Effects of Blood Sample Handling Procedures

on Measured Cytokine and Chemokine

Concentrations in Human

Serum and Plasma

Diplomarbeit

Lucian Beer

ausgeführt an der Universitätsklinik für Chirurgie

Christian Doppler Laboratory for Cardiac and Thoracic

Diagnosis and Regeneration

unter der Anleitung von

Ass. Prof. Univ.-Doz. Dr. Hendrik Jan Ankersmit

• Cytokines are cell-signaling proteins that are secreted

by numerous cell types

• Cytokine concentration patterns are alterd in a

variety of diseases

• Cytokine measurment in body fluids is popular in

research studies as well as in clinics

• Inconsistend data are available characterizing

praeanalytic factors influencing cytokine

concentrations in human serum and plasma samples

Introduction

Background

Arch Pathol Lab Med. 2008;132:1802–1806

IL-6 concentrations at 0 h, 2 h, 4 h, 8 h storage at 24°C

Background

Arch Pathol Lab Med. 2008;132:1802–1806

IL-8 concentrations at 0 h, 2 h, 4 h, 8 h storage at 24°C

EDTA Plasma

Heparin Plasma

Serum

Technical Background

• Requirements on cytokine measurement systems

• Specificity

• Sensitivity

• Simplicity

• Relialbility

• Cost

• Time

Technical Background

®Lucian Beer

Enzyme-Linked Immunosorbent Assay (ELISA)

Technical Background

Technical Background

• Does sample handling procedures alter measurable

cytokine concentrations?

• Differences between serum and plasma tubes?

• Triggers for cytokine release in vitro?

Aims of the study

Measured Cytokines• IL-8

• GRO-α

• ENA-78

• GCP-2

• MCP-1

• IL-6

• IL-1β

• TNF-α

• TGF-β

• VEGF

• IL-1RA

Experimental design

IL-8

EDTA Plasma

Serum

Heparin Plasma

Experimental design

n = 7

PBMC experiment

adapted from: http://pluriselect.com

before centrifugation after centrifugation

Diluted Blood Sample

Synthetic Polymer

Plasma – phosphate buffer mixture

Interphase with mononuclear cells

Erythrocytes, granulocytes,

dead cells

PBMC experiment

PBMC

human Fibrin

n = 8-12

Heparin Plasma

Heparin Plasma Level

0h 4h 24h

0

1000

2000

3000

4000

5000

4°RT37° *

IL-8

pg/

ml

EDTA Plasma Level

0h 4h 24h

0

200

400

600

800

10004°RT37°

IL-8

pg/

ml

Results

Whole blood experiment

* = p<0.05; ** = p<0.01n = 7

Serum Level

0h 4h 24h

0200400600800

1000

15000

20000

25000

300004°RT37°

**

*

IL-8

pg/

ml

Results

n = 7

Results

In vitro experiments

* = p<0.05; ** = p<0.01n = 12

PMBC supplemted with: autologous plasma; FCS = fetal calv serum, HI-Serum = heat inactivated serum;

autologus serum , Serum and Plasma without PBMC

Mediu

m+

20%

Plas

ma

+ 20

% F

CS

+ 20

% H

I-Ser

um+

20%

Ser

umSer

umPlas

ma

0

5000

10000

15000**

**

IL-8

pg/

ml

Results

In vitro experiments

Medium

+ 2.5% Serum

+ 5% Serum

+ 10% Serum

+ 20% Serum

0

2500

5000

7500

10000

12500

15000

*

*

**

IL-8

(pg

/ml)

n = 8* = p<0.05; ** = p<0.01

PBMC supplemented with increasing dosis of autologus serum

Fibrin(ogen)

Jennewein et al. Molecular Medicine, 2011

Fibrinogen:

•Soluble Dimer: 340 kDa

•α chain: 63 kDa

•β chain: 56 kDa

•γ chain: 47 kDa

Results

In vitro experiments

n = 8* = p<0.05; ** = p<0.01

PBMC supplemented with increasing dosis of fibrin

Medium

+ 0.25 µg Fibrin

+ 0.5 µg Fibrin

+ 10 µg Fibrin

+ 50 µg Fibrin

+ 250 µg Fibrin

+ 500 µg Fibrin

0

5000

10000

15000

20000

25000

**

**

**

**IL

-8 (

pg/m

l)

Conclusion

• Immediate processing of plasma and serum is

essential

• Non-centrifuged samples should be stored at 4°C

• EDTA plasma tubes seem to be most usable for

stability reasons

• Comparison between serum and plasma

concentrations has to be interpreted critically

• Fibrin stimulates cytokine secretion of PBMC

Special thanks

Medical University Vienna

Christian Dippler Laboratory

for Cardiac and Thoracic Diagnosis and Regeneration

Hendrik Jan Ankersmit

Michael Lichtenauer

Andreas Mitterbauer

Stefanie Nickl

Matthias Zimmerman

Department of Dermatology

Michael Mildner