Free PC Software Upgraded

From: ERIC MARTZ (emartz@titan.ucc.umass.edu)

Date: Sun Dec 20 1992 - 14:55:18 EST

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________________________________________

I have posted version 3.1b of my free PC-based list-mode

cytometry data analysis program MFI for retrieval by anonymous

FTP from flowcyt.cyto.purdue.edu in directory pub/martz.  For

detailed instructions on use of FTP, email a request to me.

Enhancements since 3.1a include

- From the gate-setting graphics screen, the ability to change

the active gate number, to clear a gate, or to turn off gating.

(Thanks to Geoff Osborne for suggesting this enhancement.)

- Help has been spruced up significantly. A new 6-page chapter

has been added entitled "How to Use MFI for the First Time".

- Overlayed histograms can now be scaled independently, making

each histogram's tallest peak reach maximum Y.  This is an

alternative to the default, in which all are on the same common Y

scale set to avoid clipping the tallest peak, and in which areas

under each curve are proportional to the number of in-gate

events.

- Dozens of bugs have been fixed.  All reported/observed bugs are

believed to be fixed.  This belief is likely to be naieve, but

much progress has been made.

--

/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -

Eric Martz              emartz@titan.ucc.umass.edu    Professor of Immunology

                   Voice 413-545-2325, FAX 413-545-1578

   Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA

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This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:22:29 EST

Pecan Code 92 (no subject)

From: Geoffrey W Osborne (gwo215@cscgpo.anu.edu.au)

Date: Mon Sep 14 1992 - 09:24:31 EST

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________________________________________

Hello,

What follows is what appears to me to be an anomoly in the output

from the version of PECAN currently available via anonymous ftp. The file

which is called 2hmlt002 only contained 5245 events, yet the "GATEDEVENTS"

for this files are substantially higher than this. Examples for two different

gates appear below, followed by the UUencoded version of the original file.

The UUencoded file may be decoded using either the standard UNIX utility or

a DOS version.

Feedback regarding these results would be greatly appreciated.

Geoffrey Osborne

Flow Cytometry (FACS Lab)

JCSMR

AUSTRALIAN NATIONAL UNIVERSITY

CANBERRA,

Australia

Email: Geoff.Osborne@anu.edu.au

Results of PECAN processed files:

Initial gate:

"FILENAME","SAMPLE ID","DATE","GATE NAME","REAGENT","GATEDEVENTS","MARKER","%(-)","<->","%(+)","<+>","

"c:\util\2hmlt001","SF9 PLUS SDS"," 8/13/92","test","LOG FL1-Height", 9019, 60, 99.0, 39.3,  1.0, 68.0

"c:\util\2hmlt002","SF9  ALPHA PLUS SDS"," 8/13/92","test","LOG FL1-Height", 7231, 60, 71.7, 35.4, 28.3,123.5

Second gate:

"FILENAME","SAMPLE ID","DATE","GATE NAME","REAGENT","GATEDEVENTS","MARKER","%(-)","<->","%(+)","<+>","

"c:\util\2hmlt001","SF9 PLUS SDS"," 8/13/92","Sf9","LOG FL1-Height", 8682, 61, 99.1, 40.2,  0.9, 70.2

"c:\util\2hmlt002","SF9  ALPHA PLUS SDS"," 8/13/92","Sf9","LOG FL1-Height", 7579, 61, 64.7, 37.2, 35.3,126.2

section 1 of uuencode 5.10 of file 2hmlt002    by R.E.M.

begin 644 2hmlt002

`

end

sum -r/size 31878/24011 section (from "begin" to "end")

sum -r/size 15010/17408 entire input file

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Need PeCAN 1.2 Source Code

From: ERIC MARTZ (emartz@titan.ucc.umass.edu)

Date: Wed Aug 26 1992 - 12:31:02 EST

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________________________________________

Both Geof Osborne (gwo215@cscgpo.anu.edu.au) and I would like to enhance

PeCAN.  We would prefer to start with the source code for version 1.2

or later.  Here is the text of a plea which I mailed to the cytometry list

on 8/1.  If anyone has the 1.2 or later source code (portions dated 8/22/88

or later) please FTP it to flowcyt.cyto.purdue.edu (into /pub/upload) and

we'll be most grateful.  It will then be there for anyone to retrieve.

