Hemo_reviewDEF6

Authors:
Prof. János Szebeni, M.D.1
and Peter Haima, Ph.D.2
1. Nanomedicine Research and Education Center, Semmelweis University and SeroScience Ltd., Budapest, Hungary
2. Life-Force biomedical communication, Netherlands
TECOmedical Clinical & Technical Review
December 2013
Hemocompatibility
of medical devices, blood products,
nanomedicines and biologicals
Testing activation of the C-system

2
ABSTRACT

Hemocompatibility testing is the evaluation of critical interactions of foreign material with blood to explore possible
adverse effects arising from the exposure of the foreign material to blood cells and proteins. Because such adverse
effects are frequent and may represent serious health risks, hemocompatability testing is very important for the in-
troduction of new medical devices, medicines, blood products and diagnostic agents. Stimulation of the immune
system can lead to an allergy-like syndrome called hypersensitivity or infusion reaction, a major and potentially lethal
hemo-incompatibility whose symptoms involve almost all organ systems. Infusion reactions can represent a real
allergy (IgE-mediated) or a “pseudoallergy”, one that involves no IgE and may arise as a consequence of activation
of the complement (C) system. Non-IgE-mediated anaphylactoid, or pseudoallergic reactions are frequent side
effects of i.v. administered nanomedicines (e.g. drug carrier systems like liposomes) and biologicals (e.g. monoclonal
antibodies). These new drugs are in the frontline of modern pharmacotherapy, but their unique toxicity problem has
not been solved to date. Regulatory guidance on the assessment of C-mediated infusion hypersensitivity has been
published recently for the case of generic liposome production. In this review we outline the theoretical foundation of
C-mediated infusion hypersensitivity to medical devices, nanomedicines and biologicals, and offer practical methods
for using C assays to predict their hemo-incompatibility.
We recommend that essentially all intravenously applied drug candidates should be tested with regard to direct
C-activation as a risk factor for infusion hypersensitivity.

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CONTENT
1 INTRODUCTION 4
1.1 The Complement system 4
1.2 Hemocompatibility and C-activation 6
Infusion hypersensitivity caused by activation of the C-system 6
1.3 Regulatory guidances for testing for C-activation 7
Medical Devices 8
Nanomedicines and biologicals 8
2 ACTIVATION OF THE C-SYSTEM BY ARTIFICIAL SURFACES, NANOMEDICINES
AND BIOLOGICALS 9
2.1 Mechanism of infusion hypersensitivity caused by C-activation 9
2.2 C-activation by artificial surfaces 9
2.3 C-activation by nanomedicines 9
2.4 C-activation by biologicals 10
2.5 Prevalence of C-activation-related pseudoallergy 11
2.6 Marketed nanomedicines & biologicals inducing C-activation-related pseudoallergy 11
Marketed Liposomal drugs 13
Marketed therapeutic monoclonals 13
Other drugs and agents on the market inducing complement
activation-related pseudoallergy 14
2.7 Recommendations to assess the C-activating capability of drugs
and drug carrier systems 14
3 METHODS AND ASSAYS TO MEASURE ACTIVATION OF THE C-SYSTEM 15
3.1 In vitro C-testing for hemocompatability in human sera 15
Experimental design of in vitro C-testing for hemocompatability 16
Protocols for in vitro C-testing for hemocompatability 17
3.2 Test models for medical devices 20
Static models with serum 20
Dynamic models with fresh whole blood 20
3.3 Flow cytometric examinations to evaluate binding of C-proteins to artificial surfaces 20
3.4 Personalized medicine: testing for hypersensitivity reactions to anticancer drugs 20
3.5 Testing C-activation in animal models 20
CxH50 21
Species-Independent C-testing for hemocompatability in animals 21
The porcine model 21
Other animal models 23
3.6 Testing of monoclonal antibodies for complement dependent cytotoxicity 24
4 REFERENCES 25
5 TECHNICAL DATA SHEETS OF COMPLEMENT PRODUCTS 27

4
1 INTRODUCTION
1.1 The Complement System
The Complement (C-) system helps, or“complements”the ability of antibodies and phagocytic cells to clear invading
cellular pathogens (e.g. bacteria) [1-3]. It is part of the immune system called the innate immune system
that is not adaptable and responds to foreign challenges in a non-specific manner. When stimulated by one of
several triggers, proteases in the system cleave other C proteins and initiate an amplifying cascade of further
cleavages. The end-result of this activation cascade is massive amplification of the response, release of cytokines,
massive acute inflammatory reactions and activation of the cell-killing membrane attack complex (MAC).
Figure 1
The complement cascade (figure was adaped from Rutkowski et al. (2010)) [4].
The classical pathway is activated by the Fc portion of immunoglobulins bound to antigen, apoptotic cells, Gram-negative bacteria, and viruses.
The C1 complex, made up of C1q, C1r, and C1s subunits, initiates the downstream classical cascade. Upon binding of C1q to an inciting stimulus,
C1r catalyzes breakage of a C1s ester bond, resulting in its activation and subsequent cleavage of C2 and C4 into their respective “a” and “b” frag-
ments. The formation of C2a4b creates C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to other C3 convertases, forming C2a4b3b,
also known as C5 convertase. It facilitates the final steps of the cascade by splitting C5 into C5a and C5b. The latter fragment is the critical first
protein that combines with C6, C7, C8, and multiple C9 proteins to form the MAC, the terminal, pore-forming c-protein complex responsible for
lysis of cells and pathogens. The MBL pathway is activated by surfaces bearing mannose groups or other pathogen-associated molecular pat-
terns. MBL or ficolin activation of mannose-associated serine proteases (MASP) results in cleavage of C2 and C4 similar to the C1 complex, with
subsequent production of C3 convertase and C- cascade activation resembling the classical pathway. Lastly, the alternative pathway is activated
by a multitude of infectious agents including various bacteria, viruses, and fungi, as well as neoplastic cells. This pathway exhibits a unique
“tickover” effect whereby low-level C3 cleavage occurs continuously. Generated C3b binds Bb, a cleavage fragment of factor B, and properdin, re-
sulting in the formation of the alternative pathway C3 convertase. Binding of additional C3b to the alternative pathway C3 convertase renders it
capable of C5 cleavage, and forms the basis for the amplification loop of the alternative pathway. Additionally, C3b generated by the alternative
pathway C3 convertase can attach to target surfaces and bind Bb, forming a C3 convertase that amplifies downstream C proteins locally at the
target surface. Although the activation and amplification of the three pathways differ initially, they commonly cleave C3 into C3a and C3b, finally
resulting in terminal formation of the MAC.

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The cascade is composed of some 30 plasma and cell membrane proteins and provides the first line of defense against
microbial or other pathogenic attacks. C-activation can proceed via three pathways (classical, alternative and lectin)
which are activated in different ways (Figure 1 [4]). They all converge in one final common pathway (Terminal Pa-
thway) at the pivotal protein C3. Mast cells, basophils, platelets and other inflammatory cells are activated liberating
inflammatory mediators (histamine, PAF, prostaglandins, etc.), which in turn, set in motion a complex cascade of res-
piratory, hemodynamic, and hematological changes, assisting the body’s self-defense. Final endpoint of the terminal
pathway is C5b to C9 being assembled into theTerminal Complement Complex (TCC, C5b-9) referred to as Membrane
Attack Complex (MAC), which causes direct lysis of invader cells by creating membrane protein channels.
While fulfilling its basic function in innate immunity, the C-system may also have serious adverse effects.The C cascade
contains some of the most powerful pro-inflammatory molecules in the body, including most notably the anap-
hylatoxins C3a, C4a and C5a [5-8] which are recognized pathogenic factors in a wide spectrum of chronic inflam-
matory diseases, including rheumatoid arthritis, glomerulonephritis, atherosclerosis, asthma, and multiple sclerosis
[9-14]. Evidence is accumulating that C proteins may also facilitate some basic processes in carcinogenesis, including
sustained cellular proliferation, angiogenesis, insensitivity to apoptosis, invasion and metastasis, and escape from
immunosurveillance [4].
Monitoring the development of C-activation is necessary to detect C-related diseases in patients and predict possible
adverse effects arising from the exposure of foreign material (medical devices, i.v. medicines, etc.) to blood cells and
proteins. A list of analytical methods available for the investigation of C-activation is shown in Figure 2.
Figure 2
Analytical methods for Complement diagnostics.

