Flow Cytometry: Principles and Applications
FLOW
CY
T
OMET
R
Y
FLOW CY
T
OMET
R
Y
Defi
n
iti
o
n:
Me
a
suring
pr
o
p
e
rti
e
s
of
cell
a
s
t
h
e
y
f
low in
a
fluid suspension
acro
s
s
an
illumi
n
ated light
path.
Ba
s
ic
me
c
hani
s
m
Biologic
a
l
sa
m
ple
L
a
b
e
l
i
t
with a
fluore
s
c
e
nt
m
a
rker
C
e
lls
mo
v
e
in a
line
a
r
stream
throu
g
h a foc
u
s
e
d
light
s
o
urce
(las
e
r bea
m
)
F
luore
s
c
e
nt
mol
e
c
u
le
g
e
ts a
c
tivated
a
n
d emits
light
th
a
t
is
filter
e
d
a
nd
d
e
te
c
ted
by
s
e
n
s
itive
lig
h
t
d
e
te
c
tors (u
s
u
a
lly
a
p
h
ot
o
m
u
ltipli
e
r
tu
b
e)
Co
n
v
e
rsion
of
a
n
alog
fluore
s
c
e
nt
si
g
n
a
ls
to digital
s
ig
n
als
Flow
Cytometry
This
me
t
h
o
d
al
l
ows
the
qu
ant
i
tat
i
ve
and
qu
a
l
it
a
tiv
e
a
n
a
l
y
sis
o
f
severa
l
p
r
o
p
e
r
ti
es
o
f
ce
ll
po
p
u
l
a
ti
o
ns
fro
m
v
i
rtu
a
l
ly
any
type
o
f
fresh
un
fix
ed
tis
su
e
o
r
body
fl
u
id.
The
p
r
o
p
e
rt
i
es
me
asu
r
e
d
i
n
cl
u
de
a
p
ar
t
i
c
le
’
s
r
e
l
ated
s
i
ze
,
r
e
l
at
i
ve
g
r
a
n
u
l
a
r
ity
o
r
i
nte
r
n
a
l
compl
e
x
i
t
y
,
an
d
r
e
l
a
tiv
e
fl
u
o
r
e
s
ce
n
c
e
i
nte
n
s
i
ty
Mo
st
c
om
m
o
n
l
y
a
n
a
l
yz
ed
m
a
t
e
rials
a
r
e:
b
l
o
od,
bo
n
e
ma
r
row
a
s
pi
r
ate
a
n
d
ly
mph
n
o
de
s
u
s
p
e
n
si
o
n
s.
Pr
i
nc
i
ple
of
Flow
Cytometry
Flow
c
y
tometer
is com
p
os
e
d
of three main
c
o
mp
o
n
e
nts:
T
h
e
F
l
o
w
s
y
s
t
e
m
(
f
l
u
i
d
i
c
s
)
Cells
in su
s
pe
n
sion
are brought in single
fi
l
e
pa
s
t
T
h
e
O
p
t
i
c
a
l
s
y
s
t
e
m
(
l
i
g
h
t
s
e
n
s
i
n
g
)
a foc
u
s
e
d
la
s
er
w
h
ich
s
c
atter
light
a
n
d emit
fluoresc
e
nce
that is
fi
l
tered
a
nd c
o
llected
T
h
e
E
l
e
c
t
r
o
n
i
c
s
y
s
t
e
m
(
s
i
g
n
a
l
p
r
o
c
e
s
s
i
n
g
)
e
m
i
t
t
e
d
l
i
g
h
t
i
s
c
o
n
v
e
r
t
e
d
t
o
d
i
g
i
t
i
z
e
d
v
a
l
u
e
s
t
h
a
t
a
r
e
s
t
o
r
e
d
i
n
a
f
i
l
e
f
o
r
a
n
a
l
y
s
i
s
The
F
low System
One
of
t
he
fun
d
am
e
ntals
of f
l
ow
c
y
tometry
is the a
b
ili
t
y
to m
e
as
u
re
the properties
of in
d
ividu
a
l
particles,
which
is ma
n
a
g
ed
by
t
he
f
luidi
c
s
s
y
ste
m
.
