Hematology

Seminars in Diagnostic Pathology (2012) 29, 31-48

Approach to peripheral blood film assessment for
pathologists

David Barth, MD, FRCPC

From the Departments of Hematology and Laboratory Hematology, University Health Network, Toronto, Canada, and the
Departments of Laboratory Medicine and Medicine, University of Toronto, Toronto, Canada.

KEYWORDS In today’s age of advanced technology used in the diagnosis of many medical problems, the blood film
Blood film; stands out as an inexpensive and quick diagnostic tool that can generally be performed in most
Morphologic laboratories. Understanding when to perform a blood film and how it is made and having an organized
assessment; sequential approach to reviewing the blood film are critical in the efficient use of this ubiquitous
Hematology diagnosis laboratory investigation. This article will review these issues, as well as common morphologic
Laboratory techniques abnormalities found on blood films, and will discuss related clinical diagnoses that they represent. The
final step of communicating the blood film findings in a clear and concise method to relay clinical
suspicions to the end user will be discussed.
© 2012 Elsevier Inc. All rights reserved.

Why do a blood film? A good approach to interpreting blood films is a valuable
skill for any diagnostician, whether in the field of pathology
In today’s age of advanced technology used in the diagnosis or clinical medicine.
of many medical problems, the blood film stands out as an
inexpensive and quick diagnostic tool that can generally be When to do a blood film
performed in most laboratories. It can be made and ready to
be assessed in 20 to 40 minutes depending on the staining Not every complete blood count (CBC) that is performed
protocol used. In some cases, the blood film can quickly will have a blood film associated with it. The most common
direct the practitioner to either the exact diagnosis or the reasons for a blood film to be made are as follows:
group of disorders that should be considered (eg, red blood
cell fragments in thrombotic thrombocytopenic purpura 1. Quantitative abnormalities: A blood film is made when a
(TTP) or circulating blasts in the peripheral blood), thereby measured parameter of the CBC falls outside a previ-
focusing the diagnostic pathway and reducing the time, ously defined range. Different consensus groups (eg, the
number, and expense of other tests that might have been International Society for Laboratory Hematology and the
performed in an attempt to make a diagnosis. In other cases, Clinical and Laboratory Standards Institute) have pub-
it quickly rules out other entities considered in the differ- lished accepted ranges for various CBC parameters.1
ential diagnosis. Each institution performing blood films may adjust these
ranges to fit their own patient requirements. For exam-
Address reprint requests and correspondence: David Barth, MD, ple, a sample with a hemoglobin of 60 g/L would trigger
FRCPC, Toronto General Hospital, 11E-410 200 Elizabeth Street, Toronto, a blood film because the hemoglobin is lower than the
Ontario, M5G 2C4, Canada. specified range generally accepted of 90 g/L.

E-mail address: [email protected].

0740-2570/$ -see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1053/j.semdp.2011.07.003

32 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

2. Qualitative abnormalities: A blood film is made when experience of the operator; however, many automated blood
morphologic features of certain cells are detected and slide makers on the market today can assess the viscosity
flagged as abnormal by the automated hemocytometer. and hematocrit of the sample and automatically adjust the
For example, immature cells such as promyelocytes or amount of blood deposited on the slide and the angle of the
blasts would trigger a blood film because their morphol- spreader to ensure a single layer of cells on the slide.
ogy does not fall in the normal range of cells expected on
a blood sample. The blood film slide should be fixed immediately and not
left unfixed for more than a few hours. This allows the
3. Delta check: In this case, a number of CBC data points preservation of good morphology and prevents the trans-
are stored in a database on an individual patient from that mission of possible infectious diseases to those personnel in
patient’s previous CBC samples. Knowing this individ- contact with the slide. A delay in fixation past a few hours
ual’s historical trends, if a quantitative abnormality oc- results in the introduction of morphologic artifact. Fixation
curs on a current CBC sample in a specific parameter, is performed with methanol. A miniscule amount of water in
but the quantitative abnormality is not past the preset the methanol can induce hydration artifact (Figures 1 and 2).
limits to trigger a blood film based on the rules from item
1 above, a blood film will still be made if the change is Staining of the slide
significant enough compared with the known historical
trend of that individual patient. The limits of change of a The blood film slide is then stained. Staining of the slide
result compared with the individual patient’s baseline allows for contrast between cellular structures. Contrast is
levels are also preset. For example, patient X has 15 the ability to visually distinguish 1 structure from another.
CBCs done, which show a hemoglobin between 145 and The Romanowsky stains are used. Although there are many
150 g/L. The next CBC reports a hemoglobin of 110 g/L. different variations of these stains (Giemsa, May Grunwald,
Although this is not less than 90 g/L as required for a Wright’s), the basic premise is that the stains are a combi-
blood film to be made by the rule in item 1 above, a nation of azure B, a cationic dye that stains nucleic acids
blood film would still be made because the delta check blue, and eosin, an anionic dye, which stains hemoglobin
rule for a hemoglobin drop of 20 g/L for that individual and eosinophil granules red/orange. Many published proto-
patient would be met. cols are available for staining procedures. The azure B:eosin
ratio, the pH of the buffer, contaminants, timing of staining,
4. Request by clinician: A CBC may be within the limits of and age of stain can all result in abnormal staining outcomes
the rules set in items 1-3 above, but the treating clinician and can be troubleshooted if staining quality is not accept-
may request a blood film to look for any abnormalities able.3
related to the case. For example, a screen for malaria or
other inclusion bodies would trigger a closer assessment The blood film can now be reviewed microscopically
of red blood cell morphology. with a thin film of oil for examination at low power and then
a drop of oil for higher power assessment. Alternatively, a
Blood sample collection

CBC samples are drawn in lavender tubes containing K2
ethylenediaminetetraacetic acid (EDTA). This allows the

blood sample to remain in a liquid state for analysis by a

hemocytometer and for a blood film to be performed. Al-

though citrated and heparinized tubes can be used, the blood

film morphology from these samples is suboptimal com-
pared with K2EDTA.2

Preparation of the slide

Blood films should be made within 12 hours of collection Figure 1 Hydration artifact (100ϫ oil). Because of inadequate
of the K2EDTA sample. Films made past this time will have drying of the blood film prior to fixation, the presence of some
significant artifact. water in the fixative, or increased humidity in the laboratory, the
red blood cells demonstrate a vacuolation type of effect. This is an
Once the decision to perform a blood film has been artifact and should not be confused with an actual clinical process.
made, a proper spread film is essential. Ideally, a large area These changes do, however, preclude a proper morphologic as-
of cells 1 cell layer thick should be obtained. A poorly sessment of the red blood cells.
spread blood film will obscure basic morphology of the cells
and make accurate assessment difficult or impossible. This
may be because of loss of intact cells (ie, smudging) or
layers of cells overlapping each other. Blood films may be
made manually and will be dependent on the skill and

