Explain the importance of identifying changes in granulocyte morphology.

Define, describe, and explain the relationship of Barr bodies to abnormal

morphology.
Discuss nuclear hypersegmentation and hyposegmentation, with respect to:
definition, appearance, composition, and significance.
Discuss nuclear hypersegmentation and hyposegmentation, with respect to:
hypersegmented granulocytes and hyposegmented granulocytes.
Identify each of the following cytoplasmic variations: Dohle bodies, Auer
rods, vacuoles, and abnormal granulation.

Variations in Morphology

Many variations in morphology may be seen when examining Wright stained

peripheral blood smears. One method of classifying these variations in white cell
morphology is based on the way the body responds to a stimulus, deficiency, or the
presence of an inherited defect. This classification falls into three groups:

Pathological:
Cells may show abnormalities in appearance and/or function. The body is
responding abnormally to a stimulus or inherited defect, resulting in
physiological impairment in the patient.

Nonpathological:
Cells may show variation in morphology but their function is normal. Their
presence does not cause physiological impairment.

Reactive:
Cells show variation in morphology but are functioning normally in response to a
specific stimulus, such as a virus or bacteria. There is a disease process in progress to
which the cells are responding. Although the morphology has varied from normal and
their presence is significant, the body is responding normally to a stimulus.
Importance of Recognition

It is important to recognize the presence of these morphology changes and to

accurately identify them for several reasons:

If the changes are pathological, their identification may aid the physician in
diagnosing a specific condition.
If the changes are not pathological, their identification alerts the physician to
the fact that the changes are present, thus avoiding a possible misdiagnosis.

If reactive, it indicates that although the cells are functioning normally, they are
reacting to a stimulus. Indicating the presence of such cells may aid in determining
the diagnosis or monitoring the course of disease once a diagnosis has been
madeHypersegmentation
A normal mature neutrophil is 10-15 m in diameter and contains 3-5 lobes or
segments. When the number of segments is increased to six or more the cell is
hypersegmented. Some hypersegmented cells will be larger than in 15 microns.
Hypersegmentation is seen most frequently in neutrophils but can also occur in
eosinophils and basophils.

The nuclear segments are composed of deoxyribonucleic acid (DNA). A defect in the
production of DNA causes the maturation process to be slower than normal which in
turn causes the nucleus to hypersegment. The cytoplasm will be normal in appearance
and function, indicating that these cells are capable of phagocytosis. These cells are
considered pathological. Conditions Associated with Hypersegmented Neutrophils

There are a number of conditions in which hypersegmented neutrophils may be seen,

such as megaloblastic anemias (including folic acid deficiency and pernicious
anemia). Individuals who are receiving chemotherapy or have long-term chronic
infections may also have hypersegmented neutrophils.

The cells seen in these conditions would be classified as pathological since the body is
responding abnormally as a result of either a deficiency of a component needed for
DNA production or because of the toxic effect that chemotherapy drugs have on
DNA. Hypersegmented Neutrophils

A hypersegmented neutrophil showing eight segments is present in this image (see

arrow) Hypersegmented Neutrophils (cont.)
Another example of a hypersegmented neutrophil is noted in the image to the right.
This particular cell has six lobes. Hypersegmented neutrophils are sometimes larger
than normal neutrophilA Normally Segmented Neutrophil

A normal segmented neutrophil having five lobes that was present on the same slide
as the hypersegmented neutrophils. Hyposegmentation

Hyposegmented cells are neutrophils with fewer than three nuclear lobes. The nucleus
may be round, peanut-shaped, band-shaped or bilobed. Since nuclear lobe
development is abnormal, the chromatin structure often appears more mature than
normal. It is sometimes very smooth, almost pyknotic

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Conditions Associated with Hyposegmented Neutrophils

The presence of hyposegmented neutrophils can be an acquired phenomenon, as a

result of severe infection, burns, malignancy, chemotherapy or other drugs such as
sulfonamides. When the causative agent is removed, the cells will return to normal.

Percentages of neutrophils affected will vary in this condition. Hyposegmented

neutrophils as an aquired phenomenon are known as pseudo-Pelger-Huet cells.
Pelger-Huet Anomaly

Pelger-Huet anomaly is the inherited form of neutrophilic hyposegmentation. Its

transmittance is autosomal dominant and the anomaly is present in about one out of
every 6000 people.

When present on a peripheral blood smear, more than 70% of the segmented
neutrophils will have bi-lobed or mono-lobed nuclei. A bi-lobed nucleus will have
two round segments of nearly equal size, connected by a thin chromatin strand. A
mono-lobed nucleus may be peanut shaped, slightly indented, or round; chromatin
appears fully mature and parachromatin is evident. Pelger-Huet anomaly in the
homozygous state has an increased number of cells with singular round nuclei and
decreased numbers of the bilobed forms.

