Blood Cell Identification Graded - College of American ... Folders... · Blood Cell Identification – Graded ... (malaria) 76 100.0 ... Allen S, Janda W, Koneman E, et al. Koneman’s

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Blood Cell Identification – Graded Case History

This peripheral blood smear is from a 39-year-old male who recently returned from a trip to Nigeria. He presented to the emergency room with a fever. He started having symptoms on the plane on the way home ten days ago. He complains now of myalgias, arthralgias, nausea, decreased appetite, cough, headache, and persistent fever. A peripheral smear is submitted for review. Laboratory data include; WBC = 5.0 x 10

9/L;

HGB = 12.6g/dL; HCT = 37.8%; MCV = 76.2fL.

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Referees Participants

Identification No. % No. % Evaluation

Plasmodium sp. (malaria) 76 100.0 4484 99.5 Good

The red blood cells identified by the arrows are infected by Plasmodium sp., as correctly identified by

100.0% of the referees and 99.5% of the participants.

There are five species of Plasmodium that cause the clinical disease known as malaria:

P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The different shapes and appearance of

the various stages of development and their variations between species are distinctive allowing

identification on thick and thin peripheral blood smears. The ring forms of all five types of malaria are

usually less than 2 μm in diameter. Potential look-alikes include platelets overlying red blood cells,

clumps of bacteria or platelets that may be confused with schizonts, masses of fused platelets that may

be confused with a gametocyte, precipitated stain, Babesia infection, and contaminating

microorganisms (bacteria, fungi, etc.).

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Blood Cell Identification – Graded

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Lymphocyte, large granular 50 56.2 2733 53.4 Good

Lymphocyte, reactive (to include plasmacytoid and immunoblastic forms)

33 37.1 1881 36.8 Acceptable

Lymphocyte 1 1.1 165 3.2 Acceptable

Monocyte 3 3.4 248 4.9 Unacceptable

Neutrophil, myelocyte 1 1.1 45 0.9 Unacceptable

Lymphocyte with phagocytized bacteria

1 1.1 6 0.1 Unacceptable

The white blood cell identified by the arrow is a large granular lymphocyte, as correctly identified by

56.2% of the referees and 53.4% of the participants. Large granular lymphocytes are defined as

medium to large cells with round nuclei, dense chromatin, and no visible nucleoli. Their cytoplasm is

moderate to abundant, clear or lightly basophilic, and contains several coarse, unevenly distributed,

azurophilic granules. These lymphocytes are found in blood smears from normal individuals and can be

increased in response to infections, such as infectious mononucleosis or in this case, malarial infection.

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Platelet, normal 89 100.0 5102 99.7 Good

The blood component identified by the arrow is a platelet, as correctly identified by 100.0% of the

referees and 99.7% of the participants. Platelets (also known as thrombocytes) are small, blue-gray

fragments of megakaryocytic cytoplasm typically measuring 1.5 to 3 μm in diameter. Fine, purple-red

granules are aggregated at the center or dispersed throughout the cytoplasm. They are typically single

but may form aggregates.

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Neutrophil, segmented or band 73 82.0 4083 80.0 Good

Neutrophil, toxic (to include toxic

granulation and/or Döhle bodies, and/or toxic vacuolization)

16 18.0 1005 19.6 Unacceptable

The white blood cell identified by the arrow is a normal segmented neutrophil, as correctly identified by

82.0% of the referees and 80.0% of the participants. Neutrophils have a segmented nucleus with

condensed nuclear chromatin. The nuclear segments are connected by thin filaments with no internal

chromatin structure. The cytoplasm is pale pink with specific granules.

Toxic changes in neutrophils include toxic granulation, Döhle bodies, and cytoplasmic vacuolization.

The neutrophil in image BCP-04 lacks toxic granulation and Döhle bodies, and contains only a few very

small cytoplasmic vacuoles, a finding likely representing EDTA degenerative change. Toxic granulation

and Döhle bodies each may be present in an individual cell without the other finding; either finding

alone is sufficient to designate the neutrophil as “toxic.” In contrast, since small cytoplasmic vacuoles

can be a degenerataive artifact of EDTA storage, cytoplasmic vacuolization is best considered toxic

only if accompanied by toxic granulation and/or Döhle bodies.