6
1.2 Hemocompatibility and C-activation
Hemocompatibility testing, a major part of biocompatibility testing, is the evaluation of critical interactions of foreign
material with blood to explore possible adverse effects arising from the exposure of the foreign material to blood cells
and proteins. Because such adverse effects are frequent and may represent serious health risks, testing of new me-
dical devices, intravenously applied medicines, blood products and diagnostic agents for hemocompatability is very
important. As for the hemocompatibility testing of medical devices, ISO 10993-4 [15] provides a list of recommended
assays (Figure 3). This list includes the testing of C-activation, as the above devices often expose large surfaces to
blood which provide surface for C deposition and, hence, C-activation.
A new class of i.v. medicines, called “nanomedicines” are of particular importance, as the hemo-incompatibility pro-
blems they can cause often arises from C-activation. Nanomedicines include a wide variety of synthetic and semi-
synthetic drugs, agents and drug carrier systems (liposomal drugs, micellar systems, polymer-conjugates of proteins,
imaging agents, drug carrier nanosystems) whose complexity and size in the nanometer range (5-250 nm) distinguis-
hes them from the traditional (Lipinski-type), low molecular weight medicines [16]. Biologicals, or biopharmaceuticals
(antibodies, cytokines, protein fragments and synthetic peptides) are also large molecular weight medicines; their
size range (8 – 20 nm) and molecular complexity would also qualify them as nanomedicines, however, for practical
purposes, only functionally modified (e.g., pegylated or conjugated) biologicals are considered as nanomedicines. A
common feature of nanomedicines and biologicals is that -while they are in the frontline of modern pharmacothe-
rapy-, they also have a unique toxicity problem: stimulation of the immune system. It is a frequent side effect, leading
to an allergy-like syndrome called infusion or hypersensitivity reaction (HSR). It is a major and potentially lethal hemo-
incompatibility whose symptoms involve almost all organ systems (Figure 4) [17].
Infusion hypersensitivity caused by activation of the C-system
Infusion reactions can represent a“real”allergy, one that arises after prior exposure of the reactogenic drug to blood
and involves immune memory in the form of specific IgE formation. The other type of infusion reactions involves no
IgE and may arise, at least in part, as a consequence of activation of the C-system. Non-IgE-mediated infusion reactions
are also referred to as non-IgE-mediated anaphylactoid or pseudoallergic reactions or C-activation-Related Pseudoal-
lergy (CARPA) [8]. In fact C-activation can be the sole cause of infusion reactions or may be a contributing factor. Most
important differences between IgE- and non-IgE mediated infusion reactions is that true allergies are observed only
after repeated exposure of the reactogenic drug to blood and they get stronger upon repeated administration, while
pseudoallergies develop at the first exposure and the reaction loses strength with time and repetition (Figure 5) [18].
Figure 3
Hemocompatability assays for medical devices [15].
Figure 4
Symptoms of infusion-induced hypersensitivity
reactions [17].

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1.3 Regulatory guidances for testing for C-activation
Figure 4 gives an overview of current regulatory guidance papers related to hemocompatibility and testing for
C-activation.
Figure 5
Distinguishing features and grading of true
and pseudoallergy [18].
Guideline
ISO 10993-4 (2009) [15]
ASTMF2567. Standard Practice
for testing for classical pathway
C-activation in
serum by solid materials (2010).
for testing for alternative path-
way C-activation in serum by solid
materials (2010).
for testing for whole C-activation
in serum by
solid materials (2010).
U.S. Pharmacopeia <1031>.
The biocompatibility of materials
used in drug containers and me-
dical devices and implants.
European pharmacopeia 6.0.
Measurement of C-activation by
intravenous
immunoglobulin preparations.

Recommended assays: C3a, C5a, Bb, iC3b, C4d, SC5b-9, CH50,
C3 & C5 convertase.
Describes procedures for exposing standard lot of human serum to
solid materials and measuring C4 depletion. Other validated tests for
specific Complement components or split products may substitute
the assays described here.
Describes procedures for exposing standard lot of C4-deficient
guinea pig serum to solid materials and measuring activation
of alternative C-activation. Other validated tests for specific
Complement components or split products of the alternative
pathway may substitute the assays described here.
Describes procedures for exposing human serum to solid materials
and measuring Complement activity remaining by classical pathway
mediated lysis of sensitized red blood cells. Other validated C- tests
may substitute the method described here.
USP monograph on hemocompatability is under development.

Defined amount of test material is incubated with a defined
amount of guinea pig Complement and the remaining Complement
is titrated; the antiComplementary activity is expressed as the
percentage consumption of Complement relative to the
Complement considered as 100 percent.
Comments on C testing
Medical devices

8
Medical devices
Exposure of foreign material to blood cells and proteins frequently has adverse effects and may represent a serious
health risk. Therefore, introduction of blood-exposed materials in clinical use is highly regulated. Official guidances
by the USA Food and Drug Administration (FDA), European Medicines Agency (EMA), ASTM International (American
Society forTesting and Materials) and other regulatory agencies are available, giving recommendations on assays that
need to be conducted by the manufacturers to assure the lack of harm caused by blood-exposed foreign material.
As for the hemocompatibility testing of medical devices, such as endovascular grafts, shunts, rings, patches, heart
valves, balloon pumps, stents, pacemakers, hemopheresis filters, ISO 10993-4 [15] provides a list of recommended
assays for testing C-activation (Figure 3). As for the C tests evaluating the hemocompatibility of i.v. medicines and
diagnostic agents, the same assays can be applied as recommended for medical devices, with appropriate adaptation
for dispersed, water soluble or insoluble molecules [18].
Nanomedicines and biologicals
Detailed guidance on the assessment of C-mediated hypersensitivity reactions to nanomedicines and biologicals has
not been included in any available regulatory document, although they can be lethal and it is possible to measure
and predict these reactions as part of the safety evaluation of i.v. applied nanomedicines and biologicals. However,
the phenomeon is gaining increasing attention. For example, the EMA released a final paper on data requirements
for intravenous liposomal products (Figure 6) [48] in which it is stated that use of in vitro and in vivo immune
reactogenicity assays such as complement (and/or macrophage/basophil activation assays) and testing for
C-activation-related pseudoallergy (CARPA) in sensitive animal models should be considered to evaluate the extent of
potential adverse event”(Figure 4).
Guideline
USDHHS, FDA, CDER.
Guidance for Industry, Immuno-
toxicology evaluation of
investigational new drugs (2002).

European Medicines Agency.
Committee for Human Medicinal
Products (CHMP), reflection paper
on the data requirements for
intravenous liposomal products
developed with reference to an
innovator liposomal product.
EMA/CHMP/806058/2009/Rev.
02, 21 February 2013“ [48]
USDHHS, FDA, CDER, CBER.
Guidance for Industry, Immuno-
genicity Assessment for Thera-
peutic Protein Products (2013)

If signs of anaphylaxis are observed in animal studies, follow-up
studies should be considered. Biochemical markers of an anaphy-
lactoid reaction can be observed in nonclinical toxicology studies
(e.g.,detection of serum anaphylactic Complement products in
animals showing signs of anaphylaxis) (Szebeni, 2001).
Careful evaluation of these reactions has resulted in valuable
information on biochemical markers used in clinical trials.
Use of in vitro and in vivo immune reactogenicity assays such
as Complement (and/or macrophage/basophil activation assays)
and testing for C-activation-related pseudoallergy
(CARPA) in sensitive animal models should be considered to
evaluate the extent of potential adverse event.

Immunologically based adverse events, such as anaphylaxis,
cytokine release syndrome, so-called“infusion reactions,”and
nonacute immune reactions such as immune complex disease
have caused sponsors to terminate the development of therapeutic
protein products. No recommendations are made to test for
C-activation in pre-clinical phase.
Comments on C testing
Intravenous Drugs
Figure 6
Overview of current regulatory guidance papers related to hemocompatability and testing for C-activation.

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2 ACTIVATION OF THE C-SYSTEM BY ARTIFICIAL
SURFACES, NANOMEDICINES AND BIOLOGICALS
2.1 Mechanism of infusion hypersensitivity caused by complement
activation
The immune cells responsible for true allergy are also responsible for CARPA [17]. These include mast cells, basophils
and macrophages that express a group of G-protein coupled receptors which bind anaphylatoxins (i.e., C3a/C5a/C5L2
receptors). Binding of C-activation byproducts, C3a and C5a to these receptors can trigger essentially the same intra-
cellular signal cascade that is activated upon the engagement of allergen to membrane-bound IgE, leading to the
release of a battery of secondary vasoactive mediators (so called“allergomedins“), including histamine, tryptase, PAF,
leukotrienes (LTB2, LTB4, LTC4, LTD4, LTE4,TXA2, PGD2 andTXD4). In the next step allergomedins bind to their respec-
tive receptors on endothelial and smooth muscle cells, modifying their function in ways that lead to the symptoms
of CARPA, which involve almost all organ systems (Figure 3). Different individuals and tissues may have very different
patterns of allergomedin receptors, and these receptors mediate different functions in different tissues. For example,
skin and cardiac mast cells respond to different allergomedin stimuli [19]. Increased vascular permeability, a hallmark
sign of severe (Grade IV) CARPA may entail the transfer of up to 50 % of intravascular fluid into the extravascular space
within 10 minutes [21].
2.2 C-activation by artificial surfaces
C-activation by artificial surfaces can occur via the alternative and/or classical pathway (Figure 7) [21]. The classical
pathway is initiated by activation of the C1 complex, leading to assembly of the classical pathway C3 convertase and
generation of C3a, C5a, and the terminal complement complex. The alternative pathway is initiated when water-
reacted C3 (which is constantly being formed at a low level) interacts with a receptive surface to form bound C3b.The
Bb fragment (the result of cleavage of Factor B by Factor D) binds to bound C3b to form the alternative pathway C3
and C5 convertases capable of generating C3a and C5a. The convertases are short lived and Bb is likely released from
the surface. The cleavage of C5 also releases C5b, which can ultimately lead to formation of the terminal complement
complex.
Figure 7
C-activation by artificial surfaces
(adapted from Gemmell [38]).
2.3 C-activation by nanomedicines
In the case of non-proteinaceous nanomedicines, which consist of normally non-immunogenic molecules or poly-
mers, it is their size (in the 50 – 200 nm range) and surface characteristics (molecular arrays or repetitive elements
which are recognized by pattern recognition receptors on immune cells) which make them resemble human patho-
genic viruses recognizable by the C-system. Figure 8 illustrates the sizes of drug carrier nanosystems, in relation to
the “window of immune vision” , i.e., the size and molecular weight thresholds of cellular and humoral immunity in