-----BEGIN QUOTE

- From emartz Sat Aug  1 00:13:08 1992

Subject: PeCAN PLUS?

To: cytometry@flowcyt.cyto.purdue.edu (Cytometry Mailing List)

Date: Sat, 1 Aug 92 0:13:08 EDT

I'm considering making some enhancements to PeCAN*, since Mike Lance was

kind enough to send me the source code, and it is in C, a language with

which I have experience.

I received from Bob Murphy a PECAN.EXE which signs on as version 1.2.

The file write date is 8/22/88, size 38,594.  The source code I

received from Mike Lance is for a version 1.0, 3/15/88, EXE size

38,864.  It would be helpful if someone can send me the source code

for the "final" version released by the authors (Morgan P. Conrad and

Ravi Mhatre, who were at B-D in 1988).

I wonder if anyone has already made enhancements, in which case I'd like

to know what has been added and I'd like to be able to start with the

most recent source code available.

Alternatively, if anyone has a "better" or alternative "free" program

for PC analysis of FCS1.0 or FCS2.0 files, I'd like to know more.

*PeCAN (PC ANalysis) is an elegant free program for determination of

percentage positive cells from FCS1.0 files for one, two, or three

color work.  It supports polygonal gates, requires EGA, and puts out

summary files in ASCII database format (e.g. suitable for Lotus).

After you have set the gate (typically on FSC vs. SSC or scatter vs.

FL3 = propidium iodide), and set a division line for positive/negative

on each color (it will automatically set the division for 99% negative

on your control cells!), it processes all files in the current

directory without further intervention.  However, it displays the dot

plot and histograms for each file for a user-specified number of

seconds during which pressing any key allows modifications.

------END QUOTE

--

/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -

Eric Martz              emartz@titan.ucc.umass.edu    Professor of Immunology

                   Voice 413-545-2325, FAX 413-545-1578

   Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA

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Availability of DOS and VMS programs via anonymous FTP

From: Robert F. Murphy (MURPHY@A.CFR.CMU.EDU)

Date: Wed Aug 19 1992 - 14:51:53 EST

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________________________________________

A number of files of use to flow cytometrists are available by anonymous

FTP from CMU.  These include programs for DOS and VMS systems. To copy

files, FTP to "a.cfr.cmu.edu" and login as "anonymous". Use your

internet mail address as a password.  You can then use the CD command to

change the working directory as needed.

A brief listing of directories follows (more information is contained in

each one):

[.DOS.FROMHP] the receiving program for HPtoIBM

[.DOS.MARTZ] FCS file utilities from Eric Martz

[.DOS.PECAN] multicolor list-mode analysis program

[.DOS.READDATA] simple FCS data file reading program from Morgan Conrad

[.DOS.TESTFILES] test FCS files

[.VMS.CALC4] list-mode calculator program

[.VMS.FCSLST] creates ASCII listing files from FCS files

I will be happy to answer questions, but all programs are provided

"as is".

Bob Murphy

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Getting modal values and ASCII listings on Consort/VAX systems

From: Robert F. Murphy (MURPHY@A.CFR.CMU.EDU)

Date: Wed Aug 19 1992 - 14:39:04 EST

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________________________________________

In response to a question from Ray Hester (as discussed by Alice Givan,

Eric van Buren and Mike Lance), there are at least three ways to find

mode values for FCS files on Consort/VAX systems.

1. Use the GI command in DISP4 followed by the H command (after

  positioning the cursor in a particular histogram).

2. Read the file into COTFIT using the INPUT command, specify which

  parameter you want to read, use the DELETE command to remove the

  cell-cycling function which is automatically assumed for FCS files,

  and then use the FIT command to get a listing of statistics.  The

  mode is found after the words "Y Peak at X=".

