Histopathology Lessons, Research And Resources

Hibiscus Sabdariffa Extract-A Progressive Nuclear Stain Substitute For Haematoxilin

2011-12-05T00:17:00.000-08:00

A recent disovery in an article published by the Journal of Histotechnology has shown that extract of Hibiscus flower can be used as a progressive stain in histopathology diagnosis and to demontrate tissues in histology.
Summary of the paper authored by Benard Solomon of the Pathology Department University Of Ilorin Teaching Hospital, Kwara State, Nigeria is as follows:

Title Of Paper:-Iron-Roselle: A Progressive Nuclear Stain Substitute For Haematoxylin.

Abstract
Roselle, the dried calyces of Hibiscus sabdariffa L., is a food colorant used in many tropical Countries. A simple hot-water extract of rosell, with added ferric chloride(FeCl3.6H20) and acetic acid, provides a progressive blue stain for cell nuclie comparable to that ssen with hemalum.
Dry, red calyces of H. sabdariffa were ground with a binatone blender. To 10g of the ground red calyces of H.sabdariffa in a conical flask, 200ml of distilled water was added and brought to boil to create the brilliant red-colored extract. It was immediately allowed to cool and filtered with a Whatman filter paper to give a clear H.sabdariffa extract.
The extract staining solution was compounded as follows:
100ml of clear H.sabdariffa extract, 5.0g of sodium chloride, 1.2ml of 10% ferric chloride solution, and 3.0ml glacial acetic acid. The solution was used to stain paraffin sections of formaldehyde-fixed tissues at 4 microns along with parallel hematoxylin and eosin (H&E) stainins for a control.
Results showed that staining of nuclei with the extract solution was comparable with those sections stained with H&E. It is therefore suggested that the extract solution could be a progressive nuclear stain substitue for hemalum in H&E procedures due to its domestic availability, ease of preparation and use, resistance to fading and above all its good nuclear staining properties.(The J. Histotechnologyy 31:57,2008)

Source-The Journal of Histotechnology/Vol. 31, No.2/June 2008.

A Case For Breast Tumour Banks To Complement Breast Cancer Research In Africa: By Benard Solomon, Pathology Dept. Unilorin Teaching Hospital, Nigeria

2011-04-08T02:16:00.000-07:00

Breast cancer is a global health challenge both to women and men. However, women are the major population at risk.
In advance Countries, concerted effort have been made to reduce mortality rate and breast cancer risk among women. Besides establishing
research Institutes, the availability of Breast Tumour Banks(BTB) has significantly improved the diagnosis and management of breast cancer.

A recent search in google, the world's most popular search engine reveals that Breast Tumour Banks(BTB) are available in most European Countries,
North America, Canada, U.S.A., Asia, Australia and the Middle East. There is none in Africa!

This poses a great health challenge to African population especially women.

It is estimated that 153,100 new cases of cancer occur in Canada every year. 1, 354 die weekly. In Australia, it is also estimated that 15,409 women are expeccted
to be diagnosed with Breast cancer.
In India, an estimated 80,000 new cases are diagnosed annually.

Africa has the following incidences:
* 3, 785 cases occur in South Africa annually

* 10,000 cases in Nigeria annually

* 20.4 per 100,000 in Harare and 16.4 per 100,000 in Kampala

* Gambia has 3.4 per 100,000 while Egypt has 10,556 annually.

Although breast cancer incidence is lower in Africa, there is a consensus among experts that there will be increase in the coming years.
Pre-emptive and concerted effort in the area of cancer research becomes imperative. Establishment of BTB is a sine qua non in this direction.

What is a Breast Tumour Bank? A breast tumour bank is a kind of bio-repository. A bio-repository is a facility that collects, catalogs and stores samples of biological
materials for laboratory research. Since breast tumour bank is a human sample, it is important that medical information be stored along with a written consent to use the samples
for laboratory studies.

Facilities for BTB as modeled by the Texas Tumour Bank include:

Tissue Collection: This is a special software called TissueStation, an informatics system developed and
maintained by the Clinical Research Information Systems. This is used for storage of tissue collection data using an Oracle database accessible over the web via an Oracle Forms Client.
Data security is enhanced by the use of firewall and automated routine back-up with user authorisation.

Any BTB to be deployed should have the ability to communicate with internal clinical data repository systems especially the pathology laboratory information systems and clinical protocol-based tissue request system.

There should be a freezer security system to continuously monitor temperature with individual cryoprobes that record ambient temperatures in each freezer.
The cryoprobes should be linked to a centralized computer system in the institutional security office and manned twenty four hours a day. An alarm system that notifies monitoring services must also be installed.

Talking about breast tumour banks data in the world, a recent bio-banks data released by the global directory shows that Europe has 79 banks, 15 in North America, more than 156 in America, 24 in Asia, 11 in Australia, 4 in Middle Ease
with Israel and Iran having 3 and 1 respectively.

A closer look at the directory shows that across Europe there are Breast Tumour Banks in Vienna, Brussels, Bern, Granada, Madrid, Lyon, Marseille, Toulouse, Genoa, Birmingham, Cambridge, Cardiff, Liverpool, London and Southampton.
Cities with BTB in North America are Toronto, Vancouver and Winnipeg. A google search also shows that there is breast tumour banks in Manitoba.

In the U.S.A., BTB exist is Tucson, Atlanat, Augusta, Worcester, Frederick, Ann Arbor, Rochester, Durham, Philadelphia, Houston, Bainbridge Island, San Antonio and Galveston.
Across Asia, BTB are found in China, India, Japan, Korea, Malasia, Singapore and Thailand.

Furthermore, it is worth mentioning that BTB in Australia exists in Auchenflower and Westmead.

In the Middle East, Breast Tumour Banks have been established in Iran and Israel.

