Refresher in Blood Cell Morphology. Tracy I. George, MD Professor of Pathology University of Utah

Transcription

1 Refresher in Blood Cell Morphology Tracy I. George, MD Professor of Pathology University of Utah

2 No financial conflicts of interest

3 Objectives Recognize non-neoplastic and neoplastic disorders in the peripheral blood smear Distinguish reactive from malignant lymphocytosis Discuss the differential diagnosis of cytopenias Triage peripheral blood specimens for appropriate ancillary testing

4 Examination of the Blood Smear A good quality smear has three zones Feathered edge Monolayer Body

5 Case 1 46 year old male with fever, myalgias, purpura, and neutrophilia for 6 months No splenomegaly or lymphadenopathy Radiologic examination negative ESR, CRP, ANA negative Cultures of bone marrow negative WBC: 49 x 10 9 /L Differential: neuts 96%, mono 1%, meta 1%, myelo 1%, pro 1% Hgb and PLT: normal

6 Case 1, Peripheral Blood Smear

7 Case 1

8 Case 1, Bone marrow aspirate smear

9 Case 1, Bone marrow biopsy

10 Case 1, Cytogenetic karyotype BCR-ABL1 negative by RT- PCR

11 Case 1, CSF3R T618I

12 Case 1 What is your diagnosis? a. Steroid effect b. Endogenous G-CSF effect c. Chronic myeloid leukemia d. Atypical chronic myeloid leukemia e. Chronic neutrophilic leukemia

13 Case 1 What is your diagnosis? a. Steroid effect b. Endogenous G-CSF effect c. Chronic myeloid leukemia d. Atypical chronic myeloid leukemia e. Chronic neutrophilic leukemia

14 Chronic neutrophilic leukemia Leukocytosis 25 x 10 9 /L >80% seg neuts, <10% immature grans, <1% blasts, <1 x 10 9 /L monos, no dysgranulopoiesis Hypercellular marrow, <5% blasts Hepatosplenomegaly Exclude underlying cause for neutrophilia or document clonality (infection, inflammation, drugs, neoplasms including plasma cell myeloma) Exclude: CML, PV, ET, PMF, PDGFRA/ PDGFRB /FGFR1 /PCM1-JAK2

15 N Engl J Med 368: , 2013

16 Model for Activation and Signaling of CSF3R Mutations. Maxson JE et al. N Engl J Med 2013;368:

17 Chronic Neutrophilic Leukemia Usually indolent prognosis Disease progression Increased neutrophilia Splenomegaly Thrombocytopenia and bleeding Infection Bone marrow failure Blast phase JAK2 inhibitor or dasatinib depending on mutation 2016 College of American Pathologists. Materials are used with the permission of the faculty. 17

18 Other Neoplastic Neutrophilias Myeloproliferative neoplasms Chronic myeloid leukemia Other MPN (PV, ET, PMF) MDS/MPN Atypical chronic myeloid leukemia Chronic myelomonocytic leukemia

19 Chronic myeloid leukemia Associated with BCR-ABL1 translocation Usually shows prominent left shift Basophilia Neutrophilic variant shows significant maturation Basophilia may be subtle Associated with p230 BCR-ABL1 transcript

20 Chronic myeloid leukemia

21 Other Myeloproliferative Neoplasms May show erythrocytosis or thrombocytosis Polycythemia vera shows decreased erythropoietin Granulocyte proliferation less than in CNL More likely to have dacrocytes Associated with mutations in JAK2 V617F JAK2 exon 12 CALR MPL

22 Polycythemia vera

23 Myeloproliferative/Myelodysplastic Atypical chronic myeloid leukemia Shows significant dysplasia >10% left shifted granulocytes Associated with more significant anemia More likely to have SETBP1 mutation syndromes

24 Chronic myelomonocytic leukemia Monocytosis >1 x 10 9 /L and 10% of WBC Increased immature monocytes Shows significant dysplasia Mutations seen are not specific but can support diagnosis Lynch DT, Hall J, Foucar K. How I investigate monocytosis. Int J Lab Hematol 2018;40:

25 Neoplastic Neutrophilias Significant elevation in neutrophils May be >50 x 10 9 /L Other CBC parameters abnormal Cytopenias Basophilia Immature forms and blasts present Sustained over time Clonal cytogenetic or molecular abnormality