3. For files acquired with ACQ8 V5.3, the PKn keywords contain the mode

  for each parameter.  There are three of these keywords for each

  parameter: PKnT contains the mode for the entire histogram of parameter

  n, PKnWL contains the mode for the portion of the histogram within

  the left window, and PKnWR contains the mode for the portion in the

  right window.

As for generating ASCII files, Eric van Buren correctly points out that

the PS command can be used.  You can write the listing to a file by

setting the logical name LP to point to a disk before you enter DISP4.

For example:

$ ASSIGN listing.ascii LP

$ DISP4

Each time the PS command is given, a new file with the assigned name

will be created.

An alternative is to use the FCSLST program (this is the program

mentioned by Alice Givan).  It will generate an ASCII file from any FCS

1.0 file (list mode, single paramter histograms, dual parameter

histograms). It is available from CMU via anonymous FTP

(address: a.cfr.cmu.edu, directory: [.VMS.FCSLST], file FCSLST.EXE).

Bob Murphy

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MACs

From: Alice.L.Givan@Dartmouth.EDU

Date: Tue Aug 18 1992 - 12:24:18 EST

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________________________________________

One more question about computers:

If people out there would like to let me know,  I am trying to compile a list

of flow users who are attempting to analyze data or organize results files on

MACs.  I would like to have the following information:

Flow cytometer:

Flow computer:

Flow software:

Remote (Mac)computer:

Remote use:  eg results files into MS Excel

What software/network/etc do you use or would you like to use to send and

receive files?

If you send me this information I will send back a compiled list.

Thanks.

Alice.L.Givan@dartmouth.edu

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PeCAN PLUS?

From: ERIC MARTZ (emartz@titan.ucc.umass.edu)

Date: Fri Jul 31 1992 - 23:13:08 EST

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________________________________________

I'm considering making some enhancements to PeCAN*, since Mike Lance was

kind enough to send me the source code, and it is in C, a language with

which I have experience.

I received from Bob Murphy a PECAN.EXE which signs on as version 1.2.

The file write date is 8/22/88, size 38,594.  The source code I

received from Mike Lance is for a version 1.0, 3/15/88, EXE size

38,864.  It would be helpful if someone can send me the source code

for the "final" version released by the authors (Morgan P. Conrad and

Ravi Mhatre, who were at B-D in 1988).

I wonder if anyone has already made enhancements, in which case I'd like

to know what has been added and I'd like to be able to start with the

most recent source code available.

Alternatively, if anyone has a "better" or alternative "free" program

for PC analysis of FCS1.0 or FCS2.0 files, I'd like to know more.

*PeCAN (PC ANalysis) is an elegant free program for determination of

percentage positive cells from FCS1.0 files for one, two, or three

color work.  It supports polygonal gates, requires EGA, and puts out

summary files in ASCII database format (e.g. suitable for Lotus).

After you have set the gate (typically on FSC vs. SSC or scatter vs.

FL3 = propidium iodide), and set a division line for positive/negative

on each color (it will automatically set the division for 99% negative

on your control cells!), it processes all files in the current

directory without further intervention.  However, it displays the dot

plot and histograms for each file for a user-specified number of

seconds during which pressing any key allows modifications.

--

/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -

Eric Martz              emartz@titan.ucc.umass.edu    Professor of Immunology

                   Voice 413-545-2325, FAX 413-545-1578

   Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - */

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Need HPTOIBM (update)

From: ERIC MARTZ (emartz@titan.ucc.umass.edu)

Date: Fri Jul 31 1992 - 15:38:36 EST

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________________________________________

Yesterday I said:

> I have received copies of HPTOIBM.CODE from both Bob Murphy and Mike

> Lance.  I have serial file transfer working to the PC but these

> apparently identical versions (#1.0, file size 17,920 bytes) contain a

> bug which makes them transmit only every 2nd file.  That this was a

> bug in an early version is confirmed by Morgan Conrad, their author.

> He says the bug was fixed but the bad version "keeps reappearing".  If

> someone has the version with this bug fixed, please let me know.

> We'll arrange to make it available to all by FTP.

