adopted to minimize or completely eradicate these errors which can give rise to damage liability and litigation. Recommended strategies include:
(1) Holding of regular audits
(2) Improving standards through clinico-pathology meetings
(3) Peer review audits using selected samples
(4) Specialist referral of difficult cases and
(5) Adoption of standard criteria and reporting guidelines.
In an attempt to bring the value of histopathology as a diagnostic tool to the front burner, this paper has briefly exposed the evolution of histopathology cum histotechnique. A better understanding of the history of histopathology and transformation over the past 50 years and beyond has thus been given.
Closely following is the role played by different professionals like medical histologists (Histotechnologists/scientists) to make diagnostic possible at microscopic level.
In order to have a broad understanding and appreciation of the various ways of obtaining biopsy samples in the theatre, sampling methods which primarily determines the quality of diagnosis to be given has been elucidated. A full knowledge of the nature of specimens and where they were taken has thus been fully established.
To make this subject clearer, different materials demonstrable in the histopathology laboratory have been discussed with brief mention of the techniques and the results expected. Also mentioned were means of interpreting microscopic slides.
Histopathology like any other medical science has its limitations. This has been highlighted as diagnostic fallibility.
Histopathology has thus been showcased as a valuable diagnostic tool in establishing the reasons, types and degree of diseases for both the living and the dead.
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