[Cytometry] Cytometry Digest, Vol 64, Issue 19

Bruce Davis brucedavis at trilliumdx.com
Mon Feb 17 12:59:28 EST 2014


Re: Antibody titration & staining index (Davis, Bruce)


I encourage review of the ICSH/ICCS Guidelines for Validation of Cell
Based Fluorescent Assays published in a special issue of Cytometry B.
This outlines proper antibody titration methods and other performance
characteristics of a fully validated laboratory developed flow cytometry
assay.  The link to this information is
http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.v84.5/issuetoc

Regards,

Bruce H Davis, MD



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On 02/17/2014, 12:00 PM, "cytometry-request at lists.purdue.edu"
<cytometry-request at lists.purdue.edu> wrote:

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>Today's Topics:
>
>   1. Antibody titration & staining index (Ina Laura Pieper)
>   2. A novel flow cytometric bacterial infection marker (Jari Nuutila)
>   3. Re: archiving data in a flow core? (Rob Salomon)
>   4. criopreservation (Jose Benito)
>   5. Re: whole blood **tech support response** (Roy Edward)
>   6. Re: Antibody titration & staining index (Barsky, Lora)
>
>
>----------------------------------------------------------------------
>
>Message: 1
>Date: Sun, 16 Feb 2014 20:39:12 +0000
>From: Ina Laura Pieper <inalaurapieper at gmail.com>
>To: Purdue list <cytometry at lists.purdue.edu>
>Subject: [Cytometry] Antibody titration & staining index
>Message-ID:
>	<CAM8oOngAqmmF3CaD3+jiuLj39gXyBdxTH8r33E0mQRFGWA+H=Q at mail.gmail.com>
>Content-Type: text/plain; charset="iso-8859-1"
>
>Hello flowers,
>
>What's the objective way of deciding on the optimal antibody concentration
>during antibody titrations? I've searched and found this description on
>the
>UC Flow blog of plotting the stain index against antibody concentration,
>http://ucflow.blogspot.co.uk/2009/06/antibody-titrations.html
>However, to calculate stain index it seems I need a positive and negative
>population in my sample, probably of the same autofluorescence, which
>isn't
>always achievable.
>
>What do I do if I want to calculate the optimal concentration in a
>titration of a CD14-antibody in whole blood? I've done overlay histograms
>of stained and unstained samples gated on monocytes (positive) and
>granulocytes (negative), so that i can visually identify the concentration
>at which the granulocytes are no longer staining unspecifically. Is this
>the right way to do it? Seems very subjective to me. From the attached
>figure I think a 1:5 dilution is the best (no unspecific binding on
>granulocytes whilst still a clear monocyte signal).
>
>What should I do when staining a homogenous sample, and there is no
>negative population?
>
>Best regards,
>Ina Laura
>
>------------------------------
>
>Message: 2
>Date: Mon, 17 Feb 2014 11:11:09 +0200
>From: Jari Nuutila <jarnuu at utu.fi>
>To: cytometry at lists.purdue.edu
>Subject: [Cytometry] A novel flow cytometric bacterial infection
>	marker
>Message-ID: <5301D22D.8050907 at utu.fi>
>Content-Type: text/plain; charset="iso-8859-1"; Format="flowed"
>
>Dear clinicians, infection researchers, and all those who are interested
>in novel flow cytometric applications,
>
>Once again I am informing you that our latest article entitled:
>"Bacterial infection (BI)-INDEX: an improved and simplified rapid flow
>cytometric bacterial infection marker" by Nuutila et al. has
>beenpublished in the Diagnostic Microbiology and Infectious Disease
>(Volume 78, Issue 2, February 2014, Pages 116--126).
>
>In this article, we are presenting the novel flow cytometric
>multiparametric marker of bacterial infection designated bacterial
>infection INDEX (BI-INDEX), which displays 90% sensitivity and 96%
>specificity in distinguishing between bacterial and viral infections
>within 1 hour. We propose that our novel rapid BI-INDEX test will be
>useful in assisting physicians to ascertain whether antibiotic treatment
>is required, thus limiting unnecessary antimicrobial usage.
>
>The article with full bibliographic details is available online at:
>
>http://authors.elsevier.com/sd/article/S0732889313005531
>
>Sincerely,