In reply, Morgan Conrad informed me that the debugged version may

still announce itself as #1.0 and may have exactly the same byte size.

The file write date is unreliable since the HP Pascal Filer/Filecopy

function changes the date to the current date.  Thus the only way to

tell if it skips files is by how it performs.

--

/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -

Eric Martz              emartz@titan.ucc.umass.edu    Professor of Immunology

                   Voice 413-545-2325, FAX 413-545-1578

   Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA

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Re: Data retrieval from FACS

From: Mike Lance (Mike_Lance@bdis.com)

Date: Wed Jul 29 1992 - 12:30:10 EST

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________________________________________

                      Subject:                               Time:9:18 AM

 OFFICE MEMO          RE>Data retrieval from FACS 44         Date:7/29/92

In reply to Ray Hester's message

>Can anyone tell me how to get data(# of cells in each channel) from

>a FACS 440 Consort 40 VAX file?  On the 440 you could always push the

>"scroll" key (an investigator wants to calculate median and mode

>for fluorescence histograms).  With the VAX I'm not sure.  Thanks.

DISP4 gives mean and mode values for histograms.  Use the graphic input (GI)

commands C (for mean and CV) and H (for mode).  To see a listing of the

counts in each channel, use the LIST command in COTFIT.

Michael Lance

Becton Dickinson

(with help from A. Parsons @BD)

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FACS VAX DATA RETRIEVAL

From: IRBH000 (@VM.CC.PURDUE.EDU:IRBH@USOUTHAL)

Date: Tue Jul 28 1992 - 09:12:00 EST

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________________________________________

There may have been a problem with transmittal of this request

yesterday, so I am going to try and send it again.

An investigator here at South Alabama wants to determine the mode

and median values of histograms acquired on a FACS 440 using the

VAX Consort 40 software. Since this software is apparently only

capable of giving a mean value, we would like to get the raw data

(# of cells in each channel) and use it to produce the values

required (mode and median).

Can anyone tell us how to get a printout of these numerical values?

Thanks in advance.

Ray Hester

Univ. South Alabama

BITNET: IRBH@USOUTHAL

FAX: (205) 460-6071

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Geometric vs. Arithmetic Mean

From: Mike Lance (Mike_Lance@bdis.com)

Date: Tue Jul 14 1992 - 15:03:42 EST

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                      Subject:                               Time:11:32 AM

 OFFICE MEMO          Geometric vs. Arithmetic Means         Date:7/14/92

> I was wondering if someone could enlighten me as to the relevance of the

>geometric mean when applied to the calculation mode for log amplified data.

In response to Goeff's message...

LYSYS II, version 1.1 and PC-LYSYS give the user the ability to generate

the mean on log data by selecting one of four different methods:

Method 1.  Geometric mean expressed as a channel value:

Data  in channel values  --> calculate mean --> report result

Method 2.  Geometric mean expressed as a linear values:

Data in channel values --> calculate mean --> convert

mean to linear value--> report result

Method 3. Arithmetic mean expressed as a linear value:

Data in channel values --> convert data to linear values

  --> calculate mean --> report result

Method 4. Arithmetic mean expressed as a channel value:

Data in channel values --> convert data to linear values

--> calculate mean --> convert mean to a channel value -->

report result

Each method will give a different result, and for the most part,

arithmetic means will have a larger value than geometric means

since the arithmetic method tends to be biased by high values in

the data.

Since it represents the population's average signal intensity, the

arithmetic mean is useful for comparing flow cytometric results

with those obtained on the same sample using a spectrofluorimeter.

For data distributions where logarithmic presentation is appropriate,

the geometric mean represents the typical signal level better than

the arithmetic mean because it is less influenced by high outliers.

For example, "loose" gating  with the inclusion of one bright

contaminant within the gate could significantly alter the arithmetic

mean.  

-----

Excerpts from an unpublished BDIS Research Note: STATISTICAL

CALCULATIONS IN BECTON DICKINSON SOFTWARE ON LOGARITHMICALLY

AMPLIFIED DATA, by D. Gandour, et. al.

