Flowctometry Wojciech Gorczyca.4th Edition(2023) برای بزرگنمایی عکسها کلیک را روی ان نگه دارید..... ■■■【19】Myelodysplastic Neoplasms FIGURE 19.1 Dyserythropoiesis: megaloblastoid changes (a), ring sideroblasts (b, iron staining), multinucleation (c-f), giant eryth- roblasts with detached nuclear fragments (f), (c-h), cytoplasmic vacuoles (h-i), binucleation (j), internuclear bridging (k), cytoplasmic blebs (1), prominent nucleoli (1, r), nuclear disintegration/karyorrhexis (m-o), intercytoplasmic bridging (p), and mitotic figures (r-s) FIGURE 19.2 Dysgranulopoiesis. Atypical granulocytes with hypolobated nuclei, pelgeroid features (arrows), increased size and hypogranular cytoplasm are present FIGURE 19.3 Megakaryocytic atypia. (a–d) Atypical megakaryocytes with hyperlobulated or hypolobated nuclei FIGURE 19.4 MDS-immunohistochemistry. The staining with CD34 (a) and CD117 (b) shows increased number of blasts indi- cating MDS-EB (even when excluding plasma cells and mast cells, it is evident that subset of blasts is negative for CD34). Aberrant phenotype of blasts is often seen in MDS cases. Myeloblasts are positive for MPO (c). The staining with CD71 (d) shows erythroid hyperplasia with leftward shift (compare with MPO) FIGURE 19.5 MDS. Granulocytes (green dots) display low side scatter (a), which may mimic blasts on CD45 versus side scat- ter display (a) or monocytic cells due to their expression of CD11b (c) and CD11c (d). Dysplastic granulocytes differ phenotypically from blasts by lack of CD34 and CD117 expression and from monocytic cells by strong CD10 (b) and CD16 (e) expression and lack of HLA-DR (f), CD14 (g), and CD64 (h) expression. Cytologic evaluation (a; inset) confirmed dysplastic (agranular) granulocytes FIGURE 19.6 Antigen expression during myeloid maturation FIGURE 19.7 Abnormal CD11c/CD16 pattern in MDS. Panel a shows CD11c/CD16 pattern in normal bone marrow. Note two distinct populations, one negative and the other positive for CD11c and CD16 with only few cells in between. In MDS (b-d), the distinction between those two populations is less visible (b, c). Panel b shows up-regulation of CD11c on CD16-negative cells (arrow), panel c shows down-regulation of CD16 and up-regu- lation of CD11c on subset of cells (arrow, compare with a), and panel d shows marked down-regulation of both markers FIGURE 19.8 MDS flow cytometry (dysgranulopoiesis). MDS-flow cytometry (dysgranulopoiesis). (a-c) CD10 expression. (a) Normal (control) sample with positive CD10 expression (arrow). (b) MDS with partial loss of CD10 expression; only minute subset of granulocytes (arrow) is CD10+. (c) MDS with complete lack of CD10 expression by granulocytes. (d-i) Aberrant expression of CD11b and HLA-DR by granulocytes. (d) Normal (control) sample with variable but mostly bright expression of CD11b and negative HLA-DR. Panels e through i show gradual increase in the severity of phenotypic atypia associated with MDS. e shows distinct subset of granu- locytes with subtle down-regulation of CD11b (arrow), whereas f shows majority of granulocytes being negative CD11b (arrow). In g and h, apart from prominent down-regulation of CD11b (arrow), subset of granulocytes shows also aberrant expression of HLA-DR (dashed arrows). Panel i shows prominent down regulation of CD11b (arrow) and up-regulation of HLA-DR (dashed arrows), creating characteristic "window" pattern FIGURE 19.9 MDS-flow cytometry. Flow cytometry analysis (different MDS cases) shows increased blasts (a-b; compare with control samples in a' and b'), decreased side scatter and down-regulation of CD10 (c; compare with control on c'), and normal side scatter and aberrant expression of CD56 (d; compare with control on d'). Gating on granulocytes/maturing myeloid precursors (e-g) shows down-regulation of CD16 (e), CD33 (f) and down-regulation of CD11b (g). Control (benign) bone marrow samples are shown in a'-d' and e'-g FIGURE 19.10 Low grade MDS (MDS-SLD) - flow cytometry. Granulocytes (grey dots) show minimally decreased side scatter and normal expression of CD45 (a). CD34 staining shows rare blasts (b; green dots). Occasional granulocytes show aberrant expression of CD56 (c; arrow). The analysis of CD11b and CD16 shows decreased expression of both markers (d; arrow) FIGURE 19.11 MDS-IB - flow cytometry. Blasts expressing CD34 and CD117 are increased (a-b; blue dots). In addition, granulo- cytes and monocytes display aberrant expression of CD56 (c-d; arrow). Significant subset of granulocytes shows also down-regulation of CD13 and CD16 (e-f) FIGURE 19.12 MDS-EIB - flow cytometry. CD45 versus side scatter (a) shows slightly increased blasts (green dots) and normal expression of CD45 by granulocytes/maturing myeloid precursors (gray dots). The side scatter of granulocytes appears normal (a). Blasts are positive for CD34 and CD117 (b-c; green arrow). Granulocytes (gray arrow) are negative for CD34 (b) and display aberrant expression of CD117 (c; compare with isotypic control on c') and show normal expression of CD10 (d), CD16 (e), and CD33 (f). Minor subset of granulocytes shows aberrant expression of CD56 (g). Bright CD45 (instead of moderate) and strong expression of both CD10 and CD16 confirms that CD117 is aberrantly expressed by granulocytes and excludes promyelocytes FIGURE 19.13 MDS-IB - flow cytometry. Histology (a) shows hypercellular marrow with increased number of megakaryocytes which display atypical cytomorphology (Inset: red cell precursor with prominent dyserythropoiesis as seen on aspirate smear). Flow cytometry (b-h) shows mixed population of myeloid cells, monocytes, lymphocytes, and blasts. Granulocytes do not display decreased side scatter (b) but show down-regulation of CD16 (e), CD10 (g), and CD11b (h), and up-regulation of CD56 (f). Blasts (b; green dots) are increased in number. They express CD34 (c) and CD117 (d) FIGURE 19.14 MDS-IB-flow cytometry. Blasts (green arrow) are increased (a). They are positive for CD34 and CD117 (b). Granulocytes display markedly decreased side scatter (a; gray arrows), partial (aberrant) expression of Cd117 (b; gray arrow), decreased expression of CD11b and CD16 (c; gray arrow), aberrant expression of CD56 (d; arrow) and completely negative CD10 (e, arrow). The right column (a'-e') shows benign bone marrow for comparison FIGURE 19.15 MDS-IB with prominent leftward shift with increased blasts (green dots) and promyelocytes (orange dots). Blasts show low side scatter and dim CD45 (a), positive CD34 (b), positive CD117 (c) and positive HLA DR (d). Promyelocytes show high side scatter (a), negative CD34 (b), positive CD117 (c), and negative HLA-DR (d). Granulocytes (gray dots) show decreased CD45 expression (a) indicating leftward shift and negative CD34, CD117, and HLA-DR (b-d) FIGURE 19.16 MDS-flow cytometry. BM analysis by flow cytometry shows increased blasts (blue dots; arrow) with aberrant phe- notype: negative CD34 (a, d), positive CD117 (b, d) and partial expression of CD11b (e). The 2 lower panels shows only mononuclear gate (without granulocytes) FIGURE 19.17 Minute PNH+ granulocytic population FIGURE 19.18 BM with parvovirus infection. (a) Aspirate smear with highly "dysplastic" erythroid precursors with typical viral inclusions. (b-c) BM core biopsy shows rare erythroid precursors infected by parvovirus (c, immunohistochemistry) FIGURE 19.19 Dysmaturation associated with growth factor treatment (G-CSG; Neupogen). Patient with chronic renal failure and recent onset of pancytopenia with significant reduction in the expression of CD10 (a, a'), CD11b (b, b'), and CD16 (c, c') and aberrant expression of CD56 (d, d') one month after Neupogen (filgrastim) treatment (upper panels, before treatment; lower panels, after treatment) FIGURE 19.20 (a-c) Transient dyspoietic features and leftward shift following recent chemotherapy (for lymphoma). Granulocytes (purple dots) show down-regulation of CD45 (a), CD10 (b), and CD16 (c). (d-i) BM analysis from patient with HIV: increased hematogones (d); non-specific adsorption of surface immunoglobulins on B-cells (e-f; it is difficult to separate kappa from lambda+ B-cells); T-cells with reversed CD4:CD8 ratio (g); and subtle phenotypic atypia of maturing myeloid precursors (h-i; arrow) FIGURE 19.21 PNH - FC analysis. PNH clones (arrow) detected by FC show down-regulation of CD59 on red blood cells (a), down- regulation of CD24 and FLAER on neutrophils (b) and down-regulation of CD14 and FLAER on monocytes (c) FIGURE 19.22 MDS differential diagnosis (part 1). (a) MM. Atypical plasma cells mimic dysplastic red cell precursors (GPHA staining is negative; CD20 staining is positive). (b) Parvovirus infection. (c) B12/folate deficiency. (d) Gaucher disease (d, core biopsy; d' aspirate smear stained with W-G; d", aspirate smear with iron staining). (e) Post-treatment dyspoiesis (atypical myeloid precursor mimicking promyelocytes). (f) Reactive erythroid hyperplasia associated with hemolysis. (g) Acute erythroid leukemia (AEL). (h) Metastatic carcinoma. (Continued) FIGURE 19.22 (Continued) MDS- differential diagnosis (part 2). (i) HCL. (j-n) Myeloproliferative neoplasms: CML (j), PV (k), ET (1), CMML (m), and mixed myelodysplastic/myeloproliferative neoplasm - MDS/MPN-SF3B1-T (n) ■■■【20】Myelodysplastic Myeloproliferative Neoplasms FIGURE 20.1 Algorithmic approach to monocytosis. Abbreviations: AML, acute myeloid leukemia; AMML, acute myelomonocytic leu- kemia; MDS/MPN-n, MDS/MPN with neutrophilia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; SCMML, secondary CMML; TB, tuberculosis; PMF, primary myelofibrosis; PV, polycythemia vera; MDS; myelodysplastic syndrome; PCM, plasma cell myeloma; MPN, myeloproliferative neoplasm; MDS/MPN-RS-T, MDS/MPN with ring sideroblasts and thrombocytosis; SM, systemic masto- cytosis; SLE, systemic lupus erythematosus; ITP, immune