Group of clonal hematopoietic stem cell disorders in which,
1. Inhibition of terminal myeloid differentiation 2. Over proliferation into STEM CELL compartment ACCUMULATION OF MYELOBLASTS IN THE BONE MARROW NORMAL HEMATOPOIETIC PROGENITOR CELL SUPPRESSED NORMAL HEMATOPOIETIC PROGENITOR CELL SUPPRESSION ANAEMIA THROMBOCYTOPENIA NEUTROPENIA All responsible for MAJOR CLINICAL COMPLICATIONS OF AML PATIENT SAMPLE COLLECTION
1. Blood Sample 2. Bone Marrow Sample Aspiration Trephine Biopsy Blood Cell Counting (P. smear) Routine microscopic examination Cytochemistry Flow cytometry & immunohistochemistry FISH PCR LABORATORY TESTS FEATURE ALL AML Leukemic blasts Lymphoblasts Myeloblasts Size Smaller, 10-15 m Larger, 12-20 m N/C Ratio High Low Chromatin Clumped Spongy Nucleoli <2; indistinct 2-5, distinct Nuclear membrane Irregular, convoluted Regular Auer Rods Not present Present in 10-20% FEATURE ALL AML MPO Negative Positive Sudan Black B Negative Positive NSE Negative Positive TdT Positive Negative PAS Positive (shows block pattern) Positive in <25% of cells Lysozyme Negative Positive CONDITION SPECIFIC MARKERS ALL T-cell CD1a, CD2, CD3, CD4, CD5,CD7, CD8, CD11b,CD25,CD45, CD56
B-cell CD10, CD19, CD20,CD21, CD22, CD23,CD79a, Sig, Ig NK cell CD16, CD56, CD57 AML CD13, CD14, CD15 ,CD33, CD41, CD61, CDw65, CD71, Glycophorin A, MPO CELL MORPHOLOGY MYELOBLASTS 1. Delicate nuclear chromatin 2. 2-4 nucleoli 3. Azurophilic, peroxidase +ve granules/Auer rods may be present. MONOBLASTS 1. Often folded/lobulated nuclei, 2. NO Auer rods 3. POD ve, NSE +ve PROMONOCYTE/ MONOCYTE Abn nuclear maturation, granulation , loss of basophilia. ERYTHROID PRECURSORS Normal/varying degrees of dyserythropoiesis BASOPHILS Rarely in AML/if present show abnormal granule formation & nuclear maturation. MEGAKARYOCYTES / . Dysplastic hyperlobated, hypolobated, multinuclear, small and blastic forms may be present. STAIN RESULT Myeloperoxidase (MPO) Identifies blast cells as myeloid. Dysplastic neutrophils may be -ve. Eosinophil granules always +ve. Monoblasts and promonocytes may be -ve. Sudan Black B Same Chloroacetate esterase (CAE) Specifically identifies cells of the granulocyte lineage. Non specific estrase (NSE) Specifically identifies cells of the Monoblast lineage. Alpha-Naphthyl Acetate esterase (ANAE) Stains monocytes and megakaryocytes at all stages of maturation. Toluidine blue Stains the granules of basophils and mast cells. Periodic Acid Schiff (PAS) It is not lineage specific but the pattern of staining may be helpful Diagnosis is confirmed by staining cells for myeloid specific surface markers CELL MARKERS Hematopoietic stem cell Progenitor CD34, CD117, TdT Myeloid cell
CD11b, CD11c, CD13, CD14, CD15, CD16, CD33, CD34, CD38, CD45, CD71,CD123 , CD163, MPO T-cell CD1a, CD2, CD3, CD4, CD5,CD7, CD11b, CD25,CD45, CD56, HLA-DR B-cell CD10, CD19, CD20,CD21, CD22, CD23,CD79a, SIg, HLA-DR, Ig Megakaryocyte CD41, CD61 Erythrocytes CD 71, CD235a, Glycophorin A Neutrophil CD11c, CD15, CDw65, MPO Monocytes/Macrophages CD4,CD33, CD64, CD 163, HLA-DR, MPO cCD79a-In AML, presence usually represents aberrant B cell antigen in leukemias of distinct myeloid linage, not biphenotypic differentiation
