Scientific Highlight
April 2014
Research Unit Microbe-Plant Interactions (AMP)
PSP-Element:
G-504600-001
Person to contact for further enquiries:
Dr. Martin Rieger, [email protected], Tel. 2772
Dr. Michael Schmid, [email protected], Tel. 3415
Prof. Dr. Anton Hartmann, [email protected], Tel. 4109
Title of the Highlight:
Total internal reflection (TIRF)-based quantification of procalcitonin for sepsis
diagnosis - A point-of-care testing application
Keywords:
Point-of-care-testing, Procalcitonin, Sepsis, TIRF
Central statement of the Highlight in one sentence:
This is the first point-of-care-testing assay for quantification of procalcitonin in
human whole blood and plasma for sepsis diagnosis.
Text of the Highlight:
A new, highly sensitive fluorescence immunoassay for a TIRF (total internal
reflection)-based point-of-care testing (POCT) device was developed for the
detection of procalcitonin (PCT), a specific and early marker for sepsis and
microbial infections. The immunoassay was performed on a bench-top system
that fulfilled all necessary characteristics of a POCT application, including a short
measurement time (< 9 minutes), no sample pretreatment requirements and
application directly near patients. New rat monoclonal antibodies targeting PCT
were screened and characterized. The best combinations of antibodies were then
integrated into single-use cartridges, and the reduction of nonspecific binding
was achieved by supplying suitable additives. Moreover, human recombinant PCT
(hrPCT) for use as a standard was developed in the native form of hPCT in
plasma (PCT1-116, PCT3-116). The assay achieves the required sensitivity range
in human plasma to allow reliable differentiation between healthy persons and
varying stages of infection severity (LOD = 0.04 ng/mL; LOQ = 0.12 ng/mL).
Furthermore, the developed PCT assay can be applied in whole human blood with
an adequate sensitivity (LOD = 0.02 ng/mL; LOQ = 0.09 ng/mL). To our
knowledge, this is the first diagnostic test for sepsis to use whole blood, which is
a crucial requirement for POCT. We were able to detect native PCT in patient
samples and showed a good correlation (R2 = 0.988) with the results of the
Kryptor® device from BRAHMS, a state of the art device for the detection of PCT.
1
Publication:
Rascher D, Geerlof A, Kremmer E, Krämer P, Schmid M, Hartmann A, Rieger
M (2014) Total internal reflection (TIRF)-based quantification of procalcitonin - A
point-of-care testing application. Biosensors and Bioelectronics.
doi:10.1016/j.bios.2014.03.052
Taking account of the HMGU mission:
Sepsis is with 155,000 cases of infections per year in Germany and a mortality
rate of over 55% for severe sepsis a significant threat for human health. We
report here for the first time a full point-of-care-testing application for the
quantification of procalcitonin in human whole blood, which allows faster
diagnosis of sepsis as well as antibiotic stewardship, a step towards personalized
medicine.
The internal HMGU co-operation partners with whom the highlight was
compiled, if appropriate:
-
2
Total internal reflection (TIRF)-based quantification of
procalcitonin for sepsis diagnosis – A point-of-care
testing application
Helmholtz Zentrum München
Research Unit Microbe-Plant Interactions, Dr. Martin Rieger
PSP: G-504600-001
Introduction, aims of the study
Introduction:
 Sepsis is defined as an invasion of microorganisms and/or their toxins in the
bloodstream, accompanied by the organism's reaction against this invasion.
A)
 With more than 155.000 new infections in Germany per year and a mortality from
over 50% for severe sepsis, on average 162 people die every day because of
sepsis.
 Moreover sepsis causes costs from about 1.7 billion Euro per year for the therapy
which is ~32% of the intensive care budget in Germany.
B)
 Golden hour: Initiation of effective antimicrobial therapy within the first
hour following the onset of septic shock-related hypotension was associated with
80% survival chance, which drops every hour per 9%.
 Moment of diagnosis and early medication reduce the high mortality!
 Procalcitonin (PCT) (A) is the recommended marker for sepsis diagnosis (B);
Several Immunoassays for PCT commercial available but none of these fulfill all
criteria for point-of-care-testing (POCT)
C)
Aim of the study:
 The aim of the study was therefore to develop a POCT assay for the quantification
of PCT in human whole blood based on monoclonal Antibodies (mAb) and a total
internal reflection (TIRF)-based POCT device (C)
Rascher D, Geerlof A, Kremmer E, Krämer P, Schmid M, Hartmann A, Rieger M (2014) Total
internal reflection (TIRF)-based quantification of procalcitonin - A point-of-care testing
application. Biosensors and Bioelectronics. doi:10.1016/j.bios.2014.03.052
POCT analyzer respons® IQ – measuring principle
A)

All necessary reagents for the
POCT-analyzer respons® IQ (A)
are contained in the singleready-to-use cartridge (B)

Complete Sandwich total
internal reflection fluorescence
immunoassay (C) is performed
on the chip
C)
B)
Capture-mAb
RFID chip
Sample input (50 µL)
Rascher D, Geerlof A, Kremmer E, Krämer P, Schmid M, Hartmann A, Rieger M (2014) Total
internal reflection (TIRF)-based quantification of procalcitonin - A point-of-care testing
application. Biosensors and Bioelectronics. doi:10.1016/j.bios.2014.03.052
POCT analyzer respons® IQ - Standard calibration curve
A)
B)
 Assay achieves required sensitivity of PCT in plasma (A) and whole blood (B) with a limit of
detection
(LOD) of 0.04 and 0.02 ng/mL, respectively and a limit of quantification (LOQ) of
A
0.12 and 0.09 ng/mL respectively (n= 8; m=3-5).
 First diagnostic test for PCT in human whole blood
Rascher D, Geerlof A, Kremmer E, Krämer P, Schmid M, Hartmann A, Rieger M (2014) Total
internal reflection (TIRF)-based quantification of procalcitonin - A point-of-care testing
application. Biosensors and Bioelectronics. doi:10.1016/j.bios.2014.03.052
POCT analyzer respons® IQ - Patient samples

7 deep frozen patient plasma
samples measured undiluted
and diluted (n = 13; m = 1-3)

PCT concentration determined
with state-of-the-art technology
Kryptor® from BRAHMs
 Signal of POCT assay correlates very well (R2 = 0.988) with PCT concentration
determined with state-of-the-art technology Kryptor® from BRAHMs
 Ready for clinical studies with large sample amounts
Rascher D, Geerlof A, Kremmer E, Krämer P, Schmid M, Hartmann A, Rieger M (2014) Total
internal reflection (TIRF)-based quantification of procalcitonin - A point-of-care testing
application. Biosensors and Bioelectronics. doi:10.1016/j.bios.2014.03.052