Identification and Diagnosis of the Acute Leukemias Morphology - Romanowsky stain, special stains (Sudan black, MPO, esterases, PAS) FAB A classification of leukemias based on morphology and predominant cell type. Myeloid M1- M7 or Lymphoid L1-L3 Immunophenotyping By Flow Cytometry Assign lineage Myeloid vs Lymphoid Subclassify lymphoid (pre B, T cell etc) Hybrid or mixed lineage

Identification and Diagnosis of the Leukemias Genetics Cytogenetics - Allows karyotyping of metaphase chromosomes Rt-PCR - Allows detection and amplification of abnormal gene sequences

Acute Leukemias Epidemiology Uncommon Disease 1 - 3/100,000 Childhood 80% ALL v.s. 20% AML Adult 20% ALL v.s. 80% AML Difference likely reflects the susceptibility lymphoid/myeloid precursors to leukemogenic events at different ages

Signs & Symptoms Leukemic cells infiltrate ® Organomegaly Dysfunction Lymph nodes ® Adenopathy Liver/Spleen ® Hepatosplenomegaly Skin/Gums CNS ® Confusion/Decrease level of consciousness Bone (rapid marrow expansion) ® PAIN

Signs & Symptoms Marrow failure due to replacement of normal precursors by leukemic blasts RBC ® Anemia ® Fatigue etc….. WBC ® Infections (Leukemic WBCs not functional) Platelets ® Hemorrhage Hyperviscosity - seen in acute leukemias with extremely high WBC (>100) Leukostasis - Confusion ® Coma Respiratory Distress

Acute Lymphoblastic Leukemia 6 year old male 2 week history of bruising fever of unexplained origin CBC LKC 32.3 x 10E9/L HB 58 g/L THR 20 x 10E9/L 95% blasts, 2% neutrophils, 3% lymphocytes

Acute Lymphoblastic Leukemia Flow Cytometry shows presence of a large population of precursor B cells CD19+CD10+ colored red (Slide 1) Absence of surface kappa or lambda light chains (Slide 2) Negative for myeloperoxidase, population of cells positive for TdT (Slide 3)

Acute Lymphoblastic Leukemia (Slide 1) Acute Lymphoblastic Leukemia CD45 CD4 CD8 SS CD19+10+ CD19 CD2 CD10 CD20

Acute Lymphoblastic Leukemia (Slide 2) CD 45 SS CD19 CD19 KAPPA LAMBDA

Acute Lymphoblastic Leukemia (Slide 3) CD45 SS CD45 CD45 MPO TdT

DNA Analysis By staining the nucleus of the cell with Propidium Iodide (PI) the DNA content relative to normal can be assessed. Normal cells have a DNA index of 1.0 Abnormal cells (aneuploid) can have an increased (hyperdiploid) or decreased (hypodiploid) flow DNA index (D.I.)

Acute Lymphoblastic Leukemia DNA Analysis Patient + Control Patient D.I. =1.0

Acute Lymphoblastic Leukemia DIPLOID ANEUPLOID DNA INDEX 1.4