The University of Texas Medical School,Department of Pathology and Laboratory Medicine

Blood Smear: Preparation and Staining

Reference:
1. Davidson, I. and Henry J., Clinical Diagnosis by Laboratory Methods, I. Davidsohn
and J. Henry, eds., W.B. Saunders Co., Philadelphia 1974, pp. 135-137.
2. Wintrobe, M.W., et al., Clinical Hematology, Lea & Febiger,Philadelphia, 1974, p.
26.
Principle:
A thin blood smear is stained with Wright-Giemsa stain to enable evaluation of white blood
cell, red cell and platelet morphology.
Specimen:
Peripheral blood or potassium EDTA anticoagulated blood (1 to 2 mg EDTA/1ml blood) may
be used. Smears of peripheral blood must be made immediately. Smears may be made from
EDTA-anticoagulated blood as long as two to three hours after collection. All specimens
must be free of clots.
Procedure:
1. Anticoagulated specimens must be checked with two applicator sticks to be sure no
clots are present. If clots are present the specimen is unsatisfactory.
2. A small drop of blood is placed on the surface of a clean glass slide near the end. If
blood is taken from the finger, care must be taken to avoid touching the slide to the
skin.
3. The slide is held between two fingers and the thumb of the left hand with the drip of
blood on the upper surface towards the right. (Reverse for the left handed individual).
4. An edge of the spreader slide is placed on the first slide to the left of the drop of blood
and is pulled to the edge of the drop. The angle between the two slides will vary
according to the size of the drop and the viscosity of the blood. The larger the drop
and the lower the hematocrit of the blood, the greater the angle must be to avoid
running off the slide when spreading. Blood with a high hematocrit must be spread
with a lower angle or the smear will be short and too thick to allow differentiation of
cells. The approximate angle for normal blood is 30 to 40 degrees.
5. The drop of blood should be allowed to bank evenly behind the spreader which is then
pushed to the left in a smooth, quick motion. The more rapid the motion, the shorter
and thicker the smear. The smear should cover approximately half the slide with a
gradual transition from thick to thin. No ridges should be present and the end (called
the "feather edge") should be smooth and even. In the feather edge the red blood cells
should not be routinely overlapped.
6. The smear is labeled on the frosted edge of the slide or the thick end of the blood with
the patient's last name, first name, specimen number and date.
7. When the smear is completely dried, it may be stained by using the automated Hema-
Tek II Slide Stainer. Automated Method:
o The air dried smear is fixed with absolute methoanol and placed on a staining
rack.
o Allow slide to dry completely.
o Prime stainer lines to remove air bubbles.
o Place dried smear on stainer.
Reagents:
Hema-Tek II Stain Pak
Quality Control:
Strict adherence to procedure and visual check of the quality of stained slides.
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Introduction to Histology
Author: Paul Kwan, Ph.D.
I. Definition
Histology is the study of microscopic anatomy dealing with the structures of cells, tissue and
organs in relation to their functions.
The first part of the course deals with basic tissues (a collection of similar cells and the
extracellular matrices surrounding them): epithelium; connective tissues, including blood,
bone and cartilage; muscles; and nerves.
The second part of the course deals with organs, systemic arrangement of tissues performing
a specific function, as of respiration, digestion etc.
Species differences will be included where appropriate. This, however, is not a course in
comparative histology.
The course deals mainly with the structural aspects of cells, tissues and organs. It also covers
the basic functions of these structures.
Although the students will have extensive course works in physiology, this course tries to
mirror the philosophy of Albert Szent-Gyrgyi, Structure and function are two sides of the
same coin: structure without function is meaningless, and function without structure does not
exist.
II. Methods of Study
The most common way to study tissues in histology is to preserve the material, make a
section thin enough for light (or electrons) to pass through it, stain it to give it some contrast,
and then observe the tissue under a microscope.
A. Routine Tissue Processing (Paraffin Sections)
FixationThe preservation of the tissues to be studied
1. Immersion fixation, most common.
Put small pieces of tissue directly into a fixative such as formaldehyde. As the fixative
penetrates into the tissue, the material is preserved (fixed). Because the rate of diffusion of
the fixative into the tissue is slow, the pieces must be quite small to be preserved properly.