Toxic granulation is the presence of large purple or dark blue granules within segmented neutrophils,

bands, or metamyelocytes (Images 1-3). In image BCP-04, very small normal primary granules are

discernable, but they do not appear enlarged and are not as basophilic in their staining characteristics

as toxic granules. Döhle bodies are found within neutrophilic cytoplasm and appear as single or multiple

blue to grey-blue inclusions of variable size and shape (Image 2, arrow), a feature lacking in this case.

These inclusions represent denatured aggregates of free ribosomes or stacks of endoplasmic reticulum.

Vacuoles within the cytoplasm of cells showing toxic granulation and/or Döhle bodies constitute toxic

vacuolization. These toxic vacuoles (Image 3) are variable in size, may coalesce, and generally are

larger and more numerous than those associated with degenerative change. EDTA storage may

produce degenerative vacuolization that is comprised of a few, small, punch-out appearing vacuoles, as

in this case.

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Blood Cell Identification - Graded

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Image 1

Image 2

Image 3

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Erythrocyte with overlying

platelet 86 96.6 4825 94.3 Good

Platelet, normal 3 3.4 260 5.1 Unacceptable

The blood cell identified by the arrow is an erythrocyte with an overlying platelet, as correctly identified

by 96.6% of the referees of the 94.3% of the participants. This artifact is important to distinguish from

red cell inclusions or parasites. Many times, platelets overlying red cells are surrounded by a thin clear

zone or halo, which is not a feature of most genuine red cell inclusions. Of interest, the red blood cell

above the red blood cell identified in this photo is infected by Plasmodium sp.

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Discussion

This case illustrates a blood smear from a patient infected with Plasmodium sp. There are five species of

Plasmodium that cause the clinical disease known as malaria: P. falciparum, P. vivax, P. ovale, P. malariae, and

P. knowlesi. Malarial infection is transmitted by infected Anopheles mosquitos. The distribution of malarial

endemic regions spans >95 countries, with ~500 million cases reported each year. Those most at risk for infection

are children <5 years of age, pregnant women, refugees from endemic countries, and non-immune travelers to

endemic regions.

The life cycle of Plasmodium begins with the bite of the vector, an Anopheles mosquito, whose saliva contains

infective sporozoites that can be injected through its proboscis. As the infected saliva enters the human host, the

sporozoites circulate in the bloodstream for 20-30 minutes before entering the liver, where they will lay dormant

as they multiply for the next 10 days. At the end of the 10 days, multiple of these small ring forms (called

merozoites) break free from the parenchymal cells of the liver and are released into the bloodstream to infect

erythrocytes.

The pathophysiology of infection belies its clinical presentation, wherein parasite-infected red blood cells

accumulate in various organs, including the heart, brain, liver, and lungs. Patients may have a non-specific

presentation of malaise, fever, and myalgias, which typically occur 7-30 days after the mosquito bite. This quickly

progresses to splenomegaly, anemia, and jaundice. In severe infections (usually caused by P. falciparum), the

patient may develop cerebral encephalopathy, hypoglycemia, hypotension, liver dysfunction, disseminated

intravascular coagulation, acute renal failure, and hemoglobinuria (“blackwater fever”).

Identification of malarial infection in humans is performed by examining Romanowsky stained (often Giemsa)

thick and thin peripheral blood smears under a 100x oil immersion objective. Ideally, the blood smears are

obtained at different intervals in the day to account for the fluctuating numbers of circulating parasites in the blood

stream. Of the five malarial species, P. vivax (most common), P. falciparum, and P. malariae are the most

commonly seen. P. ovale and P. knowlesi are rarer species.

