Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

http://www.cyto.purdue.edu/hmarchiv/index.htm

 

Here is a summary of replies and communication. I did not reply to two communications which were commercial in nature. Each thread is separated by double line (=====). Thank you very much.
Masa

------Original Question------
Dear Ma'am and Sirs:

We are having training on multiplex (10 beads) cytokine flow analysis of Th1/Th2 shift by treatment vs placebo (human). We will measure the baseline serum cytokine profiles as well as supernatants of isolated PBL or PBMC stimulated by PHA.

Does anyone have opinions about the use of autologous serum vs. FBS for the ex vivo study? As we hypothesize the baseline serum of the treatment group to be shifted to either Th1 or Th2, the use of autologous serum seems to make more sense than making this variable constant (FBS).

Any opinions about autologous serum or multiplex beads analysis will be appreciated. I will try to summarize feedbacks, but these two may have to be separate questions.

In health,

Masa Sasagawa, ND
Naturopathic Physician - Washington
Affiliate Assistant Professor - University of Washington
School of Nursing, Dept of Biobehavioral Nursing and Health Systems
Postdoctoral Research Fellow - Bastyr University
14500 Juanita Dr NE, #472
Kenmore, WA 98028
Phone: 425-602-3178
Fax: 425-602-3079
masas@bastyr.edu

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Dear Masa,

Thank you for your thoughts and references regarding autoserum vs FBS.

I just thought about other thing based on what you said about the different time points for measuring different cytokines. Although it is easy just to have several wells for collection on different days (there should be still enough cells for that), but if you have separate samples for each cytokine this diminishes the advantages of the multiplex assay, which allows for measuring a number of cytokines simultaneously. In this case, it is as easy, but much cheaper, to use ELISA. We are using not individual plates, but ELISA kits or sets that cost us $1000 for 20-40 plates, so when we compared the cost to the multiplex flow the latter could not compete.

Regarding FBS and human cerum for cultures, if there are any doubts about FBS I think it is better to use human serum. In fact, FBS can be either the same or (non-significantly different) or worse than human serum, but it can not be better, because it is unnatural and if it yields higher cytokine or other responses this will mean that it itself stimulates.

However, talking about autologous vs pooled donor serum one should take into account the fact that patient serum may have blocking activity, i.e. suppress in vitro proliferation, such as PHA induced, and cytokine synthesis. I do not know how "sick" your patients are, i.e. what diseases you treat and investigate, but still in any patients this will be a possibility, and also suppressive activity may be different in serum samples on different stages of treatment.

So, I think a serum of choice with standard activity and standard cytokine contents would be pooled donor serum. We used to obtain and pool blood donor serum from local blood services or alternatively we purchase donor AB serum from Cambrex (imported from the USA).

Another consideration about autologous serum in culture, you may know how much baseline cytokines will be in the autologous serum added to the culture (although it is just 10%) if you test serum for the same cytokines, however if then it will be cultured for 24-72 hours you will not know how much of it will loose activity during this time, will be used up (i.e. bound by receptors and internalized) or destroyed.

Best regards and Happy New Year!

Yours sincerely,

Vladislav

-----Original Message-----
From: Masa Sasagawa <masas@bastyr.edu>
To: cyto-inbox
Sent: Mon, 19 Dec 2005 10:02:41 -0800
Subject: RE: autologous serum and multiplex cytokine beads

Dear Valdislav,

Thank you very much for thoughtful response. Two responses that I received for my inquiry were from Dr. Daniela Lens who wanted to know a collective reply of this issue, and the other was from a vender of multiplex cytokine flow. I basically had no useful info except for your response.

I agree that in vitro stimulation is far from in vivo simulation, and the two experimental designs cannot be related. We have been using FBS for in vitro/ex vivo culture in order to make the variable constant, but I wanted to tweak the assay to maximize the sensitivity. However, as you mentioned, if the assay is too sensitive to be reliable, the reproducibility goes down in the drain. Multiplex cytokine flow has amazing sensitivity as well as the range of detection (from few pg/mL to 10 ng/mL) so I thought ELISPOT might be better for high sensitivity. Variables of this type of assay include day-to-day variability, individual difference, transient vs permanent change, age, genetic, diet, stress, nutritional status, etc, etc. Another variable is time point. With a strong mitogenic stimulant, IL-10 comes out very quickly while IL-2 is optimal at 24 hr and IL-12p70 takes 3 days because some are primary as well as measuring secondary and tertiary responses. I would also consider the intracellular cytokines, but it is expensive and when sample cells are limited, it is difficult to optimize.

