Successful flow cytometric immunophenotyping of body fluid specimens

Successful flow cytometric immunophenotyping of body fluid specimens Fiona E. Craig, MD Division of Hematopathology Mayo Clinic Arizona 2017 MFMER slide-1

Financial disclosure No conflicts 2017 MFMER slide-2

Objectives On completion of the workshop, participants will be able to: 1. List 3 similarities and 3 differences between flow cytometric immunophenotyping of peripheral blood and body fluid specimens 2. Describe 3 steps that can be taken to facilitate successful flow cytometric immunophenotyping of cerebrospinal fluid and vitreous fluid specimens 3. Discuss how they might design an algorithm to assist in triaging body fluid specimens for flow cytometry 2017 MFMER slide-3

Body fluids Cerebrospinal fluid (CSF) Vitreous fluid Ascitic (peritoneal) effusion Pleural effusion Pericardial effusion Breast implant seroma 2017 MFMER slide-4

Peripheral blood vs. other body fluids Similarities: Often relatively easy to collect Normal cell subsets known Can be involved by hematolymphoid neoplasms Neoplastic cells identified through restricted antigen expression and / or aberrant immunophenotype Phenotype often characteristic of a disease entity Immunophenotype can supplement, and sometimes replace morphologic evaluation 2017 MFMER slide-5

Peripheral blood vs. other body fluids Differences: Body fluids may have very few cells e.g. CSF The fluid may be toxic to the cells e.g. CSF Non-specific staining is more often a problem Contamination with peripheral blood is frequent Interpreters are less familiarity with the nonneoplastic disorders e.g. viral menginitis 2017 MFMER slide-6

Keys to successful FCI of body fluids Apply good flow cytometric practices Prevent cell loss and maximize information from a few cells Reduce non-specific staining, if possible, and deal with it, if not Consider contamination with peripheral blood before interpreting the data Become more familiar with the non-neoplastic disorders that may be encountered 2017 MFMER slide-7

CSF: History B lymphoblastic leukemia 2017 MFMER slide-8

CSF: History B lymphoblastic leukemia 2017 MFMER slide-9

CSF cell degeneration Cell number decreases within 30 minutes of sampling Likely due to ph and hypotonicity related cell death Granulocytes and monocytes more rapidly lost than lymphocytes Minimized by acquiring within 24 hours, cooling specimen, fewer centrifugation steps, and aspirating supernatant, rather than decanting Reduced by addition of stabilization medium, often serum containing e.g. RPMI +/- FBS, Earle s balanced salt solution, Transfix TM De Graaf et al., Cytometry Part B 2011:80B;43-50 2017 MFMER slide-10

Staining low cellularity specimens Don t waste cells by performing additional counts Always run one tube, and consider more if > 20,000 total cells Alternatively use 1/3 of sample for a screening tube (Kraan et al., Current Protocols in Cytometry 2008:45;6.25) Focus on disease(s) of interest Avoid intracellular stains because of cell loss and increased non-specific staining Use higher color flow cytometry, or panels with more than one antigen per fluorochrome De Graaf et al., Cytometry Part B 2011:80B;43-50 2017 MFMER slide-11

Antibody selection for CSF specimens Evaluation for aggressive B-cell NHL Collected in Transfix TM and shipped overnight 11 parameter flow cytometry: CD8 and sig lambda (FITC) CD56 and sig kappa (PE) CD4 and CD19 (PerCP-Cy5.5) CD3 (PE-Cy7) CD20 (APC) CD45 (APC-Cy7) Quijano S et al., J Clin Oncol 2009:27;1462-1469 2017 MFMER slide-12

Quijano S et al., J Clin Oncol 2009:27;1462-1469 2017 MFMER slide-13

Minimal requirements for interpretation Reviewed 43 samples: CSF (29), PB (14): leukemia (4), MS (9) headache (8), meningitis (4), hydrocephalus (5), Parkinson disease (2), dementia (3), CNS tumor (5), Guillain-Barré (1), other (2) B-cells and T-cell subsets detected if >5 cells/ul B-cells not detected in 80% with 1 5 cells/ul Minimum events to identify a cell population = 13 Subira et al., Am J Clin Pathol 2002: 117; 2017 MFMER 952-958 slide-14

CSF: History of diffuse large B-cell lymphoma thyroid 177 nucleated cells /ul 2017 MFMER slide-15

