[Cytometry] Opinions about planned experiment

Bruce H. Davis, MD brucedavis at trilliumdx.com
Thu Feb 18 06:57:36 EST 2010

Not sure what your marker is, but you might use an approach we took for the
Leuko64 assay for infection/sepsis that utilized a stable spectrally matched
bead and QuantiCalc software (Verity Software House) to measure CD64 and
CD163 on leukocytes.  We see lot to lot variability has a CV of <10%.
However, aside from suggesting median fluorescence and avoid freezing the
reagents, your approach might give reasonably close values.  Sure would be
nice if NIST still had fluorescent standard......

This is not meant to be an advertisement and happy to further help off line,
if you wish to contact me.


Bruce H. Davis, MD
Trillium Diagnostics

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Florian Uhle
Sent: Wednesday, February 17, 2010 1:58 PM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Opinions about planned experiment

Dear Flow cytometry community,

as I am a bloody beginner in the field of flow cytometry, I would like to
hear your opinion on my planned experiment, which involves quantitative
measurement of surface expression (I know, for a lot of flow operators this
seems to be a no-go).

In a clinical study, I would like to measure expression level of a monocytic
membrane receptor in the course of sepsis. To ensure comparability over time
and between patients, I am trying to minimize all sources of variance:

-       	- instrument will be calibrated using CaliBRITE Beads before
each measurement

-       	- antibody will be purchased in one large batch, directly
conjugated from the providing company

-       	- isotype com will be purchased the same way

-       	- conjugate will be APC, buffer of antibody will be

-       	- upon arrival, antibody will be stored frozen in single
experiment aliquots to avoid decrease intensity loss of fluorophore

-       	- staining protocol will be performed in whole blood; to
avoid any Fc related binding, a preincubation with human IgG will be

-       	- readout should be geometrical mean fluorescence intensity

I would very much appreciate any comment on this, no matter if positive or

Best wishes,


Florian Uhle
University Hospital Giessen
Department of Anesthesiology and Intensive Care Medicine
Rudolf-Buchheim-Straße 7
35392 Giessen

phone:   	+49-641- 99 444 77
fax:          	+49-641- 99 444 89
e-mail: 	florian.uhle at chiru.med.uni-giessen.de
URL:		http://www.uniklinikum-giessen.de/anaesthesie/

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