Wh
e
n
a s
a
mple
is
inje
c
ted into a flow c
y
tomete
r
,
it is
ordered
into a
st
r
eam
of
s
ingle
particles.
T
he
fluidic s
y
stem
c
o
n
s
ists
of a
F
L
OW
CE
L
L
(Quartz
C
h
am
b
er):
Ce
n
t
r
al
ch
a
nn
e
l/
core
-
through whi
c
h
the sam
p
le
is
inje
c
ted.
Outer s
h
e
a
th
-
c
o
ntains
faster flowing
fluid
k/a
She
a
th
f
luid
(
0.9% Saline /
P
B
S
)
, e
n
clo
s
ing
the
c
e
ntral
c
o
re.
Hydr
o
dyn
a
mic
Focu
s
ing
Once
the sample
is
injected
into
a
strea
m
of
s
he
ath
fluid
within
th
e
f
l
ow
c
hamb
e
r
,
they
are
forced
i
nto
the
center
of
the strea
m
forming
a
single
fi
l
e
by
the
PRINCIPLE
OF
HYDRODYNAMIC
FOCUSING.
'On
l
y
o
n
e
c
e
l
l
or
p
a
r
t
i
cle
c
an
pas
s
t
h
r
o
u
gh
th
e
la
s
er
b
e
a
m
at
a
giv
en
m
o
m
en
t
.'
•
Th
e
sa
m
p
l
e
pressure is al
w
ays
h
i
gh
e
r
than
the
sheath flu
i
d
pressure
,
ensuring
a
high
flow rate
a
l
l
o
w
i
ng
more cel
l
s
to
e
nter the stream
at a
given moment.
•
H
i
gh
F
l
ow
R
a
te
-
Im
m
un
o
ph
e
notyp
i
ng
an
a
lys
i
s
of cel
l
s
•
Low F
l
ow
R
a
te
-
D
N
A
A
n
a
l
ysis
OPTICS
A
f
t
er
the
c
e
l
l
de
l
iv
e
ry
system,
the need
is to e
x
cite
the
cel
l
s
usi
n
g
a
l
i
ght
source.
The
li
g
ht
source used
in
a flow cytometer:
L
a
s
e
r
(
m
o
r
e
c
o
m
m
o
n
l
y
)
A
r
c
l
a
m
p
Why
Las
e
rs
are
m
ore common?
They
are
highly
c
o
he
r
ent
and
uniform.
They
c
a
n
be ea
s
ily
focu
s
ed
on
a
v
ery
small
area
(li
k
e
a sample
str
e
am
)
.
They
are
mono
c
hro
m
atic,
emit
t
ing single
wavelengths
of
light.
A
R
G
O
N Lasers
-
48
8
nm w
a
vel
e
ngth
(blue
to
b
l
u
e green)
Wh
e
n a l
i
g
h
t
i
n
tersects a
las
e
r
b
e
am
at
the
so ca
l
l
e
d
'
inte
r
ogation
point'
two even
t
s
occu
r
:
a)
light
sca
t
tering
b)
e
m
ission
of l
i
ght
(
f
luores
c
ence
)
F
l
uorescence
is l
i
ght
e
m
itted
duri
n
g
dec
a
y
of
e
xcited e
l
ectron
to
i
ts b
a
sal
state.
OPTICS
a) LI
G
HT
S
C
A
TTER
When l
i
ght
f
r
om
a laser
i
n
terrogates a cel
l
,
that ce
l
l
scatters
l
i
ght
in
a
l
l
d
i
recti
o
ns.
The
scattered
l
i
ght
can t
r
avel
f
rom
the inter
r
og
a
tion
po
i
nt
d
o
wn
a path to
a
d
e
tecto
r
.
OPTICS
-
FO
R
W
ARD
SC
A
T
T
ER
(FSC)
•
L
i
g
h
t
that
is sc
a
ttered
in
the
forward d
i
rection
(alo
n
g
the same a
x
is
the
l
aser
is t
r
ave
l
i
n
g)
is
detected in the Forward Scatter C
h
an
n
e
l
.
•
T
h
e i
n
tens
i
ty
of
this s
i
g
n
al
h
a
s be
e
n
att
r
i
b
uted to
cell siz
e
,
ref
r
active
i
n
dex
(
m
embrane perm
eabi
l
ity
).
