1. Acute leukemia Morphology and Cytochemistry.pdf

Acute leukemia: Morphology and Cytochemistry - ALL - AML  Acute leukemia Morphology and Cytochemistry ‫الدكتـور ه ــيثم أحم ــد الربي ــعي‬ Dr. HAITHEM AHMED AL-RUBAIE )1985( ‫بكلوريوس طب وجراحة عـامة – جامعة بغداد‬ )1998( ‫ب ــورد ع ــلم األم ـراض – أم ـراض ال...

Acute leukemia: Morphology and Cytochemistry - ALL - AML  Acute leukemia Morphology and Cytochemistry ‫الدكتـور ه ــيثم أحم ــد الربي ــعي‬ Dr. HAITHEM AHMED AL-RUBAIE )1985( ‫بكلوريوس طب وجراحة عـامة – جامعة بغداد‬ )1998( ‫ب ــورد ع ــلم األم ـراض – أم ـراض ال ــدم‬ MBChB; FICMS- Pathology (Hematology) ‫ جامعــة بغ ــداد‬- ‫ كليـة الط ــب‬- ‫فرع علم االمراض والطب العدلي‬ DEPARTMENT OF PATHOLOGY AND FORENSIC MEDICINE COLLEGE OF MEDICINE – UNIVERSITY OF BAGHDAD Classification of Acute leukemia is based on: 1. Morphology of blasts. 2. Cytochemistry: SBB, PAS, MPO, Estrases…etc 3. Immunophenotyping. 4. Genetic analysis includes: Cytogenetic techniques Molecular genetic techniques: a. FISH b. RT-PCR  FAB Classification (introduced in 1976) ALL; morphological features L1 L2 L3 Cell size Mainly small Large, heterogenous Large, homogenous Nuclear chromatin Fairly homogenous, Heterogenous Finely stippled, may be condensed homogenous in some cells Nuclear shape Mainly irregular Irregular; clefting & Regular; oval or indentation round common Nucleolus Not visible or small Usually visible, Usually prominent and inconspicuous often large Amount of Scanty Variable, often Moderately Cytoplasm abundant abundant Cyto. basophilia Slight to moderate Variable Strong Cyto. vacuolation Variable Variable Often prominent  ALL L1 Monomorphic blasts, majority small, high N/C ratio, scanty cytoplasm, small or inconspicuous nucleoli. ALL-L1  ALL – L2 Heterogeneous blasts, variable sizes & N/C ratios, more prominent nucleoli, nuclear membrane irregularities.  Cytochemistry in ALL L1 – L2 PAS positive PAS negative (coarse granular or blocks in cytoplasm)  ALL L3 (Burkitt Leukemia) Monomorphic large blasts with prominent nucleoli strongly basophilic vacuolated cytoplasm  Cytochemistry in ALL L3 Oil red O staining the lipid contents that correspond the cytoplasmic vacuoles Cytochemistry in T- cell ALL Acid phosphatase  WHO classification of acute lymphoid leukemias: Mod Pathol 2000;13(2):193-207 There was a consensus that FAB terms ALL - L1, L2, and L3 are no longer relevant: ALL L1 & L2 morphology do not predict immunophenotype, genetic abnormalities, or clinical behavior. ALL L3 is generally equivalent to Burkitt lymphoma in leukemic phase and should be diagnosed as such. 1- Acute lymphoblastic leukemia/lymphoma(former FAB ALL L1/L2) 2- Burkitt’s leukemia (Former FAB ALL L3)  WHO classification of AML 2001: Blood,2002;100(7): 2292-2302 The blast threshold for diagnosis of AML is reduced from 30% in FAB to 20% blasts in the blood or marrow. In addition, patients with the certain clonal, recurring cytogenetic abnormalities should be considered to have AML regardless the blast percentage.  FAB Classification AML; morphological features Auer rods: Distinctive red staining rods seen in cytoplasm of myeloblasts, representing abnormal azurophilic granules. They are diagnostic of AML.  Criteria for Diagnosis of AML M0 : AML with minimal differentiation 1. Blasts ≥ 30% of all nucleated BM cells (ANC). 2. Blasts ≥ 30% of BM non-erythroid cells (NEC)*. 3. < 3% of blasts positive for SBB or MPO** by light microscopy. 4. Blasts demonstrated as myeloblasts by immunological markers or by ultrastructural cytochemistry. *NEC: exclude also lymphocytes, plasma cells, macrophages and mast cells from the count. **MPO; myeloperoxidase, a special stain. AML M0  Criteria for Diagnosis of AML M1 : AML without maturation 1. Blasts ≥ 30% of ANC. 2. Blasts ≥ 90% of NEC. 3. ≥ 3% of blasts positive for SBB or MPO by light microscopy. 4. BM maturing granulocytic cells (promyelocytes to neutrophils) ≤ 10% of NEC. 5. BM maturing monocytic cells (promonocytes & monocytes) ≤ 10% of NEC. AML M1 AML M1  Criteria for Diagnosis of AML M2 : AML with maturation 1. Blasts ≥ 30% of ANC. 2. Blasts 30-89% of NEC. 3. BM maturing granulocytic cells > 10% of NEC. 4. BM monocytic components (monoblasts to monocytes) < 20% of NEC, and other criteria for M4 are not met. AML M2 AML M2 AML M2  Criteria for Diagnosis of AML M3 : Acute Promyelocytic Leukemia Classical M3 :  The predominant cells here are the abnormal hypergranular promyelocytes, the granules are coarse and almost obscuring the nucleus.  