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Re: means

From: Marty Bigos (BIGOS@Beadle.Stanford.EDU)

Date: Wed Jul 15 1992 - 17:28:00 EST

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________________________________________

Both Bob Murphy and Alice Givan have presented very clear discussions of

arithmetic and geometric means. I'd like to add our two bits (no pun intended)

of experience here.

Almost all biologists who use our center have been convinced that for roughly

normal or log normal distributed signals the arithmetic or geometric mean is an

appropriate measure of central tendency. However, for distributions which vary

greatly from this, the "average" contains very little information regarding

central tendancy. Thus, many researchers here will use order statistics (median,

25th, 75th percentile) to characterize the central tendancy and spread of such a

distribution.

Order statistics have the following characteristics: (1) when data is "normally"

distributed, they agree with the mean. (2) they are not sensitive to outliers,

so gating conditions and off-scale pile-ups affect them less, (3) for list mode

data they are faster to calculate than means.

In a normal distribution, the coefficient of variation (cv) is:

cv = .742 (75th-25th)/median

This quantity also has properties (2) and (3) above, that is, it is less

sensitive than a true "CV" to gating and outliers and provides a very rapid

algorithm for monitoring cv's during machine alignment.

-Marty Bigos

Stanford Shared FACS Facility

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(no subject)

From: Geoffrey W Osborne (gwo215@cscgpo.anu.edu.au)

Date: Tue Jul 14 1992 - 10:09:15 EST

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________________________________________

Hello,

I was wondering if someone could enlighten me as to the relevance of the

geometric mean when applied to the calculation mode for log amplified data.

My understanding of the arithmetic mean is

mean = (x1 + x2 + x3 ...+ xn) / n

whereas the geometric mean is

geo_mean = nth root of (x1 * x2 * x3 ...* xn)

which yields a slightly different answer.

Any info will be appreciated.

Thanks,

Geoff Osborne

FACS LAB

JCSMR

Australian National University

Canberra AUSTRALIA.

Email: Geoff.Osborne@anu.edu.au.

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Re: Geometric Mean

From: ERIC MARTZ (emartz@titan.ucc.umass.edu)

Date: Tue Jul 14 1992 - 15:23:39 EST

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gwo215@cscgpo.anu.edu.au (Geoffrey W Osborne, Australian National U,

Canberra) writes:

> I was wondering if someone could enlighten me as to the relevance of the

> geometric mean when applied to the calculation mode for log amplified data.

> My understanding of the arithmetic mean is

> mean = (x1 + x2 + x3 ...+ xn) / n

>

> whereas the geometric mean is

> geo_mean = nth root of (x1 * x2 * x3 ...* xn)

>

> which yields a slightly different answer.

> Any info will be appreciated.

In my attempts to emulate, in my own programs, the means and medians

for log-acquired data given by the Becton-Dickinson FACSCAN and

FACSTAR software, I succeeded for the mean by calculating the log of

the arithmetic mean of the antilogs of the observations (channel

values representing logs of fluorescence intensity).  Thus, for the

mean of log-acquired data, B-D gives the log of the mean value that

would have been obtained had the data been acquired with linear,

rather than log, amplification.  The same principle applies to the

median in the case where there are an even number of observations (so

the median falls "halfway" between the middle two).  I was pleased to

see such care applied in the development of their software.

I, too, thought that the geometric mean should give the same value but

it appears to give a different value.  Hopefully someone else can clarify

this issue.

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Re: Geometric Mean

From: Robert F. Murphy (MURPHY@A.CFR.CMU.EDU)

Date: Wed Jul 15 1992 - 11:39:06 EST

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The arithmetic mean of a log distribution is equal to the log

of the geometric mean of the linear distribution.  It is appropriate

to calculate and report the arithmetic mean of a log distribution

when the distribution is log-normal.  Since this is frequently the

case, it is desireable to have the ability to calculate either arithmetic

or geometric means (i.e., be able to calculate an arithmetic mean of

a log distribution with and without conversion to linear).  I hope

that I have added to the confusion on this point.