10
recognizing particles as foreign [17].The Figure suggests that liposomes and certain carbon nanotubes are within the
spotlight of immune surveyance (blue triangle), while fullerenes, micelles, dendrimers, conjugated polymers, poly-
meric micelles or vesicles, aptamers, quantum dots, superparamagnetic iron oxide nanoparticles (SPIONs), polymeric
micelles, nanocrystals all fall beyond immune recognition, at least in monomeric form. Consequently, the immune
reactogenicity of these theoretically stealth particles probably involve interactions with blood elements or other ef-
fects which make them recognizable by the immune system.
On the other hand, most pathogenic human virus classes in the 40 – 300 nm range look very much like small unilamel-
lar (SUV) and multilamellar (MLV) liposomes, with Doxil, the first FDA-approved nanomedicine, being almost indistin-
guishable from HIV-1 [17]. In addition to their similarity to viruses in terms of size, there is another major reason why
nanomedicines are recognized by C as foreign; the absence of membrane proteins that protect cells from C attack [17]
or surface camouflaging that viruses use for C evasion.
Figure 9
Humanized antibodies activate C, after binding to their target.
Figure 8
Drug carrier nanosystems and their visibility by the
immune system [17].
2.4 C-activation by biologicals
As for the cause of C recognition of biologicals, non-self proteins normally carry numerous antigenic epitopes to
which the body responds with antibody production. Once such antibodies are formed, they bind to the foreign pro-
teins and activate C, which is one of their roles in immune defense. Different antibody classes have different capability
to activate C, with IgM being the most potent. The C binding function of IgGs is well-known in immunology as well as
in the industry of biologicals, leading developers to produce“C-stealth”humanized proteins (humanized antibodies)
for intravenous use. Nevertheless, once they bind to their target, humanized antibodies also activate C, as C binding
is an intrinsic function of certain IgG antibodies, regardless of their origin. This very basic principle is illustrated in
Figure 9, reminding of the mechanism by which even totally humanized, immunologically fully matched monoclonal
antibodies activate C if they bind to their target epitopes.

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2.5 Prevalence of C-activation-related pseudoallergy
It has been estimated that as many as 30 % of hospitalized patients may have a drug reaction of some type, with the
incidence of severe and fatal reactions being approximately 7 % and 0.3 % , respectively [22]. These statistics imply
roughly 2 million serious reactions per year with ~ 100,000 fatalities, making adverse drug reactions the fourth to sixth
leading cause of death in the USA [22]. Another analysis pointed out that about 25 % of all adverse drug reactions
are of allergic nature [39], of which about 77 % is non-IgE-mediated, i.e., represent pseudoallergy [23]. These statistics
imply approximately 400,000 severe and 20,000 fatal C-activation-related pseudoallergy events each year for the USA
only.
2.6 Marketed nanomedicines & biologicals inducing C-activation-related
pseudoallergy
There are a great number of nanomedicines and therapeutic antibodies reported to cause infusion reactions [17] for
liposomal-, micellar-, antibody based-, conjugated- and miscellaneous other drugs, respectively. There are also some
drug carrier systems whose C activating capabilities have been shown in vitro (e.g., poloxamers, carbon nanotubes)
[24-30].
Marketed liposomal drugs
Liposomes or other types of phospholipid assemblies are increasingly used in medicine for targeted or controlled
release of various drugs and diagnostic agents. At present, more than a dozen liposomal drugs are in the market, and
more in advanced clinical trials. Figure 10 lists those liposomal drugs in the market that have been reported to cause
CARPA. The frequency of reactions reported to different drugs varies between 3 % and 45 % [31]. Out of these, the
reactions to Doxil have been studied in most detail, in humans as well as animals. C-activation by Doxil, documented
in several studies [32,33] have been correlated with clinical symptoms in humans [34] and pigs [25,33,35]. The main
conclusions about C-activation by Doxil and other liposomes are summarized in Figure 11.
Figure 10
Marketed Liposomal drugs inducing C-activation-related pseudoallergy.

12
Figure 11
Features of C-activation-related pseudo-
allergy induced by liposomes.
Figure 12
Marketed therapeutic monoclonal antibodies inducing C-activation-related pseudoallergy [17].
Therapeutic monoclonals
Most, if not all of more than 20 mAb-based pharmaceuticals approved to date carry a risk for causing infusion reacti-
ons. Figure 12 provides specific information on the symptoms caused by these agents, which are essentially the same
as for liposomes, i.e. typical symptoms of infusion reactions (Figure 4). However, there are differences between mAb-
induced and nanoparticle-induced CARPA, one of which is that some mAb reaction may start later, mostly after 30
min, compared to the immediate start of symptoms in the case of liposomes and micellar drugs. This difference can
most easily be rationalized by the different kinetics of C-activation in the case of nanoparticles and mAbs. Namely,
while nanoparticles bind C almost immediately on their surface, mAbs need to undergo steric changes to become
C activators (see Figure 9). It is widely known in molecular immunology that when antibodies bind to foreign surfa-
ces, steric changes in the hinge region free up the C binding site on the Fc region. With mAb therapy, the relatively
slow kinetics of target binding will most likely control the rate of C-activation and other immune consequences of Ab
binding.

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Figure 13
Marketed protein conjugates inducing C-activation-related pseudoallergy [17].
Other drugs and agents on the market inducing C-activation-related pseudoallergy
Marketed protein conjugates that have been reported to cause CARPA are listed in Figure 13. Other marketed intra-
venously applied pharmaceuticals, including micellar drugs, radio and ultrasound contrast agents, that have been
reported to cause CARPA, are described by Szebeni et al. [17].
2.7 Recommendations to assess the C-activating capability of drugs
and drug carrier systems
Worldwide numerous nanomedicines, biologicals and other drugs with or without carrier systems are in various sta-
ges of development or are being evaluated in preclinical or clinical phase. In Figure 14 we have listed the various
categories of drug and drug carrier systems, estimate their C-activating capability and as a consequence the need for
C-testing to predict C-mediated infusion hypersensitivity. It is recommended that all drugs and carrier systems are
tested.

14
Liposomes
Carbon Nanotubes
Spidersilk Beads (Amsilk)

Fullerenes
Micelles
Dendrimers
Polymer Conjugates
Polymeric vesicles
Aptamers
Quantum dots
SPIONs
Polymeric micelles
Nanocrystals

drug
delivery

Yes

Particle size is within
immune surveyance and
membrane proteins that
protect from C attack are
absent. Many liposomal
drugs cause C-activation
(Fig. 10, 11).
In monomeric form,
particle size falls beyond
immune recognition.
Nevertheless, C attack
cannot be a priori exclu-
ded, as interactions with
plasma components or
cells may entail C-activa-
tion, as described for
micelles [17].

C-testing in animal models
and human sera for safety to
predict C-mediated infusion
hypersensitivity.

Drug, carrier
or combination
Theoretical
foundation
RecommendationAction Indication C-activation
carrier systems
antibodies
Prevents
triggering of
C cascade.
Strategy
believed to
enhance
antitumor
complement-
dependent
cytotoxicity
Block C
protection by
tumor cells

Block T-cell
activation

Cancer
Non-Hodgkin’s
Lymphoma,
Chronic lymphoid
leukemia (CLL)
(CD20)
Renal cell carci-
noma, colorectal
cancer

T1 diabetes,
auto immune

Yes

Yes

Yes
(by design)

Yes

C-testing for drug efficacy

and human sera for safety

Monoclonals targeting spe-
cific subtypes of Fc receptors
or IL-3.
(SuppreMol)
Monoclonal antibodies
against membrane-bound
proteins displayed by various
tumors. E.g. anti-CD20 (e.g.
Rituxan).

Monoclonal antibodies
against membrane bound
complement regulatory
proteins displayed by various
tumors. E.g. anti-CD46, CD55
and CD59).

Monoclonal antibodies that
are immunosuppressive (eg.
anti CD3)

mAbs may activate the C-
system, as C binding is an
intrinsic function of
IgG and IgM antibodies.
mAbs may activate the
C-system, as C binding is
an intrinsic function of
IgG antibodies

mAbs may activate the
C-system, as C binding is
an intrinsic function of
IgG and IgM antibodies
Such strategies ignore
the possibility that the
C-system promotes neo-
plastic development and
progression rather than
exclusively retarding it [4].
mAbs may activate the C-
system, as C binding is an
intrinsic function of
IgG antibodies.
various

various

Yes

Particle size is within
immune surveyance.

and human sera for safety.
Various

Improving
immunity

various

Yes

Active vaccines activate
C, but only locally. Passive
vaccines are antibodies
and therefore C-activating

Various

Interfe-
rence at
early stage of
the immune
reaction
prevents the
triggering of
the cascade.
Auto immune
disease

Unknown

Due to its size (40-72 kDa),
it may activate C.

C-testing for drug efficacy

and human sera for safety.

Soluble Fcγ receptors com-
peting with FcγRs expressed
on immune cells (Suppre-
mol)

Various
actions

Various, e.g.
cancer

Yes

Yes

Some membrane-bound
peptides and nucleic acids
are known to activate C.
DNA vectors are strong C
activators.
Can form charged repe-
titive units that bind C1q
and activate C

Peptides, DNA vector-based
vaccines, receptor agonists/
antagonists and inhibitors

> 2000 Da polymers

Receptors
Small drugs
Small drugs
(Conjugated ) proteins
Figure 14 C-inducing potential of various drugs in development or (pre)clinical evaluation and recommendations for measurement of C-
activation.

