Principles and Application of Flow Cytometry

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Principles and Application of Flow Cytometry

• Rodney Stuart, MD
• August 5,2005

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Objectives

• Basic principles of flow cytometry
• Separation of cells on appearance
• Separation based on light emitting dyes
• Eliminating bleed over between colors
• Studying the cells of interest
• Examples of difficult cases

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Flow Cytometry

• First labeled with fluorescent dyes.
• Forced through a nozzle in a single-cell stream
  passing through a laser beam
• The laser is focused to a known wavelength
• Excitation of a specific fluorochrome
• Photo-multiplier tubes detect the scattering of
  light and emission from the fluorescent dye

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Forward Scatter

• Cells pass through a small (50-300 µm) orifice
• Detection ranges from 0.5-40 µm
• the amount of light scattered in the forward
  direction (along the same axis that the laser
  light is traveling) size of cell

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Side Scatter

• the amount of light scattered to the side
  (perpendicular to the axis that the laser light
  is traveling) is detected in the side or 90o
  scatter channel
• Equates to shape and homogeneity of cells
  (AKA-granularity/complexity)

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Forward and Side Scatter
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Fluorescence Channels

• The specificity of detection is controlled by the
  wavelength selectivity of optical filters and
  mirrors

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Fluorochromes
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One Parameter Histogram

• A one-parameter histogram is a graph of cell
  count on the y-axis and the measurement parameter
  on x-axis.

Events
FITC
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Two Parameter Histograms

• A graph representing two measurement parameters,
  on the x- and y-axes, and cell count height on a
  density gradient. This is similar to a
  topographical map.

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Compensation

• The inherent overlap of emission spectra from
  antibody fluorescent labels makes compensation
  necessary.
• Use a negative control to set your High Voltages
  (HV) per detector. As a rule of thumb  set the
  High Voltages so that your negatives are in the
  1st decade of the parameters being collected.
  Lower high voltage settings will also result in
  lower compensation settings.

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Compensation

• The amount of spectral overlap is corrected by
  subtracting a percentage from the total of FL1
  fluorochrome pulse generated by the FL1 detector.
  The FL1 fluorochrome spillover detected by the
  FL2 detector can be viewed as interference. By
  subtracting a percentage of the total FL1
  fluorochrome pulse from the total pulse generated
  by the FL2 detector (eg. PE FITC interference)
  you can obtain a FL2 fluorochrome only pulse.
• Running samples that are individually stained
  with the antibody-fluorochrome components of
  your multi-color samples aids in appropriate
  compensation.

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Compensation

• FL1 FL1 - FL2 compensation slider

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Compensation
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-/
/-
-/-
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Gating

• Allow you to view cells of interest
• lets you decide which data to view and which data
  to ignore or discard.
• Gating can subsequently be changed when you
  analyze your data without any loss of
  information.

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Case Scenarios

• Non-Hodgkin B cell Lymphoma/Leukemia

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Case 1

• 31 year-old African American HIV male who
  presents with a rapidly enlarging neck mass

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(No Transcript)
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Follicular Lymphoma(FL)

• Most common NHL in the Western World (45 of
  adult lymphomas)
• Middle age distribution affecting males and
  female equally
• Morphology partial or often complete nodal
  architectural effacement by numerous closely
  packed follicular nodules that most frequently
  homogenous with numerous small cleaved follicular
  center cells/centrocytes and larger cells with
  open nuclear chromatin, several nucleoli, and
  modest amounts of cytoplasm, referred to as
  centroblasts
• Bone marrow involvement in 85 of patients
  (paratrabecular)

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Immunophenotyping

• Strong CD19, CD20
• Monotypic immunoglobulin or no surface Ig
  expression
• CD10 positive
• Negative for CD5 and CD43

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Diffuse Large B-cell(DLBCL)

• 20 of NHL
• Median age of 60 years with a male predominance
• However age range is wide (5 of childhood
  lymphoma)
• 4 variants centroblastic, immunoblastic,
  T-cell/histocyte-rich, and anaplastic
• Morphology diffuse large lymphoid cells (4-5X
  the size of small lymphocytes)with vesicular
  chromatin. Nuclear features can range from
  multilobated, cleaved, or even the appearance of
  Reed-Sternberg cells

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Immunophenotyping

• CD20 and CD19 seen in many but not all
• More common than FL to be sIg negative
• Some contain cytoplasmic Ig
• Some mark like FL (CD5-, CD10,bcl-6, bcl-2)
  but can lack CD10, bcl-6, and bcl-2

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Burkitt Lymphoma (BL)

• Rapidly dividing transformed cell lymphoma
• Adolescents or young adults
• Follicular formation in a minority of cases
• Morphology Diffuse infiltrate of
  intermediate-sized lymphoid cells.
• Round or oval nuclei with course chromatin,
  several nucleoli, and basophilic or amphophilic
  cytoplasm
• Starry sky appearance due to presence of
  numerous tingible-body macrophages

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Immunophenotyping

• CD19, CD20
• CD10
• BCL6
• Express monotypic light chains
• Cytoplasmic Ig may be present
• CD5-

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Immunophenotype Comparison
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Cytogenetics

• Follicular Lymphoma t(1418) (Ig-H and bcl-2
  gene) (90 of FL)
• Diffuse Large B cell Lymphoma variable, many
  with translocations involving chromosome 3q27
  (BCL6 locus), 10-20 contain t(1418)
• Burkitt Lymphoma c-myc associated t(814),
  t(822), t(28)

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Case 2

• 58 year old Caucasian male initially presented
  with fatigability, weight loss, and anorexia. He
  was found to have CLL and is currently on
  chemotherapy.

