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Blood Cell Identification Graded

Case History
The patient is a 28-year-old Middle Eastern male with a history of lifelong anemia and a previous
splenectomy. Laboratory data includes: WBC (corrected)=10.4 X 109/L; RBC=3.28 X 1012/L;
Hgb=9.2 g/dL; Hct=28.6%; MCV=87.3 fL; MCH=28.1 pg; MCHC=32.1 g/dL; RDW=26.2%;
PLT=714 X 109/L; NRBCs=919/100 WBCs.

BCK/BCP-11

Identification
Nucleated red cell, normal or
abnormal morphology (blood
only)

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

20

3370

1757

Good

100.0

99.6

99.3

The arrowed cell is a nucleated red cell that was correctly identified by 100.0% of referees and 99.5%
of participants. The cell is similar in size to the surrounding more mature red blood cells. The nucleus of
the cell is round, slightly eccentrically located and contains clumped chromatin without a nucleolus.
The cytoplasm is uniformly blue-grey in color, relatively similar to the surrounding erythrocytes.
Normoblasts circulating in the peripheral blood should be classified as nucleated red cells, regardless of
their stage of maturation. The presence of circulating nucleated red blood cells is unusual and most
commonly associated with moderate to severe anemia. They may also occur secondary to replacement
of the bone marrow by fibrosis or an abnormal infiltrate. Surrounding red blood cells include an
increased number of target cells.

BCK/BCP-12

Blood Cell Identification Graded

Identification

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

Lymphocyte

20

3276

1752

Good

100.0

96.8

98.7

BCK/BCP-13

The arrowed cell is a lymphocyte and was correctly identified by 100.0% of referees and 97.5% of
participants. The lymphocyte is slightly larger than the surrounding red blood cells, averaging from
7-15 m in size, and has a round to ovoid nucleus, clumped chromatin, and inconspicuous nucleus. In
contrast to the previous nucleated red blood cell, this cell has scant cytoplasm that is more basophilic
in appearance.

Identification
Howell Jolly Body (Wright
stain)

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

20

3371

1766

Good

100.0

99.6

99.6

The red blood cell identified by the arrow contains a Howell-Jolly body that was correctly identified by
100.0% of referees and 99.6% of participants. A Howell-Jolly body is a small round remnant of
nuclear material or DNA, measuring approximately one micron (m) in diameter. These bodies are
normally removed when red blood cells circulate through the spleen. However, they may remain in
circulating red blood cells if the spleen is hypofunctional or missing. Howell-Jolly bodies are usually
present singly, although multiple bodies may be seen infrequently in certain conditions such as
megaloblastic anemia.

BCK/BCP-14

Blood Cell Identification Graded

Identification

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

Erythrocyte

19

2586

76.9

1388

78.4

Good

321
171
140

9.5
5.1
4.2

149
17
102

8.4
1.0
5.8

Macrocyte, oval/round
Pre-keratocyte
Stomatocyte

100.0
-

Not Graded*
Unacceptable
Unacceptable

The arrowed cell is an erythrocyte and was correctly identified by 100.0% of referees and 77.4% of
participants. This normal red blood cell is round in shape, and has a central area of pallor that occupies
less than 1/3rd the diameter of the cell. The cytoplasm stains pink-red, consistent with adequate
hemoglobinization. The cell does not appear to have an appreciably greater cell diameter than the
adjacent red cells as would be expected for a macrocyte. It is also lacking the central linear slit or
discrete submembranous vacuoles that characterize a stomatocyte or pre-keratocyte, respectively.
Many of the surrounding red blood cells have unusual shapes including target cells and irregularly
shaped microcytes.
* Not graded by reason of scientific committee decision.

BCK/BCP-15

Blood Cell Identification Graded

Identification
Fragmented cell (schistocyte,
helmet cell, keratocyte)

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

20

3353

1738

Good

100.0

99.1

98.1

The arrowed cell is a fragmented red cell that was correctly identified by 100.0% of referees and
98.7% of participants. This small, irregularly shaped cell, without central pallor is a schistocyte.
Schistocytes are most commonly seen when red blood cells fragment in circulation and are resealed by
fusion of opposing ends.

