FAB CLASSIFICATION OF LEUKEMIAS Background Organized in 1976 in an attempt to provide a uniform means of discussing the leukemias worldwide. The classifications were based on morphology and cytochemistry : Peripheral Blood Findings 90%of patients have moderate to severe neutropenia 50%of patients have a leukocytosis 30%of patients have a leukopenia The blasts are of variable size 70-80%of the patients have normochromic, normocytic anemia 60%of the patients have hematocrits of <30% Thrombocytopenia is usually present : Bone Marrow Findings Blast count of ≥30% is diagnostic of acute leukemia : Cytochemistry Type I, II, and III myeloblasts show ≥3% positivity of blasts for myeloperoxidase, Sudan black B, or specific esterase Monoblasts and promonocytes are positive with the nonspecific esterase Erythroblasts and megakaryoblasts are positive with the periodic acid-Schiff Definitions ============================= Type I Myeloblast Size: 10-18 μ : Nuclesus Shape: Oval or round N/C Ratio: high Color: Dark purple Chromatin: Fine Nucleoli: 1-3 : Cytoplasm Color: Light to medium blue Contents Without azurophilic granules Clinical Conditions Acute myelocytic leukemia minimally differentiated (MO) Acute myelocytic leukemia without maturation (M1) • Acute myelocytic leukemia with maturation (M2) Acute myelomonocytic leukemia (M4) Erythroleukemia (M6a) Myeloproliferative neoplasms-chronic myelogenous leukemia, primary myelofibrosis ====================== Type II Myeloblast Size: 10-18 μ : Nuclesus Shape: Oval or round N/C Ratio: Slightly lower than type I Color: Dark purple Chromatin: Slightly more condensed than type I Nucleoli: 2-5 : Cytoplasm Color: Medium blue Contents: <20 azurophilic granules and may have Auer rods Clinical Conditions • Acute myelocytic leukemia without maturation (M1) • Acute myelocytic leukemia with maturation (M2) • Acute myelomonocytic leukemia (M4) • Erythroleukemia (M6a) Myeloproliferative neoplasms-chronic myelogenous leukemia, primary myelofibrosis ======================== Type III Myeloblast Size: 10-18 μ : Nuclesus Shape: Oval or round N/C Ratio: Lower than type I Location: Centrally located Color: Dark purple Chromatin: Slightly more condensed than type II Nucleoli: Less visible : Cytoplasm Color: Medium blue Contents: >20 azurophilic granules but don't obscure the nucleus Clinical Conditions Acute myelocytic leukemia with maturation (M2) ============================ Abnormal Promyelocyte Size: 18-25 μ : Nucleus Shape: Round or, more commonly, reniform or bilobed 2/N/C Ratio: 1 Color: Purple Chromatin: Relatively fine, becoming coarser Nucleoli: 2-3 varying from visible to indistinct : Cytoplasm Hypergranular type Color: Intensely basophilic Contents: Large red to purple granules; Auer rods may be numerous and intertwined, giving haystack appearance (faggot cells); may obscure the nucleus Microgranular type Color: Moderately basophilic Contents: Small, indistinct granules that are difficult to see with the light microscope; Auer rods are often found but not as abundant as those found in the hypergranular type Clinical Conditions Acute promyelocytic leukemia (M3, M3v) ============================ L1 Lymphoblast Size: 14-22 μ : Nucleus shape is regular or small cleaved and indented Purple nucleus has a homogeneous and condensed chromatin pattern Nucleoli are inconspicuous or not visible : Cytoplasm is scanty moderately basophilic and rarely vacuolated Clinical Condition Precursor lymphoblastic leukemia ======================= L2 Lymphoblast Size: 14-22 μ : Nucleus has an irregular or indented shape N/C ratio: high Nucleus is purplish-red with variable heterogeneous chromatin One to two nucleoli are often prominent : Cytoplasm is variable but occasionally intensely basophilic and rarely vacuolated Clinical Condition Precursor lymphoblastic leukemia ========================= L3 Lymphoblast Size: 14–18 μ : Nucleus is oval to round, is purple, and has a finely stippled and homogeneous chromatin pattern N/C ratio : slightly lower than type 2 One to two nucleoli are often prominent : Cytoplasm is intensely basophilic with prominent vacuolization Clinical Conditions Burkitt lymphoma Acute lymphoblastic leukemia (L3) Burkitt leukemia/lymphoma =============================== ACUTE MYELOID LEUKEMIA MO (Acute Myeloid Leukemia With Minimal Differentiation) Criteria Peripheral Blood : Blasts are agranular■ Platelets are decreased Bone Marrow : >30% blasts■ ≥20% blasts are reactive for myeloid-associated antigen ■Myeloblasts are type I ■No Auer rods ■Blasts are negative for lymphoid-associated antigens ■ Blasts are positive for