>
>Jari Nuutila, PhD
>
>
>------------------------------
>
>Message: 3
>Date: Mon, 17 Feb 2014 01:07:30 +0000
>From: Rob Salomon <r.salomon at garvan.org.au>
>To: Ruth Nissly <rah38 at psu.edu>, "cytometry at lists.purdue.edu"
>	<cytometry at lists.purdue.edu>
>Subject: Re: [Cytometry] archiving data in a flow core?
>Message-ID:
>	<16A1D934E3390D45A7C5B7895E5ACE038300BA96 at MXDB2.ad.garvan.unsw.edu.au>
>Content-Type: text/plain; charset="utf-8"
>
>Hi Ruth 
>
>We have moved from users  being responsible for their own data to the
>core doing this for them. This is mostly because trying to manage space
>on the local drive was becoming difficult and users were having to delete
>experiments in order to be able to run new ones - we find that
>encouraging users to delete data leads to problems.
>
>So to answer your questions
>-How long are users allowed to leave data on the acquisition computer?
>They are encouraged simply to export their data to a specific local hard
>drive location and we do the rest
>
>-How are data files older than this time-period removed from the
>acquisition computer?
>Automated by Beyond Sync based on time created - deleted files will be at
>least 2 weeks old
>
>-Are old files stored as backups by the core? If so, for how long are the
>backups stored?
>Yes as compressed files at this stage indefinitely
>
>Using Beyond Sync (no affiliation) we having semi automated a lot of the
>process. 
>
>This Software is allows us to:
>1.	Push exported FCS file on a local drive to a server location
>accessible to everyone (server 1).
>2.	Backup exported FCS to an additional archival position on a different
>server with limited access (server 2)
>3.	Cross check files on the local hard drive and server 1 before
>routinely deleting FCS files from the local drive.
>4.	Cross check files on server 1 and server 2 before routinely deleted
>archived FCS files from server 1.
>5.	Backup un-exported database files from the local drive to server 2
>before deletion.
>
>This covers almost every data situation except malicious deletion
>(however we hope no one would do that anyway).
>
>If you want more info then we are happy to provide the scripts we used (
>you'd just need to point them to different backup locations)
>
>Cheers 
>
>Rob
>_______________________________________________________
>Rob Salomon
>Flow Cytometry Manager/ Senior Flow Cytometry Scientist
>Garvan Institute of Medical Research
>Sydney, Australia 
>_________________________
>http://www.flow.garvan.org.au
>Ph 9295 8432
>r.salomon at garvan.org.au
>_________________________
>
>-----Original Message-----
>From: cytometry-bounces at lists.purdue.edu
>[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Ruth Nissly
>Sent: Saturday, 15 February 2014 6:17 AM
>To: cytometry at lists.purdue.edu
>Subject: [Cytometry] archiving data in a flow core?
>
>Dear flow-ers, 
>
>If you are in a flow cytometry core facility, I have a question: What are
>the policies held by your core regarding data storage & archiving?
>Specifically: 
>
>-How long are users allowed to leave data on the acquisition computer?
>-How are data files older than this time-period removed from the
>acquisition computer?
>-Are old files stored as backups by the core? If so, for how long are the
>backups stored? 
>
>We're getting to the point where it's taking a lot of time and money to
>archive data that very few people actually ever request, so your
>advice/experience would be helpful in deciding how to move forward. For
>reference, we have files collected using CXP, Diva, Spigot, and Summit --
>and we will soon also be getting imaging flow cytometry files.
>
>Thank you, 
>
>Ruth Nissly 
>
>research technologist
>Microscopy & Cytometry Facility
>The Huck Institutes of the Life Sciences Pennsylvania State University
>
>W-124A Millennium Science Complex
>University Park, PA 16802
>
>1-814-863-2762
>rah38 at psu.edu 
>
>
>
>
>------------------------------
>
>Message: 4
>Date: Mon, 17 Feb 2014 13:10:09 +0100
>From: Jose Benito <jbenito1 at hotmail.com>
>To: "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
>Subject: [Cytometry] criopreservation
>Message-ID: <DUB118-W4300842E1A6AFFF5258A788F990 at phx.gbl>