Otherwise there may be fixation artifacts (like the center of the piece not properly preserved)
in the tissue.
Because of its simplicity, this is the most commonly used method in general histology and
pathology.
2. Perfusion fixation, better preservation.
By introducing the fixative into the blood stream, the fixative can reach organs/tissue quite
quickly and achieve good fixation. This requires a more elaborate set up and skills (inserting
a canula into a blood vessel). Care must be taken to ensure proper pressure and flow of the
fixative. Otherwise the tissue may not be fixed well or distorted by too much hydrostatic
pressure applied during the procedure. Can produce beautifully preserved material. More
commonly used in research projects.
Routine immersion of tissues into an aqueous fixative such as formaldehyde removes soluble
compounds such as glycogen. To preserve materials of interest that are water-soluble, a
fixative like acetone is used.
3. Dehydration---removal of the aqueous material in the tissues.
In routine processing, the tissue will be embedded in a water insoluble material such as wax.
All the water in the tissue must first be removed. This is done by running the tissues through
a graded series of alcohol (70%, 95%, 100%). Inadequate removal of water will lead to
artifacts.
4. Embedding
The dehydrated tissue is passed through several changes of xylene and then placed in
liquified paraffin wax. The paraffin infiltrates the tissue (usually with the help of a vacuum).
The tissue is then place in a plastic mold that contains melted paraffin. Upon cooling, this
piece of paraffin containing the tissue is called a block and is ready to be sectioned into
thick sections (4-6 micrometers).
5. Sectioning
After trimming away excess material and exposing the tissues, the block is then sectioned
with a steel blade. The thickness of each section is about 4 to 6 micrometer (10
-6
m). The
sections are collected and placed on a glass slide. With the application of mild heat, the
paraffin surrounding the tissue melts away and the tissue sticks to the glass.
6. Staining
The sections cut from the blocks are thin enough for light to pass through. However, the
material has very little contrast and you cannot make out details when looking at the section
under the microscope. Staining the sections with dyes give contrast to the tissues and makes it
possible to study fine details.
The most commonly used stains for paraffin sections are hematoxylin and eosin (see later
section on stains).
Special stains are used to demonstrate a specific structure or class of compound found in the
tissue, e.g. PAS stain for glycogen in liver cells.
7. Cover Slipping
The stained section is covered with a drop of mounting medium such as Permount and then
a thin piece of glass (cover slip) is placed over the medium. When the medium is dry, the
section is preserved and protected in a plastic-like medium under a sheet of glass.
B. Plastic Sections
In some cases, after dehydration, the tissue is embedded in epoxy plastic. This very hard
material can be cut into semi-thin sections (0.5 micrometer) for light microscopy or thin
(0.1 micrometer) for electron microscopy.
Plastic sections used for light microscopy show better details than paraffin sections.
However, the numbers of stains that can be applied to plastic sections are rather limited.
C. Frozen Sections
To preserve molecules that may not withstand the action of paraformaldehyde and the heat
used in paraffin processing, a combination of acetone and low temperature embedding can be
used.
First fixation is done by immersing the small pieces of tissues in a low-temperature
embedding medium such as OCT. This syrupy material is put into a little plastic mold and
the pieces of tissue put into the medium. The mold is then put into a mixture of acetone and
dry ice. The medium freezes quickly into a solid block.
The frozen block is trimmed and then section in a cryostat, which is a refrigerated microtome.
The sections are collected on glass slides.
Depending on the protocol, the sections may or may not be fixed briefly with acetone.
The sections are stained and mounted either with a special aqueous mounting medium or,
after dehydration, with an organic mounting medium.
D. Electron Microscopy, Routine
Electron microscopy yields much better details than light microscopy and is invaluable in
histology.
Fixation for electron microscopy is usually done with glutaraldehyde or paraformaldehyde.
The tissue has to be minced into very small pieces so that the fixative can diffuse quickly into
the material.
