Flow cytometry and minimal residual disease

Question 1

Functions and applications of flow cytometry

Answer 1

1. analyse expression of cell surface and intracellular molecules
2. characterise and define different cell types in a heterogenous population
3. simultaneous multi-parameter analysis of single cell’s expression

Applications

• immunophenotyping of acute leukaemia and lymphoproliferative neoplasms
• diagnosis of PNH
• diagnosis of HS
• minimal residual disease monitoring

Question 2

Main components of flow cytometer

Answer 2

1. Fluidics system - generate single stream of cells to pass through laser beam
2. Optical system - laser beam excites cells and light scattering/fluorescence are captured and converted to electrical signals by photodetectors
3. Electronic system - digitalise voltage data

Question 3

Forward scatter and side scatter - correlations

Answer 3

Forward scatter correlates with cell size
Side scatter proportional to granularity (complexity) of cells

Can separate populations based on these 2 characteristics
e.g neutrophil/granulocyte = large and more granular –> high FS and SS
monocytes = large but less granular –> high FS but low SS
small lymphocytes = low FS and SS

FS: blasts (variable) > myeloid precursors > monocytes > large lymphocytes (reactive) > neutrophils/ eosinophils > small lymphocytes

SS: eosinophils > myeloid precursors > neutrophils > monocytes > blasts/large or small lymphocytes

Question 4

Analysing protein expression - purpose, example

Answer 4

Fluorochrome (attached to an Ab) often used to study the cell surface antigen of interest

When Ab is bound to corresponding Ag, the fluorochromes will emit light when excited by laser

e.g. reduced fluorescence intensity of red cells labelled with eosin-5-maleimide which normally binds to band 3 protein and Rh protein in RBC – HS

Question 5

Data display

Answer 5

Dot plot (display 2 parameters with each dot representing a cell/particle)

Question 6

Gating - purpose

Answer 6

Isolate single populations of interest within a heterogenous sample

e.g. using CD45 and side scatter profiles to gate out leucocytes of different lineages and degrees of maturation (higher CD45 in mature cells, lower CD45 in myeloid precursors/blasts)

Question 7

Cluster of differentiation - main types for each lineage, specific examples of use

Answer 7

Classification of antigens found on leucocyte surface (up to CD371 in humans)
–> useful in identification, isolation and phenotyping of cell types

B lymphoid: 10, 19, 20, 22, 79a
T lymphoid: 2, 3, 5, 7 (+/- 4,8)
Myeloid: 13, 33, 117, MPO
Non-lineage specific markers in progenitor cells: 34, HLA-DR, TdT

Can detect specific cases with aberrant expression e.g. B lymphoid cells expressing CD5 in CLL

FLAER, CD55, CD59 for PNH

Question 8

Minimal residual disease

Answer 8

Small number of leukaemic cells that remains in patient during or after treatment which can’t be detected under microscope

Strong prognostic factor for risk stratification and determination of treatment

• “good risk” = modest therapy and spare toxicity
• “poorer risk” = more intensive therapy

Question 9

Treatment response: assessing morphology - method, limitations

Answer 9

Traditionally assess by CBC, BM cellularity and BM blast count – <5% BM blasts = remission

• measure efficacy of treatment and can reveal leukaemia relapse risk
  BUT
• not sensitive enough (complete morphological remission may still have large MRD which means risk of relapse is still high)
• specificity (similarities in appearance of leukaemic cells and normal B cell progenitors)

==> IMMUNOPHENOTYPING AND PCR much more sensitive and accurate

Question 10

Treatment response: current methods for MRD monitoring

Answer 10

1. clonal rearrangement of Ig gene and TCR by quantitative PCR
   - acute lymphoblastic leukaemia, B/T cell lymphoma
2. gene fusion or leukaemia specific markers measured by quantitative PCR
   - BCR-ABL1 in CML, PML-RARA in APL
   - NPM1 mutation in AML
3. Leukaemia associated immunophenotype by flow cytometry
   - ALL, AML
   - MM, CLL

Question 11

MRD by clonal rearrangement of Ig gene and TCR - method, principle, advantages and limitations

Answer 11

Quantitative PCR
Good for disease with B/T lymphoid origin e.g. ALL, B/T cell lymphoma

Each leukaemia clone has unique molecular signature of rearranged (VDJ) antigen receptor genes which can be detected at diagnosis
==> patient specific probe designed against junctinoal region can be used to track clone in remission samples

Advantages: applicable to almost all ALL, sensitive
Limitations: time consuming and expensive

Question 12

MRD by fusion gene transcripts or other molecular markers - examples, advantages, disdvantages

Answer 12

q-PCR:
BCR-ABL1 in CML

PML-RARA in APL

inv (16), t(8;21) and NPM1 mutant in AML –> if slow response or decrease in transcripts = higher risk of relapse

Advantages: highly sensitive, easy to perform
Disadvantages: low applicability to ALL

Question 13

MRD by immunophenotyping - method, principle, sensitivity and limitations

Answer 13

Flow cytometry
- using leukaemia associated phenotypes to distinguish cells from normal progenitor cells

Leukaemia associated phenotypes identified at diagnosis
–> subsequent study during and after induction to detect presence of leukaemic cells

Variable sensitivity for ALL (0.01%; lower than other 2 methods), fast

Sufficient number of cells needed to be analysed

Question 14

Leukaemia associated immunophenotype - method, principle

Answer 14

Pattern of expression unique to leukaemic blast cells

• increased/decreased/normal Ag
  e. g. less CD19 in B lymphoblasts
• aberrant expression
  e. g. myeloid markers in B lymphoblasts
• maturation asynchrony
  e. g. both CD20 (mature) and CD34 (immature) expressed on same cell

==> clearly distinguishable from patterns seen on normal or regenerating marrow