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3 METHODS AND ASSAYS TO MEASURE ACTIVATION
OF THE C-SYSTEM
3.1 In vitro C-testing for hemocompatability in human sera
There are several methods to quantitate C-activation in man (Figure 2). Some are based on the measurement of
activation products (activation markers) while others measure consumption of (precursor) C proteins that can be
activated. Both approaches can be done by way of immunochemical assays, ELISA, RIA, (rocket) electrophoresis,
Western blot or hemolytic assays (which measure red blood cell (RBC) hemolysis caused by the formation of the
TCC. C-fragments express unique neo-epitopes that are detectable by specific monoclonal antibodies. Exposure of
serum (C source) to a test sample (biomaterial or therapeutic) results in production of these fragments, which can
be characterized quantitatively under standard conditions. The steps below (Figure 15) will facilitate investigation of
C-activation and identification of the exact underlying route of activation.
Step 1 Test for C3 cleavage
Complement component 3 (C3) is the pivotal protein of the C-system. The two C3 convertases, C2a4b (classical and
lectin pathway) and C3bBb (alternative pathway) cleave C3 into C3a and C3b. This step exposes a reactive thioester
group within the C3b molecule, which can result in C3b deposition on surfaces. C3a is an important anaphylatoxin
and can be detected in the fluid phase.Thus, C3 activation can be tested by detecting surface bound C3b and/or fluid
phase C3a.
C3: Special cases
In some cases, C3 cleavage products may be masked in the C-sample. For example, C3a may be adsorbed on to
specific biomaterials like polyacrylonitrile (PAN) and C3b may adhere to the surface of reactive materials like cellulose
acetate (e.g. hemodialysis circuits).Therefore it is recommended not only to test for both C3a and C3b, but to also test
for SC5B-9 and C5a (see step 2).
Figure 15
Investigation of C-activation and identification of the exact underlying route of activation.

16
Step 2 SC5B and C5a: Confirming results
C5 cleavage and subsequent formation of Terminal Complement Complexes (TCC) is evidence of activation of the
common terminal pathway in response to C-activation. There are several reasons to test for Terminal Pathway
activation:
1. Biomaterials which do not activate C3 should be confirmed (see C3: special cases).
2. To check the extent of C-activation when C3 cleavage (step 1) is positive.
SC5B-9 and C5a are unique fragments of the terminal pathway. After activation of C3, the two C5-convertases,
C4b2aC3b (Classical and Lectin pathways) and C3bBbC3b (Alternative pathway) cleave C5 into C5a (the most potent
anaphylatoxin) and C5b. C5b binds Component C6 and C7, which in turn bind C8 and C9, thereby forming the lytic
C5b-9 complex. The soluble, non-lytic complex, SC5B-9, is also formed and can be detected fluid phase together
with C5a.
Step 3 Understanding the process: test for C4d and Bb
If prior studies are positive and if the method of activation needs to be defined (for example surface characterization)
testing can be done for C4d and Bb. C4d is a fragment of C4b formed through activation of the Classical and Lectin
pathway after cleavage of C4. C4b is deposited on a reactive surface; C4d may be released by action of complement
regulatory proteins in the serum. Bb, a fragment of the alternative pathway, can be detected when Factor B is cleaved
into Bb andBa. Bb is indirectly bound to a surface via binding to C3b. Testing for these fragments will provide insight
into chemicalmodifications or other bioengineered approaches for circumventing the problem.
Experimental design of in vitro C-testing for hemocompatability
For a proper design of experiments, there are three elements to consider:
1. Complement source
2. Controls
3. Standardization
1 Complement source
Human serum is the best Complement source to test. However, the serum has to be fresh, frozen serum with low exis-
ting levels of activation fragments. EDTA, low dose Heparin and recalcified plasma can be used for C testing, citrated
plasma should be avoided. Quidel provides human serum, which is standardized and collected specially to preserve
active C (product No A113 5.0 ml or A112 2.5 ml). This human serum has been tested for infectious diseases, for
C-activation markers, and for the CH50. After testing, specimen stabilizer (Quidel A9576) must be added to the plas-
ma/blood samples to prevent any further C- activation.
2 Controls
An adequate number of controls and reference materials must be used for the respective experimental test system.
Most kits include internal controls and standards. C-activation takes place on the boundary layer of the blood-material
contact. Therefore, the relation between the size of the test surface and the volume used (serum, plasma, blood) is an
absolutely essential parameter for the standardization of the test system. In addition to the controls provided by the
kit, the following experimental controls (CONTROL) must be considered:
• CONTROL 1 Background: Serum source only
Measures the impact of the system alone on Complement
• CONTROL 2 Positive control: Serum + activator
Confirms C-activity and demonstrates strong positive signal
• CONTROL 3 Negative control: Serum + inhibitor (EDTA)
Negative controls shows any direct interference in the assay
• CONTROL 4 Buffer control (NEG): Buffer which contains the therapeutics Confirms buffer does not interfere
Activator – Since the complete sample handling is never entirely possible without artificial activations, the back-
ground activation can be quantified by using control samples and then compared to the test samples. Based on the
experimental condition, one can decide which activator to use to function as a positive control:

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• HAGG: Heat Aggregated Gamma globulin (Quidel A114). Activator of the classical pathway – C4d, however also
C3a will be activated (application therapeutic antibodies, passive gamma globulin therapies).
• Zymosan: yeast lipopolysachararide. Activator of the alternative pathway - iC3b – C3a - C5a - SC5b-9 –
Bb (application antisense oligonucleotides, charged biomaterial surfaces.)
• CVF: Cobra Venom Factor (Quidel A600). Alternative pathway: iC3b – C3a - C5a - SC5b-9 – Bb. Acts directly
on C3 and is an extremely potent C-activator to fully activate the C-system.
• Certain biomaterials (e.g. Cuprophan™). A membrane that can activate. Maybe used as activator for material
comparison - should only be used if the surface area of the test material is similar.
Note that for inhibition experiments, titrating an activator is challenging to find the right balance between activation
and inhibition using CVF. In this case, HAGG is a better option.
Temperature has an impact on C-activation. So to control for temperature and for related activators created by the
system (e.g.“nephritic”factors), keep one sample of each control (CONT 1-4) on ice.
3 Material or therapeutic
It is important to standardize for size & surface area for volume/concentration of therapeutic/biomaterial.
Please refer to figures 16a and 16b for guidelines.
Figure 16a
Biomaterial Testing Decision Tree.

18
Figure 16b
Experimental design flow charts.
Protocols for in vitro c-testing for hemocompatability
PROTOCOLS
Complement source
• Neat normal human Complement serum (A113)
• EDTA, EDTA-treated and citrated plasma are not suitable
Therapeutics
• Neat and dilutions 1:2 to 1:32
Controls
• Activator HAGG (A114) for postitive control 2: 10 μl for 1 ml (normal human Complement serum)
= Complete activation after 30 Minutes at 37 °C
• Activator CVF (A600) for positive control 2: App. 8 Units for 1 ml (normal human Complement serum)
= Complete activation after 30 – 90 Minutes at 37 °C
• Activator Zymosan for positive control 2: 1 mg Zymosan for 1 ml (normal human Complement serum)
= Complete activation after 60 Minutes at 37°C
• Inhibitor for negative Control 3: • EDTA 10 mmol final concentration
One aliquot of all controls on ice = Background activation
Incubation
• At 37 °C, Move alliquots to ice every 15 minutes and dilute 1:2 with sample stabilizer
to block Complement reaction

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Timepoints
• 15 – 30 – 45 – 60 – 120 – 240 minutes
Assay
• Dilute aliquots according to protocol – Sample is already diluted 1:2 with stabilizer
• e.g. dilution protocol 1:100 = dilute Aliquot 1:50
• Calculate concentration, include dilution factor
• Run controls included in the Kit
• Run additional external controls A115
Refining the Protocol
• Determining the proper therapeutic concentration
• Impact on activator titration
• Optimizing the incubation parameters – Time, temperature
• Optimizing dilution profiles for activators and non activators –Impact of assay type
• Additional Controls
INTERPRETING RESULTS – CONTROLS
CONTROL 1 Background: Serum source only, measures the impact of the system alone on Complement
• Control 1 – on ice = Value as QC Sheet
• Control 1 – 37 °C = Background activation due to Temperature = 2 – 3 times higher value as QC Sheet
CONTROL 2 Positive control: Serum + activator in the experimental protocol, Confirms Complement activity
and demonstrates strong positive signal
• Control 2 – on ice = Background = app. value as QC Cheet
• Control 2 – 37 °C = Total activation = app. 10 times higher value as QC Sheet
CONTROL 3 Negative control: Serum + inhibitor (EDTA), Negative controls shows any direct interference in the
assay
• Control 3 – on ice = Value as QC Sheet
• Control 3 – 37 °C = Should be negative/low – Value as QC Sheet
CONTROL 4 Buffer control (NEG): Buffer which contains the therapeutics, Confirms buffer (therapeutics) does not
interfere
• Control 4 – on ice = app. Value as QC Sheet
• Control 4 – 37 °C = Should be app. value as QC Sheet. Influence of buffer only
Protocols for the set up of assays for the measurement of C-activation of different biomaterials are availa-
ble from TecoMedical.