26 Reactive Neutrophilias Neutrophils are the most abundant leukocyte Most neutrophilias are reactive Infection Inflammation Medication Stress Pregnancy Secondary to plasma cell neoplasm Secondary to other neoplasm

27 Reactive Neutrophilia

28 Reactive Neutrophilia Smaller elevation in WBC Other CBC parameters normal or mildly abnormal Activated changes Toxic granulation Vacuoles DÖhle bodies Transitory Symptoms of inflammation Presence of other malignancy No evidence of clonality

29 Case 2 42-year-old man with 2 wk history of shortness of breath, bleeding gums, epistaxis, tingling sensation over whole body, weight loss, weakness and strong clinical suspicion for thrombotic thrombocytopenic purpura (TTP) WBC: 3.2 x 10 9 /L Hgb: 4.7 g/dl MCV: 122 fl PLT: 27 x 10 9 /L Retic: 5.9%

30 Case 2

31 Case 2

32 Case 2

33 Case 2 What is your diagnosis? a. Megaloblastic anemia b. Microangiopathic hemolytic anemia c. Myelodysplasia d. Oxidative hemolysis

34 Case 2 What is your diagnosis? a. Megaloblastic anemia b. Microangiopathic hemolytic anemia c. Myelodysplasia d. Oxidative hemolysis

35 Case 2: Additional Studies LDH: 1600 U/L [ ] Haptoglobin: <8 mg/dl [30-200] Vitamin B12: 93 pg/ml [ ] Intrinsic factor antibody: positive Parietal cell antibody: negative MMA: 3.08 umol/l [0-0.40] ADAMTS13 activity: 71% [>=67]

36 Severe vitamin B12 deficiency mimicking thrombotic thrombocytopenic purpura JK Routh, SC Koenig. ASH image bank, 9/12/2014.

37 Diagnosis of Megaloblastic Anemias 1. CBC with macrocytic anemia (pancytopenia in severe cases)* 2. Peripheral blood smear: Oval macrocytes Hypersegmented neutrophils Marked anisocytosis and poikilocytosis Can see nrbcs, schistocytes, Howell-Jolly bodies, basophilic stippling, or Cabot rings 3. Vitamin B12 4. Folate 5. Methylmalonic acid (MMA) 6. Total homocysteine *Some patients may present with neurologic symptoms (subacute combined cord degeneration) without anemia, macrocytosis without anemia, or neither macrocytosis nor anemia. MCV can be very high.

38 Vitamin B12 deficiency testing Patients with intrinsic factor antibodies can be vitamin B 12 deficient with spuriously high measured B 12 level. Measurement problems with vitamin B 12 level assays with certain testing platforms When B 12 levels low-normal, reflex to serum MMA Scarpa E et al. Undetected vitamin B12 deficiency due to false normal assay results. Blood Transfus 2013;11: Merrigan SD et al. Intrinsic factor blocking antibody interference is not detected in five automated cobalamin immunoassays. Am J Clin Pathol 2014;141: Yang DR, Cook RJ. Spurious elevations of vitamin B12 with pernicious anemia. N Engl J Med 2012;366: Carmel R, Agrawal YP. Failure of cobalamin assays in pernicious anemia. N Engl J Med 2012;367:385-6.

39 Differential diagnosis of macrocytic anemia Megaloblastic anemia Myelodysplastic syndrome Alcohol/liver disease Drugs Hypothyroidism Hemolysis Myeloma (spurious) Aplastic anemia Chronic lung disease with hypoxia Heavy smoking Physiological (normal neonates, subset of normal pregnancy)

40 Hemolysis

41 Hemolysis

42 Hemolysis Immune mediated Cold agglutinin disease Warm autoimmune hemolytic anemia

43 Hemolysis Toxic shock syndrome Hemolytic uremic syndrome Intravascular hemolysis (microangiopathic hemolytic anemia)

44 Type 1 G6PD deficiency Hemolysis Oxidative hemolysis Supravital stain Dapsone-induced hemolysis

45 Hemolysis Immune mediated Oxidative hemolysis Intravascular hemolysis (microangiopathic hemolytic anemia)

46 Hemolysis follow-up Reticulocyte count Haptoglobin LDH Total bilirubin D-dimers Fibrinogen Urine hemosiderin Direct Coombs test PT/PTT if possible DIC ADAMSTS13 if possible TTP Clinical history

