10 November 2012 Belgium

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Ann Van de Velde
BHS Educational Course Seminar, 10 Nov 2012
Seminar 8 – Non-malignant Hematology
EINDE

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Release of neutrophils from the
marrow storage pool
• 2 fold increase in the neutrophil count < 4 h
• > 1/2 of neutrophils in peripheral circulation
attached to vascular endothelium
• “Marginated" neutrophils released immediately
("demarginated") at times of stress

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WBC
• 9 days in marrow
• 3 to 6 hours in blood
• 1 to 4 days in tissues
• Total neutrophil count, as measured from
peripheral blood, represents a population that
comprises only 5 % of the total pool sampled
during a fleeting 2 % of its total transit time.

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• Mechanisms controlling release of neutrophils
from bone marrow only partially understood
- Endotoxin
- Glucocorticoids
- Leukocyte-mobilizing factor derived from the third
component of complement (C3e)
- Chemoattractants such as C5a
- Cytokines as tumor necrosis factor (TNF)-alpha
- Androgens

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Keep in mind
• As is true for the approach to any medical
problem, there is no substitute for an accurate
history and physical examination.
• However, before this process is started, the
clinician must make sure that there is no
laboratory error involved. Blood counts that do
not make sense within the context of the
clinical findings should be repeated before
extensive evaluation is undertaken.

8. Vrouw Man
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Hematocriet 37 – 44 % 40 – 54 %
Hemoglobine 12 – 16 g/dl 14 – 18 g/dl
Rode bloedcellen 4,2 – 5,5 x 10E12/l 4,4 – 6 x 10E12/l
MCV 76 – 96 fl
MCH 27- 32 pg
MCHC 30 – 35 g/dl
Reticulocyten 0 – 2 % RBC (10-100 x 10E9/l
Witte bloedcellen 4 – 10 x 10E9/l
Neutrofiele granulocyten 0–5%
- Staafkernige granulocyten 40 – 75 %
- Segmentkernige granulocyten
Eosinofiele granulocyten 1–6%
Basofiele granulocyten 0 -1 %
Lymfocyten 20 – 45 %
Monocyten 2 – 10 %
Bloedplaatjes 140 – 440 x 10E9/l

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Leukocytes

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Definitions:
Absolute Neutrophil Count (ANC)
• ANC = WBC (cells/microL) x percent
(PMNs + bands) ÷ 100
• Neutrophilic metamyelocytes and younger
forms NOT included in this calculation

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PART 1: LEUKOPENIA
Kerkyra island, Greece

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ANC <1500/microL (<1.5 x 109/L)
• Generally accepted DEFINITION of neutropenia
• TRESHOLD for neutrophil toxicity and infectious
risk following chemotherapy.
- Mild: 1000 and 1500/microL,
- Moderate: 500 and 1000/microL
- Severe: less than 500/microL.
• Leukopenia and granulocytopenia are generally used
interchangeably with neutropenia, although somewhat different.

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Leukopenia
• Refers to a low total white blood cell count
that may be due to any cause
- lymphopenia and/or neutropenia
• Almost all leukopenic patients are
NEUTROPENIC since the number of neutrophils
is so much larger than the number of
lymphocytes.

16. Vrouw Man
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Hematocriet 37 – 44 % 40 – 54 %
Hemoglobine 12 – 16 g/dl 14 – 18 g/dl
Rode bloedcellen 4,2 – 5,5 x 10E12/l 4,4 – 6 x 10E12/l
MCV 76 – 96 fl
MCH 27- 32 pg
MCHC 30 – 35 g/dl
Reticulocyten 0 – 2 % RBC (10-100 x 10E9/l
Witte bloedcellen 4 – 10 x 10E9/l
Neutrofiele granulocyten 0–5%
- Staafkernige granulocyten 40 – 75 %
- Segmentkernige granulocyten
Eosinofiele granulocyten 1–6%
Basofiele granulocyten 0 -1 %
Lymfocyten 20 – 45 %
Monocyten 2 – 10 %
Bloedplaatjes 140 – 440 x 10E9/l

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Granulocytopenia
• Reduced absolute number of ALL circulating
cells of the granulocyte series
- neutrophils, eosinophils, and basophils
• Almost all granulocytopenic patients are
NEUTROPENIC since the number of neutrophils
is so much larger than the number of
eosinophils and basophils.