Although BTB could exist in other cities yet to be included in the global directory as compilation is still on-going, it is imperative to note that those Continents and Countries have instituted pragmatic efforts at promoting breast cancer research.

The African situation is pathetic! From South Africa to Zimbabwe, Egypt, Tunisia, Ghana and Nigeria, no single BTB has been established.
It is therefore not surprising that recent breakthrough in breast cancer research such as the success stories that led to the discoveries of Tamozifen and Herceptin came from Countries with Brest Tumour Banks especially America which has the largest number
of Breast Tumour Banks.

Incidence of breast cancer is lower among African Countries when compared with their counterparts in other Continents but that cannot be taken as an excuse because breast cancer is on the increase. One of the reasons for this is the adoption of Western lifestyle by African
middle class. The future generation are therefore at higher risk. Taking pre-emptive measures by establishing Breast Tumour Banks for the promotion of research with the objective of finding home-grown remedies that are scientifically tested becomes sensible and a duty African
Governments and Non-Governmental Organizations becomes an urgent intervention strategy.

Review Of Multimedia Devices In Use In Histopathology Practice And Education-Effective Tool For Continuous Professional Development- By Benard Solomon

2010-12-14T04:42:00.000-08:00

Introduction

Since the modern transformation in Information Communication Technology, the science of histology and histopathology has not been spared the extensive influence of the power of technology. It is now possible for students to be taught not only through visual-aids but through the Internet.

Computer devices that make this possible are called multimedia. This paper will review these devices and how they have improved the science, education and practice of histopathology as a medical science.

Definitions

The Webster dictionary defines histology as ‘ the branch of biology concerned with the microscopic study of the structure of tissues.

In the same vein, the medical dictionary defines histology as ‘The study of the form of structures seen under the microscope. Also called microscopic anatomy, as opposed to gross anatomy which involves structures that can be observed with the naked eye.'

Histopathology in the order sense is defined as ‘the science concerned with the study of microscopic changes in diseased tissues.’ (09 Oct 1997)

For the purpose of this paper, histology could be defined as the microscopical study of normal tissues while histopathology is the microscopical study of diseased tissues.

Multimedia

These are applications that integrate text, pictures, motion pictures, animated graphics and sound. They are used with the computer system. In this way, the computer is not only seen as a working or educational tool but an entertainment medium. Multimedia applications relate directly to :

CPU, CD ROM/DVD drive, Sound Card, Graphic Card Display Monitor and Editors.

Multimedia applications as it relates to the study and practice of histopathology shall be discussed as follows:

1. Animations: These are moving diagrams or cartoons that move in sequence of images displayed one after the other. DiGiacinto D and Fisk G has written extensively on the effectiveness of learning when verbal instruction is combined with visual demonstrations. This becomes especially useful when dealing with a complex informaiton. O’Day D.H. also demonstrated how to provide simulated experiences to assist learners in comprehending detailed processes using powerpoint and Camtasia.

2. Audience Response System: This is a software package that work intuitively with PowerPoint to create interactive presentations that engage participants, assess learning and promote discussion. This system particularly promote real-time learning whereby lecturers can gauge the level of student comprehension. The Mayo Clinic has been exceptional in the use of this technology for its histopathology training program.

3. Web-Based Course Management System: This education system allow educational institutions to deliver online curriculum via the Intranet. Students are able to access lectures with voice-over audio recording wherever they may be and at anytime they wish once there is Internet connection. This system thus make it possible for student to consolidate knowledge gained during class instructions. It is also interesting to understand that this system allow teachers to provide online examinations which can be graded by the system and returned to students for immediate feedback. The Open, long distance learning system benefits a lot from the technology. Above all, this system also allow for knowledge acquisition by students or practitioners with different learning styles through the incorporation of pictures, text, animations and videos.

4. Camtasia Studio/Camstudio: This is an audio, voice-over recording software which could be used in synchrony with PowerPoint presentations. Lectures can be recorded and uploaded to the web-based course management system. Students can then access the lectures at their convenience and as often as they need. A powerful open source alternative to Camtasia is Camstudio which is simple to use and easy to download.

5. Adobe Contribute CS3: This web publishing tool is mainly used to create and edit web content. Weekly calendars are published and information are posted for student’s update.

6. Teleconference: This technology makes it possible for remote presentations to be made mostly by well established global organisations like the National Society for Histotechnology (NSH) or the American Society for Clinical Pathology (ASCP). It provides current trends from experts in the field of pathology in real-time.

7. Videos: Videos enhance learning through active demonstration of histopathology techniques. It could be used to assist students in understand basic concepts of the science of histopathology e.g. microscopy, types of biopsies and histopathology techniques.

From the above multimedia applications in use, it is evidently clear that the following are achieved:

1. Enhancement of educational presentations and learning through in-depth, detail both visual and verbal thus enhancing continuing education.

2. Creation of PowerPoint presentations that contain questions to evaluate the progress, performance and knowledge of students.

3. Enhancement of formalized learning through structured online courseware that can be modified to fit the needs of learners.

4. Liberty to view presentations at learner’s convenience which has contributed to continuing professional development in great measure.

5. Leverage for education team to create web pages that contain calendars, newsletters and education venues and web forms to request for credit and teleconference materials.

6. Provision of latest information pertinent to areas of specialization and education which has enhanced general understanding of topical issues.

Websites For Incorporated Multimedia Education

The following websites are a great resource for multimedia use in histopathology:

1. http://www.expressionpathology.com

2. http://www.visualhistology.com

References

1. icrocomputing and www, National Open Uninversity pg. 91

2. O’Day DH: How to make pedagogically meaningful animations for teaching and research using PowerPoint and Camtasia. Proceedings of the IPSI-2006, International Conference on Advances in the Internet, Processing, Systems, and Interdisciplinary Research, Chapter IV; February 6; Marbella, Spain. 2000b

3. DiGiacinto D: Using multimedia effectively in the teaching-learning process. J Allied Health 36:176-179, 2007.

4. Fisk G: Using animation in forensic pathology and science education. LabMedicine 39:587-592, 2008.

Issues On Diagnostic Value Of Histopathology-By Benard Solomon

2010-07-02T06:41:00.000-07:00

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.