In some cases there are giant granules or multiple Auer rods (faggot cells).  Blasts are fewer than 30%. AML M3 (Faggot cell) AML M3 (Classical)  AML M3 Faggot cells Megakaryocyte like  Criteria for Diagnosis of AML M3 : Acute Promyelocytic Leukemia M3V (variant) :  The predominant cells are with reinform, bilobed, multilobed or convoluted nucleus with sparse fine granules or agranular cytoplasm.  Sometimes the nuclei are called Dumb-bell nuclei.  Faggot cells may also be seen. There is no clear demarcation between cases of classical M3 and M3V, cases with intermediate features are seen. AML M3V  Criteria for Diagnosis of AML M4 : Acute myelomonocytic leukemia 1. Blasts ≥ 30% of ANC. 2. Blasts ≥ 30% of marrow NEC. 3. BM maturing granulocytic cells ≥ 20% of NEC. 4. Significant monocytic components (monoblasts, promono- cytes and monocytes) as shown by one of the following: 5. a. BM monocytic components ≥ 20% of NEC and, PB monocytic components ≥ 5×109/L, or b. BM monocytic cells > 20% of NEC and confirmed by cytochemistry or increased serum or urinary lysozyme*, or c. BM resembling M2, but PB monocytic cells ≥ 5 ×109/L, confirmed by cytochemistry or increased lysozyme concentration. *Lysozyme estimation should exceed 3 × times the normal values in serum (11.5 ± 4 µg/mL), or urine (2.5 µg/mL). AML M4 AML M4 Cytochemistry in AML M1 – M4 SBB positive PAS negative Cytochemistry in AML M1 – M4 Myeloperoxidase MPO  Criteria for Diagnosis of AML M5: Acute monoblastic/monocytic leukemia 1. Blasts ≥ 30% of ANC. 2. Blasts ≥ 30% of NEC. 3. BM monocytic cells > 80% of NEC. ❑ AML-M5a (Monoblastic) : o Monoblasts ≥ 80% of BM monocytic component. ❑ AML-M5b (Monocytic) : o Monoblasts < 80 % of BM monocytic component. AML M5a AML M5a AML M5a AML M5b AML M5b  Cytochemistry in AML M4 & M5 Non-specific estrase (NSE) for monocytic cells (Deep Orange) Chloracetate estrase (CAE) for myeloblasts and myeloid precursors (Blue) AML M4 AML M5  FAB Criteria for Diagnosis of AML (OLD) M6 : Acute erythroleukemia 1. Erythroblasts ≥ 50% of ANC. 2. Blasts ≥ 30% of NEC. FAB AML M6 (Erythroleukemia)  AML M6 BMA BMA with PAS stain  FAB Criteria for Diagnosis of AML (New) M6 : Pure erythroid leukemia In the 2016 WHO revision;  Erythroleukemia is classified according to the number of blast cells as a percentage of ANC and is not separately recognized.  Pure erythroid leukemia: 1. > 80% of BM cells (ANC) being erythroid with 2. > 30% erythroid cells being proerythroblasts; 3. There is no significant myeloblastic component.  FAB AML M6 (Pure Erythroid leukemia)  Criteria for Diagnosis of AML M7 : Acute megakaryoblastic leukemia 1. Blasts* ≥ 30% of ANC. 2. Blasts demonstrated to be megakaryoblasts by immunological markers, ultrastructural examination or ultrastructural cytochemistry. *At least 50% of the blasts are megakaryoblasts.  AML M7 Peripheral blood Bone marrow aspirate  AML M7 Peripheral blood. Megakaryoblasts with cytoplasmic blebs.  AML M7 BM Biopsy. AML-M7 showing BM Biopsy. AML-M7 showing increased numbers of megakaryocytes. extensive marrow fibrosis. H&E stain. Reticulin stain.  Cytochemistry: Sudan Black B (SBB) in AML M1 – M4 Positive in AML Negative in ALL SBB is a lipophilic dye that binds irreversibly to an undefined granule component in granulocytes, eosinophils and some monocytes.  Cytochemistry: Myeloperoxidase (MPO) in AML M1 – M4 MPO is located in the primary and secondary granules of neutrophils and their precursors, in eosinophil granules and in the azurophilic granules of monocytes.  Cytochemistry: Periodic Acid-Schiff (PAS) Positive in ALL Negative in AML In hemopoietic cells, the main source of positive reactions is glycogen.  Because of their lack of specificity, cytochemical stains should be regarded as redundant in the diagnosis of ALL unless immunophenotyping is unavailable, and when used, there must be a constant awareness of their lack of specificity. Summary  The first step in the diagnosis of AL starts from morphology.  Subtyping is mainly based on BM findings  Special stains help in the diagnosis when immunophenotyping is unavailable