3

CHAPTER
CHAPTER

Inclusion bodies in Erythrocytes

Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins. They
typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of
viral capsid proteins.

Normally a red blood cell does not contain inclusions in the cytoplasm. However, it may be seen
because of certain hematologic disorders.

There are three kinds of erythrocyte inclusions:

Developmental Organelles

A. Howell-Jolly bodies: small, round fragments of the nucleus resulting from karyorrhexis or nuclear
disintegration of the late reticulocyte and stain reddish-blue with Wright stain.

Appearance
This DNA appears as a basophilic (purple) spot on the otherwise eosinophilic (pink) erythrocyte
on a standard H and E stained blood smear. These inclusions are normally pitted out by the spleen
during erythrocyte circulation, but will persist in individuals with functional hyposplenia or
asplenia.

Causes
Common causes of asplenia are splenectomy due to trauma, and autosplenectomy caused by
sickle cell anemia. Ten percent of patients with Celiac disease also present with splenic atrophy
with subsequent Howell-Jolly bodies. Other causes are radiation therapy involving the spleen,
such as that used to treat Hodgkin lymphoma. Howell-Jolly bodies are also seen in: severe
hemolytic anemia, megaloblastic anemia, hereditary spherocytosis, and myelodysplastic
syndrome (MDS).
B. Basophilic stipplings - this stipplings is either fine or coarse, deep blue to purple staining inclusion
that appears in erythrocytes on a dried Wright stain.

Basophilic stippling refers to an observation found when observing a blood film, where
erythrocytes display small dots at the periphery. These dots represent accumulations of rRNA and
are always pathological.

It is associated with several conditions, including:

1. Sideroblastic anemia
2. Lead poisoning
3. Beta thalassemia (though some have questioned this)
4. Megaloblastic anemia.
C. Pappenheimer bodies - are siderotic granules which are small, irregular, dark-staining granules
that appear near the periphery of a young erythrocyte in a Wright stain.

Pappenheimer bodies are abnormal granules of iron found inside red blood cells on routine blood
stain.[1] They are a type of inclusion body formed by phagosomes that have engulfed excessive
amounts of iron. They are seen in diseases such as sideroblastic anemia, hemolytic anemia, and
sickle cell disease. They can interfere with platelet counts when the analysis if performed by
electro-optical counters.
D. Polychromatophilic red cells - young red cells that no longer have nucleus but still contain some
RNA.

E. Cabot Rings - ring-like structure and may appear in erythrocytes in megaloblastic anemia or in
severe anemias, lead poisoning, and in dyserythropoiesis, in which erythrocytes are destroyed
before being released from the bone marrow.

Cabot rings are thin, red-violet staining, threadlike strands in the shape of a loop or figure-8 that
are found on rare occasions in erythrocytes. They are believed to be microtubules that are
remnants from a mitotic spindle.

Associated conditions

Cabot rings have been observed in a handful of cases in patients with megaloblastic anemia, lead
poisoning and other disorders of erythropoiesis.

Abnormal Hemoglobin Precipitation

A. Heinz bodies - round bodies, refractile inclusions not visible on a
Wright stain film. It is best identified by supravital staining with
basic dyes.

Form and appearance

Heinz body stain of feline blood showing three distinct heinz

bodies.

Heinz bodies appear as small round inclusions within the red cell body, though when stained with
Romanowsky dyes they may appear as projections from the cell. They appear more clearly when
supravitally stained (e.g., with methylene blue or bromocresyl green).

Etiology and associated disorders

Heinz bodies are formed by damage to the hemoglobin component molecules, usually through
oxidations, or the change of an internal amino acid residue(from an inherited mutation), which
causes the damaged molecules to, often degrade and then precipitate, damaging the cell
membrane by hydrophobically adsorbing to it thereby increasing cell permeability, leading to
premature cell lysis. Damaged cells are attacked by macrophages in the spleen, where the
precipitate and damaged membrane are removed, leading to characteristic "bite cells". The
denaturing process is irreversible and the continual elimination of damaged cells leads to Heinz
body anemia.