Bob Murphy

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Re: Wavelength Shift Calibration

From: Robert F. Murphy (MURPHY@A.CFR.CMU.EDU)

Date: Mon Jul 13 1992 - 14:44:38 EST

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In response to Eric Martz's mail message regarding use of beads

for calibration of ion measurements by flow cytometry, the

first use of that method that I am aware of is described in

the paper "Endosome pH measured in single cells by dual fluorescence

flow cytometry: Rapid acidification of insulin to pH 6" (1984)

J. Cell Biol. 98:1757-1762 (see Figure 1).

For calibration of illuminated volume by a similar method and

a general discussion of discrimination of bound and free ligand

by flow, see Chapter 18 of Flow Cytometry and Sorting, Second Edition,

(Melamed, Lindmo, Mendelsohn, eds.) Wiley-Liss, Inc., 1990, pp. 355-366.

There is also a discussion of the possible effect of AC-coupling

circuits.

Bob Murphy

Carnegie Mellon University

murphy@a.cfr.cmu.edu

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means

From: Paul.M.Guyre@Dartmouth.EDU

Date: Tue Jul 14 1992 - 19:08:00 EST

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This is a somewhat simplistic explanation of geometric vs arithmetic means

that I wrote for an in-house flow lab newsletter.  It explains the difference

in the two types of statistics -- I assume that the BD software uses the

relative fluorescence intensity (i.e. 1-10,000 for a 4-log-decade amplifier

OR 1-1024 for a linear amplifier) to calculate these values,  but I haven't

checked the calculations myself.  This  may just be telling you what you

already know....

"The third value that can be used to describe a histogram distribution is the

mean.  The mean is the conventional "average" --  that is,  you add up

everybody's weight, divide by the total number of kids in the fifth grade,

and end up with the average weight of a fifth grader. The main (and serious)

complication is that statisticians talk about two kinds of means -- depending

on whether they are considering geometrically or arithmetically distributed

normal distributions (does the distribution look symmetrical when plotted on

linear graph paper or when plotted on log graph paper?).  To calculate an

arithmetic mean you add up all the values and divide by the total number of

individuals.  To calculate the geometric mean,  you multiply together all the

values and then get the nth root of the total product.  Which value is

appropriate depends on whether or not you want to weight the bright cells

according to their true (high) brightness.  By way of an example,  if you

have a population of five cells, with fluorescence intensities of 1, 100,

100, 100, and 10,000, then their arithemetic mean intensity will calculate to

be 2060;  their geometric mean will be 100.  (Their mode will be 100, and the

median value will also be 100).  It is the arithmetic mean which gives the

cell at 10,000 its "real" weight -- in much the way that one very large kid

in the fifth grade can seriously affect the calculated weight of an "average"

fifth grader. The geometric mean sees that the cell at 10,000 is 100 times

brighter than the cells at 100;  and the cell at 1 is one hundredth as bright

as the cells at 100 (i.e. the cells show a log-normal distribution -- they

look symmetrical when plotted on log graph paper).  Therefore the cell at

10,000 is given equal weight to the cell at 1 in the calculation of the

geometric mean. "

Which mean you use is,  I would think, a matter of taste.  My own opinion is

that the median is a far better flow cytometric parameter -- partly because

it avoids the problem of deciding which type of mean is relevant (it also

avoids the problem of correctly evaluating "off scale" events.

Hope this helps a bit.

Alice Givan

Dartmouth Medical School

Lebanon, NH

E-Mail c/o Paul M Guyre@dartmouth.edu

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RE: Kinetics, unrinsed?

From: MURPHY@A.CFR.CMU.EDU

Date: Fri Jan 08 1993 - 11:10:33 EST

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>Eric Martz writes:

>Can anyone direct me to publications in which the time kinetics of

>binding of directly fluorochrome-conjugated antibody to living cells

>were followed by flow cytometry?  The idea is to add the antibody to

>the cells and then immediately begin acquisition, without rinsing.

>Ideally, a single large file can be acquired over several minutes

>while binding proceeds; then medians can be taken on sequential

>"slices" of the data in the file.  Thus, the entire kinetic curve is

>generated from a single sample.