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Small Lymphocytic Lymphoma/Chronic Lymphocytic
Leukemia

• CLL Most common Leukemia in adults in the
  Western world
• SLL 4 of NHL
• Older adults
• Morphology population of small lymphocytes
  containing round to slightly irregular nuclei
  with condensed chromatin and scant cytoplasm,
  mixed with larger prolymphocytes
• Smudge cell on peripheral smear

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Immunophenotype

• CD19 and CD20
• CD23 and CD5
• Low level expression of sIg and monotypic light
  chain

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Rituximab

• anti-CD20 monoclonal antibody
• Rituxan may induce antibody-dependent
  cell-mediated cytotoxicity
• Rituxan may also induce complement-dependent
  cytotoxicity
• Causes decrease in CD20 available for detection
  ab to bind
• Either through direct binding site competition
• Or transient down-regulation of CD20

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Case 3
14 year-old Caucasian male with previously
diagnosed lymphoid neoplasm for which he was
treated with chemotherapy. He has been
clinically asymptomatic with close follow-up for
the last year. Current bone marrow biopsy
obtained is shown here
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Case 3
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Acute Lymphoblastic Leukemia (ALL)

• Consists of immature, pre-cursor B (Pre-B) (85)
  or T (Pre-T) lymphocytes
• Pre-B manifests in childhood (many subtypes by
  European Group for the Immunological
  Characterization of Leukemias)
• Pre-T manifests in adolescents
• Morphologically indistinguishable
• Immunophenotyping needed for typing

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ALL Immunophenotyping

• Terminal deoxynucleotidyl transferase (TdT)
  gt95 of time
• Pre-B
• Positive for CD19, CD22, CD79a, CD24, CD34,
  CD45, HLADR, CD38, CD9, and CD10
• Negative for surface Ig expression
• Pre-T
• Positive for CD1, CD2, CD5, CD7

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Case 3
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Hematogones

• Bone marrow B-cell precursors (detected on flow
  in 80 of bone marrow aspirates)
• Cytologic features
• Size 10-20 microns
• May appear as mature lymphocytes
• Small cell with round or oval nucleus
• May be indistinguishable from neoplastic
  lymphoblasts
• Sometimes exhibits 1 or more indentations or
  shallow clefts with condensed chromatin
• Rim of deeply basophilic cytoplasm

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Hematogones

• Most numerous in children (up to 21) of bone
  marrow in infants and declines to lt5 after 16
• Have been observed in reactive lymph nodes and in
  peripheral blood
• Usually TdT negative
• Other conditions can increase the number in
  adults
• Regenerating marrow following chemotherapy
• Autoimmune diseases
• Congenital cytopenias
• Lymphomas (usually stage 3)
• Neuroblastomas
• AIDS

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Hematogone Immunophenotype

• Always positive for
• Progenitor cell markers CD34 and CD38
• CD19
• Dim CD22

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Hematogone Immunophenotype

• Maturation markers
• Stage 1(red)
• Positive TdT and Bright CD10
• Stage 2(blue)
• Moderate CD10, dim CD20, and variable sIG
• Stage 3(Yellow)
• Dim to moderate CD10, moderate to bright CD20,
  and variable sIG

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Hematogones vs. ALL
RedLymphoblasts YellowHematogones
Concurrent CD34 and CD20 Abberant over-expression
of markers (eg. CD10)
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References

• http//biology.berkeley.edu/crl/flow_cytometry_bas
  ic.html
• Gervasi F, Lo Verso R, Giambanco C, Cardinale G,
  Tomaselli C, Pagnucco G. Flow cytometric
  immunophenotyping analysis of patterns of antigen
  expression in non-Hodgkin's B cell lymphoma in
  samples obtained from different anatomic
  sites.Ann N Y Acad Sci. 2004 Dec1028457-62.
• Kroft SH, Asplund SL, McKenna RW, Karandikar NJ.
  Haematogones in the peripheral blood of adults a
  four-colour flow cytometry study of 102
  patients.Br J Haematol. 2004 Jul126(2)209-12.
  
• S Sava an1, M Büyükavc 2, S Buck1 and Y
  Ravindranath1 . Leukaemia/lymphoma cell
  microparticles in childhood mature B cell
  neoplasms. Journal of Clinical Pathology
  200457651-653.
• Maloum K, Sutton L, Baudet S, Laurent C, Bonnemye
  P, Magnac C, Merle-Beral H. Novel flow-cytometric
  analysis based on BCD5 subpopulations for the
  evaluation of minimal residual disease in chronic
  lymphocytic leukaemia.Br J Haematol. 2002
  Dec119(4)970-5.
• Jilani I, O'Brien S, Manshuri T, Thomas DA,
  Thomazy VA, Imam M, Naeem S, Verstovsek S,
  Kantarjian H, Giles F, Keating M, Albitar
  M.Transient down-modulation of CD20 by rituximab
  in patients with chronic lymphocytic
  leukemia.Blood. 2003 Nov 15102(10)3514-20.
  Epub 2003 Jul 31