There are several pathways leading to the hemoglobin damage.

1. In α-thalassemia, lack of α subunits causes β subunits to form tetramers which
precipitate out of solution, creating Heinz bodies.
2. G6PD (Glucose-6-Phosphate Dehydrogenase) deficiency brought on by administration of
oxidant drugs (e.g., primaquine) also can result in Heinz bodies.
3. Heinz bodies can also be found in chronic liver disease.[7]
B. Hemoglobin H Inclusions - alpha thalassemia, greenish-blue inclusion bodies appear in many
erythrocytes after four drops of blood is incubated with 0.5mL of Brilliant cresyl blue for 20
minutes at 37°C.
C. Protozoan Inclusion
 1. Malaria
2. Babesia

Reticulocytes

Reticulocytes ("retics") are young, anucleate

erythrocytes, which are released from bone
marrow into the blood in increased numbers as
a response to anemia caused by hemolysis
(destruction) or loss (hemorrhage) of
erythrocytes in most species (horses are a
notable exception - see more below).
Identification of immature anucleate red blood
cells allows us to determine if the bone marrow
is responding to an anemia (given sufficient
time) by increasing red blood cell production.
This is termed a regenerative response. The
bone marrow response is evaluated by
detecting immature erythrocytes by virtue of
the presence of RNA in the form of ribosomes
and rough endoplasmic reticulum in their
cytoplasm. The more immature the cell, the
more RNA it contains. In contrast, mature red
blood cells, which are no longer synthesizing
hemoglobin, contain very small amounts or no
RNA (they are essentially small sacs of
hemoglobin).
In a Wright's-stained blood smear of a regenerative
The RNA can be detected in immature
anemia in a cat (top image), the larger purple cell (P) is
red blood cells in several ways:
a polychromatophil which corresponds to an aggregate
reticulocyte (A) in the new methylene blue-stained
 With a supravital dye, such as smear (bottom image). In contrast, the larger red cell is
new methylene blue: When a macrocyte (M) that corresponds to a punctate
immature red blood cells are reticulocyte (PR).
stained with a supravital dye,
the cytoplasmic RNA is precipitated into a reticulum-like network. Thus the term,
reticulocyte, was coined. With these dyes, two types of reticulocytes are seen - those with big
aggregates of RNA, which are also called aggregate reticulocytes and those with small dots of
RNA, which are called punctate reticulocytes(see image to the right). Since cells with more
RNA are younger, aggregate reticulocytes are more immature than punctate reticulocytes. In
most species, this does not matter because both types of reticulocytes have similar half lives
in the circulation, but in cats, aggregate reticulocytes only persist for 12-24 hours before they
mature into punctate reticulocytes. Punctate reticulocytes can persist for several days (7-10
days usually) in this species. Since punctate reticulocytes last for a while in the circulation of
cats, we only count aggregate reticulocytes when assessing whether the bone marrow is
currently responding to an anemia in this species.

 Fluorescent dyes: Fluorescent dyes that bind to RNA (and DNA) can also be used to detect
reticulocytes using laser-based technology. This is how reticulocytes are counted in dogs and
 cats at Cornell University with our hematology analyzer (see below). A common dye used for
this purpose is thiazole orange.

 Standard hematogy stains: Depending on their RNA content, reticulocytes can be detected
with routine polychrome stains (eg, Wright's, DiffQuik) used for staining blood smears. RNA
(or DNA) stains blue with these dyes, whereas hemoglobin stains red. When the immature
red blood cells contain large amounts of RNA, the blue mixes with the hemoglobin to yield a
purple color and the cells are termed polychromatophilic red blood cells. These
polychromatophils correspond to aggregate reticulocytes. Thus, we can examine a blood
smear for the presence of polychromasia to assess if the marrow is responding to an anemia
in most species, other than the horse. Indeed, the proportion of polychromatophils can be
used as a rough guide to estimate the reticulocyte count. In contrast to aggregate
reticulocytes, punctate reticulocytes will stain red and not purple because they only contain
small amounts of RNA (the hemoglobin dominates). Therefore, they are a lot harder to
identify on a regular blood smear. Punctate reticulocytes can be larger than normal (until
they are remodeled by splenic macrophages as they recirculate), i.e. they can be macrocytes,
and may be detected on routine blood smears by their larger size. Unfortunately, there are
other causes of large red blood cells so not all macrocytes are punctate reticulocytes and we
do not rely on these cells to evaluate the regenerative response in most species (the horse is
an exception).