Barth Peripheral Blood Film Assessment for Pathologists 33

Figure 2 Hydration artifact (100ϫ oil). Because of inadequate was taken, and another identifying piece of information.
drying of the blood film prior to fixation, the presence of some This could represent a unique laboratory number assigned to
water in the fixative, or increased humidity in the laboratory, dark that slide or the patient’s hospital identifier number. It is
rings (as indicated by the arrows) are seen within the red blood extremely important to review this information prior to
cells. This is an artifact and it should not be confused with the ring viewing the slide. In a busy laboratory area, it is all too easy
forms seen in malaria. Sometimes, if the ring is in the center of the to pick up the wrong patient’s slide and make the right
red cell, it can give a punched out appearance of central pallor, diagnosis on the wrong patient. In that situation 2 patients
making the red cell appear falsely hypochromic (ie, a larger central may be affected: the patient who erroneously had the diag-
pallor area). nosis made may receive treatment he or she does not need
and the patient who truly has the disease process may not be
coverslip can be mounted on the slide either temporarily treated. As well, the date on the slide is important. Because
with oil or permanently with mountant that is miscible with multiple blood films may be done on a single patient, if a
xylene. A coverslip is beneficial because it allows a slide to blood film from day 1 of a treatment course is mistakenly
be stored for years without dust and scratch damage. This assessed as being from day 7, it may be erroneously as-
can be done if the mountant is neutral and the slide is stored sumed that the treatment course is having no effect.
in the dark.4
Microscopic assessment of the blood film; initial
Prior to reviewing the blood film, the CBC report should screen
be reviewed to assess the indexes reported and to see
whether any flags or concerns were raised by the automated After all this, we are now ready to visually assess the
hemocytometer. This allows the reviewer to see whether the blood film. Just as there is an orderly method for assessing
morphologic features correlate with the reported values and electrocardiograms, chest X-rays, and histology slides, an
to possibly focus the reviewer on any abnormalities that orderly method for assessing blood films is important so as
may have been detected. not to miss both significant and subtle abnormalities. Below
is a suggested approach; however, different individuals may
It is also suggested to review the blood film before use different approaches that suit their needs or style of
looking at the clinical history supplied. This will allow the reporting. Reviewing the blood film in a similar process
reviewer to objectively comment on the changes seen with- each time ensures that certain areas of review will not be
out being biased by the clinical suspicion. It is recom- skipped and allows the reviewer to develop a fast but
mended to look at the clinical history (if supplied) once the consistent review pattern for each slide.
blood film has been reviewed to ensure one hasn’t missed a
low-level change that the clinician may be looking for and The blood film should be initially scanned quickly at low
may have pertinence to the case, although not in significant power (10ϫ). This gives a sense of whether there is a
quantity (eg, if clinical suspicion is for malaria, it would be significant macroscopic abnormality (ie, very high/low cell
necessary to go back and review the slide under higher oil counts, circulating malignant cells, problems with arrange-
magnification for a longer period to completely exclude ment of red blood cells, dual population of red blood cells,
low-level involvement and to review another slide stained platelet clumping, fibrin strands, and, rarely, circulating
with Giemsa stain). large parasites such as filariasis).

Identifying information on the slide The thick end of the blood film (Figure 3), where the
initial drop of blood was planted and then pushed to make
The slide must have identifying information on it. This the blood film, is too thick to be of morphologic use. Red
should include the patient’s name, the date the CBC sample blood cells appear clumped and overlapped and white blood
cells are contracted and dark. At the opposite end of the
slide, the thin edge (or tail) of the blood film (Figure 4),
where the end of the pushed blood finishes, demonstrates a
concentration of white blood cells, many of which may be
degenerated. The red blood cells in this area form chains
and aggregates, lack a central pallor, and have the appear-
ance of spherocytes. Although red blood cell morphology is
not assessable in this thin area, the area can be used to look
for rare circulating malignant cells, platelet clumps, large
platelets, and circulating large parasites.

The assessor should slowly move from the thin edge for
the blood film toward the thicker area until an area is found
where the red blood cells are just close but not touching
each other and the red blood cells should have a central

34 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Figure 3 Thick end of the blood film (20ϫ dry). This is the thick Figure 5 Assessable area of the blood film (100ϫ oil). In this
part of the blood film where the initial drop of blood is placed and area, the red blood cells are just separated from each other and
then pushed to form a single layer of cells for the blood film. In this have a central pallor. This is the main area of blood film assess-
area, the red blood cells are clumped and the white blood cells ment for all lineages.
appear as small, dark bodies. This area is not assessable.
clude rouleaux (Figure 6), where red blood cells appear
pallor at this point (Figure 5). This is the area of best connected or stacked together in short or long single-file
morphologic assessment. chains (as seen in high-protein states, whether reactive
or clonal) or agglutination (Figure 7), where red blood cells
Microscopic assessment of the blood film; red are clumped in ball-like formations (as seen in cold agglu-
blood cell assessment tinin disease).

Arrangement Size
Red blood cells in this area should be close to each other Whereas the mean corpuscular volume (MCV) and/or

but not touching. Abnormalities of arrangement would in- mean corpuscular hemoglobin (MCH) reported in the he-
mocytometer analysis is mainly used as an assessor of the
size of red blood cells, the blood film can yield this infor-
mation as well. Red blood cells are roughly 7.5 ␮m in
diameter.5 The size of a mature lymphocyte nucleus is

Figure 4 Thin end of the blood film (20ϫ dry). This is the thin Figure 6 Rouleaux with background staining (100ϫ oil). This
end of the blood film (or tail) where the pushed drop of blood ends. change is seen in the usually assessable area of the blood film. The
In this area, the red blood cells form a chain-like pattern and the red blood cells are stacked upon each other in a chain-like forma-
red blood cells are sphered and lack a central pallor. This area can tion; however, one can still see the outlines of each red blood cell.
be useful for assessing for platelet clusters, malignant cells, filar- In the area surrounding the red blood cells, a purplish hue is seen.
iasis, and other protozoa. It is not useful for red blood cell assess- This represents background staining (as indicated by the arrows).
ment. Care must be taken not to assume that this area represents
true red blood cell rouleaux formation or spherocytosis of the red
blood cells. Slowly moving deeper into the slide where the red
blood cells start to separate and demonstrate central pallor is
required for proper assessment of red blood cells.

Barth Peripheral Blood Film Assessment for Pathologists 35

Figure 7 Agglutination (100ϫ oil). The red blood cells are Figure 9 Polychromasia (100ϫ oil). The dark purple cells (as
clumped together and the width and height of the agglutinate are indicated by the arrows) represent polychromatophilic red blood
roughly equal (compared with the rouleaux formation, where it is cells. These cells contain enough reticulin to be stained this color
a narrow chain of red blood cells). It is more difficult to see the by the Romanowsky stain. They are larger in size than normal red
outline of each red blood cell (compared with rouleaux formation). blood cells. An increase in polychromasia on a blood film repre-
sents reticulocytosis; however, reticulocytes may not have enough
generally consistent with the size of a normal red blood cell. reticulin in each cell to give them the bluish color on Romanowsky
Larger cells can be considered macrocytic and smaller cells stain and the lack of polychromasia does not rule out reticulocy-
microcytic. The hemocytometer indexes are more accurate tosis. A reticulocyte count may be required, especially in the
in general. However, occasionally, a small population of investigation of anemia. Polychromasia, if significant, can increase
macrocytic or microcytic cells can be identified morpholog- the MCV of a sample because the cells are larger in size.
ically that may signify an early disease process that may not
be recognized by the MCV/MCH because these indexes are Chromaisia
based on averages of the larger population of red blood Chromaisia represents the amount of central pallor in the
cells. A dual population should be checked for (Figure 8).
This may represent 2 populations of red blood cells, with 1 red blood cells. The mean corpuscular hemoglobin concen-
generally being hypochromic and/or microcytic and the tration (MCHC) is the related red blood cell index; however,
other being normochromic and/or normocytic/macrocytic. the correlation may not be exact. Normal red blood cells
This could be seen in partially treated/transfused iron defi- have a central pallor approximately 1/3 to 1/2 of the red
ciency, sideroblastic anemias, and combined B12/iron defi- blood cell diameter. Hypochromasia (may have low
ciency. MCHC) represents increased central pallor, which could be
seen in iron-deficient and thalassemic states. The border
between the stained area of the red blood cell and the central
pallor area should demonstrate a gradual progression. If a
very sharp demarcation is seen, this usually represents a
“punched out” artifact that can be seen in hydration artifact
(Figure 2). Hyperchromasia (may have high MCHC) can be
seen in cells that lack central pallor such as spherocytosis,
irregularly contracted red blood cells, or some macrocytes.

