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Leukemias
Background: Leukemias
are a group of heterogeneous neoplastic disorders of white blood cells.
Based on their origin, myeloid or lymphoid, they can be divided into 2
types. Leukemias traditionally have been designated as acute or chronic,
based on their untreated course. Acute leukemias usually present with
hemorrhage, anemia, infection, or infiltration of organs.
Many patients with chronic leukemias are asymptomatic. Other leukemias
present with splenomegaly, fever, weight loss, malaise, frequent
infections, bleeding, thrombosis, or lymphadenopathy. Some chronic
leukemias enter a blast phase where the clinical manifestations are
similar to the acute leukemias.
Chronic myelogenous leukemia (CML) is characterized by an uncontrolled
proliferation of granulocytes. Usually an accompanying proliferation of
erythroid cells and megakaryocytes is present. Many patients are
asymptomatic but may present with splenomegaly, weight loss, malaise,
bleeding, or thrombosis.
Chronic lymphocytic leukemia (CLL) represents a monoclonal expansion of
lymphocytes. In 95% of cases, CLL is a predominantly malignant clonal
disorder of B lymphocytes. The remainder is secondary to a T-cell clone.
The neoplastic cell is a hypoproliferative, immunologically incompetent
small lymphocyte. There is primary involvement of the bone marrow and
secondary release into the peripheral blood. The recirculating lymphocytes
selectively infiltrate the lymph nodes, the spleen, and the liver. The
majority of patients are asymptomatic at diagnosis. As the disease
progresses, lymphadenopathy, splenomegaly, and hepatomegaly develop. A
secondary immune deficiency with hypogammaglobulinemia exists.
Acute lymphocytic leukemia (ALL) is a malignant clonal disorder of the
bone marrow lymphopoietic precursor cells. In ALL progressive medullary
and extramedullary accumulation of lymphoblasts are present that lack the
potential for differentiation and maturation. An inhibition of the normal
development of hematopoietic cell elements occurs. The clinical
presentation is dominated by progressive weakness and fatigue secondary to
anemia, infection secondary to leukopenia, and bleeding secondary to
thrombocytopenia. When 50% of the bone marrow is replaced then peripheral
blood cytopenias are observed.
Acute myelogenous leukemia (AML) is a group of neoplastic disorders of
the hematopoietic precursor cells of the bone marrow. AML is subdivided by
the French-American-British system into 6 categories depending on the
morphology. AML is not a disorder of rapidly proliferating neoplastic
cells. The time for one cell division is prolonged with respect to that of
normal bone marrow blast cells. A failure of maturation of the neoplastic
cell clone exists. The bone marrow is gradually replaced by blast cells.
Therefore, the most important complications are progressive anemia,
leukopenia, and thrombocytopenia.
Pathophysiology: In leukemias, a clone of malignant
cells may arise at any stage of maturation in either the lymphoid,
myeloid, or pluripotential stages. The cause for this clonal expansion is
understood poorly in most cases but appears to involve some rearrangement
of the DNA. External factors, such as alkylating drugs, ionizing
radiation, and chemicals, and internal factors, such as chromosomal
abnormalities, lead to DNA changes.
Chromosomal rearrangements may alter the structure or regulation of
cellular oncogenes. For instance, in the B-cell lymphocytic leukemias,
chromosomal translocations may put the genes that normally regulate heavy
and light chain immunoglobulin synthesis next to the genes that regulate
normal cellular activation and proliferation. This results in
proliferation of lymphoblasts. As the population of cells expands, the
bone marrow starts to fail. Pancytopenia is typical and results in part
from the physical replacement of normal marrow elements by the immature
cells. In addition, the abnormal cells may secrete factors that inhibit
normal hematopoiesis.
As the bone marrow becomes replaced, the abnormal cells spill into the
circulation and infiltrate other organs such as the liver, spleen, and the
eye. The ocular manifestations may be secondary to direct infiltration of
the leukemic cells, as a result of the abnormal systemic hematological
parameters or as a result of opportunistic infections.
Frequency:
- In the US: In 1999, 30,200 newly diagnosed cases of
leukemia occurred. Of the leukemias diagnosed in the US, 10,100 (33%)
were AML; 7,800 (26%) were CLL; 4,500 (15%) were CML; and 3,100 (10%)
were ALL. Incidence of AML, CML, and ALL in adults is 2.3, 1.3, and
1/100,000 people per year, respectively.
