A novel approach to IgE-sensitization of basophils and mast cells

!U N I V E R S I T Y O F C O P E N H A G E N F A C U L T Y O F S C I E N C E

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MASTER’S THESIS

A novel approach to IgE-sensitization of basophils and mast cells

Sofie Louise Degn-Petersen Biologi-Bioteknologi

Det Natur- og Biovidenskabelige fakultet - SCIENCE

Copenhagen University

Internal supervisor:

Lars K. Poulsen, PhD & DMedSci, Professor, Head of Research, Allergy Clinic, Copenhagen

University Hospital, Gentofte

External supervisor:

Per Stahl Skov, DMSc, Professor, Reflab ApS – Member of R-Biopharm Group

Submitted 01.04.2016

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Preface''

This master’s thesis ends my master degree in Biology-Biotechnology from Copenhagen University.

The thesis was carried out from March 2015 - April 2016 and comprises 60 ECTS points. The research

was performed mainly at Reflab ApS under the supervision of professor Per Stahl Skov, D.MSc. Two

months were also spent at the Laboratory of Medical Allergology, Allergy clinic, Copenhagen

University Hospital at Gentofte Hospital with Bettina M. Jensen, PhD as supervisor. Finally, two days

were spent at the Department of Dermatology and Allergy, Charité, Universitätsmedizin Berlin under

the supervision of Dr.rer.nat. Jörg Scheffel.

I would like to give a special thanks to my external supervisor Per Stahl Skov for giving me the

opportunity of being a part of the amazing working team at Reflab ApS. Further I sincerely thank you

for a year of guidance, patience and support of my project and me. I would also like to thank Sidsel

Falkencrone, Ph.D, for hours of guidance in data presentation, statistic models and for overseeing my

project. I want to thank the staff at Reflab and my two fellow master students for an unforgettable year.

I have really enjoyed working with you both academically and socially.

I furthermore want to thank my internal supervisor professor Lars K. Poulsen for being an excellent

sparring partner and for sharing his knowledge. Also, I want to thank Bettina M. Jensen for supervising

my work during my time at Gentofte Hospital. You have been an inspiration to my research and I have

really enjoyed working with you.

I also owe my thanks to professor Carsten Bindslev-Jensen for providing patient materials used in this

thesis. Furthermore, I want to thank consultant, Dr. Lene Heise Garvey, PhD and Dr. Morten Opstrup,

PhD, for the cooperation regarding the studies of chlorhexidine allergy and for providing patient

material needed for the experiments.

I want to thank Jörg Scheffel for his time during my visit in Berlin and for his inputs on my project. At

the same time I want to thank Pil Mandrup Müller, M.Sc, for preparing cell cultures and helping me

during my stay in Berlin. Additionally, I want to thank you for proofreading my assignment and for

hours of academic sparring.

Finally I want to thank Camilla Kjærgaard Hansen for creating several graphic illustrations.

__________________________________________

Sofie Louise Degn-Petersen, April 2016

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Abstract'

Passive sensitization is an in vitro assay for Immunoglobulin E (IgE)-mediated allergy detection. The

assay is mainly used to detect allergies in patients with non-responding basophils that have lost their

ability to release histamine upon IgE-mediated stimulation. Passive sensitization exploits the use of

donor basophils from buffy coat. Donor cells are treated with acid to remove most of the surface bound

IgE. The cells are then re-sensitized with patient serum or plasma and challenged with different

allergens to investigate if the patient has an allergic profile. Unfortunately, the classic method for

performing passive sensitization is not very sensitive when analyzing patients with low levels of

allergen specific IgE. Furthermore, the method has been shown to be insufficient for detecting allergy

towards some low molecular allergens that are often found in drugs. A new approach for diagnosing

patients with low specific IgE or an allergic profile for low molecular allergens is therefore needed for

passive sensitization.

A novel method called reverse passive sensitization was developed in the hope to create a more

sensitive assay. Here donor basophils were sensitized with pre-formed allergen-IgE complexes. The

method was however found to be less sensitive compared to the classic passive sensitization when

evaluated on a range of different sera. Lower sensitivity was found for both patients with low specific

IgE levels and for detection of low molecular allergens.

Following the unsuccessful implementation of a novel assay with greater sensitivity, the classic passive

sensitization assay was investigated with regards to the sensitivity to hopefully be able to optimize this.

Matrix-bound allergens located in immunoCAPs were used for allergen stimulation of sensitized cells.

This approach was however not found to increase sensitivity for either house dust mite or chlorhexidine

allergic patients. Blocking of degranulation inhibitory signalling from the IgG receptor FcγRIIb was

further examined in order to obtain higher IgE-mediated histamine release. An increased histamine

release was however not observed when sensitized cells were stimulated with allergen. An increase in

histamine release was however found upon anti-IgE stimulation of FcγRIIb-blocked cells.

Finally, a new cell source for the assay was investigated since the availability of buffy coat collected

from the blood bank is decreasing. The commercially available mast cell line LAD2 was investigated

for its applicability for the assay. LAD2 cells were able to release histamine upon anti-IgE sensitization

of hIgE-sensitized cells. Serum incubation of the cells was however found to block any release of

histamine. Passive sensitization performed on LAD2 cells was thus not possible.

Based on the work in this thesis, it was not possible to improve the existing method of passive

sensitization. A factor in serum besides specific IgE was further found to influence cell sensitization and

histamine release.

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List'of'abbreviations'

APC Antigen presenting cell

BAT Basophil activation test

CPS Classic passive sensitization

FU Fluorescence unit

HC Healthy control

HDM House dust mite (Dermatophagoides pteronyssinus)

hIgE Human IgE

HR Histamine release

IgE Immunoglobulin E

IgECH Chlorhexidine specific IgE

IgEHDM House dust mite specific IgE

IgEPE Peanut specific IgE

IgG Immunoglobulin G

ITAM Immunoreceptor tyrosine-based activation motifs

ITIM Immunoreceptor tyrosine-based inhibitory motifs

OPA Ortho-phthaliadehyde

PMA Phorbol 12-myristate 13-acetate

RPS Reverse passive sensitization

RT Room temperature

THC Total histamine content

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Table of content

Preface!.........................................................................................................................................................................!i!

Abstract!......................................................................................................................................................................!ii!

List!of!abbreviations!............................................................................................................................................!iii!

Table of content!.......................................................................................................................................................!1!

1! Introduction!........................................................................................................................................................!3!1.1! Type I allergy!............................................................................................................................................................!3!1.2! Basophils and mast cells!........................................................................................................................................!3!1.3! Allergy symptoms and treatment!........................................................................................................................!5!1.4! Allergy Diagnosis!....................................................................................................................................................!6!1.5! Basophil based allergy testing!.............................................................................................................................!6!1.6! Histamine release test!.............................................................................................................................................!7!1.7! Classic passive sensitization!.................................................................................................................................!8!1.8! Stable cell line for histamine detection!..........................................................................................................!10!1.9! Diagnosis of chlorhexidine allergy!.................................................................................................................!11!1.10! Reverse passive sensitization!..........................................................................................................................!12!

2! Aim!....................................................................................................................................................................!15!

3! Methods!............................................................................................................................................................!16!3.1! Sera!............................................................................................................................................................................!16!3.2! Buffy coat!................................................................................................................................................................!16!3.3! Stripping of buffy coat!........................................................................................................................................!16!3.4! Classic passive sensitization!..............................................................................................................................!17!3.5! Reverse passive sensitization!............................................................................................................................!18!3.6! Passive sensitization of peanut allergic patients!.........................................................................................!19!3.7! Anti-FcγRIIb blocking of basophils!...............................................................................................................!20!3.8! Passive sensitization of Chlorhexidine allergic patients!..........................................................................!20!3.9! ImmunoCAP assay!...............................................................................................................................................!21!3.10! LAD2 cells!............................................................................................................................................................!21!3.11! Data processing!...................................................................................................................................................!23!

4! Results!...............................................................................................................................................................!27!4.1! Development of the Reverse passive sensitization!....................................................................................!27!4.2! Application on clinical material!.......................................................................................................................!34!

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4.3! Chlorhexidine!.........................................................................................................................................................!35!4.4! The use of matrix-bound allergens in classic passive sensitization!.....................................................!37!4.5! Passive sensitization of LAD2 cells!................................................................................................................!38!4.6! Blocking of the inhibitory IgG receptor in passive sensitization!..........................................................!41!

5! Discussion!........................................................................................................................................................!44!5.1! Reverse passive sensitization!............................................................................................................................!44!5.2! Reverse passive sensitization compared to classic passive sensitization!...........................................!46!5.3! Chlorhexidine - an example of a low molecular allergen.!.......................................................................!47!5.4! Matrix bound allergen by immunoCAP!........................................................................................................!48!5.5! Stable cell line for passive sensitization!........................................................................................................!48!5.6! Blocking of the inhibitory IgG receptor in passive sensitization!..........................................................!49!

6! Conclusion!.......................................................................................................................................................!52!6.1! Future perspectives!...............................................................................................................................................!52!

7! Appendix!..........................................................................................................................................................!58!7.1! List of materials!.....................................................................................................................................................!58!7.2! Optimization experiments for reverse passive sensitization!...................................................................!61!7.3! Statistic data analysis!...........................................................................................................................................!62!7.4! Passive sensitization for detecting chlorhexidine allergy!........................................................................!63!7.5! Fluorescence investigations of LAD2 cells!..................................................................................................!64!7.6! Passive sensitization of FcγRIIb blocked basophils!..................................................................................!65!

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1 Introduction

1.1 Type I allergy

Experiencing an allergic reaction is very unpleasant and can in some cases be life threatening for the

patient (1). It is therefore of the outmost importance to get a quick and precise diagnosis for these

patients, preferably without causing the patient further damage. Allergic reactions are defined as

immunologically mediated hypersensitivity responses to an otherwise harmless antigen such as pollen

(2). Allergy is classified into four different types, where type I is an Immunoglobulin E (IgE)-mediated

hypersensitivity reaction. Allergen specific IgE is produced as a result of allergen exposure and act as a

recognition link between the allergen and the immune cells causing the allergic reaction.

Allergens enter the body through different routes causing various types of allergic reactions (2). They

can enter through the skin, be inhaled or ingested. Once an allergen is exposed to the immune system,

antigen presenting cells (APC) engulf the allergen and break it down to smaller fragments, which are

presented on the surface of the cell (2,3). The APC then travels to the lymph node and activate naive T

cells specific for the fragmented epitope. The activated T cells differentiate into Th2 cells that produce

cytokines and co-stimulatory signals for B cell activation. Binding of the Th2 cell to the B cell and

stimulation by secreted cytokines causes the B cell to differentiate into an immunoglobulin producing

plasma cell. Class switching to IgE secretion of the plasma cell happens through cytokine stimulation

from the Th2 cells. The half-life of free IgE is only 1-5 days, but is prolonged when bound to a high

affinity receptor on immune cells (4).

1.2 Basophils and mast cells

The primary immune cells involved in allergy type I reactions are the mast cells and basophils (5,6).

Both cells originate from hematopoietic stem cells in the bone marrow. Immature mast cells circulate in

the blood stream before entering different tissue where they mature and reside for months. Basophils on

the other hand enter the blood stream in their mature state where they circulate. They can however enter

the tissue upon inflammation. Basophils have a much shorter life span compared to mast cells and only

live for a few days (6).

Both mast cells and basophils express the high affinity IgE receptor FcεRI and have numerous IgE

molecules bound to their surface (2,6). The FcεRI receptor is composed of three subunits; the

extracellular IgE-binding ⍺-chain, the transmembrane β-chain and two γ-chains responsible for

intracellular signalling. Exposure to an allergen crosslinks cell bound IgE and causes receptor

activation, which results in down stream signalling leading to different cell responses (Figure 1). Mast

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cell and basophils contain cytosolic granules that contain preformed inflammatory mediators including

histamine, proteoglycans and proteases. Upon receptor cross-linking by IgE binding to an antigen, the

cells immediately release the preformed mediators in a process known as degranulation. The cells also

express different surface markers such as CD63 and CD203c. Neoformed mediators such as

leukotrienes, platelet activating factors and (in mast cell only) prostaglandin are also produced following

cell activation (5,7). A late phase response may occur hours after activation causing the cells to release

various cyto- and chemokines including TNF⍺ and IL-3, which support basophil differentiation (3,8).

The mast cells also release various other cytokines important for the immune response.

Figure 1: IgE-mediated activation of mast cell and basophil resulting in release of preformed and Neoformed mediators, surface marker expression and cytokine release. Selected examples of each type of response to cell activation are shown as bullet points. Camilla K. Hansen 2016.

Basophils and mast cells contain both activating and inhibitory immunoreceptors (9). Cell activating

receptors such as the high-affinity IgE receptor FcεRI and the low affinity immunoglobulin G (IgG)

receptor FcγRIIa (CD32a) contain immunoreceptor tyrosine-based activation motifs (ITAM). Upon

receptor cross-linking, the ITAMs are phosphorylated causing down stream signalling events for cell

activation. An inhibitory low affinity IgG receptor has also been discovered in both mast cells and

basophils named FcγRIIb (CD32b). This receptor contains immunoreceptor tyrosine-based inhibitory

motifs (ITIM) and was shown to have an inhibitory effect on degranulation and other cell activating

processes such as FcεRI-mediated Ca2+ mobilization and Syk phosphorylation1 (9,10). The signalling

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!1 Cross-linking of the IgE receptors recruits Lyn, which phosphorylates the receptor ITAM leading to Syk activation, which leads to downstream phosphorylation of signalling molecules resulting in Ca2+ influx and cell activation (62).

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pathway is not fully known, but studies have shown that co-aggregation between two FcγRIIb receptors

or FcγRIIb and FcεRI inhibits IgE mediated histamine release (9). It has been hypothesized that IgG

produced antibodies play a regulatory role in allergic reactions by down regulating the IgE-mediated

degranulation in mast cells and basophils (10,11). The inhibitory effect of IgG signalling could

potentially be important for both the diagnosing and treatment of allergic symptoms.

1.3 Allergy symptoms and treatment

The release of different mediators during the immediate response to cell activation causes a local

allergic inflammation (2,3). In general, blood flow and vessel permeability are increased causing

swelling and redness of the skin and helps the recruitment of additional leukocytes to the inflamed area

to eliminate the intruding allergen. Additional symptoms include bronchoconstriction, edema and

increased mucus secretion in the lung tissue and cramping, vomiting and diarrhoea in the

gastrointestinal tract. In severe cases an allergic reaction can cause systemic anaphylaxis and be life

threatening to the patient. Also, allergy can be the cause of chronic conditions such as allergic rhinitis

(hay fever), asthma and urticaria due to an on-going late-phase response.

For some allergies such as food allergies avoidance is the main way to manage an allergic profile.

Symptoms of other types of allergy such as asthma and hay fever can be reduced by inhalation of β2-

adrenoceptor agonists for bronchial smooth muscle relaxation or corticosteroids for reduced airway

inflammation (12,13). Symptom treatment by anti-histamine is also widely used for allergic symptoms

such as sneezing, itching and rhinorrhoea (runny nose) (14). Immune tolerance towards an allergen can

in some cases be obtained by immunotherapy (15,16). Patients are exposed to increasing amounts of an

allergen over a three-year period in the attempt to change their immunological response to this allergen.

The treatment has been shown to be effective in some types of allergy such as asthma and hay fever by

reducing symptoms and for some patients creating an immune tolerance (15). However, immunotherapy

is not a successful treatment for all patients. The treatment is also not risk free and can cause severe

symptoms such as anaphylactic shock.

Diagnosis of the allergic profile for each patient is important in order to choose the best treatment for

the specific patient. It is furthermore very important to provide the diagnosis immediately after the

patient shows symptoms in order to avoid further patient contact with the allergen and begin appropriate

treatment.

