New Applications of the Human Whole Blood Pyrogen Assay

New Applications of the Human Whole Blood Pyrogen Assay

______________________________
~I~-~,c'
New Applications of the Human Whole Blood
Pyrogen Assay (PyroCheck)
Stefan Fennrich, Albrecht Wendel, and Thomas Hartung
University of D-Konstanz
Summary
The absence ofpyrogens in injectable drugs is an indispensable safety control because contaminants causing fever pose a
life-threatening risk to the patient resulting in the worst case
in death by shock. When fever- inducing agents, i.e.pyrogens,
come into contact with the immunocompetent
cells in blood,
these cells release mediators which transmit the fever signal to
the thermoregulatory
centre of the brain. The Phamocopoeia
lists currently two test systems for pyrogenicity:
1. The in vivo rabbit pyrogen test which measures the fever
reaction following injection of the sample to the animals.
2. The in vitro Limulus Amebocyte Lysate assay (IAL) which
measures the coagulation in a lysate prepared from the blood
of the horseshoe crab specifically initiated by endotoxins, i.e.
cell wall components from Gram-negative bacteria.
The new test presented here (PyroCheck) exploits the reaction
of monocyteslmacrophages
for the detection of pyrogens:
human whole blood takenfrom healthy volunteers is incubated
in the presence of the test sample in any form, be it a solution,
a powder or even solid material. Pyrogenic contaminations
initiate the release of the "endogenous pyrogen" 1nterleukin1fJ determined by ELlSA after a fixed incubation time.
The technology presently listed in the Pharmacopoeia
is
limited to parenteralia (rabbit test: biologicals and pharmaceuticals, LAL: predominantly pharmaceuticals).
In the EU
Medical Devices Directivefrom
1995 the rabbit pyrogen test
for medical products is in some cases requested. (in some
cases IAL of an eluate from the device). However, pyrogentesting needs to cover also innovative high-tech products such
as medical devices (implants, medical plastic materials,
dialysis machines), cellular therapies and species-specific
agents (e.g. recombinant proteins). Here we report that the
human blood test PyroCheck is suitable for testing filters in
air quality control as well as for assessing medical devices
and biocompatibility
(dialysate fluid}, i.e. that it can be
extended to a wide spectrum of applications.
Keywords: 3R, replace, pyrogen test, Limulus Amebocyte
endotoxin, pyrogens, medical devices
146
ZusammenJassung:
Weitere Anwendungsm6glichkeiten
des
Pyrogentests mit menschlichem Vollblut (PyroCheck)
Die Abwesenheit von Pyrogenen in injizierbaren Armeimitteln
(Parenteralia) ist unabdingbar fUr die Patientensicherheit,
denn diese [ieberinduzierenden
Verunreinigungen stellen fiir
den Menschen ein erhebliches Risiko dar. 1m Extremfall
bedeuten sie den Tod durch Schock. Im Kontakt mit immunkompetenten Zellen bewirken Pyrogene die Ausschiittung von
BotenstofJen, die direkt auf das zentrale Thermoregulationszentrunt im Gehirn einwirken. Die Pharmakopoe schreibt zwei
Testsysteme zur Pyrogendetektion
vor:
1. Den in vivo Kaninchen-Pyrogen-Test,
der nach Injektion der
Prufsubstan: die Erhohung der Korpertemperatur ermittelt.
2. Den in vitro Limulus Amobozyten Lysat-Test (IAL), der die
Koagulation eines aus dem Blut des Pfeilschwanrkrebses
gewonnenen Lysates mifit, ausgelost durch Endotoxin, eines
Zellwandbestandteiles
Gram-negativer Bakterien.
Der hier vorgestellte neue Test (PyroCheck) nutzt zur Detektion die Reaktion von mensch lichen MonozyteniMakrophagen
auf Pyrogene: Blut gesunder freiwilliger Spender wird mit der
Probe, unabhangig ob es eine Losung, ein Pulver oder
irgendein solides Material ist, zusammen inkubiert. Anwesende
Pyrogene induzieren die Ausschuitung von "endogenen
Pyrogenen" wie Interleukin-1fJ, welches mit dem ELlSA
bestimmt wird.