Computed Tomography(CT)
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.

Cone Biopsy
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.

Core Biopsy
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.

Curettings
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.

Endoscopic Biopsy
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.

Excision Biopsy
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.

Incisional Biopsy
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.

Imprints
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.

Punch 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.

Shave Biopsy
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.

Smears
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.

Stereotactic Biopsy
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 Biopsy
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:

(1) Carbohydrates
(2) Mucins
(3) Pigments
(4) Lipids
(5) Elastic Fibers
(6) Collagen Fibers
(7) Reticulin Fibers
(8) Amyloid Fibrils
(9) Endocrine Glands
(10) Myelin
(11) Microorganisms
(12) RNA and DNA

Carbohydrates
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.

Mucins
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.

Pigments
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
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 Fibers
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 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 Fibers
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 Fibrils
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
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).

Microorganisms
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.

Diagnostic Fallibility
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.

Conclusion
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.

Bibliography

Michael Titford: A Short History Of Histopathology Technique, Journal of Histotechnology, Vol. 29, No. 2, June 2006. pp 99-110

Bracegirdle B: A History of Microtechnique (ed 2). Lincolnwood, IL, Science Heritage Ltd. 1986

Johannes Muller (1801-1858) Anatomist, physiologist, pathologist (Editorial). JAMA 214:2049-2051, 1970.

Cook H: Evolution of histology. Biomedical Scientist. 9:825-827m 2000

Von Waldeyer W:Untersuchungen über den Ursprung und den Verlauf des axsencylinders bei Wirbellosen und Wirbelthieren sowie über dessen Endverhalten in der quergensteiften Muskelfaser. Henle Pfeifer’s Rat Med. 20:193-256, 1863.

Pearse AGE: Histochemistry Theoretical and Applied. Vol 1 (ed 3). New York, Churchill Livingstone, 1968, pp 5, 17-19

Gruhn JG: Behind the pathologist. Pathologist 3:1-4, 1983

Taylor CR: Immunoperoxidase techniques-practical and theoretical aspects. Arch Pathol Lab Med 102:113-121, 1978

Dabbs DJ: Diagnostic Immunohistochemistry. New York, Churchill Livingston, 2002, pp3-43

Boenisch T(ed): Handbook of Immunochemical Staining Mehtods (ed 3). Carpinteria, CA, DakoCytomation, 2001

Philip Bryant: Issues about tissues, Part 3: Sampling Outside the Laboratory, The Journal of Histotechnology Vol. 29, No. 2, 81-87, 2006

Advance Breast Biopsy Instrumentation
Liberman L: Advanced Breast Biopsy Instrumentation (ABBI): analysis of published experience. AJR Am J Roentgenol 172:1413-1416,1999

Brush Biopsy
Bush A, Pohunek P: Brush biopsy and mucosal biopsy. Am J Respir Crit Care Med 162:S18-S22, 2000

Computed Tomograph
Raghu G: interstitial lung disease: a diagnostic approach. Are CT ascan and lung biopsy indicated in every patient? Am J Respir Crit Care Med 151:909-914, 1995

Cone Biopsy
Paterson –Brown S, Chappate OA, Clark SK, Wright A, Maxwell P, Taub NA, et al: The significance of cone biopsy resection margins. Gynecol Oncol 46:182-185, 1992

Core Biopsy
Liberman L, La Trenta LR, Dershaw DD, Abramson AF, Morris EA, Cohen MA, et al: Impact of core biopsy on the surgical management of impalpable breast cancer. AJR Am Jroentgenol 168:495-499, 1997

Britton PD, Flower CD, Freeman AH, Sinnatamby R, Warren R, Goddar MJ, et al: Changing to core biopsy in an NHS breast screening unit. Clin Radiol 52:764-767,1997

Crosby Capsule Biopsy
Scobie BA: Jejunal biopsy using Crosby capsule. N Z Med J 62:76-80, 1963

Endoscopic Biopsy
Levine DS, Ried BJ: Endoscopic biopsy technique for acquiring larger mucosal samples. Gastorintest Endosc 37:332-337, 1991

Excision Biopsy
Herd RM, Hunter JA, McLaren KM, Chetty U, Watson SC, Gollock JM: Excision biopsy of malignant melanomas by general pracatitioners in south east Scotland 1982-91, BMJ 305:1476-1478, 1992

Fine Needle Aspiration Cytology(FNAC)
Willen H, Akerman M, Carlen B: Fine needle aspiration (FNA) in the diagnosis of soft tissue tumours: a review of 22 years experience. Cytopathology 6:236-247, 1995

Incisional Biopsy
Bong JL, Herd RM, Hunter JA: Incisional biopsy and melanoma prognosis. J Am Acad Dermatol 46:690-694, 2002

Imprints
Ferreiro JA, Myers, JL, Bostwick DG: Accuracy of frozen section diagnosis in surgical pathology: review of a 1-year experience with 24,880 cases at Mayo Clinic Rochester: Mayo Clin Proc 70:1137-1141, 1995.