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1.4 Allergy Diagnosis

Several different methods for allergy diagnosis are available. Some of the most traditional allergy tests

are the in vivo skin prick test and in vitro measurements of specific IgE (1). These tests together with a

case history are the most common tests used for diagnosing food and inhalation allergies (1,16). The

skin prick test is performed by applying the investigated allergen on the skin of the patient and puncture

the skin with a needle (2). An allergic reaction will show in skin redness and an itchy swelling of the

skin around the needle puncture due to allergen exposure to mast cells in the outer layer of the skin. The

test can easily be performed and the result appears immediately and is visual for the patient.

Measurement of specific IgE is an in vitro test performed on a blood test drawn from the patient (16).

Specific IgE levels was previously measured by radioallergosorbent testing but is today mainly detected

by the ImmunoCAP assay. Here the allergen of investigation is bound to a solid cellulose phase in a

sponge-like structure. Patient serum is then added to the cellulose sponge and specific IgE will bind the

allergen if present in the serum. Non-bound IgE is then washed through the sponge and bound specific

IgE can be detected flourometrically by an enzyme-conjugated anti-human-IgE antibody. The patient

has been sensitized to the allergen of investigation if patient specific IgE level is above detection limit

of the method. It should be noted that in some types of allergies the specific IgE level could decrease in

the time after the allergic reaction has occurred (17). This is especially seen with some drug allergens to

which the patient is rarely exposed. The test therefore needs to be performed within the first weeks or

months after an allergic reaction is observed.

Several other in vivo tests besides the skin prick test exist such as intradermal skin test and different

provocation tests. The advantage of performing in vivo testing is a fast and visual response (2).

Furthermore, a provocation test also shows whether an allergen sensitization has clinical relevance

(16,18). A large disadvantage of in vivo testing is however the risk of compromising the patient by

causing a severe allergic reaction (18). Especially provocation test has the risk of causing anaphylactic

shock. Different in vitro allergy tests are therefore widely used as a risk free alternative. In vitro testing

also provides the possibility of testing for multiple allergens in one single blood sample (19,20).

Furthermore, the method enables testing with potentially harmful chemicals and toxins without

compromising the safety of the patient.

1.5 Basophil based allergy testing

Besides measuring specific IgE, the Basophil activation test (BAT) and the histamine release (HR) test

are examples of in vitro methods for allergy testing (18,20). These two methods are used to evaluate

basophil responsiveness upon allergen stimulation either by measuring basophil surface activation

markers by flow cytometry (BAT) or quantifying histamine released flourometrically (HR-test). The

BAT exploits the increased expression of surface markers such as CD63 and CD203c in response to

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allergen-IgE crosslinking (18). The principle of the BAT is that a patient whole blood sample is

stimulated with the allergen(s) of investigation and analysed for an increase in activation markers by

flow cytometry. A clear correlation between CD63 and histamine release by activated basophils is not

always observed, even though surface expression of CD63 is related to degranulation (21). Therefore,

the expression of the surface markers cannot be directly compared to histamine release but should be

considered separately. One of the main challenges using BAT is that the blood samples collected should

be used within 3 hours for optimal basophil responsiveness (18,22). However, in some cases the test can

be performed on blood samples up to 24 hours after collection. The BAT test is an interesting research

tool and can be used together with the HR-test as an in vitro test for a biological response to allergen

stimulation (19).

1.6 Histamine release test

The HR-test, also known as the Glass Microfibre-based Histamine Analysis, is an in vitro method used

for type I allergy detection (23,24). An allergic profile is investigated by stimulating patient basophils

by the allergen of investigation and detecting whether histamine is released. The method was invented

by Skov et al. in 1984-1985 as a potential large-scale alternative to the already existing conventional

leukocyte histamine release test, which was very time consuming since the technique required isolation

of leukocytes from whole blood followed by isolation of released histamine by the cells (25). With the

conventional method it was only possible to test few patients per day and the test was therefore

primarily used as a research tool (20). The results obtained with the HR-test showed to be almost

identical with the results of the conventional method (23). Furthermore, the HR-test was shown to be in

good agreement with other allergy tests such as radioallergosorbent test, skin prick test and bronchial

provocation test (23,24,26).

The major breakthrough for the HR-test came with the use of microtiter plates coated with glass

microfibers for natural histamine binding (20,27). Glass microfibers are crushed and fixed to the

bottom of a standard 96 micro well plate by water-soluble glue. The glass fibre plates were found to

bind approximately 90% of the histamine in a given solution when applied to the wells (20). This gives

the HR-test the advantage of capturing released histamine from allergen-exposed basophils to a solid

phase. The bound histamine is measured through a reaction with ortho-phthaliadehyde (OPA) dissolved

in NaOH. Histamine is released from the glass fibres by the increase in pH (28). OPA then binds

histamine and creates a highly fluorescent derivate. The reaction is stabilized by perchloric acid before it

is measured by a fluorometer (20). The OPA-histamine molecules are excited at 355nm and emits light

at 455nm, which is detected and converted into a histamine concentration by a chosen standard curve.

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Today, HR-testing is performed for large scale screening at Reflab. A whole blood sample from the

patient is mixed with the allergen of interest in the well of the glass fibre plate. The plate is then washed

and histamine released from the basophils is measured. The HR-test has however its limitations.

Approximately 6-12% of the investigated patients have non-responding basophils that have lost their

ability to release histamine upon IgE-mediated stimulation (29–32). The lack of responsiveness has been

linked to a Syk deficiency of the basophils (33,34). Non-responding basophils has been observed to

cycle in and out of responsiveness. However when the cells are unresponsive the allergic profile of the

patient cannot be established with the HR-test. A second disadvantage of the assay is the need of a fresh

blood sample collected within 24 hours (23). This requirement limits the distance for sample collection

and puts pressure on the transportation system. Furthermore, the method has been found to be less

sensitive when testing certain low molecular allergens such as some drug allergens (35).

Classic Passive Sensitization, a method based on the HR-test, has therefore been developed to test

histamine release in patients with non-responding basophils (29,30). Here donor basophils with the

ability to release histamine are sensitized with patient serum or plasma for allergy detection. This further

solves the practical difficulties with the HR-test since plasma and serum can be frozen and stored for

longer time before use (36).

1.7 Classic passive sensitization

Classic passive sensitization (CPS) is an extended version of the original HR-test and is primarily used

for patients with non-responding basophils (30,37). The method exploits the use of donor basophil cells

from buffy coat extracted from blood donation. The basophils are treated with an acid to remove most of

the cell bound IgE, a procedure known as stripping (38). Serum or plasma from the patient of

investigation is added to the cells allowing the binding of patient IgE to the donor basophils through the

unbound FceRI surface receptors. The cells are then challenged with allergen and histamine release

detection is performed as a standard HR-test (Figure 2).

Figure 2: Classic passive sensitization; 1) Donor basophils are treated with acid to remove cell bound IgE 2) Stripped basophils are sensitized by patient serum/plasma causing patient IgE to bind to the surface receptor, FceRI 3) Donor basophils are challenged with allergen 4) Histamine release from the cells is detected fluorometrically. Camilla K. Hansen 2015.

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Classic passive sensitization is mainly used for patients that are found to have non-responding basophils

in the HR-test (Reflab ApS). The method is also used in cases where patient material has longer than 24

hours of transportation e.g. samples from other countries. Passive sensitization has however been shown

to have some limitations. The method has shown to be less sensitive than the HR-test for detecting

allergy in patients with low specific IgE levels (30). In a study by Budde et al. (2002), patients allergic

to the house dust mite (HDM) Dermatophagoides pteronyssinus with allergen specific IgE levels

ranging from 1.2-4.2 kU/L were investigated with classic passive sensitization (32). They found that

patients with specific IgE levels lower than 2.6 kU/L could not be detected. Another limit of the assay

is a low sensitivity for detection of low molecular allergens (35). Such allergens are found in drugs such

as penicillin and chlorhexidine.

The use of donor basophils as the cell source for the assay also gives rise to difficulties. Firstly the buffy

coat is extracted from blood donation at the blood bank. A reduction in blood donations has led to a

limited accessibility of buffy coats and therefore it can take days or weeks to get a buffy coat sample

(Blodbanken at Rigshospitalet; Personal experience). Furthermore, a large number of donors cannot be

used for CPS since they either have non-responding basophils or an allergic profile. Finally, donor

variation between buffy coats makes the assay unstable since it is not always possible to predict whether

a buffy coat will perform optimally in the assay (Personal experience from Reflab ApS). It would

therefore be highly valuable to obtain a stable basophil or mast cell line with the same features as donor

basophils from buffy coat. The three different limits mentioned above for the classic passive

sensitization are addressed in the following sections.

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1.8 Stable cell line for histamine detection

1.8.1 Mast cell line

The first cell line that resembled human mast cells and could be used for long term research was the

HMC-1 cell line (39,40). The HMC-1 cells have been proven useful for studying different aspects of the

mast cell properties and functions. The cell line is however insufficient when investigating histamine

release as a result of antigen stimulation, since the cells do not express the FcεRI receptor (39,41).

Another mast cell line was published in 2003 by Kirshenbaum et al. designated LAD2 (40). The cell

line originates from a bone marrow aspirate taken from a patient with mast cell leukaemia. LAD2 cells

are cultured in serum-free medium containing SCF, which is needed for cell proliferation (40,42). One

of the most important properties of LAD2 cells is their expression of functional FcεRI receptor and their

ability to degranulate upon receptor activation. The cells have also shown to express the surface

receptors FcγRII and CD203c (42,43). In a study by Guhl et al. (2010) LAD2 and HMC-1 cells were

compared with human tissue mast cells (44). When examining FcεRI expression, they found that LAD2

cells and human tissue mast cells have resembling levels of ⍺- and γ-subunit expression, whereas the β-

subunit has a lower expression in LAD2 cell. When comparing the HMC-1 to these findings, they saw

that the ⍺- and γ-subunit was expressed at lower levels compared to the other cell types and that the β-

subunit was undetectable. The ability to release histamine upon FcεRI stimulation was also

investigated2. They saw an increased spontaneous histamine release from both cell lines when compared

to the human tissue mast cells. Specific histamine release due to cross-linking of the FcεRI receptor was

observed for LAD2 cells, whereas no release above baseline was observed for the HMC-1 cells.

Based on these findings, the LAD2 cell seems to have potential as an alternative for buffy coat in the

passive sensitization. Since passive sensitization is normally performed on donor basophils, the use of a

stable basophil cell line should also be investigated.

1.8.2 Basophil cell line

One of the most used humane basophil cell lines is KU812, which originates from a male patient with

blastic crisis of chronic leukemia (42,45). The cell line is cultured in media containing fetal calf serum

and proliferate independent of cytokine stimulation. KU812 has been shown to express the FcεRI

receptor to some extent and to contain histamine and tryptase. Jensen et al. (2005) performed a

comparative study of the features of KU812 and LAD2 cells. They found that KU812 expressed FcεRI.

The receptor expression was however 4-fold higher in LAD2 cells. Furthermore, they saw that cell

incubation with IgE resulted in a high upregulation of FcεRI expression in LAD2 cells. This

upregulation was however only observed to some extent for KU812 cells, which they hypothesized

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!2 Cells were sensitized with hIgE and stimulated with anti-IgE.

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could be due to a fast doubling time of 24h compared to a doubling time of two-three weeks for the

LAD2 cells (40,42). The number of FcεRI positive cells did not differ between the two cell types in

response to IgE incubation. Receptor functionality was shown by a rise in calcium levels due to anti-IgE

stimulation. Release of mediators including histamine was however not observed for the KU812 cells.

Other basophil cell lines have been produced but to my knowledge, no histamine releasing cell line has

yet been developed and no promising basophil cell line exists for passive sensitization.

Establishing a stable cell line for passive sensitization would solve many practical difficulties when

performing CPS. However, the method still has a low sensitivity for low molecular allergens found in

drug allergies such as penicillin and chlorhexidine. This difficulty has also been observed in other in

vitro tests.

1.9 Diagnosis of chlorhexidine allergy

In vitro diagnosing of IgE-mediated allergy toward different types of drugs can be difficult. Specific IgE

levels towards drugs such as penicillin and chlorhexidine decline in the time following a hypersensitive

reaction. In a study by Hjortlund et al. (2014) 26 patients allergic to penicillin showed a 50% decline in

penicillin specific IgE (IgEPE) within 1.6 month to 7 years after exposure (17). Furthermore, half of the

patients showed this decrease within less than a year. A decrease in specific IgE levels has also been

shown with chlorhexidine specific IgE levels (IgECH) (46). In both cases the decrease in specific IgE

was found to be donor dependent and did not correlate with the initial level of IgE or positivity of the

reaction. Therefore, it is very important to perform in vitro testing such as measuring specific IgE and

HR-testing within the first weeks to months after the hypersensitive reaction occur and before IgE levels

decrease below assay detection limit.

Chlorhexidine is a disinfectant used in numerous medical settings and cosmetic products (47). The

molecule binds phosphate-containing proteins of the bacterial cell wall, hereby penetrating the

cytoplasmic membrane causing cytoplasmic leaking of the cell (48). Chlorhexidine is a small molecule

of 0.5kDa and exists in water soluble forms as three different types of salts; chlorhexidine diacetate,

chlorhexidine digluconate and chlorhexidine dihydrochloride (47,49). Chlorhexidine has been found to

cause hypersensitive reactions and can in severe cases lead to anaphylactic shock (48,49). The

immunogenic epitope of chlorhexidine has been investigated in different blocking experiments with

molecules resembling parts of the chlorhexidine structure (48,50). Patient serum pre-incubated with

chlorhexidine resembling molecules such as alexidine, 4-chlorophenol and chlorguanide was tested for

specific IgE binding to chlorhexidine by radioallergosorbent test or other ELISA based methods.

Alexidine and chlorguanide were found to have some inhibiting ability while 4-chlorophenol had none.

However, since blocking with chlorhexidine itself showed much more efficient blocking, it has been

! 12!

suggested that the entire chlorhexidine molecule is complementary to the IgE antibody. It should be

noted that the immunogenic epitope could vary between patients. The practical work of this thesis

includes a further study of the immunogenic epitopes of chlorhexidine with three chlorhexidine

resembling molecules shown in Figure 3. The structure of Proguanil hydrochlorid resembles one arm of

the complete chlorhexidine molecule. 4-chloroanilide mimics the two 4-chlorophenol groups whereas

alexidine dihydrochloride mimics the remaining part of the chlorhexidine molecule.

Figure 3: Molecular structures of chlorhexidine, proguanil hydrochloride, 4-chloroanilide and alexidine dihydrochloride.

The golden standard for chlorhexidine allergy testing is using the skin prick test (46). However, in vitro

testing is the only ethical method in cases where patients have developed severe symptoms as a result of

chlorhexidine exposure such as cardiac arrest due to anaphylaxis (50). A study by Garvey et al. (2007)

showed that specific IgE measurement and histamine release following passive sensitization could be

used as alternative in vitro methods to some extent (46). Passive sensitization was however found to be

less sensitive than specific IgE measurement. Based on these findings the authors recommended that

more than one type of test should be used and compared with the case history (46).

Passive sensitization needs to be more sensitive to low molecular allergens found in many types of

drugs if the method should be used primarily as diagnosing method. The method also has the limit of

being unable to detect allergy in patients with low specific IgE. To solve both problems a novel version

of the method was invented called reverse passive sensitization (RPS).

1.10 Reverse passive sensitization

RPS is a novel method for investigating histamine release upon passive sensitization of basophils. The

method has so far only been used in a few pilot studies by the research group at Reflab and has not been

optimized or investigated for the ability to detect allergies. The principle of the method is to mix serum

or plasma from the patient with the allergen of investigation and incubate the mixture for complex

! 13!

formation of allergen and the allergen specific IgE. The IgE-allergen complexes are then incubated with

stripped donor basophils before detection of released histamine (Figure 4).

Figure 4: Reverse passive sensitization; 1) Allergen and IgE containing serum/plasma are mixed 2) Complex formation incubation 3) Donor basophils are acid treated to remove cell bound IgE 4) Allergen-IgE complexes are added to the stripped basophil donor cells 5) Histamine release detection. Camilla K. Hansen 2015.

