FLOW CYTOMETRY (USUALLY ADD NOTES X 15 MINS) PHYO WAI KYAW
Flow cytometry is a technique of quantitative single cell analysis. Flow cytometers were
developed in the 1970’s primarily for cell sorting and used in research laboratories
Basic Principles of Flow Cytometry
Basic Parts
Single cell or particle suspension
Fluorescent dyes or Abs that can be attached to an antigen or protein of interest
Flow cell, sheath fluid and a focused laser beam
Mechanism
Light is either scattered or absorbed when it strikes a cell. Light scatter is
dependent on the internal structure, size and shape of the flow cell.
Forward scatter = size of the cell
Side Scatter = complexity of the cell
Fluorescent dyes absorb light of a specific wavelength and reemit light of a different
wavelength. Fluorescent signals are detected by PMT and amplified. Optical filters are
used to steer light of specific wavelengths to the photo dector.
Electronics
Electrical pulses are digitized, the data is stored (‘list mode data’), analysed and
displayed through a computer system. The end result is quantitative information about
every cell analysed. Large numbers of cells can be processed quickly
Applications of flow cytometry
-Cell Sorting
-Immunophenotyping (leukaemia/lymphoma)
-Immunemonitoring (T-cell subsets in HIV)
-Leukocyte function assays to determine the functional status of cells within the immune system
-HLA-B27
-DNA Content cell cycle analysis
-Detection of PNH (CD59 and CD55)
-CD34 Enumeration
-Apoptsis
-MRD in leukaemia
-Foetal haemoglobin
-MHC Tetramer technology in combination with functional cytokine release assays has enabled
the study of highly specific T-cell subsets
IMMUNOPHENOTYPING OF LEUKAEMIC CELLS
Techniques
[Link] collection and transport
[Link] preparation and staining
[Link] QC
[Link] acquisition , interpretation
[Link]
I. SAMPLE COLLECTION
[Link]: peripheral blood, bone marrow, tissue, body fluids and FNAs
[Link]: EDTA, ACD, Heparin
[Link]: tissue culture medium
[Link]: ambient temperature 18 – 220C
[Link] : viability <70% results interpreted with caution. Every attempt should be
made to derive useful information from any specimen submitted for analysis
Specimen Storage
PB, BM collected in heparin usually stable up to 72hrs
Samples collected in EDTA are stable up to 24hrs
ACD acceptable for whole blood (72 hrs) but not recommended for BM (may cause
viability problems for small volumes of BM due to changes in pH).
Tissues, FNAs, body fluids should be processed ASAP.
[Link] PROCESSING
Whole blood lysis is generally the recommended procedure
Remove contaminating red cells, while leaving white cells intact
Assumes all leucocyte subsets are equally tolerant to the lysis method
Incubation with monoclonal antibodies can be done before or after lysis of red blood cells
Density gradient separation eg, ficol. Enrichment for MNC
Tissues: mechanical dissociation is preferred to enzyme digestion
Viability can be performed using 7AAD, trypan blue or PI.
Monoclonal antibody panels
Multiparameter immunofluorescence is preferred
Panels should be designed to resolve normal as well as malignant cells (normal cells act
as internal reference standards)
Brighter fluorochromes (eg, PE) used in expected cases of low antigen expression
Acute Leukaemia; Two Step Strategy
Screening panel can consist of:
Non lineage; CD45, CD34,HLA-DR
T cell; CD2, CD3, CD7
B cell; CD10, CD19, CD20
Myeloid; CD13, CD33, CD117
Additional Abs to identify maturation, prognostic features or aberrant phenotypes
Lymphoma / Chronic leukaemia panels
CD20/KAPPA/LAMBDA
CD10/CD19/CD5/CD23
CD20/CD103/CD25/CD11c
CD38/CD138/CD45/CD19/CD56
CD3/CD4/CD8
[Link] QUALITY CONTROL
Verification of a leukaemic population
Negative controls; unstained cells or isotype controls (background fluorescence and non
specific binding)
Positive controls confirm;
sample preparation method
reagent and staining procedures
instrument settings
Use standardised commercial cell preparations or a control normal blood sample or
residual normal cells within the test sample.
[Link] QC
-verify optics and fluidics
V. DATA ANALYSIS
Identify the abnormal population
Determine what markers the abnormal cells are expressing
Provide some descriptive information about the abnormal population
VI. REPORTING
Should consist of a descriptive comment eg,
‘There is a monoclonal population of kappa/lambda*-expressing B cells which co-
express CD5 and CD23, consistent with B-cell chronic lymphocytic leukaemia’.
or
‘No evidence of involvement with B-cell lymphoma’.