CSF: History of diffuse large B-cell lymphoma thyroid 177 nucleated cells /ul 2017 MFMER slide-16

CSF: History of diffuse large B-cell lymphoma thyroid 177 nucleated cells /ul 2017 MFMER slide-17

CSF: History of diffuse large B-cell lymphoma thyroid 3.3 nucleated cells /ul 2017 MFMER slide-18

CSF: History of diffuse large B-cell lymphoma thyroid 3.3 nucleated cells /ul 2017 MFMER slide-19

Other considerations if cellularity low Interpretation is often based on limited information Internal control populations may be lacking Use validated antibody combinations in premixed cocktails Avoid carryover, i.e. cap tubes, acquire and analyze a blank tube 1 st,, record sequence of acquisition There are often too few cells to confirm unexpected findings or investigate further 2017 MFMER slide-20

Successful CSF flow cytometry Craig, at al., Am J Clin Pathol 2011: 135(1); 22-34 2017 MFMER slide-21

Addressing non-specific staining Gate on cells of interest e.g. viable B-cells for Ig If sufficient cells, try multiple washes to remove bound immunoglobulin and other interfering substances Incubate with blocking reagent, such as immune rabbit serum, at 37 0 C Assess for antigens less often affected, e.g. aberrant antigen expression rather than light chain restriction Try intracytoplasmic staining after permeabilization Gate on cells of interest, and use internal controls 2017 MFMER slide-22

Pleural fluid: Concurrent liver mass needle core biopsy demonstrates aggressive B-cell malignancy. 2017 MFMER slide-23

Pleural fluid: Concurrent liver mass needle core biopsy demonstrates aggressive B-cell malignancy. 2017 MFMER slide-24

Ascitic fluid: PTLD, Burkitt lymphoma, EBV+ 2017 MFMER slide-25

Ascitic fluid: PTLD, Burkitt lymphoma, EBV+ 2017 MFMER slide-26

Wright PAP H&E EBER 2017 MFMER slide-27

CSF: History of plasma cell myeloma, lambda monotypic 2017 MFMER slide-28

CSF: History of plasma cell myeloma, lambda monotypic 2017 MFMER slide-29

CSF: History of plasma cell myeloma, lambda monotypic 2017 MFMER slide-30

1418 Pleural fluid: Recent diagnosis double hit lymphoma, kappa monotypic 2017 MFMER slide-31

Pleural fluid: Recent diagnosis double hit lymphoma, kappa monotypic 2017 MFMER slide-32

Pleural fluid: Recent diagnosis double hit lymphoma, kappa monotypic 2017 MFMER slide-33

FISH for MYC/IGH fusion FISH PROBES: MYC[8q24](R)/IGH[14q32](G) Courtesy Dr. L. Baughn MML FISH Laboratory 2017 MFMER slide-34

FISH for 3 BCL2/5 BCL2 Separation FISH PROBES: 3 BCL2(G)/5 BCL2(R)[18q21] Courtesy Dr. L. Baughn MML FISH Laboratory 2017 MFMER slide-35

Do positive flow studies = CNS disease? PB contamination of CSF, particularly if: PB white count high e.g. chronic lymphocytic leukemia Distinctive Immunophenotype e.g. B lymphoblastic leukemia Abnormal cells might be found at sites of inflammation, but are not primary drivers e.g. CSF positive CLL cells, but alternative neurologic diagnoses account for clinical presentation Nowakowski GS et al., Cytometry Part B 2005; 63B:23-27 2017 MFMER slide-36

CSF: History of CLL 11.1/uL nucleated cells; 2.2/uL RBC 2017 MFMER slide-37

CSF: History of CLL 6.1/uL nucleated cells; 2.2/uL RBC 2017 MFMER slide-38

Findings in non-neoplastic disorders Disease WBC Count Predominant cell type Flow Cytometric Findings None 0 5/mL Lymphocyte Acute bacterial meningitis Predominantly CD4+ T-cells B-cells < 1% 100-100,000/mL Neutrophils Granulocytes Viral meningitis 10 1,000/mL Lymphocytes Predominantly T-cells Multiple sclerosis often < 5/mL Lymphocytes Guillain-Barré syndrome Para-neoplastic neurological syndromes Increased CD4+ T-cells, B- cells, and plasmacytoid dendritic cells often < 5/mL Lymphocytes? Increased CD8+ T-cells Variable Lymphocytes Some increase T-cells (x3) Marked increase B-cells (x20) 2017 MFMER slide-39