Clinical series show variable data regarding prevalence and incidence
of ocular involvement in patients with leukemia. These differences arise
from the differences in study design. In some studies, patients were
examined at different stages of the disease. In others, ophthalmologists
examined only symptomatic patients. In most studies, no distinction is
made between the different leukemias.
Three prospective studies reveal that 14-53% of patients had ocular
manifestations of the disease prior to the start of chemotherapy.
Leukemia is responsible for 2-6% of orbital tumors in children.
Furthermore, up to 11% of children with proptosis will have some form of
acute leukemia.
Autopsy series show the highest frequency of ocular involvement. It
is presumed that dying patients have a higher disease burden. In
addition, histopathological methods allow detection of lesions that are
not clinically detectable. About 28-80% of cases have intraocular
manifestations. An autopsy study reports 8-12% have orbital involvement.
Despite changes in treatment and survival over the past decades,
ocular involvement as examined by histopathological methods has remained
fairly constant in the past 70 years.
- Internationally: It has been estimated that 231,000
new cases of leukemia were diagnosed globally during 1990. It has been
estimated that in Western countries, CLL constitutes the most frequent
type of leukemia with 25% of cases, CML represents 20%, and AML 20%.
Mortality/Morbidity: When all
leukemias are lumped together, the global 5-year survival is 20%. In
developed countries, 31% survive for 5 or more years, compared with 15% in
developing countries. This underscores the lack of access to high-tech
treatment in the developing world. In 1990, 184,000 deaths were reported
globally, secondary to all leukemias. It was estimated that in the US
during 1999 there would be a total of 22,100 deaths secondary to all
leukemias.
The breakdown of deaths according to the different subtypes is as
follows:
- Deaths secondary to ALL and AML have been reported at 1,400 and
6,900, respectively.
- In children with ALL, 90% of patients achieve a complete remission,
and up to 80% can remain disease free at 5 years following treatment. In
adults with ALL, remissions occur in 60-80%, while 20-35% will maintain
a leukemia-free survival.
- Currently, 65-70% of patients with AML attain remission. The 5-year
survival rate during the period 1989-1994 was 43%.
- Secondary to CLL 5,100 deaths have occurred, secondary to CML 2,300
deaths have occurred, and 6,400 deaths have occurred secondary to other
leukemias.
- In CLL, the natural history is highly variable. The median survival
is 6 years and the natural history is not altered by therapy. Infection
is the leading cause of death. The median survival of CML with treatment
is 5 years. Granulocytic sarcoma of the orbit, also known as chloroma,
represents an extramedullary site of AML or CML. Survival has been
reported to range from 1-30 months after the onset of ocular signs and
symptoms. Some studies suggest that the presence of intraocular leukemic
infiltrates correlates with CNS involvement and with decreased
survival.
Race: In the US, ALL and CLL are more common in whites
than in blacks.
Sex: Of the estimated 231,000 new cases diagnosed in
the world, 130,000 were males and 101,000 were females. Of the 30,200 new
cases of leukemia diagnosed in the US during 1999, 16,800 cases were
reported in males and 13,400 cases in females. During 1999, the breakdown
of new cases of leukemia by gender and category were as follows:
- CLL: 4,500 cases were reported in males and 3,300 cases in females.
- CML: 2,700 cases were reported in males and 1,800 cases in females.
The age-adjusted incidence is higher in men than in women (1.7 versus
1.0).
- ALL: 1,800 cases were reported in males and 1,300 cases in females.
Childhood ALL demonstrates a notable male predominance.
- AML: 4,900 cases were reported in males and 5,200 cases in females.
The age-adjusted incidence of AML is higher in men than in women (2.9
versus 1.9).
- In other leukemias, 2,900 cases were reported in males and 1,800
cases in females.
Age: Most childhood leukemias are acute.
- ALL is the most common malignancy in children, especially affecting
those aged 2-10 years. ALL is seen in only 20% of adult acute leukemias
and behaves more aggressively than the childhood type.
- AML constitutes 15-20% of acute leukemias in children. Incidence of
AML increases with age; younger than 65 years the incidence is 1.3, and
older than 65 years the incidence is 12.2.