myeloid-associated antigens Cytochemistry : <3%blasts are myeloperoxidase, Sudan black B, and specific esterase (chloroacetate) positive ============================ M1 (Acute Myeloid Leukemia Without Maturation) Criteria Peripheral Blood : The predominant cell is usually a type I myeloblast■Auer rods are rare Bone Marrow : >30% blasts ■≥90% or more of the nonerythroid cells are myeloblasts ■<10% promyelocytes or more mature cells of the granulocytic series Cytochemistry : Myeloperoxidase and Sudan black B are positive in >3% of the blasts ■Naphthol AS-D chloroacetate (specific esterase) may be positive■Nonspecific esterases are negative ========================= M2 (Acute Myeloid Leukemia With Maturation) Criteria Peripheral Blood : Type II myeloblasts may be the predominant cell■Auer rods typically present Bone Marrow : >30% blasts■30-89% or more of the nonerythroid cells are myeloblasts■≥10% are promyelocytes or more mature granulocytes ■Blasts are predominantly type II or type III Cytochemistry : ≥3% blasts are myeloperoxidase and Sudan black B positive■Naphthol AS-D chloroacetate (specific esterase) positive ========================= M3 (Acute Promyelocytic Leukemia- Hypergranular) Criteria 70-80% hypergranular cases about Peripheral Blood : Blasts and promyelocytes show heavy granulation and multiple Auer rods■White blood cell count is usually decreased <5.0 × 10⁹/L, but the range is 3.0-15.0 × 10⁹/L■ Auer rods range from 10 to 20 per cell (faggot cells) and the rods may be intertwined or single. Few Auer rods are possible Bone Marrow : Most of the cells are abnormal promyelocytes with heavy azurophilic granulation■Multiple Auer rods found in promyelocytes (faggot cells) ========================== M3v (Acute Promyelocytic Leukemia -Microgranular Variant) Criteria 20-30%of cases are microgranular Peripheral Blood : White blood cells are markedly increased■Promyelocytes are usually bilobed and the cytoplasm contains only a few granules Bone Marrow : Azurophilic granules are small and difficult to see with light microscopy (<250 nm)■Promyelocytes are large with a lower N/C ratio■ The nucleus is usually lobulated, irregular, folded, bilobed, or monocytoid in appearance Cytochemistry : Myeloperoxidase, Sudan black B, and specific esterase positive ========================== M4 (Acute Myelomonocytic Leukemia) Criteria Peripheral Blood : White count is usually increased■ Both myelocytic and monocytic differentiations are found■≥5 x 10⁹/L monocytes and precursors are found■ Auer rods may be present Bone Marrow : >30% myeloblasts, monoblasts, and promonocytes■≥20% granulocytic precursors■ ≥20% monocytic precursors■ If the bone marrow has <20% monocytic component, then it must have a peripheral blood monocytosis of ≥5 x 10⁹/L (monocytes and precursors)■ Blast percentage includes type I and type II myeloblasts, monoblasts, and promonocytes Cytochemistry : Myeloblasts are positive for myeloperoxidase, Sudan black B, and specific esterase and negative with nonspecific esterases■Monoblasts and promonocytes are negative or only slightly positive with myeloperoxidase and negative with Sudan black B■Nonspecific esterase is positive and inhibited by sodium fluoride ============================= M4eo (Acute Myelomonocytic Leukemia With Increased Bone Marrow Eosinophils) Criteria Peripheral Blood : WBC is usually increased (range 30 x 10⁹/L to 100 ×10⁹/L)■Abnormal eosinophils are not found■Myeloblasts and monoblasts present Bone Marrow : ≥5% and <30% abnormal eosinophils■ Atypical eosinophils with possible pseudo-Pelger-Huët features in the nuclei and abnormal basophilic granules Cytochemistry : Abnormal eosinophils are specific esterase and periodic acid-Schiff positive ========================= M5a (Acute Monoblastic Leukemia) Criteria Acute leukemia with almost total monocytic dominance Peripheral Blood : White blood cells are usually increased■Blast morphology is variable■Auer rods are usually absent Bone Marrow : <20% granulocytic precursors ■≥80% are typically monoblasts ■Auer rods are usually absent Cytochemistry : <20% are myeloperoxidase positive■ ≥80% are nonspecific esterase positive■Naphthol AS-D chloroacetate negative■Naphthol AS-D acetate esterase is ++++ (strong positivity) and inhibited by sodium fluoride (1+ or negative) ========================== M5b (Acute Monocytic Leukemia) Criteria Peripheral Blood : Monocytosis with the promonocyte as the predominant cell Bone Marrow : ≥80% immature monocytic component with the promonocyte as the predominant cell■<20% are the granulocytic component Cytochemistry : <20% are myeloperoxidase positive