>Content-Type: text/plain; charset="iso-8859-1"
>
>Dear all, this is not a question about cytometry but I am sure many of
>you deal with this issue. -I am moving in the near future to a different
>lab and need to start from the scratch with all the necessary equipment
>for a cell culture lab. Among the equipment of course a criopreservation
>system is one of the key elements of the lab. So, I am thinking between
>the classical liquid nitrogen equipment or an ultra-low freezer (-150?C).
>I wonder if any of you have any experience with one of this ultra-low
>freezer (liquid nitrogen free) in terms of stability and durability of
>sample preservation. Of course the advantage over the LN2 is the
>independence of a constant supply of LN2 and the much lower maintenance
>costs (I assume!).
>
> 
>
>Thank you very much
>
>Jose M Benito
>
>Hospital Carlos III
>
>Madrid
>
>Spain
> 		 	   		  
>
>------------------------------
>
>Message: 5
>Date: Mon, 17 Feb 2014 10:55:51 +0000
>From: Roy Edward <roy at biostatus.com>
>To: PURDUE CYTOMETRY LIST <cytometry at lists.purdue.edu>
>Subject: Re: [Cytometry] whole blood **tech support response**
>Message-ID: <CF2787B8.198EF%roy at biostatus.com>
>Content-Type: text/plain; charset="iso-8859-1"
>
>Hello Barbara,
>A couple of other alternatives might be ..
>
>Lyse, no wash:
>Bj?rnsson, Sven, et al. "Total nucleated cell differential for blood and
>bone marrow using a single tube in a five-color flow cytometer." Cytometry
>Part B: Clinical Cytometry 74.2 (2008): 91-103.
>
>
>This approach uses the hypotonic formic acid protocol for a lyse, no-wash
>procedure.  The key step is the addition of the lysis solution, 5 second
>vortex and then immediate addition of the neutralising buffer (author?s
>pers. comm.).  Nucleated cells are gated on the presence of the far-red
>cell-permeant DNA probe, DRAQ5.  Additionally, back-gating on DNA content
>for each phenotypically restricted population will then provide info on
>cell cycle, aneuploidy, s-phase fraction, ..
>
>No lyse, no wash:
>Allan, Robert W., M. A. Ansari-Lari, and Sandra Jordan. "DRAQ5-based,
>no-lyse, no-wash bone marrow aspirate evaluation by flow cytometry."
>American journal of clinical pathology 129.5 (2008): 706-713.
>
>
>This is an analysis of the merits and demerits of RBC lysis for flow
>cytometry and compared to the classical microscopic method. To avoid any
>of the risks associated with lytic reagent (most importantly lysis of late
>erythroblasts and non-specific losses from wash steps), whole BM (and
>equally peripheral blood, obviously) is diluted and stained with
>antibodies and DRAQ5. Cells are gated on DNA content (also permitting
>doublet exclusion).  Scatter properties are retained.
>
>In either case, gating on a far-red DNA dye for nucleated cells is highly
>sympathetic to the use of the new Brilliant Violet conjugates.
>
>Kind regards,
>Roy
>
>Roy Edward
>Email: roy at biostatus.com
>
>BioStatus Limited
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>
>
>On 13/02/2014 14:10, "Harvey, Shane" <HarveyS at Princeton.Huntingdon.com>
>wrote:
>
>>Barbara,
>>I have found that in the case of the lyse no wash protocol not working
>>that going back the lysing with ACK Lysis buffer does the trick.
>>
>>Stain blood with your antibodies of choice for 15-30min
>>Add 10:1 ACK/Blood volume of Lysis buffer
>>Lyse for 8-12 min, then spin down for 5min at 400g
>>Decant, Add your favorite buffer/1xPBS
>>Spin 5min at 400g, decant.
>>Add your Buffer/1xPBS Again, Spin for 5 min at 400g
>>Decant, Fix, and you are good to go.
>> 
>>
>>Shane Harvey
>>Scientist
>>
>>Huntingdon Life Sciences Inc
>>100 Mettlers Road
>>Somerset NJ 08873, USA
>>
>>HarveyS at Princeton.Huntingdon.com
>><mailto:HarveyS at Princeton.Huntingdon.com>
>> 
>>
>>Dear all,
>>
>>I would like to know whether someone can suggest how to perform
>>immunofluorescence staining on whole blood (CD3, CD56, CD16, CD19,
>>CD14). I've tried to lyse red cells with BD FACS lysis solution but it
>>seems it doesn't work very well. Can anyone suggest a protocol to
>>efficiently lyse red cells?
>>
>>Thanks a lot,
>>Barbara
>>Dr. Barbara Morandi
>>Immunology Unit
