Kirsten Kortekaas, Leiden University Medical Center
19 November 2009
De Jager et al.1 stress the need for rigorous quality control when measuring cytokines in plasma or serum in the context of clinical trials. The authors rightly state that proper timing of sampling, careful sample handling and storage, and strict quality control is crucial for reliable cytokine measurements. However, they did not point towards potential pitfalls of the early steps of blood collection and handling which may profoundly affect the results of cytokine measurements. A first problem may occur when a blood sample is collected on ice. The temperature of ice cubes can be far below the freezing point. As such, contact between the blood collection tube and the ice cubes may result in local cell lysis. This problem is avoided by placing the tubes in ice water. A second problem is the use of serum. Serum preparation depends on the induction of the coagulation cascade and thus results in thrombocyte activation and release of inflammatory mediators. Serum cytokine levels may therefore merely reflect thrombocyte count. Thirdly, separation of cellular constituents from whole blood, especially the leukocyte/platelet-enriched fraction, is important. De Jager et al.1 and Duvigneau et al.2 already showed that a delay of sample processing will lead to different cytokine profiles by either degradation, absorption, or cellular production of cytokines. An important ignored factor is the incomplete removal of cellular constituents during plasma preparation. This may lead to artificially high plasma cytokine levels due to cell fragmentation during the freeze-thawing cycle. For example, we think that this effect is a systematic problem when interpreting plasma cytokine data from studies in obese or smoking individuals. Both conditions are associated with higher leukocyte3 and thrombocyte counts which may result in elevated residual cells. This problem is avoided by using a second centrifugation or filtration step of the plasma prior to storage. Fourth, plasma must be properly separated from the pellet as soon as possible after centrifugation. Special care should be taken to not disturb the cellular pellet when removing the plasma by pipetting. Decanting the tube should be avoided. We agree with the Jager et al.1 that careful sample handling is a prerequisite for reliable cytokine measurements. The early steps in sample collections are a crucial and often ignored factor of potential bias.
K.A. Kortekaas, MSc J.H.N. Lindeman, MD PhD Leiden University Medical Center PO box 9600; 2300 RC Leiden, the Netherlands j.h.n.lindeman@lumc.nl
Reference List 1. de JW, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunol 2009; 10:52. 2. Duvigneau JC, Hartl RT, Teinfalt M, Gemeiner M. Delay in processing porcine whole blood affects cytokine expression. J Immunol Methods 2003; 272:11-21. 3. Huang ZS, Chien KL, Yang CY, Tsai KS, Wang CH. Peripheral differential leukocyte counts in humans vary with hyperlipidemia, smoking, and body mass index. Lipids 2001; 36:237-245.
Prerequisites for cytokine measurements
19 November 2009
De Jager et al.1 stress the need for rigorous quality control when measuring cytokines in plasma or serum in the context of clinical trials. The authors rightly state that proper timing of sampling, careful sample handling and storage, and strict quality control is crucial for reliable cytokine measurements. However, they did not point towards potential pitfalls of the early steps of blood collection and handling which may profoundly affect the results of cytokine measurements.
A first problem may occur when a blood sample is collected on ice. The temperature of ice cubes can be far below the freezing point. As such, contact between the blood collection tube and the ice cubes may result in local cell lysis. This problem is avoided by placing the tubes in ice water. A second problem is the use of serum. Serum preparation depends on the induction of the coagulation cascade and thus results in thrombocyte activation and release of inflammatory mediators. Serum cytokine levels may therefore merely reflect thrombocyte count. Thirdly, separation of cellular constituents from whole blood, especially the leukocyte/platelet-enriched fraction, is important. De Jager et al.1 and Duvigneau et al.2 already showed that a delay of sample processing will lead to different cytokine profiles by either degradation, absorption, or cellular production of cytokines. An important ignored factor is the incomplete removal of cellular constituents during plasma preparation. This may lead to artificially high plasma cytokine levels due to cell fragmentation during the freeze-thawing cycle. For example, we think that this effect is a systematic problem when interpreting plasma cytokine data from studies in obese or smoking individuals. Both conditions are associated with higher leukocyte3 and thrombocyte counts which may result in elevated residual cells. This problem is avoided by using a second centrifugation or filtration step of the plasma prior to storage. Fourth, plasma must be properly separated from the pellet as soon as possible after centrifugation. Special care should be taken to not disturb the cellular pellet when removing the plasma by pipetting. Decanting the tube should be avoided.
We agree with the Jager et al.1 that careful sample handling is a prerequisite for reliable cytokine measurements. The early steps in sample collections are a crucial and often ignored factor of potential bias.
K.A. Kortekaas, MSc
J.H.N. Lindeman, MD PhD
Leiden University Medical Center
PO box 9600; 2300 RC Leiden, the Netherlands
j.h.n.lindeman@lumc.nl
Reference List
1. de JW, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunol 2009; 10:52.
2. Duvigneau JC, Hartl RT, Teinfalt M, Gemeiner M. Delay in processing porcine whole blood affects cytokine expression. J Immunol Methods 2003; 272:11-21.
3. Huang ZS, Chien KL, Yang CY, Tsai KS, Wang CH. Peripheral differential leukocyte counts in humans vary with hyperlipidemia, smoking, and body mass index. Lipids 2001; 36:237-245.
Competing interests
None