thrombocytopenic purpura; NGS, next gene sequencing FIGURE 20.2 CMML. (a-b) BM aspirate smear with prominent monocytosis with cytologic atypia and only occasional immature monocytes. (c) BM core biopsy shows hypercellular marrow with monocytosis and megakaryocytic atypia. (d-i) Aggregates of plas- macytoid dendritic cells with ≥10 cells/aggregate. Core biopsy (d) shows paratrabecular cluster of plasmacytoid dendritic cells (low magnification). Clot section (e-f, H&E, low and higher magnification; g-1, immunohistochemistry) shows cluster of plasmacytoid dendritic cells which are positive for CD4 (g) and CD123 (h), and negative for CD56 (i) FIGURE 20.3 CMML - morphology and immunohistochemistry. Blood smear (a, a') shows atypical, but mostly mature-appearing monocytes. BM aspirate smear (b) shows maturing myeloid and erythroid precursors with increased monocytic cells. BM histology (c) shows markedly hypercellular marrow with trilineage maturation, atypical megakaryocytes, and increased monocytes (best visual- ized with immunohistochemistry stainings; d-i). Blasts are not increased (d-e). Staining with CD163 (f) shows only scattered positive cells, but muramidase staining is strongly positive (g). Only rare erythroid precursors are noted (h). Some of the neoplastic monocytes display dim staining with MPO (i) FIGURE 20.4 CMML. (a) BM aspirate with maturing myeloid cells and increased monocytes. (b-c) BM core biopsy is hypercellular marrow with prominent monocytosis. Immunohistochemical staining (d-g) demonstrates slightly increased blasts (d; CD34 staining), and predominance of cells positive for MPO (e), CD68 (f), and HLA-DR (g) FIGURE 20.5 CMML - immunohistochemistry. (a) BM shows prominent involvement by CMML. Neoplastic monocytes are mature with strong expression of CD68 (b), CD163 (c), and muramidase (d), and weakly positive for CD56 (e). MPO (f), CD34 (g), and CD117 (h) are negative FIGURE 20.6 CMML flow cytometry. Neoplastic monocytes (blue dots; blue arrow) are increased in number, display bright expression of CD45 (a), and have the phenotype similar to benign monocytes [with bright CD13 (b), bright CD33 (c), bright CD14 (d), bright CD64 (e), bright CD11c (f), positive HLA-DR (g), and positive CD4 (h)] with the exception of aberrant expression of CD56 (i). Granulocytes (gray dots; gray arrows) show aberrant expression of CD56 (i) FIGURE 20.7 CMML flow cytometry. BM aspirate (a) shows increased number of atypical monocytes. Flow cytometry (b-p) shows increased number of blasts (b-c; green dots) and monocytes (blue dots). Neoplastic monocytes are positive for CD13 (e), CD33 (f), CD14 (g), CD64 (h), and CD11c (j), and display aberrant expression of CD10 (i), CD16 (k), and CD56 (1). Granulocytes (b; gray dots) display aberrant expression of CD10 (m), CD56 (n), CD11b (0), and CD16 (p) FIGURE 20.8 CMML-flow cytometry. Flow cytometric analysis of the BM shows increased monocytes (a; green dots), decreased side scatter of granulocytes (a; purple dots) and increased blasts (a; blue dots). Blasts are positive for CD34 (b) and CD117 (c) and display aberrant expression of CDHLA-DR (d; variable and partially negative) and CD56 (e; blue dots). Monocytes (green dots) are positive for HLA-DR (d) and show aberrant (positive) expression of CD56 (e). The majority of monocytes are positive for both CD64 and CD14 (f), but minor population of monocytes show variable expression of CD14 with some cells being negative. Gating on granulocytes only (g-i) shows abnormal phenotype indicating dyspoiesis, in the form of down-regulation of CD16 (g), aberrant pattern of CD13 versus CD11b (h) and prominent up-regulation of CD56 (i; arrow). Panels g' to i' represent normal control to better visualize phenotypic atypia of granulocytes (show in g-i) FIGURE 20.9 CMML 2-flow cytometry. Aspirate smear shows myeloid leftward shift with blasts and monocytes (a). Flow cytom- etry (b-i) shows mixed population of blasts (green dots), monocytes (blue dots), neutrophils/maturing myeloid precursors (grey dots) and lymphocytes (red dots). Blasts are positive for CD45 (b), CD34 (c), CD117 (c-d), CD133 (d), CD13 (e) and show aberrant expression of CD33 (negative to dim; e) and CD5 (positive; i). Monocytes are positive for CD45 (b), CD13 and CD33 (e), CD11b and CD15 (f), CD14 and CD64 (g-h). Majority of monocytes are CD16-(g) FIGURE 20.10 Classification of monocytes based on CD14 and CD16 expression FIGURE 20.11 Comparison of distribution of classical CD16-/CD14bright+ monocytes (MO1), intermediate CD16+/CD14bright+ mono- cytes (MO2) and non-classical CD16+/CD14dim+ monocytes (MO3) in normal BM (a) and in CMML (b). Note the predominance of MO1 monocytes in CMML (b) FIGURE 20.12 CMML with NPM1 mutation. (a) Aspirate smear showing predominance of atypical monocytes, dysgranulopoiesis, and dyserythropoiesis (blasts and blasts equivalents are <20%). BM core biopsy (b, low and intermediate magnification) shows hyper- cellular marrow with increased monocytes, mild eosinophilia but without significantly increased blasts FIGURE 20.13 Comparison of benign monocytes (left column) with CMML (right column). Neoplastic monocytes in CMML show bright expression of CD14 (a) and CD64 (b), negative CD16 (c), and partially positive CD56 (d) FIGURE 20.14 CML (BCR-ABLI) with increased blasts and monocytes (blood). In rare cases of CML, flow cytometric analysis of peripheral blood may mimic CMML. FC analysis of blood shows blasts (blue dots) with moderate CD45 (a), positive CD34 (b) and positive CD13 (c) and numerous monocytes (green dots) with bright CD45 (a), bright CD13 (c), negative CD123 (d), bright CD11b (e), and bright CD14 (f). Cytogenetic and FISH testing for BCR-ABL1 (Philadelphia chromosome) is crucial in the differential diagnosis FIGURE 20.15 Acute monoblastic leukemia is characterized by immature and atypical monocytic cells (a) composed of promono- cytes and monoblasts. When analyzed by flow cytometry the monocytes comprise the predominate population (b; blue dots, arrow) and frequently show very aberrant phenotype, such as partially negative CD13 (c), heterogeneous ("smeary") expression of CD14 (d) and positive CD123 (e) FIGURE 20.16 MDS/MPN-RS-T. Aspirate smear shows many ring sideroblasts (a). BM core biopsy shows hypercellular marrow with megakaryocytic atypia (b) and mild reticulin fibrosis (c) FIGURE 20.17 MDS/MPN-SF3B1-T shows features of myelodysplastic syndrome (such as MDS-RS) overlapping with a MPN (such as ET or PMF). BM aspirate (a-b) and core biopsy (c-d) show hypercellular marrow with trilineage dyspoiesis. There is marked mega- karyocytosis with atypia and clustering. An iron stain on fresh aspirate (e) shows >15% ringed sideroblasts FIGURE 20.18 MDS/MPN-n. Patient underwent BM biopsy due to marked leukocytosis (90 x 109/L). H&E section from the BM core biopsy (a-b) shows hypercellular marrow with myeloid hyperplasia and leftward shift. Blood smear (c-e) shows leukoerythroblastosis. BM aspirate (f) shows myeloid hyperplasia with leftward shift without basophilia or hypolobated micromegakaryocytes. Flow cytometry analysis (g-j) shows increased myeloid to lymphoid ratio due to myeloid hyperplasia (granulocytes are represented by purple dots), aber- rant expression of CD10 on majority of granulocytes (g), increased blasts (h; blue dots), aberrant expression of CD56 on subset of granu- locytes and subset of blasts (i), and aberrant expression of CD11b on majority of blasts (j; blue dots) ■■■【21】Myeloproliferarive Neoplasms FIGURE 21.1 Algorithm for the diagnosis of classic myeloproliferative neoplasms (MPN) FIGURE 21.2 Algorithmic approach to thrombocytosis FIGURE 21.3 Algorithmic approach to neutrophilia. Abbreviations: MDS/MPN-n, MDS/MON with neutrophilia; BM, bone mar- row; CEL, chronic eosinophilic leukemia; CML, chronic myeloid leukemia; CNL, chronic neutrophilic leukemia; CMML, chronic myelo- monocytic leukemia; ET, essential thrombocythemia; FISH, fluorescence in situ hybridization; HES, hypereosinophilic syndrome; NGS, next generation sequencing; PMF, primary myelofibrosis; PV, polycythemia vera; WBC, white blood cell count FIGURE 21.4 Algorithm for the diagnosis of PV (PV, polycythemia vera; MPN, myeloproliferative neoplasm, EPO, erythropoietin; BM, bone marrow; NGS, next Gene sequencing) FIGURE 21.5 Algorithmic approach to eosinophilia. CEL is diagnosed with persistent eosinophilia with evidence of clonality by genet- ics testing and/or increased blasts (PDGFRA, PDGFRB, FGFR1, BCR-ABL, and MYH11-CBFB must be negative). HES shows sign of organ damage or involvement; It differs from CEL by lack of clonality and increased blasts. MPN with FGFR1 may be associated with T-ALL, eosinophilia, and mastocytosis. Abbreviations: CEL, chronic eosinophilic leukemia; HES, hypereosinophilic syndrome; CML, chronic myeloid leukemia (BCR-ABL+); MPN, myeloproliferative neoplasm; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia FIGURE 21.6 Myeloproliferative neoplasms (MPN) - general morphologic features: (a) blood film with thrombocytosis; (b) blood film with neutrophilia with leftward shift; (c) blood film with erythroid precursors; (d) basophilia; (e) BM aspirate with prominent megakaryocytosis with atypia; (f) BM aspirate with prominent eosinophilia; (g) BM core biopsy showing hypercellular marrow with myeloid hyperplasia and atypical hypolobated megakaryocytes; (h) BM core biopsy with atypical megakaryocytic clustering: (i) BM core biopsy with prominent reticulin fibrosis; (i) dilated sinuses with intravascular hematopoiesis FIGURE 21.7 Myeloproliferative neoplasm (MPN) - flow cytometry. Flow cytometry analysis of blood (a) shows increased granulo- cytes with leftward shift (decreased CD45 expression) and increased blasts (a-b; blue dots). Basophilia (b) may be prominent as show by CD123 staining (c). Analysis of BM