- CML constitutes less than 5% of childhood leukemias. The incidence
of CML increases slowly with age until the middle 40s, when the
incidence starts to rise rapidly.
- Incidence of CLL is over 10/100,000 for persons older than 70 years
but less than 1/100,000 for those younger than 50 years. Mean age at
diagnosis of CLL is 60 years.
History:
- In a majority of patients diagnosis of leukemia has been made before
presenting to an ophthalmologist. However, in some patients ocular
symptoms and examination lead to diagnosis of leukemia.
- Most patients do not develop symptoms as a result of intraocular
involvement.
Physical:
- Posterior segment manifestations
- The posterior segment manifestations are protean in nature and may
be secondary to direct invasion of the leukemic cells.
- They result from systemic hematological abnormalities, such as
anemia, thrombocytopenia, and hyperviscosity or opportunistic
infections secondary to the immune dysfunction.
- Direct infiltration
- Retinal grayish-white nodules that may be surrounded by
hemorrhage manifest direct infiltration.
- Perivascular sheathing may be another manifestation of a
leukemic infiltrate.
- Roth spots, white-centered retinal hemorrhages, may represent a
cluster of leukemic cells. On the other hand, septic emboli or
platelet-fibrin material gives a similar funduscopic finding.
- Rarely, pale gray swelling of the optic nerve head may indicate
optic nerve infiltration.
- Leukemic retinopathy
- Retinal hemorrhages are the most common finding in most series
and are thought to be secondary to anemia and thrombocytopenia.
These hemorrhages may be dot-shaped, flame-shaped, intraretinal,
subretinal, or subhyaloid.
- Cotton-wool spots are known to represent nerve fiber layer
infarcts. However, they are not correlated with hematological
parameters of anemia or blood viscosity.
- Retinal vein tortuosity and dilation are thought to be secondary
to hyperviscosity.
- Peripheral retinal microaneurysms and retinal neovascularization
may be seen particularly in patients with CML. They are thought to
occur as a result of peripheral nonperfusion and ischemia from
hyperviscosity.
- Sea fans reminiscent of sickle cell retinopathy may be seen.
- Neovascularization of the disc has been reported in a case where
no apparent ischemia was present. It was recognized that angiogenic
factors secreted from the tumor may play a role in the pathogenesis
of retinal and optic nerve head neovascularization.
- The vitreous seldom is involved.
- The choroid is the most commonly affected ocular structure in
pathological studies. Clinically, it often is difficult to detect
the subtle choroidal changes.
- Occasionally, serous retinal detachments and retinal pigment
epithelium (RPE) changes have been reported.
- Opportunistic infections include cytomegalovirus retinitis,
toxoplasma chorioretinitis, endogenous fungal endophthalmitis, and
herpetic retinitis.
- Anterior segment manifestations
- Anterior segment involvement in leukemia is rare but significant
because it often is an extramedullary site of relapse. Anterior
segment manifestations occur more commonly in ALL than in all the
other types of leukemia.
- A change in iris color, iris nodules, hyphema, hypopyon, glaucoma,
a sterile corneal ring ulcer, and a pannus all have been described in
leukemic patients.
- Corneal involvement is rare. Scleral, episcleral, and conjunctival
involvement usually is silent and is limited to perivascular
infiltration that can be demonstrated on pathological
sections.
- Leukemic cells may infiltrate the orbit during the course of an
acute or chronic leukemia. Unusual orbital involvement with leukemia
has been reported to include infiltration of the lacrimal gland and
drainage system, rectus muscles, and dermis.
- Orbital involvement in children is more common in acute leukemias,
whereas orbital involvement in adults is more common in chronic
leukemias.
- The leukemic infiltrate may range from insignificant, where it is
virtually asymptomatic, to a space occupying lesion with its
concomitant symptoms.
- The patient may have proptosis, ecchymosis, chemosis, diplopia,
visual disturbance, or motility disturbances.
- In children, the orbital involvement is characterized by an acute
and rapid process that may be confused with orbital cellulitis. In
general, these infiltrates are bilateral and do not destroy
bone.
- Granulocytic sarcoma of the orbit, also known as chloroma, is an
extramedullary form of myelogenous leukemia.
- Unilateral, painless proptosis develops over weeks to months prior
to diagnosis of leukemia. Eyelid redness or violaceous discoloration
may be present, which turns into ecchymosis that may be confused with
rhabdomyosarcoma or metastatic neuroblastoma. If AML or CML is already
present, then a rapid and fulminant bilateral proptosis is
characteristic.
