INTERNATIONAL SOCIETY OF LABORATORY HEMATOLOGY. 30TH INTERNATIONAL SYMPOSIUM May, 2017 / HONOLULU, HAWAII

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1 INTERNATIONAL SOCIETY OF LABORATORY HEMATOLOGY 30TH INTERNATIONAL SYMPOSIUM May, 2017 / HONOLULU, HAWAII Michael Keeney, ART, FCSMLS(D) Coordinator Special Hematology, London Health Sciences Centre, Canada Associate Scientist Lawson Health Research Institute

2 Financial Disclosure Consultant for Beckman Coulter 2016 College of American Pathologists. Materials are used with the permission of the faculty.

3 Objectives Review B cell maturation Highlight using case studies the importance of level of marker expression in addition to positive/negative CD45 negative cells a closer look 3

4 FITC PE True PE signal emission intensity 525nm bandpass filter 575nm bandpass filter wavelength Unwanted signal appearing in PE channel

5 Compensation settings determined at PMT settings on population of interest Unstained lymphocytes Unstained Lymphocytes Using single labeled lymphocytes Using multiple labeled lymphocytes UNCOMPENSATED Using multiple labeled lymphocytes COMPENSATED

6 Considerations Ideally, compensation should be performed with the same fluorochromes produced by the same manufacturer as the antibodies under investigation Instrument PMT s must be set at appropriate level (determined in single color staining) for each fluorochrome). The antibody used for compensation must be equivalent to brightest signal likely to be encountered in your assay

7 Alternative to real time compensation Files collected in listmode can be analysed offline using several software packages A control file can be run with mutually exclusive populations and then the settings from this file used to compensate any other data run with the same fluorochromes at the same PMT settings Archival data can be reanalyzed data is not lost if incorrect compensation set at time of acquisition

8 Compensation must be done on the actual fluorochrome used in the test

9 Appropriate Compensation CD34+ 36/uL CD34 ANALYSIS WITH 7-AAD VIABILITY DYE FL4-FL2 = 20% Appropriate compensation

10 Under compensation CD34+ 8/uL CD34 ANALYSIS WITH 7-AAD VIABILITY DYE FL4-FL2-10% Under compensation

11 Recognizing bad flow data

12 Time Gating

13 Doublet Discrimination DOUBLETS CAN CREATE ERRONEOUS RESULTS False CD19+CD5+ population.

14 Current 8-10 color analysis on bench top analyzers round Major advances in computing power, optics, electronics and fluorochrome technology Colors = Complexity what combinations? what fluorochromes?

15 London Health Custom Design Tube Antigen CD8 Kappa CD4 Lambda CD19 CD20 CD5 CD10 CD56 Near-IR Viability Dye CD3 CD45 Fluorochrome FITC FITC PE PE ECD PE-Cy5.5 PE-Cy7 APC APC-Alexa700 Excitation: 633nm Emission 780nm Pacific Blue Krome Orange

16 Result Reporting: Bethesda Guidelines Report percent abnormal population Antibody Distribution Negative, Positive, Partial Antibody Fluorescence - Dim Positive, uniform cell population with a median fluorescence intensity abnormally less than that seen on an antigen positive normal leukocyte population. Example CD20 in CLL - Bright Positive, uniform cell population with a median fluorescence intensity abnormally greater than that seen on an antigen positive normal leukocyte population. Example CD20 in HCL - Heterogeneous Variably intense staining, not a tight uniform cluster of positive immunofluorescence staining. Example CD38 on maturing B cells

17 Bethesda Reporting Dim CLL CD19+, CD20+(dim) CD5+(dim)Kappa+(dim) Normal

18 Discussion The availability of new fluorochromes, coupled with multilaser instruments with simplified setup and compensation makes routine ten color analysis possible for the routine clinical laboratory Knowledge of fluorochromes, antibody and antigen interactions required Full understanding of normal versus abnormal population phenotype required Sophisticated data analysis programs coupled with education essential for successful implementation for all staff involved in testing and analysis

19 Normal B Cell Maturation Wood and Borowitz (2006) Henry s Laboratory Medicine

20 Normal B cell Maturation Pattern

21 The three pearls

22 Case 1 Nine month old male seen by GP for nodules in his scalp, suggestive of lymphoid infiltrate, incidental finding of bilateral enlarged kidneys Referred to hematology as WBC 16,300/uL increased Plt WBC 12,300/uL, Hb 111g/L, Plt 536x10*9/L Normal differential Scalp lymphoid infiltrate suspicious for leukemia Bone marrow aspirate, flow ordered