OPTICS
-
SIDE
SC
A
TTER
(SSC)
L
a
ser l
i
g
h
t
that
i
s scattered
at
90
degre
e
s
to
the
a
x
is
of
the
l
aser
path is detected in the
S
i
de
Sc
a
t
t
er
C
h
an
n
e
l
.
Th
e i
n
tensity
of
this sig
n
al
is proportional
to the
amount
of
cytoso
l
ic
structure
in
the
ce
l
l
(
eg
.
g
ranu
l
es,
ce
l
l
i
n
cl
u
si
o
n
s,
etc.)
Side
scat
t
er
detect
o
r
M
easuring
cell
gr
a
nul
a
rity
F
S
C
D
e
t
e
c
t
o
r
C
o
l
l
e
c
t
i
o
n
L
e
n
s
S
S
C
D
e
t
e
c
t
o
r
L
a
s
e
r
B
e
a
m
M
o
n
o
c
y
t
e
s
R
B
C
s
,
D
e
b
r
i
s
,
D
e
a
d
C
e
l
l
s
FSC
Study of F
S
C and
S
SC a
l
l
o
ws
us
to k
n
ow
the
d
i
f
ferenti
a
tion
of
d
i
f
ferent
types
o
f ce
l
ls.
SSC
G
r
a
n
u
l
o
c
y
t
e
s
L
y
m
p
h
o
c
y
t
e
s
Why FSC &
SSC?
Th
e
light
sca
t
tered
i
n
the
forw
a
rd
d
i
rection
is
proport
i
on
a
l
to
the
square
o
f
the
r
a
d
i
u
s
o
f
a
sphere,
and so to
the
size
o
f
the cell
o
r
particl
e
.
Th
e
cells
are
l
a
belled
antibo
d
ies
o
r
s
tained
w
i
th
with
fluo
r
o
c
hrom
e
-li
n
ke
d
f
l
u
oresc
e
nt
membrane,
cytop
l
asm
i
c
o
r
n
u
cl
e
ar
dye
.
Com
m
only
used
Fluorochromes
Opti
c
s
B)
EMIS
S
ION
OF
F
L
UO
R
E
S
C
E
NT
LIGHT
(FLUOR
E
S
C
E
N
C
E
)
As the
fluorescent mo
l
ecu
l
e
present
i
n
or
o
n the
particle is
i
nterrogated
by the
l
a
ser
l
i
g
h
t,
it
w
i
ll
a
b
sorb
e
n
ergy
f
r
om
the laser
l
i
ght
and
rele
a
se
the
a
bsorbed
en
e
rgy
at
l
o
ng
e
r
w
a
ve
l
e
ngt
h
.
Em
i
tted
p
h
otons
p
a
ss through
the
co
l
l
e
ction
l
e
ns a
n
d
are spl
i
t
and
steered down
spec
i
fic
chan
n
e
l
s
w
i
th the
u
se of filters.
Optic
s
-
Filters
Di
f
ferent
wav
ele
ngths
o
f
light
are
scattered simu
l
taneous
l
y
f
r
om
a cell
Need
to
s
p
l
it
the
l
i
g
ht
i
n
to
its
specific
wave
l
engths
in
order
to
m
eas
u
re
and
q
u
antify
th
e
m
independent
l
y
.
Th
i
s
i
s
don
e
w
i
th
filters.
The
system
of
filt
e
r
s
e
n
sures
th
at
e
a
ch
p
h
ot
o
d
e
tector
receives
light
bands of
vario
us
wavel
e
n
g
th
s
.
Optic
a
l
fil
t
ers
reflect
s
o
me
others.
T
yp
e
s
o
f
filters
1.
Lon
g
P
a
ss
3
.
B
a
nd
P
a
ss
are
desig
n
ed
s
uch
that
t
h
ey
abso
r
b
or
w
a
v
e
len
g
ths
o
f
l
i
g
ht,
w
h
ile
t
r
ansmi
t
ti
n
g
2
.
S
h
ort P
a
ss
4
.