Figure 8 Dual population (40ϫ dry). This blood film demon- Color
strates a mixture of normocytic, normochromic red blood cells Because of the eosin component of the Romanowsky
admixed with hypochromic, microcytic red blood cells. This fea-
ture can be missed if the slide is not scanned at low power initially. stains, normal red blood cells appear pink in color. Poly-
chromasia (Figure 9) is the bluish/purplish/grayish color of
red blood cells that contain an increased amount of reticulin.
They represent reticulocytes. Although a certain amount of
reticulin is required to demonstrate polychromasia on Ro-
manowsky stains, reticulocytes with less reticulin will not
demonstrate polychromasia. Therefore, a reticulocyte count
performed either by flow cytometric or by manual staining
technique is necessary to accurately quantitate reticulocytes.

36 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Figure 10 Spherocytes (100ϫ oil). Spherocytes (as indicated by Figure 12 Oxidative process (100ϫ oil). Irregularly contracted
the arrows) are smaller red blood cells that lack central pallor and red blood cells (as indicated by the solid arrows) are smaller red
are completely round in shape. (Color version of figure is available blood cells that lack central pallor. Unlike spherocytes, they are
online at www.semdiagpath.com.) irregular in shape and not spheres. Blister cells (as indicated by the
dotted arrows) are seen. They demonstrate a thin rim of red cell
Shape membrane over an area of pallor at the edge of the red blood cell.
Red blood cells are between a round and an oval shape. These 2 forms are both part of the same process. (Color version of
figure is available online at www.semdiagpath.com.)
Many variations in shape can occur and have clinical rele-
vance to underlying medical processes. Because different Smaller red blood cells lacking a central pallor
morphologic cell types can have overlapping features,
sometimes making distinction between different cell types Spherocytes (Figure 10) are smaller red blood cells that
difficult, the presentation below arranges red blood cell lack a central pallor; however, they are completely round
morphologic variants by their common overlapping features and smooth in shape. Cells overlying each other can arti-
and then demonstrates the differentiating features. Exam- factually look like spherocytes. Spherocytes can be seen in
ples of various shapes and a few (but not exhaustive) related hereditary spherocytosis, autoimmune hemolytic anemia,
clinical diagnoses include the following. and acute hemolytic transfusion reaction. Microspherocytes,
which are even smaller spherocytes, can be seen in Clos-
Figure 11 Fragments (100ϫ oil). Fragments (as indicated by the tridium perfringens infection and in thermal injury (eg,
solid arrows) are smaller red blood cells that lack central pallor. burns).
They are jagged in shape and appear as a piece of red blood cells.
Note how an occasional spherocyte is seen (indicated by the dotted Fragments (Figure 11) are smaller red blood cells that
arrows) and a rare, irregularly contracted RBC (indicated by the lack a central pallor; however, they have sharp angulated
thick arrow) is seen. In a microangiopathic (fragmenting) process, edges and may have variable sizes. Red blood cell frag-
occasional spherocytes and irregularly contracted cells are seen; ments have received specific names because of their shape
however, this does not represent 2 or 3 separate processes because (eg, comet cell, triangular cell, helmet cell, and schisto-
the fragments are in the majority. (Color version of figure is cytes); however, they represent the same cell type and
available online at www.semdiagpath.com.) generally the same clinical significance. They can be seen in
TTP, disseminated intravascular coagulopathy (DIC), he-
molytic– uremic syndrome (HUS), malignant hypertension,
vasculitis, and hemolysis, elevated liver enzymes, low
platelet count (HELLP) syndrome.

Irregularly contracted cells (Figure 12) are smaller red
blood cells that lack a central pallor; however, unlike the
smooth round shape of spherocytes, they have irregular
edges and sometimes a darker color.

Blister cells are smaller red blood cells that lack a central
pallor. The blister appears as a vacuole at the red blood cell
surface. A thin rim of cytoplasm appears to enclose this
vacuole.

Bite cells are smaller red blood cells that lack a central
pallor and appear to have an oval bite taken out of the red
blood cell. Some cells have more than 1 bite per cell. Bite,

Barth Peripheral Blood Film Assessment for Pathologists 37

blister, and irregularly contracted red blood cells can be Figure 14 Echinocytes (100ϫ oil). Also called burr cells. These
seen in oxidative hemolysis (eg, G6PD deficiency) and cells have a central pallor and small bumpy projections equally
unstable hemoglobinopathies. distributed around the periphery of the red blood cell. (Color
version of figure is available online at www.semdiagpath.com.)
In the processes that cause the above red blood cell
morphologic changes, more than 1 red blood cell type can around the surface of the red blood cell. They are most
be seen. In the process of red cell fragmentation, occasional commonly seen in recently transfused red blood cells.
irregularly contracted red blood cells, bite cells, or sphero- Elongated cells
cytes can be seen along with the predominant population of
fragments. In these cases, the red blood cell morphologic Eliptocytes are elongated cells whose length is greater
change that is in the majority should be used to make the than 2ϫ its width (Figure 15).6 Sometimes these cells are
diagnosis (eg, microangiopathic hemolytic anemia). referred to as pencil cells. They have rounded edges. They
can be seen in hereditary eliptocytosis, hemoglobinopathies,
Spiculated red blood cells and iron deficiency.

Acanthoctes (spur cells) Ovalocytes are elongated cells whose length is less than
These cells do not have central pallor and are smaller 2ϫ its width.6 These cells have rounded edges and can be
seen in megaloblastic states.
than the normocytic red blood cell. They have long, thin
projections, some with bulbous or pointy edges, which are Teardrop cells are elongated cells that have 1 end tapered
distributed unevenly around the surface of the red blood and ending in a blunt tip. These cells can be seen in bone
cell. Acanthocytes (Figure 13) can be seen in microcytic marrow infiltration, fibrotic bone marrow, medication ef-
disorders such as iron deficiency and hemoglobinopathies fect, megaloblastic states, and postchemotherapy.
and in normocytic/macrocytic disorders such as liver dis-
ease and hyposplenic states.

Echinocytes (burr cells)
These cells do have central pallor and a diffuse, even

distribution of short bumpy projections with rounded edges
around the surface of the red blood cell (Figure 14). They
resemble a bottle cap viewed from above. They can be seen
in renal disease, hypophosphatemia, and blood film artifact.

Spiculated spherocytes
These cells lack a central pallor and look like sphero-

cytes with medium-size, thin projections evenly distributed

Figure 13 Acanthocytes (100ϫ oil). Also called spur cells. Figure 15 Eliptocytes (100ϫ oil). These cells have a length that
Acanthocytes are smaller red blood cells that lack a central pallor is greater than 2ϫ its width. (Color version of figure is available
and have long extensions that are spaced irregularly around the online at www.semdiagpath.com.)
circumference of the red cell, occasionally ending in a bulbous
formation. (Color version of figure is available online at
www.semdiagpath.com.)

38 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Figure 16 Sickle cells (100ϫ oil). These cells are elongated and Figure 18 Hyposplenic changes (100ϫ oil). The solid arrow is
have sharp, pointed ends (as indicated by the solid arrow) com- demonstrating a Howell–Jolly body, which is a single, round,
pared with the rounded ends of the eliptocytes in Figure 15. Note smooth, dark blue inclusion body seen in this red blood cell. It
the occasional target cell (as indicated by the dotted arrow) and represents DNA. The dotted arrow indicates Paphenheimer bodies,
Howell–Jolly body (as indicated by the thick arrow). The target which are multiple, jagged and irregular, light blue inclusion
cell and Howell–Jolly bodies represent the hyposplenic state bodies seen in this red blood cell. It represents iron deposition.
caused by autoinfarction of the spleen in many sickle cell patients. Both these inclusion bodies can be seen in hyposplenic states. Note
The presence of sickle cells in the blood film means this person has the target cells and acanthocytes seen in the background as part of
a sickling disorder because sickle cells would not be seen in the the hyposplenic changes. (Color version of figure is available
blood film of a person with sickle cell trait. (Color version of figure online at www.semdiagpath.com.)
is available online at www.semdiagpath.com.)
outside hemoglobinated ring, creating a target-like appear-
Classic sickle cells are elongated cells with tapered, ance. These cells can be seen in microcytic processes of iron
sharp tips (Figure 16). Variant sickle forms such as oat and deficiency and hemoglobinopathy and in normocytic/mac-
boat cell forms may have more rhomboid shapes and appear rocytic processes of liver disease and biliary obstruction.
to have “chopped off” edges.
Other shapes Inclusion bodies

Target cells (Figure 17) have a central area of hemoglo- Inclusion bodies inside red blood cells can be composed
bin surrounded by a clear area and then are encircled by an of various materials, each with diagnostic specificity. Some
examples of inclusion bodies, their content (in brackets),
Figure 17 Target cells (100ϫ oil). These cells have a rim of and related diagnoses include the following:
hemoglobin with a central area of hemoglobin seen within the area
of central pallor, giving them the appearance of a target. (Color Howell–Jolly bodies (DNA)
version of figure is available online at www.semdiagpath.com.) Howell–Jolly bodies are single, round, smooth purplish-

blue inclusion bodies (Figure 18). They are not refractile of
light when adjusting focus and are seen in hyposplenic
states.

