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@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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