Revised FAB classification of AML S.NO CLASS INCIDENCE
1. M0 Minimally differentiated AML 2-3% 2. M1 AML without differentiation 20% 3. M2 AML with maturation
30-40% 4. M3 Acute promyelocytic leukemia 5-10% 5. M4 Acute myelomonocytic leukemia 15-20% 6. M5 Acute monocytic leukemia 10% 7. M6 Acute erythroleukemia 5% 8. M7 Acute megakaryocytic leukemia 1% MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPING 1. CLASS 1. CRITERIA M0 1. Myeloid blasts >20% of nucleated Bone marrow cells 2. MPO +ve on ultrastructural cytochemistry 3. <3% blasts +ve for Sudan Black B 4. CD 13, 33, 117 M1 1. Myeloid blasts >90% of BM nonerythroid cells (i.e also excluding lymphocytes, plasma cells, mast cells & macrophages from the count) 2. Maturing granulocytic cells (i.e promyelocytes to polymorphonuclear cells) <10% of nonerythroid cells 3. Promonocytes <10% of nonerythroid marrow cells 4. >3% blasts +ve for Sudan Black B 5. CD13, 33, 117, MPO M2 1. Blasts 20-89% of bone marrow nonerythroid cells 2. Maturing granulocytic cells (i.e promyelocytes to polymorphonuclear cells) >10% of nonerythroid cells 3. Monocytic cells (monoblasts to monocytes) <20% of nonerythroid cells & not meeting other criteria for M4 M3 1. Promyelocytes (hypergranular) >20% of BM nucleated cells M3 variant 1. Promyelocytes (hypogranular) >20% of BM nucleated cells 1. CLASS 1. CRITERIA M4 1. Blasts >20% of BM nucleated cells 2. Monocytic cells,precursors and neutrophils ,precursors are each more than 20%. 3. MPO +ve >3% blasts. 4. NSE +ve in cells of monocytic lineage 5. CD13, 33 (myeloid); CD14, LYSOZYME (monocytic) M5 1. >80% cells in the bone marrow are monocytic (monoblasts, promonocytes & monocytes). 2. There is intense NSE +vity 3. CD 14, 36, 64, 11c M6 1. More than 20% of non-erythroid cells are myeloblasts and more than 50% of all nucleated cells are erythroblasts Or 2. More than 80% of marrow cells are erythroblasts with no significant Myeloblastic component 3. PAS stain gives a diffuse or block positivity in erythroblasts. 4. MPO +ve in myeloblasts 5. Erythroblasts react with monoclonal antibody against glycophorin A M7
1. Megakaryoblasts are 20% or more in the marrow 2. Marked bone marrow fibrosis. 3. Blasts are platelet peroxidase, CD41 (glycoprotein IIb/IIIA) & CD61 (glycoprotein III a) +ve MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE Blasts >20% of nucleated BM cells No evidence of maturation CYTOPLASM : Scant Grey to light blue in color No granules No Auer rods Lack definitive cytological & cytochemical markers of myeloblasts .
NEGATIVE FOR ALL (<3% +ve for MPO,SB) CD 13 CD33 CD34 CD38 CD117 HLA-DR
*T-cell marker CD7 - frequently positive e.g of biphenotypic, mixed lineage, or hybrid acute leukemias. M0
Bone marrow aspirate smear, Wright-Giemsa stain M0 M0, MPO +ve <3% MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE 1. Blasts vary in size 2. Nuclei is round to oval/ irregular 3. Scant, Agranular usually, blue- grey cytoplasm 4. Auer rods few 5. In this setting, if Auer rods are seen, the diagnosis of M1 AML is established.
*By morphology alone, M1 blasts cannot be distinguished from M0, agranular M2 blasts or L2 blasts.