Die gegenwiirtig in der Pharmakopoe vorgeschriebene
Pyrogentestmethodik
ist begrenri auf den Nachweis von
Parenteralia (Kaninchen- Test: biologische und pharmazeutische Arzneimittel; LAL: hauptsdchlicb pharmazeutische
Arzneimittel). Die Priifung von medizinischen Materialien ist
im EU Gesetz fiir Medizinprodukte
seit 1995 geregelt und
schreibt auch z. T. den Kaninchentest vor (sonst IAL des
Eluates eines Materials). Pyrogentestung sollte daruber hinaus
innovative high-tech Produkte einschliefJen, wie Medizinprodukte (Implantate, medizinische Materialien, Dialyseprodukte), zellulare Therapeutika und spezifische Therapeutika wie
rekombinante Proteine. Hier wird gezeigt, dafJ der humane
Pyrogen-Test PyroCheck geeignet ist zur Testung von Filtern
zur Qualitiitskontrolle der Luft, sowie Medirinprodukten
und
zur Prufung auf Biokompatibilitat
(Dialyseflussigkeiten}.
Es
zeigt sich, dafJ diese neue Technik fiir ein sich ausweitendes
Anwendungsspektrum
praktikabel ist.
Lysate Test, human whole blood test, PyroCheck,
Interleukin-Ifi,
ALTEX 16, 3/99
~~
FENNRICH
ET AL.
--~~---------------------------~c'
1 Introduction
Immediate recognition of bacterial infection is mandatory for the survival of the
hnman organism. Evolution of mankind,
therefore, has created a highly sensitive
detection system which identifies bacteria and other germs based on those structural components they do not share with
the human body. Most effectively, the human system reacts to structures which are
highly conserved among different bacterial strains. The most potent stimuli are
endotoxins from Gram-negative bacteria.
These lipopolysaccharides (LPS) of the
outer cell membrane induce a cascade of
defense mechanisms known as inflammation and fever. Due to their inherent capability to induce fever, such stimuli are also
termed pyrogens (for review see Pearson,
1985). When cells of the immune system,
namely blood monocytes and rnacrophages (c.f. title page of this issue of ALTEX),
come in contact with pyrogenic contamination, they release mediators which transmit the fever reaction within the organism.
The most prominent representative of these endogenous pyrogens is the pro-inflammatory cytokine Interleukin-IB (IL-IE).
The new pyrogen test (Hartung and
Wendel, 1995, 1996) is based on this principle: The sample to be tested is incubated with a small amount of blood taken
from a healthy donor. Any pyrogenic activity, independent of its chemical nature,
induces the formation ofIL-IE which can
be determined by ELISA. Currently, a
national collaborative prevalidation of this
test is performed between the University
of Konstanz, a Federal Institute and industrial partners.
Table 1: Comparison of the application spectra of the rabbit pyrogen test, the Limulus
Amebocyte Lysate assay (LAL) and the human whole blood assay (PyroCheck).
Tests
Principles and Applications
Rabbit
Priciple of test
Detectable
pyrogens
Applications
LAL
cytoxine
reease (1L-1B)
Gram-neg.
+
+
Gram-pes.
+
Fungi
+
Biologicals
+
-
Pharmaceuticals
+
+
+
Medical Devices
-
(+)
+
(+)
(+)
-
+
Air quality
Blood products
blood assay is based. A standardised version of the test in form of a kit (PyroCheck, DPC Biermann, Bad Nauheim)
is now commercially available as the result of a successful technology transfer
to a industry partner. Preliminary results
of the evaluation of feasibility of the test
for pharmaceuticals and biologicals have
been reported previously (Fennrich et al
1998, 1999). In table 1, the present state
of the properties of the human whole
blood assay is compared with the ones
of the rabbit test and the LAL. It is evident that among the two in vitro tests,
Gram-positive and fungal (Zimmermann,
1999) contaminations escape detection
by the LAL while the blood assay detects
them.
Human Fever Reaction
+
+
+
+
Our current research activities are focussed on expansion of the test to hitherto uncovered gaps of product safety, such
as applications to biocompatibility, e.g.
of dialysis fluids. A second field encompasses testing of medical devices, e.g. plastics or filters. The goal of this research
line was to identify applications that can
neither be covered by rabbit pyrogen test
nor by the LAL or can replace the animal
test in a wider range of applications.
