Exam 2 Hematology

Neoplasm of hematopoietic tissue characterized by uncontrolled proliferation of neoplastic blood cells with replacement of normal bone marrow elements with neoplastic blood cells

• Attributed to mutation and altered expression of onocogenes and tumor suppressor genes, which results in unregulated cell proliferation

All ages

Adults

sudden

insidious

< 6 months

2-6 months

immature

mature

mild to severe

mild

mild to severe

mild

Variable

increased

mild

prominent

• CBC:
• Morphology of the blood cells
• Expression of various cell surface antigens
• chromosomal abnormalities

• Wright-Giemsa
• Cytochemical
• Iron

• Hematoxylin and eosin
• PAS or Leder
• Iron
• Reticulin

200 nucleated cells

500 nucleated cells

AML-M2, favorable prognosis

AML-M4, long remission

AML-M3, long survival

CML, ALL

Burkitt leukemia

ALL poor prognosis

AML/MDS poor prognosis

ALL (precursor B-cell type)

T- cell lineage leukemia

• Fatigue/Malaise
• Splenomegaly/Hepatomegaly
• Myelofibrosis with progression
• Extramedullary hematopoiesis
• Insidious course
• Blast crisis

Chronic Myelogenous Leukemia (CML)

a myeloproliferative neoplasm of pluripotent bone marrow stem cell

MPN (15-20%) of all cases of MPN

Philadelphia (Ph) chromosome and/or the BCR-ABL1 fusion gene

Chr 9 and 22

Chr 9 to Chr 22

(BCR/ABL1): produces a phosphoprotein (p190 and p210) with high tyrosine kinase activity

Exons: 12-16

Fusion proteins: p210 (CML)

Exons: 1-2

p190 (ALL)

Exons: 17-20

p230 (CML)

• Symptomatic or asymptomatic
• Fatigue, weakness, headache, fever, bone pain, night sweats
• Fullness due to hepatosplenomegaly

• Anemia (sometimes), sustained leukocytosis, thrombocytosis (>440k/uL)
• Circulating granulocyte precursors, absolute basophilia

low

<5%

Stable phase with leukocytosis, blasts <5%

• Presistent or increasing WBC (>10k/uL)
• Increasing splenomegaly
• Persistent thrombocytosis (>10^6/ul) or persistent thrombocytopenia
• clonal cytogenic evolution
• basophilia (>20% in PB)
• Blasts (10-19% in blood or marrow)

• Blasts >20% (Myeloid 70%; Lymphoid 30%)
• Extramedullary blast proliferation

• 70-90% respond to imatinib (Gleevac)
• 5-year progression free survival and OS- 80-95%

• R/O reactive leukocytosis (LAP score)
• Other myeloproliferative neoplasm (cytogenetics)
• Atypical chronic myeloid leukemia BCR-ABL1 (aCML) (not really the same disease)

• Leukocyte alkaline phosphate
• LAP activity widely distributed in human tissues and present in neutrophils
• decrease LAP in CML
• May be increased during remission
• Responsible for a depression in phagocytic function of neutrophils

1. Marked hypercellularity, increased M:E ratio (>10:1)
2. Myelocyte bulge
3. Small hypolobated megakaryocytes
4. Reticulin fibrosis may be present

Lymphoproliferative disorders

Leukemia that are not acute lymphoblastic leukemias:

• Chronic lymphocytic leukemia
• Prolymphocytic leukemia (B- or T-cell)
• Hairy cell leukemia
• T- cell large granular lymphocytic leukemia
• Adult T-cell leukemia

CLL (30% of all cases)

mature B-cell involving peripheral blood, bone marrow, spleen and lymph nodes

Small lymphocytic lymphoma (SLL)

>5X10^9/L monoclonal lymphocytes with a CLL phenotype in the peripheral blood

• Asymptomatic in the early stage
• Fatigue
• Generalized lymphadenopathy
• Splenomegaly
• Anemia from decreased production, marrow infiltration, or autoimmune process
• Infection: although B-lymphocytes are increased, they are not functional, and in fact patient has hypogammaglobulinemia

• Leukocytosis 10,000 to 600,000: absolute lymphocytosis/relative (or absolute) neutropenia
• Anemia: normocytic normochromic, if present, unless something else is going on
• Thrombocytopenia
• Anemia and htrombocytopenia usually indicated advanced disease

• CD19+, CD5+, CD23+, B-Cells
• CD20 expressionis lower than on normal B-cells
• Surface Ig is usually "dim"
• CD38 and ZAP-70 are important prognosis markers

CLL

• Some present with lymphadenopathy and no peripheral lymphocytosis
• Lymph node may be infiltrated by cells but they're identical to CLL cells
• SLL is pathologist diagnosis
• Many patients have both circulating component and a lymph node component

• Median survival 4-5 years
• 30% of patients have a 10 year survival
• Survival is highly variable
• Death may be due to infection: Richter's transformation
• Many die of unrelated illness to CLL

older adults

CLL

• form of chronic B-lymphocyte leukemia
• Named for hair-like, irregular cytoplasmic projections on cells in PB
• Nuclear morphology tends to be characteristics
• Pancytopenia
• Splenomegaly
• Marrow Fibrosis (Dry Tap)