20
3.2 Test models for medical devices
Several in vitro or ex vivo test models may be used for preclinical assessment of new components (artificial devices)
regarding their clinical usability for blood contact. The quality of the blood components used (whole blood, platelet
rich plasma, plasma, serum) and the choice of suitable anticoagulants (Citrate, EDTA, heparin, hirudin) are of decisive
importance for all these models. Therefore, the following elimination criteria must be observed when selecting test
individuals: no smokers and no intake of medication 14 days prior to blood donation (especially haemostasis- influen-
cing pharmaceuticals such as acetylsalicylic acid (ASA), oral contraceptives, non-steroidal antiphlogistics, etc). Equally
important are gentle blood drawing and rapid processing. Dynamic models using fresh human whole blood should
be used for testing complete hemocompatibility, including C-activation. Blood, plasma, serum extraction and hepa-
rinization to test heart-lung machines, etc., a large amount of blood is needed. For other tests a defined serum that
has been prepared and tested accordingly should be used. If no fresh whole blood is available, frozen NHS (normal
human serum) should be used (e.g. A113 or A112). This serum should be specially harvested to preserve the C factors
and tested for infectious diseases, C-activation and CH50. EDTA plasma or recalcificated plasma are not suitable.
Note: NHS must be placed on ice at all times prior to activation.
Heparin (low dose) or hirudin are to be used for plasma or whole blood. After testing, EDTA must be added to the
plasma/blood samples to rule out any further in vitro activation.
Use of fresh blood
Figure 17
Chandler loop model for testing
new coatings stents etc.
(http://chandlerloop.com)
Dynamic models with fresh whole blood
Chandler loop model
In an in vitro“closed loop”model (modified Chandler loop), 30 ml heparinized (1 IE/ml Heparin) fresh human blood is
recirculated (20 U/min, 37 °C) in circular closed tube sections.This test arrangement can be employed through various
modifications; for testing new coatings, new pharmaceuticals (anticoagulants, inhibitors, etc.), stents, etc. This model
can be used to quantify activation products directly via traditional ELISAs (C3a, C5a, SC5b-9) and the adsorbed plasma
proteins can be detected directly on the surface using a newly developed modi-
fied ELISA technique (e.g. C3a). For this purpose, the tube sections to be examined
will be rinsed directly after completion of the Chandler circulation, fixed (4 % para-
formaldehyde in PBS [w/v], pH 7.4), cut into 2 cm long pieces and frozen. The tube
sections will then be closed on one side and used directly as large “ELISA well”.
Using appropriate antibodies, different plasma proteins can be detected directly
on the test surface (e.g. C3a). Using incubation times of different length in the
Chandler loop model, the kinetics of the adsorption behavior of plasma proteins
can be determined. In addition, the soluble C-activation markersC3a, C5a, SC5b-9,
etc. can be measured in the systemic blood.
perfect health, no infectious diseases
no known factor deficiency
no coagulation disorder
no medication (especially haemostasis-influencing
pharmaceuticals)
skin disinfection
brief congestion
gentle puncture of the large cubital vein on the arm by
an injection cannula (e.g. Venisystems Butterfly-M9)
use of pre-prepared neutral monovettes (Heparin low
dose, Hirudin, etc.)
rejection of the first 3 ml blood
Important criteria for blood collection:Important criteria for blood donors:

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Heart-lung machine model
In this rather complex model, fresh human blood is recirculated in a short-circuited system, using oxygenation,
defined tube length and a build-up of an arterial counter pressure (i.e. the same conditions as for a patient during
cardiopulmonary bypass surgery). The activation potential of foreign surfaces with respect to the different parame-
ters of haemostaseological systems can be checked, without endogenous counterbalances trying to compensate for
these effects [36]. Concentrations of C3a, C5a, SC5b-9, etc. can be determined from the blood drawn at different times
and thus the kinetics of the C
3.3 Flow cytometric examinations to evaluate binding of C-proteins to
artificial surfaces
Assessment of C-activation by artificial surfaces can be performed by using ELISAS to measure the products of C-
activation. However, measurement in the fluid phase only could be misleading as was the case for acrylonitrile hol-
low-fiber and plate dialyzers [37] where C3a andC5a were surface adsorbed which explained for the lack of incre-
ase of plasma C3a and C5a during hemodialysis. Using artificial surfaces in the form of microspheres, Gemmell [38]
demonstrated that there are significant levels of surface bound Bb andC4d, C3d, and iC3b and that the membrane
attack complex (SC5b-9) was surface bound on all surfaces with its concentration increasing to levels as high as 0.5
mg/cm2. Further, the surface bound represented a substantial percentage of the total generated.
After incubation of the microparticles in serum, C proteins adsorbed on the surface were marked using fluorescent
dyes. The fluorescence intensity, in addition to other parameters, could be detected in the flow cytometer. Compared
with standardized fluorescent particles, Gemmell obtained information on the absolute amount of adherent C
proteins and their composition after various incubation times.
3.4 Personalized medicine: testing for hypersensitivity reactions to
anticancer drugs
Anticancer drugs that contain nanoparticles (liposomes, micelles), as well as therapeutical monoclonal antibodies
(mAbs) often cause hypersensitivity reactions (HSRs) as a consequence of their Complement (C) activating effect.
Liposomes (e.g., Doxil), micellar drugs (e.g.,Taxol andTaxotere), and monoclonal antibodies (mAbs), such as rituximab
(Rituxan, MabThera) are known to induce hypersensitivity reactions (HSRs) in a relatively high percentage (10-80 %)
of patients [15, 39]. A study by Szebeni et al. suggested a relation between C-activation caused by these reactogenic
anticancer drugs in vitro (in sera of cancer patients), and the HSRs they cause in vivo upon infusion. Study conclusions
were following:
1. Measurement of SC5b-9 in human serum or whole blood in vitro following incubation with reactogenic drugs
may serve as a predictive test for HSRs. Details and validation of the assays need to be elaborated for each drug.
2. In case of Rituxan and other drugs, whose therapeutic targets are blood cells, whole blood C assays may better
mimic the in vivo C response than serum assays.
3. FACS analysis of the deposition and subsequent proteolysis of C3b on lymphocytes provides direct evidence for,
and a measure of cell-associated C-activation by Rituxan.
4. On the basis of the known role of C-activation in the rise of specific immunity, in vitro C tests might also be used
to predict the immunogenicity of Rituxan and other protein therapeutics.
3.5 Testing C-activation in animal models
To quantify the C activating capability of test materials (e.g. liposomes, other nanoparticles, antibodies, etc), experi-
ments can be performed using animal models. For example, the EMA recommends to use immune reactogenicity
assays such as C assays to test intravenous liposomal products for C-activation-related pseudoallergy (CARPA) in sen-
sitive animal models. Rat, mouse and porcine C5a can be measured by specific commercially available ELISA kits.
However, the use of C5a as a marker for in vivo C-activation is problematic because of the rapid clearance of C5a from
blood by C5aR-carrying cells (WBC, platelets, macrophages, etc), while specific ELISAs for the measurement of stable
byproducts are not available.
The classical C hemolytic (CH50) assay can be used to measure total C-activation through the ability of the C-system to
lyse sensitized sheep red blood cells.The CH50 reflects the ability of the C in a sample to activate; the more activation,
the higher the CH50 signal. However this signal is not a direct measurement of C-activation. Moreover, the technique

22
is very sensitive to serum matrix irregularities, requires normalization of the serum, shows high inter-lab and inter-
assay variation and is very insensitive.
CxH50
The traditional hemolytic assay can be made more specific assay by focusing on a single protein. This assay, called
CxH50, detects the functional activity of a specific Complement component (for example,. C3H50 for C3 function).The
method utilizes specific Complement depleted sera as a source of C proteins. A test specimen can reconstitute the
specifically depleted protein in the test matrix, thereby restoring the C activity.Thus, all lysis of the erythrocyte (minus
background lysis of depleted serum and erythrocytes) is a function of the specific C protein from the test specimen.
Studies have shown that non-human serum can serve to replace the depleted protein and reconstitute Complement
function. The CxH50 is available in many test systems such as:
• C2 (C2H50)
• C3 (C3H50)
• C4 (C4H50)
• C5 (C5H50)
• C1q (C1qH50)
Protocols for the set-up of CxH50 assays are available from TecoMedical.
Species independent c-testing for hemocompatability in animals
Unfortunately, most of the commercially available ELISAs are human specific ([40] leaving an expanding need for
animal testing of C-activation largely unmet. To fill this gap Tecomedical and Quidel have recently introduced a pan-
specific C3 (PS-C3) converter kit (MicroVue Pan-Specific C3 kit) represents a novel approach of animal-specific C
ELISAs (patent pending). The C3 Complement Matrix (CCM) and the C3 Converter Reagent (CCR) in the kit convert the
activity of C3 in the animal specimen to human SC5b-9 that is detectable with the traditional human SC5b-9 ELISA kit.
Thus the method allows sensitive and quantitative measurement of C3 in animal blood, plasma or serum, which, to
the extent C3 had been consumed prior to the assay, also provides a measure of prior C-activation.
The MicroVue Pan-Specific C3 kit expands the methodical arsenal of C analysis in animals, enabling, among many
applications, preclinical immune toxicology testing of C-mediated (pseudoallergic) adverse drug effects. The PS-C3
method is a three-step procedure. In the first step animals are treated – or their sera or plasma incubated - with
the test drugs or device to explore possible C-activation. Samples are collected at appropriate times for the second
step:conversion of animal C3 to human SC5b-9. C3 conversion is performed as specified in the MicroVue Pan-Specific
C3 kit. In the third step SC5b-9 is measured using the human SC5b-9 ELISA kit. Figure 18 gives an example of results
obtained with this new approach
Figure 18
C3 consumption in rats measured by the
Pan-Specific C3 kit. Rats were injected
with cobra venom factor and zymo-
san, known activators of the C-system
in animals. The graph shows the effects
of these agents, triggering major C3
consumption with kinetics consistent
with the C activating effect of these
agents in vivo.
More information can be found in the Tecomedical review: Species Independent Measurement of
C-activation in Animals (August 2013).