Liquid Based Cytology
Bergeron C, Bishop J, Lemarie A, Cas F, Ayivi J, Huynh B, et al: Accuracy of thin-layer cytology in patients undergoing cervical cone biopsy. Acta Cytol 45:519-524, 2001

Loop Electrosurgical Excision Procedure
Livasy CA, Maygarden SJ, Rajaratnam CT, Novotny DB: Predictors of recurrent dysplasia after a cervical loop electrocautery excision procedure for CIN-3: s study of margin, endocervical gland and quadrant involvement. Mod Pathol 12:233-238, 1999

Punch Biopsy
Todd P, Garioch JJ, Humphreys S, Seywright M, Thomson J, du Vivier AWP: Evaluation of the 2mm punch biopsy in dermatological diagnosis. Clin Exp Dermatol 21:11-13, 2996

Shave Biopsy
Gambichler T, Senger E, Rapp S, Alamouti D, Altmeyer P, Hoffman K: Deep shave excision of macular melanocytic nevi with the razor blade biopsy technique. Dermatol Surg 26:662-666, 2000

Smears
Underwood JCE: Macroscopy, microscopy and sampling. In introduction to Biopsy Interpretation and Surgical Pathology.

Stereotactic Biopsy
Sickles EA: Breast imaging: from 1965 to the present. Radiology 215:1-16, 2000

Trephine Biopsy
Bain BJ: Bone marrow trephine biopsy. J Clin Pathol 54:737-741, 2001

Mucins
Cook HC: Carbohydrates, in Bancroft JD, Stevens A(ed). Bloxham, UK, Scion Publishing, 2008, pp 286, 276-281

Pigments
Wilson POG, Chalk BT: The neuroendocrine system, in Bancroft JD, Stevens A(ed). London, Churchill Livingstone, 1990.

Lipids
Bayliss High OB: Lipids, in Bancroft JD, Stevens A(ed): Theory and Practice of Histological Techniques (4th ed). New York, Churchill Livingstone, 1990.

Elastic Fibers
Henwood A: Current applications of orcein in hsitochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining. Biotech Histochem 78:303-308, 2003.

Reticulin Fibers
Lynch Medical Lab Tech. pg 994.

Amyloid
Wright JR, Caulkins E, Humphrey RL: Potassium permanganate reaction in amyloidosis. Lab Invest 36:274-281, 1977.

Myelin
Lowe J, Cox G: Neuropathological techniques, in Bancroft JD, Stevens A(ed). London, Churchill Livingston, 1990.

Microorganisms
Bishop PJ: The history of the Ziehl-Neelson stain. Tubercle 51:196-206, 1970.

Culling CFA: Handbook of Histopathological Techniques (2nd ed). London, Butterworths, London, 1963. pg 319-320.

Drury RAB, Wallington EA: Carlton’s Histological Technique (4th ED). New York, Oxford University Press, 1967. pg 335

RNA and DNA
Sheehan DC, Hrapchak BB: Theory and Practice of Histotechnology (End ed), St. Louis, MO, C.V. Mosby Company, 1980. pg. 150-151, 234, 235, 239

Philip Briant. Issues About Tissues, Part I: The Objectives of Histopathology: Journal of Histotechnology, Vol. 28, No. 2, 63-66, 2005.
Role Of Autopsy
National Confidential Enquiry into Perioperative Deaths (NCEPOD). 35-43, Lincoln’s Inn Fields, London, 2002.

Benbow EW, Roberts ISD. The autopsy: complete or not complete? Histopathology 42:417-423,2003

Barendregt WB, De Boer HHM, Kubat K: Autopsy analysis in surgical patients: a basis for clinical diagnosis, Br J Surg 79:1297-1299, 1992.

Diseases, Diagnoses and Biopsy Interpretation
Philips J. Murray P, Kirk P: The nature of disease. In: The Biology of Disease. 2nd ed. Blackwell Science, Oxford, 2001.

Sobin LH, Wittekind C(eds): TNM Classification of Malignant Tumours. 6th ed. Wiley-Liss, New Yourk, 2002.

Diagnostic Fallibility
Underwood JCE: Diagnosit histopathology. In: Introduction to Biopsy Interpretation and Surgical Pathology. 2nd Ed. Springer-Verlag, London. 1987, pp 1-16

Ramsay AD; Errors in histopathology reporting: detection and avoidance. Histopathology 34:481-490, 1999.

Nordrum I,Johansen M, Amin A, Isaksen V, Ludvigsen Ja; diagnostic accuracy of second-opinion diagnoses based on still images. Hum Pathol 35:129-135, 2004.

Progress In The Development Of Diagnostic Histopathology Staining-By Benard Solomon

2009-07-16T02:17:00.000-07:00

Dyes were the foundation for diagnostic histopathological staining. Most of the early methods for histopathological analysis of tissue sections have been overtaken by modern and safer methods like immunohistochemistry. Over time, some of the dyes used in the past are no longer available and the chemicals used now consider hazardous. Professionals who were familiar with these techniques are also rare to come by. New entrants to the field of histotechnology and pathology deserve to know the progressive development of histopathological diagnostic staining techniques and their wide range of applications.
This article attempt to bridge the information gap between the old and new generation of practitioners in the field of histopathology and pathology as regards staining techniques and their evolution.

Microscopical Staining Development In Histopathology Diagnosis

Naturally occurring substances such as madder, saffron and indigo were used to color tissues by early surgeons and pathologists. The staining techniques were developed by seventeenth century amateur scientists like Antoin Leeuwenhoek. The first microscopical staining was credited to Joseph Von Gerlach in 1858. He stained cerebellum with ammoniacal carmine. The mid 1800s brought about the development of improved microscopes with corrected spherical and chromatic aberration. The creation of the aniline dye industry in 1856 accelerated the development of histopathology staining.