Pilot studies of RPS have indicated that the method might be more sensitive than CPS when a patient

with low specific IgE levels towards HDM was tested. The result has however not been validated before

this thesis. The theory of why the reversed form of passive sensitization could be more sensitive than

classic passive sensitization lies within the possibility of prolonged IgE-allergen complex formation.

The handling time of the basophils is critical when performing CPS (Experience from our laboratory). If

the cells are not stored at optimal cell conditions they will be unstable resulting in degranulation and

apoptosis. The available time for the complex formation of IgE with allergen is therefore limited to one

hour. This might not be enough time if specific IgE is present in low concentrations or if the specific

IgE has a low affinity for the allergen of investigation.

Figure 5 presents the hypothesized theory of complex formation over time depending on patient specific

IgE status. If serum contains IgE with high affinity towards an allergen, or the concentration of specific

IgE is high, it is hypothesized that complex formation will happen immediately and will be able to

cross-link many IgE receptors resulting in a high level of histamine release. On the other hand if the

serum contains IgE with low affinity or the concentration of specific IgE is low, the complex formation

of IgE and allergen might take longer time to form. If such serum was to be tested after one hour of

complex formation there might not be a lot of complexes to cross-link the receptors and cause histamine

release. A prolonged incubation of the serum with allergen could theoretically enable a level of

! 14!

histamine release that would resemble the amount release from serum with high specific IgE affinity or

concentration. Reverse passive sensitization is therefore thought to have the potential of being more

sensitive for patients with low levels or affinity of specific IgE compared to classic passive sensitization.

Figure 5: Theory of IgE-allergen complex formation depending on time. The two curves represent the affinity of IgE towards an allergen or the concentration og IgE in serum which can be high (red) or low (blue). The arrows indicate the time point for maximal complex formed.

To my knowledge, the sensitization of basophils with previously complexed IgE and allergen has not

yet been investigated in vitro. IgE has however been described to exist both free and bound in different

immune complexes in the blood stream (51). These immune complexes can both be formed by binding

of auto-antibodies towards the IgE itself or by binding of different immunoglobulin classes to the same

specific allergen. It is therefore likely that cell sensitization by already formed IgE-allergen complexes

happens naturally in vivo.

! 15!

2 Aim

The aim of the thesis was to address the limitations of the passive sensitization assay, namely the poor

sensitivity of the assay when testing patients with low concentrations of specific IgE and detecting

allergic reactions towards low molecular allergens. Additionally an apparent limitation of the passive

sensitization assay is the need for a continuous and stable supply of donor basophils from buffy coats.

Initially the approach was to develop a novel method for passive sensitization designated reverse

passive sensitization. By implementing a step where IgE is pre-incubating with allergen before cell

sensitization, it was hypothesised that the assay sensitivity could be increased due to a more efficient

receptor cross-linking. Reverse passive sensitization was compared to the classic method for passive

sensitization and investigated on clinical material.

Another approach was to optimize the sensitivity of the classic passive sensitisation assay by exploiting

matrix-bound allergens in immunoCAPs for allergen stimulation of passive sensitized basophils.

Additionally, blocking of the inhibitory signal from the IgG activated receptor FcγRIIb to increase the

level of released histamine upon IgE mediated degranulation was explored.

Finally, to address the limits of available donor basophils the use of a commercially available mast cell

line was investigated for their applicability to the passive sensitization assay.

A flow diagram of the thesis work is presented in Figure 10 in the result section.

! 16!

3 Methods

A list of materials and selected apparatus used for the experiments is shown in Appendix 6.1. The list

includes product name, manufacture, catalogue number and distributor.

3.1 Sera

Sera from peanut allergic patients were generously provided by Carsten Bindslev-Jensen (Odense

Universitetshospital). All sera were ethically approved for research purposes. Furthermore, sera from

chlorhexidine allergic patients were kindly provided by Lene Heise Garvey and Morten Obstrup

(Copenhagen University Hospital at Gentofte Hospital). All experiments performed with these sera are

well described in an additional ethical protocol for existing projects at the department of the Laboratory

of Medical Allergology, Allergy clinic, Copenhagen University Hospital at Gentofte Hospital. Sera

from house dust mite allergic patients and healthy control subjects were willingly given from the staff at

Reflab Aps and collected as described. A blood sample was collected and left at room temperature (RT)

for 30-60min for proper blood clotting. The sample was centrifuged for 10min at 2000G 20°C. Serum

separated from the remaining blood sample was saved and kept at -20°C until used.

3.2 Buffy coat

Buffy coat was collected at Rigshospitalet bloodbank and screened for nine of the most common

allergies by a standard HR-test (Reflab ApS). In short, 1mL blood was incubated with 4mL Pipes buffer

(Ampliqon) and IL-3 (2ng/mL, R&D systems Europe Ldt.) for 20min at 37°C and added to a

reconstituted standard screening microfiber plate (Reflab Aps) before analysed as described in section

3.4. The buffy coat was also screened for non-standard allergens if used for passive sensitization to that

specific allergen. Anti-IgE (KPL) response was further investigated to assess the cells ability to release

histamine through the FcεRI pathway. 12.5mL selected buffy coat was mixed with 20mL RPMI 1640-

media (Sigma-Aldrich) and IL-3 (10pg/mL) in a 50mL centrifugation tube. The cells were kept at 5°C

until used.

3.3 Stripping of buffy coat

Stored buffy coat was washed twice by adding NaCl2 (9mg/mL, Region Hovedstadens Apotek) to a final

volume of 50mL followed by centrifugation at 1000G for 5min at 11°C and removal of the supernatant.

5°C cold Stripping buffer (Ampliqon) was added to a final volume of 50mL. The sample was

centrifuged as before and the supernatant was removed. The cells were washed in Pipes buffer,

centrifuged as previously and resuspended in Pipes buffer to a final volume of 12.5mL.

! 17!

3.4 Classic passive sensitization

Sensitization

Stripped buffy coat was mixed with patient serum as shown in Table 1. A sample with serum from a

healthy control (HC) donor was included. All samples were incubated at 37°C for 1h after which the

cells were diluted in Pipes buffer as shown in Table 1.

Final volume Buffy Coat Serum Incubation Pipes buffer

1815µl (48wells) 250µl 65µl 37°C 1h 1.5mL

3630µl (96 wells) 500µl 130µl 37°C 1h 3mL

7260µl (192 wells) 1000µl 260µl 37°C 1h 6mL

Table 1: Ratio between buffy coat, serum and Pipes buffer for sensitization reactions of different volumes.

Allergen challenge

A histamine glass microfiber plate (Reflab ApS) was prepared by adding 25µl Pipes buffer to non-

sample wells. The allergen of investigation was diluted to different concentrations in Pipes buffer and

25 µl was added to selected wells in the plate. Samples containing Pipes buffer or anti-IgE (5µg/mL)

were included as sample background and positive control, respectively. 25µl of serum sensitized buffy

coat was added to the wells of the glass microfiber plate. The plate was incubated at 37°C for 1h.

Histamine detection

The microfiber plate was washed once using a microplate washer (Reflab ApS). 150µl SDS (0.4%,

Region Hovedstadens Apotek) was added to each well and the plate was incubated at 37°C for 30min

and washed twice by microplate washer. Histamine was released from the glass microfiber by adding

75µl 0.5mg/mL OPA (Reflab ApS) dissolved in NaOH (50mM, Ampliqon) to the plate, incubating it for

10min at RT and adding 75µl HClO4 (0.59%w/w, Ampliqon). This was performed using a multidrop

(Thermo Scientific). Finally the histamine release was measured flourometrically by a Histareader 501

(Reflab ApS).

3.4.1 Total histamine content

Total histamine content (THC) of the basophils was measured by diluting stripped buffy coat in Pipes

buffer to the same cell concentration as the investigated sample. 200µl blood sample was mixed toughly

with 60µl HClO4 (7% w/w, Region Hovedstadens Apotek) and incubated for 30min at 37°C. 200µl was

added to the sample before centrifugation at 9800G for 5min. 25µl supernatant was transferred to a

reconstituted microfiber plate for quantitative detection of histamine (Reflab ApS). The plate was

prepared by adding 25µl Pipes buffer in all wells and 25µl HClO4 (0.9% w/w) to non-sample wells.

! 18!


All experiments performed to optimize and investigate RPS compared to CPS were performed on two

sera from patients allergic towards the house dust mite Dermatophagoides pteronyssinus HDM with

specific IgE (IgEHDM) of 0.79kU/L and 9.2kU/L3 unless otherwise described. These sera were chosen

since they were accessible at all times and since their characterization in histamine release tests are well

known (Reflab Aps). Specific IgE levels had been determined by the immunoCAP assay. The HDM

allergen Dermatophagoides pteronyssinus extract (GREER allergy immunotherapy) was used as

allergen stimulation for the two patients. A flow diagram of the RPS is shown in Figure 6.

Figure 6: An overview of reverse passive sensitization shown in the three main phases; Complex formation, Sensitization and Histamine detection.

3.5.1 Original protocol

Complex formation

The allergen of investigation was diluted to several concentrations in Pipes buffer to obtain an

appropriate concentration range for HR-testing. 10µl allergen dilution was mixed with 10µl patient

serum and incubated at 5°C for 24h.

Sensitization

50µl stripped buffy coat was added to each sample followed by incubation at 37°C for 1h. Samples were

subsequently diluted in 250µl Pipes buffer.

Histamine detection

A standard histamine glass microfiber plate was prepared by adding 50µl Pipes buffer to non-sample

wells. The remaining wells intended for cell samples received 25µl Pipes buffer. 25µl sample was

transferred to the prepared microfiber plate. The plate was incubated at 37°C for 1h, before the

histamine content was detected as described in section 3.4.

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!3 1kU/L IgE = 2.4ng/mL IgE (3)

! 19!

3.5.2 Optimized protocol

Complex formation

A serial dilution of the allergen of interest specific for each experimental setup was made by addition of

Pipes buffer. 25µl allergen dilution was added to a 96 microwell plate (450µl, Thermo Scientific)

together with 25µl patient serum (serum can be diluted 50%). A sample with HC serum was included in

the study as a negative control. Further samples without serum and/or allergen were prepared. Cell

activity was measured by stimulation with Anti-IgE (5µg/mL). The plates were incubated at 5°C for 1h.

Sensitization

50µl stripped buffy coat was added to each well and the plate was incubated at 37°C for 1h. All samples

were diluted in 250µl Pipes buffer.

Histamine detection

The content of the microwell plate was mixed on a microplate shaker (Flow Laboratories DSG-Titertek)

for 30s. 25µl of all samples was transferred to a prepared (as described in the original protocol) standard

histamine glass microfiber plate. The histamine plate was incubated at 37°C for 1h, before histamine

detection was performed as described in section 3.4.

3.5.3 Sample filtration

In some experiments RPS samples were filtrated before added to a microfiber plate. Sample filtration

was performed with a Viva spin 0.5mL concentrator with 3kDa or 5kDa molecular weight cut-off value

(Viva science Sartorius group). Blood cells were removed from the sample by centrifugation before

filtration. 200µl samples were added to the concentrator following centrifugation at 9800G for 5min

(RT). The flow through was used for histamine detection as described in section 3.4.

3.6 Passive sensitization of peanut allergic patients

CPS and RPS was performed on 23 sera from peanut allergic patients with IgEPE ranging from 1.8-

252kU/L. CPS was performed as described in section 3.4 and RPS was performed as described in

section 3.5.2 with a serum concentration of 50%. The start concentration of peanut allergen (Arachis

hypogaea, GREER) was 90ng/mL. Allergen for CPS stimulation was diluted to 12 different

concentrations by 3.5-fold dilutions (concentrations are shown in Table 2). Allergen for RPS was

diluted to five different concentrations by 10-fold dilutions (concentrations are shown in Table 2). The

lowest allergen concentration causing a specific HR compared to the HC serum was evaluated for all

patients with both methods and grouped into HR classes according to concentration as shown in Table

2. This is explained further in the data processing, section 3.11.

! 20!

CPS dilutions RPS dilutions Allergen conc. (µg/mL) HR class C1 C1 90 1

C2

30 C3 C2 9 2 C4

3

C5 C3 0.9 3 C6

0.3

C7 C4 0.09 4 C8

0.03

C9 C5 0.009

5 C10

0.003 C11

0.0009

C12

0.0003

Table 2: CPS and RPS performed with different peanut allergen concentrations (C) grouped into HR class. CPS was performed with 12 allergen concentrations (3.5-fold dilutions) and RPS with 5 allergen concentrations (10-fold dilutions). Allergen concentration (µg/mL) is shown for all dilutions and further grouped into five HR classes.

3.7 Anti-FcγRIIb blocking of basophils

CPS was performed with the anti-FcγRIIb antibody [AT10] (Abcam®). Anti-CD23b was diluted in

Pipes buffer to a stock concentration of 1mg/mL and stored in aliquots of 2.5µl pr. tube at -20°C. Thaw

and freeze cycles were avoided for all experiments. CPS was performed as described in section 3.4 with

and without anti-FcγRIIb (2.5µg/µl) in the sensitization step. The assay was performed on two HDM

allergic patients (IgEHDM 0.79kU/L and 9.2kU/L) and one HC serum.

3.8 Passive sensitization of Chlorhexidine allergic patients

Serum samples from nine chlorhexidine allergic patients were used in different setups for RPS, CPS or

both performed as previously described. All nine patients were previously diagnosed positive for

chlorhexidine allergy by skin prick test in combination with a case history. The allergens used were

chlorhexidine (Sigma-Aldrich), Instillagel (Lidocainhydrochlorid 20mg/g + chlorhexidingluconat

0.5mg/g, Farco-Pharma GmbH), Chlorhexidine digluconate solution (Sigma-Aldrich), chlorhexidine

dihydrochloride (Sigma-Aldrich), chlorhexidine acetate (Sigma-Aldrich), Proguanil hydrochlorid

(Sigma-Aldrich), Alexidine dihydrochlorid (Sigma-Aldrich) and 4-chloroanilide (Sigma-Aldrich). All

allergens except instillagel were tested in a concentration range from 0.05mg/mL to 0.05ng/mL with a

dilution factor of 3.5 between the different concentrations. Instillagel was tested from 0.01mg/mL to

0.01ng/mL referring to chlorhexidigluconat content of the gel (3.5-fold dilutions).

IgE binding properties of chlorhexidine resembling molecules

A mix of Proguanil hydrochlorid (0.05mg/mL), Alexidine dihydrochlorid (0.05mg/mL) and 4-

chloroanilide (0.05mg/mL) was made (Experimental mix). Serum was pre-incubated with experimental

mix (360µg/mL) or Pipes buffer for 1h at 5°C before classic passive sensitization was performed as

! 21!

described in section 3.4 with chlorhexidine concentrations of 0.05mg/mL-0.05ng/mL in a 3.5 fold

dilution. A HC serum was included in the study. The individual experimental molecules were also tested

by pre-incubating serum with Proguanil hydrochlorid (360µg/mL), Alexidine dihydrochlorid

(360µg/mL), 4-chloroanilide (360µg/mL) or Pipes buffer.