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The porcine model
Pigs are particularly sensitive for nanoparticle induced cardiopulmonary distress and cutaneous changes, a
feature that may be related to the presence of pulmonary intravascular macrophages (PIM cells) in this, as well as
in other even-toed (hoofed) animals (Artiodactyla), including sheep and goats [41].
Protocol [17]
• Animals. Pigs (15– 40 kg) of both sexes are sedated within intramuscular ketamine (500 mg) and
anesthetized with 2 % isoflurane, using an anesthesia machine, or with intravenous nembutal (30 mg/kg).
• A pulmonary artery catheter is advanced via the right internal jugular vein through the right atrium into
the pulmonary artery to measure pulmonary artery pressure (PAP).
• A catheter is advanced in the femoral artery to measure systemic arterial pressure (SAP).
Treatment
Liposomes or other test materials are diluted in phosphate buffered saline (PBS) and injected into the pulmonary
artery as a bolus, via the pulmonary arterial catheter. The bolus is flushed into the circulation with 5 – 10 mL PBS.
Endpoints
Note:
• The cardiopulmonary symptoms of pig CARPA mimics the most severe, grade IV or V allergic reactions in
humans(Figure 4) which involves hypotensive shock with cardiac arrhythmias, ventricular fibrillation and cardiac
arrest, as well as the skin symptoms (flushing and rash). Thus, the model is appropriate for studying the
mechanism and conditions of severe HSRs in hypersensitive man.
• The individual variation of hemodynamic reaction in pigs is relatively small.
• The pig CARPA model is uniquely capable to screen out immune reactive nanoparticles that may cause severe
reactions in hypersensitive individuals.
• Although its oversensitivity implies false-positivity in terms of predicting reactions in normal humans, it needs
to be remembered that infusion reactions cause real harm on lying hypersensitive individuals, who are also not
“normal ”in their response to nanoparticles.
CAS score.

Classical C-hemo-
lytic assay (CH50).

Porcine C5a assay.

MicroVue Pan-
Specific C3 kit.

CAS 0, no response;
CAS 1, minimal;
CAS2, mild; CAS3,
moderate; CAS4,
severe; CAS5, lethal.
Total complement
activity is abnor-
mal if C-system is
activated.
% increase is mea-
sure of activation
of C-activation.

% consumption of
C3provides mea-
sure of C-activation.
The symptoms spe-
cifying different CAS
values are described
in Szebeni et al. [25].
Assay is insensitive,
cumbersome and
shows high inter-
lab and inter-assay
variation.
Use of C5a as a
marker is problematic
because of the rapid
clearance of C5a from
blood by C5aR-
carrying cells (WBC,
platelets, macropha-
ges, etc).
Species-independent
sensitive method.

Measure of the severity of cardiac electric, circulatory (systemic
and pulmonary), and skin changes.

Traditional method for determination of functional complement
activity. Measures the ability of the test sample to lyse 50% of a
standardized suspension of sheep erythrocytes coated with anti-
erythrocyte antibody. Both the classic activation and the terminal
complement components are measured in this reaction.
Measure increase of activation fragment C5a as measure of
formation of Terminal Complement Complex, indicating
activation of thecommon terminal pathway.

Sensitive and quantitative measurement of C3 (% consumption
of C3).

endpoint score commentmeasurement

24
Other animal models
C-activation-related pseudoallergy can be induced by i.v. injection of liposomes and other nanoparticles in many
other species, with sensitivity and symptoms differing from those seen in pigs [27y]. The sensitivity to liposomal
CARPA usually decreases in the following order: pig, dog, human (hypersensitive), rabbit, sheep, rat and mouse. The
methods of detection and endpoints are similar or the same as described above, with obvious adaptations to the size
differences among different animals. Figure 19 tabulates some characteristic features of CARPA in pigs, dogs, goats,
rats, rabbits and mice.
Figure 19
Liposome-induced CARPA in different
species: Dose dependence and symptoms.
Up and down arrows indicate the rise or fall
of the parameter specified,while the size of
arrows is proportionate with the strength of
response [42-45].
Figure 21
Scoring of Complement-Dependent Cytotoxicity reactions [47].
3.6 Testing of monoclonal antibodies for
Complement dependent cytotoxicity
Antibodies against defined cell surface antigens are being widely explored as the-
rapies for cancer and other disease. Complement-Dependent Cytotoxicity (CDC)
is a mechanism of killing cells in which the C protein C1q binds an antibody trig-
gering the C cascade, leading to the formation of the membrane attack complex
(MAC) at the surface of the target cell. The activation of the Classical pathway
results in lysis of the target cell (Figure 20).
Testing for CDC potential of monoclonal antibodies is performed by plating target cells and treating them with serial
dilutions of the antibody. Normal Human Serum Complement (Quidel P/N A112 or A113) is also added to the cells as
a qualified source of intact C. Following incubation, cell lysis is measured and used to calculate percent toxicity [46].
Protocol
• Plate target cells (Raji, WIL2-2, etc.), 50 μL of 10 X 106 cells/mL density per well
• Prepare serial 3 –fold dilutions of test antibody, Add 50 μL/well
• Dilute Normal Human Serum Complement 1:4, Add 50 μL/well
• Incubate 2 hours at 37°C (7.5% CO2 incubator)
• Measure cell lysis (using Alamar Blue, 51Cr release, etc)
Endpoints
Calculate toxicity based on percent lysis (Figure 21)
Figure 20
Complement-Dependent
Cytotoxicity.

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[31] Cavadas M.
Pathogen-mimetic stealth nanocarriers for drug delivery:
a future possibility
Nanomedicine: nanotechnology, Biology and Medicine
2011;7:730–43.
[32] Szebeni J.
The interaction of liposomes with the C-system
Crit Rev Ther Drug Carrier Syst 1998;15:57 – 88.
[30] Szebeni J, Baranyi B, Savay S, Lutz LU, Jelezarova E, Bunger
R, et al.
The role of C-activation in hypersensitivity
to pegylated liposomal doxorubicin (Doxil ®)
J Liposome Res 2000;10:347 – 61.
[33] Szebeni J, Bedöcs P, Rozsnyay Z, Weiszhár Z, Urbanics R,
Rosivall L, et al.
Liposome-induced C-activation and related
cardiopulmonary distress in pigs: factors promoting
reactogenicity of Doxil and AmBisome.
Nanomedicine NBM 2012;8:176 – 84.

26
[34] Chanan-Khan A, Szebeni J, Savay S, Liebes L, Rafique NM,
Alving CR, et al.
C-activation following first exposure to
pegylated liposomal doxorubicin (Doxil): possible role
in hypersensitivity reactions
Ann Oncol 2003;14: 1430 – 7.
[35] Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS,
Dobbins DE, et al.
Hemodynamic changes induced by liposomes and
liposome-encapsulated hemoglobin in pigs: a model for
pseudo-allergic cardiopulmonary reactions to liposomes.
Role of Complement and inhibition by soluble CR1
and anti-C5a antibody
Circulation 1999;99:2302 – 9.
[36] Colman RW, Scott CF, Schmaier AH, Wachtfogel YT, Pixley RA,
Edmunds LH, Jr. (1987)
Initiation of blood coagulation at artificial surfaces
In: Ann.N.Y.Acad.Sci. Leonard,E.F.; Turrito,V.T.; Vroman,L.;
Edtrs.p. 253-67
[37] A. Kandus, R. Ponikvar, J. Drinovec, S. Kladnik,
and P. Ivanovich.
Anaphylatoxins C3a and C5a adsorption on acrylonitrile
membrane of hollow-fiber and plate dialyzer,
an in vivo study
Int. J. Artif. Organs, 13, 176–180 (1990).
[38] Gemmell, C.H. (1997)
A flow cytometric immunoassay to quantify adsorption
of C-activation products (iC3b, C3d, SC5b-9)
on artificial surfaces
J Biomed Mater Res, 37, 474-48
[39] Harker LA, Ratner BD, Didisheim P.
Cardiovascular biomaterials and biocompatibility
CardiovascPathol 993;2(Suppl):1S –224S.
[40] Quidel.
Assays for Complement Antigens
http://quidelcom/products/product_
listphp?cat=10&by=product_family&group=2. (2013).
[41] Winkler GC.
Pulmonary intravascular macrophages in domestic animal
species: review of structural and functional properties
Am J Anat 1988;181:217 – 34.
[42] Szebeni J, Bunger R, Baranyi L, Bedocs P, Toth M, Rosivall L, et al.
Animal models of Complement-mediated hypersensitivity
reactions to liposomes and other lipid-based nanoparticles
J liposome Res 2007;17:107 – 17.
[43] Hugli TE, Morgan EL.
Mechanisms of leukocyte regulation by Complement-
derived factors
Contemp Top Immunobiol 1984;14:109 – 53.
[44] Hugli TE.
Structure and function of anaphylatoxins
Spring SeminImmunopathol 1984;7:193 – 219.
[45] Marceau F, Lundberg C, Hugli TE.
Effects of anaphylatoxins on circulation
Immunopharmacol 1987;14:67 – 84.
[46] European Pharmocopoeia 6.0. 2.6.17:
Test for antiComplementary activity of immunoglobulin
(2008).19 International
[47] Lebeck, KL and Garavoy, MR.
Histocompatibility testing and organ sharing
In: Immunogenetics Laboratory Manual, 3rd Edition,
Chapter 8 (1997).
[48] Committee for Human Medicinal Products (CHMP),
Reflection paper on the data requirements for intravenous
liposomal products developed with reference to an
innovator liposomal product.
EMA/CHMP/806058/2009/Rev. 02, 21 February 2013“

