human CBA flex set

Hello flowers

We want to measure cytokines in plasma/serum from patients using the CBA flex set. Currently, we tried the following cytokines IL-3, IL-6, IL-10, IL-β and TNF-α but we noticed some problems. For each cytokine and every sample we got a 0.00 value except for IL-6 but these values were very low (mean 1.53 pg/ml range 0.00-6.12). The standards were very nice and we even made two extra dilutions to increase sensitivity. We believe it is strange that everything is 0.00, we should at least be able to measure something even if it is very low. 

For sample collection we used BD: serum tubes with red cap and	plasma: Li-heparin tubes with gel (light green cap). 

With TNFalpha we tested several things on fresh samples: 
1. serum tubes or plasma tubes gave the same results (null values) 
2. when we leave samples for 2 days on RT before measurement: null values 
3. we performed a TNF-α Elisa on the same samples: same results as CBA (null values) 
4. we tried samples with CRP>10 (meaning patients had infections): all null values 
5. we used TNF- α protein we had in the –80°C freezer to spike in PBS and we got nice values! 
6. In the past we used already the CBA TH1/Th2 kit and we got nice results, although the values were very low. At that time we used other blood tubes (Serum tubes Terumo). 

We thought the blood tubes were responsible for our problem. Therefore we tested TNF alpha and IL1 on other blood tubes: BD EDTA tubes and BD p100 tubes. We measured on fresh material (immediately after sampling) and we left tubes without centrifugation on RT for 2 days. We could only measure very small amounts in the p100 tubes left 2 days at RT (EDTA was 0.00, the fresh material for both tubes was 0.00). We also tried polystyrene and polypropylene tubes for incubation, without any difference. Again, the standards are always very nice and Elisa gives comparable results.
So, are these results normal or do we have a problem here? Is there anybody who used the human CBA flex set that had comparable results?  Are there actually reference values available?

Thank you very much !!!

Best regards, Karen 


Karen Hensen, PhD 
Laboratory of Experimental Hematology 
Virga Jesse Hospital 
Stadsomvaart 11 
3500 Hasselt 
Belgium 
Phone +32-11-309700 
Fax +32-11-309750