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Agranulocytosis
• Literally means ABSCENCE of granulocytes
• Often incorrectly used to indicate severe
neutropenia (ie, ANC <500/microL)

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Two fundamental issues
1. Is the patient at increased RISK for infection
because of neutropenia?
2. Does the presence of neutropenia indicates a
SERIOUS UNDERLYING DISORDER that is
secondarly effecting the neutrophil count?

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Febrile neutropenia

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Infection propensity
• Only 3% of neutrophils circulating in peripheral blood
• Vast majority in BONE MARROW RESERVE POOL and remainder is in TISSUE and
MARGINATED POOL attached to lining of blood vessels
• Standard complete blood count (CBC) is sampling very smallest compartment of
neutrophils and does not accurately reflect body’s capacity to protect against
bacterial infection.
• Most important issue is whether adequate neutrophils get to the site of infection.
• No good clinical laboratory test available to quantitate tissue neutrophil
delivery.
• Adequacy of the marrow reserve pool is most critical determinant of
propensity to infection.
- NORMAL MARROW CELLULARITY
- NORMAL MATURATION OF THE NEUTROPHIL SERIES

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Bone marrow reserve status
• If the bone marrow reserve pool is completely adequate, there is no
relationship between the degree of neutropenia and propensity to
infection.
• Most physicians are aware of the extreme danger present in patients
with significant fever and very low absolute neutrophil counts based
upon their experiences during training with patients who have received
chemotherapy or who have bone marrow failure syndromes. These
patients have NO bone marrow reserve.
• Patients with immune mediated neutropenia but normal bone marrow
reserve are on the other end of the spectrum and are at NO increased
risk of infection because of the neutropenia.

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Bone marrow reserve status
• If a neutropenic patient has a frank abscess or purulent
exudate, he or she can get neutrophils to tissue and likely
has a normal marrow.
• The presence of mucosal ulcerations and severe gingivitis
suggests inability to deliver neutrophils. However,
immune disorders can directly cause similar lesions in the
presence of normal reserve neutropenia.

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Etiology of isolated neutropenia
• Neutropenia results from four basic mechanisms:
- decreased production
- ineffective granulopoiesis
- shift of circulating PMNs to vascular endothelium or tissue pools
- enhanced peripheral destruction.
• Confirmation of one of these mechanisms requires leukokinetic studies
employing bone marrow cultures, radionuclide tagging of blood PMNs,
and other monitoring devices not readily available outside the research
laboratory.
• Various interactions between subtle genetic differences and
environmental factors. Apoptosis of marrow precursors is now recognized
as a common mechanism for many acquired and congenital
neutropenias.

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Acquired neutropenias
• There are many acquired causes of
neutropenia
- Infection
- Drugs
- Immune disorders

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Infectious neutropenias
• Most common cause of acquired isolated neutropenia
• Bacterial, viral, parasitic and rickettsial infections.
• Short-lived - rarely results in bacterial superinfection
• Mechanisms
- Redistribution
- Sequestration and aggregation
- Destruction by circulating antibodies.
• More severe and protracted neutropenia
- Hepatitis B virus
- Epstein-Barr virus
- Human immunodeficiency virus:

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Drug-induced neutropenia and
agranulocytosis
• Adverse idiosyncratic reaction
• Second most common cause of neutropenia
• Requires that the drug have been administered within 4
weeks of onset neutropenia.
• Drugs with highest risk of inducing severe neutropenia
- clozapine
- thionamides (antithyroid drugs)
- sulfasalazine
• Mechanism
- Immune-mediated destruction of circulating neutrophils
by drug-dependent or drug-induced antibodies
- Direct toxic effects upon marrow granulocytic precursors

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Nutritional neutropenia
• B12 and folate deficiency, as well as inborn errors of B12
metabolism
• B12 and folate deficiency best detected by measuring
methylmalonic acid (MMA) and homocysteine (HcY). Both are
elevated with B12 deficiency and HcY alone is elevated in folate
deficiency.
• Copper deficiency and subsequent low ceruloplasmin.
- Malabsorption
- post-gastric bypass surgery
- Critically ill patients who have prolonged hospitalizations