Findings in non-neoplastic disorders Vafaii P & DiGiuseppi JA. Cytometry Part B 2014; 86B:106-110 2017 MFMER slide-40

CSF: Recent diagnosis of high grade B-cell lymphoma with rearrangements of MYC and BCL-2 2017 MFMER slide-41

CSF: Recent diagnosis of high grade B-cell lymphoma with rearrangements of MYC and BCL-2 2017 MFMER slide-42

Keys to successful FCI of body fluids Apply good flow cytometric practices Prevent cell loss and maximize information from a few cells Reduce non-specific staining, if possible, and deal with it, if not Consider contamination with peripheral blood before interpreting the data Become more familiar with the non-neoplastic disorders that may be encountered 2017 MFMER slide-43

Utility of CSF Flow Cytometry Aggressive B-cell lymphoma (Burkitt or DLBCL): CSF protein, WBC, RBC and LDH not helpful Flow more sensitive than Cytology Low % tumor cells impeded Cytology detection Flow (+) 45% relapsed in CNS despite active treatment Flow (-) 8% relapsed in CNS despite less intensive prophylactic therapy Hegde et al., Blood 2005: 105(2); 496-502 2017 MFMER slide-44

Utility of CSF Flow Cytometry Staging DLBCL, Burkitt, other NHL, AML, B-LL, CML, other CSF+ clinical features suspicious 80%, but NOT all CSF+ more likely to have pleocytosis and increased protein, but NOT all Flow (+) 44/219: Cytology (+) 15, suspicious 5, (-) 24 Cytology (+) 19/219 patients: Flow (+) 15, (-) 4 Flow more sensitive than cytology, but both are necessary Bromberg et al., Neurology 2007: 68; 1674-1679 2017 MFMER slide-45

Utility of CSF Flow Cytometry CSF evaluation for aggressive B-NHL Flow (+) 27/123 (22%) specimens Cytology (+) 6%, suspicious 2% Flow (+) in all Cytology (+), except one false positive Flow (+), Cytology (-) lower cell count than (+)/(+) Cutoff > 20% tumor cells or >1 neoplastic cell/ml A single 11 parameter, 6-color, tube could detect B- NHL regardless of subtype Quijano et al., JCO 2009:27(9); 1462-1469 2017 MFMER slide-46

Utility of CSF Flow Cytometry Demonstrated utility in staging for hematolymphoid neoplasms More sensitive than cytology, especially when cellularity is low Patients with CNS disease may be asymptomatic WBC count, RBC count, protein, and LDH are of limited value NCCN recommends routine use of flow cytometry, in conjunction with cytology, for the diagnosis of CNS lymphoma 2017 MFMER slide-47

CSF: Previous diagnosis high grade B-cell lymphoma, with rearrangements of MYC and BCL6 2017 MFMER slide-48

CSF: Previous diagnosis high grade B-cell lymphoma, with rearrangements of MYC and BCL6 2017 MFMER slide-49

Successful CSF flow cytometry Craig, at al., Am J Clin Pathol 2011: 135(1); 22-34 2017 MFMER slide-50

Indications for body fluid FCI Craig FE et al., Am J Clin Pathol. 2011; 135:22-34: 4.8% (11 of 230 specimens) All but 1 had a previous diagnosis of malignancy Collie AM et al., Am J Clin Pathol 2014; 141:515-21: Positive flow 8.2% (41 of 501 specimens) All had history hematologic malignancy or atypical morphology (atypical lymphocytes or blasts) 2017 MFMER slide-51

Indications for body fluid FCI Requirement in evaluation for known hematolymphoid malignancy, particularly highgrade lymphoma Controversy about use as a screening tool, particularly without a morphologic correlate: False positive CSF monotypic populations in multiple sclerosis Low frequency of positive result if no clinical suspicion hematoplymphoid neoplasm Discuss with frequent requesters and determine best algorithm 2017 MFMER slide-52

Successful flow cytometric immunophenotyping of body fluid specimens Fiona E. Craig, MD Division of Hematopathology Mayo Clinic Arizona 2017 MFMER slide-53