D
i
chroic
Opti
c
s
-
Lon
g Pa
s
s Filters
T
ransmit
a
l
l
w
a
ve
l
e
n
gths
greater than
sp
e
cif
i
ed
w
a
vel
e
ngth
E
x
ample:
50
0
LP
w
i
ll
t
r
ansmit
a
l
l
w
a
vel
e
ngths
greater
than 5
0
0
n
m
400nm
500nm
600nm
700nm
T
ran
s
mi
t
tan
c
e
Or
i
gin
a
l
f
rom
C
y
t
om
ation
T
raining
M
anual
Opti
c
s
-
Short
Pa
s
s
Filt
e
r
T
ransmits
a
l
l
w
a
ve
l
e
n
gths
l
e
ss than sp
e
cif
i
ed
w
a
vel
e
ngth
E
x
ample:
6
00
S
P
w
i
ll
t
r
ansmit
a
l
l
w
a
vel
e
ngths
l
e
ss
than 6
0
0
n
m.
400nm
500nm
600nm
700nm
T
ran
s
mi
t
tan
c
e
Or
i
gin
a
l
f
rom
C
y
t
om
ation
T
raining
M
anual
Opti
c
s
-
Band Pa
s
s Filter
T
ransmits
a sp
e
cif
i
c
b
a
nd
of
w
a
ve
l
e
n
gths
E
x
ample:
5
50/20
B
P
F
i
lter w
i
ll
t
r
ansmit
w
ave
l
en
g
ths
of
l
i
g
ht
betw
e
en
54
0
nm
and
56
0
nm (550/20
=
5
50+
/
- 1
0
,
n
ot 550+/-
20)
400nm
500nm
600nm
700nm
T
ran
s
mi
t
tan
c
e
Or
i
gin
a
l
f
rom
C
y
t
om
ation
T
raining
M
anual
Opti
c
s
-
Dichro
i
c
Filters
L
o
ng
p
a
ss or short p
a
ss filters
P
l
aced
at a
4
5º ang
l
e
of
i
nci
d
ence
P
a
rt
o
f the
l
i
ght
is refl
e
cted at
9
0º ,
and
part of the
l
i
ght
is
t
r
ansmitted
a
nd conti
n
ues.
Dichroic
Fil
t
er
Detector
1
Detector
2
OPTICS -
DETEC
T
ORS
Th
e p
h
otod
e
tectors
co
n
vert
the
p
h
otons
to
e
l
ectrical
impu
l
ses.
T
wo
common
t
ypes of
detectors
used
i
n
flow
cytometr
y
:
P
h
otodi
o
de
used
for
st
r
ong
sign
a
ls,
w
h
en
saturati
o
n
is a
potential prob
l
em
(
e
g
, forward scatter
detector
)
.
P
h
otomultip
l
i
e
r
tube (PMT)
more
sens
i
tive
than photod
i
ode
but
can be
destroyed by e
x
pos
u
re
to too
much li
g
ht.
used
for
si
d
e
scatter
and
fluorescent
si
g
no
l
s.
ELE
C
TRONICS
The
electronic
subsystem co
n
verts
ph
o
tons
to
p
h
otoe
l
ectrons.
Meas
u
res ampl
i
tude,
area and w
i
dth
of
p
hotoe
l
ectron
p
u
ls
e
.
It
ampl
i
fies
pu
l
se
e
i
ther
l
i
ne
a
rly
or
l
og
a
rithmic
a
l
l
y
and then dig
i
taliz
i
ng
the amp
l
ifi
e
d
pu
l
se.
T
i
m
e
Electroni
c
s
-
Crea
t
ion
of
a
V
ol
t
a
g
e P
u
lse
Data
Ana
l
y
s
i
s
-
Plot
T
ypes
There are
several
pl
o
t
cho
i
ces:
S
i
ng
l
e
C
o
l
o
r
H
i
stogram
F
l
uorescence
i
n
tensity
(FI)
versus the number of
ce
l
ls counted.
T
wo
Col
o
r
D
o
t
P
l
o
t
FI
of
p
arameter 1
versus
F
I of
P
a
rameter 2
T
wo
Col
o
r
C
o
ntour P
l
ot
C
o
nc
e
ntric
rin
g
s
form
arou
n
d
p
o
p
u
l
a
tio
n
s.