cells■Promonocytes may show some weak positivity with myeloperoxidase and are Sudan black B negative■ ≥80% of the cells are nonspecific esterase positive ■ ≥80% of the cells are nonspecific esterase negative with sodium fluoride inhibition ============================== M6a (Erythroleukemia) Criteria Usually exhibits three phases and there is more myeloid involvement as the disease progresses Peripheral Blood : Normocytic normochromic to macrocytic/normochromic anemia■ Anisocytosis, poikilocytosis, basophilic stippling, and nucleated red blood cells Bone Marrow : Acute and abnormal proliferation of erythroid and myeloid precursors■ >50% erythroblasts (all nucleated cells)■ ≥30% myeloblasts (nonerythroid cells) type I and type II■Trilineage dysplasia common-dyserythropoiesis, dysmegakaryopoiesis, and dysgranulopoiesis Cytochemistry : Periodic acid-Schiff positive in early erythrocytic precursors■Myeloperoxidase and Sudan black B show >3% positive in myeloblasts =========================== M6b (Pure Erythroid Leukemia) Criteria Erythroid cell line malignancy with no myeloid involvement Peripheral Blood : Usually a macrocytic anemia■Platelets are decreased Bone Marrow : ≥80% of the cell are of erythroid lineage (≥30% must be proerythroblastic) Cytochemistry : Myeloperoxidase, nonspecific esterase, and Sudan black B negative■Block positivity with the periodic acid-Schiff ========================== M7 (Acute Megakaryoblastic Leukemia) Criteria Peripheral Blood : Variable white blood cell count but usually decreased■Normocytic/normochromic anemia■Platelets are variable, bizarre, and atypical Bone Marrow : >30% blasts (usually hard to get an aspirate for quantitation of blasts)■ ≥50% megakaryocytic cells (megakaryoblasts, promegakaryocytes, and megakaryocytes)■ Megakaryoblasts are highly pleomorphic ■Small round cells with scant cytoplasm and dense heavy chromatin or larger vacuolated blasts Cytochemistry : Myeloperoxidase and Sudan black B negative■Periodic acid-Schiff positive■ Nonspecific esterase (acetate) positive■Nonspecific esterase (butyrate) negative ============================== ACUTE LYMPHOBLASTIC LEUKEMIA L1 (Precursor Lymphoblastic Leukemia) Criteria Mutation of a single lymphoid stem cell causing proliferation of malignant lymphoblasts Peripheral Smear : White blood cells may be increased decreased or normal■Normocytic/normochromic anemia■Decreased platelets Bone Marrow : Hypercellular■≥25% blasts that are predominantly small blasts, up to twice the size of a normal small lymphocyte, nucleoli are not present and the cytoplasm is scant and only slightly or moderately basophilic Cytochemistry : Sudan black B, peroxidase, specific esterase, and nonspecific esterase are negative■Large block positivity with the periodic acid-Schiff ■Focal positivity with acid phosphatase in T-cell blasts ■ Terminal deoxynucleotidyl transferase is positive in 90- 95% in L1 and L2 and negative in L3 =========================== L2 (Precursor Lymphoblastic Leukemia) Criteria Mutation of a single lymphoid stem cell causing proliferation of malignant lymphoblasts Peripheral Blood : White blood cells may be increase decreased or normal■Normocytic/normochromic anemia■Platelets are often decreased Bone Marrow : The blasts are larger than L1, heterogeneous in size, the nucleus is irregular with clefting, and nucleoli are present Cytochemistry : Sudan black B, peroxidase, specific esterase, and nonspecific esterase negative■Large block positivity with the periodic acid-Schiff■Focal positivity with acid phosphatase in T-cell blasts■ Terminal deoxynucleotidyl transferase is positive in 90- 95% in L1 and L2 and negative in L3 =========================== L3 (Burkitt Type) Criteria The lymphoblasts are similar in appearance to those found in Burkitt lymphoma.Constitutes about 3-4% of precursor lymphoblastic leukemias in children and adults Peripheral Blood : White blood cells may be increased decreased or normal■ Normocytic/normochromic anemia■Decreased platelets are often seen■ Blasts are larger than L1 and have round to oval nucleoli with fine, homogenous chromatin, and one or more nucleoli may be seen■Cytoplasm of the blasts is deeply basophilic and vacuolated Bone Marrow : Hypercellular with blasts that are larger than L1, have a round-to oval-shaped nucleus with fine, homogenous chromatin, and one or more nucleoli present■Cytoplasm of the blasts is deeply basophilic and vacuolated Cytochemistry : Sudan black B, peroxidase, specific esterase, and nonspecific esterase negative■Periodic acid-Schiff negative■ Terminal deoxynucleotidyl transferase negative ■Oil red