Papenheimer bodies (iron)
Papenheimer bodies are generally multiple but seen in

focal clusters, are irregular in shape and light blue, and
generally are at the periphery of the red cell (Figure 18).
These are seen in sideroblastic anemia and hyposplenic
states.

Basophilic stippling (RNA)
Basophilic stippling are bluish dots diffusely spread

throughout the red blood cell (Figure 19). They may be faint
and require focusing up and down to see them. They are
seen in thalassemic states, hemolytic processes, and dyspla-

Barth Peripheral Blood Film Assessment for Pathologists 39

Figure 19 Basophilic stippling (100ϫ oil). Basophilic stippling cases in assessing clinical relevance. For example, observing
is indicated by the arrows. The stippling is fine and seen diffusely and reporting 1 or 2 target cells on the whole blood film may
dispersed across the red blood cell. One of the cells at the top of not have any clinical relevance and, if constantly reported in
this field demonstrates a much lighter stippling; however, under many blood film reports, may lessen the sensitivity of the
the microscope, focusing up and down on this cell would bring the consumer of the blood film to this feature when clinically
stippling into much stronger view, like the stippled cell in the relevant. There are a number of different grading systems in
middle of the field. (Color version of figure is available online at the literature using various methods to quantify and relate the
www.semdiagpath.com.) degree of red cell morphologic changes. Some use a 4-point
grading scale from 1ϩ to 4ϩ that is tied to a percentage of red
sia. More coarse basophilic stippling has been described in cell abnormality. The clinical utility of reporting a grade of
lead poisoning. morphologic changes is less clear. International Society for
Laboratory Hematology guidelines suggest changes greater
Nucleated red blood cells: Nuclei (DNA) than or equal to 2ϩ are clinically relevant; however, these are
Nucleated red blood cells appear with larger, round, related to automated hemocytometer flags.7 Some pathologists
use greater than 5% as a clinically significant general cutoff for
pyknotic, dark purplish nuclei. They can have more imma- red cell abnormalities. It should be clearly understood that in
ture nuclei or dysplastic morphology. They are seen in some cases a much lower percentage of red cell changes can be
hemolysis, blood loss, and marrow infiltration. critical and clinically relevant (eg, in patients with thrombocy-
topenia, fragments of 1% to 2% can signify a microangiopathic
Infectious agents hemolytic process such as TTP) and that early recognition of
Malaria may appear in different stages of maturation and this process can be life saving in many cases. The percentage
of parasites seen (eg, malaria) should be reported at any level.
different species will have varying morphology. The red Familiarization with an accepted grading system is recom-
blood cell inclusion in Plasmodium falciparum (Figure 20) mended.
may demonstrate thin, bluish rings with 1 or 2 small, red
nuclei. Babesiosis can also be seen in red blood cells. White blood cell assessment

Substances appearing as red blood cell inclusions When discussing white blood cells, 5 subtypes are in-
Stain deposit or debris sometimes mimics these inclusions; volved: neutrophils, lymphocytes, monocytes, eosinophils,
and basophils. These subtypes are listed in order of fre-
however, they are not usually smooth and round and tend to quency seen in adults. In children, lymphocytes predomi-
refract light on adjustment of focus with respect to Howell– nate from after the first week of life until age 5 to 6.8
Jolly bodies and are darker than Papenheimer bodies. Platelets
overlying a red blood cell can be mistaken for infectious Figure 20 Falciparum malaria (100ϫ oil). Single and multiple
substances, but the granulation of the platelet should distin- falciparum malaria inclusions are seen within many red blood
guish this. Hydration artifact (Figure 2) can form a thin, dark cells. These parasites have a single or double red nucleus and a thin
ring in the middle of a red blood cell, which could mimic blue ring. The percentage of parasites should be reported and the
malaria; however, no nucleus is seen and the ring is dark rather percentage of involvement on the peripheral blood film may
than light blue. change the clinical management of the patient. This degree of
involvement seen on this slide is at critical levels and requires
Grading red blood cell morphologic abnormalities urgent intervention. (Color version of figure is available online at
www.semdiagpath.com.)
Assessing the degree of blood film involvement by a spe-
cific red blood cell morphologic change is important in some

40 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Figure 21 Pelgerized neutrophil (100ϫ oil). The neutrophil on Figure 23 Toxic changes (100ϫ oil). The neutrophils on this
the left (indicated by the solid arrow) is hypolobulated and has a film demonstrate increased dark red––purple granulation, consis-
mononuclear form. This is considered pelgerized. The neutrophil tent with toxic granulation. A blue dohle body in the cytoplasm at
on the right (indicated by the dotted arrow) is normal. (Color the border of the cytoplasm is indicated by the arrow. A third
version of figure is available online at www.semdiagpath.com.) component of toxic changes is not seen on this film, toxic vacuo-
lation, which are vacuoles in the cytoplasm of the neutrophil.
Neutrophils (Color version of figure is available online at www.semdiagpath.
Arrangement com.)

Neutrophils occasionally are seen in aggregates in blood cytes, which can be observed in normal individuals, mega-
samples that are older prior to having blood films being loblastic anemia, and medication effect.
made. This can also be seen in infectious process and
immune disorders. It is not generally clinically significant. Nuclei
Size Neutrophils have multilobed nuclei connected by thin chro-

Neutrophils are approximately 3 to 4 times the size of matin bridges. The chromatin pattern is clumped and normally
normal red blood cells and are 12 to 15 ␮m in diameter.9 there are 3 to 5 nuclear lobes. Two lobes or less are considered
Generally, they do not vary in size. Occasionally, large hypolobulated or pelgerized (Figure 21), which can be seen in
hypersegmented neutrophils are seen called polymacro- myelodysplastic disorders or congential disorders. Six or more
lobes or 3% to 5% of neutrophils with 5 lobes or more are
Figure 22 Megaloblastic changes (100ϫ oil). A hyperseg- considered hyperlobulated10 (Figure 22), which can be seen in
mented neutrophil with greater than 6 nuclear segments is seen, B12/folate deficiency, myelodysplastic disorder, medication
surrounded by macrocytic red blood cells that have oval forms effect, and occasionally in reactive neutrophilia.
(oval macrocytes). (Color version of figure is available online at
www.semdiagpath.com.) Cytoplasm
The cytoplasm of neutrophils is pinkish with reddish gran-

ules. Increased dark red and purple granules, along with vac-
uolization and dohle bodies, can be seen in toxic changes
(Figure 23) in cases of infection, inflammation, trauma, and
G-CSF administration. Dohle bodies without the other toxic
changes can be seen in hereditary platelet disorders and preg-
nancy. Abnormal large granulation can be seen in hereditary
disorders such as Chediak–Higashi syndrome. Hypogranula-
tion (Figure 24) is the absence of granulation, and in fact the
cytoplasm has the same color as the background between cells.
This can be seen in myelodysplastic disorders. It is important
to not mistake light staining of neutrophils for hypogranula-
tion. In some cases the granules of lightly stained neutrophils
will not be seen but the cytoplasm will have a pinkish color.
Some neutrophils will have a corner of hypogranular cyto-
plasm on a background of normally granulated cytoplasm,
which is normal.