MPO,SB PAS NS E Bu CD 13 CD33 CD34 CD19 CD117 HLA-DR
>3% - - - M1
Bone marrow aspirate smear, Wright-Giemsa stain M1, MPO+ve M1 MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE Maturation down the granulocytic line Cells beyond Promyelocyte Differentiated neutrophils, some eosinophils , and rarely basophils Maturing cells are dysplastic (mostly) Nuclear chromatin coarser, clumped Type II blasts significant in number Unlike normal promyelocytes and early myelocytes, the Golgi apparatus is poorly developed. Nuclear maturation lags behind the cytoplasmic maturation. Auer rods are frequently visible.
MPO,SB PAS NSE CAE CD 13 CD33 CD34 CD99 CD117 HLA-DR
CD56 CD19 >3% - - + A variant associated with eosinophilia The eosinophils may show mild atypia, particularly in the more immatures ones, characterized by the appearance of coarse cytoplasmic granules ranging in color from deeply basophilic, resembling primary or basophil granules to those that have a salmon-like color. The more mature eosinophils usually are not atypical.
A very rare subtype, is also an example of AML with differentation- in this case down the basophil lineage. The leukemic blasts are type II and the cytoplasmic granules are coarse and basophilic, resembling those in normal mature basophils, and the cytoplasm is basophilic in color and may contain vacuoles. Some of the more mature forms often are dysplastic M2 AUER ROD M2
Bone marrow aspirate smear, Wright-Giemsa stain M2 AML with eosinophilia (M2Eo)
Bone marrow aspirate smear, Wright-Giemsa stain Sudan black B +ve blasts, stronger in dysplastic metamyelocytes. Inset: Chloroacetate in maturing granulocytes. MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE Arrest at the promyelocyte - late myelocyte stage Cell size & shape vary Poorly developed Golgi apparatus Variable nuclear:cytoplasmic ratio Cytoplasmic color - light blue to pink, variable number of granules that may have a pink, red, or dark purple color. The granules may obscure the nucleus. Auer rods & Faggot cells seen Round, oval, indented, reniform, Angel wing like nucleus The nuclear chromatin -coarse, clumped Nucleoli may /may not be visible.
MPO,S B CAE PAS NSE Bu CD2 CD4 CD11c CD13 CD33 CD34 CD45 CD56 CD64 CD117 + + - - - HYPERGRANULAR HYPOGRANULAR / MICROGRANULAR Most common subtype, 80% of cases Cytoplasmic granulation prominent Large (giant) granules may be seen Blasts vary in size Auer rods are common Granulation sparce, in some cells granules are not seen on a Wright- Giemsa stain Distinct morphological features such as bilobed nucleus Auer rods less frequent (confused with monoblast) M3 AML with Auer rods
Bone marrow aspirate smear, Wright-Giemsa stain M3
Bone marrow aspirate smear, Wright-Giemsa stain Sudan black B +ve Typical heavy cytoplasmic positivity Promyelocyte CAE +ve (blue) Inset: atypical ANAE +ve 1% of M3 cases. MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE Appear as typical myeloblasts or monoblasts/promonocytes, & cells that are difficult to categorize as belonging to either lineage . Depending upon which lineage they appear like the following varies, 1. Size & shape 2. The cytoplasm- Scant- Myeloblasts like Abundant- Monocytic like 3. N:C ratio 4. Nucleus variable in size and shape 5. Nucleoli prominent in those with monoblastic and promonocytic features. In the more differentiated cells, cytoplasmic granules that resemble promonocytes (fine granulation with a "salt and pepper" appearance) Auer rods may be seen
MPO,S B CAE PAS NSE Bu Myeloid lineage CD13+ CD33
Monocyte lineage CD4 CD14+ CD116 CD11c Lysozyme + + - + + 1. May represent up to 10% of all adult AMLs 2. Usually occurs at a younger age 3. Variable number of differentiating eosinophils present 4. Eosinophils are atypical (dysplastic). More mature eosinophils are less atypical 5. Coarse basophilic granules seen with a variable number of eosinophilic granules. 6. The morphologic picture is diagnostic and is associated with the inv(16) cytogenetic abnormality.