TECOmedical27
5.2 Technical information on Complement elisa’s
CIC-C1q MicroVue™ Quidel®
QUANTIFICATION OF CIRCULATING IMMUNE COMPLEXES
Cat. No. A001
Tests 96
Method ELISA
Range 10 - 30 µg Eq/ml
Sensitivity 1.0 µg Eq/ml
Incubation time 2 hours
Sample volume 10 µl (dilute 1:50)
Sample type Serum and plasma
Sample preparation Specimens should be collected aseptically and prepared using standard techniques or
clinical laboratory testing. Do not heat-inactivate the specimens. Sample may be stored at
2-8 ºC for up to 7 days. For longer periods store below -20 ºC.
Reference values One hundred six (106) sera were collected from normal, asymptomatic subjects.
The average CIC concentration was 2.1 µg Eq/mL (S.D.=1.9)
Specifity Complement-CICs are bound to immobilized to C1q-Protein

Species Human, African green monkey, Cynomolgus Macaque, Rhesus macaque, Baboon
Intended use The CIC-C1q Assay is designed for the detection of circulating immune complexes (CIC)
in human serum or plasma. CIC have been measured in a variety of conditions: infections,
autoimmune disorders, trauma and neoplastic proliferative diseases. In addition the
measurement of CIC can be important for the evaluation of certain forms of rheumatoid
arthritis and for monitoring the effectiveness of therapy.
CIC Control
Cat. No. A013
1 Set (2 levels)
5 COMPLEMENT ELISA’S
5.1 Overview of available Complement elisa’s

28
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A002
96
ELISA
5 - 48 µg Eq/ml
S4 µg Eq/ml
2 hours
10 µl (dilute 1:50)
Serum and plasma
All specimens should be collected aseptically. Do not heat-inactivate the specimens.
Samples may be stored on ice for up to 6 hours. For longer periods store below -70 ºC.
Normal ≤ 15 µg Eq/ml
Abnormal ≥ 20 µg Eq/ml
Immobilized monoclonal anti human C3 fragments capture C3d containing immune
complexes.
Human
The CIC-RCR Assay is designed for the detection of circulating immune complexes (CIC) in
human serum or plasma. CIC have been measured in a variety of conditions: infections, au-
toimmune disorders, trauma and neoplastic proliferative diseases. In addition the measure-
ment of CIC can be important for the evaluation of certain forms of rheumatoid arthritis and
for monitoring the effectiveness of therapy.

CIC-C3d MicroVue™ Quidel®
Raji-Cell-Replacement
QUANTIFICATION OF CIRCULATING IMMUNE COMPLEXES WITH C3 ACTIVATION
FRAGMENTS

TECOmedical29
Cat. No.
Tests
Method
Range
Incubation time
Sample volume
Sample type

Reference values
Specifity
Species
Intended use

A037
96
ELISA
Concentrations of Functional C1-INH are expressed as Mean Percentage. Range 35 - 100 %.
2 hours
Serum and EDTA plasma
Sample preparation: Specimens should be collected aseptically and prepared. EDTA plasma
sample may be held at room temperature (15-30 ºC) for up to 24 hours. Serum sample should
not be stored at room temperature for longer than 6 hours. If exceeded, the plasma or se-
rum must be stored frozen (-20 ºC or below). Avoid freezing and thawing of the sample.
Concentrations ≥ 68 % Mean Normal is considered normal.
C1-INH-Reactant, binds specific to functional active C1-INH.
Human, Rhesus macaque, Baboon
The C1-Inhibitor assay measures the amount of functional C1 inhibitor protein (C1-INH) in
human plasma or serum. This protease inhibitor has enzyme regulating functions. A defi-
ciency of functionally active C1-INH may lead to life-threatening angioedema. Two major
forms of C1-INH deficiency have been reported: the congenital form, termed hereditary an-
gioedema (HAE), and the acquired form, which is associated with a variety of diseases inclu-
ding lymphoid malignancies. Hereditary angioedema is characterized by transient but recur-
rent attacks of nonpruritic swelling of various tissues throughout the body.
C1-Inhibitor Plus MicroVue™ Quidel®
FUNCTIONAL C1 INHIBITOR PROTEINS

30
Cat. No.
Tests
Method
Range
Incubation time
Sample volume
Sample type

Sample preparation
Specifity
Species
Intended use

A006
96
ELISA
0.2 - 2.0 µg/ml
1.5 hours
100 µl (after dilution 1:25 – 1:200)
Serum and EDTA-plasma
The proper collection and storage of specimens is essential, since C3 is highly susceptible to
proteolysis and hydrolysis. Serum or EDTA plasma specimens should be collected aseptically
using standard techniques. They should be tested immediately or stored at 4 ºC or on ice
until assayed. This should not exceed 4 hours. For long-term storage, freeze at -70 ºC within
2 hours after collection.
Anti-iC3b monoclonal anti body specifically binds iC3B and not to C3.
Human
The iC3b enzyme immunoassay measures the amount of the iC3b fragment in human plas-
ma or serum, as well as in other biological fluids, experimental samples or mixtures.
The levels of iC3b can be significantly elevated in the serum and plasma of some patients
with complex-associated diseases such as rheumatoid arthritis and systemic lupus erythe-
matodus. iC3b levels may also be elevated in body fluids from other patients with infections,
burns, myocardial infarctions, glomerulonephritis, and acute respiratory distress syndrome.

iC3b MicroVue™ Quidel®
QUANTIFICATION OF THE IC3B FRAGMENT OF C3 PROTEIN

TECOmedical31
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A027
96
ELISA
0.06 - 0.65 µg/ml
LOD: 0.018 µg/ml
LLOQ: 0.033 µg/ml
1.5 hours
100 µl (after dilution 1:10 for plasma, 1:20 for serum)
Serum and plasma
Specimens should be tested immediately or stored at 4 ºC or on ice until assayed.This should
not exceed four hours.
For long-term storage, freeze at -70ºC within two hours after collection.
in Plasma 0.49 - 1.42 µg/ml (+2 SD)
in Serum 0.80 - 6.26 µg/ml (+2 SD)
Monoclonal mouse-antibody, specifically binds Bb.
Human, cynomolgus macaque, baboon, rhesus macaque
The Bb fragment enzyme immunoassay measures the amount of the Complement fragment
of Bb, an activation fragment of Factor B in human plasma or serum, as well as in other
biological fluids, experimental samples or mixtures. This measurement allows a quantitative
assessment of the extent of activation of the alternative pathway of Complement in the test
sample. Measurement of alternate pathway activation aids in the diagnosis of several kidney
diseases (e.g. chronic Glomerulonephritis, lupus nephritis), as well as several skin diseases
(e.g. dermatitis herpetiformis and pemphigus vulgaris). Other diseases in which activation
of the alternate pathway of Complement has been observed include rheumatoid arthritis,
sickle cell anemia, and gram-negative bacterial infections.
Bb Plus MicroVue™ Quidel®
BB FRAGMENTS OF FACTOR B OF THE ALTERNATIVE COMPLEMENT PATHWAY

32
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use
C4a MicroVue™ Quidel®
QUANTIFICATION OF THE C4A FRAGMENT
A036
96
ELISA
5 – 40 ng/ml
LOD: 0.29 ng/ml
LLOQ: 5 ng/ml
ULOQ: 61 ng/ml
2 hours 15 minutes
10 µl (dilute 1:40 for plasma, 1:80 for serum)
Human and primate plasma or serum, other biological fluids.
Sample collection is critical. Care must be taken to avoid C4a generation in the sample.
For optimal plasma results K2- or K3 EDTA collection tubes are recommended. Serum and
EDTA plasma specimens should be collected aseptically using standard techniques. Samples
should be tested immediately or stored on ice for no longer than 4 hours. For long-term
storage samples should be frozen at -70 ºC, or below.

Monoclonal mouse-antibody, specifically binds human C4a and C4a-des Arg.
Human, primate
The C4a Enzyme Immunoassay Kit measures the amount of the Complement fragment C4a,
an activation fragment of Complement protein C4 in human and primate plasma, serum and
other biological fluids. Measurement of C4a in human plasma or serum provides evidence for
the involvement of the classical or lectin pathway of Complement.
Under normal conditions, activation of the classical or lectin Complement pathways results
in the cleavage of the Complement protein C4 into C4a and C4b by the protease C1s. C4a is
rapidly cleaved to its more stable, less active form C4a-des Arg by endogenous serum car-
boxypeptidase N enzyme. Thus quantitation of C4a (C4a plus C4a-des Arg) should provide a
reliable measurement of classical or lectin pathway activation that has occurred in the test
samples.
• Rheumatoid Arthritis
• Systemic Lupus Erythematosis (SLE)
• Lyme Disease
Sample n Mean (ng/ml) Range (ng/ml)
EDTA-Plasma 32 1694.65 383.5 – 8168.17

Serum 44 1098.00 20.92 – 4437.24

TECOmedical33
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A008
96
ELISA
0.025 - 0.25 µg/ml
LOD: 0.01 µg/ml
LLOQ: 0.022 µg/ml
1.5 hours
10 µl (dilute 1:70 for normal Samples)
Serum, EDTA plasma or other biological fluids
The proper collection and storage of specimens is essential, since C4d is highly susceptible to
proteolysis. Serum or EDTA plasma specimens should be collected aseptically using standard
techniques. They should be tested immediately or stored at 4 ºC or on ice until assayed. This
should not exceed four hours. For long-term storage freeze at -70 ºC within two hours after
collection.
EDTA plasma 0.7 µg – 6.3 µg/ml (+2SD)
Serum 1.2 µg – 8.0 µg/ml (+2SD)
Monoclonal mouse-antibody, specifically binds C4d.
Human, cynomolgus macaque, baboon, rhesus macaque
The C4d fragment enzyme immunoassay measures the amount of the C4d-containing
activation fragments of C4 (C4b, iC4b, and C4d) present in human serum, EDTA plasma and
other biological or experimental samples.
The levels of C4d, when normalized for the presence of endogenous C4, can be significant-
ly elevated is plasma specimens obtained from some patients with rheumatoid arthritis,
hereditary angioedema, systemic lupus erythematosis and other illnesses. Cd4 may also be
elevated in body fluids and plasma samples obtained from patients in which classical
Complement pathway activation is known to occur, e.g. from patients with a variety of
humoral autoimmune diseases, septicemia, thermal injury, multiple organ trauma, myocar-
dial infarction, hereditary angioedema, glomerulonephritis and acute respiratory distress
syndrome.
C4d MicroVue™ Quidel®
QUANTIFICATION OF C4D-CONTAINING FRAGMENTS OF ACTIVATED C4 OF THE
CLASSICAL COMPLEMENT PATHWAY