Causes:
- Etiology of the leukemias appears to be multifactorial. Genetic,
viral, and environmental factors, such as ionizing radiation, drugs, and
chemicals all have been implicated in the pathogenesis of
leukemia.
- It is believed that the final common pathway is damage to the DNA in
one way or another. This damage may rearrange the genetic material
allowing previously silent oncogenes to be expressed.
- Patients with an abnormal number of chromosomes (eg, trisomy 21) and
chromosomal translocations are at an increased risk of developing
ALL.
- Risk factors implicated in the development of AML include the
following:
- Myelotoxic agents (eg, ionizing radiation, benzene, alkylating
agents)
- Chromosomal abnormalities (eg, Down syndrome, chromosomal
instability syndromes)
- Predisposing hematological disorders (eg, aplastic anemia, chronic
myeloproliferative disorders, paroxysmal nocturnal
hemoglobinuria)
- Chromosomal abnormalities, especially trisomy 12, are common in
patients with CLL. Familial case clusters have been reported in CLL.
HTLV-1 infection also has been implicated in CLL.
- Damage to the bone marrow by agents, such as benzene and ionizing
radiation, may cause CML.
- Of patients with CML, 90% have an acquired chromosomal abnormality,
the Philadelphia chromosome, which is a translocation of half of the
long arm of chromosome 22 to another chromosome, usually chromosome
9.
DIFFERENTIALS
Cellulitis, Orbital
Central
Retinal Vein Occlusion
Other Problems to be Considered:
Rhabdomyosarcoma
Metastatic neuroblastoma
Hyperviscosity
retinopathy
Childhood iritis
WORKUP
Lab Studies:
- CBC is the most useful initial laboratory test in patients
suspected of having leukemia. Most patients will show some abnormality
in the CBC and some blasts will be seen in the peripheral smear in
patients with acute leukemias.
- To diagnose CLL, a lymphocytosis of greater than
5000/mm3 must be present. The absolute neutrophil count
usually is normal and red cell and platelet counts are mildly
decreased. In addition, the peripheral smear or bone marrow should
show normal mature small lymphocytes with less than 55% atypical or
blast forms.
- CML is defined by its peripheral WBC count. Typically,
leukocytosis is in excess of 100,000/mm3. The differential
count shows that neutrophil precursors are present. This is
accompanied by a basophilia and eosinophilia. Unlike those in AML,
these cells are mature and functional.
- Bone marrow aspiration establishes diagnosis of leukemia. The
morphology of blasts usually can differentiate between ALL and
AML.
- In ALL, a homogeneous infiltrate of lymphoblasts replaces the
normal bone marrow elements. Lymphoblasts usually are small and
measure approximately 14 µm in diameter. They have scant cytoplasm
with no granules. The nucleus has no nucleoli or a small indistinct
one.
- For the diagnosis of AML, 30% of the nucleated cells in the
aspirate must be blast cells of myeloid origin. Multiple large
nucleoli, delicate chromatin, gray-blue cytoplasm, and Auer rods
characterize myeloblasts. The presence of Auer rods is virtually
diagnostic of AML, because these condensed lysosomal cytoplasmic
azurophilic rod-shaped structures do not appear in ALL.
- In CLL, bone marrow infiltration exceeds 30% lymphocytes. The
lymphocytes are mature with less than 55% atypical or blast forms. The
nuclei are round, cytoplasm is scant, chromatin is compact, nucleoli
are inconspicuous, and mitotic figures are rare.
- Immunophenotyping using multiparameter flow cytometry following
labeling with monoclonal antibodies to cell-surface antigens
identifies the B or T cell origin of the lymphoblasts.
- Based on the expression of B lineage-restricted antigens and
clonal rearrangements of immunoglobulin heavy and light chain genes,
it has been estimated that up to 80% of ALL cases arise from B-cell
precursors. The majority possesses a common ALL antigen (CALLA) that
is present only on leukemic cells.
- T-cell ALL possesses receptors for sheep erythrocytes, and when
these are combined they form E-rosettes.
- A final subset of ALL lacks B- or T-cell characteristics and is
referred to as null-cell ALL.
- Certain myeloid-specific antigens, such as CD13, CD33, and CD41,
have been used to diagnose AML.
