Advanced Flow Workshop Shumway Murphey

Workshop 2: Advanced Flow Workshop-
Wayne Shumway, CHT, CHS and
Cathi Murphey, MT(ASCP), CHS, BLM
XM: DONOR + RECIPIENT SELECTION
Best donor choice = least
fluorescence from
secondary antibody
3
detected.
FCXM #1
3
The quantity of signal
XM:
above background is of
- Bkgnd:
36
200% of background
significance (because it
Aby:
3
(77 ch. . Shift)
directly reflects the
Y
number of patient
FCXM #2
1
antibodies bound), the
XM:
1
proportion of background
- Bkgnd:
12
200% of background
is IRRELEVANT*!
Aby:
1 (77 ch. . Shift)
*with regard to the number of antibodies present
Y
Y
Y
Y
Y
Y
Y
Channel Shifts and the Illusion of Increased
Sensitivity When Using Pronase
Y Y
The same number of
Untreated B Cells
antibody molecules
XM:
binding against a lower
- Bkgnd:
background produce a
36
greater channel shift – Aby:
3
but remain the same
77 ch. . Shift
number of antibody
molecules!
Pronase Treated B Cells
Linear data reflects the
XM:
true quantity above
- Bkgnd:
14
background – a value
Aby:
3
that is independent of
154 ch. . Shift
the background.
Y Y
Y
Y
Y
3
Y
Y
1
Y
Y
Y
3
background
1
background
Relative Linear Values (RLV) are frequently referred to as MFI (Median
or Mean Fluorescence Intensity) values. It is these RLV / MFI values
that are “numbers” in the conventional sense (NOT the channel
values!), they have simply been plotted on a log scale. Events with an
MFI value of 600 are twice as bright as those with an MFI of 300 (the
expected, conventional, “linear” relationship). HOWEVER, if an event
reads at channel 600, an event half as bright will read at channel 523.
So how do we convert the channels to
the “numbers”?
CHANNEL VALUE
Calculate “R” = the
channel value divided
by channels per decade
512
512/256 = 2
End of 1 st decade
End of 2 nd decade
End of 3 rd decade
256 512 300 768 1024
200 400 600 600 800
End of 4 th decade
1000
10 R = RELATIVE
LINEAR VALUE
(RLV – aka MFI)
10 2 = 100
1
RLV
10 100 1000
Fluorescence Intensity
10,000
W H E N I N L O G “M M O D E” E :
CHANNEL VALUES
BD = “Log Data”
Coulter = “Linear Data”
RELATIVE LINEAR VALUES
BD = “Linear Data”
Coulter = “Log Data”
End of 1 st decade End of 2 nd decade End of 3 rd decade
End of 4 th decade
256 512 768 1024
RELATIVE LINEAR VALUE
is a much better term than
Median Fluorescence Intensity
1
RLV
200 400
600 800 1000
10 100 1000 10,000
5