Historical Facts Behind 3 Important Dyes In Histopathology Staining

1. Carmine: This is a commonly used dye in histology with wide applications. Carmine is most commonly used in an ammoniacal solution to study microscopic tissue structures. The colored compound is obtained by grinding the dried bodies of the female Coccus cacti insect found in Mexico. Cochineal from the insect contains carminic acid. Further treatment with alum and calcium salts results in carmine. Rudolph Virchow, the ‘Father of Pathology’ used carmine in his microscopy studies. Boiling carmine with acetic acid creates aceto-carmine used in staining plant chromosomes. In 1896, Mayer devised the mucicarmine stain by the addition of aluminium as mordant. The method was modified by Southgate in 1927. The mucicarmine stain is used to identify mucin production in adenocarcinomas, in signet ring carcinomas and some mucin-containing fungi such as Cryptococcus neoformans. Carmine was combined with potassium salt to stain glycogen as in Best’s carmine method published in 1906.
2. Haematoxylin and Haematin: Haematoxylin is a natural substance with useful properties and long history in histopathology. It was first used by Wilhelm von Waldeyer in 1863. It is obtained from the logwood tree , Haematoxylon campechianum, found mainly in Central America. It is a weak dye and the different staining solutions are based on its oxidized form, hematein. Hematein when combined with a mordant, an oxidizer and sometimes a differentiating agent, can be used to identify a wide variety of cellular components. Solutions prepared from hematein are usually called ‘hematoxylin’. Alum based hematoxylin solutions include Ehrlich-1886, Mayer-1903 and Harris-1900 just to mention a few. Weirgert’s iron hematoxlyin was introduced in 1904 and has a wide application as a nuclear stain in trichrome methods. This progressive nuclear stain is resistant to subsequently applied acidic solutions, making it suitable for procedures with multiple steps. Other hematoxylin solutions are Verhoeff elastic method –1908, Weil myelin method-1928 and PTAH-1897.
3. Silver Nitrate: Silver nitrate has extensive use in histopathology. The von Kossa reaction first published in 1901 was used in the demonstration of calcium carbonate and calcium phosphate. The Masson Fontana argentaffin reactions originated in 1914. Silver nitrate in ammoniacal solution is reduced by argentaffin cells found in the epithelial lining of intestine and lungs, melanin, chromaffin and some lipofuscins. Other substances stain with the ammociacal silver solution hence a need for confirmatory tests. Silver nitrate also react with argyrophils without being reduced as in Bielschowsky method published in 1904. External substance such as formalin is used to reduce the silver. Additional silver nitrate methods have been tailored for certain substances like Gomori reticulin method-1837, Grocott method for fungi-1955, Gomori method for basement membrane-1946, Steiner an dSteiner method for organisms-1944, Holmes method for nerves-1947. The Gordon and Sweets method-1936 is very popular in the United Kingdom.

Staining Methods And Other Substances Demonstrated In Histopathology.Hematoxylin And Eosin: This method was devised by Wissowzky in 1876. Numerous H&E methods exist but they follow the same general staining procedure of staining the nuclei in an alum or iron mordanted hematoxylin, differentiation in dilute acid alcohol and bluing in running tap water or slightly alkaline water. The cytoplasmic components are then stained in aqueous or alcoholic eosin. Standardization efforts has been limited in H&E staining as a result of variation in personal tastes.

Romanowsky Stains-Giemsa StainsThe Romanowsky stain-1891 replaced the Ehrlich’s stain as a popular multicolored stain in the field of hematopathology. Ehrlich created the field of hematopathology when he demonstrated that certain white blood cell structures had different affinities for the same dyes based on their acid or alkaline composition.
The Romanowsky method uses eosin, methylene blue, and oxidation products of methylene blue to stain blood smears, resulting in a multicolor of pink, through orange, to red-purple and an almost black color. The boiling or aging of the methylene blue(polychromin) generates products of demethylation(azures) that provide a purple rather than blue coloration of leukocyte nuclei when present in a solution that also contains eosin.
Modifications were made to the Romanowsky method and include, Unna-1891, Jenner-1899, Lieshman-1901, Wright-1902, and Giemsa-1902. Giemsa is the most popular. Some of these stains are adaptable to formalin-fixed , paraffin-embedded sections and used for cell maturation studies in bone marrow biopsies, organism detection etc.

Acid Fast Bacilli
Robert Koch in 1882 first developed a working method for the demonstration of Mycobacterium tuberculosis. His method laid the foundation for all other methods. Paul Erhlich stained AFB with a solution of either aniline basic fuchsine or aniline methylene violet and decolorizing with a mineral acid. Franz Ziehl recommended the use of carbolic acid(phenol) to aid in dye penetration and Rindfleisch(1882) recommended heating the slide. Neelsen in 1883 joined Erhlich’s basic fuchsine stain and Ziehl’s phenol. This method is still widely used today and applicable to paraffin sections. Faraco in 1938 recommended olive oil to retain acid-fastness of the organism lost in routine paraffin processing. Fite etal in 1947 recommended 30% vegetable oil such as cotton seed or peanut oil for dewaxing slides during the staining of Mycobacterium leprae. Later in 1952, Wade recommended rectified turpentine mixed with liquid petroleum. In the modern histopathology laboratory, a fluorescent method using auramine-rhodamine is favored for its ability to expose sparse, acid fast bacilli. It is seen as a reddish yellow organism on a green-black background.

Mallory’s Phosphotungstic Acid Hematoxylin(PTAH)This method was devised by Mallory in 1897. This method is used to demonstrate muscle striations, intercalated discs, nervous tissue and fibrin using ripened hematoxylin which stains polychromatically with phosphotungstic acid as mordant.

Gram Stain
Christian Gram devised this method in 1884. This method is widely used and applicable to paraffin sections. Modifications of the Gram method used in histopathology laboratories include the Gram-Twort method-1924, Brown and Brenn method-1931 and the Brown and Hopps method-1973.

Other Organism StainsThe Warthin-Starry method for Syphilis was introduced in 1920. Fungi could be demonstrated by the Gridley stain-1953 and Grocott-Gomori method-1955. Inclusion bodies such as cytomegalovirus(CMV) and rabies is demonstrated by Macchiavello’s stain developed for Rickettsia in 1937. The Leung method –1996 is recommended for Helicobacter pylori.