Following is what I found on PubMed:

Compared to autosera, FBS induces more differentiated and less stable transcriptional profiles on bone marrow mesenchymal stem cells: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16081661&query_hl=11

Autologous serum provides more accurate immunological profile of DCs: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16118326&query_hl=7

DC immunotherapy can be maintained either by autosera or FBS: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10813531&query_hl=3


Isolated lymphocytes lose responsiveness upon stimulation when precultured with FBS, but not with auto-serum: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10679815&query_hl=9


IFNg production by PBMNC stimulation was altered by the use of autologous serum or the concentration of FBS in media: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2977292&query_hl=5


Osteoblastic differentiation is more stable when using autologous serum, and for ex vivo immunotherapy, the potential introduction of bovine proteins into the patient should be avoided:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12715921&query_hl=1


I will wait few more days to compile responses. Thank you again and happy holidays.

Sincerely yours,
Masa

________________________________

From: rozenkov@netscape.net [mailto:rozenkov@netscape.net]
Sent: Monday, December 19, 2005 4:01 AM
To: cyto-inbox
Subject: Re: autologous serum and multiplex cytokine beads

Dear Masa,

I have not seen any replies to your question in the mailing List, possibly people e-mail you directly and I believe they may have more specific suggestions.

I think the main thing is that PHA will stimulate cytokine production very strongly, so in vitro supernatant levels will be very different from serum levels. Therefore, adding autoserum or FBS to culture media should not have distinguishable effect on PHA stimulated cytokine levels. (I guess that was your question, i.e. what serum is better to use in cultures.)

Some people believe that FBS may have some stimulatory effect on cell culture and cytokine production and, therefore, suggest human serum (donor) instead of FBS. Pooled human donor serum can be an alternative to FBS with constant composition of cytokines and other factors. However, again, I think that PHA will stimulate so strongly that serum influence will not be important.

One problem that we had while analysing multiple cytokines in the same assay using serum and control and stimulated culture supernatants was that the levels of different cytokines in the same sample might vary from low or undetectable to very high. So we could not use the same dilutions of standards/controls and needed to repeat the same samples with different controls suitable for different cytokines. However, this will depend on your kits, their dynamic ranges and standards.

I do not know what treatment you use and how much it can stimulate or shift cytokine production, but in our clinical trials using quite influencing cellular therapies we saw very transitory elevation of serum cytokine levels, and intracellular testing of cytokines failed to reveal any spontaneous production (without stimulation) before or after treatment. We could stimulate cytokine production (IFNg and IL-4), we did not use PHA, but PMA/Ionomycin. However, cytokine production stimulated with strong non-specific mitogens is a different test, as opposed to non-stimulated production, because it releases any possible cell capacity of cytokine production, so I think it does not really reflect the functional status of patients' cells, but shows the changes in circulating lymphocyte composition, i.e. if there are more or fewer Th1 or Th2 cells in peripheral blood at the time of sampling.

Good luck in your research.

Yours faithfully,
Vladislav

Vladislav Rozenkov, M.D., Ph.D.
Division of Medicine
University of Queensland
Australia
rozenkov@netscape.net

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Dear Jorge, sorry for delay in reply.

We just had training on FlowCytomix (manual attached). Beckman Coulter is giving us 10% discount, but it still costs >$2000 per 80 samples (8 for standards). It is very easy to use. We are going to optimize the assay by varying the range of standards so that we can maximize the sensitivity. By the way, we had to pay for partial cost for the training kit. You should negotiate to get free training on their kits--may not be the one that you want to use though.

Write me if you have any specific questions.
Best wishes and happy holidays,

Sincerely,
Masa
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Dear Masa,

I am very interested in TH1/TH2 shifts in humans in another project. Could you please tell me which bead manufacturer/distributor are you using? We do have a Calibur and a Luminex, so equipment is no problem.
Thanks for any help you could give.
Best regards,

Jorge

Jorge Neumann, MD
Lab of Transplantation Immunology
Santa Casa Hospital
Porto Alegre, Brasil
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We have been using fetal bovine serum instead of autologous serum or pooled human serum for a number of years. This is to avoid handling any potential pathogens in the sera of subjects as well as in the "screened" lots of pooled human serum. It also keeps the variable (serum) constant. We screen the fetal bovine serum for its ability to support control (apparently healthy normal individual) T cell proliferation to PHA. We pick the best lot of serum that is not toxic. In addition, in transplantation settings (islet), fetal bovine serum is also being tested instead of using human serum albumen (FDA recommended) as a protein source for culture of isolated islets prior to transplantation into Type 1 diabetes patients. Although it is generally thought that culturing in media containing fetal bovine serum may sensitize T cells to xenogeneic (bovine) antigens, there is no compelling evidence for this contention.

Sundararajan Jayaraman, Ph.D.

Associate Professor

Dept. of Surgery

University of Illinois College of Medicine

909 South Wolcott Avenue

Chicago, IL 60612

Phone: 312 355-5133 (Office); 312-413-0366 (Lab)

Fax:: 312-355-1497

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If you get any private replies, I would appreciate receiving any info on this matter as well. Thanks,

Daniela

Dra. Daniela Lens. MD, PhD

Profesor Agregado

Departamento Básico de Medicina

Hospital de Clínicas-Facultad de Medicina Avda Italia s/n CP 11600 Montevideo Uruguay Tel/FAX (5982)4800244

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