The main function of neutrophils is phagocytosis. This function is not affected in

either the acquired or the inherited anomaly. Since inherited Pelger Huet anomaly is
associated with functionally normal neutrophils, the neutrophils are considered a
nonpathological variant. Normal Band Forms vs. Pelger-Huet Bands

Recognition and diagnosis of the inherited form of Pelger-Huet anomaly is important

because many of these Pelger-Huet neutrophils may be classified as bands, therefore;
increased numbers of bands might be erroneously reported in these patients.

Since increased numbers bands frequently indicate infection, reporting Pelger-Huet

cells as normal band forms could result in inappropriate treatment for infection.
Pelger-Huet cells have denser nuclear chromatin than neutrophilic band formsBilobed
Neutrophil
This image shows an example of a bilobed neutrophil, often referred to as the "pince-
nez" or an "eye-glasses" formPelger-Huet Cells

The arrowed cells are typical of the bilobed cells seen in Pelger-Huet anomaly. Notice
the dense nuclear chromatin, showing little parachromatin. Single-Lobed Pelger-Huet
Cells
This mature neutrophil has a single nuclear lobe. The nuclear chromatin is dense.
Cells such as this and the previously shown bilobed "pince-nez" form may be seen
with inherited Pelger-Huet or acquired pseudo Pelger-Huet.

*Note: When examining a slide made from an EDTA tube of normal blood, an
occasional cell containing a round pyknotic nucleus and neutrophilic-appearing
cytoplasm may be seen. Rare cells such as these do not indicate the presence of
Pelger-Huet anomalyBarr Body

A Barr body (see image) appears as a small drumstick-like projection on one of the
lobes of some neutrophils in females. Barr bodies are attached to the nuclear lobe by a
single narrow stalk, which distinguishes them from other thicker projections,
sometimes referred to as "clubs." Since Barr bodies are the morphological expression
of the inactivated X chromosome, Barr bodies (one per neutrophil) can be seen in up
to 3% of the neutrophils on a female's peripheral blood slide. In rare chromosome
disorders in which three or more X chromosomes are present, two to three Barr bodies
per neutrophil can be seen.

Barr bodies must also be distinguished from hair-like projections sometimes seen in
the band form, following irradiation or in patients with a malignant tumor that has
metastasized.
Recognition of a Barr body in a neutrophil is important in order to avoid reporting it
as abnormal (unless two or more per neutrophil are seen). The Barr body is
considered nonpathological unless associated with rare chromosome disordersDhle
Bodies

Dhle bodies are small, blue, irregular-staining patches of cytoplasm seen near the
edge of the cell in granulocytes in certain conditions. Electron microscopic
examination reveals that they are composed of aggregates of rough endoplasmic
reticulum remaining from an earlier stage of development.

The Dhle body in this cell can be seen near the edge of the cytoplasm. Notice that
the cytoplasmic granules are somewhat heavier and darker than normalDhle Bodies,
continued

Dhle bodies are seen in a number of conditions, including:

infections
burns
measles
leukemia
chemotherapy

Dhle bodies are only present when the body is responding to unusually severe stress
or stimulus. This severe stress may cause the cytoplasm of some cells to mature
improperly.

Their presence does not aid in the diagnosis of the disorders in which they are found,
but they are frequently seen along with toxic granulation and/or vacuoles in cases of
infection or burns.

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Unusually Darkly Staining Dhle Bodies

Unusually dark-staining Dhle bodies are seen in the cytoplasm of this cell, along
with bacteria, an infrequent finding.

The bacteria are indicated by the red arrows and stain almost black. The Dhle bodies
are patches of dark-blue stained material, indicated by the blue arrows. Toxic
Granulation

Hover over the image to zoom in.

Toxic granulation is manifested by the presence of large granules in the cytoplasm of

segmented and band neutrophils in the peripheral blood. The color of these granules
can range from dark purplish blue to an almost red appearance. Toxic granules are
actually azurophilic granules, normally present in early myeloid forms, but are not
normally seen at the band and segmented stages of neutrophil maturation. These
granules contain peroxidases and hydrolases.

Toxic granulation is seen in cases of severe infection, as a result of denatured proteins

in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is
the most frequent cause of toxic granulation. This phenomenon may be seen in cells
which also contain Dhle bodies and/or vacuoles. Cells containing toxic granules may
have decreased numbers of specific granules.