23 Case 1 34% BM 14% B cells. 5% CD10dim/neg CD20 neg,cd38 bright.?plasma cells

24 Case 1 BM

25 Case 1 Bone marrow morphology Cellular marrow Erythropoeisis 20-25% Granulopoesis 40-50%, orderly maturation Lymphocytes 30%, 10-15% blast like Increased megakaryocytes

26 Case 1 2 week follow up 77% B cells 90% CD10 dim/neg CD20 neg, bright CD38 pb-all

27 Case 1 BM 2 week follow up

28 Case 1 Cytogenetics Complex karyotype Structural abnormalities in chromosome 1,2,3,4,9,14,17 and 19 t(17;19), rare in B cell acute lymphoblastic leukemia

29 Case 1 Follow up On high risk protocol AALL 0232 for infantile B-ALL MRD 0.08% at Day 42 Cord blood transplant Relapse 5 months post transplant Pearl of Pathology Differentiating precursor B cells (hematogones) from B- ALL can be challenging. Standardized analysis and clear understanding of normal essential, including arrested maturation

30 Case 2 4 year old girl referred to pediatric hematology with pancytopenia WBC 6,800/uL(Neuts 400/uL), Hb 42g/L Plt 28? Aplastic anemia Bone marrow ordered, aspirate, biopsy, flow and cytogenetics

31 Case 2 BM flow cytometry Polyclonal B cells, helper suppressor T cells No increase in blasts Maturing myeloid population

32 Case 2 CD45 neg B-ALL

33 Case 2 BM

34 Case 2 CD45neg CD19+CD10+ Cytogenetics Gain of X chromosome and 4,6,8,9,11,13,14,15,17,18,21 CD20(partial,dim) CD34 neg Sig&cIg neg Diploid DNA Index 1.22

35 Case 2 Currently in remission after completion of AALL0932 standard risk B-ALL therapy

36 Case 2 Pearls of Pathology: CD45 negative B-ALL is not uncommon Always look at ungated set of plots for each marker E.g. SS versus each fluorescence parameter

37 Case 3 58 year old male with pararenal mass WBC 4,000/uL, Hb 98g/L, Plt 229 blasts reported on blood film Peripheral blood sent for flow cytometry

38 Case 3 PB flow cytometry Dim CD45, CD19+CD10+ CD34- Sig neg

39 Case 3 PB flow cytometry Dim CD45, CD19+CD10+, CD20(partial)CD5 neg, SIg, cig neg

40 Case 3 BM flow cytometry

41 Case 3 BM biopsy

42 Case 3 BM biopsy

43 Case 3 BM biopsy report Diffuse infiltrate large lymphoid cells Scant to moderate cytoplasm Large irregular nuclei with prominent nucleoli Increased mitotic activity Positive for BCL-2, BCL6, CD10 and MUM1 Negative for TdT CD5 Cytogenetics, FISH on para renal mass C-MYC and BCL2 rearrangements detected

44 Case 3 Double/triple hit lymphoma (DHL/THL) Morphologically heterogeneous B cell neoplasms characterized by rearrangement of the MYC protooncogene with concurrent BCL2 and/or BCL6 gene rearrangement WHO 2008 criteria - most cases of DHL and THL classified DLBCL or B-cell lymphoma, unclassifiable with features intermediate between DLBCL and Burkitt lymphoma (BCLU).

45 Case 3 DHL/THL Retrospective study from one institution, features of 13 cases (9 DHL/4 THL). median age was 59 years (range 30-74) eight females, five males. enlarging lymphadenopathy/masses (11 patients) abnormal peripheral blood findings (2 patients). TdT positivity (four cases, two THL/two DHL); dim CD45 expression (7 cases), lack of CD20 (2 cases), or lack of sig (3 cases) and blastoid morphology (2 cases). Conclusion: Expression of TdT in a B-cell lymphoma with mature features or expression of surface light chain in a case otherwise suggestive of B-lymphoblastic leukemia/lymphoma should prompt an expedited evaluation for DHL/THL. Leuk Lymphoma Feb 18:1-10. [Epub ahead of print] Double- and triple-hit lymphomas can present with features suggestive of immaturity, including TdT expression, and create diagnostic challenges. Moench L, Sachs Z, Aasen G, Dolan M, Dayton V, Courville EL.