Barth Peripheral Blood Film Assessment for Pathologists 41

malignant lymphocytes because they can be seen in reactive
cases. Nuclear clefting is unusual in normal lymphocytes
and should arouse suspicion for lymphoproliferative disor-
ders; however, smokers may have cleft or bilobed lympho-
cytes. Complex nuclear folding or nuclear lobation should
arouse suspicion for lymphoproliferative disorders. Nuclei
are generally found centrally in lymphocytes. Eccentric
positioning of nuclei as in plasmacytoid lymphocytes may
be seen in high-protein states and reactive lymphocytes.

Figure 24 Dysplastic neutrophil (100ϫ oil). This neutrophil Cytoplasm
demonstrates 2 changes. Its cytoplasm is nearly absent of granules Mature lymphocytes generally have scant, pale, purple–
and has almost the same color and appearance as the background.
This is a hypogranular neutrophil. As well, this neutrophil is blue cytoplasm and have a high nuclear:cytoplasmic (N:C)
hypolobated with 2 lobes and considered pelgerized in formation. ratio. Reactive lymphocytes may have a slightly lower N:C
(Color version of figure is available online at www.semdiagpath. ratio and a darker blue cytoplasm. These lymphocytes can be
com.) seen in infections and inflammatory states. Atpical lympho-
cytes generally have a low N:C ratio with a spread out, dark
Inclusion bodies blue cytoplasm. These generally represent viral infections,
Aside from the inclusion bodies such as dohle bodies and classically infectious mononucleosis (Epstein–Barr), or cyto-
megalovirus and rarely acute seroconversion of human immu-
increased granulation in Chediak–Higashi syndrome listed nodeficiency virus. Occasionally, atypical lymphocytes (Fig-
above, infectious inclusion bodies can be seen in neutro- ure 26) have a spread out cytoplasm that invaginates around
phils. This could include bacteria and fungal elements. adjacent red blood cells; however, this phenomenon can be
seen occasionally in lymphoproliferative disorders as well.
Left shift Large granular lymphocytes, both reactive and clonal, have a
Immature forms of neutrophils can be seen in the peripheral pale gray cytoplasm with low N:C ratios, with scattered red-
dish fine granulation. Hair-like projections may be seen in
blood. Starting from segmented neutrophils and progressively malignant lymphocytes; however, polar cytoplasmic projec-
becoming more immature are bands, metamyelocytes, myelo- tions in the same direction on the majority of lymphocytes may
cytes, and promyelocytes. Generally, a left shift is considered be an artifact feature.
when myelocyte forms or more immature forms are seen. This
may represent a reactive process such as infection or marrow Figure 25 Small, mature lymphocytes (100ϫ oil): The small
infiltration. An increase in band forms alone has been used in lymphocytes on this film (as indicated by the solid arrows) dem-
pediatric populations as an indicator for infection but does not onstrate round nuclei with clumped chromatin and a high nuclear-
seem to hold for adult patients. to-cytoplasmic ratio with bluish, agranular cytoplasm. The nucleus
of these cells can be used roughly to assess size of the red blood
Lymphocytes cells (ie, same-size red blood cells ϭ normocytic; smaller-size red
blood cells ϭ macrocytic; larger-size red blood cells ϭ macro-
Size cytic). A smudge cell is seen in the lower left corner as indicated
Lymphocytes are approximately 1 to 1.5 times the size of by the dotted arrow. (Color version of figure is available online at
www.semdiagpath.com.)
normal red blood cells, 10 to 12 ␮m. Larger lymphocytes
(12 to 16 ␮m) can be seen in atypical or reactive lympho-
cytosis or large granular lymphocytosis, as described be-
low.11

Nuclei
Lymphocytes are mononuclear. Mature lymphocytes

have a round nucleus with clumped chromatin and generally
have a uniform shape (Figure 25). Lymphocyte nuclei may
have irregular contours. Atypical lymphocytes may have a
significant pleomorphism in nuclear size and shape. Nucle-
oli may be seen. Nucleoli alone are not pathognomic for

42 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

considered promonocytes (Figure 27), an immature precur-
sor of monocytes, which are counted as blasts in monocytic
leukemias.

Cytoplasm
The monocyte cytoplasm is grayish-purple and spread

out. A low N:C ratio is seen. Cytoplasmic vacuoles are
noted with occasional fine reddish granules. Inclusions may
be seen such as infectious agents, vacuoles in inherited
metabolic disorders, and occasionally erythrocytes.

Eosinophils

Figure 26 Atypical lymphocyte (100ϫ oil). This lymphocyte is Size
larger than a normal, small, mature lymphocyte. It has an irregular Eosinophils are approximately 3 to 4 times the size of
larger nucleus with clumped chromatin and an increase in dark
blue, spread out cytoplasm (a lower nuclear-to-cytoplasmic ratio). normal red blood cells, 12 to 16 ␮m.13 They generally do
Atypical lymphocytes have varying morphology and are not as not have significant size variation.
monomorphic as lymphoma cells. (Color version of figure is
available online at www.semdiagpath.com.) Nuclei
Eosinophils normally have 2 to 3 nuclear lobes. The
Smudge cells
Smudge cells represent disintegrated cells (Figure 25). nuclei demonstrate clumped chromatin connected by chro-
matin bridges. Hyposegmented eosinophils have 1 lobe and
Although these may represent lymphocytes, other cells may can be seen in hereditary, dysplastic, and myeloproliferative
smudge as well. Smudge cells can be seen in small numbers disorders. Hypersegmented eosinophils have 4 or more
on normal blood films. Lymphoproliferative and leukemic lobes. This feature can be seen in both reactive and malig-
disorders and occasionally reactive processes may have nant eosinophils and does not distinguish between the 2
increased numbers of smudge cells as well, but should have processes. Occasionally, ring nuclear forms can be seen in
other features suggestive of these processes. both reactive and malignant eosinophilic disorders.

Inclusion bodies Cytoplasm
Immunoglobulin deposits with the appearance of a bunch Eosinophil cytoplasm contains large, chunky, orange

of grapes (called Mott cells) may be seen in the cytoplasm granules. Eosinophils may degranulate in the peripheral
of plasmacytoid lymphocytes in high-protein states. Large
inclusion bodies can be seen in the cytoplasm of lympho-
cytes in hereditary disorders such as Chediak–Higashi syn-
drome. Vacuolization of the cytoplasm could be seen in
inherited metabolic disorders.

Monocytes

Size Figure 27 Abnormal monocytes (100ϫ): This slide demonstrates
Monocytes are approximately 4 to 5 times the size of promoncytes (as indicated by the solid arrows), which are mononu-
clear cells with chromatin that is more open than a mature monocyte,
normal red blood cells, 12 to 20 ␮m.12 They generally do but slightly more clumped than a blast and may have prominent
not have significant size variation. nucleoli, or in this case folded or convoluted nuclei. The dotted line
represents a monoblast with more open chromatin and a higher
Nuclei nuclear cytoplasmic ratio. The cell with pinker cytoplasm and multi-
Monocytes are mononuclear. However, their nuclear ple dark separate nuclei is a pyknotic neutrophil. (Color version of
figure is available online at www.semdiagpath.com.)
shape is generally a horseshoe-like, thick band. The chro-
matin is clumped. In some cases the nucleus folds in on
itself, giving the appearance of a mononuclear shape. Dys-
plastic monocytes may have abnormal nuclear segmentation
or lobation. Monocytes with more open chromatin, round/
oval, folded or convoluted nuclei, and nucleoli should be