The PMC-LEARN-MN Structures And Substances Demonstrated In Histopathology
These are
P-Pigments
M-Mucins
C-Carbohydrates
L-Lipids
E-Elastic Fibres
A-Amyloid
E-Endocrine Glands
R-RNA&DNA
N-Nerves
M-Myelin
N-Neuroglia

Pigments: Pigments can be found in the human body normally. Others are introduced pathologically. The use of nonbuffered formalin creates formalin pigment in tissues especially in blood containing organs. Mercuric and dichromate pigments are introduced when tissues are fixed in those chemicals. Exogenous pigments such as carbon is found in lungs of smokers and Asbestos fibers found in the lungs of shipyard workers. Endogenous pigments are of most interest to pathologists. These include hemosiderin, melanin, chromaffin substance, lipochrome, lipofuschin, hemoglobin, and Dublin-Johnson pigment.
The Perls’ Prussian blue method for hemosiderin was published in 1867. Melanin demonstration method of Fontana was published in 1912 and Modified by Masson in 1914. Melanin is a naturally occurring pigment found in the skin and substancia nigra of the midbrain.
The argentaffin cells of the intestinal and respiratory tract epithelium can form carcinoid tumours. Diazo method was introduced for the demonstration of argentaffin cells in 1931. Generally, the argyrophil cells are demonstrated by silver methods. These include the Hellman method-1960, the Grimelius method-1968, the Churukian and Schenk method 1979.
Bile pigment is demonstrated by Fouchet method introduced in 1917. The rubeanic method for cupper was introduced in 1958 while the rhodamine method was developed in 1969. The leuco patent blue method for hemoglobin was developed in 1938 while the benzidine method was introduced in 1941.

Mucins: Hale’s colloidal iron method introduced in 1946was the first solution for the demonstration of mucins. Other modifications of this method exist. In 1965, the high iron diamine method was introduced to identify sulfated mucins with a brown black color.
Steedman in 1950 developed the alcian blue method for acid mucins. Alcian blue method combine well with other techniques such as H&E, PAS, Trichrome methods etc.
Carbohydrates: Histopathologists look for the absence or increase in carbohydrates, its staining reations and distribution. Recently, John Kiernan classified them into polysaccharides, proteoglycans and glycoproteins. In 1946, the P.A.S. was developed by McManus for carbohydrates which became one of the most useful stains in histology.

Lipids: In the histology community, lipids stains are commonly called ‘fat stains’.Normally, fats are found surrounding certain organs of the body and in the skin as packing. When found inside any organ, it becomes a pathological process. Sudan III was the first fat stain to be introduced in 1896 by Daddi. In 1901, Sudan IV was introduced; Oil red O in 1926 and Sudan black in 1935.
Most fat stains work on preferential solubility. The stain is more soluble in the fat than in the organic solvent. 70% was initially used as solvent for fat stains but it has been found to dissolve out most of the fat. Alternative methods were devised using other solvents. Such methods include Lillie and Ashburn’s method published in 1943 using a supersaturated solution of isopropyl alcohol and the Chiffelle and Putt’s method of 1951 which uses propylene glycol as solvent.
Having a good understanding of lipids classification is important in the choice of staining methods. Lipids are often classified as simple lipids, compound lipids and derived lipids. Baker’s acid hematein method was introduced in 1946 for phospholipids. The Luxol fast blue method was also published in 1953 for phospholipids and neuronal storage disorders. The Nile blue sulphate method was published in 1947 for the staining of neutral lipids and free fatty acids. More complicated lipids methods exist. They include the OTAN(osmium tetroxide alpha naphthylamine) for cholesterol esters was published in 1959 and the gold hydroxamate method for phosphoglycerides was published in 1963.

Elastic Fibers: The Orcein elastic stain was developed in 1890. It was very popular in early times and highly recommended for delicate elastic fibers in skin and was favored by dermatopathologists. Verhoeff’s elastic method was introduced in 1908 and is the most popular stain used today in histopathology laboratories. It is often combined with Van Gieson.

Amyloid Fibrils: Amyloid fibrils are demonstrated in amyloidosis-a group of diseases resulting in the deposition of insoluble protein in the interstitial spaces of blood vessel and various organs. Congo red is the most popular routine stain for amyloid. It was developed in 1922. The thioflavine T fluorescence method for amyloid was introduced in 1959.

Endocrine Glands:Chromaffin reaction is the method of choice for adrenal glands. For the pancreas, the Gomori’s chrome alum hematoxylin phloxine stain introduced in 1952 stains beta cells blue and alpha cells red. The aldehyde fuchsin method introduced in 1950 stains beta cells deep blue. The orange fuchsine green method of 1961 is suitable for pituitary staining. The Tripas method of Pearse introduced in 1949 stains beta cells magenta, acidophil cells orange and nuclei blue-black.

RNA and DNA:The feulgen reaction was the earliest method for nucleic acids. It was introduced in 1924. In 1932, Einarson introduced the chrome alum gallocyanine method to stain nucleic acid. The methyl green-pyronine method devised in 1899 and improved by Unna in 1902 stains DNA and RNA differentially and invaluable in study of cell population of tumours.

Myelin: At one time in the past, hematoxylin was used to demonstrate myelin in tissue sections. Later, the Weigert pal method developed in 1886, Kultschitsky method of 1890 and Loyez method of 1910 were commonly used. PTAH method of 1897 also demonstrates myelin. In modern times, common methods include Luxol fast blue –1953, Solochrome cyanine-1965, Swank and Davneport-1935 and Luxol fast blue/oil red O method of 1956.

Nerve Cells, Axons, and Neurofibrils: The classical stain for neurofibrils in the Bielschowsky silver method published in 1904. Other methods are Marsland, Glees and Erikson-1954. The Palmgren method was published in 1948. More methods include
Davenport-1936 and Holmes-1947. The Gally’s silver stain published in 1971 is said to demonstrate more pathological lesions than any other method.