A stepwise approach to the laboratory characterization of malarial species in blood smears is recommended, as

microscopic examination remains the “gold standard” for detection. As P. falciparum causes the most virulent of

the malarial infections, with potential for severe symptoms and rapid progression to death, it is imperative to first

exclude this species. The following features will be seen in a P. falciparum infection (see Image 1): a) banana- or

crescent-shaped gametocytes are pathognomonic (but uncommonly seen); b) tiny ring forms (trophozoites),

which occupy <1/3 the diameter of the red blood cell; c) multiple ring forms in one red blood cell, with 2 nuclei in

the same ring; d) “heavy” malarial load, with >20% of erythrocytes infected by parasites, indicating a severe

infection; e) “applique” effect, whereas ring forms appear “stuck-on” the red blood cell membrane.

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Image 1

If none of the above 5 features are seen, a search for evidence of other malarial organisms, including P. vivax, is

undertaken. Features of P. vivax infection include (see Image 2): a) somewhat irregularly-shaped erythrocytes,

which are enlarged, pale, and contain pink-red staining granules (so-called Schuffner’s dots); b) “ameboid” quality

of erythrocyte, wherein mature trophozoites begin to fill the entire red blood cell; c) merozoites with 12-14 or more

individual schizonts (early segmented form with more than one nucleus); d) large and circular gametocytes

(arrowhead) which fill more than half the cell diameter; e) finely granular and brownish pigment in both the

gametocyte and schizont.

Image 2

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When blood smears are positive for parasites, a quantification/percentage of erythrocytes by parasites must be

provided to the clinician to provide an index of malarial load. Other laboratory tests available include the rapid

antigen tests or rapid diagnostic tests (RDTs) which utilize a qualitative immunochromatographic assay/stain

method -- useful in the rapid diagnosis or exclusion of P. falciparum. This rapid screen does not detect

parasitemia levels less than 0.5%. All positive RDTs must be confirmed on microscopy. Nucleic acid testing is

very sensitive and specific, using PCR (polymerase chain reaction) testing as its platform. Often, results are not

available quickly enough in the routine laboratory setting, limiting its utility in the rapid diagnosis of acutely ill

patients. For now, its use is confined to confirmation of the specific species of malaria. Serologic testing for IgG

malaria antibody using an ELISA method is limited to screening for chronic malaria or retrospectively diagnosing

malaria in a previously non-immune individual.

References:

1. Centers for Disease Control and Prevention: Malaria References and Resources. Centers for Disease

Control and Prevention. Atlanta: Georgia [8 Feb 2010; Accessed: 8 Jan 2011].

2. Collins WE, Jeffrey GM. Plasmodium malariae: parasite and disease. Clin Microbiol Rev 2007;20:579-

592.

3. Winn W, Allen S, Janda W, Koneman E, et al. Koneman’s Color Atlas and Textbook of Diagnostic

Microbiology, 6th ed. Philadelphia: Lippincott Williams and Wilkins, 2006.

4. World Heath Organization: Malaria fact sheet. World Health Organization. Geneva: Switzerland

[Accessed: 8 Jan 2011].

Joan E. Etzell, MD, Vice Chair

Maria Vergara-Lluri, MD

Hematology and Clinical Microscopy Resource Committee

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Blood Cell Identification – Ungraded

Case History

This peripheral blood smear is from a 17-year-old patient who presents to the emergency room with ecchymoses. Laboratory data include: WBC = 58.2 x 10

9/L; HGB = 6.6 g/dL; PLT = 6.6 x10

9/L; Fibrinogen = 63

mg/L; Prolonged PT and PTT; and markedly elevated D-dimer level.

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Lymphocyte 78 89.7 4466 88.7 Educational

Lymphocyte, reactive (to include plasmacytoid and immunoblastic forms)

4 4.6 162 3.2 Educational

Nucleated red cell, normal or abnormal morphology


Neutrophil with dysplastic nucleus and/or hypogranular cytoplasm


The white blood cell indicated by the arrow is a lymphocyte, as correctly identified by 89.7% of the referees

and 88.7% of the participants. Lymphocytes may exhibit variation in size ranging from 7 to 15 µm and in

N:C ratio from 5:1 to 2:1. Most lymphocytes have round to oval nuclei that may be slightly indented or

notched. The chromatin is diffusely dense or coarse and clumped. Most lymphocytes have a scant amount

of pale blue to moderately basophilic, agranular cytoplasm. Some lymphocytes show a perinuclear clear

zone or halo which surrounds the nucleus. Also of note, the cell in the lower left of this image is an

abnormal promyelocyte with multiple Auer rods, a finding that is common in acute promyelocytic leukemia

with t(15;17).

