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Clinical and Diagnostic Laboratory Immunology, March 2002, p. 295-298, Vol. 9, No. 2
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.2.295-298.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay

Christian Schultz,* Christina Rott, Petra Temming, Julia von Puttkammer, and Peter Bucsky0

Department of Pediatrics, Medical University of Lübeck, Lübeck, Germany

Received 27 March 2001/ Returned for modification 20 June 2001/ Accepted 6 November 2001

Intracytoplasmic detection of cytokines by flow cytometry has become a powerful tool in the characterization of cytokine-producing cells. However, it is not known to what extent specimen age and the use of various negative controls may influence the amount of cytokine-positive cells. We therefore compared different times of storage and the use of several negative controls in the determination of intracytoplasmic levels of cytokines. There was a substantial decline of interleukin-2- and gamma interferon-positive lymphocytes after 20 h and especially after 48 h of storage. The precision of intracytoplasmic interleukin-6 determination decreases after long-term storage compared to 2 h of storage, whereas the amount of interleukin-8-positive monocytes remained rather stable. Therefore, we recommend performing the analysis as fast as possible after the blood sample is drawn. Under consideration of isotype-matched antibodies and nonstimulated cells as negative controls instead of the purified antibody-blocking control, strikingly higher amounts of interleukin-2-, gamma interferon, interleukin-6-, and interleukin-8-positive cells were found. For a meaningful interpretation of data these differences have to be kept in mind. Further studies should evaluate the exact specificity of these controls.

* Corresponding author. Mailing address: Department of Pediatrics, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Phone: 49-451-500 2567. Fax: 49-451-500 3767. E-mail: ChrSchultz{at}aol.com.

Clinical and Diagnostic Laboratory Immunology, March 2002, p. 295-298, Vol. 9, No. 2
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.2.295-298.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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