Blood Cell Identification Ungraded


Case History
The patient is a 26-year-old sailor with chills and fever. Laboratory data includes: WBC=8.5 X 109/L;
Hgb=12.6 g/dL; MCV=87 fL; PLT=186 X 109/L.

BCK/BCP-16

Identification
Plasmodium sp. (malaria)
Parasite seen, referred for
definitive identification

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

15
1

2756
444

1311
306

Educational
Educational

93.8
6.2

84.5
13.6

77.5
18.1

The arrow designates a red blood cell infected with a malaria parasite (Plasmodium sp.). For this
ungraded challenge 93.8% of referees and 82.1% of participants choose this identification. The
arrowed red blood cell differs from the surrounding erythrocytes by its enlarged size and by the
presence of an intracellular organism. As malaria parasites mature, their morphology shows variations.
Growing Plasmodium trophozoites can have cytoplasmic extensions resembling pseudopods. This is
illustrated in the arrowed cell, which has a pseudopod or amoeboid appearance.

17

BCK/BCP-17

Blood Cell Identification Ungraded

Identification
Plasmodium sp. (malaria)
Parasite seen, referred for
definitive identification

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

15
1

2769
432

1314
305

Educational
Educational

93.8
6.2

84.9
13.3

77.7
18.0

The arrow demonstrates another erythrocyte infected with the malaria parasite (Plasmodium sp.) as
correctly identified by 93.8% of referees and 82.4% of participants. The arrowed red blood cell differs
from the other erythrocytes in size and appearance. This cell illustrates a maturing Plasmodium
organism with increased cytoplasm and characterized by a complex looping configuration with
vacuolation and purplish chromatin.

18

BCK/BCP-18

Blood Cell Identification Ungraded

Identification
Erythrocyte with overlying
platelet
Platelet, normal

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

15

93.8

3156

96.4

1654

96.5

Educational

6.2

40

1.2

25

1.5

Educational

The arrowed cell shows a platelet overlying an erythrocyte that was correctly identified by 93.8% of
referees and 97.7% of participants. A platelet, superimposed on a red blood cell, can resemble an
intracellular organism or an inclusion body. A distinguishing feature of a superimposed platelet is its
halo or clear zone as illustrated in the arrowed cell.

19

BCK/BCP-19

Blood Cell Identification Ungraded

Identification
Lymphocyte, reactive (to
include plasmacytoid and
immunoblastic forms)
Lymphocyte, large granular
Lymphocyte

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

14

87.5

3009

91.9

1574

91.8

Educational

2
-

12.5
-

88
144

2.7
4.4

46
78

2.7
4.6

Educational
Educational

The arrowed cell is an example of a reactive lymphocyte that was correctly identified by 87.5% of
referees and 91.8% of participants. Reactive lymphocytes can vary in size and shape. They can consist
of ovoid to irregular cells with an approximate range in size from 10-25 micrometers. Nuclei are round
to oval with coarse or moderately condensed red-purple chromatin. Nucleoli are not prominent. The
cytoplasm can have pseudopods or be amoeboid in appearance. The outer margin of a reactive
lymphocyte can have a dark edge that is different from the rest of the cytoplasm. Another
characteristic feature of a reactive lymphocyte involves the cytoplasm surrounding adjacent red blood
cells. As the arrowed cell illustrates, the reactive lymphocyte appears to adhere to the contours of
neighboring red blood cells.

20

BCK/BCP-20

Blood Cell Identification Ungraded

Identification
Echinocyte (burr cell, crenated
cell)
Acanthocyte (spur cell)

Referees
No.
%

BCK
Participants
No.
%

BCP
Participants
No.
%

Performance
Evaluation

14

87.5

3206

97.8

1685

98.2

Educational

12.5

63

1.9

25

1.5

Educational

The arrowed cell is an example of an echinocyte (red blood cell with multiple short blunt projections).
This identification was correctly choosen by 87.5% of referees and 98.0% of participants. These
projections are evenly sized and shaped and evenly distributed over the red blood cells surface. An
echinocyte can be distinguished from an acanthocyte, which is a red blood cell showing sharp unevenly
distributed projections on its surface. Echinocyte means sea urchin, and alternate nomenclature
includes burr cell or crenated cell. An echinocyte has central pallor and multiple homogeneous
projections. Etiology can be medical or artefactual. Echinocytes can be seen in patients with uremia.
Echinocytes are considered artefactual when present in aged blood or in an environment of high
humidity.

