Quick Guide

Microscopy in Hematology
Authors: Dr. Michael Gögler, Anke Koenen
Carl Zeiss Microscopy GmbH, Germany
Date: November 2022

Talking about hematology is talking about blood, blood-forming organs and blood diseases. In clinical
laboratories, hematologists diagnose and treat blood disorders and malignancies, including types of
hemophilia, leukemia, lymphoma and sickle-cell anemia.
To do so, hematologists routinely investigate peripheral blood smears on glass slides with a microscope
to find any abnormalities indicating such hematological diseases or to look for blood parasites, such as
those found for malaria and filariasis.

Blood – circulation and blood cells

What is blood? • Arteries carry oxygenated blood from
Whole
Blood is a body fluid that performs the heart to the rest of the body. Blood
a variety of transport and regulatory • Veins transport the blood back to the
functions. The heart pumps it constantly heart and lungs, so it can get more
around the body (fig. 1) of humans and oxygen to send back to the body via
animals providing the cells with necessary the arteries.
substances such as nutrients and oxygen.
It also carries carbon dioxide and other How much blood is circulating through
Centrifuged
waste materials to the lungs, kidneys, and the body depends on a person’s size. The Blood
digestive system to be removed from the larger a person is, the more blood volume
body. Blood fights infections and carries they have. An average heavy, healthy, adult
hormones around the body. person has a blood volume of about eight
percent of his or her body weight. A person
Blood circulation weighing about 70 kilograms therefore has Plasma (55%)
The circulatory system is made up of two about five to six liters of blood.
White Blood Cells &
types of blood vessels:
Platelets (4%)
The color of blood
Blood in the human body is red, the Red Blood Cells (41%)

shade of red may vary. The red color itself

comes from the hemoglobin. This is a
protein molecule in the red blood cells. Figure 2 Composition of blood

Hemoglobin contains iron, which reacts

with oxygen, giving blood its typical red
color. The level or amount of oxygen in Fun fact – most animals also have red
the blood determines the shade of red. blood. However, there are exceptions:
As blood leaves the heart and is oxygen-
rich, it is bright red. When the blood • Some types of octopus, squid, and
returns to the heart, it has less oxygen crustaceans have blue blood due to a
and is darker. high concentration of copper. When
copper reacts with oxygen, it gives
Blood Components and their function their blood its blue color.
Blood is made up of solid components, • The skink, which is a type of lizard, has
the blood cells and a liquid intercellular green blood due to biliverdin, which is
Figure 1 The circulatory system, substance, blood plasma. a by-product of the liver.
courtesy of: CC BY-SA 2.5*

* https://commons.wikimedia.org/w/index.php?curid=741255

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 Platelets
(thrombocytes) What are blood types?
Even though everyone’s blood contains the same elements, not
everyone’s blood is alike. There are different blood types based
on the absence or presence of specific antigens and antibodies
White blood cells
(lymphocytes) on the surface of the red blood cells.

Red blood cells

(erythrocytes) There are eight blood types, described using the letters A,
B, and O. People with the blood type A have an A-antigen
on their red blood cells, people with type B blood have a
B-antigen. Some people have both. People with O blood type
do not have either A or B antigens on the red blood cells.
Type O is the most common blood type in the world. Besides
Figure 3 Blood cells getting a letter or two, a person’s blood is either “positive”
or “negative.” This is a way of keeping track of whether
The yellowish plasma makes up about half of the content of blood. someone’s blood has a protein called Rh protein.
Composed of 92% water, it also contains nutrients such as glucose,
proteins helping blood to clot, hormones, and waste products.

The other half of blood volume is composed of blood cells.

There are mainly three types of blood cells.

Granulocytes Agranulocytes

Neutrophil Eosinophil Monocytes

A antigen B antigen Rhesus D factor

Lymphocytes Figure 5 Blood types

Basophil
If your blood is positive, you have this protein. If it’s negative,
you don’t. It is more common to have Rh-positive blood than
Rh-negative. Either way is totally fine.

Figure 4 White blood cell

Red blood cells (erythrocytes) White blood cells (leukocytes) Platelets (thrombocytes)
Diameter: 7.5 μm Diameter: 7 – 20 μm Diameter: 2 – 3 μm
Formed in the bone marrow Formed in the bone marrow or spleen,
thymus, and lymph nodes
Contain the protein hemoglobin that carries Part of the immune system, which helps the When a blood vessel breaks, platelets gather
oxygen from the lungs into the organs and body defend itself against infection. Occur in the area and help seal off the leak. Platelets
tissues and carbon dioxide from the tissues in various types including granulocytes, work with proteins called clotting factors to
into the lungs. Hemoglobin is responsible for monocytes, and lymphocytes. Different types control bleeding inside our bodies and on our
the red color. fight different germs such as bacteria, viruses. skin.
Require about 9 days to develop and have a Depending on the type of leukocyte, life span Have a life span of about 7 to 9 days.
life span of about 4 months can vary from a few days to years.
Insufficient number of red blood cells can Depending on the type of leukocyte, a Thrombocytes are responsible for blood
cause paleness, fatigue, shortness of breath, distinction is made between non-specific coagulation and haemostasis.
and other symptoms. This is referred to as (phagocytosis) and specific (formation of
anemia. antibodies) immunity.