T
h
e more
de
n
se
the
p
op
u
l
a
tion,
the
c
l
o
ser
the r
i
ngs
are
to
e
ach
other
T
wo
Col
o
r
D
e
nsity
P
l
ot
Areas of h
i
gh
e
r
de
n
sity wi
l
l
have
a di
f
ferent
col
o
r
than
other areas
Plot
T
ypes
C
o
nt
o
ur
Pl
o
t
D
e
n
sity
P
lo
t
Gre
y
sca
l
e
D
e
ns
i
ty
D
o
t
Pl
o
t
ww
w
.tr
ee
st
a
r
.
c
om
H
i
sto
g
ram
D
A
T
A
ANA
L
YSIS
-
G
A
TING
Gati
n
g
i
s
in
e
s
s
e
nce
e
l
e
ctro
n
i
c
wind
o
w
th
at
sets
uppe
r
an
d
l
ower
lim
i
ts
on
t
h
e
typ
e
and
am
o
u
n
t
of
mater
i
al
tha
t
pas
s
es
th
r
ou
gh.
Selec
t
i
o
n
of
only
a
ce
rta
in
po
p
u
l
at
i
on
of
ce
lls
for
a
n
a
l
y
sis
on
a
p
l
ot.
Allows
the
a
b
il
i
ty to
l
ook
at
p
a
r
a
met
ers
sp
e
cif
i
c
to
o
n
ly t
hat
su
b
s
et.
Interpret
a
tion
of Graphs
An
im
p
o
r
tant
tool
for
ev
a
lu
a
ting
d
a
ta
is
the dot
pl
o
t
.
The
inst
r
u
m
ent
d
e
tects
e
a
ch
cell as
a
p
o
int
on
an
X
-Y
gr
a
ph.
This
form
of
data
pr
e
sen
t
a
t
ion
lo
o
ks
at
two
p
a
r
a
m
e
ters
of
the
sa
m
ple
at
the
sa
m
e
tim
e
.
Three
common
modes
for
dot
plots
are:
Forw
a
rd
scatter
(FS
C
) vs. s
i
de
scatter
(SS
C
)
T
o
l
o
ok
at the
d
i
st
r
i
b
ution
of c
e
l
l
s
bas
e
d
up
o
n
size
&
granularity
S
i
ng
l
e
col
o
r
vs. s
i
de scatter
T
o
visu
a
l
i
ze
the expression
of the
fluo
r
escence
of the
cel
l
s
T
w
o
-co
l
or
flu
o
resce
n
ce
p
l
ot.
T
o
d
i
f
f
erentiate
betw
e
en
those cel
l
s
that
express
on
l
y one
of the
particul
a
r
fluorescent
m
arkers,
t
hose
t
hat
express
ne
i
the
r
,
and
t
hose
t
hat
express
bot
h
.
used
to
d
i
scri
m
inate
de
a
d
cel
l
s
f
r
om
t
he
l
i
ve
on
e
s
t
hat
are expressing
the
d
esired
fluorescence
.
How
to
di
f
ferentia
t
e
dim
&
bright
exp
r
es
s
ion
of
an
antigen?
Dim
:
cells
are
p
r
es
e
nt
m
o
re
tow
a
r
d
s
the
o
r
ig
i
n
(
0)
on
x
(
red
)
-
y
axis
(
p
i
nk
)
Brig
h
t
:
cel
l
s
a
r
e
p
r
es
e
nt
aw
a
y
from
the
o
r
ig
i
n
(
0)
on
x
(
g
r
ee
n
)
& y
(
yel
l
o
w
)
axis.