aspirate may show mast cells (d). Granulocytes display aberrant expression of CD56 (e; compare with healthy control shown on e'), decreased side scatter (f) and down-regulation of CD10 (f; compare with healthy control shown on f'). In contrast to CMML, monocytes are not increased but display aberrant phenotype (CD56 expression; g) FIGURE 21.8 Major characteristics of CML: blood smear (a) with neutrophilia and leftward shift, FISH (b) and cytogenetic (c) with BCR-ABL1 rearrangement (Philadelphia chromosome). Bone marrow biopsy (d-e) and aspirate smear (f) showing myeloid hyperplasia and atypical hypolobated dwarf megakaryocytes FIGURE 21.9 Flow cytometric features of CML in peripheral blood. The features differentiating CML from reactive neutrophilia include increased blasts expressing CD34, HLA-DR, and CD117 (a–c), increased basophils, expressing CD123 (d), decreased side scat- ter of granulocytes (e-g), down-regulation of CD10 (e; compare with control shown on e'), up-regulation of CD56 on granulocytes (f; compare with control shown on f') and down-regulation of CD16 on granulocytes (g; compare with control shown on g') FIGURE 21.10 CML - blood. FC analysis shows increased basophils (a; green dots; green arrow). Granulocytes (gray dots; gray arrows) display moderate CD45 and slightly decreased side scatter (a), bright CD11b (b-c) and CD13 (b), and variable expression of CD16 (majority of granulocytes show bright CD16, but subset is CD16-negative; c). Basophils show slightly brighter expression of CD45 than neutrophils (a), low side scatter (a), moderate CD11b (dimmer than in neutrophils; b-c), bright CD13 (b), negative CD16 (c), and partially dim CD22 (d) FIGURE 21.11 Flow cytometric features of CML. Rare cases of CML show small but distinct population of circulating immature B-cells ("hematogones-like") with the following phenotype: CD45+ (a), HLA-DR+ (b), CD34- (c), CD117- (d), CD22+ (e), CD10+ (f), and CD19+ (f). It is uncertain whether this population represents an emerging B-ALL blast crisis FIGURE 21.12 CML - FC analysis of blood. FC shows down-regulation of CD45 on subset of granulocytes (gray dots), resembling FC from bone marrow sample rather than blood (granulocytes at different stages of maturation), and presence of CD45- (a) and CD34+ (b) B-lymphoblasts. FISH studies confirmed the diagnosis of CML (BCR-ABL rearrangement; c). Immature B-cells are positive for CD19 (d), TdT (e), and CD10 (f) FIGURE 21.13 FC features of CML in the BM aspirate. Granulocytes/maturing myeloid precursors (purple dots) display aberrant pattern of maturation including down-regulation of CD10 (a; compare with control on a'), CD13 versus CD16 (b; compare with control on b') and CD11b versus CD13 (c; compare with control on c') FIGURE 21.14 CML - accelerated phase (AP). The core biopsy shows hypercellular bone marrow (a) with increased blasts on core biopsy (a, histology; b, immunostaining with CD34). Flow cytometry analysis (c) revealed increased blasts (green dots) FIGURE 21.15 CML - myeloid blast phase. Aspirate smear (a) shows blasts. FISH studies (b) show increased number of BCR-ABL1 copies. Flow cytometry (c-g) shows blasts (green dots; Arrow) expressing CD45 (c), CD34 (d), CD13 (f; Dim expression), and CD33 (g; Bright expression). CD117 is negative (e). Residual granulocytes (gray dots) have higher Side scatter (c) and lower forward scatter (d-g) when compared to blasts FIGURE 21.16 CML in blast phase. Blasts (green dots) show moderate CD45 (a), partial expression of CD34 (b), dim positive CD117 (c), negative to dim HLA-DR (d), and positive expression of CD13 (e) and CD33 (f) FIGURE 21.17 CML in chronic phase (in the BM) and "localized T-lymphoblastic blast crisis" (inguinal adenopathy with T-lymphoblastic lymphoma diagnosed at the time of CML diagnosis). Patient had no prior history of CML or other malignancy. The morphologic, flow cytometric, and FISH analysis of the BM showed CML in chronic phase with extra copies of BCR-ABL1. Concurrent analysis of the lymph node showed involvement by T-lymphoblastic lymphoma with extra copies of BCR-ABLI. Lymphomatous cells in the lymph node were positive for CD2, CD5, CD7, cytoplasmic CD3, TdT, and partially CD10. Surface CD3 was not expressed. The flow cytometric analysis of the BM shows marked lymphopenia (no blasts, including T-lymphoblasts have been identified). Granulocytes displayed aberrant expression of CD56 on subset. FISH: aqua, arginosuccinate synthetase 1 gene on 9q34; green, BCR gene on chr.22; and red, ABL1 gene on chromosome 9; yellow signal indicates BCR-ABLI fusion FIGURE 21.18 CML (after treatment) - blood. FC analysis shows a minute population of blasts (blue dots; arrow) with aberrant phenotype: CD19 (a), CD38 (a), CD34+ (b), CD13+ (c), CD10+ (d), and CD20 (d). Some of the FC features resemble hematogones (vari- able forward scatter, CD10 positivity, and CD19 versus CD38 pattern) FIGURE 21.19 PV. (A) BM core biopsy shows hypercellular marrow with increased m:E ratio and megakaryocytosis. (b) High magnifi- cation displays numerous atypical megakaryocytes including hyperlobulated forms mimicking ET. (c-e) Immunohistochemical staining shows scattered red cell precursors (c), myeloid hyperplasia (d), and megakaryocytosis (E). Reticulin fibrosis is mildly increased (f) FIGURE 21.20 Post-polycythemia myelofibrosis (PPMF). (a-b) histology (intermediate and high magnification) shows megakaryo- cytic hyperplasia with marked cytologic atypia. (c) reticulin staining shows grade 3 fibrosis FIGURE 21.21 Essential thrombocythemia (ET). (A) Blood film with thrombocytosis. (b) hypercellular BM aspirate with promi- nent megakaryocytes. (c-e) BM core biopsy showing mildly hypercellular BM with myeloid hyperplasia and megakaryocytic atypia in the form of loose clusters and large megakaryocytes with hyperlobated nuclei. (f) Silver staining depicts mild diffuse increase in reticulin fibers. (g-i) Immunohistochemistry shows predominance of myeloid cells (g), atypical megakaryocytes (CD61 staining; h) and rare blasts (CD34 staining; i) FIGURE 21.22 PMF. (A) blood smear with red blood cell poilkilocytosis and tear-drop cells. (b) hypocellular, aspicular BM aspi- rate (c) hypercellular marrow with osteosclerosis and megakaryocytosis. (d) the cells appear to "stream", due to fibrosis. (e) increased reticulin fibers (silver staining). (f-g) clusters of atypical megakaryocytes FIGURE 21.23 Early PMF-flow cytometry of BM. Blasts (a-b; green dots) are slightly increased. They are positive for CD34 and CD117 (b). Granulocytes (gray dots; arrows) show aberrant expression of CD117 on minor subset (b) and aberrant expression of CD56 on prominent population (c) FIGURE 21.24 Chronic neutrophilic leukemia (CNL). (a-b) blood smear shows marked neutrophilia (high magnification shows typical toxic appearance of neutrophils with numerous cytoplasmic granules. (c) BM aspirate smear shows markedly increased m:E ratio due to myeloid hyperplasia. (d-e) core biopsy shows hypercellular marrow with predominance of granulocytic cells without increased number of blasts. There is mild reticulin fibrosis (f) FIGURE 21.25 Chronic eosinophilic leukemia (CEL). Hypercellular BM with predominance of mature-appearing eosinophils (a-b, H&E section at low and high magnification; c, giemsa staining) FIGURE 21.26 Chronic eosinophilic leukemia (CEL) - flow cytometry analysis. Eosinophils have high side scatter (a-f), are posi- tive for CD45 (a), CD13 (b), CD33 (c), and CD11b (e). They do not express CD10 (d) and CD16 (f) FIGURE 21.27 Comparison of eosinophils (a) with granulocytes and non-viable cells based on forward scatter versus viability staining. Eosinophils (a) have much lower forward scatter and show non-specific staining with viability dye, mimicking non-viable elements (b; negative control shows truly necrotic cells with much brighter expression of the viability dye, aqua V450 (arrow) FIGURE 21.28 HES (blood). (A) Blood with prominent eosinophilia (WBC = 104 k/uL). (b) BM biopsy shows hypercellular mar- row with eosinophilia and without increased blasts. (c-e) Flow cytometry shows eosinophils (arrow) with increased side scatter (c), negative CD16 (d) and dim CD33 (e). Cytogenetics, FISH (BCR-ABL, PDGFRA/B, FGFR1), and next gene sequencing studies were negative ■■■【22】AML defined by Differentiation FIGURE 22.1 AML with minimal differentiation. (a-d) BM is totally replaced by blasts (a), which are negative for MPO (b) and posi- tive for CD34 (c) and CD117 (d). (e) Aspirate smear shows small to medium-sized blasts with scanty basophilic cytoplasm and round to occasionally irregular nuclei with fine chromatin pattern. (f-k) Flow cytometry analysis shows blasts (green dots) with SCC, negative CD45 (a), bright CD34 (b), negative to dim CD117 (c), negative CD13 (d), dim CD33 (e), and positive HLA-DR (f) FIGURE 22.2 Minimally differentiated AML. Blasts (green dots) show the following phenotype: dim CD45+ (a), CD13+ (b), CD15- (c), CD33- (d), CD34+ (e), CD117+ (f), CD133+ (g) and HLA-DR+ (h) FIGURE 22.3 AML without maturation. The BM section (a) shows sheets of blasts. There is no evidence of maturing marrow ele- ments. Blasts are negative for CD34 (b), positive for CD117 (c), MPO (d), and CD43 (f). CD15 is not expressed (e) FIGURE 22.4 AML without maturation. Blasts (green dots) are characterized by low side scatter (a) and high forward scatter (b-n) and show the following phenotype: CD45+ (moderate expression, a), CD34- (b), CD117+ (c), HLA-DR+ (d), CD7+ (e), CD11c+ (dim/partial expression, f), CD13+ (g), CD15 (h), CD33+ (i), CD71- (j), CD81+ (k), CD123+ (1), CD133 (m), and CD200-(n) FIGURE 22.5 AML without maturation. Blasts (green dots) show low side scatter and moderate CD45 (a), positive CD117 (b), and aberrant expression of CD11b on subset (c; arrow) FIGURE 22.6 AML with maturation. (a) BM