• HCL cells have abundant clear cytoplasm that separates one cell from another, creates a "fried egg" appearance
• Spleen also has a characteristic morphology: splenectomy not common any more

Tartrate-resistant acid phosphate (TRAP) positive- most characteristic cytochemical feature

• Addition to morphology, immunophenotype is highly characteristic
• Bright surface Ig
• Bright CD20/22/11c
• Fried eggs

• Abrupt onset of signs and symptoms
• Weakness, pallor, fatigue
• Bleeding abnormalities
• Flu-like symptoms
• Bacterial infections
• Hepatosplenomegaly/Lymphadenopathy
• Bone Pain

• Morphology
• Cytochemistry
• Immunophenotyping: Flow cytochemistry, immunostain
• Cytogenetic

• Adults
• Median age is 63 years

• Acute nonlymphocytic leukemia (ANLL)
• Myeloblastic leukemia
• Myelogenous, myelocytic, or granulocytic leukemia

• CBC findings
• Anemia
• White cell count: variable
• Platelet count: decreased
• White blood cell differential (immature cells); PB >20% blasts

• Aspirate smear: By FAB classification >30% blasts, WHO criteria >20% blast
• Well prepared smears of bone marrow aspirate are required for morphological and cytochemical stains
• Biopsy specimen

• Myeloblast
• Promyelocytes

Myeloblast with immature cytoplasm with or without granules, some with Auer rods, loose open chromatin, some with distinct nucleoli

• AML (5%)
  
• Acute Myeloid Leukemia with no evidence of myeloid differentiation by morphology or cytochemistry
• Primitive leukemic blasts
• Negative for B or T cell markers
• CD34, CD38, HLA-DR, CD13 and/or CD117

• AML without maturation
• High number of blasts w/o more mature cells (<10%)
• Fine lacy, chromatin, distinct nucleoli
• MPO/SBB/NCAE +
• Auer rod may be present
• CD33, CD13, CD117, CD34 and HLA-DR

• AML with maturation
  
• Maturation to or beyond the promyelocytic stage
• Promyelocytes and later forms make up >10% of immature cells
• MPO/SBB/NCAE +
• t(8;21)
• CD33, CD13, CD15, CD34, HLA-DR, CD117

• Acute Promyleocytic leukemia
• Blasts and abnormal promyelocytes:
• FAB (>30%), WHO (APL >20%)
• Abundant cytoplasm
• Heavy Granulation (hypergranular)
• Auer rods frequently present

• DIC association
• Coagulation abnormalities
• Presence of schistocytes on PB smear
• Thrombocytopenia
• t(15;17)
• CD33, CD13, CD117
• MPO, SBB, NCAE +
• = CD34 and HLA-DR

• Microgranular variant
• Primary granules of leukemia of leukemic cells are not visible on smears
• Reniform or bi-lobed nucleus
• High WBC count
• MPO/SBB/NCAE +
• t(15;17)

Diffusely infiltrated by promyelocytes

• Acute myelomonocytic leukemia
• Common form of AML
• Granulocytic and monocytic differentiation
• MPO/SBB/NCAE, NSE positivity
• Monocytes in PB
• Increase serum lysozyme (muramidase)
• CD33, CD11b, CD13, CD14, CD15
• Blast + for CD34 and/or CD117

• Associated with BM eosinophilia with abnormal and immature forms
  
• Associated with abnormal chromosome 16 (inversion)
• Longer median survival than M4

• Poorly differentiated
• Predominance of monoblasts (>80%)
  
• Abundant cytoplasm
• Distinct nucleolus

• Well differentiated
  
• Promonocytes and monocytes
• Predominant cells in PB are monocytes

• Monocytes migrate to extramedullary sites: Hepatosplenomegaly, Lymphadenopathy, Skin and gum involvement, CNS involvement
• Increased serum and urinary lysozyme
• CD33, CD11, CD13, CD14, CD15
• Positive: NSE
• MPO/SBB: =

• Erythroleukemia
• Abnormal proliferation of myeloid and erythroid precursors
• Hypercellular bone marrow with marked erythroid hyperplasia
• Megaloblastic changes/dyserythropoiesis/ineffective erythropoiesis
• Myeloblasts and promyelocytes increase
• Ringed sideroblasts with iron stain

M6

Positive

• Acute megakaryoblastic leukemia
  
• Neoplastic proliferation of megakaryocytes and atypical megakaryocytes in BM
• Presence of megakaryocytic fragments and micromegakaryoblasts in PB
• MPO, SBB negative
• CD41, 42b, 61 positive

• Primary granules of myeloid cells
• Stains: Promyelocytes to neutrophils
• +: M1, M2, M3, M4, M6

• Stains lipids in cells
  
• Most sensitive stain for granulocytic cells
• similar reaction as MPO
• +: M1, M2, M3, M4, M6