One of the characteristics of the whole
blood assay is that the direct contact between the relevant cells (monocyte/macrophage) and with the surface of any material of interest is the key event that initiates the signal that is taken as the final readout. In other words, a heterogeneous
Rabbit Pyrogen Test
2 Material and methods
.../'!
pyrogen
See earlier publications (Hartung and
Wendel, 1995, 1996; Fennrich et al, 1998)
For testing medical devices it is easy to
bring the diluted whole blood in contact
with the material either by moving the
material into the blood or by rinsing it with
blood. The dilution procedure and the test
time are the same as described earlier for
testing drugs.
human blood tests
fever reaction defense mechanism
mammal
arthropoda
~
test semple
leucocyte
~
~
whole blood
FEVER
cytoxines (1L-1B)
~
cytoxines (1 L-1 B)
~
ELISA
3 Results
Human Py ro 9en Test
The diagram in figure 1 illustrates the
principle on which the human whole
Figure 1: The basic principles of the rabbit pyrogen test and the human whole blood
assay PyroCheck.
ALTEX 16,3/99
Human Whole Blood
147
_F_ENN
__ ~_C_H_E_T_AL_.
~~~
_
.' 0'
phase reaction is the basic physicochemical principle of the assay. In contrast, the
LAL requires a homogeneous reaction
phase, i.e. it is restricted to LPS in soluble
form. Hence, the contaminant to be tested
has to be eluted from the original test material- a technical complication of the procedure with unknown quantitative doseeffect relationships. A well-known characteristic property of pyrogens is that they
tend to firmly attach to material, even test
tubes, while in contrast the surface itself
can induce the immune system to react.
The experiments shown in figure 2 provide evidence that endotoxins soaked into
filters and dried are also capable of initiating a concentration-dependent release of
IL-l from whole blood. Under these conditions, we blindly tested air filters which
were used in the air conditioning of a veterinary facility for sheep. Contaminated
filters were identified and later microbiological analysis confirmed this classification (data not shown). These findings suggest that where neither the test material
nor the surface can be brought into solution, the blood test is the alternative of
choice as an indicator of biohazard.
The data shown in figure 3 demonstrate
that bacteria and fragments of their membranes can be detected in a very sensitive
way with the blood assay, independent of
whether they had been killed before by heat
or exposed to antibiotics. Taking this into
account suggests that such conditions to be
tested as contaminants reflect the more realistic situation because crude endotoxins are
more likely to be expected in test samples
than purified LPS in a sterile environment.
4000
E 3000
OJ
So
.•..
~ 2000
1000
ng
_
Figure 3: Release of IL-1 B from of the human whole blood initiated by bacteria killed by
heat or antibiotics.
4 Discussion
The absence of pyrogens (fever inducing
contamination) represents an indispensable
safety control in products given to patient
via a parenteral way.The rabbit pyrogen test
has served for drug safety control for more
than fifty years (Flint, 1984). Due to improved manufacturing procedures, contaminated end-products have become rare and
the adequacy of a test consuming larger
animals is doubted. However, the level of
safety reached needs to be maintained since
infusion or injection of contaminated drugs
to often critically ill patients is life-threatening. Innovative high-tech products such as
medical devices (implants, medical plastic
heat inactivated
c::::J treated
_live
20000
with peniCillin/streptomycin
bacteria
""-e-
~
10000
n
0
10'
10'
10'
10'
Bacteria/ml
[x3,24]
II
10'
II
10'
10'
Figure 2: Concentration-dependent
release of IL-1B from of the human whole blood
initiated by nitrocellulose filters contaminated with defined LPS-concentrations
(representative for medical device testing).
148
~g
LPS/filter
E. coli: Incubation with human whole blood
30000
]
Cl
~
Medical device testing: IL-1 B-release from whole blood
by incubation of NC-filters coated with LPS
materials, dialysis machines), cellular therapies and species-specific agents (e.g. recombinant proteins) will continuously expand this product segment in the future.
In special prescriptions for medical devices (EU Medical Device Directive) it is
regulated to use the rabbit pyrogen test.