Table 1

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Microscopic Blood It is imperative to use the proper technique when preparing

Examination the smear. If it is poorly made the stained smear will have no
value. If the spreader is moved too slowly or the angle used is
Role of microscopy too small, a thick blood film will be produced, and it will not
Laboratory blood analysis is one of the most important be readable. If the spreader is moved too rapidly or the angle is
routine diagnostic procedures in a clinical lab. Hematologists too large, a thin film will be produced, resulting in many of the
routinely investigate peripheral blood smears on glass slides leukocytes becoming aggregated at the feathered edge. Other
with a microscope to find abnormalities in morphological causes of poor blood smears are Hemolysis of cells due to moist
characteristics of cells and tissues indicating hematological fingers, scratches or holes.
diseases or to look for blood parasites, such as those found
for malaria and filariasis. A microscopic image can deliver Bone marrow examination
information on cell types based on their morphology, about the In situations where a blood test is abnormal or does not
quantity and composition of blood cells. Light microscopy with provide enough information about the suspected problem,
a magnification of up to 1,000× is employed to recognize and a bone marrow examination can be required. It is used to
count the various cell types within a monolayer and document diagnose and monitor certain diseases of the blood or the blood
the results with a digital camera. This way, many types of building system. Fragments from the bone marrow are typically
blood diseases or the developmental stages of parasites can separated or concentrated and then aspirated on several glass
be visualized. In certain cases, the microscopic examination of slides. These fragments are squeezed gently to generate the
peripheral blood smears is complemented with a bone marrow smears. Complementary, bone marrow blood smears are
examination. Key microscopy techniques include brightfield, prepared.
darkfield, DIC, fluorescence, immunocytochemistry and
immunohistochemistry.

Blood smear
A blood smear is used to look for abnormalities in blood cells, be it
their morphology or quantity. It can help to detect, diagnose, and
monitor deficiencies, diseases, and disorders involving blood cell
production, function, and lifespan. Normally a microscopic analysis
of a thin blood smear is done when the complete blood count or
the differential white blood count deliver abnormal results. [1]
1. Place a clean glass slide on a flat surface. Add a small drop of
blood from, for example, the fingertip.
2. Place the cover slip in an angle of about 30 – 45°.
3. G ently wipe the edge of the coverslip over the blood and
produce a smear.
4. T he smear is air-dried, fixed to the slide with methanol, and
stained to distinguish the various cell types. Figure 7 Representative image of a Auer rods visible in a bone marrow sample
ZEISS Axioscope 5

Figure 6 Preparation of a blood smear Figure 8 Representative image of bone marrow in brightfield,
ZEISS Axioscope 5, ZEISS Axiocam 208 color, 40× objective

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Staining methods in hematology
Blood components Coloration
In hematology most staining methods
Erythrocytes Pink
are based on panoptic stains according
Nuclei of leukocytes and nucleated Reddish purple
to Pappenheim and Romanowski stains erythrocytes
such as Wright’s stain, Leishman stain, Eosinophilic granules Brick red to reddish brown
or Giemsa stain. These allow for the Basophilic granules Dark purple to black
detection of erythrocyte, leukocyte or Neutrophilic granules Light purple
thrombocyte abnormalities. It is imperative Lymphocyte cytoplasm Light blue
that the slides are dried completely Monocyte plasma Gray-blue
before any staining. Otherwise there
is the risk of denaturation of the white Table 2

blood cells, which can hamper a proper Wright’s staining*

differentiation. The drying should be solution. The cell nuclei appear reddish- This technique is named for James Homer
completed as soon as possible after the purple in the sample, the plasma of Wright, who modified the Romanowsky
blood smear is produced. lymphocytes and monocytes bluish, the stain. For Wright’s staining there are
plasma of granulocytes pale pink. several variations in technique available
Pappenheim staining* because of different behavior of the stain
Artur Pappenheim (1870 – 1916) was a Giemsa staining* and buffer and because of differences in
german hematologist. The Pappenheim Giemsa is a Romanowsky type stain, film thickness. So, the choice of recipe
or May-Grünwald-Giemsa staining is named after Gustav Giemsa, a german is not as important as the consistency
a panoptic differential staining, which chemist. It is a differential stain, in the staining technique. It is classically
makes use of the behaviour of the containing a mixture of Azure, Methylene a mixture of eosin (red) and methylene
Giemsa and May-Grünwald staining. blue, and Eosin dye. The stain is used blue dyes and offers a coloration similar
It depicts all microscopic phenomena to obtain differential white blood cell to the panoptic method. If performed
used in routine diagnostics, strongly counts, to differentiate nuclear and correctly, the red blood cells should
and differently colored on a clean cytoplasmic morphology of the various look yellowish-red and the neutrophils
background, so that even fine deviations blood cells. It stains red blood cells should have dark purple nuclei, reddish-
can be easily detected. In Pappenheim pink, platelets pale pink, lymphocyte lilac granules, and pale pink cytoplasm.
staining, the preparations are fixed with cytoplasm blue and leucocyte chromatin This method is widely used to perform
concentrated May-Grünwald solution, magenta. Giemsa can also be used to differential white blood cell counts, which
stained with diluted May-Grünwald stain blood parasites such as malaria are routinely ordered when conditions
solution and, after rinsing with Aqua and other spirochete and protozoan such as infection or certain diseases are
destillata, counterstained with Giemsa microorganisms. suspected.