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Primary immune disorders
• ANTINEUTROPHIL ANTIBODIES mediate neutrophil destruction
- by splenic sequestration of opsonized cells
- by complement-mediated neutrophil lysis
• Antineutrophil antibodies
- Infections
- drug exposure
- immune deficiencies.
- specific PRIMARY immune disorders
• Propensity to infection may be more related to the underlying immune
disorder than to the neutropenia !
• Vasculitis, leading to mucosal ulcers.
- Oral symptoms completely resolve with treatment of the underlying vasculitis
with no change in the ANC, proving that the mucositis and the neutropenia are
not related.

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• Isoimmune neonatal neutropenia — Moderate to severe neutropenia
in newborn infants secondary to transplacental passage of IgG antibodies
directed against neutrophil specific antigens inherited from the father of
the infant. ~ Rh hemolytic disease. Otherwise normal infant and patients
do well.
• Chronic AUTOIMMUNE neutropenia — primarily in infants and
children under age four and is also called CHRONIC BENIGN
NEUTROPENIA OF INFANCY AND CHILDHOOD. Specific treatment is not
required. Many patients remain free of infections and maintain normal
lifestyle with no or minimal medical intervention. Spontaneous remission
with disappearance of autoantibodies is common.
• Chronic IDIOPATHIC neutropenia — BENIGN CHRONIC
NEUTROPENIA, no obvious cause. Serologic abnormalities and evidence
of antibody production have been found in 30 to 40 %. Benign course
despite degree of neutropenia. Presence of normal marrow reserve may
explain the lack of significant infections.

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• Pure white cell aplasia — rare disorder characterized by complete disappearance
of granulocytopoietic tissue from bone marrow. Often associated with thymoma
and is due to presence of antibody mediated GM-CFU INHIBITORY ACTIVITY. No
marrow reserve and at risk for infection.
• Other autoimmune disorders — T-GAMMA LYMPHOCYTOSIS (large granular
lymphocyte syndrome) and FELTY'S SYNDROME. Often associated with
RHEUMATOID ARTHRITIS. LGL has markedly decreased marrow reserve as well as
autoimmune vasculitic components.
• Complement activation — Exposure of blood to artificial membranes, as in
dialysis and extracorporeal membrane oxygenation, may result in complement
activation in vivo. Neutrophil aggregation and adherence to endothelial surfaces,
often in the lung. Neutropenia and cardiopulmonary symptoms typically occur
shortly after exposure to the membrane. Can be prevented during hemodialysis by
using BIOCOMPATIBLE MEMBRANES.

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Hypersplenism
• Enlargement of the spleen from any etiology
• Splenic trapping
• Severity of neutropenia is related to the size of
the spleen
• Rarely sufficient to result in severe infection

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Cyclic neutropenia
• Recurrent mouth infections
• Regular oscillations in numbers of blood
neutrophils, monocytes, eosinophils,
lymphocytes and reticulocytes at
approximately 21-day intervals.
• Usually in childhood, as a familial syndrome
• Treatment is largely supportive and G-CSF has
been effective in preventing infection and
reducing symptoms.

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(Bone marrow disorders)
• Aplastic anemia
• Leukemias
• Myelodysplasia
• Post-chemotherapy
- not an isolated defect
- associated with varying degrees of anemia and
thrombocytopenia.
• Examination of peripheral smear and bone marrow
aspirate/biopsy are indicated when more than one
cell line is involved.

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Diagnostic approach
• First step in the approach to the patient with
neutropenia is CONFIRMATION OF THE DIAGNOSIS.
• Review of a Wright—Giemsa stained peripheral blood
smear will confirm reduced number of neutrophils.
• In all cases in which the white blood cell differential
count has been generated by automatic counters, it
should be repeated manually.
• Pseudoneutropenia
- If blood is left standing for a prolonged period of time
- Paraproteinemia
- Anticoagulants that can cause cellular clumping

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Diagnostic approach
• Monitoring of blood counts for 8 to 12 weeks if
there are no other important clinical factors
present.
• According to this schema, if the patient
develops clinical symptoms related to
neutropenia or changes in other cell lines in
the blood count, a full evaluation should be
undertaken.

