Pathology Practical Book Harsh Mohan
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1TECHNIQUES IN PATHOLOGY
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ANTHONY VAN LEEUWENHOEK (1632-1723)
Born in Holland, draper by profession, during his spare time invented the first ever microscope by grinding the lenses himself and mad 400 microscopes. He also first introduced histological staining in 1714 by using saffron to examine muscle fibre.
2Section Contents
Microscopy
03
Routine Histopathology Techniques and Staining
07
Frozen Section and its Staining
13
Special Stains and Immunohistochemistry
15
Surgical Pathology Request Form
19

MicroscopyExercise 1

Objectives
  • ➢ To understand the working of various parts of a light microscope and learn how to maintain and operate a light microscope.
  • ➢ To enumerate various other types of diagnostic microscopy and understand the basic principles of their working and applications.
Microscope is the most commonly used apparatus in the diagnostic laboratory. It produces greatly enlarged images of minute objects. Most commonly used is a light microscope; this is described first, followed by various special types of microscopy.
 
Light Microscope
The usual type of microscope used in clinical laboratories is called light microscope that employs visible light as source of light. A light microscope can be a simple or a compound microscope.
Simple microscope. This is a simple hand magnifying lens. The magnification power of hand lens is from 2x to 200x.
Compound microscope. This has a battery of lenses fitted in a complex instrument. One type of lens remains near the object (objective lens) and another type of lens near the observer's eyes (eyepiece lens). The eyepiece and objective lenses have different magnifications (described below). The compound microscope can be monocular having single eyepiece (Fig. 1.1), or binocular which has two eyepieces (Fig. 1.2).
A usual compound microscope has mechanical, electrical and optical parts. These include: stand, body, optical system, and light/illumination system.
 
STAND
This is horseshoe-shaped in monocular microscope and gives stability to the microscope. Binocular microscopes have a variety of shapes of stand.
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FIGURE 1.1: Monocular light microscope, Model YS 50 (Photograph courtesy of Nikon, Japan through Towa Optics India Pvt. Ltd., Delhi).
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FIGURE 1.2: Binocular light microscope, Model E 200 (Photograph courtesy of Nikon, Japan through Towa Optics India Pvt. Ltd., Delhi).
 
BODY
Microscope body consists of a limb which arises from the joint with the stand. It helps in moving microscope in comfortable position from one place to another. Nowadays, microscope body is built in an ergonomic shape to avoid excessive strain on the back and neck of the user.
The stand and the limb of the body carry the following parts: body tubes, mechanical stage, and knobs for coarse and fine adjustment.
Body tubes. There are two tubes for optical path of the microscope. External tube at its lower end has a revolving nosepiece having slots for screwing in objective lenses of different magnifications. According to the number of objective lenses required to be fitted on the nosepiece, it may be triple, quadruple (4 slots), quintuple (5 slots) or sextuple (6 slots) nosepiece. The other end of the optical tube has an internal tube which is a draw tube with eyepieces placed at the upper end.
Mechanical stage. This is a metallic platform having slide holder that accommodates glass slide having object to be seen. Stage is attached to the limb just below the level of objective lenses. It has an aperture in its centre which permits the light to reach the object. Movement of the glass slide on the stage is regulated by two knobs attached to slide holder on the side of the stage. By this, the slide can be moved forward-backwards as well as left-right sides; it is also possible to make measurements on the object in the slide by graduated scale provided on the stage in both x and y axis.
Just below the stage is substage which consists of condenser through which light is focused on the object. The substage can be moved up and down. The substage has an iris diaphragm; its closing and opening controls the amount of light reaching the object with maximum resolution and minimum glare. Viewing object under higher magnification of objective lens requires more light and hence opened up diaphragm, and vice versa while viewing under lower magnification of objective lens.
Knobs for coarse and fine adjustment. For focusing of the object, knobs are provided on either side of the body—bigger knobs for coarse adjustment and smaller knobs for fine adjustments. In earlier models of microscopes, in order to focus the object on the glass slide to be viewed, coarse and fine adjustment knobs moved the body tube along with objective lenses up and down, while the microscope stage remained fixed. However, currently available models of microscopes provide for up and down movement of the microscope stage in order to bring the glass slide in focus near the objective lens, while the latter remains fixed. The fine focus is graduated and by each division objective moves by 0.002 mm.
 
OPTICAL SYSTEM
Optical system is comprised by different lenses which are fitted into a microscope. It consists of eyepiece, objectives and condensers.
Eyepiece. A monocular microscope has one eyepiece while a binocular microscope has two. Eyepiece has two planoconvex lenses. Their magnification can be 5x, 10x, or 15x. Commonly, 10x magnification is used.
Objectives. These are made of a battery of lenses with prisms incorporated in them. Their magnification power provides varying range. Usually 4x, 10x, 40x and 100x (oil immersion) objectives are used. However, other magnifications such as 2x and 20x are also available. These lenses are of various types, e.g. achromat, apochromat, planapoachromat, etc.
Condenser. This is made up of two simple lenses. As discussed above, it condenses light on to the object on the slide by up or down movement, and by opening or closing of the diaphragm.
 
LIGHT/ILLUMINATION SYSTEM
For daylight illumination, a mirror is fitted at the base of the microscope which is plane on one side and concave on the 5other side (Fig. 1.1). Plane mirror is used in sunlight while concave in artificial source of light. Currently, most of the microscopes have in-built electrical illumination fitted in the base with illumination ranging from 20 to 100 watts (Fig. 1.2).
 