aspirate shows large blasts with abundant granular cytoplasm, irregular nuclei and several nucleoli (inset: positive staining with MPO). (b) Histology section shows replacement of the marrow by immature mononuclear cells. Blasts are positive for CD34 (c), HLA-DR (d), and MPO (e) by immunohistochemistry FIGURE 22.7 AML with maturation. Blasts (blue dots) show dim CD45 (a), positive CD34 (b), positive CD117 (c), negative CD11b (d), and positive CD13 (e) and CD33 (f). Granulocytes (purple dots) are strongly positive for CD11b (d) and do not express CD34 or CD117 FIGURE 22.8 AML with maturation and aberrant phenotype (CD13-/CD15+/CD34-). Blasts (green dots) show low side scatter and moderate expression of CD45 (a). Neutrophils/maturing myeloid precursors (gray dots, a, arrow) show high side scatter and brighter expression of CD45 when compared to blasts. Blasts show the following phenotype: CD13- (b), CD15+ (c), CD33+ (d), CD34 ̄ (e), CD117+ (f), CD133+ (g), and HLA-DR+ (h) FIGURE 22.9 AML with FLT3-ITD. Blasts are rather poorly differentiated with irregular nuclei (a). Metaphase cytogenetics (b) shows normal karyotype (46,XX). Flow cytometry analysis (c-g) shows blasts with the following phenotype: CD45+ (c), CD34- (d), CD117+ (e), HLA-DR- (f), and CD13+ (g). PCR analysis was positive for an internal tandem duplication (ITD) between FLT3 exons 14 and 15 (h) FIGURE 22.10 AMML. (a-b) Cytology shows mixed population of blasts and atypical monocytes. Blasts are MPO+ (c). (d-e) Histology section shows hypercellular marrow with predominance of blasts and monocytes FIGURE 22.11 AMML (cytology). BM aspirate smear show mixed population of blasts and monocytes at different stages of maturation FIGURE 22.12 AMML is characterized by presence of blasts (≥20%) and monocytes (≥20% ), Blasts (green dots) have moderate CD45 (a) and low side scatter (a), and monocytes (blue dots) have bright CD45 and minimally increased side scatter (a). Blasts show the following phenotype: CD34+ (b), CD117+ (b), CD133+ (c), HLA-DR (c), CD13+/CD33+ (d), CD14-/CD64 (e), CD11b /CD15- (f), CD11c dimly (g), and CD14-/CD16- (h). Monocytes show the following phenotype: CD34-/CD117+/CD133+ (b-c), HLA-DR+ (c), CD13+/ CD33+ (d), CD14+/CD64+ (e), CD11b+/CD15+ (f), CD11c bright (g), and CD14+/CD16- (h) FIGURE 22.13 Acute myelomonocytic leukemia (AMML) - flow cytometry. Blasts (green dots) and monocytes (blue dots) predom- inate. Blasts show low side scatter and moderate CD45 (a), positive CD117 (b), negative CD34 (b; rare blasts are positive), negative CD14 and CD64 (c), negative CD11b (d), dim CD11c (e), positive CD13 (f), and positive CD33 (g). Monocytes show bright CD45 (a), negative CD34 and CD117 (b), bright CD64 (c), negative CD14 (c), positive CD11b (d), bright CD11c (e), dim CD13 (f), and bright CD33 (g) FIGURE 22.14 AMML flow cytometry. Blasts (green dots) show low side scatter and moderate CD45 (a), dim CD33 (b), bright CD34 (c), negative CD64 (d), negative CD14 and CD16 (e), negative CD11b and CD15 (f) and positive CD200 (g). Monocytes (blue dots) show bright CD45 (a), bright CD33 (b), aberrant expression of CD34 (c), bright CD64 (d), positive CD14 (e), partially positive CD16 (e), bright CD11b (f), and positive CD81 (g). Note presence of classical and non-classical monocytes (e) FIGURE 22.15 Acute monoblastic leukemia - cytology and cytochemistry. Examples of acute monoblastic leukemia (a-e). Note abundant cytoplasm, occasional cytoplasmic vacuoles and prominent nuclear irregularities. (f) Cytochemistry shows strong staining with NSE (inset negative MPO) FIGURE 22.16 Acute monoblastic leukemia - histology and immunohistochemistry. BM is hypercellular (a) and on high magnifi- cation shows predominance of atypical mononuclear cells replacing normal elements (b). Aspirate smear (c) shows monoblasts with irregular nuclei and nucleoli (inset: positive NSE). Immunohistochemistry (d-i) shows positive muramidase (d), HLA-DR (e), CD68 (f) and CD56 (g) and negative CD34 (h) and MPO (i) FIGURE 22.17 Acute monoblastic leukemia with prominent phenotypic atypia. (a) Aspirate smear shows highly atypical large monoblasts with nucleoli and cytoplasmic vacuoles. (b) BM core biopsy shows marrow replacement by sheets of monoblasts. (c-h) Immunohistochemistry: monoblasts are negative for CD34 (c) and CD117 (d) and positive for MPO (e), CD56 (f), CD68 (g), and muramidase (h). (i-k) Flow cytometry analysis shows bright CD33 expression (i), bright CD56 expression (j), and positive CD64 (k) FIGURE 22.18 Acute monoblastic leukemia - flow cytometry. Monoblasts (blue dots) are positive for CD45 (a), HLA-DR (b; bright), CD117 (d), CD11b (e; variable expression), CD56 (h), and CD64 (i). They do not express CD34 (b), CD14 (f), and CD16 (g) FIGURE 22.19 Acute monocytic leukemia. The smear (a) shows atypical monocytes with irregular nuclei and vacuolated cyto- plasm, which are positive for non-specific esterase (NSE; b). Monocytes are positive for CD45 (c), CD11b (d), CD11c (e), CD14 (f), CD15 (g), CD33(h), CD64 (i), CD117 (j), and CD133 (k) FIGURE 22.20 Acute monoblastic leukemia - flow cytometry. Monoblasts (green dots, arrow) show bright expression of CD45 and slightly increased side scatter (a). CD34 and CD117 are not expressed (b-c). HLA-DR is positive (d) and both CD33 and CD64 are brightly expressed (g-h). There is aberrant expression of CD11b (e: variable with subset negative and dimly positive), CD13 (f: mostly negative, only minor population is positive) and CD14 (h: mostly negative with only minute population showing variable expression) FIGURE 22.21 Acute monocytic leukemia with aberrant (partial) loss of CD45 expression (flow cytometry). Monoblasts (green dots) are negative to partially dimly positive CD45 (a), negative for CD34 (b) and CD117 (c), positive for HLA-DR (d) and CD11c (e),mostly negative for CD14 (f; minor subset is dimly), and positive for CD64 (g) and CD56 (h) FIGURE 22.22 Acute monoblastic leukemia from a four-year-old boy. Flow cytometry analysis shows immature monocytic cells expressing CD45 (a) and CD117 (b), lack of CD34 (c), positive HLA-DR (d), CD64 (e), CD11b (f), CD56 (g), and CD4 (h). Metaphase cytogenetics (i) shows unusual three-way translocation involving MLL gene: 46,XY,t(2;10;11)(q23;p11.2;q23). FISH studies confirmed rearrangement of MLL at 11q23 (j). The three-way translocation involving MLL gene is rare, the most common being t(9;11). AML with 10p12/11q23 rearrangements are more common in males and mainly found in infants and children FIGURE 22.23 Acute monoblastic leukemia with trisomy 8. (a) Aspirate smear shows predominance of monoblasts and promono- cytes. B-h) Immunohistochemistry performed on clot section shows the following phenotype: muramidase (b), CD123+ (c; dim), CD68+ (d; focal), CD56+ (e), CD117-(f), MPO- (g), and CD71- (h). (i) FISH testing showed trisomy 8 (CEP8 red probe) FIGURE 22.24 Cytologic differential diagnosis of acute monoblastic leukemia: (a) Burkitt lymphoma; (b) high grade B-cell lym- phoma with MYC and BCL2 rearrangement (HGBL-R, double hit lymphoma); (c) T-cell lymphoma with unusual "blastoid" appearance; (d) plasma cell leukemia with abundant cytoplasm mimicking monoblasts; (e) hairy cell leukemia with unusual "blastoid" cytologic features; (f) small cell carcinoma; (g) blastic plasmacytoid dendritic cell neoplasm (BPDCN); (h) acute erythroid leukemia (AEL); (i) hypogranular APL; anaplastic large cell lymphoma (j); alveolar rhabdomyosarcoma (k); acute megakaryoblastic leukemia (1) and diffuse large B-cell lymphoma with leukemic blood involvement (m-m', cytology; m", flow cytometry) FIGURE 22.25 Comparison of monoblasts (a-e) with hypo- granular APL (a'-e'). In contrast to hypogranular promyelocytes in APL, monoblasts are usually CD117 negative (a), HLA-DR posi- tive (b), CD34 negative (c), CD11b positive (d) and CD64 brightly positive (e). Hypogranular APL show positive CD117 (a'), negative HLA-DR (b'), positive CD34 (c)', negative CD11b (c'; minor subset of cells show non-specific staining), and dim (not bright) CD64 (e'). Since neoplastic promyelocytes in APL tend to display non-specific staining, correlation with isotypic controls and with markers known to be negative in AMLs (such as CD8, CD20, kappa, lambda etc.) is recommended for reliable analysis of phenotypic profile FIGURE 22.26 Comparison of monoblasts (a-e; blue dots) and myeloblasts (a'-e'; green dots). Monoblasts differ from myelo- blasts by brighter expression of CD45 a-a'), bright expression of CD64 (b), often negative CD117 (c) and CD34 (d), and brighter expression of CD11c (e). Myeloblasts are either CD64 negative (b') or show dim CD64, are usually positive for CD117 (c') and CD34 (d') and show negative or dim expression of CD11c (e') FIGURE 22.27 Comparison of monoblasts (a-e) with BPDCN (a'-e'). The phenotype of monoblasts (blue dots) and blastic plasma- cytoid dendritic cells (green dots) is similar. Monoblasts are often positive for CD4 (a) and CD56 (b), expression of which is the hallmark of BPDCN (a'-b'). In contrast to monoblasts, BPDCNs are negative for CD11c (c-c') and CD11b (not shown). CD123 is typically positive in BPDCN but maybe also expressed in acute monoblastic leukemia (d-d'). Rare BPDCNs may be positive for CD33 (e-e') FIGURE 22.28 DLBCL mimicking acute monoblastic leukemia on aspirate smear (a). Flow cytometry (b-c) shows clonal (lambda+) B cells FIGURE 22.29 Pure erythroid leukemia - cytology. Numerous immature erythroid precursors with cytoplasmic vacuoles and occasionally irregular cytoplasmic borders are present FIGURE 22.30 AEL with ring sideroblasts. The iron staining of the aspirate smear (a) and clot section (b) shows numerous ring sideroblasts. BM is packed with immature erythroid cells (c) FIGURE 22.31 AEL with erythroid precursors resembling myeloblasts (a). Flow cytometry