HEMATOLOGY�Microscopy Session

Karen B. Damian, MD, FPSP

Department of Pathology

UP College of Medicine

Learning Objectives:

1. Recognize different morphologic changes in peripheral blood (PB) and bone marrow (BM)
2. Correlate the morphologic findings with the blood counts
3. Explain the morphologic findings in relation to the pathophysiology
4. Understand the development and consequences of hematologic neoplasms

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Outline

1. Review of hematopoiesis
2. Disorders of red cells
3. Disorders of platelets
4. Disorders of white cells
5. Summary

I. Hematopoiesis

II. Disorders of RBC

1. Anemias
1. Anemias of blood loss
• Acute
• Chronic
2. Hemolytic anemias
• Thalassemia syndromes
3. Diminished erythropoiesis
• Aplastic anemia *
• Iron deficiency anemia *
• Megaloblastic anemia
2. Polycythemias

Anemias of Blood Loss

• Acute blood loss (massive)
• Leukocytosis
• RBC: normochromic, normocytic
• Reticulocytosis: 10-15% after 7 days
• Thrombocytosis

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Anemias of Blood Loss

• Chronic blood loss
• Rate of loss exceeds regenerative capacity of BM
• Iron reserves are depleted
• Iron deficiency anemia

Thalassemia Syndromes

• β – Thalassemia Major
• PBS findings: anisocytosis, poikilocytosis, microcytosis, hypochromia
• Target cells, basophilic stippling, fragmented RBC
• β - Thalassemia Minor
• PBS findings: hypochromia, microcytosis
• Basophilic stippling, target cells

PBS – Thalassemia patient

II. Disorders of RBC

• Aplastic anemia
• Syndrome of chronic primary hematopoietic failure
• PB: pancytopenia (macrocytic normochromic anemia, neutropenia, thrombocytopenia)
• BM: markedly hypocellular; replaced by fat cells (appears “empty”)

Aplastic Anemia

Normal marrow�(20-year old)

Aplastic anemia

II. Disorders of RBC

• Iron deficiency anemia
• Depletion of iron stores 🡪 low Hb, Hct
• PB: hypochromic, microcytic red cells, poikilocytosis
• BM: increased erythropoiesis, low stainable iron

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Normochromic normocytic RBCs

Hypochromic �microcytic RBCs

Normal CBC

Iron deficiency anemia

Megaloblastic anemias

• Pernicious anemia (Vit B12 deficiency)
• Folate deficiency anemia
• PBS: macro-ovalocytes, anisocytosis and poikilocytosis, hypersegmented and large neutrophils
• BM: markedly hypercellular, megaloblastoid erythroid precursors, giant metamyelocytes and band forms

PBS- Megaloblastic anemia

II. Disorders of Platelets

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• Bleeding disorders related to thrombocytopenia
• Chronic Idiopathic Thrombocytopenic Purpura (ITP)
• Thrombocytosis *

Chronic idiopathic thrombocytopenic purpura

• Autoantibodies to platelets occurring in the setting of a variety of predisposed conditions and exposures
• PBS: megathrombocytes
• BM: increased number of megakaryocytes (immature forms present).

Megathrombocyte

BMA of an ITP patient

Thrombocytosis

• Refers to increase in platelet count
• May be reactive (e.g. chronic infection) or neoplastic (e.g. essential thrombocythemia)

IV. Disorders of WBC

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• Leukopenia
• Reactive (inflammatory) proliferation
• Neoplastic
• Lymphoid
• Myeloid

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Leukopenia

• Abnormally low WBC count
• Neutropenia more common than lymphopenia
• BM: compensatory hypercellularity due to increase in granulocytic precursors

Reactive proliferation

• Increase in the number of WBC in the blood
• Common reaction to inflammatory states
• Neutrophilia, eosinophilia, basophilia, monocytosis, lymphocytosis
• In sepsis, morphologic changes in neutrophils may be seen: toxic granulations, cytoplasmic vacuoles, Dohle bodies

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Toxic granulations

Cytoplasmic vacuoles

Dohle body

Lymphoid neoplasms

• B- or T- cell acute lymphoblastic leukemia
• Chronic lymphocytic leukemia/Small lymphocytic lymphoma
• Myeloma
• Lymphomas
• Precursor B- and T- cell
• Peripheral B- and T-, NK- cell
• Hodgkin

Lymphopoiesis

Acute Lymphoblastic Leukemia/Lymphoma

• Neoplasm of precursor B- or T- lymphoid cells
• 85% are B-ALLs
• PB: ↑or ↓ WBC, anemia, ↓ platelets
• BM: hypercellular with numerous lymphoblasts
• Lymphoblasts: scanty basophilic cytoplasm, delicate nuclear chromatin, inconspicuous nucleoli, convoluted appearance

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BM biopsy: ALL showing hypercellular marrow replaced by tumor cells.

BM aspirate: ALL showing numerous lymphoid blasts with round nuclei, inconspicuous nucleoli, fine chromatin, scant cytoplasm.

Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma

• Neoplasm of mature B-lymphoid cells
• PB: leukocytosis with lymphocytosis (>4000/mm³)
• BM: lymphoid aggregates or infiltrates

PB: CLL showing numerous mature lymphocytes with round to slightly irregular nuclei, inconspicuous nucleoli, clumped chromatin, scant cytoplasm.

Multiple Myeloma

• Neoplasm of plasma cells with production of monoclonal immunoglobulins
• Diagnosis based on clinical, radiographic and laboratory features
• PB: anemia (due to marrow replacement)
• BM: plasmacytosis (>30%)

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BM biopsy: MM showing numerous plasma cells replacing almost the entire marrow.

BM aspirate: MM showing numerous mature and immature plasma cells, including abnormal multinucleated forms.

Rouleaux formation: commonly seen in conditions with increased plasma fibrinogen or globulins, such as multiple myeloma.

Acute lymphoblastic lymphoma

• Neoplasms composed of immature B or T cells
• B-ALL > T-ALL
• Most common cancer of children
• BM: hypercellular composed mostly of lymphoblasts

Lymphoblastic lymphoma

Flow cytometry- B-ALL

Non-Hodgkin lymphoma

Flow cytometry- DLBCL

Hodgkin lymphoma

Hodgkin lymphoma

CD30 and CD15 staining in

RS cells

Myeloid neoplasms

• Acute myeloid leukemias
• Myeloproliferative neoplasms
• Myelodysplastic syndromes

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Acute Myeloid Leukemia

• Neoplasm of hematopoietic progenitors caused by acquired oncogenic mutations that impede differentiation
• PB: ↑or ↓ WBC, anemia, ↓ platelets
• BM: > 20% myeloid blasts in the BM
• Myeloblasts: delicate nuclear chromatin, 2-4 nucleoli, moderate cytoplasm with granules, Auer rods

PB: AML showing numerous blasts with irregular nuclei, multiple prominent nucleoli, fine chromatin, occasional Auer rods.

• Auer rod
• Formed by fusion of azurophilic granules
• Definitive evidence of myeloid differentiation
• Found in only 20% of AML’s

Flow cytometry - AML

Acute Promyelocytic Leukemia

• Type of AML with repressed myeloid maturation beyond promyelocytic stage
• t(15:17); PML-RARA fusion gene
• Cells may contain numerous Auer rods
• High incidence of DIC

PB: APML showing promyeloytes with multiple Auer rods (known as “faggot cells”).

BM: hypercellular in AML

BM: replaced by immature myeloid cells

Myeloproliferative neoplasms

• Chronic myeloid leukemia
• Polycythemia vera
• Primary myelofibrosis
• Systemic mastocytosis
• Chronic eosinophilic leukemia
• Stem cell leukemia

Chronic Myeloid Leukemia

• Neoplasm of terminally differentiated myeloid cells
• Distinguished from other MPNs by the presence of bcr-abl gene (Philadelphia chromosome)
• PB: leukocytosis (>100,000 cells/mm³), blasts, thrombocytosis
• BM: markedly hypercellular with granulocytic

(and often megakaryocytic) hyperplasia

PB: CML showing blast, myelocyte, metamyelocyte, and neutrophils.

BM aspirate and biopsy: CML showing granulocytic and megakaryocytic hyperplasia.

Cytogenetics: CML showing t(9;22), a defining characteristic to differentiate from other chronic myeloproliferative disorders (PV, ET, PMF).

Polycythemia vera

• Increased marrow production of RBC, granulocytes and platelets (panmyelosis)
• JAK2 mutation in >97% of cases
• BM: hypercellular due to increase in red cell, granulocytic precursors and megakaryocytes
• Spent phase: marrow fibrosis

Essential thrombocythemia

• JAK2 mutation in 50% of cases
• Elevated platelet count
• No polycythemia and marrow fibrosis
• BM: mildly hypercellular, markedly increase megakaryocytes with large abnormal forms
• PB: large platelets

Essential thrombocythemia

Primary myelofibrosis

• Obliterative marrow fibrosis
• JAK2 mutation in 50-60% of cases
• BM: initially, hypercellular with large, dysplastic megakaryocytes, minimal fibrosis
• Progression of disease: hypocellular and diffusely fibrotic, atypical megakaryocytic clusters, osteosclerosis
• PB: nRBC, left shift, dacryocytes

Primary myelofibrosis

Myelodysplastic syndromes

• Group of clonal stem cell disorders characterized by maturation defects that are associated with ineffective hematopoiesis
• High risk of transformation to AML
• BM: hypercellular (sometimes normo- or hypo-), dysplastic differentiation, myeloid blasts <20% of cellularity
• PB: pseudo-Pelger Huet cells, giant platelets, macrocytes, poikilocytes, myeloid blasts <10% of WBC count.

Dyserythropoiesis

Dysgranulopoiesis

Dysmegakaryopoiesis

v. Summary

• Identifiable morphologic features are associated with distinct entities
• Blood and bone marrow findings are explained by underlying pathophysiology
• Neoplasms may arise at any maturation stage involving one or more cell lines

QUESTIONS?