MAGNIFICATION AND RESOLVING POWER OF LIGHT MICROSCOPE
Magnification power of the microscope is the degree of image enlargement. It depends upon the following:
  1. Length of the optical tube
  2. Magnifying power of the objective lens used
  3. Magnifying power of the eyepiece
With a fixed tube length of 160 mm in majority of standard microscopes, the magnification power of the microscope is obtained by the following:
Magnifying power of objective × Magnifying power of eyepiece.
Resolving power represents the capacity of the optical system to produce separate images of objects very close to each other.
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Where
  • λ is wavelength of incidental light; and
  • NA is numerical aperture of lens which is generally engraved on the body of the objective lens.
Resolving power of a standard light microscope is around 200 nm.
 
HOW TO USE AND MAINTAIN A LIGHT MICROSCOPE?
  1. The microscope should be kept on stable even surface and in comfortable position.
  2. It should be kept dust free, taking care to use separate cleaning paper/cloth for mechanical and optical parts.
  3. Obtain appropriate illumination by adjusting the mirror or intensity of light.
  4. When examining colourless objects, condenser should be at the lowest position and iris diaphragm closed or partially closed.
  5. When using oil immersion, 100x objective should dip in oil.
  6. After using oil immersion, the lens of the objective should be cleaned with tissue paper or soft cloth.
 
NEWER APPLICATIONS OF LIGHT MICROSCOPE
In the recent times, computers and chip technology have helped in developing following newer applications of light microscope:
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FIGURE 1.3: A penta-headed light microscope, for simultaneous use by one primary user and 4 other users seated nearby.
 
Teaching Microscopes
It is possible to modify a modern light microscope by various attachments for the purpose of teaching, training and group discussions (Fig. 1.3). These include attaching multiple viewing stations for simultaneous viewing by more users seated across or on the side (dual headed, multiheaded). Alternatively, the image can be projected simultaneously on the monitor by attaching a camera; still further improvement may be by interface of a CPU to click and store selected images as well.
 
Image Analysers and Morphometry
In these techniques, microscopes are attached to video monitors and computers with dedicated software systems. Microscopic images are converted into digital images and various cellular parameters (e.g. nuclear area, cell size, etc.) can be measured. This quantitative measurement introduces objectivity to microscopic analysis.
 
Telepathology
It is the examination of slides under microscope set up at a distance. This can be done by using a remote control device to move the stage of the microscope or change the microscope field or magnification called as robotic telepathology. Alternatively and more commonly, it can be used by scanning the images and using the high-speed internet server to transmit the images to another station termed as static telepathology. Telepathology is employed for consultation for another expert opinion or for primary examination.
 
OTHER TYPES OF MICROSCOPY
 
DARK GROUND ILLUMINATION (DGI)
This method is used for examination of unstained living microorganisms, e.g. Treponema pallidum.6
Principle. The microorganisms are illuminated by an oblique ray of light which does not pass through the microorganism. The condenser is blackened in the centre and light passes through its periphery illuminating the living microorganism on a glass slide against dark background.
 
PHASE CONTRAST MICROSCOPY
Like DGI described above, phase contrast microscopy is also used for examination of unstained structures, most often living cells for assessing their functions at the level of organelles such as mobility, phagocytosis, etc.
Principle. Illumination of varying phase and amplitude is passed through unstained cells which assesses the difference in refractive indices of various organelles; the organelles shine differently based on whether they are dense/dark (higher refractive index) or less dark (low refractive index).
 
POLARISING MICROSCOPE
This method is used for demonstration of birefringence, e.g. amyloid, foreign body, hair, etc.
Principle. The light is made plane polarised. Two discs made up of prisms are placed in the path of light, one below the object known as polariser and another placed in the body tube which is known as analyser. Polariser sieves out ordinary light rays vibrating in all directions allowing light waves of one orientation to pass through. The lower disc (polariser) is rotated to make the light plane polarised. During rotation, when analyser comes perpendicular to polariser, all light rays are cancelled or extinguished. Birefringent objects rotate the light rays and therefore appear bright in a dark background.
 
FLUORESCENT MICROSCOPE
This method is used for demonstration of naturally-occurring fluorescent material and other non-fluorescent substances or microorganisms after staining with some fluorescent dyes, e.g. Mycobacterium tuberculosis, amyloid, lipids, elastic fibres, etc. Light source of low wavelength (UV light) for illumination is used, most often mercury vapour lamp or xenon gas lamp.
Principle. Fluorescent microscopy is based on the principle that illumination of a substance with a low wavelength (UV region, i.e. invisible spectrum) emits light at a higher wavelength (visible spectrum), thus localising the substance with fluorescence in the visible range. Fluorescent dyes are used depending upon the type of material to be visualised, e.g. fluorescein isothiocyanate (FITC), thioflavin, etc.
 
ELECTRON MICROSCOPE (EM)
EM is used for study of ultrastructural details of the tissues and cells. For electron microscopy, tissue is fixed in 4% glutaraldehyde at 4°C for 4 hours. Ultrathin microsections with thickness of 100 nm are cut with diamond knives.
Principle. By using an electron beam of light, the resolving power of the microscope is increased to 50,000 to 100,000 times and very small structures can be visualised. In contrast to light microscopy, resolution of electron microscopy is 0.2 nm or less.
There are two types of electron microscopy:
  1. Transmission electron microscopy (TEM)
  2. Scanning electron microscopy (SEM)
 
Transmission Electron Microscopy (TEM)
TEM helps visualise cell's cytoplasm and organelles. For this purpose, ultrathin sections are required. TEM interprets atomic rather than molecular properties of the tissue and gives two dimensional image of the tissue.
 
Scanning Electron Microscopy (SEM)
SEM helps in the study of cell surface. In this three-dimensional image is produced. The image is produced on cathode ray oscillograph which can also be amplified. SEM can also be used for fluorescent antibody techniques.