3.9 ImmunoCAP assay

Buffy coat was stripped as described in section 3.3 and used for passive sensitization with 12 different

sera. Serum originated from nine patients with an allergic profile of chlorhexidine, two patients with

HDM allergy and one HC subject. 65µl serum and 500µl stripped buffy coat was incubated for 1h at

37°C following the addition of 1 mL Pipes buffer (Reflab ApS). 40µl of each sample was placed on a

prepared immunoCAP sponge (Thermo Fisher Scientific) containing glycine, anti-IgE and

chlorhexidine or HDM dependent on the allergic profile of the tested serum. The HC was tested for both

allergens. The immunoCAP was prepared by vacuum filtering 200µl Pipes buffer 10 times through the

sponge by a vacuum pump (Biorad) followed by centrifugation at 600G for 10sec. For measuring total

histamine release 40µl sample with 0.3ng/µl Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich)

and 20ng/µl ionomycin (Sigma-Aldrich) was furthermore added to a second glycine immunoCAP. All

immunoCAPs were incubated for 30min at 37°C in a moist chamber (closed plastic box with wet gauze

in the bottom). The immunoCAPs were further incubated 5min with 100µl Pipes buffer. The

immunoCAPs were centrifuged at 300G for 5min at room temperature (Acceleration 5, break 3) and

flow-though was collected in a 96-well v-shaped plate (Greiner bio one). 50µl of the supernatant of the

flow though was transferred twice to a histamine plate prepared by addition of 50µl Pipes in non-sample

wells. The histamine plate was incubated for 30min at 37°C before it was analyzed as described in

section 3.4

3.10 LAD2 cells

3.10.1 Cell medium and culturing

LAD2 medium was prepared by adding 200mL StemPro®-34 SFM medium (Life Technologies), 5.2mL

StemPro®-34 SFM supplement (Life Technologies), 2mL L-glutamine (2mM, Sigma-Aldrich), 2mL

Penicillin:Streptomycin (10.000U/mL:10.000µg/mL, Sigma-Aldrich) and 200µl rhSCF (100µg/mL,

Peprotech) to a 250mL stericup-GP with a 0.22µm filter (Milipore). The medium was vacuum filtrated

and stored at 2-8°C until use. LAD2 cells were kindly provided by Dr. A. Kirschembaum (National

Institute of Health) and were thawed in pre-warmed LAD2 medium. The cells were isolated by

centrifugation at 300G for 10min at RT and resuspended in LAD2 medium. Cell number and viability

was assessed by mixing 50µl cell culture with 50µl tryphan blue (Sigma-Aldrich) and placing the

sample in a hemocytometer. The cells were counted manually by using a light-microscope. Cell

concentration was adjusted to 2.5-4·105 cells alive/mL by further addition of LAD2 medium. The cells

were transferred to a culture flask at a surface density of 5-10·104 cells/cm2. The flask was incubated at

! 22!

37°C with 5% CO2, humidified. Cell media was changed once a week by harvesting the cells (300G,

10min, RT) and resuspending them in a new incubation flask with pre-warmed LAD2 medium at the

concentration and surface density previously described. The cells were used for experiments after 6-13

weeks of culturing.

3.10.2 Anti-FcεRI activation of LAD2 cells

1.4·105 cells were isolated from a LAD2 culture by centrifugation at 300G for 10min at RT. The cells

were resuspended in 1.4mL HR buffer (Pipes + 0.5% Human serum albumin (CSL Behring, USA)).

50µl of the cell culture was mixed with 50µl CRAI anti-FcεRI antibody (Biolegend) in final

concentrations of 10-1000ng/mL in a 96 well V-shaped cell plate. The cell plate was incubated at 37°C

for 1h.

Supernatant and lysing microplate preparation

HR and total histamine content of each sample were investigated as illustrated in Figure 7. The cell plate

was centrifuged 300G for 10min at 11°C. 25µl supernatant was transferred to a standard microfiber

plate prepared by addition of 25µl Pipes buffer to all wells. The microfiber plate was incubated at 37°C

for 30min, washed in deionized water and kept dry and dark. The cell plate was further used for

estimating total histamine content by adding 25µl HClO4 (3.5%, Reflab ApS) to all wells. The cell plate

was incubated for 30min at 37°C. 100µl Pipes buffer was added to each well and the cell plate was

centrifuged at 1000G for 10min at 12°C. 25µl supernatant was transferred to a standard histamine

microfiber plate prepared by addition of 25µl Pipes buffer to all wells. The histamine plate was

incubated at 37°C for 30min. Histamine detection was performed on all histamine plates as described in

section 3.4

Figure 7: HR detection of cell supernatant directly from sample and from the sample after cell lysing by acid treatment and further buffer dilutions. Modification of figure by Bettina M. Jensen (2016).

3.10.3 Detection of IgE receptor on LAD2 cells

LAD2 cells were harvested and resuspended in warm LAD2 medium to obtain a concentration of 4·105

cells/mL. 1mL cell culture was incubated in a 12 well culture plate O/N with 1mg/mL human IgE

(Calbiochem). The cells were washed in 15mL HR buffer and resuspended to 1·105 cells/mL. 2·105 cells

were washed in cold MACS buffer (500mL PBS (Sigma-Aldrich), 12.5mL HSA (20%), 2mL EDTA

0.5M (RH apotek)) to a final volume of 1.5mL. The cells were centrifuged at 300G, 5min, RT and

resuspended in 200µl cold MACS buffer for a final concentration of 1·106 cells/mL. 100µl cell

! 23!

suspension was incubated 30min at 4°C with either 5µl PE anti-human IgE (Biolegend) or a PE mouse

IgG1, k antibody (Biolegend) as isotype control. 0.5mL MACS buffer was added to both samples

following centrifugation for 5min at 300G at 10°C. The cells were resuspended in 100µl MACS buffer.

A glass slide was inserted into a cytofunnel (Thermo Scientific) and placed in a cytospinner (Thermo

Scientific). 100µl cell solution was placed on the cytofunnel and transferred to the glass slide by

centrifugation at 41G for 5min. A vector shield (Vector Laboratories) was stained with DAPI

(Invitrogen) 1:10 and was placed on the slide, which was analysed on a Zoe fluorescence cell imager

(Biorad).

3.10.4 Serum incubation of LAD2 cells

Serum from four patients with different IgE levels and known allergy status was diluted in pre-warmed

LAD2 medium to obtain a serum concentration of 20% and 50%. 100µl LAD2 medium, 20% or 50%

serum dilution was transferred to a 96 well culture plate. LAD 2 cells were harvested by centrifugation

at 300G for 10min (RT) and resuspended in warm LAD2 medium to a final concentration of 1·106

cells/mL. 100µl cell suspension was added to each well on the culture plate and incubated at 37°C, 5%

CO2, humidified overnight. Each culture was thoroughly inspected in a light microscope and cell

viability was assessed manually by a tryphan staining and hemocytometer. Each cell culture was moved

to a centrifugation tube and pelleted by 300G for 10min at RT. 25µl supernatant was transferred to a

standard histamine plate prepared by addition of 25µl Pipes in sample wells and 50µl Pipes in non-

sample wells. LAD2 medium with serum (10% or 25%) was also included as background measurement.

The plate was incubated at 37°C for 30min and histamine detection was performed as described in

section 3.4.

3.10.5 LAD2 sensitization and histamine release

LAD2 cells were harvested by centrifugation at 300G for 10min at RT and resuspended in warm LAD2

medium to a final concentration of 1·106 cells/mL. 0.5mL cell culture was sensitized with 0.5mL LAD2

medium containing serum (25%), hIgE (1µg/mL), mIgE-DNP (1µg/mL, Sigma-Aldrich) and anti-

FcγRIIb (7.5µg/mL) in different combinations. The plate was incubated O/N, before washed in 10 mL

HR buffer, centrifuged 300G, 10min, RT and resuspended in 5mL HR buffer. 50µl cell suspension was

added to a 96 well V-shaped plate containing 50µl stimuli consisting of either anti-IgE (8-2000µg/mL,

KPL) or DNP-HSA (Sigma-Aldrich), HDM allergen (0.03-10µg/mL, GREER allergy immunotherapy)

or PMA:Ionomycin (8-2000 µg/mL:4-1000µg/mL). All stimuli were diluted in HR buffer. The cell

plates were incubated 37°C for 1h before analyzed as 3.10.2 Anti-FcεRI activation of LAD2 cells.

3.11 Data processing

Fluorescence data from histamine detection using the HR-test system was evaluated by a standard curve

of four measurements of 0 and 50ng/mL histamine integrated in each histamine microfiber plate as

shown in Figure 8. The setup of the histamine microfiber plate included empty wells in row A and B.

! 24!

Row A was used as rinse wells and row B was used for background measurements and as an estimation

of the assay variation. The remaining wells on the plate were used for samples.

Figure 8: Standard histamine plate (Reflab ApS). Setup: Column 1) Histamine standard curve (ng/mL), Row A) Rinse wells, Row B) Assay background. The remaining wells is for sample application.

The fluorescence unit (FU) pr. ng histamine and assay background was calculated in order to find the

histamine in ng/mL present in each sample well. The calculations were performed in three steps:

• FU pr. ng histamine = (mean (50ng/mL containing wells) – mean (0ng/mL containing wells))/50

• Assay background FU = mean (rowB)

• Histamine content of sample = (Sample FU - Assay background FU) / FU pr. ng histamine

The general detection limit of the assay is 10ng/mL histamine (Reflab ApS). This means that if a

microfibre plate is analyzed several times with day-to-day variation, the assay background values will

be ≤10ng/mL. 10ng/mL histamine is furthermore the lowest concentration used for calibration of the

fluorometer (Histareader). Sample data below 10ng/mL histamine needs to be evaluated carefully.

When data was evaluated clear outliers were removed from the data set. Clear outliers were assessed

manually and validated by double samples and/or dose response curve before they were removed from

the data set.

Passive sensitization is used to test patients positive or negative of a given allergen. A HC serum with

no specific IgE for the investigated allergen was always included to evaluate whether the histamine

released was specific for the allergen. A positive outcome relies on two parameters: A) if a dose-

respond curve is observed in response to increase in allergen concentration and B) if the HR observed

from the patient is significantly higher than for the HC serum. A significant HR is calculated as the

histamine detected for the HC serum plus three times the assay background of the plate. Patient samples

with significantly more HR than the HC sample but with a HR value lower than the detection limit of

the assay were defined as borderline positive. This definition was used because the assay variation of

that specific plate was found to be much lower than the general assay variation. These results are

marked in the result section and interpreted with extra care.

! 25!

The HR data are illustrated in different ways throughout the results part depending on the experiment

and wanted take-home massage. THC and maximal HR were sometimes measured to be able to

compare data performed on different basophil donors and cell amounts. Below is a list of histamine

designations shown as an overview over the different data forms:

• HR (ng/mL): Histamine content of the wells when converted from FU to ng/mL.

• HR% of total content: the percentage of sample histamine compared to total histamine content of

the cells found by cell lysing with HClO4.

• HR% of max release: the percentage of the sample histamine compared to histamine release induced

by PMA-ionomycin stimulation.

• HR classes: histamine released according to allergen concentration grouped into classes. The HR

class shows the lowest concentration to which a significant HR is obtained as illustrated below:

HR-class 0 Negative to all allergen concentrations

HR-class 1 Positive to C14

HR-class 2 Positive to C1 and C2

HR-class 3 Positive to C1, C2 and C3 ect.

THC of samples was measured in two ways. For CPS and RPS using basophils, one THC was measured

as described in section 3.4.1. Since the THC was measured on a sample with the same basophil

concentration, data was directly compared to this value.

When measuring THC for passive sensitization using LAD2 cells an individual value was measured for

each sample as described in section 3.10.2. Calculation of HR as a percentage of total histamine content

is illustrated in Figure 9. In short, histamine content was presented as ng/mL for both supernatant and

lysed samples. The removed 25µl sample for supernatant measurement was then taken into account

before comparing the HR with THC.

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!4 C1 is the highest tested concentration; C2 is lower in concentration, etc.

! 26!

Figure 9: Calculation of HR as a percentage of total histamine content in LAD2 assays by comparing supernatant samples with lysate samples. Bettina M. Jensen (2016).

Statistical data analyses were performed on selected experiments where the same experimental setup

was used with more than three buffy coat donors. The analyses were made using SigmaPlot (v 11.0,

Systat Software, San Jose, USA). Data for the different buffy coat donors were either tested individually

or pooled according to serum profile. Data were evaluated for normal distribution by the Shapiro-Wilk

normality test. If data were normal distributed, a t-test was performed. For not normally distributed data

the Wilcoxon Signed Rank Test was used.

! 27!

4 Results

The overall aim of the experimental work included in this thesis was to optimize the sensitivity of the

passive sensitization assay. The initial aim was approached by exploring a novel method for passive

sensitization called reverse passive sensitization. Subsequently new approaches for optimizing the

classic passive sensitization assay were explored as well. Figure 10 presents the experimental

approaches in a flow diagram. RPS was initially developed and optimized. The assay was then

compared to CPS and investigated for clinical application. Different approaches were then investigated

for the optimization of the CPS including stimulation with matrix-bound allergen, the use of a stable cell

line and blocking of the IgG receptor FcγRIIb.

Figure 10: Flow diagram of the result presentation. The main goal was to optimize passive sensitization for allergy detection. The results are shown in two main groups; 1) RPS including development of the method, comparison to CPS and application on clinical material. 2) CPS including investigations of matrix-bound allergens, a new cell source and blocking of FcγRIIb.

4.1 Development of the Reverse passive sensitization

RPS was developed in the search for a method more sensitive than CPS for patients with low specific

IgE. When performing CPS the donor basophils are initially sensitized with IgE and then exposed to the

allergen of investigation. On the other hand, RPS exploits pre-incubation of allergen and IgE to form

complexes before sensitizing the donor cells. This provides a prolonged time for complex formation and

is hypothesized to make the assay more sensitive when low amounts of specific IgE are present in the

serum.

The following section displays some of the most important findings from the development of the

method. A table of the main optimization experiments performed in this study are listed in Appendix 6.2

showing different parameters investigated and display the overall work flow. All experiments performed

to optimize the RPS assay were performed on serum from two HDM allergic patients. One serum

! 28!

contained 9.2kU/L IgEHDM and the other serum contained 0.97kU/L IgEHDM. The sera will be referred to

as the patient with high and low IgEHDM, respectively.

4.1.1 Optimizing of the complex formation

Based on the findings in initial experiments, the volume for the complex formation was chosen to be

50µl by mixing 25µl allergen with 25µl serum. This both minimized the amount of serum needed for

the assay while avoiding sample variation due to pipetting errors (data not shown). The optimal

incubation time point and temperature was investigated for both HDM allergic patients. The result for

the patient with high IgEHDM is shown in Figure 11. The results showed a small decrease or no

difference in maximal histamine release as the time of incubation was prolonged. For each time point a

decrease of approximately 5-10% HR was detected as the temperature was raised from 5°C to room

temperature and from room temperature to 37°C. The optimal time point and temperature for complex

formation was found to be 5°C for 1h. No HR above detection limit was observed for the patient with

low IgEHDM. Two additional experiments were performed with incubation of either 1h or 24h at 5°C. No

difference was observed between the two time points (data not shown).

Figure 11: RPS performed on serum from the patient with high IgEHDM. Specific histamine release (%) of total content is shown for incubation time 1, 6 and 24h in combination with incubation temperature 5°C (white bars), Room temperature (RT, grey bars) and 37°C (black bars). Standard deviations (SD) illustrates sample variation of duplicates.

4.1.2 Reverse passive sensitization performed with varying serum concentrations using six different

buffy coats donors.

RPS was performed on six different buffy coats with two HDM allergic patients and serum

concentrations in a range of 0-100%. One of the buffy coats differed from the remaining by a poor

histamine release when RPS was performed with the positive sera from the patient with high IgEHDM

levels. The only histamine released from this buffy coat above detection limit was found when using a

Complex formation at different time points

Time (hours)

1h 6h 24h

HR

of t

otal

con

tent

%

0

10

20

30

40

50

60 5 CRT37 C

! 29!

serum concentration of 100%. The buffy coat was excluded from the comparative study since a dose-

response curve above detection limit was not observed.

The results of all the RPS experiments are shown in Figure 12 together with the results of the five

selected buffy coats pooled. Statistic data analysis was performed on the data to evaluate whether HR

was significantly different between the tested serum concentrations. P-values are shown in Appendix

6.3 table A.

Figure 12: RPS performed on six different buffy coat donors. Serum containing IgEHDM levels of 0.79kU/L (red) and 9.2kU/L

(green) was used in a concentration range of 0-100%. HR is shown in percentage of total histamine content. A) Individual HR

(n=6); B) Pooled HR (n=5). DL = detection limit based on general assay cut off.