Application Examples

Figure 9 Representative image of Trypanosoma Figure 10 Representative image of blood smear, Giemsa staining
brucei gambiense parasite in human blood smear.
Giemsa staining.

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Figure 11 Representative image of blood vessels Figure 12 Representative image of blood with
leucocytes

Figure 13 Representative image of polychromatic erythrocytes, abnormal as Figure 14 Representative image of erythrocytes
this should be part of the bone marrow and not the blood

Figure 15 Representative image of blood vessel. Azan stain; Orange: Figure 16 Representative image of uncovered blood smear (human).
cytoplasm, Red: nuclei, Blue: collagen, Plan-Apochromat 20× / 0.8 Wright stain, Achroplan 100× / 1.25 Oil

Figure 17 Representative images of blood cells shown in a STEM image taken with the EVO ® STEM detector at 20 kV.

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Suitable Microscope Equipment
When examining a peripheral blood ZEISS Axiolab 5 ZEISS Axioscope 5
smear, a hematologist starts with a low Axiolab 5 is made for the clinical routine Axioscope 5 with Axiocam 208 color
magnification objective, typically a 20× or work that goes on every day in your he- makes documenting your hematology
10×. The hematologist gets an overview matology lab. The white LED illumination samples very efficient. Plan Neofluar
of red and white blood cell densities, the with very high color rendering index is objectives provide the necessary visu-
number of erythrocytes, its color and ideal to visualize peripheral blood smears alization of subtle color nuances, even
rough morphology and if obvious cellular and bone marrow samples in true color. for challenging samples such as bone
inclusions are present. In higher magnifi- marrow smears. The color impression in
cation (typically 60× or 100×, sometimes the camera image shows up exactly the
40× for bone marrow), a manual differen- same as it appears through the eyepieces.
tial cell count is performed and red and This smart microscope makes automatic
white blood cell morphology including adjustments for brightness and white bal-
the presence of inclusions or pathogens ance to keep digital documentation tasks
are visualized. A very good differenti- in your clinical routine easy.
ation of cell types and clearly visible
cellular details are absolute prerequisites
in hematology. Hematologists rely on
crystal-clear images to visualize morpho-
logical details such as delicate granules,
rod-shaped inclusions, irregularities of
the nuclear membrane or clefted nuclei. The constant color temperature of the LED
Furthermore, highest color fidelity is facilitates system operation and digital
key when examining blood smears and documentation. Combine Axiolab 5 with
bone marrow preparations. Apart from the microscope camera Axiocam 208 col-
brightfield microscopy, phase contrast or and take full advantage of the smart
and polarizing microscopy techniques are micro­s copy concept: you’ll be experienc-
also used for certain types of samples. ing a completely new form of digital doc-
While hematological stains* result in a umentation in your daily clinical routine.
good transparency of the sample and Just focus your sample and press a single All you have to do is focus on your sam-
specific staining of cellular features, it is button for crisp images in true color. The ple, press the ergonomic Snap button on
the optical quality of the microscope, the 4k live image will look exactly like you the microscope, and that’s it. To store
fidelity of the attached camera for digital see it through the eyepieces, with all the images with the respective patient ID the
documentation, and the ergonomic­ details and subtle color differences clearly camera can be integrated with laboratory
design of the instrument that can make visible. Manual camera parameter ad- information systems that support Twain.
all the difference when examining patient justments are not necessary which saves
samples. time. Discussions of critical cases with
a colleague can now be done with ease * T he user is responsible for correct sample
using the high resolution live image. preparation and application of staining
methods.

References
[1] https://www.cdc.gov/dpdx/diagnosticprocedures/blood/specimenproc.html

Related White Papers

[1] https://p.widencdn.net/rg433b/EN_wp_microscopic-examination-blood
[2] https://p.widencdn.net/8azb8g/EN_wp_Axio-Lab-A1-Malaria
[3] https://p.widencdn.net/zuwbem/EN_wp_EVO_python-blood-analysis

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