47 Case 3 60-year-old woman with WBC: 8 x 10 9 /L Lymphocytes 52% Mild neutropenia and anemia Long history of rheumatoid arthritis Recurrent bacterial infections

48 Case 3

49 Case 3 Flow cytometry T-cell gene rearrangement studies showed a clonal rearrangement

50 Case 3 What is your diagnosis? a. Reactive T-cell expansion due to autoimmune disease b. Reactive T-cell expansion due to infection c. T-cell large granular lymphocytic leukemia d. Chronic NK cell leukemia e. Sezary syndrome

51 Case 3 What is your diagnosis? a. Reactive T-cell expansion due to autoimmune disease b. Reactive T-cell expansion due to infection c. T-cell large granular lymphocytic leukemia d. Chronic NK cell leukemia e. Sezary syndrome

52 T-cell Large Granulocytic Leukemia Persistent, clonal proliferation of T-cell large granular lymphocytes 2-3% of chronic lymphocytic leukemias Associated with autoimmune disorders Rheumatoid arthritis in 25% of patients Associated with bone marrow transplant Likely due to chronic antigenic stimulation Develops following benign proliferation of T-LGLs

53 T-cell Large Granulocytic Leukemia LGLs >2 x 10 9 /L >15% of WBCs Neutropenia common May have anemia or thrombocytopenia Flow immunophenotype CD3, CD57, CD16 Usually CD8, TCR α/β May show loss of normal T-cell antigens

54 T-cell Large Granulocytic Leukemia Normal cytogenetics in almost all cases STAT3 mutation in 40% of cases Not specific T-cell gene rearrangement studies positive Testing is required in majority of cases

55 T-cell Large Granulocytic Leukemia Heterogeneous course Some cases spontaneously regress Others progress and require treatment Progressive cytopenias Recurrent infections Other immune disfunctions

56 Other Chronic Lymphoproliferative Disorders B-cell disorders Chronic lymphocytic leukemia Monoclonal B-cell Lymphocytosis T-cell disorders Sezary syndrome T-cell prolymphocytic leukemia Adult T-cell leukemia/lymphoma NK cell disorders Chronic lymphoproliferative disorders of NK cells

57 B-Cell Disorders Chronic lymphocytic leukemia Primary differential diagnosis in older adult with lymphocytosis Usually shows small, mature lymphocytes Clumped chromatin CD5+ B-cells

58 T-Cell Disorders Sezary Syndrome Overlap with mycosis fungoides Primary presentation in blood or bone marrow Cerebriform nuclear appearance CD4 positive in majority of cases

59 T-cell prolymphocytic leukemia Similar incidence to T-LGL leukemia Rapidly rising, very high WBC count Bulky lymphadenopathy CD4 positive or CD4/CD8 double positive Inv(14)(q11;q32) in 90% of cases T-Cell Disorders

60 Adult T-cell leukemia Very rare with endemic disease distribution Associated with chronic HTLV-1 infection Usually CD4 and CD26 positive, CD57 negative T-Cell Disorders

61 Chronic lymphoproliferative disorders of NK cells Morphology indistinguishable from LGLs CD2 and CD56 positive, surface CD3 negative T-cell clonality studies negative KIR receptor restricted NK-Cell Disorders

62 Reactive Granular Lymphocyte Expansions Viral infection EBV, CMV most common Morphologic spectrum of lymphocytes with reactive features Following stem cell transplant Associated with CMV infection With autoimmune disease Due to chronic immune stimulation With other neoplasm Solid tumor Lymphoma

63 Reactive vs. Neoplastic Lymphocytosis Features Favoring Reactive Young age Pleomorphic morphology Small mature cells, larger activated cells, LGLs, immunoblasts Clinical history consistent with infection Features favoring neoplastic Adult patients Monomorphic morphology Small, round nuclei Folded or cleaved nuclei Convoluted nuclei Villous cytoplasm Plasmacytoid Granules Prominent nucleoli Large cells

64 Case 4 75-year-old man presenting to ED with abdominal pain, nausea, and vomiting. Abdominal X-ray concerning for possible abdominal aortic aneurysm. WBC: Normal Hgb: 9.9 g/dl MCV: 84 fl PLT: 56 x 10 9 /L

65 Case 4

66 Case 4

67 Case 4

68 Case 4

69 Case 4

70 Case 4 What is your diagnosis? a. Acute myeloid leukemia b. Autoimmune hemolysis c. Intravascular hemolysis d. Myelodysplastic syndrome