>I'm particularly interested in the unrinsed mode described above, but

>I'd also be interested in any publications using a more conventional

>approach in which binding is stopped by rinsing at various times in

>different samples, and then each sample is analyzed as a single time

>point.

>Finally, I'd like to know what software is available (commercial or

>otherwise) to slice up the events in a single list-mode file.  (I have

>some home-grown software under development if anyone is interested in

>using it.)  Do any of the commercial packages (e.g. from Phoenix, Verity,

>B-D, or Coulter) do time-slicing?

The references below review the types of measurements Eric described,

give references to original work by Bohn & Mmanske, Crowell & Salzman,

Finney & Sklar and others, and give protocols for both equilibrium

methods and dilution methods.  The KINPRO program in the Consort/VAX

package calculates gated means and standard deviations for time-slices

of FCS files and creates a file for plotting of these means versus time.

If CHRONOS is still supported for the HP, it does similar things.

R. F. Murphy (1990). Ligand Binding, Endocytosis, and Processing. In:

Flow Cytometry and Sorting, Second Edition, M. R. Melamed, T. Lindmo, M.

L. Mendelsohn (eds.), Wiley-Liss, Inc., New York, pp. 355-366.

R. F. Murphy (1992) Scatchard analysis by flow cytometry. In: Flow

Cytometry and Cell Sorting, A. Radbruch (ed.), Springer Verlag, Berlin,

pp. 59-62.

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Image Analysis - Help Please!

From: paul@vphb.vet.purdue.edu

Date: Fri Feb 05 1993 - 07:55:55 EST

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- From Paul Robinson, Purdue University:

My group are in the process of making a significant purchase in the area of

image analysis instrumentation. Our studies have previously used flow cytometry

extensively

and  will continue to do so, however, it is clear that there are many areas we

need to have a capability to do image analysis. I would appreciate some advice

from the group.

Our general needs are:

Evaluation of ion fluxes (Calcium etc,) pH

Oxidative burst such as HE, DCF, and similar

Membrane potential, fluidity studies

confocal microscopy, multiple color phenotyping, dual excitation & emission

Special options might be FRAP, cell-cell communication and similar

Video output, electronic image camera,

GOOD SOFTWARE ESSENTIAL (is this asking too much?)

There are a number of instruments out there that do many of the above. My

perception is that some systems can do lots of things but perhaps not so well

and

others can do, say, excellent confocal, but lack good software for ion flux

measurement.

I would appreciate advice from people who know something about particular

instruments or are image users to give me advice.  

Instruments under consideration:

Biorad confocal

Leica Confocal

Meridian Instruments ACAS with confocal

and some others...... ????

Help please, this is our one opportunity to get some decent image equipment. I

don't want to blow it (too badly!).  Call me, fax me, email me, help me......

Thanks,  Paul Robinson      PHONE: 317- 494 6449       FAX: 317-494 0517

Purdue University Cytometry Laboratories

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Image Analysis Decisions

From: J. Paul Robinson (paul@flowcyt.cyto.purdue.edu)

Date: Thu Feb 25 1993 - 09:47:18 EST

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I recently asked a question about image analysis instrumentation and received

many useful comments and lots of advice. Thank-you.

I am now more confused than ever, and somewhat concerned about spending lots

of money and not being able to accomplish the tasks that I need to.

Several others have indicated to me that they are about to purchase similar

image analysis equipment. Would there be interest to organize a meeting at

the ISAC conference for those who would like to share the information, bias,

quotations???? is this ethical?) and anything else that has impressed them

while looking at various systems.

This meeting would be a roundtable discussion format (possibly close to a

bar!) but would include only those seriously interested in purchasing image

instrumentation. Perhaps it would end up with us sharing our ignorance, but

that too is useful.

Please indicate to me if anyone is interested and I will organize it.

I am aware of at least 8 groups that are about to spend in the $200-400,000

range within the year. It seems that we all have a lot to gain, and even

more to lose by making inappropriate decisions.

Paul Robinson

Purdue University Cytometry Laboratories

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