>>Istituto Nazionale per la Ricerca sul Cancro
>>CBA- L.go R.Benzi, 10
>>16132 - Genova, Italy
>>Tel :39 (010) 5558-221
>>Fax: 39 (010) 354282
>>E-mail: morandibarb at yahoo.it
>>
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>------------------------------
>
>Message: 6
>Date: Mon, 17 Feb 2014 15:40:16 +0000
>From: "Barsky, Lora" <lbarsky at med.usc.edu>
>To: Ina Laura Pieper <inalaurapieper at gmail.com>
>Cc: Purdue list <cytometry at lists.purdue.edu>
>Subject: Re: [Cytometry] Antibody titration & staining index
>Message-ID: <5E5E084C-CCCF-49A5-ACF6-92F5A271046A at med.usc.edu>
>Content-Type: text/plain; charset="us-ascii"
>
>I've always thought that homogenous populations like monocytes could be
>properly titrated by preparing two groups of tubes, one group stained
>with a serial dilution of CD14 (8 tubes) and another group stained with a
>serial dilution of isotope control (8 tubes).   It is critical that the
>two antibodies, CD14 and isotope control, be matched isotype class and
>have an identical beginning concentration.  The tubes are stained and
>washed separately.
>
>After processing, the matched pairs are combined (16 tubes reduce to 8
>tubes) and acquired.  Also acquire an unstained sample.  The unstained
>sample should be processed as the others were, except no antibody is
>added.  This will introduce the same artifact to the negative which may
>now be present in the antibody labeled tubes.  Artifact may come from
>processing reagents.
>
>Now your data set contains positive and negative populations and a stain
>index or  alternatively a signal to noise ratio be calculated. The
>negatives now show at what concentration the cells bind to excess
>antibody concentration while the positives will show decreasing intensity
>of label.   The point at which the negatives and positives show the
>largest separation is chosen as correct titer.
>
>Remember not to use lymphocytes as your internal negative.  They and
>monocytes have differing autofluorescence intensities, therefore become
>inadequate pairs for determining stain index or signal to noise ratio.
>Also, monocytes have been shown to bind with higher background fluors
>containing Cy- fluors, but I figure you know this already.  Choosing to
>correct this issue by finding staining tigers.
>
>Best wishes, 
>Lora Barsky
>Director, USC flow cytometry core facility
>University of Southern California
>
>Sent from my iPhone
>
>Sent from my iPhone
>
>> On Feb 17, 2014, at 6:41 AM, "Ina Laura Pieper"
>><inalaurapieper at gmail.com> wrote:
>> 
>> Hello flowers,
>> 
>> What's the objective way of deciding on the optimal antibody
>>concentration
>> during antibody titrations? I've searched and found this description on
>>the
>> UC Flow blog of plotting the stain index against antibody concentration,
>> http://ucflow.blogspot.co.uk/2009/06/antibody-titrations.html
>> However, to calculate stain index it seems I need a positive and
>>negative
>> population in my sample, probably of the same autofluorescence, which
>>isn't
>> always achievable.
>> 
>> What do I do if I want to calculate the optimal concentration in a
>> titration of a CD14-antibody in whole blood? I've done overlay
>>histograms
>> of stained and unstained samples gated on monocytes (positive) and
>> granulocytes (negative), so that i can visually identify the
>>concentration
>> at which the granulocytes are no longer staining unspecifically. Is this
>> the right way to do it? Seems very subjective to me. From the attached
>> figure I think a 1:5 dilution is the best (no unspecific binding on
>> granulocytes whilst still a clear monocyte signal).
>> 
>> What should I do when staining a homogenous sample, and there is no
>> negative population?
>> 
>> Best regards,
>> Ina Laura
>> _______________________________________________
>> Cytometry mailing list
>> Cytometry at lists.purdue.edu
>> https://lists.purdue.edu/mailman/listinfo/cytometry
>> Search the list archive at  http://tinyurl.com/cytometry
>
>
>
>------------------------------
>
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>End of Cytometry Digest, Vol 64, Issue 19
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