analysis shows moderate expression of CD45 (b) and positive CD71 (c) FIGURE 22.32 Acute erythroid leukemia (AEL). Aspirate smear (a) and BM core biopsy (b) shows predominance of proerythro- blasts. They are positive for CD71 (c), e-cadherin (d) and focally CD117 (e). They do not express CD34 (f), GPHA (g), and HLA-DR (h) FIGURE 22.33 Pure erythroid leukemia. Cytology (a) shows immature erythroblasts with cytoplasmic vacuoles. Histology (b, low power; c, high magnification) shows replacement of the BM by sheets of atypical erythroid precursors. Flow cytometry (d-f) shows positive expression of CD117 and glycophorin (d) and negative expression of CD13 and CD33 (e-f). Immunohistochemistry shows the following phenotype: GPHA+ (g), CD34- (h), MPO- (i), CD117+ (j), EMA+ (k), CD43+ (1), and CD45- (m) FIGURE 22.34 AEL - immunohistochemistry. BM core biopsy shows predominance of erythroid blasts and atypical megakaryo- cytes (a). Blasts are positive for CD117 (b; weak staining), CD71 (c, very strong staining) and EMA (d). CD34 (e), MPO (f), GPHA (g), and CD45 (h) are negative FIGURE 22.35 Pure erythroid leukemia - flow cytometry (three cases). (a-h) Neoplastic erythroid precursors (a, aspirate smear) are negative for CD45 (b, orange dots), CD34 (c), CD117 (d), HLA-DR (e), CD13 (f), CD33 (g), and CD64 (h). (i-1) Some erythroid leuke mias may be positive for CD45 (I; blue dots), CD71 (j), and partially CD117 (1). GPHA is negative (k). (m-p) Erythroblasts (green dots) show minute population with CD34 expression (m), positive CD117 (n; dim expression), positive CD71 (o) and aberrant expression of CD56 on majority of blasts (p) FIGURE 22.36 Acute erythroid leukemia. Aspirate smears shows immature erythroid precursors with vacuolated cytoplasm (a). Histology shows total BM replacement by erythroid precursors (b) Immunohistochemistry (c-g) shows positive expression of CD117 (c), CD71 (d), and E-cadherin (e), and negative GPHA (f), and MPO (g). Flow cytometry (h-k) shows lack of MPO (h), bright CD71 (i), partial CD34 expression of JO and positive CD117 (k) FIGURE 22.37 Acute erythroid leukemia. Histology (a) shows predominance of blasts, which express CD71 (b). Flow cytometry (c-e, arrow) shows bright expression of CD235a (GPHA) and CD71, and negative MPO FIGURE 22.38 AEL with deletion of 5q and 7q. (a) Histology shows sheets of immature mononuclear cells replacing normal marrow elements. (b-c) Immunohistochemistry shows strong expression of E-cadherin and CD117. (d–f) Flow cytometry shows blasts (orange dots) with negative CD45 (d), positive CD117 (e) and bright expression of CD71 (e). FISH show deletion of 5q (f) and deletion of 7q (g) FIGURE 22.39 AEL-differential diagnosis. (a) Erythroid hyperplasia with megaloblastoid features (B12/folate deficiency). (b-c) MDS (b, MDS with ring sideroblasts; c, MDS with excess blasts 2. (d) Plasma cell myeloma (PCM). (e) Acute megakaryoblastic leukemia. (f) Metastatic carcinoma. (g) Anaplastic large cell lymphoma (ALCL). (h) Reactive erythroid hyperplasia (patient with hemolytic anemia) FIGURE 22.40 Acute megakaryoblastic leukemia – cytology (two cases). (a-a") BM aspirate smear shows numerous blasts with basophilic cytoplasm with distinct blebs or pseudopod formation. (b-b') Blood smears show leukoerythroblastosis with large blasts, giant platelets, and immature nucleated erythroid cells FIGURE 22.41 Acute megakaryoblastic leukemia - histologic features of four different cases. Histologic features in acute mega- karyoblastic leukemia vary from case to case. In some cases, poorly differentiated blasts predominate (a). In other, there is mixed population of highly atypical megakaryocytes, megakaryoblasts, and blasts (b-c). In some leukemias, highly dysplastic (hypolobated) megakaryocytes predominate (d) FIGURE 22.42 Acute megakaryoblastic leukemia - flow cytometry. Although the phenotypic pattern may vary, most cases show the following phenotype: low side scatter (a, arrow), CD34+ (moderate expression; b), negative to dim expression of CD117 (c), negative HLA-DR (d), negative CD13 (e), bright expression of CD33 (f), dim expression of CD64 (g), and co-expression of CD41a and CD61 (h-i) FIGURE 22.43 Acute megakaryoblastic leukemia (flow cytometry). Blasts (green dots) show negative to dim CD45 expression (a), and positive CD41 (b) and CD61 (c) FIGURE 22.44 Acute panmyelosis with myelofibrosis. BM is hypercellular with myeloid and erythroid leftward shift (a-b), increased blasts (c-d), megakaryocytic hyperplasia with atypia (e), and diffuse reticulin fibrosis (f) FIGURE 22.45 Acute panmyelosis with myelofibrosis in patient with COVID infection. (a) Blood smear shows thrombocytosis with prominent cytologic atypia of platelets. (b) BM core biopsy shows hypercellular marrow with megakaryocytic hyperplasia and increased blasts. (c-d) Flow cytometry. Blasts (green dots) are increased; they are CD34+ (c) and CD117+ (d) FIGURE 22.46 Acute basophilic leukemia - cytology. Numerous immature cells with basophilic granules are present FIGURE 22.47 CML - myeloid blast phase with basophilia (a,cytology; b, histology) FIGURE 22.48 Extramedullary myeloid tumor (granulocytic sarcoma) - lymph node. (a) Atypical large cell infiltrate in the inter- follicular area. (b-c) Higher magnification shows mononuclear cells with blastoid appearance. Neoplastic cells have the following phenotype: CD20 (d), CD3 (e), MPO+ (f), CD15+ (g), CD117+ (h; dim), CD34+ (i) FIGURE 22.49 Extramedullary myeloid tumor (monoblastic sarcoma) skin. (a) Dense infiltrate in the dermis without epider- motropism. (b) High magnification shows mononuclear cells with irregular nuclei. Tumor cells are positive for CD45 (c), negative for CD34, positive for CD68 (e), CD56 (f), and HLA-DR (g) FIGURE 22.50 Extramedullary myeloid tumor (granulocytic sarcoma) involving small intestine. Histology (a-b) shows diffuse large cell infiltrate immunoreactive with CD34 (c) and MPO (d). Flow cytometry (e-f) shows blasts expressing CD33, HLA-DR, CD34, and CD117 FIGURE 22.51 EMT with the phenotype of AML/T-ALL - lymph node. (a) Low magnification shows interfollicular/paracortical infiltrate. (b) High magnification shows blastic appearance of neoplastic cells. The phenotype of blasts fulfilled the criteria proposed by European Group for the Immunologic Classification of Leukemia for acute biphenotypic leukemia. Immunohistochemistry (c-f) shows positive expression of CD5 (c), MPO (d), CD34 (e), and TdT (f) FIGURE 22.52 EMT with features of biphenotypic acute leukemia (B-ALL and AML with monocytic differentiation). (a) Histology with atypical mononuclear infiltrate (mostly large cell type on the left, and small cell type on the right). B-lymphoblastic component is positive for CD34 (b) and CD79a (c), whereas myeloid component expresses CD56 (d), CD4 (e), and TdT (f) FIGURE 22.53 AML-MRC. Bone marrow (a) is hypercellular with myeloid and erythroid leftward shift. Aspirate smear (b-c) shows increased blasts and prominent dyserythropoiesis (arrows). Flow cytometry (d-k) shows increased blasts (green dots), increased monocytes (dark blue dots) and neutrophils and maturing myeloid precursors (gray dots) with low side scatter. Blasts are positive for CD45 (d), CD34 (e), CD117 (f), CD133 (g), CD13 (h), and CD33 (k). Monocytes are positive for CD13 (h), CD14 (i), and CD33 (k). Neutrophils/maturing myeloid precursors show aberrant down-regulation of CD16 (j) FIGURE 22.54 Therapy-related myeloid neoplasm - flow cytometry. Flow cytometry of the BM aspirate from patient with a his- tory of B-cell lymphoma after cytotoxic therapy shows increased blasts (red arrows), increased CD45-negative erythroid precursors (a; black dots), down-regulation of CD45 on granulocytes (a), increased side scatter of CD34+ (b), CD117+ (c), and HLA-DR+ (d) myelo- blasts (positive CD34 and HLA-DR excludes promyelocytes). Granulocytes (green arrows) can be separated from blasts be positive CD11b (e) and CD16 (f), and lack of CD34 (b), CD117 (c), and HLA-DR (d) FIGURE 22.55 Therapy-related myeloid neoplasm: flow cytometry and cytogenetics. Bone marrow from patient with history of follicular lymphoma (stage IV) after cytotoxic therapy. Flow cytometry (a-e) shows increased blasts (blue dots; a-c) and phenotypic features of dyspoiesis on granulocytes (purple dots), including decreased side scatter (a), down-regulation of CD10 (d), and aberrant pattern of CD13 versus CD16 expression (e). Metaphase cytogenetics (f) shows complex changes including monosomy 5 and mono- somy 7: 42-44,XX,add(4)(q21),-5,add(6)(p22), 7, t(8;17)(p22;q12), t(9;16)(q22;q22), add(12)(p11.2), 18 FIGURE 22.56 Therapy-related myeloid neoplasm (AML after therapy for breast carcinoma) - flow cytometry. Blasts (green dots) are positive for CD34 (a), CD117 (b), CD33 (d), CD11c (d), CD19 (e), and partially CD56 (f). Cytogenetic and FISH studies were negative for t(8;21)/RUNX1-RUNXITI ■■■【23】AML defined by Genetic FIGURE 23.1 Acute promyelocytic leukemia (APL) - cytology. Aspirate smear with hypergranular promyelocytes and promyelo- cytes with numerous Auer rods FIGURE 23.2 APL-v cytology. Hypogranular (microgranu- lar) variant of APL is characterized by agranular cytoplasm and bilobed nuclear shape (four different cases) FIGURE 23.3 APL-v-cytology. Even in hypogranular (microgranular) variant of APL occasional promyelocytes with Auer rods can be identified (a, arrow). Another characteristic feature helpful in identification of APL-v is presence of asymmetric cytoplasmic protrusions (which occasionally may contain granules or Auer rods; b-c) FIGURE 23.4 APL – histomorphology (three different APL cases). BM is replaced by abnormal promyelocytes with abundant pale cytoplasm and conspicuous borders (fried egg or plant-like cells; a), promyelocytes with eosinophilic cytoplasm (b), and APL-v (c) FIGURE 23.5 APL - metaphase