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Neutropenia in absence of recurrent
or protracted infection
• Most causes are benign, especially if the ANC is >
800/microL.
• Thus, a period of observation is indicated if the patient
is asymptomatic and there are no other significant
clinical features, particularly if there is a recent history
of viral infection or a medication has been taken that is
known to be associated with neutropenia.
• Examination of the oral cavity is important, since the
presence of gingivitis or tooth abscess suggests
presence of symptomatic neutropenia.
• If neutropenia resolves, patient should be followed for
one year with complete blood count being obtained
whenever fever occurs.

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Moderate to severe neutropenia with
recurrent infection
• Bone marrow aspiration with evaluation of cellularity and morphology
- Late myeloid arrest
- Myeloid hypoplasia
• Late arrest
- idiopathic or autoimmune neutropenia, most often associated with
antineutrophil antibodies
- collagen vascular diseases
- some drug-induced neutropenias
- chronic infection
• Myeloid hypoplasia
- toxic drug-induced neutropenias
- pure white cell aplasia
- T-gamma lymphocytosis (large granular lymphocyte syndrome)
- severe congenital neutropenia
- myelodysplastic syndrome

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+ Anemia
• If anemia, particularly normocytic or
macrocytic anemia, or thrombocytopenia is
found, hematological consultation should be
requested immediately and examination of the
peripheral smear along with a bone marrow
aspiration should be performed unless the
cause is clear.

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Isolated neutropenia
• Tests for collagen vascular disease and nutritional
disorders first, prior to marrow examination.
- Antinuclear antibodies and complement
- Antineutrophil antibodies
- Immunoglobulins and immune evaluation
- Screen for HIV infection
- Methylmalonic acid and homocysteine levels
- Serum copper and ceruloplasmin levels

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Cyclic neutropenia
• Episodic infections: twice weekly measurement of
the ANC for at least six weeks to confirm diagnosis
• Decreased marrow cellularity one week before the
nadir
• Rare syndrome
• Symptoms every 21 days
• Family history
• Bone marrow aspiration is NOT helpful in this
disorder

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Antibiotic therapy (1/2)
• gastrointestinal tract - skin
• Rapid onset of overwhelming sepsis.
• Febrile patients with neutropenia related to marrow suppression
- treated immediately, following culture of body fluids, with broad-spectrum parenteral
antibiotics for coverage of both Gram-positive and Gram-negative bacteria.
• ANC >1000/microL: outpatient
• ANC of <500/microL and marrow aplasia : inpatient treatment with parenteral
antibiotics.
• Routine reverse isolation procedures are of no benefit and serve to decrease
contact with medical personnel !
• When a patient first presents with HIGH FEVER and has a VERY LOW ANC,
one must assume that the patient is high risk and has inadequate marrow
reserve.

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Antibiotic therapy (2/2)
• Monitoring of C-reactive protein level and sedimentation rate daily
• Often, cultures are negative and treatment is empiric.
• Rapid response can indicate that appropriate antibiotics have been selected, and poor response
or increase in ESR after a response can indicate that a change in antibiotics is necessary.
• Antibiotics continued for several days after fever has subsided and sedimentation rate
normalized. If ANC has risen > 500/microL on several measures, antibiotics may be discontinued
as long as no source of infection is apparent.
• If fever persists or there is no clear response to treatment, other therapies should be considered.
• If fever and neutropenia persist beyond 7 days in the immunosuppressed patient, antifungal
treatment should be considered in post-chemotherapy patients; not in patients with benign
neutropenia.
• Granulocyte transfusions to patients with Gram-negative sepsis who have not shown a clinical
response to antibiotics within 24 to 48 hours. Waned due in part to difficulties in procurement, to
better antibiotics, and to the use of bone marrow growth factors.