Evaluation of the bone marrow response is an initial step in characterizing anemia. If the bone
marrow is responding to an anemia, it is a regenerative anemia and the cause of the anemia is
hemorrhage or hemolysis. If the anemia is regenerative, there will be increased numbers of
reticulocytes in peripheral blood and we will observe polychromasia in the peripheral blood in all
species, other than the horse. The horse releases very few reticulocytes into blood in response to
a hemorrhagic or hemolytic anemia, so performing a reticulocyte count or looking for
polychromasia is not helpful in this species. They do, however, release macrocytes (which may not
be punctate reticulocytes) and we look for these larger red blood cells to determine if horses are
responding to an anemia. Anemias without increased reticulocytes are termed non-regenerative
and are due to conditions which decrease the production of erythrocytes by the marrow.

We can accurately count reticulocytes (as a percentage of total erythrocytes) using supravital or
fluorescent dyes, however this is only performed routinely in the dog and cat. In the latter
species, the reticulocyte percentage alone can be misleading, because a result above the
reference interval may not indicate an adequate regenerative response, since the percentage
alone does not take into account the severity of the anemia. To account for the degree of anemia,
we usually convert the reticulocyte percentage into an absolute reticulocyte count (see below)
and use this number to assess whether an anemia is adequately regenerative or not. In species
other than the dog and cat, we do not routinely quantify reticulocytes but just examine a
peripheral blood smear for evidence of a regenerative response, i.e. the presence of
polychromatophils (or in horses, macrocytes).

With the hematology analyzer used in Clinical Pathology (ADVIA 2120),, reticulocytes are counted
by staining blood a fluorescent dye (Oxazine 750). The immature red blood cells are then detected
by the degree of fluorescence (mature erythrocytes will not fluoresce). We have validated this
technique for the dog and cat. In addition, low numbers of reticulocytes can also be detected in
the blood of horses with regenerative anemias due to experimental blood loss or after
erythropoietin treatment. However, horses do not release sufficient reticulocytes for this to be
clinically useful (there is a large overlap in the reticulocyte counts between non-anemic horses
and horses with a regenerative anemia). In other species, reticulocytes can be counted manually
using a new methylene blue stain, although this is not done routinely.

Absolute Reticulocyte Count

To determine the adequacy of the regenerative response in dogs and cats, absolute reticulocyte
counts can be calculated. This is the method used at Cornell University for determining the adequacy
of a regenerative response as it takes into account the severity of the anemia but does not make
assumptions about a "normal" hematocrit or correction factors for reticulocyte release. The
reticulocyte percentage alone is not as useful as it does not account for the severity of the anemia.
The absolute reticulocyte count is calculated automatically in our laboratory from the reticulocyte % in
dogs and cats (whenever a reticulocyte count is automatically added to a hemogram or a reticulocyte
count is requested for a sample that also has a red blood cell [RBC] count). To calculate an absolute
reticulocyte count, we use the following formula:

Abs retic count (thou/µL) = reticulocyte % (#/100) x RBC count (mill/µL) x 1000

Outlined below are guidelines for assessing the degree of regeneration in anemic dogs and cats, along
with examples of using the results. Remember that cats have 2 different types of reticulocytes,
aggregate and punctate. Only the aggregate reticulocytes are included in a reticulocyte count (as they
are the most immature and more reflective of current bone marrow production).

