differences in blood collection

Keith Bahjat me at keithbahjat.com
Tue Mar 12 00:20:09 EST 2002


Miguel,

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.

Best of luck

Kb

--
Science is built with facts as a house is with stones--but a collection of
facts is no more a science than a heap of stones is a house. -Jules Henry
Poincare (1854-1912)

Keith Bahjat, PhD
Applications Engineering Manager
Cytomation, Inc
Fort Collins, CO
(970) 226-2200 x223
keithb at cytomation.com

on 3/11/02 10:04 AM, Marty Bigos at mbigos at gladstone.ucsf.edu wrote:

>
> Information from Jeff Harris related to this topic. Please contact
> him if you need further information.
>
> Marty
>
>
>
>> X-Webmail-User: jharris at itsa.ucsf.edu
>>
>> I don't know the references off the top of my head, but it is definitely
>> described that 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.
>>
>>
>>
>> On Sat, 9 Mar 2002 19:55:35 -0800 Marty Bigos wrote:
>>
>>>  Have you ever looked at this or anything similar?  Marty
>>>
>>>> Date: Thu, 07 Mar 2002 17:03:50 -0400 (EDT)
>>>> From: MIGUEL-ANGEL PERALES <mperales at OPAL.TUFTS.EDU>
>>>> Subject: differences in blood collection
>>>> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>>>> X-PMFLAGS: 34078848 0 1 18543.cnm
>>>>
>>>>
>>>> Not exactly a flow question, but somewhat relevant: We recently did a
>>>> study looking at T cell responses to a vaccine by Tetramer. Some
>>>> patients had green tops collected and others underwent leukapheresis.  It
>>>> seemed that T cell responses were higher in green top samples.  We didn't
>>>> compare the 2 methods in the same patient however.  Has anyone had a
>>>> similar experience or tested this?  Also, has anyone compared T cell
>>>> reponses in samples collected in green top vs, CPT tubes.
>>>>
>>>> Thanks,
>>>>
>>>> Miguel Perales
>>>
>>>
>>
>>
>>
>> Jeffrey M. Harris, M.D., Ph.D.
>> Asst. Adj. Professor, Pediatrics
>> University of California, San Francisco
>> Visiting Investigator
>> Gladstone Institute of Virology and Immunology
>
> --
> Marty Bigos
> Director, Flow Core
> Gladstone Institute of Virology and Immunology
> Building 3 SFGH Rm 509
> 415-695-3832
>

--
Science is built with facts as a house is with stones--but a collection of
facts is no more a science than a heap of stones is a house. -Jules Henry
Poincare (1854-1912)

Keith Bahjat, PhD
Applications Engineering Manager
Cytomation, Inc
Fort Collins, CO
(970) 226-2200 x223
keithb at cytomation.com







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