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Myeloid growth factors
• G-CSF therapy not indicated for all causes of neutropenia.
• Helpful in neutropenia associated with early myeloid
arrest
• Reserved for patients with demonstrated infectious
morbidity related to the neutropenia
• Use of G-CSF in patients with chemotherapy-induced
neutropenia
- use as primary or secondary prophylaxis
- neutropenia without fever
- neutropenic fever

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Neutropenia summary
1. Absolute neutrophil count <1500/microL
2. Peripheral blood white blood cell count does not accurately
reflect the body’s capacity to deliver neutrophils to tissues and
protect against bacterial infection.
3. Infection, drugs, and immune disorders are the most common
acquired causes
4. Management includes the prevention of infection, regular dental
care, use of antibiotic mouthwashes, aggressive antibacterial
therapy for fever, and the judicious use of myeloid growth
factors in selected patients

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PART 2: LEUKOCYTOSIS
Picture: Sagrada Familia by Antoni Gaudi, Barcelona

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Leukocytosis and leukemoid
reaction*
• Total white blood cell (WBC) count more than
two standard deviations above the mean, or a
value >11,000/microL in adults.
• *Leukocytosis >50,000/microL, when due to
causes other than leukemia

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Neutrophilic leukocytosis
= neutrophilia
• Total WBC > 11,000/microL along with an
absolute neutrophil count (ANC) > 2 standard
deviations above the mean (greater than
7700/microL (> 7.7 x 109/L).
• Infection, stress, smoking, pregnancy,
following exercise, (chronic myeloproliferative
disorders, such as polycythemia vera (PV) and
chronic myeloid leukemia)

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Lymphocytic leukocytosis
= lymphocytosis
• Total WBC >11,000/microL primarily due to an
absolute lymphocyte count > 4800/microL (>
4.8 x 109/L).
• Infections such as infectious mononucleosis
and pertussis (or in lymphoproliferative
disorders such as the acute and chronic
lymphocytic leukemias)

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Monocytic leukocytosis
= monocytosis
• Total WBC > 11,000/microL primarily due to
an absolute monocyte count > 800/microL (>
0.8 x 109/L).
• (Acute and chronic monocytic variants of
leukemia) and acute bacterial infection or
tuberculosis.

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Eosinophilic and basophilic leukocytosis
= eosinophilia and basophilia
• Total WBC > 11,000/microL due primarily to
an absolute eosinophil or basophil count >
450/microL (> 0.45 X 109/L ) or 200/microL (>
0.2 x 109/L)
• (Variant forms of chronic leukemia, solid
tumors), infection with helminthic parasites,
allergic reactions, and following treatment with
Interleukin-2.

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Basophilia
- Myeloproliferative neoplasms
- Basophilic leukemia, mastocytosis, hypereosinophilic
syndrome, atypical acute and chronic leukemias,
myelodysplastic syndrome
- Allergic or inflammatory reactions, including
hypersensitivity reactions, ulcerative colitis,
rheumatoid arthritis
- Endocrinopathy, including hypothyroidism
(myxedema), administration of estrogens
- Infections, including viral infections, tuberculosis,
helminth infections

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Left shift in the WBC differential
• Band form count >700/microL, often called "BANDEMIA"
• In infection, cells as immature as metamyelocytes are
often seen on the peripheral smear,
• It is unusual to see more immature cells (myelocytes,
promyelocytes, and blasts). When these latter cells are
present, they indicate a "severe left shift", most likely due
to the presence of an acute or chronic myeloproliferative
disorder
- chronic myeloid leukemia
- idiopathic myelofibrosis
- acute leukemia

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Major causes of leukocytosis
• Any active inflammatory condition or infection
• Cigarette smoking, most common cause of mild neutrophilia
• Pregnancy and uncomplicated spontaneous or cesarean delivery
• Previously diagnosed hematologic disease
• Certain medications (glucocorticoids, catecholamines)
• Presence of, and treatment for, chronic anxiety state, panic
disorder, rage, or emotional stress
• Recent vigorous exercise, thermal burn, electric shock, surgery,
or trauma
• Laboratory artifact (platelet clumping, cryoglobulinemia)

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Leukopenia and leukocytosis
Take home messages
1. Laboratory error
2. Marrow storage pool
3. Collagen vascular disease
4. Bone marrow if + anemia and/or +
thromboctopenia
5. Bone marrow if severe left shift

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This "pink meanie" jellyfish feeds on other jellyfish species, such as moon jellies.
In 2011, with the help of DNA sequencing, researchers discovered that this large,
pink-hued jelly was, in fact, a new species in an entirely new family.
Photo: Mary Elizabeth Miller, Dauphin Island Sea Lab

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THE END