Note: Cells containing only a few specific granules, with or without toxic granules,
are said to be degranulated. The nucleus in degranulated cells may often be round-
bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will
disintegrate soon. Increased basophilia of azurophilic granules simulating toxic
granules may occur in normal cells with prolonged staining time or decreased pH of
the stain.
The blue arrow in the image points to a neutrophil with toxic granulation. Dhle
bodies are also present in the cell, indicated by the red arrowsToxic Granulation

Hover over the image to zoom in.

Toxic granulation is manifested by the presence of large granules in the cytoplasm of

segmented and band neutrophils in the peripheral blood. The color of these granules
can range from dark purplish blue to an almost red appearance. Toxic granules are
actually azurophilic granules, normally present in early myeloid forms, but are not
normally seen at the band and segmented stages of neutrophil maturation. These
granules contain peroxidases and hydrolases.

Toxic granulation is seen in cases of severe infection, as a result of denatured proteins

in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is
the most frequent cause of toxic granulation. This phenomenon may be seen in cells
which also contain Dhle bodies and/or vacuoles. Cells containing toxic granules may
have decreased numbers of specific granules.

Note: Cells containing only a few specific granules, with or without toxic granules,
are said to be degranulated. The nucleus in degranulated cells may often be round-
bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will
disintegrate soon. Increased basophilia of azurophilic granules simulating toxic
granules may occur in normal cells with prolonged staining time or decreased pH of
the stain.

The blue arrow in the image points to a neutrophil with toxic granulation. Dhle
bodies are also present in the cell, indicated by the red arrowToxic Granulation and
Vacuolation
Hover over the image to zoom in.

Vacuoles are frequently seen in conditions such as infection or burns when toxic
granulation is also present. The cell in this image exhibits toxic vacuolation as well as
toxic granulation.

Note: Toxic vacuolation and toxic granulation are classified as reactive and not
pathologic since the body is responding normally in an effort to rid itself of infection
caused by bacteriaCytoplasmic Vacuolation

Vacuoles are areas of the cytoplasm which do not stain with Wright's stain and appear
as holes in the cytoplasm. Their composition may vary; some will contain remnants of
bacterial digestion, autodigestion in an aging cell, while others may contain fat. It is
not possible to differentiate the various types of vacuoles on Wright stained smears
using light microscopy.

Vacuoles may be seen occasionally in an aging granulocyte (degenerative

vacuolation), but are seen more frequently and are significant in cases of bacterial
infection and septicemia. Neutrophil with Normal Granulation
Hover over the image to zoom in.

This image is an example of a neutrophil showing normal granulation. Notice that on

close examination the granules are present but not prominent. Some normal
neutrophils may have granules slightly more prominent than this exampleAuer Rods

Auer rods are red staining, needle-like bodies seen in the cytoplasm of myeloblasts,
and/or progranulocytes in certain leukemias. Auer rods (see arrow in image) are
cytoplasmic inclusions which result from an abnormal fusion of the primary
(azurophilic) granules. Single or multiple Auer rods may be seen in the cytoplasm of a
cell. If more than one is present, they are frequently close together and may even be
overlapping.

Their identification is very important because, if found, they can confirm the presence
of myeloblasts indicating the presence of a non-lymphocytic (myeloid) leukemia.
They can also be seen in myeloid blast crisis in chronic granulocytic leukemia. Auer
rods are never seen in lymphoblasts. This differentiation is important because the
treatment of lymphoblastic and myeloblastic leukemia are different.
Auer Rods are always classified as pathological

Chediak-Higashi Anomaly

Chediak-Higashi anomaly is a rare autosomal recessive disorder. It results from a

mutation of the gene LYST which encodes a protein with multiple phosphorylation
sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic
granules.

Early in neutrophil maturation normal azurophilic granules form, but they fuse
together to form megagranules. Later during the myelocyte stage, normal specific
granules form. The mature neutrophils contain both normal specific granules and
abnormal azurophilic granules. These large abnormal granules can be seen in the
cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes.

These abnormal granules are able to kill bacteria in neutrophils and monocytes;
however, the process is much less effective than in normal cells in part, because these
neutrophils have impaired locomotion. For these reasons, individuals with Chediak-
Higashi have recurrent infections.
An accelerated lymphoma-like phase occurs, with lymphadenopathy,
hepatosplenomegaly, and pancytopenia. Death often occurs at an early age

Albinism and Chediak-Higashi Anomaly

Albinism, one of the striking physical characteristics of Chediak-Higashi anomaly, is

also thought to be related to the general dysfunction of cells. Albinism has been
specifically related to the aggregation of melanosomes, the pigment producing cells in
the bodyChediak-Higashi Anomaly vs. Toxic Granulation

The neutrophils found in Chediak-Higashi must be differentiated from toxic

granulation. In conditions causing toxic granulation, the granules are smaller and
more numerous. In toxic granulation, neutrophils are usually the only cells affected.

