03-10-2012

1
Flow cytometric analysis involves 3 stages:
Preanalytical - Specimen handling & processing,
including Ab staining
Analytical - Running sample through the flow
cytometer & acquiring data
Postanalytical - Data analysis & interpretation
Dr.GarimaGoel,MAMC
2 broad groups:
Liquid specimens
Peripheral blood
Bone marrow aspirate (BMA)
Body fluids
Dr.GarimaGoel,MAMC
2 broad groups:
Solid tissue specimens
Lymph nodes
Bone marrow biopsy
Tonsils/adenoids
Spleen
Dr.GarimaGoel,MAMC
Peripheral blood & BMA samples:
EDTA - anticoagulant of choice
Hemogram
Smears can be prepared
Specimen processed within 24hrs
Heparin
Specimen processed within 48 hrs
Dr.GarimaGoel,MAMC
Solid tissue samples:
Submitted as thin slices (<2mm thick) in sterile
tissue culture media at 4

C.
Dr.GarimaGoel,MAMC
03-10-2012
2
Ideally sample - processed within 48 hours of
collection
For tumors with high turnover rate - time
window
Dr.GarimaGoel,MAMC
WBC count, differential & unstained blood
film - accompany each specimen
Specimens - 18

-22

C in a leak proof
container
Temp. <4C & >30C - avoided
Dr.GarimaGoel,MAMC
Poor sample collection major source of
potential unsatisfactory FCM analysis
Factors affecting viability of cells
Time elapsed between sample collection &
delivery to lab
Environmental conditions during transport of
sample to lab
Dr.GarimaGoel,MAMC
Samples unacceptable for analysis:
Exposure to extreme temperature
Presence of blood clots
Grossly hemolysed sample
Dr.GarimaGoel,MAMC
Dr.GarimaGoel,MAMC
Sample volume (peripheral blood & BMA) -
0.5 to 1ml in each tube
Concentration of nucleated cells in each tube
for test - 0.5 to 2x10
6
/ml (MC - 1x10
6
/ml or
1x10
3
/l)
TLC of sample - <10
4
/l
If more dilute the sample with PBS
If less take more sample to decrease dilution
Dr.GarimaGoel,MAMC
03-10-2012
3
Lyses of RBCs
Removal of lysed RBCs
Staining with Abs
Dr.GarimaGoel,MAMC
Nucleated cells - separated from RBCs
RBC lysis - before or after staining with Abs
Lysing agents
Can be with or without fixative
E.g. - Ammonium chloride
Dr.GarimaGoel,MAMC
Failure of RBC lysis
Reticulocytes in peripheral blood
Lipids in serum
Dr.GarimaGoel,MAMC
Lysed RBCs
Removed by multiple (at least 2) washings with
isotonic fluid
Isotonic fluid e.g. Phosphate Buffered Saline
Dr.GarimaGoel,MAMC
Staining with desired Abs -before or after RBC lysis
2 types of antigens
Cell surface antigen ( )
e.g. CD19, CD10,
CD13, CD33, CD117,
CD38 etc.
Intracellular antigens ( )
e.g. TdT, cytoplasmic
light chains, cCD3,
cCD22, MPO, bcl-2 etc.
Dr.GarimaGoel,MAMC
Cell surface Ag
Staining done on
viable unfixed cells
Intracellular Ag
Staining requires cell
fixation &
permeabilization
Dr.GarimaGoel,MAMC
03-10-2012
4
To maintain structural integrity
Fixative
Preserve epitope of intracellular antigen
Without aggregation of cell suspension
E.g. formaldehyde
Dr.GarimaGoel,MAMC
To allow Abs to reach appropriate
intracellular targets
Permeabilizing agent - low concentration non
ionic detergents like saponin
Dr.GarimaGoel,MAMC
Simultaneous detection of intracellular & cell
surface Ag - sensitivity of diagnosis
Ensure stability of AgAbfluorochrome
complexes on cell surface
Preservation of targeted intracellular
antigens
Dr.GarimaGoel,MAMC
E.g.
CD19 & 10 with TdT
CD22 & cCD3
Dr.GarimaGoel,MAMC
For each sample analyzed one control tube
is required
Cells are mixed in the presence of an isotypic
control
Determines the level of non specific binding &
autofluroscence
Dr.GarimaGoel,MAMC
100l sample + Abs
Vortex +20mins incubation in dark
500l lysing agent
2 washes with isotonic fluid
Resuspendin 1ml isotonic fluid
Vortex +10mins incubation in dark
Dr.GarimaGoel,MAMC
03-10-2012
5
50 50l sample + surface Abs
15mins incubation in dark
100l fixative + vigourous vortex +15mins incubation
1 wash with isotonic fluid +100l permeabilizing agent 1 wash with isotonic fluid +100l permeabilizing agent
Resuspendin 0.5ml isotonic fluid
DO NOT VORTEX +5mins incubation in dark DO NOT VORTEX +5mins incubation in dark
2 washes with isotonic fluid
Add Intracellular Abs +15mins incubation in dark Add Intracellular Abs +15mins incubation in dark
Dr.GarimaGoel,MAMC
Type of Ab monoclonal or polyclonal
Ab isotype - IgG1, G2 or M
Ab clone
Dye conjugation
Stoichiometry between Ab & fluorochrome
Dr.GarimaGoel,MAMC
Dyes- accept light energy at a given wavelength
& re-emit it at a longer wavelength
Different fluorochromes
FITC Fluoresicein Isothiocyanate
PE Phycoerythrin
ECD PhycoerythrinTexas Red
PC5 &7 - Phycoerythrin-Cyanin5 &7
APC Allophycocyanin
Dr.GarimaGoel,MAMC
Depends on
Fluorochrome brightness
Ag density
Background staining of Ab
Amount of compensation required between
conjugates
Single or multiple laser
Dr.GarimaGoel,MAMC
Brighter fluorochrome used for
Dull Ab
Low Ag expression
For staining cells with high autofluoroscent
background
E.g. PE is used with CD25
Dr.GarimaGoel,MAMC
Duller fluorochrome used for
Bright Ab
High Ag expression
E.g. FITC with anti kappa, anti lambda, CD45
Dr.GarimaGoel,MAMC
03-10-2012
6
Simultaneous use of >1 flurochrome
Saves time & sample
Exponential in information
Helps identify new/rare cell populations
no. of tubes in Ab panel
no. cells required for entire FCM analysis
Provides internal controls
Dr.GarimaGoel,MAMC
Carefully choose combinations of
flurochrome conjugates
Non availability of all reagents in all colours
Greater potential for errors in compensation
Requirement of proper controls
Dr.GarimaGoel,MAMC
Conjugated antibodies
Stored between 2 & 8

C
Protected from light
Dr.GarimaGoel,MAMC
Do not use reagent beyond expiry date
Do not freeze reagent or sample
Let reagent come down to room temp before
use
Dr.GarimaGoel,MAMC
Minimize exposure to light
Avoid microbial contamination of reagents
Absolutions like anti kappa & anti lambda
contain sodium azide& should be handled
carefully
Dr.GarimaGoel,MAMC Dr.GarimaGoel,MAMC