DIM
BRIGHT
Y
-a
x
is
CD4
X
-a
x
is
CD8
W
H
A
T
IS
U
NIQ
U
E
IN
FLO
W
CY
T
OME
T
R
Y
MU
L
TI
P
ARAMETRIC
RAP
I
D
ANA
L
Y
S
IS OF
LARGE NUMBER
OF
CEL
L
S
INFORM
A
TION
A
T
A
SINGLE
CELL
LEV
E
L
DET
E
CTION
OF
RARE
C
E
LL
P
O
PUL
A
TI
O
NS
ALLOWS
PHY
S
ICAL
ISOL
A
TION
OF
CEL
L
S
OF
INTERE
S
T
USES
O
F
FLO
W
CY
T
OMET
R
Y
AP
P
LIC
A
TIONS
ANA
L
Y
S
IS
Immu
n
o
p
h
e
n
o
typi
n
g
D
y
es
that
bind
t
o
n
u
cl
e
ic
a
c
ids
(DN
A
,
R
N
A)
F
u
n
c
tional
a
s
s
a
ys
CELL
COUNTING
CELL
SO
R
TING
CLINI
C
AL
AP
P
LIC
A
TIONS
•
A
b
so
l
ute
C
D
4
co
u
nts
HIV/A
I
DS
•
H
L
A
B27
ass
a
y
J
o
int
Pain
•
D
i
a
g
n
o
sis
a
n
d
C
l
ass
i
ficati
o
n
•
D
e
tecti
o
n
of MRD
Hem
a
tologi
c
al
Malig
n
a
n
cies
•
D
N
A
P
lo
i
d
y
•
S Ph
a
se
fracti
o
n
Solid
T
u
m
o
u
rs
•
T
B
N
K
•
P
h
a
g
oc
y
tic
fu
n
ction
d
e
fect
Primary
Immun
o
d
e
ficiency
dis
or
d
ers
Cont..
•
Reticuloc
y
te
count
•
PNH
•
O
smot
i
c fragi
l
ity assay
Hemolytic
a
n
a
e
mia
•
Fe
t
o
-
m
a
t
er
n
al Hemo
r
rh
a
ge
•
t
rea
t
ment
response
in
S
i
ckle
Ce
l
l
Anem
i
a
Fet
al
H
b
d
e
tec
t
ion
•
P
l
a
t
e
l
et
recep
t
or
assa
y
s
(Pla
t
e
l
et
count,
G
T
,
BSS)
•
P
l
a
t
e
l
et
f
unction assay
(C
D
62
P
,
P
A
C
-
1)
•
CD34
S
T
EM
CELL
C
O
UN
T
S
•
Res
i
dual
WBC
count
in
leukodep
l
eted
b
l
ood
packs
•
F
l
ow
c
y
tometry
Cross
m
a
t
ch
•
S
u
rface
mark
e
rs in PMN, Mo
n
oc
y
tes
•
C
y
toki
n
e
res
p
o
n
se
Ble
e
ding
Disorders
T
ransf
u
sion
a
n
d
T
ransplant
Host
Imm
u
ne
resp
o
nse
in
Se
p
sis
References
•
THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC NEOPLASIA
Brent L. Wood, Michael J. Borowitz. Henry’s, 22nd edition, Chapter 34
•
https://www.bosterbio.com/protocol-and-troubleshooting/flow-
cytometry-principle
•
https://www.abcam.com/protocols/introduction-to-flow-cytometry
•
Video Link:
•
https://www.youtube.com/watch?v=5IdYFgYb9ls
•
https://www.youtube.com/watch?v=7bCZx5xPwt0&t=94s
Flow cytometry is a powerful technology used to analyze the properties of cell populations. By measuring size, complexity, and fluorescence intensity, this method provides valuable insights into various biological samples. The process involves labeling cells with fluorescent markers, passing them through a laser beam for activation, and detecting emitted light for analysis. With its ability to quantify and qualify cellular characteristics, flow cytometry is widely used in fields such as hematology, immunology, and oncology.
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FLOW CYTOMETRY
FLOW CYTOMETRY Definition: Measuring properties of cell as they flow in a fluid suspension across an illuminated light path.
Basic mechanism Biological sample Label it with a fluorescent marker Cells move in a linear stream through a focused light source (laser beam) Fluorescent molecule gets activated and emits light that is filtered and detected by sensitive light detectors (usually a photomultiplier tube) Conversion of analog fluorescent signals to digital signals
Flow Cytometry This qualitative analysis of several properties of cell populations from virtually any type of fresh unfixed tissue or body fluid. method allows the quantitative and The properties measured include a particle s related size, relative complexity, and relative fluorescence intensity granularity or internal Most commonly analyzed materials are: blood, bone marrow aspirate and lymph node suspensions.
