RE: Flow--Counting anything

Caroline,

 

Counting MAb-tagged events using known numbers of highly fluorescent
beads is a proven method that has been used in flow cytometry for
decades.  This method has been and is used in determining CD4 counts in
HIV patients, critical in stage disease classification.  There are many
publications on the subject, and I suggest you cite some of these to
defend your project.  One of our published works (Moss and Arrowood,
2001, "Quantification of Cryptosporidium parvum oocysts in mouse fecal
specimens using immunomagnetic particles and two-color flow cytometry",
J. Parasitol., 87: 406-412) showed that MAb-tagged oocysts could be
reliably quantified using TruCount tubes (Becton Dickinson).  For counts
at least a few million per ml, the TruCount tubes were as reliable as a
hemacytometer, but for counts less, known number of fluorescent beads in
with your unknown and the use of a flow cytometer is more accurate than
a hemacytometer.  However, I must warn you that the smaller the number
of unknown, the larger the CV and SD, very low numbers of what you are
counting are not normally distributed.	When numbers are in the hundreds
per ml, you run the risk of exceeding your 20% limit of the expected
value.	They are other factors to consider such as integrity of your
unknown throughout the staining and flow process, quality of your MAb,
stickiness of your unknown to material of tube(s), uniformity of the
unknown suspension, and volume accuracy of your unknown.  

 

As a disclaimer, we have no financial ties to Becton Dickinson.
Although their product is very expensive, we found that their TruCount
tubes were more convenient and reliable than bead suspensions purchased
from other manufactures.

 

    

Delynn Moss

Centers for Disease Control and Prevention

Division of Parasitic Diseases

Mail Stop F-13

Bldg 23, Room 1026

4770 Buford Hwy, NE

Atlanta, GA 30341

ph 770-488-4041

FX 770-488-4108

e-mail dmm3@cdc.gov

 

  _____  

From: ckohler@bi-vetmedica.com [mailto:ckohler@bi-vetmedica.com] 
Sent: Monday, January 30, 2006 10:10 AM
To: cyto-inbox
Subject: RE: Flow--Counting anything

 

Hello Everyone,

 

Recently a project I had been working on was discontinued for me on the
assumption that what I had proposed to do, wouldn't work. I was
attempting to count bacteria. I am using a monoclonal for detection and
had planned on using counting beads. I am not allowed to be more
specific. In short, I need some testimony to the ability of the flow
cytometry technology to detect and count..anything!...within 20% of the
final expected value. I am curious as to what the experience of the
users are, to date, and what accuracy you are experiencing. I have so
many ideas on how to make this work...

 

I am indebted to you all.

Thanks,

Caroline