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Fragmented red cell

(schistocyte, helmet cell, keratocyte, triangular cell)

87 100.0 4958 98.9 Educational

The red blood cell identified by the arrow is a fragmented red cell, as correctly identified by 100.0% of the

referees and 98.9% of the participants. This cell is also known as a triangulocyte or a schistocyte. Other

fragmented red blood cells include helmet cells and keratocytes (horn cells). Fragments should not have

central pallor; such cells are best considered non-specific poikilocytes. Fragmented cells are seen in

severe burns, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TIP)

and other microangiopathic hemolytic anemias. Patients with acute promyelocytic leukemia, as in this

case, are at risk for disseminated intravascular coagulation (DIC). When present in large numbers, red cell

fragments may cause the MCV to fall into the microcytic range or interfere with platelet enumeration.

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Neutrophil, segmented or band 84 96.6 4836 96.4 Educational

Neutrophil with dysplastic nucleus and/or hypogranular cytoplasm


The arrowed cells are neutrophils, as correctly identified by 96.6% of the referees and 96.4% of the

participants. The upper arrowed cell slightly to the right is a segmented neutrophil while the lower arrowed

cell would be considered a band neutrophil. The nucleus of a band neutrophil is indented to more than half

the distance to the farthest nuclear margin, but in no area is the chromatin condensed to a single filament.

The nucleus can assume many shapes: band-like, sausage-like; S, C, or U-shaped; and twisted and

folded on itself. The cytoplasm has specific granules predominating in the pale cytoplasm. Band

neutrophils comprise 5-10 percent of the nucleated blood under normal conditions, but may be increased

in the blood in a number of physiologic and pathologic states. Band neutrophils comprise 10-15% of the

nucleated cells in the bone marrow. The other leukocytes in this image are abnormal promyelocytes.

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Referees BCP Participants Performance


Platelet, normal 83 95.4 4870 97.1 Educational

Platelet, hypogranular 3 3.5 119 2.4 Educational

Stain precipitate 1 1.1 4 0.1 Educational

The blood component identified by the arrow is a platelet, as correctly identified by 95.4% of the referees

and 97.1% of the participants. Platelets, also known as thrombocytes, are small, blue gray fragments of

cytoplasm measuring 1.5 to 3 µm in diameter. Fine, purple-red granules are aggregated at the center or

dispersed throughout the cytoplasm. Platelets may vary in shape but most are round or elliptical. They are

typically single but may form aggregates.

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Neutrophil promyelocyte,

abnormal with/without Auer rod(s)

55 64.7 3208 64.1 Educational

Myeloblast with Auer rod 20 23.5 1075 21.5 Educational

Neutrophil, myelocyte 3 3.5 207 4.1 Educational

Eosinophil, any stage 2 2.4 108 2.2 Educational

Leukocyte with phagocytized bacteria


Monocyte, immature (promonocyte, monoblast)

1 1.2 1 <0.1 Educational

Neutrophil, promyelocyte 1 1.2 31 0.6 Educational

Plasma cell, abnormal (malignant, myeloma cell)


The arrowed cell is an abnormal promyelocyte containing multiple Auer, as correctly identified by 64.7% of

the referees and 64.1% of the participants; these cells are also sometimes referred to as myeloblasts with

Auer rod as identified by 23.5% of referees and 21.5% of participants. Auer rods are inclusions which

represent a crystallization of azurophilic (primary) granules. A cell containing multiple Auer rods is referred

to as a faggot cell (from the English faggot, meaning a cord of wood). Faggot cells are most commonly

seen in acute promyelocytic leukemia.