O positive ============================== MYELOPROLIFERATIVE NEOPLASMS Chronic Myeloid Leukemia, BCR ABL1 Positive Laboratory Features-Chronic Phase Peripheral Blood :White Blood Cells, Granulocytic leukocytosis with shift to the left (entire maturation series of granulocytes is seen)■<5% blasts■Basophilia and often eosinophilia■ No granulocytic dysplasia or toxic changes■Red Blood Cells, No or mild anemia■ Rare nucleated red blood cells■Platelets, Normal to elevated count■ Atypical large platelets, megakaryocytic cytoplasmic fragments, or megakaryocytic nuclei Bone Marrow : ≥95% cellularity■Myeloid:erythroid ratio ≥10:1■<5% blasts■Minimal or no granulocytic dysplasia■Increased small and monolobulated megakaryocytes ■Pseudo-Gaucher cells and sea-blue histiocytes can be observed if there is increased cell turnover Cytochemistry : Neutrophils in the chronic phase have markedly decreased leukocyte alkaline phosphatase score (score is usually ≤10) Laboratory Features-Accelerated Phase Peripheral Blood : White Blood Cells ,Persistent and increasing white blood cell count■ 10-19% myeloid blasts■ Entire maturation series of granulocytes is seen but notoxic changes■Basophilia ≥20%■Red Blood Cells, Normocytic/normochromic anemia■Occasional nucleated red blood cells■Platelets, Persistent thrombocytosis (>1000 x 10⁹/L) or persistent thrombocytopenia (<100 x 10⁹/L) Bone Marrow : 90-100% cellularity■Myeloid:erythroid ratio 10:1-50:1■ Increased small abnormal megakaryocytes■Pseudo-Gaucher cells and sea-blue histiocytes can be observed if there is increased cell turnover Laboratory Features-Blast Phase Peripheral Blood/Bone Marrow : >20% blasts■Extramedullary proliferation of blasts(hepatosplenomegaly)■Presence of large clusters of blasts in the bone marrow ■ In the blast phase, the blasts may have strong, weak, or no myeloperoxidase activity, but express antigen associated with granulocytic, monocytic, megakaryoblastic, and/or erythroid differentiation ■ 20% of blast phase leukemias are lymphoblastic (typically B cell) Genetics : Translocation of material from the long arm of 22 to the long arm of 9 and from 9 to 22■Fuses the BCR gene from chromosome 22 with regions of the ABL gene on chromosome 9 (BCR-ABL1 fusion protein) ================================= POLYCYTHEMIA VERA Laboratory Features Peripheral Blood :White Blood Cells, Increased in about two-thirds of patients■ Immature forms usually not seen■Basophils may be increased■Leukocyte alkaline phosphatase increased in three quarters of cases■Red Blood Cells ,Hemoglobin level increased■ Hematocrit level increased■ Red blood cell mass increased■Platlets, Normal to increased Bone Marrow : Hyperplastic■Erythroid hyperplasia■ Increased megakaryocytes ■ Granulocytic hyperplasia■ Increased reticulin in postpolycythemic myelofibrosis and myeloid metaplasia■ Iron stores are often depleted World Health Organization Criteria for Diagnosis Meet all three major criteria or the first two major and minor criteria Major Criteria Major:■Hemoglobin >16.5 g/dL in men and 16.0 g/dL in women or hematocrit >49% in men and >48% in women or increased red cell mass(>25% above mean normal predicted value)■Bone marrow showing panmyelosis with pleomorphic megakaryocytes■Presence of JAK2 V617F or JAK2 exon 12 mutation Minor Criteria Minor:■ Decreased serum erythropoietin level ================================= ESSENTIAL THROMBOCYTHEMIA Laboratory Features Peripheral Blood :White Blood Cells,Usually normal or mildly increased■Basophilia absent or minimal■Red Blood Cells Normocytic/normochromic anemia Microcytic, hypochromic anemia if there is gastrointestinal tract blood loss■Red blood cell mass not elevated■ No dacryocytes or leukoerythroblastosis■Platelets Thrombocytosis with counts ≥450 x 10⁹/L but typically higher■Platelets vary in size■Bizarre shapes with agranular forms are not uncommon Bone Marrow : Normocellular or moderately hypercellular■Increased numbers of large to giant megakaryocytes Megakaryocytes mass increased■ Increased megakaryocytes arranged in clusters or evenly dispersed■Blasts <5%■Absent or minimal reticulin fibrosis■Normal iron stores Immunophenotype : No abnormal phenotypes Genetics : 60% of cases harbor JAK2 V617F (exon 14) mutation■20-25% of cases have CALR mutations■3-5% of cases have MPL mutations■JAK2 exon 12 mutations absent Diagnostic Criteria for Essential Thrombocythemia Diagnosis of ET requires meeting all four major criteria or the first three major and minor criteria Major Criteria Major: ■Persistant platelet count ≥450 × 10⁹/L■Bone marrow biopsy findings: Megakaryocytic proliferation