21

Discussion
The patient, described in this case, was ultimately diagnosed with malaria (Plasmodium vivax). Because the
patient was a sailor who had recently traveled to South America and who presented with fever and chills,
the clinician considered an infectious etiology. As part of the clinical evaluation, a malaria screen was
ordered. This case is notable for several reasons. The patients laboratory data (WBC count, hemoglobin,
MCV, and platelets) were within normal limits. These hematology results would not routinely trigger a
peripheral smear review in most clinical laboratories. The patients symptoms and travel history prompted
concern for infection, and a malaria screen was ordered. The medical technologist identified rare malaria
organisms on thick and thin films. Given this low level of organisms on the blood smear, the malaria
parasites could have been overlooked. However, the medical technologists skill and diligence in reviewing
the blood films led to the correct diagnosis. This patient received an appropriate treatment regiment for
malaria and recovered from this potentially deadly disease.
Malaria is caused by parasites of the genus Plasmodium. The four species of Plasmodia that cause malaria
include: P. falciparum, P. vivax, P. ovale, and P. malariae. Individuals with malaria often report flu-like
symptoms including fever and chills and sweating, nausea and vomiting, malaise, and headaches. If
malaria is undetected and untreated, patients may die from the disease.
The Anopheles mosquito transmits the malaria parasite. Malaria has a wide geographical distribution
including Africa, Asia, Central and South America, and the Middle East. Although malaria is most
commonly acquired through travel to or residence in an endemic area, the disease can be transmitted in
other ways. Malaria can be acquired through blood transfusion or organ transplant. A mother can transmit
malaria organisms to her fetus during pregnancy or to her neonate during delivery. For these less common
modes of transmission, clinicians may not consider malaria as a possibility in a patients initial diagnostic
workup. Since the parasites may be detected serendipitously on blood smear review, it is important that
laboratory staff recognize Plasmodium organisms.
Laboratory evaluation of well stained blood smear(s) is crucial in making the diagnosis of malaria.
Examination of thick and thin smears is currently considered the diagnostic gold standard. However,
establishing a diagnosis of malaria may not be straightforward as experienced laboratory personnel can
attest. Examination of thick and thin smears is time consuming. Laboratory personnel must be vigilant in
reviewing smears, particularly when there are low levels of parasites. In many laboratories, clinical requests
for malaria smear review are infrequent. Since malaria parasites may be uncommonly seen and show
variations in morphology, identification of these parasites can be problematic. In thick smears, red blood
cells are destroyed and white blood cells, platelets, and malaria are visible. Malaria parasites may appear as
tiny chromatin dots with wispy cytoplasm on thick smear review. On thin smears, the morphology of both
the red blood cells and the malaria organisms remains intact. Preservation of intact erythrocytes and
parasites is useful in species identification.
Ring forms characterize the early or young phase of all Plasmodium species. When a smear contains only
ring form trophozoites, further distinction of specific Plasmodium species cannot be made. Of note, the
ring forms of malaria can resemble those of Babesia microti, another intracellular parasite.
As the malaria parasite matures, it assumes variations in size and appearance during its life cycle. There are
morphological differences among the Plasmodium species. If these distinctive features are recognized on a
blood smear, identification of the Plasmodium species can be made. This is an important aspect in the
clinical management of a patient with malaria. For example, infection with Plasmodium malariae can cause
serious renal damage (nephrotic syndrome). Plasmodium falciparum is the most severe form of malaria
since infected individuals can have severe cerebral complications and die; this is the form that must be
recognized immediately for appropriate treatment. Features of Plasmodium falciparum infection include a
high number of organisms in the peripheral blood with predominantly ring forms (mature forms are
sequestered in the peripheral vasculature). Multiple rings may be present within the same red blood cell
and infected red blood cells are not enlarged in size. Gametocytes with distinctive crescent/banana shapes
may be found but are rare in untreated patients. In the other plasmodia, various stages of the parasite life
cycle are more commonly observed, often with a predominance of one stage. A patient can be infected
22

with more than one type of Plasmodium species. When reviewing a blood smear, the possibility of a mixed
malaria infection should be considered.
Although a comprehensive review of the life cycle phases of the malaria parasite is beyond the scope of
this discussion, highlights are provided.