46 Case 4 55 year old female Previous history hyperparathyroidism Parathyroidectomy in 1999 Mild persistent hypercalcemia WBC 4,800/uL, hemoglobin 121 g/l, Plt 296 Total protein 90g/L, IgG 34g/L, monoclonal peak 20.9g/L

47 Case 4 Monoclonal IgG patient Polyclonal B cells, With maturing hematogones helper suppressor T cells, less than 3% blasts, Maturing myeloid population

48 Case 4 BM Ungated

49 Case 4 cytoplasmic immunoglobulin Need K/L

50 Case 4 BM Multiple Myeloma

51 Case 4 follow up Smouldering myeloma Recently developed CRAB (hypercalcemia, Renal insufficiency, Anemia, Bone lesions, Serum free light chain ratio 123 Bone marrow aspirate >60% plasma cells Currently on treatment pending bone marrow transplant

52 Case 5 65 year old male Slight lymphocytosis detected on routine CBC analysis (lymph 4,500/uL) Asymptomatic, sent for flow cytometry

53 Case 5 PB

54 Case 5 flow cytometry K/L ratio 0.625

55 Case 5 Monoclonal B cell lymphocytosis K/L 1.8 K/L 0.56

56 Case 5 Monoclonal B cell lymphocytosis WHO defines CLL as abnormal lymphocyte count >5x10E9/L (CD19+CD5+ light chain restricted) in the absence of other clinical symptoms Patients with small B cell clones are often detected as an incidental finding WHO 2016 CD19+CD5+ <0.5x10E9/L Low count MBL» 0.5-5x10E9/L High count MBL» Non CLL type phenotype MBL also recognized

57 Monoclonal B cell Lymphocytosis CLL immunophenotype - vast majority (75%) Atypical MBL normal levels both CD5 and CD20 with variable expression of CD23 (always exclude Mantle-cell Lymphoma in CD23 negative cases) Non-CLL-phenotype (CD5 ) MBL Lacking CD5 expression and CD10, likely resembling B- cell lymphomas of marginal-zone origin.

58 Case 5 What is the normal K/L Ratio? What is the normal or abnormal kappa:lambda ratio for entire population of all B cells? Traditional view Kappa: Lambda 2:1 or 3:1 ( 1:2-1:3) Range

59 Case 5 What is the normal K/L ratio? Wrong question It doesn t matter how many of the B cells are clonal but rather that their exists a clonal population of B cells within the total population Need to Identify malignant B cells within a population of normal B cells In order to interpret a pathological immunophenotype, it is necessary to refer to quantitative and qualitative values of normal B-cell subpopulations Pearl Identify the abnormal phenotype first then look at K/L ratio

60 Case 6 12 year old male presents to ER with fatigue bruising. Lkc 2,200/uL Hb 84g/L Plt 32 Diff neutropenia, no blasts noted, few atypical cells RBC non specific changes? Leukemia

61 Case 6 PB

62 Case 6 BM

63 Case 6 BM aspirate Hypocellular relative lymphocytosis, marked reduction in myeloid lineages.?hypoplastic/aplastic anemia Flow Cytometry less than 3% blasts, maturing myeloid, 68% lymphocytes, polyclonal B, helper, suppressor T. Unusual pattern of CD13/CD16 staining noted

64 Case 6 flow cytometry Aplastic Anemia Normal Patient Normal Patient Normal Patient

65 Case 6 RBC Test for PNH CD235a v s CD59

66 Case 6 Neutrophil test for PNH clone FLAER, CD24, CD45,CD15 18% PNH clone

67 Case 6 Follow up Patient on steroids, acyclovir and anti-thymocyte globulin PNH clone: Neutrophils 9.6%, RBC 1% Nov 2015 In remission as of April Lkc 5,500/uL (Neuts 2.3) - Hb 129g/L - Plt 208 X10e9

68 Summary B cell malignancies are the most common hematological disorders seen in flow cytometry Many have complex overlapping phenotypes Important to look at ungated plots as well as gating on specific subpopulations Level of expression of markers is as important as positive or negative (think CD13 vs CD16)! 68

69 References Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood Apr 15;111(8): Wood BL. Principles of minimal residual disease detection for hematopoietic neoplasms by flow cytometry. Cytometry B Clin Cytom Apr 23. Shanafelt TD, Ghia P, Lanasa MC, Landgren O, Rawstron AC. Monoclonal B-cell lymphocytosis (MBL): biology, natural history and clinical management. Leukemia Mar;24(3): Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, Advani R,Ghielmini M, Salles GA, Zelenetz AD, Jaffe ES. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood May 19;127(20): Moench L, Sachs Z, Aasen G, Dolan M, Dayton V, Courville EL. Double- and triple-hit lymphomas can present with features suggestive of immaturity, including TdT expression, and create diagnostic challenges. Leuk Lymphoma 2016;57: Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom Jul;82(4):