cytogenetics with t(15;17) FIGURE 23.6 APL - FISH. FISH analysis shows PML-RARA fusion (yellow signal, arrow). Inset shows negative control (note normal pattern with two red and two green signals) FIGURE 23.7 APL - immunohistochemistry. (a-b) Histologic section shows hypercellular marrow with atypical promyelocytes. Neoplastic cells are negative for CD34 (c) and HLA-DR (e) and are positive for MPO (d) and CD117 (f) FIGURE 23.8 Sixty-six-year-old female patient with clinical diagnosis of neutropenia and negative work-up. Flow cytometry of blood (a-a") showed 1.7% blasts with slightly increased SSC (a), bright CD33 (a') and positive CD117 (a"). Note the typical for APL very bright expression of CD33 (much brighter than in benign neutrophils). Based on flow data, APL was suspected, which was confirmed by PCR for PML-RARA (b). Subsequent BM biopsy showed extensive involvement by APL with typical cytologic (c) and histologic (d) findings FIGURE 23.9 Rapid progression of APL within 5 days (blood sample). First flow cytometry analysis of blood due to pancytopenia. (day 1, a) shows only very few blasts (green dots, arrow) with slightly increased SSC (a), positive CD117 (a') and bright CD33 (a"). Repeated flow cytometry analysis of blood after few days shows numerous neoplastic promyelocytes (b) with typical high SSC (b), positive CD117 (b') and bright CD33 (b"). Presence of event very minute population of blasts in blood with positive CD117 and brighter expression of CD33 than in neutrophils (a") should prompt immediate molecular testing for PML-RARA rearrangement FIGURE 23.10 APL-flow cytometry of hypergranular variant (pattern 1). Neoplastic promyelocytes are characterized by very high SSC and moderate CD45 (a), negative CD34 (b), positive CD117 (c), negative HLA-DR (d; compare with negative staining with the same fluorochrome for CD8, inset), negative CD10 (e), positive CD13 (f), negative CD16 (g), and dim CD64 (h) FIGURE 23.11 APL - flow cytometry. Neoplastic promyelocytes (green dots) show increased SSC (a) and moderate expression of CD45 (a). They are positive for CD117 (b), negative for CD11b (c), CD11c (d) and CD15 (e), positive for CD13 (f), CD33 (g), CD64 (h), and CD123 (i) and do negative for CD34 (j) and HLA-DR (k) FIGURE 23.12 Neoplastic promyelocytes in APL are char- acterized by high (non-specific) background fluorescence (left column; compare the staining of lymphocytes and APL cells). The classification of each marker to negative or positive category needs to be based on careful correlation with isotypic controls. Based on background staining of promyelocytes (gray dots, black dotted line), the expression of CD34 is negative (a-a'), CD117 is positive (b-b'), CD133 is negative (c-c') and HLA-DR is negative (d-d'). If one would evaluate expression of each marker based on controls set up on lymphocytes (red dotted line), CD34 and HLA-DR would be classified mistakenly as positive FIGURE 23.13 APL-v (hypogranular variant) - flow cytometry (pattern 2). Neoplastic promyelocytes show slow SSC (a) with the following phenotype: CD34 partially+ (b; minor population), CD117+ (c), HLA-DR (d), CD13+ (e), CD3+ (f), CD56+ (g), and CD64+ (h) FIGURE 23.14 APL-v (hypogranular variant) - flow cytom- etry (pattern 2). Neoplastic promyelocytes (blue dots) are char- acterized by low SSC (a), positive CD45 (a), positive CD34 (b), positive CD2 (c), dimly positive CD64 (d), positive CD13 (e), and lack of HLA-DR (f) FIGURE 23.15 APL-v with typical phenotype and aberrant expression of CD56: CD2+ (a), CD13+ (b), CD33+ (c), CD34+ (d), CD56+ (e), CD64+ (f), CD117+ (g), and HLA-DR- (h) FIGURE 23.16 APL with pattern 3 (partial involvement). Flow cytometry analysis of blood (a) shows mixed population of blasts (green dots), neutrophils (gray dots), monocytes (dark blue dots) and lymphocytes (red dots) [inset show typical hypogranular pro- myelocyte with bilobed nucleus]. Blasts (promyelocytes) show the phenotype of APL-v: CD2+ (b), CD11c (c; neutrophils are positive), CD13+ (d), CD33+ (e), CD34+ (f), CD117+ (g), and HLA-DR- (h) FIGURE 23.17 APL-flow cytometry (pattern 3). Two cases of APL (a and b) showing partial bone marrow involvement. Neoplastic promyelocytes (green dots; arrow) are CD117+ (a', b'). Neutrophils (gray dots) are CD117 FIGURE 23.18 APL (pattern 3). Flow cytometric analysis of blood (a) shows neoplastic promyelocytes (green dots) with high SSC, mixed with benign blood elements (neutrophils, monocytes, and lymphocytes). Blood smear shows scatter hypergranular promy- elocytes (b-c). APL cells shows typical bright CD33 expression (much brighter than in benign neutrophils (d), negative HLA-DR (e, compare with negative isotypic control, inset), positive (dim), and negative CD11b (f) FIGURE 23.19 APL (pattern 4). Unusual variant of APL composed of hypergranular promyelocytes with high SSC (a; magenta dots) and hypogranular promyelocytes with low SSC (a; green dots). Both populations express CD117 (b) and are brightly positive for CD33 (c). CD34 is expressed only by hypogranular subset of neoplastic promyelocytes (d-e; green dots). In contrast to neutrophils and benign myeloid precursors, hypergranular promyelocytes (magenta dots) are negative for CD11b). Apart from CD34 expression, both popula- tions differ in the expression of CD2 (h) and CD15 (i) with hypogranular promyelocytes being positive for CD2 (h, green dots) and hypergranular promyelocytes being positive for CD15 (I; magenta dots). Histology section of the bone marrow core biopsy shows diffuse infiltrate of neoplastic promyelocytes (j). Aspirate smear (k-1) shows two distinct promyelocytes, hypogranular cells with irregular nuclei and basophilic cytoplasm without granules, and hypergranular blasts with numerous cytoplasmic granules and occasional Auer rods FIGURE 23.20 Atypical myelomonocytic population (CD117/CD11b+/CD33+/CD64+/PML-RARA+) after treatment with ATRA com- patible with "differentiation syndrome" (recent history of APL). Flow cytometry shows very atypical myelomonocytic cells with high SSC, bright CD45 (a), negative CD34 (b) and CD117 (c), bright CD33 (d), bright CD11b (e), partial CD14 (f) and CD16 (g), and bright CD64 (h). FISH studies (i) showed rearrangement of RARA at 17q21 (48%). PML-RARA rearrangement was also confirmed by PCR (j) FIGURE 23.21 APL (left column; BM sample at the time of diagnosis) and APL after treatment (right column; blood sample 30 days after therapy). Both samples are positive for PML-RARA by FISH analysis. Neoplastic promyelocytes show typical flow cytometric fea- tures of APL: high SSC (a), negative CD10 (b), CD11b (c), CD11c (d) and CD16 (e), brightly expressed CD33 (f), positive CD117 (g) and negative HLA-DR (h; compare with isotypic negative control showing high background, non-specific fluorescence). "Differentiation syndrome" shows atypical myelomonocytic cells with high SSC (a), negative CD10 (b), positive CD11b (c'), positive CD11c (d'), nega- tive CD16 (e'), positive CD33 (f'), negative CD117 (g), and negative HLA-DR (h') FIGURE 23.22 APL after therapy with atypical myelomonocytic cells compatible with "differentiation syndrome". Aspirate smear shows highly atypical immature cells with monocytic and myelocytic features (a–c). Based on flow cytometry analysis (d-h), the cells show phenotype overlapping between neutrophils and monocytes with partially positive CD11b (d) and CD16 (e), positive CD33 (f) and CD64 (g), and negative CD117 (h). FISH studies were positive for PML-RARA rearrangement FIGURE 23.23 Acute monoblastic leukemia. Monoblasts (a-b) have irregular nuclei, which look similar to hypogranular promyelo- cytes. In contrast to APL, monocytic cells are NSE+ (c) and MPO- (d) FIGURE 23.24 Acute monocytic (monoblastic) leukemia. Neoplastic monocytes (green dots) show slightly increased SSC and mod- erate CD45 (a), negative CD117 on majority of cells (b; minor population is positive), positive HLA-DR (c), positive CD4 (d), positive CD11b (e), positive CD11c (f), positive CD33 (g) and positive (bright) CD64 (h). Neoplastic promyelocytes (APL) are positive for CD117, negative for CD4, CD11b, and CD11c, show brighter CD33 and dimmer CD64 FIGURE 23.25 AML with increased SSC. Occasional cases of AML may "mimic" APL by displaying increased SSC (a; green dots), negative CD34 (b), and positive CD117 (c). In contrast to APL, minor population of blasts in this AML shows dim HLA-DR expression (d) and major population of blasts is CD11c+ (e) FIGURE 23.26 Comparison between APL (left column) and NPM1+ AML (right column). APL and subset of NPM1+ AML (a; aspirate smear) may show similar phenotype, including lack of CD34 (b), positive CD117 (c) lack of HLA-DR (d), and positive CD33 (f). The subtle flow cytometric differences include the pres- ence of CD11c expression by NPM1+ AML (e) and brighter expres- sion of CD33 by APL (f). The threshold for positive versus negative expression is established based on isotypic negative controls, as APL blasts tend to show high non-specific background fluores- cence with many of the fluorochromes. Molecular testing for PML- RARA and NPM1 is crucial in establishing the final diagnosis FIGURE 23.27 Plasmablastic plasma cell myeloma (a) with aberrant phenotype mimicking hypogranular APL. Plasma cells (green dots) display the following phenotype: CD45+ (b), CD34 (c), CD117+ (d), HLA-DR- (e), CD33+ (f), CD38+ (g), and CD56+ (h). Lack of HLA-DDR, strong expression of CD33 and positive CD117 may suggest APL based on flow data alone FIGURE 23.28 AML with RUNX1-RUNX1T1 - flow cytometry. Blasts are large with nucleoli and occasional Auer rods (a; smear from flow sample). Flow cytometry (b-g) reveals phenotypic features of AML with maturation (b; blasts are represented by blue dots and maturing myeloid precursors by purple dots). Blasts are positive for CD45 (b), CD34 (c), CD117 (d), CD33 (e), CD19 (f; partial), and CD56 (g; partial). Granulocytes show aberrant CD56 expression on subset (g) FIGURE 23.29 AML with t(8;21) - flow cytometry. Myeloblasts have granular cytoplasm (a). They are positive for CD13 (b; green dots), CD33 (c), CD34 (d), CD19 (e), CD56 (f), and CD117 (g) FIGURE 23.30 AML with t(8;21)/RUNX1-RUNX1T1. The flow cytometry pattern is often similar to AML with maturation. Blasts (blue dots; arrow) are positive for CD45 (a), CD34 (b), CD117 (c), HLA-DR (d), and CD13 (e). Blasts in AML with t(8;21)/RUNX1- RUNX1T1 often show aberrant expression of CD19 (f) and CD56 (g; partial positivity). FISH studies (h) revealed fusion between RUNX1-RUNX1T1 (yellow signal) FIGURE 23.31 AML with t(8;21) [RUNX1-RUNX1T1] translocation (a, cytogenetics; b, FISH) FIGURE 23.32 AML with inv(16)(p13q22. Aspirate smear (a) shows abnormal eosinophils with immature, large basophilic col- ored granules. Histologic section (b) shows mixed population of blasts, maturing myeloid precursors and eosinophils. FISH stud- ies (c) shows inv(16) FIGURE 23.33 AML with CBFB-MYH11. Aspirate smear (a-b) shows large blasts with irregular nuclei and prominent cytoplasmic gran- ules and occasional Auer rods (mimicking APL). BM core biopsy (c) shows hypercellular marrow with sheets of blasts and increased eosino- phils. Flow cytometry analysis (d-h) shows blasts (green dots) with low SSC (d), dim CD33 (e), positive CD34 (f), CD117 (g), and HLA-DR (h) FIGURE 23.34 AML with inv(16) (a, FISH; b, cytogenetic) FIGURE 23.35 AML with CBFB-MYH11 - flow cytometry. Flow cytometry analysis shows increased blasts (blue dots) and mono- cytes (green dots). In addition, maturing myeloid cells (gray dots) and atypical eosinophils (pink dots) are present. Blasts show dimmer CD45 when compared to monocytes (a); they are positive for CD34 (b), CD117 (b), and HLA-DR (c). Monocytes are negative for CD34 and CD117 (b) and are positive for HLA-DR (c; brighter expression than in blasts), CD14 (d), CD64 (d), and CD11b (f). Atypical (imma- ture) eosinophils are positive for CD123 (e) and CD11b (f; dimmer expression than in monocytes) FIGURE 23.36 AML with t(3;3). BM (a-d, at low intermediate and high magnification) shows clusters of blasts (CD34+, e) and prominent megakaryocytic hyperplasia (CD61+, f) FIGURE 23.37 AML with NPM1 mutation (hypogranular APL-like flow cytometry pattern). Blasts (green dots) show low SSC (a), negative CD34 (b), dim (partial) CD117 (c), negative HLA-DR (d), dim CD11c (e), dim CD33 (f), positive CD33 (g), and negative CD64 (h) FIGURE 23.38 AML with NPM1 mutation (AMML-like flow cytometry pattern). Blasts (green dots) show low SSC (a), negative CD34 (b), and positive CD117 (c). Monocytes (blue dots) show bright CD45 (a), negative CD34 (b), negative CD117 (c), and bright CD64 (d) FIGURE 23.39 AML with NPM1 mutation. Flow cytometry analysis (a-g) shows acute monoblastic leukemia-like pattern. Blasts are positive for CD45 (a), HLA-DR (d), CD33 (e), CD64 (f; bright), CD14 (g; variable expression), and CD56 (g). CD34 and CD117 are negative (b-c). Metaphase cytogenetics shows normal karyotype (e; 46,XY) and molecular results are negative for CEBPA (i) and FLT3- ITD (j) mutations and positive for NPMI mutations (k) FIGURE 23.40 AML with NPM1 mutation (BPDCN-like flow cytometry pattern). Blasts (green dots) show low SSC (a), dim CD45 (a), negative to dim CD4 (b), negative CD13 (c), negative to dim CD33 (d), negative CD34 (e), positive CD56 (f), negative to dim CD117 (g), and negative HLA-DR (h) FIGURE 23.41 AML with NPMI mutation and eosinophilia. Blasts (green dots) show low SSC (a) and high FSC (b-h). Eosinophils show high SSC (a) and low FSC (b-h) and rare neutrophils show high FSC (b, d). Apart from low FSC, eosinophils are characterized by negative CD10 (b), bright CD13 (c), negative CD16 (d), and positive CD33 (e). Blasts are positive for CD13 (c; dim expression), CD33 (e) and CD117 (g, partial expression), and are negative for CD34 (f) and HLA-DR (h) FIGURE 23.42 Myeloid neoplasm with the translocation t(6;9). BM sample from 29-year-old patient with severe anemia (6 months after delivering a baby). BM core biopsy (a) shows hypercellular marrow with myeloid and erythroid leftward shift and decreased M:E ratio due to erythroid hyperplasia. Aspirate smear (b-b'''') shows marked dyserythropoiesis (arrows) and leftward shift of both myeloid and erythroid precursors (myeloblasts comprised <5% of marrow elements confirmed with CD34 immunostaining on both core and clot sections, not shown). Metaphase cytogenetic (c) shows the translocation t(6;9), known to be associated with DEK (located at 6p22) and NUP214 (CAN located at 9q34) gene rearrangement. Flow cytometry (d-i) shows rare blasts (green dots, d) positive for CD34 (e) and CD117 (f). Neutrophils and maturing myeloid precursors (brown dots) show decreased SSC (d), partial expression of CD117 (f), and down-regulation of CD10. The expression of CD11b, CD11c, CD15, CD16, and HLA-DR by myeloid cells did not show overt abnormalities ■■■【24】Residual AML FIGURE 24.1 AML without aberrant phenotype (negative for LAIP). Blasts (green dots) show low side scatter (a), moderate expression of CD45 (a), negative CD10 (b), positive CD13 (c), negative CD15 (d), negative CD16 (e), positive CD33 (f), positive CD34 (g), positive CD38 (h), negative CD56 (i), positive CD64 (j), positive CD71 (k), positive CD117 (1), positive CD133 (m), positive CD200 (n), and positive HLA-DR (o) FIGURE 24.2 AML positive for LAIP. Blasts (green dots, arrow) show low side scatter and moderate CD45 (a), partial/dim CD117 (b), negative CD34 (b), positive CD56 (c), dim/partial CD13 (d), and positive CD15 (e) FIGURE 24.3 MRD AML after treatment. Simplified illustration of the strategy for analysis of MRD in AML. (a) BM sample is gated on the viable CD45+ cells (without cells with low FSC, non-viable cells, and erythroid cells). The staining with CD34 (b) and CD117 (c) help to identify the final position of the blasts in the CD45dim region (b, c). The CD45dim+ cells are back gated in the CD45/ SCC and FSC/SSC plots. Blasts (green dots) defined by CD45dim+ cells, CD34+ cells and CD117+ cells are analyzed for the expression of different markers to identify aberrant phenotype of blasts. In this case, blasts showed partially dim expression of CD7 (d), minimally positive expression of CD11b (e), normal expression of CD13 (f), CD15 (g), CD19 (h) and CD33 (i), aberrant expression of CD56 (j), and CD123 (k), indicating presence of measurable minimal AML. The threshold for positive and negative expression of an antigens are set up based on staining with isotypic control antibodies on lymphocytes, blasts, and myeloid cells FIGURE 24.4 MRD+ APL after treatment. Neoplastic promyelocytes (green dots; arrow) are positive for CD117 (a-c) and CD33 (b), and do not express CD34 (a) and HLA-DR (c) ■■■【25】Ambigus AML FIGURE 25.1 AUL. Blood (a) and BM aspirate smear (b) shows large blasts with irregular nuclei and prominent nucleoli. BM biopsy (c) shows replacement of the BM by sheets of immature cells. Based on immunohistochemistry (d-k) and flow cytometry (l-u), blasts are positive for CD45 (1), HLA-DR (0), CD123 (h, p), CD38 (r), and partially CD10 (j, s). Few blasts show CD117 expression (n) FIGURE 25.2 Minimally differentiated AML with dim CD45 (a, green dots), positive HLA-DR (b), positive CD117 (b), dim CD33 (c), mostly negative CD13 (c) and positive CD7 (d). Both AUL and AML with minimal differentiation are MPO negative but expression of two of myeloid markers (CD13, 33 and/or CD117) support the diagnosis of minimally differentiated AML FIGURE 25.3 Poorly differentiated acute leukemia with phenotypic features overlapping between MPAL and AUL. Blasts (green dots) show the following phenotype: CD45+ (a), CD2+ (b), surface and cytoplasmic CD3- (c-d), CD13-(e), CD19+ (f), CD33- (g), CD34+ (h), CD64- (i), cytoplasmic CD79a partially positive (j), CD117+ (k) and MPO- (1). NGS studies showed mutations in PHF6, TP53, and NRAS genes FIGURE 25.4 Bilineal mixed phenotype acute leukemia (MPAL; AML/B-ALL) – BM (flow cytometry). Side scatter versus CD45 (a) shows two populations of blasts: CD45+ blasts (green dots) and CD45 blasts (orange dots). CD45+ blasts have phenotype compat- ible with B-ALL: CD34+ (b and d), bright CD10+ (b-c), CD20+ (c), CD33- (d), and CD22+ (e). The other population of blasts (CD45-) is CD34+ (b and d), CD33+ (d), and negative for B-cell markers (c, e) FIGURE 25.5 MPAL (AML/T-ALL) - BM. (a) Hypercellular bone marrow showing complete replacement by large blasts. (b) Aspirate smear showing blasts and rare erythroid precursors. (c) Blasts are positive for MPO. (d-h) Flow cytometry analysis shows the following phenotype of blasts: HLA-DR+ (d), CD117+ (d), CD34+ (e), CD10+ (e), CD7+ (f), cytoplasmic CD3+ (f), CD13+ (g; dim), and CD33+ (h) FIGURE 25.6 MPAL (AML/T-ALL) - flow cytometry. Blasts have the following phenotype: CD45+ (a), CD34+ (b), CD33+ (c), CD7+ (d), cytoplasmic CD3+ (e), and cytoplasmic MPO+ (e-f; partial). Note bright expression of CD7 and variable ("smeary") expression of CD33 (g) FIGURE 25.7 MPAL (AML/B-ALL; BCR-ABL1+)- flow cytometry and FISH. Blasts are positive for CD45 (a), CD34 (b), CD117 (c; partial expression), HLA-DR (d), CD13 (e), CD33 (f), CD19 (g), CD22 (h), cytoplasmic CD79a (i), and partially MPO (j). FISH studies showed BCR-ABLI fusion (k; aqua, arginosuccinate synthetase 1 gene on 9q34; green, BCR gene on chr.22; and red, ABL1 gene on chromosome 9; yellow signal indicates BCR-ABLI fusion) FIGURE 25.8 Bilineal MPAL composed of blasts with AML phenotype and separate population of blasts with B-ALL phenotype. Blasts (green dots) show low side scatter and moderate CD45 (a). They are positive for CD34 (b), with blasts of lineage showing slightly brighter expression. Blasts