Neuroglia: These are the supporting cells of the CNS. Early methods for glia demonstration are Holtzer’s stain introduced in 1921, Victoria blue method introduced in 1929. Also introduced was the Cajal’s gold chloride method of 1913 and the Hortega’s silver carbonate method of 1918.

Trichrome Stains: The most popular trichrome stain in recent time is Masson Trichrome Stain published in 1929. The Mallory stain was introduced in 1949 while the MSB method was published in 1962.

Conclusion: Different staining methods in histopathology and how they evolved has been highlighted. Future changes are expected but practitioners now have a deeper knowledge of the history behind the techniques they employ in their day-to-day practice. Such knowledge could serve as a foundation for future discoveries and improvements.

A Short History Of Histopathology Technique-Benard Solomon

2009-05-07T03:38:00.000-07:00

Very few students and teachers of histopatholgy understands the history of this medical specialization. A clear cut understanding of history and evolution is vital for the individual's confidence and upkeep of the profession. We can only build on any concept when the history and evolutionary trend are well understood.
The specialty of histopathology tecnique could be traced to the works of Johannes Muller in 1838. This followed the construction of the first compound microscope in 1591. The development of the simple microscope was credited to Anton van Leeuwenhoek who started the devolopment of simple microscopes with single lenses. This occured in 1673.
Microtome for sectioning animal tissues was first constructed in 1848. In mid 1800s, paraffin wax for infiltration and embedding was introduced. Formalin, widely used for fixation today was first used in 1893.
In 1945, automated tissue processors replaced hand processing while cryostats were first manufactured in 1951. During the last 50 years, enzyme histochemistry, electron mocroscopy and polarizing microscopy have all become diagnostic tools in histopathology.
In the 1980's, the widespread use of immunohistochemistry revolutionized cancer diagnosis and it is still evolving.
What other development of histopathology techniques occured in the United States and United Kingdom from the historical sense? These are the issues presented by Michael Titford in 'A Short History Of Histopathology Technique'.

Within the last 30 years, histotechnologists have seen the introduction of microcomputer-controlled tissue processors, immunohistochemistry (IHC), in situ hybridization.

22 Histopathology Technical Categories And Historical Background
Histopathology like any other specialization developed over time. How it happened is explained under 22 technical categories as follows:

1. Microscope Development: As far back as 1591, Zacharias Jansen made the first compound microscope in collaboration with his father in Holand. These microscopes suffered from spherical and chromatic aberration, low magnification and limited to reflected light. Anton van Leeuwenhoek made simple microscopes with better resolution in 1673 though rudimentary to today’s standard. In those days, researchers were more interested in the microscopes that the specimen. In 1827, Joseph Lister corrected the spherical and chromatic aberrations observed in early microscopes and together with Thomas Hodgkin for the first time published descriptions of smooth and skeletal muscle and the lack of a nucleus in human red blood cells. Their effort was the tonic needed for the commercial production of microscopes starting in Germany, France and Italy. The use of the microscope was further facilitated by the works of Amici and Chevalier who developed the achromatic lens for color correction in the 1830s. By the late 1870s, Ernst Abbe had further advanced the science of microscopy with the development of apochromatic lens, substage condenser to gather light and immersion lenses. The modern binocular microscope was developed in 1902.

2. Fixatives: Early researchers had made effort to harden biological specimens for thin sections to be cut. Heat fixation was employed then by Malpighi. Later, others used chemical agents such as alcohol, acetic acid, chromium trioxide and potassium dichromate to preserve tissues. Following this was the use of compound fixatives. Muller’s fluid in 1860, Zenker’s in 1894 and Carnoy’s in 1887. Formalin, the now most popular fixative was discovered by Blum in 1893.

3. Microtome Development: Hill in 1770 introduced the first hand microtome to cut sections of twig. Early microtomes were meant for plant tissues and were called ‘cutting engines’. Not until 1848 did the use of microtome for animal tissues began. The Cambridge rocker was developed in 1885 by Horace Darwin and the sledge developed by Jung in 1910. Over the years, microtomes have improved though the basic design remains the same. Motorized microtomes are the modern machines made to reduce the carpal tunnel syndrome caused by repetitive motion.

4. Embedding Media: Paraffin wax is the most popular medium for embedding. It was developed over a period of time by several researchers. Its discovery has been variously attributed to Klebs-1869, Born & Sickler –1871, Giesbrecht-1881 and Butshili-1881.

5. Histopathology As A Field: Francois Bichat of France(1771-1802) was aptly described as the ‘Father of Histology’. The specialty of Pathology first came into fruition in Germany in the 1800s. Johannes Muller can be called the ‘Father of Histopathology’. He was credited for attracting gifted students such as Rudolph Virchow, Jacob Henle, Karl Reichert, Karl Kuppfer and others who made contributions to histology in the late 1800s. Rudolf Virchow(1821-1902) was the most influential pathologist of all times. In England, the Royal Microscopical Society was formed in 1839. In 1848 John Quekett was the first to decalcify tissue. In 1845, Hermann Lebert reported that the microscope could be used to differentiate malignant and benign tissue by examining the cellular morphology.

6. Development of Staining: Carmine and Saffron were the early dyes used in the field of microscopy. Haematoxylin was reportedly first used successfully by Wilhelm von Waldeyer in 1863. William Perkin prepared the first synthetic dye from aniline in 1856. Basic fuchsine was first manufactured in in 1865 and aniline blue in 1868. Schwarz devised the first double stain in 1867. Differentiation was first used in 1868. Haematoxylin solutions were later developed in rapid succession. Delafield’s –1885, Ehrlich’s –1886, and Heidenhain’s-1892. The H&E method was developed in 1875 by Wissowzky. Ziel Nelson Acid fast stain was developed in 1883, Gram stain appeared in 1884, Congo red in 1886 and the fat stain Sudan III in 1896. Mallory’s phosphotungsti acid hematoxylin was introduced in 1897. The Giemsa stain appeared in 1902, PAS in 1946 by MacManus and the alcian blue method of Steedman in 1950.Silver stains also need being mentioned. The original von Kossa method in 1901, Masson method for argentaffin in 1914 with Fontana’s modification in 1925. Bielschowsky’s method for axon and neurofibrils-1904 etc

7. Automatic Stainers: In the United kingdom, Shandon Company introduced their first automated stainer in 1965. In the United States, The Stainomatic manufactured in the 1970s by Gam Rad Incorporated was widely used. In the late 1990s special multiple, simultaneous stainers were introduced such as the Artisan from CytoLogix.