RPS performed on the patient with low specific IgE levels caused a mean HR of 2-4% (red). No

significant difference was seen in HR between any of the different serum concentrations used. A mean

HR response of 40-60% was detected for the patient with high IgEHDM (green). All samples with serum

had a significantly higher HR than when no serum was added to the assay. Maximal histamine release

was observed when 50% or 100% serum was used. No significant difference was found between the two

concentrations. RPS performed on the patient with high IgEHDM on the six individual buffy coats,

including the one below detection limit, was further evaluated. The maximal histamine release was

found at serum concentration 50% in two buffy coat donors whereas the remaining four buffy coats had

maximal histamine release at 100% serum. Based on these findings, a serum concentration of 100%

would be recommended for this assay. A serum concentration of 50% can however be used if less serum

is available from the patient.

4.1.3 Buffy coat dilution

One of the varying factors when performing passive sensitization on different buffy coat donors is the

leukocyte count, which is not estimated for each donor sample. Different buffy coat dilutions were

therefore investigated for the patient with low IgEHDM to examine whether a change in cell concentration

Individual

Serum conc. (%)

0 20 40 60 80 100

HR

of t

otal

con

tent

%

0

20

40

60

80

Low IgEHDM

High IgEHDM

DL

Pooled

Serum concentration (%)

0 20 40 60 80 100

HR

of t

otal

con

tent

%

0

20

40

60

80

Low IgEHDM

High IgEHDM

DL

***

****** ***

a) b)

! 30!

could make the assay more sensitive for patients with low specific IgE levels. RPS was performed on

two different buffy coat donors. Buffy coat and serum dilutions were tested in a range of 0-100%. The

result is shown in Table 3 for both buffy coats as HR%. The maximal HR was found when using

undiluted buffy coat in both donors. The results furthermore showed values under the detection limit of

the assay when the buffy coat was diluted 12.5-25% (indicated with a “#” in Table 3). Maximal specific

HR was seen with serum concentration 50% and 25-50% for the buffy coat donors 1 and 2, respectively.

Based on these results, the optimal cell concentration is found when buffy coat is not diluted.

Buffy coat dilutions

12,5% 25% 50% 100%

Buffy coat 1 25% serum 0,9 # 0 # 0 0

50% serum 1,1 # 0,6 # 2,5 7,3

100% serum 0 1,1 0 2,3

Buffy coat 2 25% serum 0,7 3,5 3,5 9,4

50% serum 3,0 # 2,4 # 3,9 9,3

100% serum 1,2 4,1 2,3 5,8

Table 3: RPS performed with buffy coat conc. 12.5%-100% in combination with serum conc. 12.5%-100%. The analysis was performed with two different buffy coats on the patient with low IgEHDM levels. Data are shown as histamine release in percentage of total cell content. (#) indicates data below detection limit of the assay.

4.1.4 Interference and unspecific histamine release

High unspecific HR was observed when RPS was performed without allergen stimulation. This HR was

believed to be due to serum interference, spontaneous HR from cells or possibly unspecific serum

induced HR. The cause of unspecific HR was addressed in the following experiments in the attempt to

decrease interference in the assay and hereby be able to detect the patient with low specific IgE.

Figure 13: RPS performed on the patient with low IgEHDM levels. Buffy coat conc. 12.5%-100% in combination with serum conc. 12.5%-100% was tested. SD illustrates variation between duplicate samples.

RPS without allergen challenge

Buffy coat dilution

No cells 12,5% 25% 50% 100%

His

tam

ine

dete

ctio

n (n

g/m

L)

0

5

10

15

20

No serumSerum 25%Serum 50%Serum 100%

DL

! 31!

The main part of the unspecific HR was found to be caused by serum in a dose dependent manner

(Figure 13). Data from the experiments with different buffy coats and serum concentrations explained in

the previous section were evaluated to identify the source of unspecific HR more closely. Histamine

detected in samples without allergen stimulation is illustrated in Figure 13. Histamine detection was

observed from cell-free samples prepared with different serum concentrations and was found to increase

with increasing amounts of serum. Serum concentrations of 100% with no cells present resulted in

histamine detection of 12.8ng/mL. Histamine was also detected from samples with cells and no serum

present and was found to increase with an increase in cell number. A cell concentration of 100% showed

a histamine detection of 11.8ng/mL when no serum was present.

When a serum concentration of 100% was mixed together with undiluted buffy coat the histamine

detection was 18ng/mL, whereas 25-50% serum only showed 12 ng/mL. It is important to note that

most data are lower than detection limit of the assay and should be interpreted with caution.

Figure 14: figure shows RPS handled samples with serum from the patient with low IgEHDM or buffy coat or both. HR is shown before and after filtration of the samples.

To further investigate the cause of unspecific HR, RPS was performed on samples with serum and cells

alone and in combination as illustrated in Figure 14. Samples containing cells were centrifuged before

analysis to separate the cells from the supernatant containing histamine released in the assay. Samples of

each of the three conditions were further filtrated through a 5kDa filter by centrifugation. Histamine

content was detected for all samples and the results are shown in Figure 14. Serum measured alone with

no cells showed a histamine detection of 9.6ng/mL before filtration and 1.8ng/mL after filtration.

Performing RPS on cells without addition of serum showed a histamine detection of 2.5ng/mL before

filtration and 2.1ng/mL after filtration. Finally, RPS with both serum and cells (but without allergen)

showed a histamine detection of 13.6ng/mL before filtration and 6.6ng/mL after filtration. The

! 32!

experiment was repeated with serum from the patient with high IgEHDM level confirming the interference

pattern (data not shown). Based on these results, unspecific HR from serum was found to be due to

molecules larger than 5kDa present in the serum. Unspecific HR from cells on the other hand did not

change before and after filtration and this was found to be due to molecules smaller than 5kDa. Samples

containing both cells and serum showed higher histamine detection than detected from serum

interference and cell interference. This was guessed to be due to serum induced histamine release and

was further investigated.

A larger experiment was executed to establish the donor variation between different sources of

unspecific HR; serum interference, spontaneous HR from cells and possibly unspecific serum induced

HR. Serum from four different individuals with level of total IgE of 2.13kU/L, 33kU/L, 359kU/L and

2178kU/L (two atopic and two non-atopic) were tested with three different buffy coat donors. RPS was

performed with serum alone (concentration 25-100%), cells alone and serum together with cells.

Serum interference was found to increase with serum concentration for all five sera as previously seen

for the patient with low IgEHDM (Figure 15). Mean histamine detection for all sera increased from

5.2ng/mL to 8.3ng/mL to 11.7ng/mL for the serum concentrations 25%, 50% and 100% respectively.

Spontaneous HR from cells alone was observed to be between 1.3-5.4ng/mL depending on the buffy

coat donor (data not shown). The experiment also investigated if serum actually induced HR but this

was however not seen for any of the sera. Based on these findings it was concluded that the unspecific

histamine detection of the assay was mainly caused by the presence of serum while spontaneous HR

from the cells accounted for a limited increase in HR.

Figure 15: Serum interference level measured on sera from 4 individuals and 1 urticaria. Each dot represents one serum measured in triplicate assays (n=3). Data is shown in histamine detection (ng/mL) at sera concentrations 25-100%. The assay detection limit is indicated by the dashed line at 10 ng/ml.

Serum interference

Serum concentration

25% 50% 100%

His

tam

ine

dete

ctio

n (n

g/m

L)

5

10

15

DL

! 33!

4.1.5 Removal of serum interference by filtration for reverse passive sensitization

Histamine is a small amine that will pass through the filter whereas larger serum components will not

(52). The following experiment was performed to investigate whether a filtration step could improve the

specific HR signal of the assay by removing the predominant part of serum-induced interference.

Histamine detection from serum was distinctly reduced upon filtration with a 5kDa filter as illustrated in

Figure 14.

RPS was performed on the two HDM allergic patients with a serum concentration of 100%. Half of the

sample was filtrated with a 5kDa filter just before the histamine detection step. Histamine content was

then detected for both parts of the sample. The results are shown in Table 4 as histamine detection in the

samples before and after filtration. The results of the experiment are based on one sample for each

condition. A decrease in histamine detection after filtration was observed for all samples, with and

without allergen for both patients. The decrease was found to be the same whether allergen was present

or not. The specific release was hereby not increased by filtration despite removal of some interference

from the samples. A similar experiment was performed with a serum concentration of 50% showing the

same results.

Patient: IgEHDM = 0.79kU/L Patient: IgEHDM = 9.2kU/L

+ allergen - allergen + allergen - allergen

Before 15.2 14.2 33.5 11.1

After 4.4 3.8 30.3 5.1

Difference 10.8 10.4 3.2 6.0

Table 4: RPS on serum from two HDM allergic patients with IgEHDM levels of 0.79kU/L and 9.2kU/L (serum concentration 100%) The analysis was performed with and without allergen, before and after filtration. Data are shown as HR (ng/mL). The difference between not filtrated and filtrated samples is also shown.

! 34!

4.1.6 Comparison of Reverse passive sensitization and classic passive sensitization

RPS was compared to CPS to evaluate the method sensitivity. Both methods were performed on four

different buffy coat donors with the two HDM allergic patients with high and low levels of IgEHDM. The

results are shown in Figure 16 and statistic data evaluation is found in Appendix 6.3 table B.

Figure 16: RPS (A) & CPS (B) performed on the two HDM allergic patients with high (green) and low (red) IgEHDM level and a healthy control serum (grey). Both RPS & CPS was performed on four different buffy coat donors (n=4) stimulated with HDM allergen concentration from 0.01-10µg/mL.

The patient with high IgEHDM levels tested positive for HDM allergy in both CPS and RPS and maximal

histamine release was obtained using 10µg/mL HDM allergen in both methods. The main difference

between the two methods for this patient was a lower sensitivity of the RPS compared to the CPS. A

positive test was obtained with allergen concentrations of ≥1ng/mL for the RPS, whereas a positive test

for CPS was obtained with allergen concentrations ≥0.1ng/mL. Additionally, the HR variation between

buffy coat donors was higher when using RPS than CPS. The HDM allergic patient with low IgEHDM did

not show any significant HR response in either CPS or RPS. The comparison of the two methods

revealed that RPS has a lower sensitivity compared to CPS for the two patients investigated.

4.2 Application on clinical material

RPS was evaluated on a larger number of patients. Since the initial development of the assay was based

on only two HDM allergic patients, the method could possibly have a greater potential when testing

other allergens. The specific IgE level necessary for allergy detection by RPS was therefore further

investigated.

CPS

HDM allergen conc. (µg/mL)

0,01 0,1 1 10

HR

of t

otal

con

tent

%

0

10

20

30

40

50

60HC Low IgEHDM High IgEHDM

**

** **

RPS

HDM allergen conc. (µg/mL)

0,01 0,1 1 10

HR

of t

otal

con

tent

%

0

10

20

30

40

50

60HCLow IgEHDM

High IgEHDM

**

a) b)

! 35!

4.2.1 Classic passive sensitization and reverse passive sensitization examined on 23 peanut allergic

patients.

CPS and RPS was performed on 23 sera from peanut allergic patients in order to investigate the

sensitivity of RPS when using peanut allergen and a wider range of IgE levels. A serum concentration of

50% was used, since no significant difference between 50% and 100% serum was found in previous

studies (Figure 12). HR detected for all patients in the two methods was converted to HR project class

(as described in 3.11 Data processing) to compare the sensitivity of the two methods.

CPS and RPS: 23 Peanut allergic patients

IgE levels (kU/L)

1.8 2.3 2.7 3.1 3.7 4 4.3 4.3 5.1 5.3 9.2 13.5 21.8 50.5 52.7 70.3 70.8 83.4 155 171 217 219 252

HR

pro

ject

cla

sses

0

1

2

3

4

5RPSCPS

Figure 17: Classic passive sensitization (grey bars) and reverse passive sensitization (black bars) performed with serum from 23 peanut allergic patients with IgEPE levels in the range of 1.8-252kU/L. HR project classes shows the lowest allergen concentration to which the cell released histamine above cut-off value 1) 90-30µg/mL 2) 9-3µg/mL 3) 0.9-0.3µg/mL 4) 0.09-0.03µg/mL 5) 0.009-0.0003µg/mL.

The results of the two analyses are shown in Figure 17. We were able to detect allergy in 21 out of 23

patient when using CPS. All patients with IgEPE levels above 4kU/L tested positive and only two

patients with 2.3kU/L and 3.7kU/L IgEPE tested negative. When using RPS, we were only able to detect

allergy in 17 out of 23 patients. Furthermore, patients with IgEPE levels above 13.5kU/L all tested

positive. Six patients including the two not detected by CPS tested negative. In conclusion this shows

that RPS was less sensitive for detecting peanut allergic patients compared to CPS in patients with low

specific IgE levels.

4.3 Chlorhexidine

Chlorhexidine is a low molecular compound used as a disinfectant in medical settings and in many

cosmetic products. Chlorhexidine can cause severe type I allergic reactions in some patients. Passive

sensitization has been shown to be less sensitive for detecting low molecular allergens including

chlorhexidine. Detection of chlorhexidine allergy by RPS was compared to the CPS assay in three

chlorhexidine allergic patients to compare sensitivity of the two methods.

! 36!

4.3.1 Comparison of classic passive sensitization and reverse passive sensitization with a low

molecular allergen

CPS and RPS were performed on three sera from chlorhexidine allergic donors with IgECH levels of

98.6kU/L, 16.8kU/L and 8kU/L. Maximal HR is shown in Table 5 for both methods. The result of the

CPS showed HR above detection limit for all three patients, whereas the patient with 8kU/L IgECH was

borderline positive. The results from the RPS on the other hand, only showed HR above detection limit

for the patient with the highest IgECH. The patient with the lowest specific IgE had no difference in HR

compared to the healthy control, whereas the patient with 16.8kU/L IgECH only showed a small increase

in HR.

Chlorhexidine specific IgE CPS (HR%) RPS (HR%) HC 0,0 5,9 8kU/L 10,2 5,9 16.8kU/L 24,9 11,4 98.6kU/L 80,1 54,9

Table 5: CPS and RPS performed on sera from four chlorhexidine allergic patients. Serum contained 8kU/L, 16.8kU/L and 98.6kU/L IgECH. A healthy control (HC) serum was included. Histamine release is shown in HR of total histamine content (%).

A comparison of the maximal HR of the two methods showed a decrease of 60% in maximal histamine

release when performing RPS compared to CPS. RPS was thereby less sensitive for detecting allergy to

low molecular allergens when compared to CPS. No further investigations were performed with the

novel method since it did not appear to improve detection of allergies by passive sensitization.

4.3.2 Classic passive sensitization for detecting chlorhexidine allergy

Chlorhexidine is found in many cosmetic and medical products as three different salts. CPS was

performed to investigate whether the sensitivity of the assay differed between chlorhexidine and the

salts. CPS was performed on eight sera from chlorhexidine allergic patients with IgECH levels of 1.14-

42.7kU/L to test the response to chlorhexidine. The sera were further tested for HR to chlorhexidine and

a mix of the three salts of chlorhexidine; chlorhexidine degluconate, chlorhexidine dehydrochloride and

chlorhexidine acetate. Six patient sera with IgECH levels of 1.14-5kU/L tested negative for both

chlorhexidine and a mix of the salts (data shown in Appendix 6.4 table A). One patient with IgECH

levels of 16.8kU/L was borderline positive and another patient with IgECH levels of 42.7kU/L was a

clear positive for both chlorhexidine and the salts. The two patient sera that were tested positive for the

mix of salts were tested on each of the salts individually. No difference in response was found in HR

between the three tested salts (Appendix 6.4 table B).

4.3.3 The immunogenic epitope of chlorhexidine

To further investigate the binding properties of IgE to chlorhexidine, CPS was performed with three

chlorhexidine-like molecules, proguanil hydrochlorid, alexidine dihydrochlorid and 4-chloroanilide

! 37!

