MFI's Primary (Text) Results

MFI's primary (text) results tabulate the corrected median fluorescence intensities for a run. However, they include additional information to help judge whether the intensities are reliable, and whether the graphics screens need to be scrutinized for certain files. The results below were produced from this run organization.

The text results shown in the box below right can be displayed on the screen as the files are processed, or printed directly from MFI, or saved to a plain text file.

In order to keep the results compact, yet include all the necessary information, they look complicated at first. With a little experience, finding the information you need will become easy. The sample results below have been colored as a guide to help beginners to see their structure, but these colors will not appear in your MFI results.

The results include complete information about how the medians were calculated. That is, given the listmode data files, the information in the results would enable the run to be repeated even if the configuration file for MFI were unavailable. In other words, all program settings and gates are explicitly stated in the results.

MFI (Median Fluorescence Intensity) ver. 3.4kb8 (c) 1992-95 by Eric Martz.
DISCLAIMER: By the act of using this free and unsupported program, you
accept full responsibility for verifying its accuracy and suitability for
your purposes.  No warranty is made nor implied. Verification methods are
outlined in the on-line help.  Report bugs to
Use anonymous ftp to put problem data files in directory
/incoming at

8 input files: GDD49001 ... GDD49015

Peaks detected when excursion >5% using 32-channel histograms.

Abbreviations: Md = median, Sp = spew = mean/median.
H/T = high/total, R/T = right/total (ratios of events in High [top] or Right
  half of gate to Total events in gate).
'par 5-8:' labels the lines containing results for the 5th-8th parameters.
FACStar only: +/- after gains indicates fine gain on/off (e.g. FSC-H=2-).
  FL2, FL3 in data files, histo's/med'ns not shown.

Run 2: Titration of TS1/18 Ascites on JY1 Cells
gate  R/T   H/T     FSC Md (Sp)    SSC Md (Sp)    FL1 Md (Sp)

KMESF = (FL1)(1.57)
FACScan: FSC(1024)=E00,491  SSC(1024)=283,100  FL1(1024)=476,LOG  
5000 events acquired with threshold FSC=52 on 14-JAN-93
System: H.P. PASCAL version 3.1 FACScan Research Software  2/88

GDD49001: 20 sec from 18:44:20, 250 events/sec:
#LABEL:                                       FITC Standard Beads #3, 212 KMESF	
100%                274  (1.05)    232  (0.90)    543  (0.98)
Linear medians:                                   132        
SSC has 2 peaks:
  19% of events, peak median 12 (valley = channel 96)
  81% of events, peak median 240 (valley = channel 1023)
FL1 caution: 18% of events are in 1st occupied channel, 0.
FL1 has 2 peaks:
  94% of events, peak median 542 (KMESF 206 , valley = channel 581)
  6% of events, peak median 617 (KMESF 404 , valley = channel 1023)

GATE CHANGED TO Active Gate: #1 "JY Cells",
  FSC = 169-374, SSC = 154-409
KMESF = (FL1)(1.57)
********************* CHANGE IN ONE OR MORE PARAMETERS **********************
FACScan: FSC(1024)=E-1,682  SSC(1024)=307,100  FL1(1024)=476,LOG  
5000 events acquired with threshold FSC=52 on 14-JAN-93
System: H.P. PASCAL version 3.1 FACScan Research Software  2/88

GDD49002: 18 sec from 18:47:10, 277 events/sec:
#LABEL:                                                          JY1 cells only
62%   0.42  0.44    263  (1.01)    267  (1.02)    124  (1.06)
Linear medians:                                   3.05       

Control file set to GDD49004.

GDD49004: 17 sec from 18:49:10, 294 events/sec:
#LABEL:                                           2nd Ab only on JY1, A437 GAMG
62%   0.42  0.44    265  (1.01)    267  (1.03)    129  (1.07)
Linear medians:                                   3.19       
LinMd - control:                                  KMESF 0.00 

GDD49011: 17 sec from 18:55:14, 294 events/sec:
#LABEL:                                             TS1/18 Ascites 1/300 0.333%
56%   0.44  0.42    267  (1.01)    264  (1.03)    542  (1.01)
Linear medians:                                   131        
LinMd - control:                                  KMESF 201  

GDD49012: 17 sec from 18:56:06, 294 events/sec:
#LABEL:                                             TS1/18 Ascites 1/900 0.111%
57%   0.45  0.42    267  (1.01)    264  (1.02)    534  (1.01)
Linear medians:                                   122        
LinMd - control:                                  KMESF 186  