34
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A029
96
ELISA
10 -170 ng/ml
LOD: 3.7 ng/ml
LLOQ: 8.8 ng/ml
2 hours
50 µl (dilute 1:40 for serum)
10 µl (dilute 1:10 for plasma)
Serum, EDTA plasma, spinal fluid or other biological fluids
The proper collection and storage of specimens is essential since SC5b-9 may be generated
in improperly handled specimens. Serum or EDTA plasma specimens should be collected
aseptically using standard techniques. They should be tested immediately or stored at 4 ºC
or on ice until assayed. This should not exceed four hours. For longer-term storage freeze at
-70 ºC. Plasma concentrations better reflect in vivo concentrations in comparison to serum
concentrations.
Serum 334 - 1672 ng/ml
EDTA Plasma 127 - 303 ng/ml
Monoclonal mouse-antibody, specifically binds SC5b-9.
Human, African green monkey, cynomolgus macaque, baboon, rhesus macaque, Pigtail
monkey
The SC5b-9 enzyme immunoassay measures the amount of SC5b-9 present in human
plasma, serum and other biological or experimental samples.
The Terminal Complement Complex (TCC, SC5b-9) is generated by the assembly of
C5 through C9 as a consequence of activation of the Complement system by either the
classical, lectin or alternative pathway. The membrane attack complex (MAC), a form of TCC,
is a stable complex that mediates the irreversible target cell membrane damage associated
with C-activation.
SC5b-9 Plus MicroVue™ Quidel®
QUANTIFICATION OF THE SC5B-9 COMPLEX

TECOmedical35
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A032
96
ELISA
0.05 – 5 ng/ml
LOD: 0.012 ng/ml
0.023 ng/ml
2 hours 15 minutes
10 µl (dilute 1:200 for plasma, 1:5000 for serum)
Human plasma, serum or other biological fluids.
Sample collection is critical. Care must be taken to avoid C3a generation in the sample. For
plasma, blood samples should be collected with disodium EDTA as anticoagulant and should
be centrifuged at 2000xg at 2-8 °C.
The entire operation must be completed immediately. Samples should be prepared and as-
sayed immediately or stored on ice for up to 2 hours.
For long-term storage freeze at -70 ºC with stabilizing solution.
Serum 71.0 – 589.2 ng/ml
EDTA Plasma 33.8 – 268.1 ng/ml
Monoclonal mouse-antibody, specifically binds C3a-desArg.
Human, African green monkey, cynomolgus macaque, baboon, rhesus macaque
The C3a enzyme immunoassay measures the amount of C3a-desArg in human EDTA plasma,
serum and other research samples.
Under normal conditions, activation of the classical, alternative or lectin Complement
pathways results in the formation of a C3 convertase muli-molecular enzyme capable of
cleaving C3 to C3a and C3b. C3a is a low molecular weight (approximately 9 kD) protein frag-
ment of 77 amino acids. C3a is rapidly metabolized by the serum enzyme, carboxypeptidase
N, to the more stable, 76 amino acid form, C3a des-Arg.
The quantitation of C3a des-Arg therefore provides a reliable measurement of the level of
C-activation in the test sample.
C3a Plus MicroVue™ Quidel®
QUANTIFICATION OF THE C3A FRAGMENT

36
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A025
96
ELISA
0.1 - 1 ng/ml
LOD: 0.01 ng/ml
LLOQ: 0.050 ng/ml
2 hours 15 minutes
20 µl (dilute 1:20 for plasma)
10 µl (dilute 1:50 for serum)
Serum, EDTA- and citrated plasma.
The proper collection, storage and shipment of specimens are essential. Test immediately
or stored up to 4 hours at 2-8 ºC or on ice. For long-term storage the samples should kept
frozen at -70 ºC with stabilizing solution (Cat. No. A9576), dilute samples 1:1 with stabilizing
solution.
EDTA Plasma 0.37 – 74.33 ng/ml
Serum 13.37 – 179.23 ng/ml
C5a and C5a des-Arg
Human
C5a is generated as a result of cleavage of the terminal Complement protein C5,during
activation of the Complement system via the classical, alternative or lectin pathway. C5a is
a low molecular weight (approximately 9 kD) protein fragment of 74 amino acids. C5 a is
rapidly metabolized by the serum enzyme carboxypeptidase to more stable, less active, 73
amino acid form, C5a des-Arg.
Research has associated elevated levels of fluid phase and adsorbed C5a with hemo-incom-
patibility of some biomaterials, particularly in extracorporeal circuits. Levels of C5a have also
been associated with pathogenesis of a variety of disease states, including myocardial infarc-
tion, stroke, as well as vascular leak syndrome and associated kidney injury. The role of C5a
in the pathogenesis of malaria and other infectious diseases, as well as sepsis, is likewise well
documented.
C5a MicroVue™ Quidel®
MEASUREMENT OF TERMINAL COMPLEMENT PATHWAY ACTIVATION IN EXPERIMENTAL
SAMPLES

TECOmedical37
Cat. No.
Tests
Method
Range
Incubation time
Sample volume
Sample type

Sample preparation
Reference values
Specifity
Species
Intended use

A018
96
ELISA
Appr. 0 - 300 U Eq/ml
3.5 hours
Serum ONLY. Plasma CANNOT be used.
The proper collection, storage and shipment of specimens are essential, since Complement
may be activated in improperly handled specimens. Assay immediately or keep on ice for
testing within 4 hours, up to 3 days at 4°C. For long-term storage, freeze at -70 ºC. Maximum
6 freeze/thaw cycles.
133 ± 54 U Eq/ml
The monoclonal antibody specific to terminal Complement complexes arising as the result
of the activation step in the test.
Human, cynomolgus macaque
The binding of C1q component of C1 to immune complexes triggers the classical Comple-
ment pathway. This activation results in a cascade of enzymatic and non-enzymatic reacti-
ons, culminating in the formation of terminal Complement complexes (TCC). Under standard
conditions, the level ofTCC that can be generated in serum is a quantitative expression of the
serum’s total classical Complement activity. The MicroVue CH50 Eq EIA is designed to mea-
sure the total classical Complement pathway activity in human serum samples. The measu-
rement of CH50 allows detection of deficiencies of one or more Complement components
(C1 through C9).

CH50 Eq MicroVue™ Quidel®
TOTAL CLASSICAL COMPLEMENT PATHWAY ACTIVITY

38
Cat. No.
Tests
Method
Range
Sensitivity
Incubation time
Sample volume
Sample type
Sample preparation
Reference values
Specifity
Species
Intended use
Application
A034
96 ELISA
0.05 – 2.1 ng/ml
LOD: 0.011 ng/ml
LLOQ: 0.033 ng/ml
3 hours
10 µl (dilute plasma 1:1000, serum 1:2000), 25 µl urine (dilute 1:15)
EDTA Plasma, serum, urine
Serum / Plasma: The Ba fragment of Factor B is susceptible to proteolysis. For optimal plasma
results, K2 EDTA should be used. Collect blood sample and centrifuge immediately at 2-8°C.
Assay immediately, do not store longer than 2 hours at 2-8°C. For longer storage -70°C.
Urine: Collect preservative-free first Morning void (FMV) or second morning void (SMV)
before 10:00 am. Store sample refrigerated (2-8°C) for less than 1 day, or freeze the sample at
-70°C for longer storage. Maximum 5 freeze and thaw cycles.

Monoclonal mouse-antibody, specifically to capture the Ba fragment.
Human, African green monkey, cynomolgus monkey, rhesus monkey, canine
By quantifying the amount of Ba, the extent of alternative pathway activation at the time of
sample collection can be determined.
Activation of the alternative pathway has been associated with a variety of disease states
including SLE, chronic glomerulonephritis, rheumatoid arthritis, sickle cell anaemia and gram
negative bacterial infections.
The activation of the alternative Complement pathway can be triggered by a variety of
substances including microbial polysaccharides or lipids, gram-negative bacterial lipopolys-
accharides, surface determinants present on some viruses, parasites, virally infected mam-
malian cells, and cancer cells. In autoimmune
diseases, the alternative Complement pathway may contribute directly to tissue damage.
Alternative Complement pathway activation may also be an indicator of haemo-incompati-
bility of biomaterials.
• kidney diseases
• chronic glomerulonephritis
• lupus nephritis
• skin diseases
• dermatitis herpetiformis
Ba MicroVue™ Quidel®
QUANTIFICATION OF THE COMPLEMENT BA FRAGMENT
Sample n Mean (ng/ml) Range (ng/ml)
EDTA-Plasma 35 658 226 – 2153

Serum 29 1642 436 – 3362
Urine 167 7.7 0.6 – 27

© 11/2013 | TECOmedical Group, Switzerland
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