(shown in the theory section Figure 3). The assay was performed on sera from the same eight patients as

described in section 4.3.1 and stimulated with a mix of the three experimental molecules. None of the

sera from allergic patients showed an increase in HR compared to a serum from a healthy control

subject (data shown in Appendix 6.4 table A).

The binding properties of IgE to chlorhexidine were further examined by incubating serum with the

chlorhexidine-like molecules before performing passive sensitization. If the IgE binds the

chlorhexidine-like molecules a decrease in histamine release is expected since fewer specific IgE will be

free for binding and cross-linking to chlorhexidine itself. CPS was performed on serum from a

chlorhexidine patient with IgECH of 42.7kU/L that had been pre-incubated with each of the experimental

molecules before cell sensitization and stimulation with chlorhexidine. No blocking ability of any of the

three molecules was observed after pre-incubation since HR levels due to chlorhexidine stimulation

were the same with and without pre-incubation with the experimental molecules (Figure 18).

Figure 18: CPS performed with serum pre-incubated with buffer (●-●), proguanil hydrochlorid (○-○), alexidine dihydrochlorid (△-△) or 4-chloroanilide (▼-▼) before cell sensitization and stimulation with chlorhexidine. The assay was performed on serum from a chlorhexidine allergic patient with specific IgE levels of 42.7kU/L.

4.4 The use of matrix-bound allergens in classic passive sensitization

A new way of presenting allergen in CPS was investigated to increase the assay sensitivity. Serum

sensitized cells were applied to an immunoCAP containing the allergen of investigation bound to a

cellulose matrix in the hope of a more efficient allergen cross-linking. The assay was performed on 12

different sera and the results are illustrated in Figure 19. Nine of the sera from chlorhexidine allergic

patients had IgECH levels ranging from ≤0.35kU/L (negative control) to 126kU/L shown in Figure 19.

Two sera were collected from patients with HDM allergy and contained 9.2kU/L and 0.79kU/L IgEHDM,

respectively. One serum was obtained from a healthy control donor. All sera sensitized cells were

stimulated with an immunoCAP for anti-IgE, allergen and PMA-ionomycin response. An immunoCAP

containing glycine was used to measure spontaneous histamine release from the cells during the assay.

Furthermore a glycin immunoCAP was used for PMA-ionomycin stimulated cell for maximal cell

Pre-incubation with chlorhexidine-like molecules

Chorhexidine Log/(ng/mL)

1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5

HR (n

g/m

L)

0

10

20

30

40

50

60

70BufferProguanil4-Chloro.Alexidine

! 38!

release. This value was used to calculate histamine released due to allergen stimulation as a percentage

of the maximal histamine release of the cells.

ImmunoCAP assay

HR

of m

ax re

leas

e %

05

10152025

Anti-IgEAllergen stimulation

sIgE (ku/L)

Chlorhexidine HDM HC

<0.35 <0.35 1.49 3.99 4.89 4.95 5.0 16.8 126 0.79 9.2 --

Figure 19: ImmunoCAP assay performed on; 9 sera from chlorhexidine allergic patients with allergen specific IgE in the range of undetectable to 126kU/L, two sera from HDM allergic patients with allergen specific IgE of 9.2kU/L and 0.79kU/L respectively, and a healthy control serum. The sensitized cells were stimulated with anti-IgE (grey bars) and allergen according to the allergic profile (dark grey bar).

Mean spontaneous HR was found to be 15ng/mL for all samples. Spontaneous HR was subtracted from

the observed values in the remaining immunoCAP stimulations shown in Figure 19. All sera induced

histamine release above the assay background upon anti-IgE stimulation. Serum 10 (9.2kU/L IgEHDM)

further showed 12% HR compared to maximal HR when stimulated with HDM allergen. The remaining

sera did not show a difference in allergen stimulated histamine release and the assay background. The

use of matrix-bound allergen for passive sensitization was thus not found to make patients with low

specific IgE levels more detectable. Additionally, the assay did not show any ability to detect

chlorhexidine allergy.

4.5 Passive sensitization of LAD2 cells

The requirement of buffy coat is essential when performing passive sensitization. However, a decrease

in blood donations has made the access limited (Blodbanken, Rigshospitalet). Thus another cell source

would be very valuable. The LAD2 mast cell line was investigated for this purpose.

! 39!

4.5.1 General investigations

Presence of the FcεRI receptor on the cell surface was illustrated by fluorescence microscopy shown in

Figure 20 (More pictures in Appendix 6.5). A cytospin was performed on IgE sensitized LAD2 cells

stained with PE conjugated anti-IgE antibody.

Figure 20: Cytospin of LAD2 cells stained with PE-conjugated anti-IgE. Scale of the picture is 26µm.

Histamine release due to receptor cross-linking was investigated to assess whether the cell line could be

used for passive sensitization. Cell stimulation by the FcεRI crosslinking antibody CRA 1 only showed

a weak HR level (data not shown). However, when cells were sensitized with isolated humane-IgE

(hIgE) the cells released histamine upon anti-IgE stimulation.

4.5.2 Serum incubation of LAD2 cells

LAD2 cells were incubated with four different sera to evaluate the effect of serum in the cell

environment. Serum sensitized cells did not show any changes in cell survival. The cells did however

show a change in morphology compared to cells incubated without serum (data not shown). Cells in a

serum free environment were observed as round cells with a clear edge whereas cells incubated in serum

were often oddly shaped with long tread-like structures on their surface. Spontaneous HR by LAD2

cells incubated with and without serum was found to be the same and was estimated to be 0.5pg/cell

(data not shown).

4.5.3 Passive sensitization of LAD2 cells

Passive sensitization was performed with LAD2 cells sensitized with either hIgE or serum containing

9.2kU/L IgEHDM. The cells were subsequently stimulated with either anti-IgE, HDM or PMA-

ionomycin. Both the cells sensitized with hIgE and the cells sensitized with serum were able to release

histamine upon PMA-ionomycin stimulation (Figure 21, c). Cells sensitized with hIgE released 70% of

total histamine content when stimulated with PMA-ionomycin whereas cells sensitized with serum only

! 40!

released 53% of total histamine content. Cells sensitized with hIgE released 26% of total histamine

content upon anti-IgE stimulation (Figure 21, a). No histamine release was observed for the serum-

sensitized cells when they were stimulated with anti-IgE. None of the sensitized cells released histamine

upon HDM stimulation (Figure 21, b). The same result was observed for anti-IgE and PMA-ionomycin

stimulations when repeating the experiment with two other sera (data not shown).

Since the LAD2 cells only released histamine upon anti-IgE stimulation when sensitized with pure IgE

and not when sensitized with serum, a potential inhibitory effect of the serum was investigated. LAD2

cells were sensitized with hIgE with and without serum and stimulated with anti-IgE or PMA-

ionomycin. Cells incubated with hIgE showed an anti-IgE response of 21% HR (Figure 22, a). Cells

incubated with both hIgE and serum did not show a specific release. Furthermore PMA-ionomycin

stimulation of the two cell cultures showed 100% and 90% HR for cells sensitized with hIgE alone or

with hIgE and serum, respectively (Figure 22, b). These results indicated an inhibitory property of the

serum either upon IgE sensitization or upon the cells ability to release histamine upon receptor cross-

linking.

Anti-IgE

anti-IgE Log/(ng/mL)

10 100 1000

HR

of t

otal

con

tent

%

0

20

40

60

80

SerumhIgE

a) House dust mite

HDM Log(µg/mL)

0,1 1 10

HR

of t

otal

con

tent

%

0

20

40

60

80

SerumhIgE

b)

PMA-ionomycin

PMA-Ionomycin Log/(ng/mL)

10 100 1000

HR

of t

otal

con

tent

%

0

20

40

60

80

SerumhIgE

c)

Figure'21:'Passive'sensitization'performed'on'LAD2'cells'sensitized'with'serum'(●-●)'or'hIgE'(○-○).'Sensitized'cells'were'stimulated'with'a)'antiDIgE'b)'House'dust'mite'c)'PMADionomycin.'

! 41!

Another FcεRI activating system was also tested to ensure that serum inhibition of histamine release

was not due to unspecific interaction with the hIgE or anti-IgE. Passive sensitization was performed

with mIgE-DNP for sensitization and DNP-HSA as stimulus, with and without serum present in the

sensitization. The results showed the same pattern as previous experiments (data not shown).

Figure 22: Passive sensitization performed on LAD2 cells sensitized with hIgE (●-●) or hIgE mixed with serum (○-○). Sensitized cells were stimulated with a) anti-IgE b) PMA-ionomycin.

4.6 Blocking of the inhibitory IgG receptor in passive sensitization

Blocking of the inhibitory IgG receptor FcγRIIb was investigated in order to increase the sensitivity of

passive sensitization for detection of patients with low specific IgE levels. The goal was to get a higher

histamine release by blocking any potential inhibitory effect of specific IgG in the patient serum.

4.6.1 Basophil FcγRIIb blocking in classic passive sensitization

CPS was performed on donor basophils sensitized with serum including the FcγRIIb blocking antibody

or none. The sera used for sensitization were collected from either one of two HDM allergic patients

(0.79kU/L and 9.2kU/L) or a healthy control serum. Anti-IgE stimulation caused FcγRIIb blocked

basophils to release 86% of total histamine content in average whereas cells without blocking had an

average release of 24% (Figure 23). No real difference was found between the three sera within same

pre-incubation conditions.

The IgG blocked cells were also stimulated with HDM. Serum from the healthy control subject and the

serum with 0.79kU/L IgEHDM did not show any HR above assay detection limit. Serum with 9.2kU/L

IgEHDM showed the same dose response curve with and without addition of the blocking antibody (data

shown in Appendix 6.6). The same result was found when the experiment was repeated using another

buffy coat as basophil donor. Whereas the first basophil donor showed a 2-fold increase in HR when

blocking the IgG receptor before stimulation with anti-IgE, the second donor showed a 3-4-fold increase

Anti-IgE

Anti-IgE (ng/mL)

10 100 1000

HR

of t

otal

con

tent

%

0

20

40

60

80

100 hIgEhIgE + Serum

a) PMA-ionomycin

PMA-ionomycin (ng/mL)

10 100 1000

HR

of t

otal

con

tent

%

0

20

40

60

80

100 hIgEhIgE + Serum

b)

! 42!

in HR after IgG receptor blocking. However, no increase in sensitivity of the assay was found in regards

to allergen response.

Figure 23: CPS performed with and without FcγRIIb antibody blocking of the basophils in the sensitization step. The cells were sensitized with sera from two HDM allergic patients with specific IgE levels of 0.79kU/L (black bars) and 9.2kU/L (light grey bars) and a healthy control serum (dark grey bars). The cells were stimulated with anti-IgE (5ng/mL).

4.6.2 Passive sensitization of LAD2 cells with FcγRIIb blocking

Blocking of the IgG receptor was further explored in passive sensitization of LAD2 cells to see if the

same result was obtained with the cell line. LAD2 cells were sensitized with hIgE or serum, with and

without addition of FcγRIIb antibody. Stimulation with anti-IgE showed HR of approximately 40% of

total histamine content for cells sensitized with hIgE (Figure 24). A small difference was observed

between cells with and without FcγRIIb blocking. Regardless of whether cells were blocked with the

FcγRIIb or not, serum sensitized cells showed a HR of 11% or less. All cell sensitizations showed

almost 100% release of total histamine content when stimulated with PMA-ionomycin. The experiment

was repeated and a similar HR result was observed (data not shown). The increase in HR obtained when

blocking IgG receptor on donor basophils while stimulating with anti-IgE was not observed when using

LAD2 cells as an alternative cell source.

Anti-IgE stimulation of CD32b blocked basophils

Pre-incubation

Buffer +CD32b

HR

of t

otal

con

tent

%

0

20

40

60

80

100

120IgEHDM 0.79kU/LIgEHDM 9.2kU/LHC

! 43!

Anti-IgE

Anti-IgE Log/(ng/mL)

100 1000

HR

of t

otal

con

tent

%

0

10

20

30

40

hIgE + anti-FcyRIIbhIgESerum + anti-FcyRIIbSerum

Figure 24: CPS performed on LAD2 cells sensitized with hIgE (○-○), hIgE and anti-FcγRIIb (●-●), serum (△-△) or serum and anti-FcγRIIb (▼-▼). Sensitized cells were stimulated with anti-IgE (0-2000ng/mL).

! 44!

5 Discussion


Reverse passive sensitization is an alternative sensitization method to the classic where allergen-IgE

complexes are formed before sensitization of the basophil cell. Initial studies of the method showed

promising results for detecting IgE mediated hypersensitivity in the serum of one patient with a low

level of allergen specific IgE towards house dust mite. A weak histamine release was observed when

analyzing patient serum by reverse passive sensitization, which was not observed for the classic passive

sensitization (unpublished data from Reflab ApS). The initial aim of this thesis was therefore to

investigate if reverse passive sensitization was more sensitive for diagnosing patients with low specific

IgE levels compared to the classic passive sensitization.

5.1.1 Complex formation

In the attempt to optimize the initial reverse passive sensitization method every step of the protocol was

carefully evaluated. The initial step where IgE-allergen complexes are formed in serum was investigated

for optimal concentrations of allergen and serum, reaction volumes and incubation conditions. Allergen

concentration for maximal histamine release was found to vary between patients and buffy coat donors

(Figure 16). This is probably due to a variation in cell number and the level of FcεRI expression by the

donor basophils in combination with the level of specific IgE in patient serum. The allergen in question

should therefore always be tested in several concentrations. The optimal serum concentration was found

to vary depending on the buffy coat donor. The final serum concentration recommended for reverse

passive sensitization was 100% based on two observations. Firstly, buffy coat with low ability to release

histamine were in some cases able to release histamine when using 100% serum during complex

formation but not at lower serum concentrations (Figure 12). Secondly, reverse passive sensitization

performed on the serum with low specific IgE showed a tendency to have a higher histamine release

when using 100% serum (data not shown). These two observations indicate that using 100% serum

promotes the IgE-allergen complex and hereby the histamine release response compared to lower serum

concentrations. The findings could be explained by different factors. If patients have low specific IgE

levels further dilution of the serum cause a complex formation, which too sparse to induce basophil

activation. On the other hand, when serum specific IgE levels are high but the donor basophils are less

responsive due to decreased signalling or low IgE-receptor number, a high number of IgE-allergen

complexes are needed to activate the basophils. Hence a serum concentration of 100% is preferred but

lower serum concentrations might be an option if serum material is limited. Interestingly it should be

noted that when serum from the patient with high specific IgE levels of 9.2kU/L was diluted to 12.5%

the serum was still able to cause a significant histamine release response compared to no serum present.

The concentration of specific IgE at 12.5% is 1.15kU/L, which is almost equal to the specific IgE level

! 45!

of the low level serum (0.97kU/L). However no histamine release was found from IgE-allergen

complexes formed from the serum with low IgE level. This indicates that factors other than the level of

specific IgE are involved in the sensitization process.

Further, the optimal time and temperature for the complex formation were investigated. The initial

theory of why reverse passive sensitization might be more sensitive is that optimal complex formation is

time dependent. Varying the time and temperature for complex formation can test this theory. This is

not possible by the classical method where prolonged incubation of allergen (up to 24 hours) with

sensitized basophils result in high spontaneous histamine release. Comparing different time points for

the complex formation did however not confirm this theory since no difference in histamine release

levels was observed between the different incubation intervals of 1-24 hours (Figure 11). The optimal

incubation temperature was found to be 5°C and lower at room temperature and 37˚C (Figure 11). It is

widely accepted that the IgE levels in serum is very stable when serum is kept at room temperature for a

couple of days. It is therefore more likely that the allergen or other serum components are affected by

the increase in incubation temperature causing a decrease in histamine release.

5.1.2 Interference and analytical variations

Increased histamine release was observed in the reverse passive sensitization assay when samples

without allergen were investigated compared to the classical method. This histamine release might be

due to 1) histamine present in the serum, 2) interference of serum with the histamine detection assay, 3)

spontaneous histamine release of the cells and 4) unspecific histamine release induced by serum itself.