Principle of Flow Cytometry Flow cytometer is composed of three main components: The Flow system (fluidics) Cells in suspension are brought in single file past The Optical system (light sensing) a focused laser which scatter light and emit fluorescence that is filtered and collected The Electronic system (signal processing) emitted light is converted to digitized values that are stored in a file for analysis
The Flow System One of the fundamentals of flow cytometry is the ability to measure the properties of individual particles, which is managed by the fluidics system. When a sample is injected into a flow cytometer, it is ordered into a stream of single particles. The fluidic system consists of a FLOW CELL(Quartz Chamber): Central channel/ core - through which the sample is injected. Outer sheath - contains faster flowing fluid k/a Sheath fluid (0.9% Saline / PBS) , enclosing the central core.
Hydrodynamic Focusing Once the sample is injected into a stream of sheath fluid within the flow chamber, they are forced into the center of the stream forming a single file by the PRINCIPLE OF HYDRODYNAMIC FOCUSING. 'Only one cell or particle can pass through the laser beam at a given moment.'
The sample pressure is always higher than the sheath fluid pressure, ensuring a high flow rate allowing more cells to enter the stream at a given moment. High Flow Rate - Immunophenotyping analysis of cells Low Flow Rate - DNAAnalysis Sample Tube Sample Pressure (Variable) Sheath Pressure (Constant) Waste T ank Sheath T ank Vacuum Line Pressure
OPTICS After the cell delivery system, the need is to excite the cells using a light source. The light source used in a flow cytometer: Laser (more commonly) Arc lamp Why Lasers are more common? They are highly coherent and uniform. They can be easily focused on a very small area (like a sample stream). They are monochromatic, emitting single wavelengths of light. ARGON Lasers - 488nm wavelength (blue to blue green)
When a light intersects a laser beam at the so called 'interogation point' two events occur: a) light scattering b) emission of light (fluorescence ) Fluorescence is light emitted during decay of excited electron to its basal state.
OPTICS a) LIGHT SCATTER When light from a laser interrogates a cell, that cell scatters light in all directions. The scattered light can travel from the interrogation point down a path to a detector.
OPTICS - FORWARD SCATTER (FSC) Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel. The intensity of this signal has been attributed to cell size, refractive index (membrane perm eability).
OPTICS - SIDE SCATTER (SSC) Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel. The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.) Side scatter detector Measuring cell granularity
FSC Detector Laser Beam Collection Lens SSC Detector
Why FSC & SSC? Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC Study of FSC and SSC allows us to know the differentiation of different types of cells.
The light is proportional to the square of the radius of a sphere, and so to the size of the cell or particle. scattered in the forward direction The antibodies cytoplasmic or nuclear dye. cells are or labelled stained with fluorescent fluorochrome-linked membrane, with
Commonly used Fluorochromes FLUOROCHROMES EMISSION MAXIMUM Fluorescein Isothiocynate (FITC) 530nm Phycoerythrin (PE) 576nm Peridin-chlorophyll alpha complex (PerCP) 680nm Allophycocyanin (APC) 660nm T exas red 620nm ECD( PE - T exas Red T andem) 615nm PC5 (PE - cyanin 5 dye tandem) 667nm
Optics B) EMISSION OF FLUORESCENT LIGHT (FLUORESCENCE) As the fluorescent molecule present in or on the particle is interrogated by the laser light, it will absorb energy from the laser light and release the absorbed energy at longer wave length. Emitted photons pass through the collection lens and are split and steered down specific channels with the use of filters.
Optics- Filters Different wavelengths scattered simultaneously from a cell Need to split the light into its specific wavelengths in order to measure and quantify them independently. This is done with filters. of light are The system of filters ensures that each photodetector receives light bands of various wavelengths. are designed such that they absorb or wavelengths of light, while transmitting Optical filters reflect some others. Types of filters 1. Long Pass 3. Band Pass 2. Short Pass 4. Dichroic
Optics- Long Pass Filters Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm Transmittance 400nm 700nm 600nm 500nm Original from Cytomation Training Manual
Optics- Short Pass Filter Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths less than 600nm. Transmittance 400nm 700nm 600nm 500nm Original from Cytomation Training Manual
Optics- Band Pass Filter Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/- 10, not 550+/-20) Transmittance 400nm 700nm 600nm 500nm Original from Cytomation Training Manual
Optics- Dichroic Filters Long pass or short pass filters Placed at a 45 angle of incidence Part of the light is reflected at 90 , and part of the light is transmitted and continues. Detector 1 Detector 2 Dichroic Filter
OPTICS - DETECTORS The photodetectors convert the photons to electrical impulses. Two common types of detectors used in flow cytometry: Photodiode used for strong signals, when saturation is a potential problem (eg, forward scatter detector). Photomultiplier tube (PMT) more sensitive than photodiode but can be destroyed by exposure to too much light. used for side scatter and fluorescent signols.
