In the early 80's there was a letter in one of the journals describing a reverse in the CD4/CD8 ratio in blood that was stored at 4C. It turns out that the methodology at that time was to separate the PBMC on a density gradient; this was the source of the artifact. Apparently the density of the CD4 T cells changes with temperature. Since the advent of whole blood lysing methods, this problem with cold blood has gone away. We have also looked at different anticoagulants and temperatures and it depends on the combination of anticoagulant and temperature which gives results the same as fresh blood. Generally, we got similar results with cold and RT blood with most anticoagulants. However, to have a better handle on this matter, it is necessary to do a larger study than we did. -----Original Message----- From: Jose A. Stoute [mailto:stoutej@net2000ke.com] Sent: Friday, February 11, 2000 4:41 AM To: cyto-inbox Subject: Whole Blood Preservation Dear All, the topic of which is the best anticoagulant to collect/preserve whole blood for later analysis is of particular interest to my group. I tried to elicit a discussion on this a few months back but did not receive many responses. Therefore, I wish to add my two cents on the matter after the question by Lee Anne and Peter Chapple's comments. I am interested in measuring leukocyte surface markers in patients with and without disease. In real life we are looking at processing samples from 24 to 48 hrs after collection. We have compared sodium heparin at room temperature and 4 oC, as well Alsever's and citrate phosphate dextrose. The results so far suggest that, although no method is perfect, the samples are more stable for surface markers when kept at 4oC in heparin. I am aware of the fact that for PBMC isolation the standard recommendation is to keep the sample at room temperature in heparin but I do not know if this applies for the purpose of studying surface markers. I should also say that there are several articles published in Cytometry on this subject by McCarthy et al. (Cytometry 17:39-49,1994, J Immunol Methods 163:155-160,1993). These investigators recommend the use LDS 751 vs SSC to identify populations and argue for the use of PMSF as anticoagulant without fixation or lysis. In a separate message, Ph. Bourin stated that BioErgo had a product being sold for this purpose. I would like to know what data, if any, there are on this product to document its suitability for this application. I also would like to know other workers' opinion on the above. Regards, Jose A. Stoute "Chapple, Peter" wrote: > Lee Anne, > > Just a few thoughts on this topic - which are mostly anecdotal but probably > reliable :-) > > EDTA is not an ideal (read "suitable") anticoagulant for storage of samples, > particularly if you are doing a whole blood lysis technique - can you ask > for > Heparin instead (or as well if you want the EDTA for cell counts)? > > Even in Heparin the max storage should be 48 hours - the Friday to Monday > interval is pushing things a little. > > Whenever storing samples we do so at room temperature, in their native > plasma (in the original collection container), they seem to like this the > best > (for whole blood lysis preparation up to 48 hours); it is sterile and well > buffered. > > I'm interested in the thoughts of the rest of the group ... > > Peter Chapple > Melbourne AUSTRALIA > > >I have a basic question. I am receiving Rat and Dog blood in 3 ml > vacutainer > >tubes with EDTA. We are looking at basic cell surface markers. If I > >receive blood on a Friday, how can I preserve the blood to analyze on > >Monday? Can I leave it in the tube it was drawn in? Should I take it out > >of the vacutainer and preserve it another way? Should I stain and fix it > >for later analysis? And would preservation be best at room temp or 4*C? I > >have read some literature on this subject but have not found my exact > >situation, or what I think is my situation. > > > >If any one would know, you all would. Thanks in advance. Any suggestions > >would be helpful. > > >Respectfully, > > >Lee Anne Talbot > >Scientist > >Boehringer Ingelheim Pharmaceuticals > >Ridgefield, CT > >USA > >203-798-4133 -- Jose A. Stoute, MD Unit 64109, Box 401 USAMRU-Kenya APO AE 09831-4109 e-mail: stoutej@net2000ke.com Nairobi Tel 254-2-729303, Fax 254-2-714592 Kisumu Tel 254-35-22942, Fax 254-35-22903 Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:55:31 EST