In Chediak-Higashi anomaly, eosinophils, basophils, lymphocytes and monocytes are

affected. In eosinophils larger than normal eosinophilic granules may be seen,
basophils may exhibit larger than normal basophilic granules, lymphocytes, large
azurophilic granules. Larger pale granules/bodies may appear in monocytes. Chediak-
Higashi Anomaly Inclusions
In the center of this slide is a neutrophil from a patient with Chediak-Higashi
syndrome. Notice that some of the granules are very large and purpleUngraded
Practice Question

Chediak-Higashi anomaly is characterized by which of the following?

You answered the question correctly.

The correct answer is highlighted below

Myeloblasts containing auer rods

Large abnormal lysosomes (granules) in the cytoplasm of leukocytes
The presence of large vacuoles and Dhle bodies in neutrophils

Feedback

Chediak-Higashi anomaly is an autosomal recessive disorder characterized by large

abnormal granules in the cytoplasm of leukocytes (granulocytes, lymphocytes, and
monocytes) May-Hegglin Anomaly

May-Hegglin anomaly is an autosomal dominant condition characterized by the

presence of pale blue inclusions in neutrophils called May-Hegglin bodies. May-
Hegglin bodies, which are described as Dhle-like, are prominent and randomly
distributed throughout the cytoplasm; inclusions can also occur in eosinophils,
basophils, and monocytes. Additional findings include giant platelets, and occasional
thrombocytopenia.

Acquired forms of this anomaly may also occur as a result of the use of cytotoxic
drugs.

The blue arrow in the image points to a May-Hegglin body . The red arrow indicates a
giant platelet, a characteristic that also helps to identify the anomalyAlder Anomaly
(Alder-Reilly Anomaly )
Alder anomaly is a rare autosomal recessive disorder in which the basic defect
involves protein-carbohydrate complexes called mucopolysaccharides. The
accumulation of partially degraded (broken down) protein-carbohydrate complexes
within the lysosomes account for the larger than normal purple-staining inclusions
seen in all types of mature white blood cells, and sometimes in earlier cells.

The granules may occur in clusters, rather than diffusely, throughout the cytoplasm as
in toxic granulation. These inclusions may be seen in the bone marrow more
frequently than in peripheral blood. The physical characteristics associated with this
disorder include gargoylism and dwarfism. The function of the cells involved is not
affected.

This morpholical change would be classified as pathological since the body is

responding abnormally even though the function is not affectedAlder Anomaly,
continued

This image shows a peripheral smear from another patient with Alder anomaly.
Notice that neutrophil seen in the image has granulation which is much heavier than
what would be classified as normal. The amount of granulation may vary from cell to
cell, with some cells being unaffected. A lymphocyte showing abnormal granules is
also present on this slide. Ungra
ded Practice Question

Alder anomaly inclusions may be found in which of the following white blood cell
types?

You answered the question incorrectly.

The correct answer is highlighted below

Neutrophils only
Granulocytes only
All types of mature white blood cells

Feedback

Alder anomaly inclusions may be seen in all types of mature white blood cells, and
sometimes in earlier cellsUngraded Practice Question

Which of the following inclusions has a similar appearance on a Wright's stained

smear to a Dhle body?

You answered the question correctly.

The correct answer is highlighted below

Auer rod
May-Hegglin body
Vacuole

Feedback

May-Hegglin and Dhle bodies can appear quite similar on peripheral blood smear
examinations. They are both types of white cell inclusions that stain pale blue and
tend to be observed in the periphery of the cellular cytoplasm. May-Hegglin bodies
are found in May-Hegglin anomaly while Dhle bodies can be found in various
conditions, especially infection/inflammatory processes

Ungraded Practice Question

The inclusions that are frequently seen on the same peripheral blood smear with toxic
granulation include: (Choose ALL that apply)

That's partially correct.

The correct answers are highlighted below

 May-Hegglin bodies
Vacuoles
Auer rods
Dohle bodies

Feedback

Toxic granulation is most commonly observed in patients with severe bacterial

infections. This phenomenon is also highly associated with toxic vacuolization and
the presence of Dohle bodies.

Ungraded Practice Question

What type of inclusions are present in the cell that is indicated by the arrow?

You answered the question correctly.

The correct answer is highlighted below

Auer rods
Chediak-Higashi anomaly inclusions
Toxic granulation

Feedback

The fused, abnormally large granules in this neutrophil are Chediak-Higashi anomaly
inclusions