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Discussion

Acute promyelocytic leukemia with t(15;17)(q22;q21); PML-RARA

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia characterized by the

t(15;17)(q22;q21) resulting in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor

alpha (RARα) gene (PML/RARα). The fusion product PML-RARα homodimerizes, binds to DNA and works as a

transcriptional repressor which inhibits expression of target genes necessary for granulocytic differentiation.

Variant chromosomal translocations, such as t(11;17), t(5;17), can be detected in up to 2% of APL cases.

This leukemia accounts for approximately 10-15% of acute myeloid leukemia cases in the United States. Most

patients present with leukopenia along with a coagulopathy, fibrinolysis and proteolysis, which can be life

threatening and requires emergent therapy along with prompt diagnosis by integration of morphologic findings,

immunophenotype and cytogenetic/FISH studies. This type of leukemia is very uncommon in children less than

10 years of age, and its incidence increases steadily until it plateaus during early adulthood and remains constant

until it decreases after age 60.

The two main morphologic types of APL are the classic hypergranular or typical variant, as in this case, which

comprises the majority of APL cases, and the microgranular or hypogranular variant, comprising approximately

15-20% of APL cases. Leukopenia is typically seen in association with the hypergranular variant, with rare

circulating leukemic promyelocytes found in the peripheral blood. A leukocytosis is more commonly seen in the

microgranular variant. The microgranular subtype is also associated with the short bcr 3 type PML-RARA fusion

gene and FLT3 mutations.

Leukemic promyelocytes of either subtype can be classified as blasts in a peripheral blood or bone marrow

differential. The leukemic promyelocytes of the hypergranular variant demonstrate nuclear irregularity and

variability with reniform or bilobed nuclei. Nucleoli are variably prominent. The cytoplasm is abundant and

characterized by the presence of numerous large azurophilic cytoplasmic granules that can often obscure the

nucleus. These leukemic promyelocytes can also contain numerous bundles of Auer rods (faggot cells) as seen in

images BCP-06, -09 and -10. Myeloblasts which may or may not contain single Auer rods may also be seen. The

Auer rods of the hypergranular variant are usually larger than in other types of AML. The microgranular variant

blast contains a primarily bilobed, reniform or monocytoid like nucleus; the cytoplasm appears agranular or may

contain fine dust like azurophilic granules. Multiple Auer rods are less frequently seen in this variant.

The immunophenotype of hypergranular APL blasts demonstrates bright expression of CD33 and cytoplasmic

myeloperoxidase, variable CD13 expression and largely lack of expression of HLA-DR and CD34. Blasts of the

hypogranular variant demonstrate similar findings although may also show dim expression of HLA-DR and CD34;

the blasts also commonly express the T-lineage associated marker CD2, a finding which has been associated

with a less favorable prognosis. CD56 expression can be seen in 15-20% of APL and has been associated with a

less favorable clinical course. CD117 expression can be seen in both variants. The blasts do not typically express

CD15 or CD11b. Lack of expression of CD34 and HLA-DR is not specific for a diagnosis of APL, and integration

of ancillary studies, such as conventional cytogenetic or FISH results, should be used in confirming a diagnosis.

Very strong myeloperoxidase expression is seen in the abnormal promyelocytes by cytochemistry as well as by

flow cytometry which can be helpful in distinguishing these blasts, especially the hypogranular blasts, from

monoblasts. Sudan black B and chloroacetate esterase cytochemical staining are strongly positive in the blasts of

both variants.

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This prognosis of APL is favorable, especially when compared to other subtypes of acute myeloid leukemia. The

current standard approach to therapy is with the concomitant administration of targeted terminal granulocytic

differentiation therapy with all-trans retinoic acid (ATRA), and anthracycline based chemotherapy. This

combination leads to complete remission in 90-95% of patients. Arsenic trioxide (ATO) is also an effective therapy

in patients who have relapsed. Minimal residual disease (MRD) monitoring can be performed by sequential

quantitative reverse transcription polymerase chain reaction (RT-PCR). This can be used to predict relapse,

especially in cases with evidence of morphologic remission, and to direct preemptive therapy.

Measures to counteract the coagulopathy associated with APL should be instituted immediately if this diagnosis is

considered, as intracerebral and pulmonary hemorrhages can occur prior to and during induction of therapy.