• Stains specific enzymes in neutrophils
• similar to MPO and SBB
• Not sensitive
• +: M1, M2, M3, M4, M6

• Alpha-naphthyl acetate and alpha-naphthyl butyrate
• stains non-specific enzymes in monocytes and macrophages
• Neg: granulocytes and lymphoid cells
• +: M4 and M5

classified under the WHO category of AML with recurrent genetic abnormalities

classified under the WHO category of AML with recurrent genetic abnormalities

mixture of erythroid and myeloid cells

pure erythroid leukemia:

>80% pronormoblasts

also known as true erythroleukemia

Good

Good

better prognosis

• Cord blood treatment
  
• Bone Marrow transplant
• Chemotherapy

• Absence of leukemic blast
• Neutrophils > or = 1.5x10^9/L
• Platelets: >100x 10^9/L
• BM cellularity: >20%
• Normal maturation of cell lines

reappearance of blasts in the blood or more than 5% leukemic blasts in BM

• Primarily a disease of young children (2-5 yo)
• Malignant proliferation of lymphoid stem cells
• predominant cells in the bone marrow and peripheral blood are the lymphoblast

• 3,800 new cases per year in US
• 2,400 cases effect children
• comprises 75% of childhood leukemias

• Abrupt onset
• Fatigue, pallor, fever, weight loss
• anemia, thrombocytopenia, neutropenia
• bone pain in 80% of patients
• splenomegaly, hepatomegaly, lymphadenopathy

• N/N
• Thrombocytopenia
• Variable WBC
• BM: hypocellular >20% blasts
• Lymphoblast in BM and PB

small, uniform lymphoblast

large, pleomorphic lymphoblasts

Burkitt's type (vacuolated and deeply basophilic cytoplasm in lymphoblasts)

L1

L2

Burkitt Lymphoma

• Early precursor B-ALL
• Common ALL
• Pre B ALL

Burkitt Lymphoma

• t(8;14)
• t(2;8)
• t(8;22)

B-ALL

• Childhood cases exhibit L1 morphology
• Adult cases exhibit L2 morphology

blood/bone marrow

• Mediastinal mass
• increase WBC (50%)
• hepatosplenomegaly

T-Cell ALL

• CD19
• cCD79a
• cCD22
• CD10
• PAX5
• TdT
• CD34 (variable)

• TdT
• CD1a
• CD2
• cCD3
• CD7
• CD5
• CD4/CD8
• CD10

CD3

TdT

• HLA-DR
• TdT
• CD19
• CD20
• CD10

• HLA-DR
• TdT
• CD19
• CD20
• cIg
• 
  

• HLA-DR
• CD19
• CD20
• CD22
• CD10
• sIg

• HLA-DR
• CD1
• CD3
• CD2
• CD4/CD8
• CD5
• CD7
• CD10
• CD34
• TdT

PAS

L3

ALL from AML

Children

L3

Chemotherapy and Bone Marrow transplant

• Peroxidases are present in the primary granules of myeloid cells
  
• Promyelocytes and subsequent stages (pos)
• Monocytes a weak positive or negative

• Stains phospholipids, neutral fats, and sterols
• Phospholipids in both in primary adn secondary granules of granulocytic cells and to a lesser extent in monocytic granules
• Most sensitive stain for granulocytic cells

• Naphthol AS-D Chloroacetate esterase
• Present in neutrophils, basophils, and mast cells so will test positive
• Parallels the peroxidase and SSB stain, not as sensitive

• Alpha-naphthol acetate or butyrate
• Identifies monocytic cells
• Positive: monocytes, megakaryocytes, and platelets

monocytes, confirms AMonoL

• Stains for glycogen, mucoprotein, glycoprotein, glycolipids, and polysaccharides
• Abnormal erythroid precursors are positive
• Lymphocytes, granulocytes, monocytes, and megakaryocytes, may be positive

• Stains all hematopoietic cells

• Present in T-lymphocytes, dot-like appearance

• ID lymphoblast in acute T- cell lymphocytic leukemia

• Tartrate-resistant acid phosphatase stain
• Positive stain: HCL
• Stain specific for Hairy cells

• Used to differentiate CML from other disorders such as leukemoid reaction
• LAP enzyme present in granules of neutrophils
• LAP activity increases with stage of neutrophil maturity
• LAP is decreased in CML and increased in leukemoid reaction

CML from leukemoid rxn

AML or AMML from ALL

AML or AMML from ALL

AML or AMML from ALL

Monoblasts vs. myeloblast

ALL from AML

• heavy chain (IgM)
• Marker: B ALL
• Fluorescent microscope is used to determine if cytoplasmic Ig is present
• Direct immunofluorescence using fresh Blood smears or BM smears

• Marker of B lymphocytes and mature form of B-ALL
• Direct Immunofluorescence
• A fluorescent microscope is used to determine if surface immunoglobulin is present

• CD33

• CD13

• CD117

• CD64

• MPO

• CD79a
• PAX5
• CD19
• CD22
• CD20