71 Case 4 What is your diagnosis? a. Acute myeloid leukemia b. Autoimmune hemolysis c. Intravascular hemolysis d. Myelodysplastic syndrome

72 Best Practices: blood smear morphology Well prepared blood smear is key! 200-leukocyte differential Percentage of blasts Auer rods in blasts: yes or no Dysplasia: type and degree Granulocytes Red blood cells Platelets

73 WHO 2016/2017 Updates in MDS WHO Tumour Classification 4 th edition, IARC 2008, Arber D, Orazi A, Hasserjian R et al, Blood 2016;127: Chart by David Steensma, MD

74 WHO 2016/2017 Updates in MDS Ring sideroblasts 15% of erythroid precursors or 5% if SF3B1 mutation is present WHO Tumour Classification 4 th edition, IARC 2008, Arber D, Orazi A, Hasserjian R et al, Blood 2016;127: Chart by David Steensma, MD

75 WHO 2016/2017 Updates in MDS Dysplastic lineages Cytopenias RS BM, PB blasts MDS-SLD <15/<5%* <5%, <1% MDS-MLD <15/<5%* <5%, <1% MDS-RS-SLD <15/<5%* <5%, <1% MDS-RS-MLD <15/<5%* <5%, <1% Del(5q) <5%, <1% MDS-EB %, 2-4% MDS-EB %, 5-19% or Auer rods MDS-U + 1% blasts <5%, 1% +SLD+pancytopenia <5%, <1% Based on cytog only <15% <5%, <1% RCC none <5%, <2% Adapted from Arber et al. Blood 2016.

76 But dyspoiesis can be seen with: Stress erythrocytosis Autoimmune disease Hemoglobinopathies/thalassemias Aplastic anemia Nutritional deficiencies Alcohol Medications/Toxins Infections DP Steensma. Dysplasia has a differential diagnosis: distinguishing genuine myelodysplastic syndromes (MDS) from mimics, imitators, copycats and imposters. Curr Hematol Malig Rep 2012;7:

77 Acquired sideroblastic anemia secondary to alcoholism Hgb: 8.5 g/dl MCV: 88 fl RDW: 27.5% Normal WBC, PLT, ANC.

78 Acquired Pelger-Huet anomaly Neutropenia and morphologic changes resolved 1 month after reducing MMF dose. Etzell JE, Wang E. Acquired Pelger- Huet anomaly in association with concomitant tacrolimus and mycophenolate mofetil in a liver transplant: a case report and review of the literature. Arch Pathol Lab Med 2006;130:93-6. Courtesy of Joan Etzell, MD

79 Other causes of cytopenias: a long list! Drugs Collagen vascular diseases Autoimmune disorders Sequestration Nutritional deficiencies Infection Inflammatory disorders Hemolysis Congenital disorders Neoplasms

80 ICUS: idiopathic cytopenias of undetermined significance MDS is possible, but not proven Age-related clonal hematopoiesis of indeterminate potential (CHIP) MDS-associated somatic mutations and clonal hematopoiesis are common in ICUS Clonal cytopenias of undetermined significance (CCUS) MDS-associated somatic mutations alone are not diagnostic of MDS Malcovati L, Cazzola M. The shadowlands of MDS: idiopathic cytopenias of undetermined significance (ICUS) and clonal hematopoiesis of indeterminate potential (CHIP). Hematology Am Soc Hematol Educ Program. 2015;

81 Clonal hematopoiesis of indeterminate potential Traditional ICUS MDS by WHO 2008 Non-clonal ICUS CHIP CCUS Lower Risk MDS Higher Risk MDS Clonality Dysplasia Cytopenias Overall Risk BM Blast % < 5% < 5% < 5% Very Low(?) Very Low Low (?) < 5% < 19% Low Treatments Obs/BSC Observation Obs/BSC/GF Obs/BSC/GF IMiD/IST High HMA/HCST Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126:9-16. Clonal Cytopenias

82 References D Chabot-Richards, TI George. Leukocytosis. Int J Lab Hematol 2014;36(3): TI George. Malignant or Benign Leukocytosis. Hematology Am Soc Hematol Educ Program. 2012: I Pereira, TI George, MD, DA Arber. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Wolters Kluwer, Lippincott Williams and Wilkins, Inc., Philadelphia, PA, 2012.