Relapse can occur in 10-20% of patients. Patients at high risk for relapse include those with an elevated WBC

(>10000/uL or 10 x 109/L) at diagnosis, age over 55 years, expression of CD56 on the blasts as well as the

predominance of the PML-RARAbcr3 isoform. Overall survival is approximately 80%.

References:

1. Akagi T, Shih L, Kato M, et al. Hidden abnormalities and novel classification of t(15;17) acute

promyelocytic leukemia (APL) based on genomic alterations. Blood, 2009; 113: 1741-1748.

2. Cassinat B, de Botton S, Kelaidi C, et al. When can real-time quantitative PT-PCR effectively define

molecular relapse in acute promyelocytic leukemia patients? Leukemia Research, 2009; 33: 1178-1182.

3. Chauffaille M, Borri D, Proto-Siqueira R, et al. Acute promyelocytic leukemia with t(15;17): frequency of

additional clonal chromosomome abnormalites and FLT3 mutations. Leukemia and Lymphoma, 2008;

49: 2387-2389.

4. Collins S. Retinoic acid receptors, hematopoiesis and leukemogenesis. Current Opinion in Hematology,

2008; 15: 346-351.

5. Dunphy CH, Polski JM, Johns G, et al. Acute promyelocytic leukemia, hypogranular variant, with

uncharacteristic staining with chloroacetate esterase. Leukemia and Lymphoma, 2001; 42: 215-219.

6. Foucar K,Reichard K, Czuchlewski D. Bone Marrow Pathology. ASCP Press: Chicago, 2010.

7. Grimwade D, Jovanovic JV, Hills RK, et al. Prospective minimal residual disease monitoring to predict

relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy. Journal of

Clinical Oncology, 2009; 27: 3650-3658.

8. Jaffe ES, Harris NL, Vardiman JW, Campo E, Arber DA. Hematopathology. Saunders/Elsevier:

Philadelphia, 2011.

9. Kelaidi C, Chevret S, de Botton S, et al. Improved outcome of acute promyelocytic leukemia with high

WBC counts over the last 15 years: the European APL group experience. Journal of Clinical Oncology,

2009; 27: 2668-2676.

10. Lin P, Hao S, Medeiros J, et al. Expression of CD2 in acute promyelocytic leukemia correlates with short

form of PML-RAR transcripts and poorer prognosis. American Journal of Clinical Pathology, 2004; 121:

402-407.

11. Nowak D, Stewart D, Koeffler HP. Differentiation therapy of leukemia: 3 decades of development. Blood,

2009; 113: 3655-3665.

12. Ravandi F, Estey E. Jones D, et al. Effective treatment of acute promyelocytic leukemia with all-trans-

retinoic acid, arsenic trioxide, and gemtuzumab ozogamicin. Journal of Clinical Oncology, 2009; 27: 504-

510.

13. Sanz M, Grimwade D, Tallman MS, et al. Management of acute promyelocytic leukemia:

recommendations from an expert panel on behalf of the European LeukemiaNet. Blood, 2009; 113:

1875-1891.

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14. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. World Health

Organization Classification of Tumours of Hematopoietic and Lymphoid Tissues. IARC Press: Lyon, 2008,

p112-113.

15. Tallman MS.Treatment of relapsed or refractory acute promyelocytic leukemia. Best Practice & Research,

2007; 20: 57-65.

16. Tallman, M. What is the role of arsenic in newly diagnosed APL? Best Practice & Research, 2008; 21:

659-666.

17. Tallman M, Altman J. Curative strategies in acute promyelocytic leukemia. American Society of

Hematology, 2008; 391-399.

18. Wang Z, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood, 2008; 111:

2505-2515.

Lydia C. Contis, MD

Alice L. Werner, MD

Hematology and Clinical Microscopy Resource Committee

Documents

Blood Cell Identification Graded - College of American ... Folders... · Blood Cell Identification – Graded ... (malaria) 76 100.0 ... Allen S, Janda W, Koneman E, et al. Koneman’s