of myeloid lineage are positive for CD117 (c) and CD13 (d), negative for CD19 (e), and positive for HLA-DR (f). Blasts with B-phenotype are negative for CD117 (c), positive for CD13 (d), positive for CD19 (e), and positive for HLA-DR (f) ■■■【26】Other Neoplasms FIGURE 26.1 Blastic plasmacytoid dendritic cell neoplasm cytology. Bone marrow aspirate with predominance of blasts. Tumor cells have irregular nuclei, inconspicuous nucleoli and pale basophilic cytoplasm FIGURE 26.2 BPDCN - skin. (a–c) Histology shows diffuse infiltrate of immature mononuclear cells. (d-g) Immunophenotyping (flow cytometry and immunohistochemistry) shows expression of CD56 (d, f) andCD4 (e, g) FIGURE 26.3 BPDCN - lymph node. (a) Low magnification shows dense interfollicular infiltrate. (b-c) Higher magnification shows predominance of blasts with round nuclei, prominent nucleoli, and scanty to moderate cytoplasm. (d) Blasts are strongly positive for CD56 FIGURE 26.4 BPDCN - BM involvement. Poorly differentiated cells resembling myeloblasts and/or monoblasts are present in the BM aspirate smear (a). BM core biopsy shows diffuse replacement of the marrow elements by immature cells (b). Tumor cells are nega- tive for MPO (c) and CD117 (d) and express CD4 (e and g) and CD56 (f) [c-f, immunohistochemistry; g-h, flow cytometry] FIGURE 26.5 BPDCN - flow cytometry of the BM aspirate. Neoplastic cells (a; aspirate smear) are positive for CD56 (b), CD4 (c), CD7 (d), CD33 (e; partial), HLA-DR (f), CD123 (g), and do not express CD34 (h), CD117 (i) and CD14 (j). Metaphase cytogenetics (k) shows complex karyotypic abnormalities: 44-46,XY, add(1)(q23),-2, del(5)(q13q33), add(7)(p11.2), -11, del(12)(p11.2p13), del(13) (q12q14), add(15)(q22), add(17)(q21), add(22)(q13),+2mar FIGURE 26.6 BPDCN-flow cytometry (BM). Blasts (green dots) show dim to moderate CD45 and low side scatter (a), positive CD4 (b), CD56 (c), CD123 (d), and positive HLA-DR (e), as well as aberrant expression of CD33 (f). CD34 (g) and CD117 (h) are negative FIGURE 26.7 BPDCN (skin) - flow cytometry. Neoplastic cells are positive for CD4 (a) and CD56 (b). Pan-T-cell markers are negative (c–f) FIGURE 26.8 BPDCN (blood sample from 76-year-old patient with skin lesion and leukocytosis). Blood smear (a) shows numerous blasts. Flow cytometry (b-j) shows the following phenotype of blasts (magenta dots): CD45+ (b), CD4+ (c), CD7++ (d), CD33-(e), CD34- (f), CD56+ (g), CD117- (h), CD123+ (j), and HLA-DR+ (k) FIGURE 26.9 Skin with monoblastic sarcoma. (a) Histology shows diffuse infiltrate by large blasts. (b-g) Immunohistochemistry. Similar to BPDCN, acute monoblastic leukemia (monoblastic sarcoma) is often positive for CD4 (b) and CD56 (c), but presence of monocytic and myeloid markers, including CD33 (d), CD69 (f), and muramidase (f) and lack of CD123 expression (g) help to exclude BPDCN and confirm monocytic differentiation FIGURE 26.10 Follicular dendritic cell sarcoma. (a–c) Histology. (d) Immunohistochemistry: tumor cells are positive for CD21 FIGURE 26.11 Follicular dendritic cell sarcoma. (a) Low magnification shows nests of atypical plump cells with abundant eosino- philic cytoplasm. (b) High magnification shows pleomorphic cells with abundant eosinophilic and somewhat granular cytoplasm. Nuclei have prominent nucleoli. (c-d) Immunohistochemistry: tumor cells are positive for CD21 (c) and negative for CD45 (d) FIGURE 26.12 Follicular dendritic cell sarcoma of the nasopharynx. Histology section (a-b) shows atypical infiltrate of irregular and spindle cells. Expressing BCL1 (cyclin D1, c), fascin (d), CD21 (e), and CD23 (f) FIGURE 26.13 Histiocytic sarcoma - gallbladder. (a-b) Histologic sections show diffuse infiltrate of large pleomorphic cells with abundant cytoplasm and highly atypical nuclei. (c-e) Immunohistochemistry: tumor cells are positive for CD45 (c) and histiocytic/ macrophage markers (CD68 and HAM56; d-e) FIGURE 26.14 Langerhans cell histiocytosis - bone marrow. (a-b) Bone marrow aspirate with large cells with abundant cytoplasm, prominent nucleoli, and occasional nuclear irregularities. (c-d) Bone marrow core biopsy. Diffuse large cell infiltrate replacing normal bone marrow elements (c). High magnification shows characteristic nuclear irregularities (d). Scattered eosinophils are present FIGURE 26.15 Langerhans cell histiocytosis-lymph node. (a) Low magnification shows prominent interfollicular/paracortical infiltrate. (b) High magnification displays typical nuclear grooves (folding). (c-d) Immunohistochemistry: tumor cells are positive for CDla (c) and S100 (d) FIGURE 26.16 LCH involving the BM. (a-b) Aspirate smear show numerous atypical Langerhans cells with abundant cytoplasm. (c-d) Histoloy section (BM core biopsy) shows aggregates woth Langerhans cells with irregular, elongated nuclei. Tumor cells are positive for both $100 (e) and CDla (f) FIGURE 26.17 Langerhans cell sarcoma (axillary lymph node from 88-year-old male patient). (a-c) Flow cytometry analysis shows large CD45+ cells (a), which are negative for CD3 (b) and positive for CD1a (c). Histology sections (d-f) show highly atypical and pleo- morphic infiltrate of large cells with focal intrasinusoidal distribution (e). Tumor cells express CDla (g), S100 (i), CD43 (j), and CD45 (k). CD163 is negative (h) FIGURE 26.18 Lymph node with Langerhans cell sarcoma. (a) Histology (low magnification) shows expansion of paracortical area. (b) Touch smear shows atypical Langerhans cells with irregular nuclei. (c-k) Flow cytometry analysis shows tumor cells (purple dots) with high side scatter (a) and forward scatter (d-k), positive CD45 (c), HLA-DR (d), CD11b (e), CD11c (f), CD13 (g), CD33 (h) and CD64 (i), and negative CD34 (j) and CD117 (k). (1) High magnification of lymph node section shows diffuse infiltrate by atypical oval to spindle to irregular cells with mitotic figures. (m-r) Immunohistochemistry shows tumor cells expressing CDla (m), S100 (n), CD4 (0), CD33 (p) with negative CD163 expression (r) FIGURE 26.19 Bone marrow - metastatic tumors: mucinous adenocarcinoma (a–a"), lobular breast carcinoma (b-b'), ductal breast carcinoma (c-c'), melanoma (d-d'), small cell carcinoma (e-e'), prostate carcinoma (f-f"), neuroblastoma (g), GIST (gastrointestinal stromal tumor; h-h'), alveolar rhabdomyosarcoma (i-i"""") FIGURE 26.20 Metastatic tumors to the bone marrow. Two examples of positive flow cytometry in case of metastatic tumor to BM. (a) Metastatic large cell neuroendocrine carcinoma (a, smear with cohesive clusters of large cells with some cytoplasmic vacuoles, a' flow cytometry). Flow cytometry shows CD45-negative population (arrow) with increased side scatter. Metastatic carcinomas are often positive for CD56 and may be positive for CD38 (dim) and CD117, mimicking plasma cell neoplasms. (b) Metastatic rhabdomyosarcoma to the bone marrow (b, touch smear; b', flow cytometry). Rhabdomyosarcoma cells are negative for CD45 (b, arrow) and may be positive for CD56 and CD117. On aspirate smear or touch smear preparation of bone marrow, metastatic tumors need to be differentiated form plasma cell myeloma, melanoma (c), acute erythroid leukemia (d), anaplastic large cell lymphoma (e), acute megakaryoblastic leukemia (f), diffuse large B-cell lymphoma (g), plasmablastic lymphoma (h), and Burkitt lymphoma (i) or other high-grade lymphomas FIGURE 26.21 Composite hematopoietic neoplasm: AML with NPM1/FLT3 mutations (BM) and high-grade B-cell lymphoma, double- hit (brain + blood). Patient with brain mass and leukocytosis. Flow cytometry analysis of blood showed predominance of large B-cells with moderate expression of CD45 (a, blue dots), positive CD10 (b) and CD20 (c). Smear showed large lymphoid cells with cytoplasmic vacuoles (d). There was no evidence of acute leukemia or circulating CD34+ and/or CD117+ blasts (e-f). Concurrent analysis of brain mass showed clonal B-cells (kappa+, g-h, blue dots), with similar phenotype to that observed in blood sample. BM send subsequently for staging, showed AML with monocytic component (i) positive for NPM1 and FLT3 mutations. BM did not reveal involvement by high grade B-cell lymphoma (j-k) and showed CD117+ (1) and CD33+ (m) blasts (green dots) and increased monocytes (CD33+ and CD14*, m-n, pink dots) compatible with AML FIGURE 26.22 Blood from 89-year-old patient with composite hematopoietic neoplasm: chronic lymphocytic leukemia (CLL) and peripheral T-cell lymphoma (PTCL). CLL cells (red dots) show dimmer expression of CD45 when compared to PTCL (a), are positive for CD19 (b) and CD23 (c) and lack the expression of surface light chain immunoglobulins (d). PTCL (magenta dots) are positive for all pan T-cell markers (e-h) with dim expression of CD5 (g) and partial expression of CD7. CLL cells are also dim CD5+ (g) FIGURE 26.23 Composite B-cell lymphoma: diffuse large B-cell lymphoma (CD10+) and MZL. MZL cells are kappa (a–d) and DLBCL cells are lambda+ (a–d). The expression of CD19 is brighter in MZL than in DLBCL and the expression of CD20 is brighter in DLBCL than in MZL. Both lymphomas can be easily distinguished based on forward scatter (e–h). DLBCL is dim CD19+ (e) and MZL is dim CD11c+ (f). The expression of CD81 is positive in DLBCL and expression of CD200 is negative in both lymphomas (T-cells are dim CD200+, h) FIGURE 26.24 PNH analysis by flow cytometry. PNH analysis includes evaluation of red blood cells (identified by bright GPHA expres- sion; a-b), neutrophils (identified by positive CD15 expression; c-d) and monocytes (e-f; identified by bright CD64 expression. The results of patient's sample for each lineage are compared with negative controls (presented in a, c, and e panels). Flow cytometry revealed the presence of major PNH clone: 5% of CD59 red blood cells (b), 64% of CD24-/FLAER neutrophils (d), and 22% of CD14-/FLAER monocytes (f)