Canine Reticulocytes Feline Aggregate

Degree of Regeneration
(thou/µL) Reticulocytes (thou/µL)
Inadequate or no regeneration < 95 < 60
Mild 95-150 60-100
Moderate 150-300 100-200
Marked > 300 > 200

A dog with a hematocrit (HCT) of 15% and a RBC count of 1.5 mill/µL has a reticulocyte count of 3%,
which is above our reference interval of 0-1.5%. Based on the reticulocyte percentage alone, you
would consider the severe anemia is regenerative, however the absolute reticulocyte count is 45
thou/µL (3/100 x 1.5 x 1000), which indicates inadequate or no regeneration based on the above
table. This indicates that the bone marrow is not responding appropriately to the severe anemia (as
long as the bone marrow has had sufficient time to respond, i.e. 3-5 days) and that decreased
production of erythrocytes is contributing to or is the main cause of the severe anemia. In contrast, a
different dog with the same low HCT of 15% and RBC count has a reticulocyte count of 8%, which is
also above our reference interval. The absolute reticulocyte count is 120 thou/µL (8/100 x 1.5 x 1000),
which indicates a mild regenerative response and you would consider causes of blood loss or
hemolysis for the severe anemia.

The Reticulocyte production index (RPI, also called a corrected reticulocyte count) is a calculated value
used in the diagnosis of anemia. This calculation is necessary because the raw reticulocyte count is
misleading in anemic patients. The problem arises because the reticulocyte count is not really a count
but rather a percentage: it reports the number of reticulocytes as a percentage of the number of red
blood cells. In anemia, the patient's red blood cells are depleted, creating an erroneously elevated
reticulocyte count.

Physiology

Reticulocytes are newly-produced red blood cells. They are slightly larger than totally mature red
blood cells, and have some residual ribosomal RNA. The presence of RNA allows a visible blue stain to
bind or, in the case of fluorescent dye, result in a different brightness. This allows them to be detected
and counted as a distinct population.[2]

The idea of the RPI is to assess whether the bone marrow is producing an appropriate response to an
anemic state. Reticulocyte production should increase in response to any loss of red blood cells. It
should increase within 2-3 days of a major acute hemorrhage, for instance, and reach its peak in 6-10
days.[3] If reticulocyte production is not raised in response to anemia, then the anemia may be due to
an acute cause with insufficient time to compensate, or there is a defect with red blood cell
production in the bone marrow. Marrow defects include nutritional deficiencies (i.e. iron, folate, or
B12) or insufficient erythropoietin, the stimulus for red blood cell production. Reticulocytopenia, or
"aplastic crisis", is the medical term for an abnormal decrease of reticulocytes in the body

The reticulocyte percentage index may find new use as a more reliable detector of erythropoietin-
doping in athletes. The use of this method is referred to as "biological passport."

Calculation

Reticulocyte Production Index is calculated as follows:

1.

A value of 45 is usually used as a normal hematocrit. [4]

2.The next step is to correct for the longer life span of prematurely released
reticulocytes in the blood--a phenomenon of increased red blood cell production. This
relies on a table:

Hematocrit (%) Retic survival (days) = maturation correction

36-45 1.0
26-35 1.5
16-25 2.0
15 and below 2.5

So, in a person whose reticulocyte count is 5%, hemoglobin 7.5 g/dL, hematocrit 25%, the RPI would
be:

→ RPI 1.4

Alternatively some books provide the following formula:

Interpretation

 The reticulocyte index (RI) should be between 1.0% and 2.0% for a healthy individual.
 RI < 2% with anemia indicates loss of red blood cells, but decreased production of
reticulocytes (ie, and inadequate response to correct the anemia) and therefore red blood
cells.[2]
 RI > 3% with anemia indicates loss of red blood cells (from causes such as destruction,
bleeding, etc.), with an increased compensatory production of reticulocytes to replace the
lost red blood cells.[2]

Interpretation of these values are not standard and vary based on specific laboratory values and
clinical context. </ref http://www.sysmex.ru/files/articles/Xtra_online_reticulocytes.pdf>