8. Frozen Sections: Francois Raspail(1794-1878) was regarded as the ‘Founder of Histochemistry’ and to have used frozen sections, Stilling in 1842 was reputed to be the first person to use frozen sections. Welch was the first to use a frozen section to diagnose breast cancer in 1891. Commercial production of cryostats began in the United Kingdom in 1951 by Bright Instrument Company. Pearse was the fist professor of enzyme histochemistry in the University of London.

9. Tissue Processors: The first automated tissue processor was made in Germany in 1909. In 1945, Edwin Weiskopf, founder of the Technicon Corporation commercially produced popular tissue processor in the United States. In the U.K., Shandon began manufacture of its tissue processor in 1951. In 1979, two companies started marketing fluid transfer tissue processors and the Tissue Tek processor. In 2004 a throughput tissue processor was marketed.

10. Development of Other Histology Procedures: In the 1950s, paraffin waxes of different melting points were made available to facilitate sectioning. In the 1960s, plastic polymers were added to control hardness and improve sectioning. Cooling the blocks further hardens the wax and improves sectioning.

11. Slides, Coverslips and Mountants: In 1839, the 1 inch by 3 slides was adopted by the Microscopical Society of London which became the global standard. In 1840, the glass coverslip was developed by the Chance Glass Company of Birmingham, England. In 1985, the automatic glass coverslipper was introduced by Hacker Instruments.. Canada Balsam was the early mountant used in histopathology. It as first used in 1835. Synthetic mountants became popular in 1960s.

12. Other Laboratory Ware and Consumables:The Coplin jar was invented by W.M. Coplin in 1897. Early Histotecnologists used paper molds for embedding or Leuchart’s type molds. It is still in use in most laboratories of developing countries. Paraffin blocks were then mounted onto wooden blocks or a microtome stage for sectioning. Floating Bath was introduced by Gaskell in 1890. Embedding Rings were introduced by Dr. James B. McCormick in 1958. The cassettes system also invented by Dr. McCormick was introduced in 1968. Color Code cassettes were introduced in the 1980s. In 1978, the disposable microtome blades were introduced by the Feather Company. This eliminated the use of steel knives which required frequent honing and stropping.

13. Alternative Embedding Media: Celloidin was first used in 1877. Ester wax was developed in 1947 by Steedman and recommended for hard tissues, incests and cortical bone.

14. Plastic Sections: In the mid 1980s, Glycol Methacrylate (GMA) embedding was introduced for the production of thin biopsy sections. It is however, limited to academic and research environments

15. Enzyme Histochemistry: Gomori and Takamatsu developed methods for demonstrating alkaline phosphatase in frozen sections in 1939. Enzyme histochemistry is now largely limited to diagnosing muscle biopsies such as neuromuscular disorders, acetylcholinesterase in Hirschsprung’s disease, some enzyme deficiency disorders and other uncommon uses.

16. Electron Microscopy: Ernst Ruska developed the first EM in the late 1920s. Siemens built the first commercial electron microscope in 1939. Although found primarily in medical centers and university settings, electron microscopy came into use in the late 1950s and early 1960s. By the 1970s, they became widely spread.

17. Polarizing Microscopy: No clear details about how it was introduced into clinical laboratories though has wide applications. It is most useful in viewing birefringent materials such as collagen fibres, bone, striated muscle, hair and cholesterol and amyloid deposits.

18. Fluorescence Microscopy: This technique was developed by Coons, Creech and Jones in 1941. Though expensive, it is useful in the auramine rhodamine method for acid fast bacilli.

19. Immunohistochemistry(IHC): Researchers first used DAB-3,-3 diaminobenzidine to create a stable colored compound at the site of protein in the tissue. In 1970, Sternberger etal developed the peroxidase antiperoxidase method (PAP). In 1974, Taylor and Burns developed IHC method for formalin-fixed, paraffin-embedded tissues. IHC became available to routine histopathology laboratories in the U.S. in the early 1980s.

20. Laboratory Information Systems(LIS): Arrived in clinical laboratories starting from mid 1970s. Workable packages for pathology laboratories came up later.

21. Books And Literature: Arthur Bolles Lee published ‘The Microtomist’s Vad-Mecum in 1885. Mallory And Wright Published ‘Pathological Techniques’ in 1901. Harry Carlton published ‘Histopathological Techniques in 1926. Culling published Handbook of Histopathological Techniques in 1957. Pearse published Histochemistry-Theoretical and Applied in 1953. In 1980, Dezna Sheehan and Barbara Hrapchak published Theory and Practice of Histotechnology. More recently, John Kiernan’s Histological and Histochemical Methods-Theory and Practice published in 1981 with recent updates.

22. Present State Of Histopathology Technique: Less harzadous fixatives and newer IHC methods now take front stage. Microwave processors are now used for processing and staining in some laboratories. New instruments such as Sakura Finetek Xpress processes thin slices of tissue rapidly using proprietary chemicals. The use of tissue microarrays for gene expression profiling gaining increased interest.

Reference
Michael Titford: A Short History Of Histopathology Technique: The Journal of Histotechnology/vol.29, No 2./June 2006 pg 99-110