ELECTRONICS The electronic subsystem converts photons to photoelectrons. Measures amplitude, area and width of photoelectron pulse. It amplifies pulse either linearly or logarithmically and then digitalizing the amplified pulse.
DataAnalysis- Plot Types There are several plot choices: Single Color Histogram Fluorescence intensity (FI) versus the number of cells counted. Two Color Dot Plot FI of parameter 1 versus FI of Parameter 2 Two Color Contour Plot Concentric rings form around populations. The more dense the population, the closer the rings are to each other Two Color Density Plot Areas of higher density will have a different color than other areas
Plot Types Density Plot Contour Plot Histogram Greyscale Density Dot Plot www.treestar.com
DATAANALYSIS - GATING Gating is in essence electronic window that sets upper and lower limits on the type and amount of material that passes through. Selection of only a certain population of cells for analysis on a plot. Allows the ability to look at parameters specific to only that subset.
Interpretation of Graphs An important tool for evaluating data is the dot plot. The instrument detects each cell as a point on an X-Y graph. This form of data presentation looks at two parameters of the sample at the same time.
Three common modes for dot plots are: Forward scatter (FSC) vs. side scatter (SSC) To look at the distribution of cells based upon size & granularity Single color vs. side scatter To visualize the expression of the fluorescence of the cells Two-color fluorescence plot. To differentiate between those cells that express only one of the particular fluorescent markers, those that express neither, and those that express both. used to discriminate dead cells from the live ones that are expressing the desired fluorescence.
How to differentiate dim & bright expression of an antigen? Dim : cells are present more towards the origin(0) on x(red) - y axis (pink) BRIGHT DIM Y-axis CD4 Bright : cells are present away from the origin(0) on x(green) & y(yellow) axis. X-axis CD8
WHAT IS UNIQUE IN FLOWCYTOMETRY MULTIPARAMETRIC RAPIDANALYSIS OF LARGE NUMBER OF CELLS INFORMATIONATASINGLE CELL LEVEL DETECTION OF RARE CELLPOPULATIONS ALLOWS PHYSICAL ISOLATION OF CELLS OF INTEREST
USES OF FLOWCYTOMETRY
APPLICATIONS ANALYSIS Immunophenotyping Dyes that bind to nucleic acids (DNA, RNA) Functional assays CELLCOUNTING CELLSORTING
CLINICALAPPLICATIONS Absolute CD4 counts HIV/AIDS HLAB27 assay Joint Pain Hematological Malignancies Diagnosis and Classification Detection of MRD DNAPloidy S Phase fraction Solid Tumours Primary TBNK Phagocytic function defect Immunodeficiency disorders
Cont.. Reticulocyte count PNH Osmotic fragility assay Hemolytic anaemia Feto- maternal Hemorrhage treatment response in Sickle CellAnemia Fetal Hb detection Platelet receptor assays (Platelet count, GT , BSS) Platelet function assay (CD62P , PAC-1) Bleeding Disorders Transfusion and Transplant CD34 STEM CELLCOUNTS Residual WBC count in leukodepleted blood packs Flow cytometry Crossmatch Surface markers in PMN, Monocytes Cytokine response Host Immune response in Sepsis
References THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC NEOPLASIA Brent L. Wood, Michael J. Borowitz. Henry s, 22nd edition, Chapter 34 https://www.bosterbio.com/protocol-and-troubleshooting/flow- cytometry-principle https://www.abcam.com/protocols/introduction-to-flow-cytometry Video Link: https://www.youtube.com/watch?v=5IdYFgYb9ls https://www.youtube.com/watch?v=7bCZx5xPwt0&t=94s
