method of blood collection (Thanks and summary)

Oughton, Julie julie.oughton at orst.edu
Thu Mar 21 12:41:33 EST 2002


Thanks for posting the summary to the entire group. Even though I'm more
interested in mouse blood, the information is quite pertinent and very
interesting.

However, I would like to entertain a follow-up question.

I am involved in a project with the SMILE program (Science and Math
Investigative Learning Experiences) where we will be introducing flow
cytometry to a group of high school girls. We will examine the blood for
various leukocyte phenotypes and compare the results with blood smears and
WBC.

Typically, I collect and phenotype mouse blood all in one day. Due to the
lack of time, we will need to collect blood samples the day before the SMILE
project. Hence, we need a good anti-coagulant which will allow us to hold
samples overnight.

I've searched the email archives for the most appropriate anticoagulant for
blood storage. From what I could glean in the archives, sodium heparin may
be our best choice. Any comments or suggestions?

Thanks in advance,
Julie Oughton
Oregon State University



-----Original Message-----
From: MIGUEL-ANGEL PERALES [mailto:mperales at OPAL.TUFTS.EDU]
Sent: Tuesday, March 19, 2002 12:29 PM
To: cyto-inbox
Subject: Re: method of blood collection (Thanks and summary)





     Question: Does the method of blood collection (green tops, CPT,
     leukapheresis) influence results of T cell assays such as Elispot?

     Conclusions: ACD may be better for straight phenotype, but heparin is
     probably better for T cells used in functional assays.  Based on our
     experience, this may also be true for T cells used for Tetramer
     assays.

     Responses:
     Russ Wick:  Apheresis instruments always collects blood in ACD.
     Heparin tubes are used for various immunological assays. Examples

     include mixed lymphocyte culter and mitogen response. My suspecion is
     that ACD has some effect of lymphocyte response. Only a guess...

     Jeffrey Harris: the sodium heparin green top tubes can activate
     lymphocytes- that's why many labs use lavender/purple tops (using EDTA
     as anticoagulant) or yellow top ACD (acid citrate/dextrose - citrate
     is also an anticoagulant) tubes for culture/stimulation methods and
     even phenotyping when you are worried about a labile marker - I must
     say however, that for robust assays I don't think the heparin is a
     huge effert, because we use them for the CFCs in the core lab and our
     "non-stimulated" controls for cytokine secretion are quite negative,
     but there is some small background activation by markers like CD69;
     similarly we sort samples based on CD62L, which is also quite labile
     and sensitive to stimulation/temperature/etc. but we try to use the
     blood freshly and move quickly to separate the lymphocytes by Ficoll
     and keep them cold on ice to avoid surface marker changes - whereas
     the core and clinical labs use only EDTa-containing tubes for
     phenotyping for this reason.

     Keith Bahjat: It is true that sodium heparin vacutainers may contain
     varying amounts of endotoxin, leading to unwanted cellular activation,
     especially of APCs. I remember hearing a while back that BD was
     supposed to be working on certified endotoxin-free vacutainers, but I
     haven't seen anything yet. But in my experience, this kind of
     contamination is minimal to nonexistent. When blood is collected and
     then leukopheresed, CPDA is the anticoagulant used. CPDA, like EDTA
     and ACD, is a calcium chelator. Lymphocytes collected in
     calcium-chelating agents are extremely difficult to activate (we all
     know what role calcium mobilization plays in cellular activation, so I
     won't elaborate). Several rounds of washing with a calcium containing
     medium can restore some of this potential, but they still fall far
     short of cells which have never been exposed to calcium-chelators. I
     wouldn't recommend use of calcium-chelators for any
     activation-dependant experiments, no matter how many washing steps
     were involved.





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