The unspecific histamine detection was addressed in the attempt to gain a higher specific histamine

release in the reversed passive sensitization. Serum was found to cause the main part of unspecific

histamine release in the assay, which increased in a dose-dependent manner with the serum

concentration. When serum was filtered through a 5kDa filter, the histamine detection of the filtrate was

observed to decrease considerably (Figure 14). These findings indicate that serum interferes with the

assay through particles ≥5kDa present in the serum. It was hypothesized that proteins from serum might

get trapped in the glass fiber matrix of the plates. Upon histamine detection, OPA might bind amine-

groups of the proteins causing a fluorescent derivate that results in histamine detection by the

flourometer. Low amounts of unspecific histamine were observed for the cells during reverse passive

sensitization (Figure 13). This was caused by particles smaller than 5kDa and was hereby thought to be

due to unspecific histamine release from the cells during the assay procedure (Figure 14). Finally,

unspecific histamine release induced by serum itself was investigated since spontaneous histamine

release or serum interference could not explain all the unspecific histamine detected in the method.

Histamine release due to unspecific serum stimulation of the cells was however not observed. In

conclusion, the assay variation is considered minor compared to the impact of serum interference.

! 46!

5.1.3 Filtered reverse passive sensitization

Since filtration of serum was found to decrease serum interference, reverse passive sensitization was

performed including a filtration step immediate before analyzing the sample flourometrically. The

motivation behind decreasing the assay interference was to gain a higher histamine release specific to

the allergen stimulation from the sera with low specific IgE level. A reduction in unspecific histamine

detection was observed from filtered samples. Removing of serum interference did however not cause

an increase in allergen induced histamine release (Table 4). Exclusion of particles larger than 5kDa from

serum did hereby not make the assay more sensitive for detecting of allergy.

To sum up, reverse passive sensitization was found to be optimal for histamine detection when IgE-

allergen complex formation was performed with 100% serum and a serial dilution of allergen 1h at 5°C,

following sensitization with undiluted buffy coat. Removal of interference in the assay by filtration did

not improve the sensitivity of the assay.

5.2 Reverse passive sensitization compared to classic passive sensitization

The optimized protocol of reverse passive sensitization was compared to the classic passive

sensitization by performing both assays on the two house dust mite allergic patients using four different

buffy coat donors (Figure 16). Reverse passive sensitization was found to have a lower sensitivity and a

higher sample variation compared to the classic passive sensitization and was hereby not found to have

an improved sensitivity for detecting allergy in the serum with low specific IgE as hypothesized.

Reverse- and classic passive sensitization was further compared on 23 sera from peanut allergic patients

with specific IgE levels ranging from 1.8-252 kU/L (Figure 17). The classic assay was able to detect

peanut allergy in all sera with specific IgE levels ≥4kU/L. The assay was further able to detect 3/5

patients with specific IgE levels between 1.8-4kU/L. In a previous study by Budde et al. (2002) the limit

for specific IgE levels of the classic passive sensitization was found to be 2.6kU/L in house dust mite

allergic patients. Based on these findings the classic passive sensitization seems to have a detection limit

of 2-4kU/L specific IgE for well-documented high molecular allergens. The sensitivity of the reverse

passive sensitization was found to be lower than the classic approach when evaluating the sera from

peanut allergic patients. Patients with specific IgE levels ≥13.5kU/L could all be detected using the

reversed assay (Figure 17). Out of the remaining eleven patients with specific IgE levels ranging from

1.8-13.5kU/L only five tested positive. No pattern in specific IgE level was found for these patients,

which could indicate that another factor of the serum affects the sensitization and/or histamine release of

the basophils. Based on these finding the reverse passive sensitization was found to be less sensitive for

patients with low specific IgE levels compared to the classic passive sensitization.

! 47!

5.3 Chlorhexidine - an example of a low molecular allergen.

Classic passive sensitization has previously been shown to have a low sensitivity for low molecular

allergens such as some types of drugs (35). In this study, the assay sensitivity of the low molecular drug

chlorhexidine was investigated for both classic and reverse passive sensitization (Table 5). Reverse

passive sensitization showed to have a markedly reduced sensitivity for detecting chlorhexidine allergic

patients compared to classic passive sensitization. The method does hereby not show an improved

sensitivity for low molecular allergens and was not further used in this study. Instead, optimization of

assay sensitivity of the classic passive sensitization was pursued. The level of specific IgE in serum

needed for a positive result in the classic passive sensitization for chlorhexidine was found to be

approximaltly 8kU/L. This level was found to be lower for the high molecular allergens of house dust

mite (4kU/L) and peanut (2kU/L) (32). These results support the previous finding that classic passive

sensitization is less sensitive for detecting low molecular allergens (35).

Chlorhexidine exists as three different salts in medical settings and in cosmetic products; chlorhexidine

diacetate, chlorhexidine digluconate and chlorhexidine dihydrochloride (47). Some patients therefore

might only be sensitized to one or two of these salts of chlorhexidine and might only respond when

tested by this specific salt of chlorhexidine. If all sera are tested with only one type of salt when

investigated by passive sensitization this could result in an increase in false negative result and hereby

lead to a lower sensitivity for the allergen. However, no difference in histamine release was observed

when passive sensitization was performed on two sera from chlorhexidine allergic patients with the

different salts of chlorhexidine (Appendix 6.4 table B). Testing individually with all three salts of

chlorhexidine would hereby not improve the assay sensitivity. It should however be noted that this

observation is only based on two patients. A larger study material is needed before the theory can be

disproved. Such a study was however not possible due to lack of sera with high specific IgE levels for

chlorhexidine.

The immunogenic epitope of chlorhexidine was further investigated. Sera from two chlorhexidine

allergic patients were analyzed for histamine release following passive sensitization with three

molecules with structures that resemble parts of the chlorhexidine molecule. Proguanil hydrochlorid

resembles one arm of the complete chlorhexidine molecule, 4-chloroanilide mimics the two 4-

chlorophenol groups and alexidine dihydrochloride mimics the remaining part of the chlorhexidine

molecule (Figure 3). None of the three molecules were able to cause histamine release above detection

limit (data not shown). Further, no blocking ability was found from either of the molecules when pre-

incubated with serum before passive sensitization was performed with chlorhexidine as allergen

stimulation (Figure 18). Blocking ability of alexidine and chlorguanide has in previous studies been

found to some extent when performing radioallergosorbent test or ELISA (48,50). Chlorhexidine was in

! 48!

these experiments found to have a much higher blocking ability, which could indicate that IgE binding

of chlorhexidine involves the entire molecule for optimal binding.

5.4 Matrix bound allergen by immunoCAP

Since reverse passive sensitization was found to be less sensitive than the classic passive sensitization,

new approaches were studied for optimizing assay sensitivity of the original method. Binding of

allergen to surface bound IgE happens in a solution when performing classic passive sensitization. It

was hypothesised that a higher degree of cross-linking could be obtained if the allergen was bound to a

solid phase. Upon binding of IgE to an allergen the cell would stay at the solid phase surface where

multiple allergens are present for further crosslinking. Passive sensitization using immunoCAPs was

therefore investigated (Figure 16). The new approach did however not provide an increased sensitivity

for either patients with low specific IgE levels or for cell exposure to the low molecular allergen

chlorhexidine. The result furthermore suggests that cross-linking of IgE by chlorhexidine only happens

when the allergen is soluble since chlorhexidine allergic patients with high specific IgE only tested

positive in the classic passive sensitization but not in the immunoCAP-assay. This might be due to the

size of the molecule. As previously mentioned the immunogenic epitope of chlorhexidine has been

suggested to include the entire molecule (48,50). Binding of chlorhexidine to the matrix of the

immunoCAP might remove one out of two possible binding sites on the molecule. Hence eliminating

cross-linking of two IgE molecules to the same chlorhexidine allergen.

5.5 Stable cell line for passive sensitization

Since the availability of buffy coat for passive sensitization is limited, a new cell source for the assay

would be very valuable. The use of the mast cell line LAD2 cells for passive sensitization was

investigated. The cells were found to tolerate serum incubation but showed a change in morphology.

Serum-incubated cells had thread-like structures on their surface, whereas the cells without serum

showed a clear round edge. This change indicated that the serum affects the cells through an unknown

mechanism. The functionality of the morphological change is however unknown to the best of my

knowledge. Since mast cells are tissue resident in vivo it might be speculated whether this mechanism

occurs naturally in the body.

No histamine release was detected when passive sensitization was performed with serum sensitized

LAD2 cells (Figure 21). This was observed when the cells were exposed to either the allergen of

investigation or anti-IgE. The cells did however release histamine upon PMA-ionomycin stimulation,

which was included as a positive control to be sure that the cells contain histamine-containing granules.

The findings indicate that IgE from serum is not properly bound or that serum somehow blocks IgE

! 49!

activation of the receptor. This was further investigated by sensitizing LAD2 cells with both pure IgE

and serum. Sensitization of cells with pure IgE and subsequent incubation with anti-IgE resulted in

histamine release whereas none was found upon anti-IgE stimulation when cells were sensitized with

serum (Figure 22). This indicates that serum blocks IgE mediated signalling or IgE binding to the

receptor in the mast cell line. Passive sensitization can therefore not be performed using LAD2 cell

before this blocking factor has been identified.

A histamine release blocking factor in serum was investigated in the 1980’s by a German research group

(53–55). They found that transferrin present in human serum inhibited histamine release of rat

peritoneal mast cells and human basophils. Transferrin is a transport molecule of iron, that is otherwise

toxic in its free form (56). Iron bound to transferrin can then be delivered to the cells though transferrin

receptors (CD71) expressed on cell surface of all haematopoietic cells including basophils. Inhibition of

histamine release happened though an unknown process that was hypothesized by the authors to be

independent of IgE-mediated activation (54). This was thought since both IgE-dependent and IgE-

independent stimuli by Ca-ionophore A23187 and melittin were equally affected by the blocking factor.

Based on these findings it can be speculated whether transferrin could be the blocking factor of

histamine release in LAD2 cells. It is however important to note that serum did not block histamine

release from the LAD2 when stimulating IgE-independently with PMA-ionomycin, since Ca-ionophore

A23187, melittin, PMA and ionomycin all have calcium triggering working mechanisms which all can

result in histamine release (57,58). It is nevertheless still possible that the serum blocking of LAD2 cells

due to transferrin can be bypassed by PMA-ionomycin stimulation in our study. The concentration of

transferrin in plasma has been found to vary between 2-3.6mg/mL (59). In the study by the German

research group they saw a blocking effect of transferrin levels between 0.2-1mg/mL (54). Transferrin

should therefore be further investigated as a candidate for the blocking factor.

Another mast cell source for the assay was also considered during this thesis work. Primary cells were

collected from transgenic mice bone marrow and cultured with IL-3 and SCF to obtain mast cells only

(Department of Dermatology and Allergy, Charité, Universitätsmedizin Berlin). The murine mast cells

have a humanized FcεRI alpha chain, which bind humane IgE. Passive sensitization was performed

twice using the mast cells without any success. No further work was performed with the cells due to

practical issues. This type of primary cells does however open up for new potential strategies for

generating a functional cell line for passive sensitization.

5.6 Blocking of the inhibitory IgG receptor in passive sensitization

The low affinity IgG receptor FcγRIIb has been found to inhibit degranulation upon receptor

crosslinking either to a similar receptor or to FcεRI (9). Since serum contains both IgE and IgG with

! 50!

affinity for the same allergen, it was hypothesized that IgG inhibition of degranulation could lead to less

histamine released when performing passive sensitization. It was furthermore hypothesized that patients

with low specific IgE levels might show a higher histamine release if the negative regulation of

degranulation was eliminated. A blocking antibody for FcγRIIb was used in order to test this hypothesis.

The antibody binds the FcγRIIb and blocks IgG binding without activating the receptor. A schematic

drawing of the theorized effect of using the blocking antibody for passive sensitization is shown in

Figure 25.

Figure 25: Mast cell degranulation dependent on IgE and IgG regulation in normal situation and when an antibody blocks the IgG receptor. Cross-linking of FcεRI receptor by IgE bound allergen promotes degranulation. Cross-linking of FcγRIIb by IgG bound allergen inhibits FcεRI activated degranulation. A blocking antibody blocks but does not activate IgG binding to the inhibitory FcγRIIb resulting in a higher histamine release. Camilla K. Hansen 2016.

Passive sensitization of donor basophils with and without the blocking antibody did not show any

difference in histamine release when cells were stimulated with allergen. This was observed for both the

serum with high and low levels of house dust mite specific IgE. These results suggest that inhibition of

degranulation by IgG signalling is not a problem when performing passive sensitization. Further testing

on a larger sera material is however necessary to rule out any effect on using the blocking antibody in

passive sensitization.

A difference in histamine release was observed when FcγRIIb-blocked cells were stimulated with anti-

IgE (Figure 22). Blocking of the FcγRIIb caused a large increase in histamine release for three different

sera investigated. This finding was very surprising since no allergen was present to cross-link the IgG

receptor causing an inhibitory response. The results thus indicate that anti-IgE activate the IgG receptor

through an unknown mechanism causing an inhibitory signal from the FcγRIIb receptor. A study of the

FcγRIIb regulation of IgE-mediated signalling in human basophils by MacGlashan et al. (2014)

! 51!

confirmed these results (60). Here they observed an increase in phosphorylation of the FcγRIIb receptor

upon increasing stimulation with goat polyclonal anti-hIgE in isolated human basophils.

Phosphorylation of the receptor due to either non-specific IgG from goat or human or from stimulation

with a mouse IgM anti-IgE was not found. These results indicate an activation of FcγRIIb caused

specifically by goat anti-IgE. To further confirm these results they stimulated basophils with goat anti-

IgE together with a FcγRIIb blocking antibody and saw a decrease in receptor phosphorylation upon

increasing concentrations of the blocking antibody. Furthermore, an increase in histamine release was

observed with increasing concentrations of the blocking antibody. Based on these and our results, it can

be speculated whether goat anti-IgE have an unspecific binding to FcγRIIb. This is not optimal for the

histamine releasing assays where anti-IgE operates as a positive control of the cells’ ability to release

histamine upon IgE-mediated activation. The results obtained using the FcγRIIb blocking antibody do

however suggest that using this antibody could reverse this unwanted effect. Anti-IgE from another

manufacturer or animal species seems to be an option to avoid the unspecific inhibition caused by goat

IgG.

The blocking antibody was also investigated when using LAD2 cells for passive sensitization (Figure

24). Blocking of FcγRIIb and stimulating with anti-IgE did however not cause an increase in histamine

release in cells sensitized with either serum or pure IgE. In contrast, a tendency to decreased histamine

release was observed for cells incubated with the blocking antibody. These results indicate a different in

receptor activation or signalling for LAD2 cells compared to basophils from buffy coat. It can be

speculated whether the difference is found within the mast cell line only or if it applies to mast cells in

general. LAD2 cells have been found to express FcγRII receptors but investigations of the signalling

pathway has to the best of our knowledge not been performed (42,43).

! 52!

6 Conclusion This master project sought to establish a more sensitive method for detecting type I hypersensitivity by

passive sensitization of patient serum that contains low serum IgE levels. Furthermore, an increased

sensitivity to low molecular allergens often found in drugs was explored. The aims were investigated

through different approaches and the following was established:

• Reverse passive sensitization was less sensitive than the classic passive sensitization

• Stimulation with matrix-bound allergens decreased the sensitivity of classic passive

sensitization

• Negative signalling by FcγRIIb did not make the assay less sensitive for allergy detection

• Histamine release through passive sensitization with serum was not applicable to LAD2 cells

whereas pure IgE resulted in cells responding to an IgE stimulus

Based on the above findings it was not possible to improve the existing method of passive sensitization

within the time frame of this thesis. The experimental work of the thesis furthermore showed that the

level of specific IgE is not the only factor determining sensitization since serum can inhibit IgE

sensitization.