After rinsing away the first fixative, the tissue is usually fixed further with a solution of
osmium tetroxide.
After dehydration, the pieces of tissue are embedded in epoxy plastic.
A diamond knife is used to make thin sections from the plastic blocks. The sections are
picked up on small copper grids.
Staining of the thin sections usually involve the use of uranyl acetate solution followed by a
lead citrate solution. These stains, being of electron-opaque heavy metals, impart contrast to
the very thin sections.
Because of the way the tissue is fixed and embedded, very few special staining methods are
available for thin sections. The most common one is immunohistochemistry, using gold
particles as markers. These particles, bound to structures in the cells, appear as little dark
spheres under the electron microscope.
E. Electron Microsopy, Scanning
This uses an electron beam to scan the surface of a structure and gives a three dimensional
topographic view of the subject with amazing details.
III. Common Artifacts Encountered with
Tissue Sections
Folds --- paraffin sections, after being cut on a steel blade, are placed gently in a bath of
warm water. The sections float on the water surface and stretch out like a ribbon. The most
common artifact, folds, occurs when the sections did not stretch out completely. This can also
happen when the sections are picked up and transferred to glass slides.
Debris --- dirt and debris in the water bath are common and the water surface needs to be
cleaned regularly during sectioning.
Knife marks --- nicks in the knife lead to drag marks in the sections.
Shrinkage / swelling --- the tissues may swell or shrink during processing.
Staining artifacts --- sometimes structures can be over-stained or under-stained. Other times
the structure may stain inappropriately.
It is important to recognize these artifacts when studying a specimen so as not to make the
wrong conclusion or worse, diagnosis.
IV. Routine Stains for Paraffin Sections
The way a tissue component stains depends on the interaction of the component with certain
dyes.
The most common stain used in routine paraffin sections is called H&E. This stands for
hematoxylin and eosin.
Hematoxylin is a dye extracted from longwood. In routine processes, it stains the nuclei of
cells purple/blue.
Eosin is a dye that usually stains the cytoplasm orange-red.
A. Basic/Cationic Dyes
Hematoxylin --- blue/blue-black. Stains heterochromatin in nuclei, rough endoplasmic
reticulum, proteoglycans etc.
Methylene blue --- blue. Stains anionic compounds in the cell as well as in the extracellular
matrix. Metachromatic with certain polyanions such as those in the granules in mast cells.
Also used in semi-thin plastic sections.
Toluidine blue --- blue. Similar to methylene blue.
Azures --- blue. Tissue components that interact with and are stained by these dyes are
described as being basophilic. For example, DNA in cell nuclei and RNA in the cytoplasm
stain bluish with these dyes and you will hear comments such as note the basophilia of the
chromatin material or note the basophilic in the basal cytoplasm.
Metachromasia (change color) High acid material (polyanion rich) can change the normal
color of a dye (orthochromatic) to a different (metachromatic) color. Mast cell granules,
when stained with toluidine blue, show up as purplish droplets. The extracellular matrix of
cartilage is also metachromatic due the presence of chondroitin sulfate molecules. This kind
of reaction is attributed to the way the dye molecules are bound in proximate array to tissue
polyanionic polymers, such as glycosaminoglycans.
B. Acidic/Anionic Dyes
Eosin - orange-red/pink
Anilin blue - blue
Orange G - orange
Picric acid - yellow
Tissue components that interact with and are stained by these dyes are described as being
acidophilic or eosinophilic. For example, certain secretory materials appear bright red/orange
and are described as acidophilic granules in the cytoplasm. Collagen is a typical
eosinophilic substance in the tissue.
C. Mixed Dyes
In routine staining, the dyes are applied in sequence with washes in between. In some cases,
however, a mixture of dyes is applied to the tissue.
Romanowsky stains is the best know example of this type of mixed dyes and contain
combinations of eosin and methylene blue / azures. They are usually used for the staining of
blood or bone marrow smears.
Giemsa stain and Wright stain are common examples of Romanowsky stains.
For more information, see the chapter on Blood.