Barth Peripheral Blood Film Assessment for Pathologists 43

blood. This feature can be seen in both reactive and malig- Figure 29 Platelet satelitism (100ϫ oil). Platelets are clustered
nant eosinophils and does not distinguish between the 2 around the neutrophils, giving the same clinical effect as platelet
processes. Purplish/orange granulation can be seen in eo- clustering (Figure 28). Neutrophils may be seen clustered together
sinophils as part of acute myeloid leukemia associated with as well in cases of platelet satelitism. (Color version of figure is
inversion/translocation of chromosome 16 and in chronic available online at www.semdiagpath.com.)
myelogenous leukemia. Abnormal inclusion bodies can be
seen in hereditary disorders such as Chediak–Higashi syn- blood samples taken in K2EDTA. Platelet clustering can
drome. lead to false low platelet counts; however, most automated
hemocytometers are able to detect this. A repeat blood
Basophils sample in a citrated blood collection tube should resolve
platelet clumping. Platelets may also be seen ringing neu-
Size trophils, known as platelet satellitism (Figure 29). This
Basophils are approximately 3 to 4 times the size of event is caused by an in vitro reaction seen in blood samples
taken in K2EDTA and can be resolved in the same fashion.
normal red blood cells, 10 to 14 ␮m.14 They generally do
not have significant size variation. Size
Platelets are usually about 1/7 the size of a normal red blood
Nuclei
Basophils normally have 2 to 3 nuclear lobes. The nuclei cell and approximately 1 to 3 ␮m in size.15 Platelets approx-
imately greater than half the size of a normal red blood cell are
demonstrate clumped chromatin connected by chromatin called large platelets. Giant platelets are considered the same
bridges and are commonly obscured by the heavy basophil size or larger than the size of a normal red blood cell. Large
granulation. and giant platelets can be seen in peripheral platelet destruction
processes, bone marrow infiltrative processes, myelodysplastic
Cytoplasm and myeloproliferative processes, and certain hereditary plate-
Basophil cytoplasm contains large, dark purple/black let disorders. Given the wide range of causes of large and giant
platelets, this feature alone is not very useful in making a
granules. Basophils may degranulate in the peripheral diagnosis without other clinical, laboratory, and morphologic
blood. This feature can be seen in both reactive (eg, allergic features. Normal-sized platelets in a situation of thrombocyto-
reactions) and malignant basophils and does not distinguish penia may suggest platelet production abnormality but further
between the 2 processes. Degranulating basophils can objective assessment is required to confirm this. Small platelets
sometimes be mistaken for neutrophils with toxic granula- may be seen in rare hereditary disorders such as Wiskott–
tion. Aldrich syndrome.

Platelet assessment

Arrangement
Platelets are normally seen individually scattered about

the blood film. Platelets can occasionally be seen in clusters
(Figure 28). This is generally an in vitro reaction seen in

Figure 28 Platelet clustering (100ϫ oil). A large cluster of Granulation
platelets are seen together. (Color version of figure is available Platelets have a purplish–reddish granulation. Hy-
online at www.semdiagpath.com.)
pogranular platelets (Figure 30) can be seen in myelodys-
plastic and myeloproliferative processes and certain hered-
itary platelet disorders. It can also be seen in traumatic
blood draws and in activated platelets.16

44 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Figure 30 Various platelet sizes and morphologies (100ϫ oil). Figure 32 A myeloblast with an Auer rod (100ϫ oil). This blast
The solid arrow indicates a giant hypogranular platelet. The dotted demonstrates a mononuclear nucleus with open chromatin pattern,
arrow indicates a giant granular platelet. Giant platelets are larger than an inconspicuous nucleolus, and an Auer rod (indicated by the
the size of a normal red blood cell. The small multiple arrows together arrow). The presence of the Auer rod indicates that this blast is
indicate a large platelet, which is greater than half the size of a normal malignant and of myeloid etiology. (Color version of figure is
red blood cell, and the thick arrow indicates a normal-size platelet. (Color available online at www.semdiagpath.com.)
version of figure is available online at www.semdiagpath.com.)
have detected other cells present. If not, a quick scan at the
Bare megakaryocyte nuclei end of reviewing the slide is suggested to exclude other
Bare megakaryocyte nuclei cells are only rarely seen in cells.

the peripheral blood (Figure 31). They are large with a Blasts
purplish, lobated nucleus with condensed chromatin and no Blasts are immature cells with open nuclear chromatin.
cytoplasm. They can be seen in reactive processes and
myeloproliferative disorders and should not be mistaken for They may vary in size from the size of small mature
abnormal malignant cells. lymphocytes to large cells. Although many blasts have a
Other cells and features high N:C ratio, some blasts will have a low or variable N:C
ratio. Nuclei may be irregular in shape, with clefting or
At this point, you will have reviewed the features of the lobation. Nucleoli may or may not be seen. Other features
red and white blood cells and platelets. An initial scan may could include cytoplasmic granulation or vacuolization and
Auer rods. Blasts are best seen on the lateral edges of the
Figure 31 Bare megakaryocyte nucleus (100ϫ oil). A bare blood film. Blasts greater than 20% of all nucleated blood
megakaryocyte nucleus is rarely seen in the peripheral blood. It cells on the peripheral blood film are consistent with acute
should not be mistaken for a circulating tumor cell because it lacks leukemia. Blast percentages less than 20% could still rep-
cytoplasm and has the nuclear features of a megakaryocyte, with resent acute leukemia (when confirmed with a bone mar-
dark purple and convoluted nucleus. (Color version of figure is row), myelodysplastic syndromes, marrow infiltrative, or
available online at www.semdiagpath.com.) reactive processes (generally seen as part of left-shifted
neutrophils). When Auer rods are seen in blasts, they always
signify that the blast is of malignant etiology and represent
a myeloid phenotype (Figure 32).

Plasma cells
Plasma cells are generally 1.5 to 2 times the size of a

normal red blood cell, but can sometimes be much larger, 8
to 20 ␮m.17 They are mononuclear with clumped chromatin
and have variable N:C ratios and eccentric nuclei with
bluish cytoplasm. A paranuclear hof can be seen. Plasma
cells in small numbers can be seen in the peripheral blood in
reactive/inflammatory processes; however, they can be seen
in plasma cell dyscrasias and lymphoproliferative disorders
as well. Malignant plasma cells may have a centralized
nucleus that can sometimes confuse morphologic assess-

Barth Peripheral Blood Film Assessment for Pathologists 45

Figure 33 Circulating plasma cells (100ϫ oil). Multiple plasma Figure 35 Microcytosis with hypochromasia (100ϫ oil). Hypo-
cells are seen. They have round, eccentrically placed nuclei with chromic and microcytic red blood cells are seen with occasional
clumped chromatin and blue cytoplasm. One of the plasma cells target cells. The small, mature lymphocyte can be used to size the
has a more central nucleus, which can be seen as a malignant red blood cells. Many of the red blood cells have increased central
feature. Note the rouleaux formation in the red blood cells. (Color pallor (hypochromia). (Color version of figure is available online
version of figure is available online at www.semdiagpath.com.) at www.semdiagpath.com.)

ment. Malignant plasma cells may also have nuclear bud- Background staining
ding, prominent nucleolus, elongated nuclear shape, and A purplish haze surrounding red blood cells can be seen
multiple nuclei. Clonal plasma cells Ͼ20% in the peripheral
blood are consistent with plasma cell leukemia (Figure 33). in high-protein states along with red blood cell rouleaux.
Mast cells This may represent clonal and nonclonal states such as
myeloma or inflammatory states.
Mast cells are generally 20 to 30 ␮m in size.18 They have
dark purple granules that, unlike basophils, do not obscure the Cryoglobulins
nucleus. The nucleus is round– oval. Mast cells are uncommon Cryoglobulins (Figure 34) are abnormal proteins that
in the peripheral blood. Malignant mast cells may have oblong
football-shape or lobulated nuclei and may degranulate. appear as light purplish clouds between red blood cells.
Occasionally, they may overlay a red blood cell, giving it
Figure 34 Cryoglobulinemia (100ϫ oil). Cloud-like pale purple the appearance of a bite or blister cell. In some cases the
cryoglobulin deposits are seen in between and overlaying the red light purplish clouds are very difficult to see and their
blood cells. When the cryoglobulins overlay the red blood cells, overlay on top of red blood cells may be the only morpho-
they can give the false appearance of irregularly contracted and logic feature.
bite cells, especially if the cryoglobulins are small and not darkly
stained. (Color version of figure is available online at www. Infection
semdiagpath.com.) Various stages of malaria may only be seen outside of

red blood cells (gametocyte form). Bacteria, fungi, proto-
zoa, and microfilaria may be seen extracellularly.