Life cycle of a malaria parasite involves the Anopheles mosquito and human being.
Plasmodium parasites are most commonly transmitted to a human being by the bite of female
Anopheles mosquitoes. In the human being, the infectious life cycle of the malaria parasite
involves liver, spleen, and red blood cells.
The life cycle of a malaria parasite consists of the following: ring form trophozoite, mature
trophozoite, immature schizont, mature schizont (containing merozoites), microgametocyte, and
macrogametocyte. Each of these stages has unique morphology.
Maturation and asexual reproduction of malaria parasites occur in the human being and consist
of an exoerythrocytic phase that occurs in the liver and an erythrocytic phase.
P. vivax and P. ovale organisms can persist in the liver for several years in a dormant stage. If
untreated, relapse of malaria infection can occur several years after the initial presentation of the
disease.
After infecting the liver, malaria organisms enter the bloodstream by rupturing the liver cells
(hepatocytes). Malaria parasites invade red blood cells where they grow and develop. The
organisms essentially feed on hemoglobin with the formation of brownish gold pigmentation that
can be sometimes seen on blood smear examination.

Malaria parasites mature within red blood cells, but then cause their rupture. As the red blood cells become
damaged, cytokines and cellular products are released which result in an infected individual developing
fever and chills. These symptoms can be severe. Rupture of red blood cells can cause anemia. Damaged
red blood cells become sequestered in the spleen, which clinically can become enlarged (splenomegaly).
Malaria parasites can damage the liver, and infected individuals may be jaundiced. Renal failure and
neurological symptoms can also occur in malaria.
In the current case, the patient presented with fever, profuse sweating, and shaking chills. The clinician
suspected malaria given these symptoms and the patients recent travel to an endemic region. A malaria
screen was ordered, and malaria organisms were identified. As previously mentioned, species identification
of Plasmodium organisms is important in treating infected patients. The sailor was diagnosed with P. vivax.
The Duffy red cell antigen is thought to be a receptor for infection by P. vivax organisms. P. vivax has a
propensity to invade reticulocytes, and infected cells are larger. Red blood cells, infected with P. vivax, can
show fine pink granules, known as Schuffner stippling.
A salient feature of P. vivax is its dormancy within the liver (hypnozoites). Relapse of malaria can occur
when hypnozoites are released into the bloodstream. Malaria, caused by P. vivax, can recur over weeks to
months to years.
In summary, it is imperative that laboratory personnel be well trained to identify the various morphologies
of the Plasmodium species. Currently, blood smear examination of well-stained slides is vital in making the
diagnosis of malaria. Alternate means for detection of Plasmodium organisms using rapid diagnostic tests
and molecular detection are being evaluated for future use.

23

References :
1. Hematology, Clinical Microscopy, and Body Fluids Glossary-Surveys 2007 College of American
Pathologists, pages 16-18.
2. Mahon CR, Manuselis G. Textbook of Diagnostic Microbiology. Philadelphia, PA: W.B. Saunders
Company: 2000.
3. Underwood JCE, ed. General and Systematic Pathology. New York, NY: Churchill Livingstone:
2004.
4. Rubin, E. Rubins Pathology: Clinicopathologic Foundation Of Medicine. Philadelphia, PA: Lippincott,
Williams, and Wilkins: 2005.
5. Setencich O, Jackson RB. Malaria is still a major global transfusion concern. JAB: The Journal of
American Red Cross Blood Services. 2006:49-51.
6. Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, PA: W.B.
Saunders: 2001.
Relevant Web Sites :
1. World Health Organization http://www.who.int/topics/malaria
2. United States Department of Health and Human Services CDC- Centers for Disease Control and
Prevention http://www.cdc.gov/malaria

Patricia Devine, MD
Hematology and Clinical Microscopy Resource Committee

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