M4 Some blasts with myeloblastic features and some with monoblastic
Bone marrow aspirate smear, Wright-Giemsa stain, M4Eo AML with atypical young eosinophils M4E0 M4 AML MPO +ve M4 AML Sudan Black B +ve M4, NSE +ve CATEGORY M5a MONOBLASTIC, Undifferentiated M5b MONOCYTIC, Differentiated 1. Predominant cell type 2. Size 3. Cytoplasm
4. Vacuole 5. Auer rods 6. Nucleus 7. Chromatin 8. Nucleoli 9. Erythrophagocytosis Monoblast 1-1.5 times neutrophil size Abundant, color ranges from a medium to dark blue
Present Ab Varies in shape n size Fine 1 or <, Prominent Ab Promonocyte More variable Abundant, light blue to blue- grey, fine granules "salt and pepper appearance
More Prominent Ab Varies, Convoluted Clumped 1 or <, Less Prominent Present TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE M5a Vacuoles are PAS +ve MPO,SB ANAE PAS NSE Bu Stem cell/hematopoietic progenitor cell CD34- HLA-DR - Monocytic lineage CD4, CD14 CD11c CD64 CD68 CD116 lysozyme Myeloid lineage CD33 CD13 CD117 - + - + + M5b Both nuclear & cytoplasmic differentiation +/- + - + + M5a M5 AML Non-specific esterase (NSE) stain M5b TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE Dysplastic proerythroblasts predominate Varied-size and shape Presence of bizarre, giant forms Megaloblastic & dysplastic nuclear features prominent, including multilobated nuclei with Howell-Jolly bodies Marked lag in nuclear maturation wrt cytoplasmic Vacuoles prominent On Fe stain, ring sideroblasts A myeloblastic component may be present, tendency to transform to either an M1, M2, or M4 subtype with time MPO,SB CAE PAS NSE GlyA Stem cell/hematopoietic progenitor cell CD34- HLA-DR- Erythroid lineage Glycophorin A +ve Myeloid lineage CD13+ CD33+ CD117+ Monocytic lineage CD4+ CD14+ CD11b+ CD11a+ CD64+ +/- (M6a) - (M6b) +/-
- +
+ +/- (M6a) - (M6b) +
+ M6a : Proerythroblasts mixed with myeloblasts M6b : Proerythroblasts M6a AML with myeloblasts & erythroblasts
Bone marrow aspirate smear, Wright- Giemsa stain M6b AML with multiple Howell-Jolly bodies- M6a AML - myeloblasts & erythroblasts and with many cytoplasmic vacuoles M6a PAS+ve M6b Giant multinucleate late normoblasts PAS +ve Granular in proerythroblasts Homogeneous in normoblasts TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE Variable Microblasts like lymphoblasts a. Irregular cytoplasmic blebs b. Membrane projections c. Fine cytoplasmic granules (micromegakaryoblastic subtype) or very heterogenous population of blasts including, a. Large megakaryoblasts b. Blue cytoplasm c. With/without nuclear lobation & cytoplasmic granules On a biopsy specimen, fibrosis, ranging from reticulin to collagenous seen. MPO,SB PAS NSE AP Stem cell/hematopoietic progenitor cell CD34- HLA-DR- *CD45- Myeloid lineage(may be +ve) CD13 CD33 Megakaryocyte lineage CD41 (glycoprotein IIb/IIIa) CD61 (glycoprotein IIIA) CD36 (glycoprotein IIIb)
Platelet Peroxidase +ve - + + + *Common Leukocyte Antigen CD45 is NEGATIVE M7 AML Micromegak -aryoblastic subtype
Bone marrow aspirate smear, Wright-Giemsa stain M7 M7 AML with large, immature and bilobed megakaryoblast