V. Special Stains and Reactions
A number of stains are commonly encountered in histology. These are used to bring out
certain structures / components of interest.
Elastic stains for elastin --- Verhoeff (black), resorcin fuchsin (purple), and orcein (red) ---
for walls of blood vessels etc.
Feulgen reaction, a form of Schiff reaction. Used for DNA in cell nuclei. Magenta. The
staining is specific for DNA and not RNA. The degree of staining is proportion to the amount
of DNA present. This property is employed in the densitometric quantitation of DNA in
tissues.
Lipid Stains that dissolve in or stabilize lipid materials --- Sudan black, oil red O, osmium
tetroxide (black) --- used for lipid droplets in liver and other tissues. Used with frozen
sections because lipids are not extracted as during paraffin processing involving solvents.
Masson trichrome, Mallorys --- used for connective tissues. Collagen, blue-green.
Cytoplasm, red-pink. Nuclei, blue-black.
PAS --- periodic acid-Schiff reaction, demonstrates 1,2-glycol groups present in
carbohydrates. Stains polysaccharides, oligosaccharides, and some glycoproteins. Most
common example, glycogen in liver and striated muscle cells. Magenta.
Silver --- used for Golgi apparatus, basal lamina, reticular fibers, and neurofibrils. Black-
brown.
Gold --- in the form of gold chloride, useful in neurology to demonstrate astrocytes, axons
and nerve endings.
Vital stains --- trypan blue, Indian ink--- used for differentiating viable versus dead cells as
well as for demonstrating cells with phagocytic capabilities.
VI. Other Methods of Investigation
A. Immunohistochemistry
Detection of specific antigens (usually proteins) in the tissue using antibody and colored
markers. Example: presence of oncogene protein products in a specimen. Used extensively in
pathology.
B. In situ hybridization
Detection of specific nucleic acid sequences in the tissue using labeled complimentary
probes. Example: strain-specific diagnosis of viral infection.
C. Autoradiography
Use of radioactively labeled tags and photographic emulsion to localize compounds in the
cell. Example: labeled amino acids to trace the path of protein synthesis and packaging in the
exocrine pancreas.
D. Fluorescence Microscopy
Instead of visible light, this technique uses ultraviolet light waves for illuminating the subject.
Dyes that fluoresce under UV light are used to stain different object of interest. A routine use
is the study of the basement membranes in glomeruli in renal diseases. Some intracellular
materials, such as lipofuscin pigment granules in neurons, are autofluorescent and will appear
bright under UV light without the application of dyes.
E. Confocal Microscopy
The confocal microscope has a much greater death of field than conventional microscopes. A
thicker slide of tissues can be studied and the architecture of the tissue shows up better with
confocal microscopy.
VII. 2- and 3-Dimensional Perspectives
The study of routine histology involves the use of many glass microscope slides containing
tissue sections. It is important to understand that a section is but a small part of the
tissue/organ and may not yield an accurate picture of the tissue/organ being studied. You
actually lose a dimension when you take a slice of a three-dimensioned structure (the
thickness of a histological section is so small that it usually is insignificant).
For a three-dimensioned structure, sectioning will reduce it to two dimensions. For example,
a section of an orange is represented as a slice.
For a two-dimensioned structure, sectioning will reduce it to one dimension. For example, a
profile of a sectioned fiber is represented as a dot most of the time.
When examining a histological section, try to visualize what the original organ/tissue looks
like in 3 dimensions. This takes understanding and some practice. Another issue to keep in
mind is the plane of section. Depending on the plane of section, a familiar shaped structure
can take on odd shapes. For example, you generally expect to see a section of a tube to appear
as a ring. However, a section of a tube can appear as a ring, an oval, an eclipse and other
shapes depending on the plane of section. Keep this in mind when examining your slides.
Slice a hard boiled egg thinly along the long axis, lay out the slices and look at them. Can you
reconstruct the egg if you only have three or four slices? If some slices of the egg you have
were cut along the long axis while others were cut along the short axis, how difficult will the
job be?
Understanding these concepts will help you make the proper interpretation of the images you
are studying and be able to relate to the real tissue/organ.
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