with loose cluster formation,Enlarged, hyperlobulated megakaryocytes, No significant granulocytic or erythroid proliferation or granulocytic shift to the left, No significantgranulocytic or erythroid dysplasia,No significant reticulin fibrosis■Exclusion of other myeloproliferative neoplasms t(9;22)(q34.1;q11.2); BCR-ABL1 negative■Presence of JAK2, CALR, or MPL mutation Minor Criteria Minor:■Another clonal abnormality or exclusion of reactive =================================== Myeloifibrosis Laboratory Features Peripheral Blood : White Blood Cells,Count is usually <30.0 x 10⁹/L Immature cells in the myeloid series■Red Blood Cells,Nucleated red blood cells■Normocytic/normochromic anemia■ Dacryocytes (tear-shaped red blood cells) are present■Platelets ,Normal, decreased, or increased Morphology may be abnormal Bone Marrow : In the early/prefibrotic phase, the aspirate is hypercellular, trilineage hematopoiesis present, and megakaryocytic atypia■Blasts <5%■During the fibrotic phase, it is inaspirable or results in a dry tap Cytochemistry : Reticulin stain is increased Immunophenotype : No abnormal phenotypic features Genetics : 60% of patients have the JAK2 V617F mutation■CALR mutations in 25% of cases■MPL mutations in 6-7% of cases■May have additional mutations but no Philadelphia chromosome or BCR-ABL1 fusion gene WHO Diagnostic Criteria for Early/Prefibrotic Primary Myelofibrosis Diagnosis requires meeting all three major criteria and at least one minor criterion Major Criteria Major:■Hypercellular bone marrow with megakaryocytic hyperplasia and atypia, granulocytic hyperplasia, and normal or decreased erythropoiesis with grade 0 or 1 (of 3) reticulin fibrosis■Exclusion of other WHO-defined myeloid neoplasms ■Presence of a JAK2, CALR, or MPL mutation or presence of other clonal marker and absence of other causes of reactive reticulin fibrosis Minor Criteria Minor:■Leukocytosis ≥11 x 10⁹/L■ Increased serum lactate dehydrogenase ■Anemia (not attributable to other underlying condition) ■ Palpable splenomegaly WHO Diagnostic Criteria for Overt Primary Myelofibrosis Major Criteria Major:■ Megakaryocytic proliferation and atypia with grade 2 or 3 (of 3) reticulin fibrosis or collagen fibrosis■ Exclusion of other WHO-defined myeloid neoplasms ■Presence of JAK2, CALR, or MPL mutation or presence of other clonal marker and absence of other causes of reactive reticulin fibrosis Minor Criteria Minor:■Leukocytosis ≥11 × 10⁹/L■Increased serum lactic dehydrogenase■ Anemia (not attributable to other underlying condition)■ Palpable splenomegaly■ Leukoerythroblastosis ================================= MATURE B-CELL NEOPLASMS CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA Laboratory Features Peripheral Blood : White Blood Cells count is increased to 20-200 × 10⁹/L■ Absolute lymphocytosis■Typical, small lymphocytes, with a hypermature- appearing nucleus■Smudge cells present■ <10% prolymphocytes■Red Blood Cells, Normocytic/normochromic anemia■Platelets ,Normal ,Often decreased with disease progression Bone Marrow : >30% lymphocytes ■ <10% prolymphocytes Immunophenotype : CD5, CD19, CD23, and CD79b positive■CD20 and sIg weak Genetics : 13q14.3 deletion, which is the most common chromosomal abnormality■Most common mutated genes are NOTCH1, SF3B1,TP53, ATM, BIRC3, POT1, and MYD88 ===================================== B-CELL PROLYMPHOCYTIC LEUKEMIA Laboratory Features Pripheral Blood : White Blood Cells,Typically> 100 x 10⁹/L■>55% prolymphocytes and usually >90% (WHO)■ Medium-sized cells that contain a large, vesicular nucleolus and condensed nuclear chromatin and have lower N/C ratio■Red Blood Cells Normocytic/normochromic anemia■Platelets Decreased Bone Marrow : Same types of prolymphocytes seen in the peripheral blood Immunophenotype : CD19, CD20, CD22, CD79a, CD79b, FMC7, and sig positive■CD5 and CD23 positive in less than one-third of cases Genetics : Exhibit heavy- and light-chain Ig gene rearrangements■Cells express much more sIg than do chronic lymphocytic leukemia cells■ MYC abnormalities are common■ May see TP53 deletions■ Must lack t(11;14)(q13;q32), which is found in mantle cell lymphoma ============================ HAIRY CELL LEUKEMIA Laboratory Features Peripheral Blood : White Blood Cells Usually decreased■ Presence of hairy cells■ Monocytopenia Neutropenia■Red Blood Cells, Moderate normocytic/normochromic anemia■Platelets, Thrombocytosis in 80% of patients Bone Marrow : Cannot be aspirated in more than half of the cases because of reticulin fibers■Small to medium-sized lymphoid cells with oval or bean-shaped nucleus■ The pale blue cytoplasm