[Adaptation of the TriTEST TM for the investigation of blood samples for a multicenter study taken to a single laboratory].
BACKGROUND
Immunotoxicological studies in humans are usually carried out via the determination of some selected immune parameters in subjects occupationally and/or environmentally exposed to immunotoxic substance. One of the most often measured parameters is the determination of lymphocyte subsets, which needs to be carried out in a very short time (a few hours) after blood collection. This is the major problem limiting the determination of lymphocyte subpopulations in field studies, where samples are usually collected directly at the workplace, and very often at the end of the workshift. Unfortunately, these collection modalities significantly prolong the time between collection and analysis. The problem is more evident in multicentric studies, where a further problem is represented by the time needed to send samples to the laboratory.


OBJECTIVE
Since an immune evaluation was planned, including the determination of lymphocyte subpopulations CD4 (T-helper), CD8 (T-suppressor cytotoxic) and CD16/CD56 (natural killer) in the project "Assessing health effects in man from exposure to low doses of inorganic mercury in environmental and occupational settings", a method was developed for performing cytofluorimetric analysis in "field studies".


METHODS
The method is based on commercially-available kits, and involves in loco treatment. Whole blood is labeled with monoclonal antibodies, and fixed samples immediately after collection. After the treatment, the samples are ready for flow cytometric analysis, which may be performed after a two-day period from sample collection.


CONCLUSIONS
The method described is adequate for immunotoxicity testing in field studies because it prolongs the maximum latency time from collection and cytofluorimetric analysis up to 48 hours. A second interesting characteristic of the method is the possibility of using whole blood, without any need of either complex manipulations or particular equipment.
Mammone
T
T
International Centre for Pesticide Safety, Busto Garolfo, Milano. teresa.mammone@icps.it
Birindelli
S
S
Guarneri
D
D
Messa
A
A
Schiavulli
N
N
Carrus
A
A
Colombi
A
A
Soleo
L
L
Colosio
C
C
Maroni
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D001793:Blood Preservation; D001800:Blood Specimen Collection / Q000379:methods*; D002338:Carotenoids / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D007753:Laboratories; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets; D015337:Multicenter Studies as Topic / Q000379:methods; D010799:Phycoerythrin / Q000032:analysis; D015800:Protozoan Proteins / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D014186:Transportation
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D015800:Protozoan Proteins; D011933:Reagent Kits, Diagnostic; C073227:peridinin chlorophyll-a protein, Dinophyceae; D010799:Phycoerythrin; D002338:Carotenoids; D016650:Fluorescein-5-isothiocyanate