These are essential parts of DIN-, EN- or
ISO standards for testing medical products
like syringes, catheters, stylets and machines or parts of them (Zamow and Spielmann, 1998). It is regulated to prepare an
eluate that has to be injected into the rabbit,
like the classical pyrogen test claim it. The
possibility of testing cell culture plastic
material directly by exposing it to human
blood was shown in a collaboration with a
industrial partner and will be first demonstrated on the 3rd world congress on alternatives and animal use in the life sciences
in Bologna 1999.
Taken together, it seems possible to reduce or replace the rabbit test in the regulation (DIN, EN, ISO).
On the other hand, the blood test is much
more versatile than the in vivo rabbit test
with respect to applications to medical devices, blood products and air quality. These applications demonstrate the putative
use of the blood test in the control of biological hazards in e.g. aircraft or hospital
ventilation systems. Further applications
include air quality control during work in
waste recycling, or microbiological air
quality assessment in animal mass production facilities.
ALTEX
16, 3/99
r:"\
FENNRICH ET AL.
--~----------------------------------For most biologicals, especially blood-derived drugs, the rabbit animal experiment
still represents the only choice at an annual expense of hundreds of thousands of
animals in the European Union. In addition, this test is laborious and expensive.
However, for new therapies such as recombinant human proteins or cellular therapeutics, the rabbit assay is not applicable because in many cases false-positive
results will be obtained due to the speciesspecificity of the immunological recognition of these agents. The only in vitro alternative presently available, the Limulus
amebocyte lysate test, which is based on
the coagulation of horseshoe crab blood,
detects only one class of pyrogens - endotoxins (lipopolysaccharides, LPS) from
Gram-negative bacteria - leaving patients
at risk originating from undetected "nonendotoxin" pyrogens such as Gram-positive bacterial toxins, viruses and fungi
(Diamond, 1989; Kayser, 1998). Thus,
only an assay based on the human fever
reaction will close these "safety gaps".
The introduction ofthis kit version enables international availability, distribution of the methodology and interlaboratory comparisons. It was very helpful, to
use this version inside of the prevalidation network and the feedback of experience with this method was included in the
test procedure for all participants.
In recent years, several alternative cellular assays have been suggested in order to replace the rabbit test and offer testing in the relevant species (man) (Eperon, 1997; Gommer, Peterbauer, 1999;
Poole, 1988; Taktak, 1991; Werner, 1998;
Werner-Pelmayer, 1995). All of them are
based upon the response of human leukocytes (principally monocytes), which
release inflammatory mediators (endogenous pyrogens) in response to pyrogenic
contamination
(exogenous pyrogens).
However, the cell-based in vitro assay systems differ with regard to the cells employed (isolated primary blood leukocytes or whole blood or immortalised monocyte cell lines), the mediator determined (interleukin-I, interleukin-6, tumor
necrosis factor, neopterin, or NO) and the
precise set-up of the test. Using human
blood implies considerably simplified
handling, reliability and reproducibility
of the assay. Now, this blood test will be
evaluated in comparison to the other human cell approaches.
ALTEX 16, 3/99
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Acknowledgements
A national collaborative prevalidation of this
test is performed between the University of
Konstanz, the Paul-Ehrlich-Insitut (Langen),
the University of Heidelberg and some the
commercial enterprises Centeon (Marburg),
Biotest (Dreieich), PharmaHameln (Hameln),
Phytos (Neu-Ulm) and Biochem (Karlsruhe).
Sponsored by the German Minister of Education, Science, Research and Technology
(BMBF) the suitability of the new method to
replace the rabbit pyrogen test for biologicals
is currently evaluated. In addition, an evaluation of the test for pharmaceuticals sponsored
by Boehringer Mannheim on behalf of the
European Pharmakopoea is presently carried
out.
A standardised version of the test in form of
a kit (PyroCheck) is now commercially available by DPC Biermann, Bad Nauheim, Germany.
The fruitful help and technical assistance of
Astrid Leja, Dona Kindinger and Gregor Pinski are gratefully acknowledged.
Correspondence
to
Dr. Dr. Thomas Hartung
University of Konstanz
Biochemical Pharmacology
P.O. Box M 668
D-78457 Konstanz
149