is abundant and has “hairy” projections Cytochemistry : Tartrate-resistant acid phosphatase positive■Specific esterase (naphthol AS-D chloroacetate esterase) and myeloperoxidase reactions are negative Immunophenotype : CD103, CD20, CD19, CD22, CD11c, CD25, CD200, and annexin A1 positive Genetics : About 85% of cases demonstrate IGHV genes with somatic hypermutation■BRAF V600E mutations ============================== MATURE T-CELL NEOPLASMS T-CELL LARGE GRANULAR LYMPHOCYTIC LEUKEMIA Laboratory Features Peripheral Bood : White Blood Cells,Persistent neutropenia■Lymphocytosis in the range of 4.0-10.0 x 10⁹/L■ Presence of large granular lymphocytes, generally >2.0 × 10⁹/L■ Predominant lymphocytes have moderate to abundant cytoplasm and fine or course azurophilic granules■Red Blood Cells,May have a macrocytic anemia■Platelets Normal to decreased Bone Marrow : Lymphocytic infiltration is variable■Left-shifted granulocytic maturation is common Immunophenotype : CD2, CD3, CD8, CD16, and CD57 positive Genetics : T-cell receptor gene clonally rearranged■STAT3 mutation is present in about one-third of cases =============================== ADULT T-CELL LEUKEMIA/LYMPHOMA Laboratory Features Peripheral Blood : White Blood Cells,May be only a few abnormal cells in the peripheral blood■Cells have highly convoluted nuclei with deep multilobulated indentation■Cell size and N/C ratio are larger than that of normal lymphocytes■Red Blood Cells,Normocytic/normochromic anemia■Platelets,Normal to decreased Bone Marrow : Presence of infiltrates and evidence of bony remodeling and fibrosis Immunophenotype : CD2, CD3, and CD5 are positive■CD7 is negative Genetics : Rearrangement of TR genes ============================== SéZARY SYNDROME Laboratory Features Peripheral Blood: White Blood Cells ,Hyperconvoluted lymphoid cells in peripheral blood■Red Blood Cells ,May have a normocytic/normochromic anemia■Platelets, Normal to decreased Bone Marrow : Eosinophilia, Monocytosis, Plasmacytosis Rare infiltrates of Sézary cells Cytochemistry : Focal positivity with acid phosphatase■Myeloperoxidase, alkaline phosphatase, and specific esterase (chloroacetate esterase) negative Immunophenotype : CD3 and CD4 positive■CD8, CD7, and CD26 negative■Flowcytometry:CD4+/CD7-in >30% of cases or CD4+/CD26- in >40% or T-cell population Genetics : T-cell receptor genes are clonally rearranged■Overexpression of PLS3, DNM3, TWIST1, and EPHA4 ================================ MYELODYSPLASTIC SYNDROMES Criteria Group of clonal hematopoietic stem cell diseases characterized by ■Ineffective hematopoiesis ■<20% blasts in peripheral blood and bone marrow ■Dysplasia in ≥10% of cells in one or more myeloid lineages■Cytopenia in at least one hematopoietic lineage■ Persistent cytopenias: The increased degree of apoptosis within the bone marrow progenitors contributes to the cytopenias : Dyserythropoiesis Peripheral blood Bone marrow : Dysgranulopoiesis Peripheral Blood Bone marrow : Dysmegakaryopoiesis Dysplasia (≥10% based on evaluation of ≥30 megakaryocytes) Peripheral Blood Bone marrow MYELODYSPLASTIC SYNDROME WITH SINGLE-LINEAGE DYSPLASIA (MDS-SLD) Criteria if≥10% dysplastic cells in affected cell lineage■ Red blood cells-hemoglobin concentration <10 g/dL ■White blood cells-absolute neutrophil count <1.8 × 10⁹/L■Platelets-platelet count <100 × 10⁹/L■Erythroid precursors contain <15% ring sideroblasts if no SF3B1 mutation and <5% if SF3B1 mutation Laboratory Features Peripheral Blood : White blood cells if affected neutropenia(<1.8×10⁹/L),<1% blasts■Red Blood Cells Normocytic/normochromic or macrocytic/normochromic anemia(<10 g/dL),Anisochromasia or dimorphic population■Platelets, If affected , thrombocytopenia (<100 × 10⁹/L) Bone Marrow : <5% blasts■no Auer rods■Markedly decreased to markedly increased erythroid precursors■Dysplasia present in ≥10% of single lineage■ Hypercellular or normocellular■Ring sideroblasts may be present but account for■<15% of the erythroid precursors or <5% if SF3B1 mutation is present■ Iron stores may be increased Genetics : 50% have cytogenetic abnormalities but they are not specific■Del(20q), gain of 8, and abnormalities of 5 and 7■60-70% of cases have somatic driver mutations that affect the stem cell■ Most commonly mutated genes are TET2 and ASXL1 ========================= MYELODYSPLASTIC SYNDROME WITH RING SIDEROBLASTS AND SINGLE-LINEAGE DYSPLASIA (MDS- RS-SLD) Laboratory Features Peripheral Blood : White Blood Cells,<1% blasts■Red Blood Cells Normochromic, macrocytic or normochromic,normocytic anemia Dimorphic pattern with hypochromic microcytes and normocytic