GDD49013: 17 sec from 18:56:52, 294 events/sec:
#LABEL:                                           TS1/18 Ascites 1/2700 0.0370%
58%   0.44  0.41    267  (1.00)    264  (1.02)    503  (1.02)
Linear medians:                                   92.2       
LinMd - control:                                  KMESF 140  

GDD49014: 18 sec from 18:57:45, 277 events/sec:
#LABEL:                                          TS1/18 Ascites 1/8100 0.00123%
59%   0.45  0.41    267  (1.01)    262  (1.03)    441  (1.01)
Linear medians:                                   52.8       
LinMd - control:                                  KMESF 77.9 

GDD49015: 15 sec from 18:58:29, 333 events/sec:
#LABEL:                                         TS1/18 Ascites 1/24300 0.00411%
59%   0.45  0.43    267  (1.01)    265  (1.03)    358  (1.02)
Linear medians:                                   25.0       
LinMd - control:                                  KMESF 34.3 

gate  R/T   H/T     FSC Md (Sp)    SSC Md (Sp)    FL1 Md (Sp)
HIGH VALUES:   (excluding control-corrected values)
75.9% 0.45  1.00    274  (1.05)    267  (1.03)    617  (1.07)
Linear medians:                                   257        
In gate 1:75.9%
LOW VALUES:   (excluding control-corrected values)
56.1% 0.00  0.41    263  (1.00)    12   (0.90)    124  (0.98)
Linear medians:                                   3.05       
In gate 1:56.1%
File range began at 18:44 and ended 0 hours and 14 minutes later.

The gray block at the top lists information about the run that is always given before the quantitative results. It includes the version of the program, the disclaimer, the range of files in the run, the initial gate ("Ungated" in this case), the settings for peak detection, the meanings of abbreviations used later in the results (a configuration option can suppress printing these), the description of the run, the "table heading" for the results (in green and red), the type of cytometer and its settings.

The "raw" results are given in a series of rows and columns embedded in the results. Here, these are colored in green and red. Notice how the table heading (green and red line in the top gray block) lines up with columns of values given below for each file.

The sample labels are shown along the right margin of the results, making them easy to spot (here in blue). Also shown in blue below are the final median intensities for FL1, after conversion to a linear scale, conversion to KMESF units, and subtraction of the control (blank) ("LinMd - control").

Colored here in magenta are important transitions, or warnings about possibly unexpected situations, such as the occurrence of two peaks in the first sample, and the "end pileup" of off-scale events in FL1 for the first sample.

Items shown in green concern the gate. First is the percentage of events in the gate. Next are R/T and H/T. These show the fraction of events in the gate that to the right of center, and above center. If the gate is well centered around a symmetrical cloud of events, R/T and H/T will be close to 0.5. If the R/T and H/T values change significantly during the run, it is a warning that the cloud of events has shifted relative to the gate. This, or a drop in percentage in-gate, might be reason to adjust the gate boundaries.

The raw median intensities, given and channel values, are shown in red. In parentheses is the spew (Sp) value. Spew is explained in the top gray block, in the list of abbreviations, as mean/median. Spew is close to 1.0 for symmetrical peaks; a spew significantly different from one signals an asymmetrical peak. In this run, the spew farthest from 1.0 is the value 0.90 for SSC in the first sample (beads). This resulted from the bimodality of SSC, here resolved automatically into two peaks.

The gray block at the bottom summarizes the ranges of values for the run. In this case, the inclusion of the ungated bead sample in this run, with its extreme values, has rendered the summary of little use. The solution is to re-do this run after untagging the first sample (to exclude it from the run). The summary block that results is shown below.
gate  R/T   H/T     FSC Md (Sp)    SSC Md (Sp)    FL1 Md (Sp)
HIGH VALUES:   (excluding control-corrected values)
62.1% 0.45  0.44    267  (1.01)    267  (1.03)    542  (1.07)
Linear medians:                                   131
In gate 1:62.1%
LOW VALUES:   (excluding control-corrected values)
56.1% 0.42  0.41    263  (1.00)    262  (1.02)    124  (1.01)
Linear medians:                                   3.05
In gate 1:56.1%
File range began at 18:47 and ended 0 hours and 11 minutes later.
Here is the summary of the ranges of results for the same run (Run #2), but with the sample of standard beads omitted (by simply untagging it). This version of the run includes only the samples for the antibody titration. This summary makes it easy to see that all ranges are small (except, of course for FL1), and that all spews are close to 1.0. Such a summary provide reassurance that it is unnecessary to scrutinize graphics for every file.