In some cases, multiple morphologic features can be
combined for a unifying diagnosis. Below is a limited list of
some of these cases:

Howell–Jolly bodies, acanthocytes, and target cells:
hyposplenic states (Figure 18)

These can be associated as well with occasional sphero-
cytes, neutrophilia, lymphocytosis, and thrombocytosis.

Microcytosis, hypochromasia, target, teardrop, and
pencil cells: acanthocytes and rare fragments: iron
deficiency and/or hemoglobinopathies (Figure 35)

Whereas thalassemic states may have basophilic stip-
pling, not all cases do. Thrombocytosis may be seen in iron
deficiency.

46 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

Target cells and acanthocytes
This can be seen in liver disease; however, in microcytic

cases, it can represent iron deficiency and or hemoglobi-
nopathy states. Thrombocytopenia and macrocytosis may
be seen in liver disease.

Mild–moderate thrombocytopenia with large platelets
and neutrophils with dohle bodies and no other toxic
changes

This includes hereditary thrombocytopenic disorders
such as May–Hegglin anomaly.

Future technologies

Figure 36 Leukoerythroblastic picture (100ϫ oil). A nucleated Whereas dependence on a microscope to review blood films
red blood cell is indicated by the solid arrow. The dotted arrow has been the historical precedent, newer technologies are avail-
indicates the myelocytes and the thick arrow indicates the promy- able that will change this practice in the near future. Telepa-
elocytes (as part of the left-shifted granulopoiesis). (Color version thology, the scanning of a pathology slide, and digitizing the
of figure is available online at www.semdiagpath.com.) image are currently available and constantly improving in
quality, speed, and price. With good oil scanning techniques, a
Nucleated red blood cells and left-shifted neutrophils reliable digital image of a blood film can be made. This image
(myelocytes and more immature forms): can be viewed remotely from the site of the scan on a computer
Leukoerythroblastic picture (Figure 36) screen. The operator is able to move to different areas on the
slide and magnify these areas with magnifications from 4ϫ to
This may represent a reactive picture in extreme blood 100ϫ oil, just as they would a physical microscope. This type
loss, hemolysis, trauma, or infection or could represent of technology allows for blood films to be quickly reviewed
marrow infiltration. When combined with teardrop cells, from remote sites (eg, in areas where there is no pathologist,
this may represent a myelophistic picture of marrow infil- for a clinician at the bedside, for a pathologist offsite, or to get
tration or fibrosis. a quick second opinion on a case from other pathologists
without having to send a slide physically). This will only
Neutrophilia with left shift, eosinophilia, or improve the quality and efficiency of the reporting pathologist
basophilia, with thrombocytosis and improve turnaround times, especially for remote areas.
This will also revolutionize teaching of a new generation of
This may represent a myeloproliferative neoplasm, spe- pathologists who will not be tied to physical slide sets for
cifically chronic myelogenous leukemia. education.

Macrocytic cells with a mixture of microcytic cells Digital imaging has also been used in automated white
This may represent a combined B12/folate and iron de- blood cell differentials. These devices use computers to scan a
slide, identify the different subtypes of white blood cells, give
ficiency state. In cases of malabsorption caused by celiac a differential and then place them in groups for a technologist
disease, hyposplenic changes from functional hyposplenism to review. The technologist can review the identifications of
may be seen. the white blood cells by the computer, make changes when
cells are not accurately identified, and accept the differential.
Oval macrocytosis with hypersegmented neutrophils: This technology improves accuracy of the differential, im-
Megaloblastic changes (Figure 22) proves speed and therefore turnaround time, and allows tech-
nologists to send unidentified cells electronically for second
This can be seen in B12/folate deficiency, medications opinions very quickly. It is a useful learning and teaching
affecting folate metabolism, and myelodysplastic disorders. device because digital libraries of cells for comparison are
usually included in these programs.
Dual population of mainly normocytic/macrocytic red
blood cells with a subpopulation of microcytic Reporting of findings
hypochromic cells and papenheimer bodies:
sideroblastic anemia Once a blood film has been assessed morphologically, a report
is required to be issued. There are different ways to approach
This can be seen in myelodysplastic disorders, alcohol- this task. The major emphasis of issuing a report is to be able
ism, medication effect, and hereditary disorders. to impart the significant findings of the blood film in words to

Fragmentation of red blood cells with
thrombocytopenia (Figure 11): microangiopathic
hemolytic anemia with thrombocytopenia

This can be seen in TTP, HUS, DIC, HELLP syndrome,
and vasculitis.

Barth Peripheral Blood Film Assessment for Pathologists 47

Table 1 A list of common blood film abnormalities and examples of blood film reports that can be used in these cases

Microcytosis: suggest iron studies to exclude iron deficiency and hemoglobinopathy investigation to exclude hemoglobinopathies.
Dual population: a combination of microcytic and normocytic red blood cells is seen on this peripheral blood film. This may