- The malignant cells in CLL correspond to a minor subpopulation of
B cells that express cell surface immunoglobulins IgM/IgD and the
T-cell associated antigen CD5.
- Histochemical stains for myeloperoxidase (Leder stain) and
nonspecific esterase have a strong affinity for myelogenous precursors
but fail to stain lymphocytic forerunners.
- Demonstration of nuclear DNA polymerizing enzyme terminal
deoxynucleotidyl transferase (TdT) is indicative of a lymphoid origin.
However, up to 2-5% of patients with AML exhibit this enzyme.
Exceptions may occur when a malignant clone arises from multipotent
cells that may express both myelogenous and lymphocytic
characteristics.
- Chromosomal analysis
- Chromosomal analysis also plays an important role. The diagnosis
of CML is established by identifying cytogenetically or molecularly a
clonal expansion of a hematopoietic stem cell possessing a reciprocal
translocation between chromosomes 9 and 22.
- Chromosomal analysis of the leukemic cell currently provides the
most important pretreatment prognostic information in AML.
Imaging Studies:
- Fluorescein angiography may reveal myriad diffuse leakage points at
the level of the RPE. This pattern also may be seen in
Vogt-Koyanagi-Harada, diffuse choroidal melanoma, metastatic tumors, and
posterior scleritis.
Histologic Findings:
Histopathological studies have shown the choroid to be the ocular
structure most commonly involved by leukemia. The choroid is thickened,
especially at the posterior pole. The RPE may be hyperplastic, atrophied,
or hypertrophied. Photoreceptor loss, drusen formation, serous detachment,
and cystoid retinal edema may be present.
Immature white blood cells infiltrate the retina and when they
accumulate, nodular masses may be seen. The retinal vessels usually are
packed with immature leukocytes. Capillary nonperfusion may result due to
massive accumulation of cells. Diffuse infiltration of the iris and
ciliary body commonly is seen. The infiltrates usually are denser near the
sphincter and the base of the iris. The trabecular meshwork may be clogged
with leukemic cells leading to glaucoma.
Histopathological studies indicate that leukemic infiltration in the
orbit most often was mild and diffuse as opposed to massive and tumorous.
A chloroma of the orbit is composed of immature granulocyte cells, which
contain large amounts of the enzyme myeloperoxidase, giving the tumor a
greenish hue on gross examination. Because of the poorly differentiated
nature of this tumor on histological examination and often unremarkable
CBC, it may be misdiagnosed as a lymphoma.
Histological diagnosis of lymphoma in a rapidly growing orbital mass of
a child is unlikely because orbital lymphomas in children are quite rare.
Medical Care: The
treatment of leukemia is in constant flux, evolving and changing rapidly
over the past few years. Most treatment protocols employ systemic
chemotherapy with or without radiotherapy. The basic strategy is to
eliminate all detectable disease by using cytotoxic agents. To attain this
goal 3 phases usually are employed, as follows: remission induction phase,
consolidation phase, and maintenance therapy phase.
Chemotherapeutic agents are chosen that interfere with cell division.
Tumor cells usually divide more rapidly than host cells, making them more
vulnerable to the effects of chemotherapy. Primary treatment will be under
the direction of a medical oncologist, radiation oncologist, and primary
care physician. Although a general treatment plan will be outlined, the
ophthalmologist does not prescribe or manage such treatment.
- Initial treatment of ALL employs various combinations of
vincristine, prednisone, and L-asparaginase until a complete remission
is obtained.
- Maintenance therapy with mercaptopurine is continued for 2-3 years
following remission.
- Use of intrathecal methotrexate with or without cranial irradiation
to cover the CNS varies from facility to facility.
- Daunorubicin, cytarabine, and thioguanine currently are used to
obtain induction and remission of AML.
- Maintenance therapy for 8 months may lengthen remission. Once
relapse has occurred, AML generally is curable only by bone marrow
transplantation.
- Presently, treatment of CLL is palliative.
- CML is characterized by a leukocytosis greater than 100,000 cells.
Emergent treatment with leukopheresis sometimes is necessary when
leukostastic complications are present. Otherwise busulfan or
hydroxyurea may control WBC counts. During the chronic phase, treatment
is palliative.