or macrocytic cells Bone Marrow : Increase in erythroid precursors with dyserythropoiesis ■ No significant dysplasia in nonerythroid lineages■<5% myeloblasts in nucleated bone marrow cells ■no Auer rods Cytochemistry : Prussian blue stain,≥15% ringed sideroblasts as defined by ≥5 iron granules encircling one-third or more of the nucleus (≥5% if SF3B1 mutation is present) Immunophenotype : Aberrance in the immature erythroid progenitor compartment■ CD34+ cells (blasts) are typically <5% Genetics : SF3B1 mutation detected in 64-83% of cases =========================== MYELODYSPLASTIC SYNDROME WITH MULTILINEAGE DYSPLASIA (MDS-MLD) Criteria One or more cytopenias■Dysplasia of ≥10% of cells in two or more lineages■<1% blasts in peripheral blood and <5% in bone marrow■ No Auer rods■No monocytosis■Erythroid precursors contain <15% ring sideroblasts if no SF3B1 mutation and <5% if SF3B1 mutation Laboratory Features Peripheral Blood : White Blood Cells,Dysgranulopoiesis,Absolute neutrophil count <1.8 × 109/L,<1% blasts,No Auer rods, <1 x 10⁹/L monocytes■Red Blood Cells,Hemoglobin <10 g/dL,Dimorphic population■Platelet count <100 x 10⁹/L,May have abnormal morphology Bone Marrow : Normocellular or hypercellular but hypocellular can be seen■Dysplasia in ≥10% of the cells in two or more myeloid cell lines (erythroid, granulocytic, and megakaryocytic) ■<5% blasts■No Auer rods■Megakaryocyte abnormalities may be seen : Cytochemistry : Prussian blue stain to evaluate presence of ring sideroblasts and increased iron stores Immunophenotype: CD34+ cells (blasts) are typically <5% Genetics :Abnormalities include trisomy 8, monosomy 7, del(7q) monosomy 5, del(5q), and del(20q)■ Several gene mutations can be present ============================= MYELODYSPLASTIC SYNDROME WITH RING SIDEROBLASTS AND MULTILINEAGE DYSPLASIA (MDS- RS-MLD) Laboratory Features Peripheral Blood : White Blood Cells , <1% blasts, Dysgranulopoiesis■Red Blood Cells, Normochromic, macrocytic or normochromic normocytic anemia, Dimorphic pattern with hypochromic microcytes and normocytic or macrocytic cells■Platelets Abnormal platelet morphology Bone Marrow : Increase in erythroid precursors with dyserythropoiesis■ Granulocytes or megakaryocytes show ≥10% dysplastic forms■<5% myeloblasts in nucleated bone marrow cells and■ no Auer rods Cytochemistry : Prussian blue stain ≥15% ringed sideroblasts as defined by ≥5 iron granules encircling one-third or more of the nucleus (≥5% if SF3B1 mutation is present) Immunophenotype : Aberrance in the immature progenitor compartment■Abnormal maturation in granulopoiesis, the monocytic compartment, and erythropoiesis CD34+ cells (blasts) are typically <5% Genetics : SF3B1 mutation detected in 57-76% of cases MYELODYSPLASTIC SYNDROME WITH EXCESS BLASTS (MDS-EB) Criteria Characterized by 2–19% blasts in the peripheral blood or 5-19% myeloblasts in the bone marrow Subcategories■MDS-EB-1: 2-4% blasts in peripheral blood or 5-9% blasts in bone marrow■MDS-EB-2: 5-19% blasts in peripheral blood , or 10-19% blasts in bone marrow, If Auer rods are present it is MDS EB-2 regardless of blast numbers Laboratory Features Peripheral Blood : White Blood Cells ,Neutropenia,Dysgranulopoiesis■Red Blood Cells Anisopoikilocytosis with macrocytes, Dimorphic population, Decreased reticulocytes■Platelets Decreased, Large, giant, or hypogranular Bone Marrow : Usually hypercellular; may be hypocellular or normocellular■Clusters or aggregates of blasts■ Erythropoiesis, granulopoiesis, and megakaryopoiesis may be increased with variable dysplasia Cytochemistry : Peroxidase stain is positive Immunophenotype : CD34, CD117, or CD33 positive Genetics : 30-50% of cases have clonal cytogenetic abnormalities and can include +8, −5, del(5q), -7, del(7q), and del(20q)■ Splicing gene mutations are common (SRSF2) MYELODYSPLASTIC SYNDROME WITH ISOLATED DEL(5Q) Criteria Anemia with or without other cytopenias■Del(5q) occurs either in isolation or with one other cytogenetic abnormality other than monosomy 7/del(7q)■<1% of the peripheral blood leukocytes and <5% blasts of nucleated cells in bone marrow■ Auer rods are absent Laboratory Features Peripheral Blood : White Blood Cells Normal■Red Blood Cells Macrocytic anemia ,Hemoglobin level often <8.0 g/dL■Platelets Normal or elevated count Bone Marrow : <5% blasts of nucleated cells■ No Auer rods■ Increased megakaryocytes, which are normal to slightly decreased in size■ Dysmegakaryopoiesis Monolobated or hypolobated nuclei in megakaryocytes ■Hypercellular or normocellular■May have dysplastic erythroid precursors but less pronounced Cytochemisty : Prussian blue stain revealing ring sideroblasts may be present Genetics : Deletion of bands q31-q33 on the long arm of chromosome 5■ Cases with one additional cytogenetic abnormality except monosomy 7 or del (7q) have similar outcome ================================= /MYELODYSPLASTIC MYELOPROLIFERATIVE NEOPLASMS CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) Criteria Persistent peripheral blood monocytosis defined as >1.0 × 10⁹/L and >10% monocytes■ Absence of Philadelphia chromosome, BCR-ABL1 fusion, or myeloproliferative neoplasm■Absence of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2■<20% blasts or blast equivalents in peripheral blood or bone marrow■Dysplasia in one or more of the myeloid lineages .Subcategories: ■CMML-0,Blasts <2% in peripheral blood and <5% in bone marrow and no Auer rods■CMML-1, Blasts 2-4% in peripheral blood or 5-9% in bone marrow and no Auer rods■CMML-2, Blasts between 5% and 19% in peripheral blood or 10-19% in bone marrow or Auer rods are present Laboratory Features Peripheral Blood : White Blood Cells Usually normal to decreased■ Monocytes range from 2 to 5 x 10⁹/L but may be above 80 x 10⁹/L■Monocytes are >10% of the leukocytes■Monocytes are mature but can exhibit abnormal granulation or nuclear lobulation■ Blasts and promonocytes are <20% of the white blood cell count■ There are <10% neutrophil precursors■Dysgranulopoiesis is common■ Basophilia is typically mild but rare cases of increased eosinophils have been described■Red Blood Cells ,Anemia is usually normocytic but sometimes macrocytic, Dimorphic population■Platelets, Decreased count, Abnormal forms may be found Bone Marrow : Usually hypercellular■ Granulocytic proliferation■Slight dysgranulopoiesis■<20% blasts and promonocytes■ Dyserythropoiesis■ Slight dysmegakaryopoiesis■Increased monocytic precursors Cytochemistry : Nonspecific esterase positive for monocytic cells ■Myeloperoxidase and Sudan black B positive in granulocytic cells■ Periodic acid-Schiff negative Immunophenotype :Expresses the myelomonocytic antigen such as CD33 and CD13■Variable expression of CD14, CD68, and CD64 Genetics : 60% of cases show TET2 mutations ■ 50% of cases have SRSF2 mutations■ 40% of cases have ASXL1 mutations ============================== • Atypical Chronic Myeloid Leukemia (aCML) BCR-ABL1 Negative Laboratory Features Peripheral Blood : Leukocytosis with counts ≥13.0 x 10⁹/L but most have counts from 24.0 to 96.0 x 10⁹/L■ Immature and dysplastic 10-20% immature cells (promyelocytes, myelocytes, and metamyelocytes)■ Blasts are usually <5% but must be <20%■Monocytes are usually <10%■Basophilia <2%■Dysgranulopoiesis is pronounced with pseudo Pelger- Huët cells, abnormally clumped chromatin, or bizarre segmentation■Red Blood Cells, Anemia ,Dyserythropoiesis ,Macro-ovalocytosis may be present■Platelets Count is variable but decreased numbers are common Bone Marrow : Hypercellular due to increased neutrophils and their precursors■ Increased myeloid to erythroid ratio with >10:1 common■Blasts are typically <5% but always <20%■ Dysgranulopoiesis, dyserythropoiesis, and dysmegakaryopoiesis■ Some cases have over 30% erythroid precursors Cytochemistry :No diagnostic abnormalities Immunophenotype : Neutrophils and precursors are positive for CD33, CD13, and CD15 Genetics : SETBP1 and ETNK1 mutations are relatively common ■ The CSF3R mutation is present in <10% of cases ■ Common cytogenetic abnormalities are inv(17q),trisomy 8, and deletion of long arm of chromosome 20 ========================== JUVENILE MYELOMONOCYTIC LEUKEMIA (JMML) Laboratory Features Peripheral Blood : White blood cell count varies from 25.0 to 30.0 × 10⁹/L■Mainly neutrophils with some immature cells such as promyelocytes and myelocytes■Monocytes are increased (1.0 x 10⁹/L)■Blasts and promonocytes usually account for <5% and always <20%■Red blood cells,Nucleated red blood cells are frequent■Marked increased in hemoglobin F■Platelets variable but may be decreased and may be severe Bone Marrow : Hypercellularity Granulocytic proliferation but rarely erythroid precursors can predominate■Monocytes account for about 5-10% or cells Blasts and promonocytes are <20%■No Auer rods■ Dysplasias are minimal but pseudo Pelger-Huët cells or hypogranular forms may be seen■Megakaryocytes are often decreased Cytochemistry : Nonspecific esterase stain is positive in monocytic precursors■Myeloperoxidase and Sudan black B positive in granulocytic cells Immunophenotype : No specific immunophenotypic abnormalities have been reported Genetics : 25% of cases have monosomy 7■65% of cases have a normal karyotype