represent partially treated iron deficiency (eg, transfused or treated with iron therapy) or a sideroblastic process. Clinical and
laboratory correlation is required.
Fragments and thrombocytopenia: red blood cell fragments are seen approximately ___% with thrombocytopenia. This is
suggestive of a microangiopathic hemolytic process with thrombocytopenia. Clinical and laboratory correlation are suggested to
exclude the following entities: DIC, TTP, HUS, HELLP, vasculitis, sepsis, malignant hypertension, disseminated malignancy, etc.
Spherocytosis: peripheral blood demonstrates spherocytosis. Suggest hemolytic investigation including reticulocyte count,
haptoglobin, bilirubin, and LD. A direct antiglobulin test should be performed to look for evidence of immune-mediated
hemolysis. Hemolytic transfusion reaction and hereditary spherocytosis should be considered.
Red blood cell agglutination: peripheral blood film demonstrates red blood cell agglutination. Suggest hemolytic investigation
including reticulocyte count, haptoglobin, bilirubin, and LD. A direct antiglobulin test should be performed to look for evidence
of immune-mediated hemolysis, along with a cold agglutinin screen to assess the clinical relevance of this finding.
Rouleaux: peripheral blood film demonstrates rouleaux. Suggest serum immunoelectrophoresis to look for a monoclonal protein.
Nonclonal high-protein states should be excluded as well (inflammation, infection).
Oxidative changes: peripheral blood film demonstrates oxidative changes. Suggest hemolytic investigation including reticulocyte
count, haptoglobin, bilirubin, and LD. Heinz body test could be performed. G6PD deficiency, medication, and toxin effects
should be considered along with rare unstable hemoglobinopathies.
Leukoerythroblastic picture: blood film demonstrates a leukoerythroblastic picture. Clinical and laboratory correlation is suggested
to look for a peripheral cause (eg, hemorrhage, hemolysis, infection, inflammation, postsurgery, trauma) versus a bone marrow
abnormality (eg, malignant infiltration, fibrosis).
Left-shifted neutrophils: blood film demonstrates left-shifted neutrophils. Clinical and laboratory correlation are suggested to look
for a peripheral cause (eg, hemorrhage, hemolysis, infection, inflammation, postsurgery, trauma) versus a bone marrow
abnormality (eg, malignant infiltration, fibrosis).
Isolated neutropenia: blood film demonstrates neutropenia. Suggest excluding infectious process, medication effect, immune
process, endocrinopathies, and bone marrow abnormalities.
Macrocytosis: suggest excluding B12/folate deficiency, medication effect, liver and thyroid disease, alcohol use, reticulocytosis,
and bone marrow abnormalities.
Isolated thrombocytopenia: isolated thrombocytopenia with no platelet clumping or red blood cell fragmentation. Suggest
excluding idiopathic thrombocytopenic purpura, medication effect, infectious process, bone marrow abnormality, peripheral
destruction, and hypersplenism.
Thrombocytopenia with platelet clumping: platelet clumping is seen. This may result in an inaccurate reporting of platelet count
(false thrombocytopenia may be reported). Suggest repeating the CBC sample in a citrated blood collection tube for accurate
platelet count reporting.
Toxic changes: peripheral blood demonstrates toxic changes. Infectious and inflammatory states should be excluded. Cytokine
administration (eg, granulocyte colony-stimulating factor) should be excluded as well.
Mild lymphocytosis with no morphologic atypia: peripheral blood demonstrates mild lymphocytosis. Suggest clinical correlation
(eg, reactive process, infection, smoking, lymphoproliferative disorder). If persistent, then suggest flow cytometry to phenotype
lymphocytes.
Significant lymphocytosis or lymphocytosis with morphologic atypia: peripheral blood demonstrates significant lymphocytosis (with
morphologic atypia). Suggest flow cytometry to phenotype lymphocytes.
Thrombocytosis: peripheral blood demonstrates thrombocytosis. Suggest clinical and laboratory correlation for reactive causes (eg,
iron deficiency, inflammatory states, postsurgery, marrow infiltration) versus primary causes (eg, myeloproliferative neoplasm).
Target cells (normocytic or macrocytic): target cells seen. Suggest excluding liver disease, biliary obstruction, and
hemoglobinopathy (eg, hemoglobin C trait).
Target cells (normocytic or macrocytic) and acanthocytes: target cells and acanthocytes seen. Suggest excluding liver disease or
biliary obstruction.
Target cells, acanthocytes, and Howell–Jolly bodies: consistent with hyposplenic changes.
Sickle cells: sickle cells are seen on this blood film. This is consistent with a sickling disorder. Suggest hemoglobinopathy
investigation to subtype.
Echinocytes: echinocytes seen. Suggest exclude renal disease, hypophosphatemia, and artifact.
Blasts greater than 20% of all nucleated cells on the blood film: blasts seen at approximately __% (morphologically describe the
blasts). This is likely consistent with acute leukemia. Suggest flow cytometry to phenotype the blasts and correlation with bone
marrow and cytogenetic studies.
Blasts less than 20% of all nucleated cells on the blood film. Blasts seen at approximately __% (morphologically describe the
blasts.) Because the blasts are less than 20% of all nucleated cells on the peripheral blood sample, this could represent
myelodysplastic syndrome, acute leukemia, marrow infiltration, or a reactive process. Suggest clinical correlation and correlation
with bone marrow examination.
Malaria parasites seen: malaria seen at approximately __%. (If possible, speciate malaria subtype or at least, if possible, suggest
falciparum vs nonfalciparum subtype. If not comfortable with this subtyping, send out sample for speciation.)

48 Seminars in Diagnostic Pathology, Vol 29, No 1, February 2012

those individuals reading the report who have not seen the approach to the various parts of the blood film assessment and
blood film themselves. The report should be clearly worded reporting so that specific areas are not missed. Last, good
and concise. It cannot be assumed that the reader of the report communication in the blood film report is essential in translat-
has knowledge of some of the clinical significances of all the ing the morphologic features seen into clinical correlation.
morphologic changes being described and, therefore, attaching Keep in mind that the blood film review is always done with a
a comment on the possible clinical significances of the mor- patient’s clinical care as the major priority. In today’s age of
phologic findings becomes extremely important. more complex and expensive laboratory investigations, timely,
accurate, clear, and concise reporting with good clinical cor-
In the event of a normal-appearing blood film, the com- relation make the blood film an inexpensive, easily accessible,
ment “blood film agrees with CBC report” could be con- quick, and powerful laboratory diagnostic test that can alone
cisely used to suggest that no additional information is sometimes make a clinical diagnosis or help in including or
gained from the blood film. excluding other entities on the diagnostic pathway.

If a specific abnormality is seen in 1 cell line, a simple References
comment on that individual abnormality may be reasonable.
1. ISLH Guideline: Suggested Criteria for Action Following Automated
Some may divide the report into red blood cells, white CBC and WBC Differential Analysis. Stokie, IL, International Society
blood cells, and platelets and comment on each of these cell for Laboratory Hematology, 2009
lines individually. This approach may be helpful to describe
abnormalities if found in multiple cell lines. 2. International Council for Standardization in Hematology 1993 Rec-
ommendations of the International Council for Standardization in
Although it is important to describe the relevant morpho- Haematology for Ethylenediaminetetraacetic Acid Anticoagulation of
logic changes seen on a blood film, a comment as to possible Blood for Blood cell Counting and Sizing. International Council for
clinical significance of these findings is suggested (eg, if Standardization in Haematology: Expert Panel on Cytometry. Am J
spherocytosis is seen, a comment such as “Spherocytosis. Clin Pathol 100:371-372, 1993
Suggest hemolytic investigations including DAT, reticulocyte
count, haptoglobin, bilirubin and LDH. Possibilities could in- 3. Bain B, Lewis M: Preparation and staining methods for blood and bone
clude autoimmune hemolytic anemia, transfusion reaction and marrow films, in Lewis SM et al (eds): Dacie and Lewis Practical
hereditary spherocytosis” could be made). Although an ex- Hematology (Ed 9). London, UK, Churchill Livingstone, 2001, pp 48-49
haustive differential of the morphologic feature is beyond the
scope of a blood film report and not generally required, expla- 4. Bain B: Morphology of blood cells, in Bain BJ (ed): Blood Cells, A
nation for those reading the report who may not understand the Practical Guide (ed 3). Oxford, UK, Blackwell Science, 2002, p 13
clinical significance of “spherocytosis” in this case allows for
clear communication of the more common clinical suspicions 5. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
that may arise morphologically and make for a useful blood American Pathologists, 1998, p 2
film report.
6. Bain B: Morphology of blood cells, in Bain BJ (ed): Blood Cells, A
In some cases, multiple morphologic changes may be Practical Guide (ed 3). Oxford, UK, Blackwell Science, 2002, p 65
seen on a single blood film. Sometimes, the set of changes
can be correlated with 1 disease process (eg, “microcytosis, 7. Padmore R: Grading Red Blood Cells. Broadsheet: Quality Manage-
hypochromasia, teardrop, pencil and target cells with rare ment Program. Laboratory Services, 2010, pp 1-122
acanthocytes are seen”). This could be reported, followed
by the comment “suggest exclude iron deficiency and/or 8. Geaghan S: Normal blood values: selected reference values for neo-
hemoglobinopathy,” or some may choose to just report the natal, pediatric and adult populations, in Hematology, Basic Principles
clinical significance comment alone, especially if a technol- and Practice (ed 4). Philadelphia, Churchill Livingstone, 2004, p 2733
ogist report on this sample has reported those changes
already. 9. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 24
Table 1 outlines some morphologic scenarios and examples
of possible reports that could be used in these situations. 10. Bain B: Morphology of blood cells, in Bain BJ (ed): Blood Cells, A
Practical Guide (ed 3). Oxford, UK, Blackwell Science, 2002, p 79
Conclusions
11. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
This article is a brief overview and practical guide to the American Pathologists, 1998, p 218-222
process available on this subject. It is important for the blood
film reviewer to have or to establish a routine, sequential 12. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 36

13. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 30

14. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 28

15. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 198

16. Bain B: Morphology of blood cells, in Bain BJ (ed): Blood Cells, A
Practical Guide (ed 3). Oxford, UK, Blackwell Science, 2002, p 112

17. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 238

18. Glassy E: Color atlas of hematology. CAP. Northfield, IL, College of
American Pathologists, 1998, p 328