- When CML converts to the blastic phase, approximately one third of
cases behave as ALL and respond to treatment with vincristine and
prednisone. The remaining two thirds resemble AML but respond poorly to
AML therapy.
- Allogeneic bone marrow transplant is the only curative therapy for
CML. However, it carries a high early mortality rate.
- Leukemic retinopathy usually is not treated directly. As the
hematological parameters normalize with systemic treatment, many of the
ophthalmic signs resolve. There are reports that leukopheresis for
hyperviscosity also may alleviate intraocular manifestations.
- When definite intraocular leukemic infiltrates fail to respond to
systemic chemotherapy, direct radiation therapy is recommended.
- Relapse, manifested by anterior segment involvement should be
treated by radiation. In certain cases, subconjunctival chemotherapeutic
agents have been injected.
- Optic nerve head infiltration in patients with ALL is an emergency
and requires prompt radiation therapy to try to salvage some
vision.
Consultations: A multidisciplinary approach is
required in the treatment of a leukemic patient.
Complications:
- Posterior segment complications from bone marrow transplants were
seen in 13% of the patients. Among the complications seen were
vitreous hemorrhage, infectious retinitis, cotton-wool spots, and
retinal detachment.
- Radiation retinopathy has been reported to occur in patients
undergoing bone marrow transplant and high-dose chemotherapy and who
received low-dose teletherapy. High-dose chemotherapy may lower the
threshold for radiation retinopathy.
- Ocular ischemia evidenced by optic disc and retinal
neovascularization may lead to tractional retinal detachment following
chemotherapy and radiation therapy.
- Dry eye, keratitis, and cataracts may be sequelae of external beam
radiation therapy with 3000-4000 rads. However, as little as 1150 rads
may cause lenticular opacities.
- In one study, 82 ALL survivors and 15 AML survivors were followed
for an average of 3 years. All of the AML survivors had a normal
ocular examination. Cataracts developed in 52% of ALL survivors.
However, only 1 patient suffered significant visual dysfunction as a
result of ALL or its treatment.
Prognosis:
- Leukemic retinopathy usually is seen in patients who show a relapse
and does not imply a bad prognosis.
- Leukemic infiltration portends a poor prognosis and usually is
associated with CNS involvement.
- Optic nerve head infiltration is associated with CNS disease and a
poor prognosis.
- Prognosis of ALL is age dependent. Children have a much better
outlook than adults. See Mortality/Morbidity.
IMAGES:
PICTURES
Caption: Picture 1. A
4-year-old boy presented with sudden proptosis of his left eye.
Caption: Picture 2. A
CBC revealed anemia (Hb 8.6 mg/dL), thrombocytopenia (64,000), and
leukocytosis (12,900). The peripheral smear revealed the presence of
blasts 28%, lymphocytes 44%, segmented 14%, monocytes 6%, bands 2%,
metamyelocytes 1%, and myelocytes 1%. The patient was diagnosed with
AML type M4-M5 chloroma of the left orbit.
Caption: Picture 3. CT
scan reveals infiltration in the left orbit. Notice that the bone is
uninvolved. An lumbar puncture revealed that the cerebral spinal
fluid was clean of leukemic cells.
Picture Type:
CT
Caption: Picture 4.
Systemic chemotherapy was instituted and the proptosis resolved.
Unfortunately 4.5 months later, the child passed away secondary to
multiorgan failure.
Caption: Picture 5. A
76-year-old man was discovered on routine examination to have an
impending bilateral central retinal veins obstruction. Further
workup revealed a WBC of 709,000, a hemoglobin of 12 mg/dL, and a
platelet count of 104,000. The patient eventually was diagnosed with
CML. The following is a red-free photograph of the right fundus.
Notice the intraretinal hemorrhages.
Caption: Picture 6.
The same patient as above. This is a red-free photograph of the left
eye showing intraretinal hemorrhages.
Caption: Picture 7. 14
year old boy with a past medical history of ALL who complained of a
sudden loss of vision OD. Visual acuities were count fingers OD and
20/20 OS. Notice the macular hemorrhage responsible for the loss of
vision. Picture courtesy of Dr Rafael Jiménez.
Caption: Picture 8.
Same patient as above. His hematological work up revealed a
hemoglobin level of 5.6, WBC of 1800 and platelets of 3000. Picture
courtesy of Dr Rafael Jiménez.
REFERENCES
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