6.1 Future perspectives

On different occasions during this thesis it was found that the level of specific IgE did not correlate with

the histamine released upon passive sensitization. A histamine release-blocking factor was further

observed from serum when passive sensitization was performed on LAD2 cells. It is therefore necessary

to identify the regulating property of serum upon IgE-sensitized basophils and mast cells to optimize the

method further. We suggest that this could be investigated by fractionating serum by filtration to

identify the size of the inhibiting factor(s) of serum.

Transferrin should furthermore be investigated for its ability to affect histamine release of basophils and

mast cells based on the literature (53–55). This could be examined by neutralizing the effect of

transferrin in serum by blocking the binding of transferrin to its receptor by an already commercially

available antibody targeting transferrin. Transferrin could also be separated from IgE by filtration of

serum before performing passive sensitization, since transferrin has a size of 80kDa and IgE has a size

of 190kDa (59,61). Removal of a histamine release-blocking factor in serum could make passive

! 53!

sensitization more sensitive for patients with low levels of specific IgE. Performing the assay on LAD2

cells might become possible by this approach.

Passive sensitization is mainly performed when patients are shown to have non-responding basophils in

the HR-test (Reflab ApS). HR-test has been shown to have a higher sensitivity for patients with low

sIgE levels and low molecular allergen detection compared to passive sensitization. A different

approach for detecting these patients could therefore be to develop a method for making non-responding

basophils respond. SYK is an important enzyme in the FcεRI activated signalling that has been found to

be deficient in non-responding basophils (33,34). A different approach for optimizing allergy

diagnosing by histamine release detection could therefore be to investigate a way to restore SYK

expression in these cells.

Another approach to make non-responding basophil respond to allergen stimulation could be by

stimulating the enzyme protein kinase C with PMA. A study by Skov et al. 1987 showed that histamine

release from non-responding patients could be detected when the basophils were primed with small

concentrations of PMA (37). No histamine release was observed without PMA-priming of the cells.

Reversal of the non-responding profile of patients would hereby make the direct HR-test a diagnostic

useful detection method in most cases.

! 54!

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7 Appendix

7.1 List of materials

Patient material Patient material Provider Sera from peanut allergic patients Professor, Dr.med Carsten Bindslev-Jensen, Odense Universitetshospital

Sera from chlorhexidine allergic patients

Consultant, PhD Lene Heise Garvey and PhD Morten Obstrup, the Laboratory of Medical Allergology, Allergy clinic, Copenhagen University Hospital at Gentofte Hospital

Sera from House dust mite allergic patients Reflab ApS Sera from healthy control subjects Reflab ApS

Used at the laboratory of Reflab ApS: Product Manufactor Catalouge no. Distributor

RPMI 1640-medium Sigma-Aldrich L0501-500 VWR

NaCl 9mg/mL Region Hovedstadens Apotek 805975 Region Hovedstadens Apotek

Pipes Buffer pH 7.4 Ampliqon AMPQ97350.1000 VWR

OPA disc 0.5mg/mL Reflab RLR013 Reflab

Natriumhydroxid 50 mM Ampliqon AMPQ99681.1000 VWR

HClO4 0.59% w/w (0.059 mol/L) Ampliqon AMPQ99682.1000 VWR

HClO4 7% Region Hovedstadens Apotek 863987 Region Hovedstadens Apotek

SDS 0.4% Region Hovedstadens Apotek 865448 Region Hovedstadens Apotek Strippe buffer pH 3.55 (Natriumhydrogenphosphat, Kaliumchlorid, Destilleret H2O) Ampliqon AMPQ44779.1000 VWR

Recombinant human IL-3 R & D systems Europe Ldt 203-IL-010 R & D systems Europe Ldt

96 microwell plate (450µl) Thermo scientific 7322661 VWR

Anti-IgE (5µg/mL) KPL 01-10-04 KPL

Anti-CD32 antibody [AT10] abcam ab41899 abcam Histamine standard glass microfiber plate Reflab RLA210 Reflab

Screening standard glass microfiber plate Reflab RLA217 Reflab Quantitative screening glass microfiber plate Reflab RLA212 Reflab

Peanut extract, Arachis hypogaea

GREER allergy immunotherapy XPF171D3A2.5

GREER allergy immunotherapy

House dust mite Dermatophagoides pteronyssinus extract

GREER allergy immunotherapy CPB82D3A2.5


Chlorhexidine diacetate, European Pharma Sigma-Aldrich C1520000 Sigma-Aldrich

Chlorhexidine digluconate solution Sigma-Aldrich PHR1294-1G Sigma-Aldrich

Chlorhexidine dihydrichloride Sigma-Aldrich C8527-5G Sigma-Aldrich

Proguanil hydrochloride Sigma-Aldrich G7048-50MG Sigma-Aldrich

4-Chloroaniline, 98% Sigma-Aldrich C22415-5G Sigma-Aldrich

Chlorhexidine, 99.9+% Sigma-Aldrich 282227-1G Sigma-Aldrich

Alexidine dihydrochloride Sigma-Aldrich A8986-50MG Sigma-Aldrich

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Instillagel 20mg/g+0.5mg/g Lidocainhydrochlorid/Chlorhexidin gluconate Farco-Pharma GmbH N/A

Clinic of allergy, Gentofte Hospital

Vivaspin 0.5mL concentrator, 5000MWCO PES Viva Science Sattorius Group 512-3745 VWR Vivaspin 0.5mL concentrator, 3000MWCO PES Viva Science Sattorius Group 512-3745 VWR

Apparatus Manufactor

Microplate shaker DSG-Titertek 140-1400RPM Flow Laboratories

Flourometer, Histareader 501, RLA501 Reflab ApS

Microplate washer, Trippel-washer Reflab ApS

Multidrop Drop DW, RLU100/101 Fisher Scientific Used at the laboratory: Laboratories for Medicinsk Allergologi, Gentofte Hospital Product Manufactor Catalouge no. Distributor

LAD2 Cells NIH/Dr. A. Kirshembaum Recieved: June 2003 NIH/Dr. A. Kirshembaum

StemPro-34 SFM Life Technologies 10640-019 N/A

StemPro-34 SFM supplement Life Technologies 10641-025 N/A

Penicillin/Streptavidin Sigma-Aldrich P0781 N/A

L-glutamine Sigma-Aldrich G7513 N/A

rhSCF Peprotech 300-07 N/A

Tryphan blue (0.4%) Sigma-Aldrich T8154 N/A

96-well V-shaped microplate Greiner bio one 651161 Invitro A/S

Histamine plate Reflab RLA210 Reflab

Pipes buffer Reflab RLA706 Reflab

Perchloric acid 0.59% RH Apotek 86 35 86 RH Apotek

Perchloric acid 7% Reflab RLR012 Reflab

Sodium hydroxide 50mM RH Apotek 850713 RH Apotek Sodium Dodecyl Sulfate (SDS) stock solution 20.4% Reflab RLR007 Reflab

OPA disk Reflab RLR013 Reflab Ionomycin calcium salt, from streptomyces conglobastus stock solution 10mg/mL in 99% EtOH Sigma-Aldrich I0634 Sigma Phorbol 12-myristate 13-acetate (PMA) Stock solution 1 mg/mL in 99% EtOH Sigma-Aldrich P8139 Sigma

Human Serum Albumin (20%) CSL Behring, USA 109697 N/A

PBS Sigma-Aldrich D8537 Sigma

EDTA RH apotek 863200 RH apotek House dust mite Dermatophagoides pteronyssinus extract allergen GREER allergy immunotherapy CPB82D3A2.5


Human IgE (1.145mg/mL) Calbiochem 401152 Merck Chemicals

Anti-CD32 antibody [AT10] Abcam ab41899 Abcam

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Affinity Purified Goat anti-Human IgE (epsilon) 1mg/mL KPL 01.10.14 VWR International

CRAI (anti-FcepsilonRI antibody) Biolegend 334602 Biolegend

mIgE-DNP Sigma-Aldrich D8406 Sigma

DNP-HSA Sigma-Aldrich A6661 Sigma

PE anti-human IgE Biolegend 325506 Biolegend

PE mouse IgG1, k isotype control Biolegend 400113 Biolegend

Shandon single cytofunnel Thermo Fisher Scientific 12076679 Thermo Fisher Scientific

VECTAshield Antifade mounting medium Vector Laboratories H-1000 Vector Laboratories SlowFade Gold antifade mountant with DAPI Invitrogen S36938 Thermo Fisher Scientific

ImmunoCAP® Allergen d1, HDM, Derm. Pter. Thermo Fisher Scientific d202 Thermo Fisher Scientific

ImmunoCAP® Allergen SAS, U363, glycine Thermo Fisher Scientific U363 Thermo Fisher Scientific

ImmunoCAP® Allergen c8, chlorhexidine Thermo Fisher Scientific c8 Thermo Fisher Scientific

ImmunoCAP® specific IgE, Anti-IgE Thermo Fisher Scientific Total IgE Thermo Fisher Scientific

Apparatus Manufactor

Stericup-GP, 0.22µm, Polyethersulfone, 250mL, radio-sterilized Milipore, cat. # SCGPU02RE

Cytospin 4 Thermo Scientific

Zoe flourescence cell imager Biorad

Vacuum pump, Vacuum Module V.2 Biorad

Flourometer, Histareader 501, RLA501 Reflab ApS

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7.2 Optimization experiments for reverse passive sensitization

No. Date Serum conc.

Serum vol. Allergen conc.

Allergen vol.

Incubation time and temp.

Buffy coat conc. Filter

1 20.05.15 10-100% 10µl 10µg/mL 10µl 24h 5C 100% -

2 26.05.15 1-100% 10µl 0.1-10µg/mL 10µl 24h 5C 100% -

3 01.06.15 10-100% 10-50µl 10µg/mL 10-50µl 24h 5C 100% -

4 01.06.15 20-100% 50µl 10µg/mL 50µl 1/6/24h 5C/RT/37C 100% -

5 09.06.15 20% 25µl 0.01-100µg/mL 25µl 1h 5C +/- 100% -

6 15.06.15 5-100% 25µl 100µg/mL 25µl 1/24h 5C 100% -

7 16.06.15 5-100% 10µl 10µg/mL 10µl 1/24h 5C 100% -

8 26.08.15 12.5-100% 25µl Buffer 25µl 1h 5C 12.5-100% -

9 03.09.15 12.5-100% 25µl 10µg/mL 25µl 1h 5C 12.5-100% -

10 04.09.15 +/- 100% 25µl Buffer 25µl 1h 5C +/- 100% +

11 15.09.15 +/- 100% 25µl Buffer 25µl 1h 5C +/- 100% +

12 15.09.15 50-100% 25µl 10µg/mL 25µl 1h 5C 100% +

13 01.10.15

12.5-100% 25µl 10µg/mL 25µl 1h 5C 100%

- 14 05.10.15

15 06.10.15

16 13.10.15

17 06.10.15

25-100% 25µl Buffer 25µl 1h 5C +/- 100%

+ 18 08.10.15

19 13.10.15

20 14.10.15

100% 25µl 0.01-10µg/mL 25µl 1h 5C 100% -

21 15.10.15 Table: An overview of the main experiments performed for RPS optimization. RPS was performed with HDM allergen on two allergic patients with high and low levels of allergen specific IgE.

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7.3 Statistic data analysis

12.5% 25% 50% 100%

Patient low IgEHDM 0% 0.079 0.91 0.132 0.064

12.5% - 0.159 0.171 0.991

25% - - 0.810 0.991

50% - - - 0.875¤

Patient high IgEHDM 0% <0.001*** <0.001*** <0.001*** <0.001***

12.5% - 0.001** 0.008** 0.056

25% - - 0.160 0.587

50% - - - 0.703

Table A: The HR data illustrated in figure 2 was compared statistically. If data was normally distributed a t-test was used. For data not normally distributed a Wilcoxon Signes Rank Test was used (¤).*p<0.05 **p<0.01 ***p<0.001. (n=5)

Difference compared with healthy control sera

HDM 0.01 HDM 0.1 HDM 1 HDM 10

RPS Low IgEHDM 0.546 0.334 1.000¤ 1.000¤

High IgEHDM 0.795 0.080 0.027* 0.034*

CPS Low IgEHDM 0.077 0.439 0.867 0.750

High IgEHDM 0.028 0.008** 0.005** 0.002** Table B: The HR data illustrated in Figure 16 was compared statistically between the two HDM allergic patients and the healthy control serum. If data was normally distributed a t-test was used. For data not normally distributed a Wilcoxon Signed Rank Test was used (¤). *P<0.05.(n=4)

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7.4 Passive sensitization for detecting chlorhexidine allergy

Chlorhexidine (solid) Chlorhexidine Salt mix Chlorhexidine Exp. Mix

Patient IgEch (kU/L) Max HR Result Max HR Result Max HR Result A 4,95 4,6 Neg 0,9 Neg 4,1 Neg B 42,70 20,8 Pos 18,7 Pos 0,0 Neg C 16,80 8,4 Pos * 8,6 Pos * 3,5 Neg D 1,14 2,4 Neg 2,4 Neg 4,2 Neg E 3,99 10,1 Neg 3,9 Neg 1,9 Neg F 1,49 2,6 Neg 4,0 Neg 5,7 Neg G 4,89 2,8 Neg 3,5 Neg 4,7 Neg

H 5 3,1 Neg 2,3 Neg 4,6 Neg Healthy control - 3,1 Neg 1,1 Neg 0,0 Neg Table A: Result of Classic passive sensitization analysis of eight sera from chlorhexidine allergic patients and one serum from a healthy control. The sera were analysed for response to chlorhexidine, a mix of three salts of chlorhexidine and a mix of chlorhexidine resembling molecules. Maximal histamine release is shown in HR% of total content. Chlorhexidine specific IgE levels are further shown for each patient. Positive results are marked in green. * Positive when evaluating on individual cut off, not the general cut-off

HC IgECH = 16.8kU/L IgECH = 42.7kU/L

Chlorhexidine digluconate 0.0ng/mL 31.6ng/mL 64.5ng/mL

Chlorhexidine dehydrochlorid 1.6ng/mL 32.0ng/mL 69.3ng/mL

Chlorhexidine diacetate 3.3ng/mL 29.7ng/mL 60.1ng/mL Table B: Passive sensitization performed with two sera containing 16.8kU/L and 42.7kU/L chlorhexidine specific IgE and a healthy control sera. The sera was investigated for response to chlorhexidine digluconate, chlorhexidine dehydrochlorid and chlorhexidine diacetate. Histamine release in ng/mL is shown in the table.

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7.5 Fluorescence investigations of LAD2 cells

Figure: cytospin of IgE-sensitized LAD2 cells stained with PE conjugated anti-IgE antibody (top row) or an isotype control (lower row). The cells were further stained with DAPI show in the first column of pictures. Second column of pictures show anti-IgE staning. Third column shows DAPI and PE anti-IgE antibody merged. The scale of all pictures is 100µm expect DAPI stain of the isotype stained cells with is 48µm.

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7.6 Passive sensitization of FcγRIIb blocked basophils

Figure: Classic passive sensitization performed on basophils incubated with buffer (●-●) or FcyRIIb blocking antibody (○-○). The cells were stimulated with house dust mite allergen in a concentration range 0.01-10µg/mL. The sensitization was performed on sera from two chlorhexidine allergic patients with specific IgE levels of a) 0.97kU/L and b) 7.9kU/L and C) a healthy control sera.

a) b)

c)

Patient: IgEHDM 0.97kU/L

House dust mite allergen (µg/mL)

0,001 0,01 0,1 1 10 100

HR

% o

f tot

al c

onte

nt

0

20

40

60

80

100 BufferFcyRIIb blocked

Patient: IgEHDM 7.9kU/L


0,001 0,01 0,1 1 10 100

HR

% o

f tot

al c

onte

nt

0

20

40

60

80

100 BufferFcyRIIb blocked

Healthy control


0,001 0,01 0,1 1 10 100

HR

% o

f tot

al c

onte

nt

0

20

40

60

80

100 Buffer FcyRIIb blocked

A novel approach to IgE-sensitization of basophils and mast cells

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