Blood-to-blood Immunological Compatibility Test: A Possibility with

Fluorescent Immuno-Biochips

K. Charrière

, A. Guitton

, V. Ratié

, L. Sighi-Dumoulin

,V. Bourcier

, P. Morel

L. Pazart

and B. Wacogne

1,4

Centre Hospitalier Universitaire de Besançon, Centre d’Investigation Clinique,

INSERM CIC 1431, 25030, Besançon, France

Haemovigilance Service, Besançon University Hospital, 25000 Besançon, France

Etablissement Français du Sang Bourgogne/Franche-Comté, 25000 Besançon, France

FEMTO-ST Institute, Univ. Bourgogne Franche-Comte, CNRS,

15B avenue des Montboucons, 25030 Besancon, cedex, France

{vanessa.ratie, lydia.sighidumoulin, pascal.morel}@efs.sante.fr, bruno.wacogne@univ-fcomte.fr

Keywords: Crossmatch, Human Red Blood Cells, Fluorescence, Biochip, Immuno-detection.

Abstract: One of the most feared transfusion accident is the haemolytic reaction. A majority of countries imposes a

compatibility test before each transfusion, at the patient’s bedside or in laboratory. Regardless of the test

performed, it does not prevent human errors and nothing ensures an “error free” procedure. Complete

crossmatch is the only test ensuring a complete blood compatibility between donors and patients. It relies on

the direct or indirect detection of agglutinations which occur when the patient’s plasma is mixed with the red

cells to be transfused. It requires extracting plasma. The work described here will help avoid all the

immunologic incompatibilities by the use of a compatibility test without plasma extraction. It relies on an

immuno- biochip technology in a microfluidic environment with fluorescence detection. This position paper

presents preliminary results obtained with artificial samples together with comments on the state of industrial

competition and the new device market positioning.

1 INTRODUCTION

In France every year, more than 3 million labile blood

products, 80% of which were red blood cells (RBC),

are given to more than 550 000 patients (ANSM

2016). French health institutions have seen a rise in

deliveries of RBC of + 26.3% in total between 2000

and 2014 (EFS, 2014). In 2016, according to the

haemovigilance report by ANSM (National Agency

for Drug Safety), 6780 adverse effects in recipients

(AER) related to transfusion were reported. In total,

255 adverse effects in receivers related to transfusion

were recorded due to immunological incompatibility.

The situation seems to be similar in countries with the

same level of safety as France.

In most countries, a crossmatch (a compatibility

test between blood for transfusion and the receiver's

blood) is carried out in a laboratory prior to

transfusion. The current techniques to carry out a

crossmatch are either manual, with blood reagents

and samples being mixed in tubes or being placed on

gel columns before centrifuging, or automated. As far

as these automated systems are concerned, the

analyzers may only be used in a laboratory and the

analyses are time consuming which can delays the

delivery of the blood product. This test is even

avoided in some emergency situations.

Currently, the final pre-transfusion test at the

patient's bedside consists mainly of an identity check

(identity of the red cell concentrate (RCC) and

identity of the patient). This method cannot guarantee

that there will be no transfusion accident because

50% of the reported adverse effects are due to human

errors, most of them being “wrong blood in tube”

(SHOT 2011). Therefore, in spite of increasingly

effective safety systems, it is currently impossible to

completely eliminate the risks due to human errors,

both in the laboratory and at the time of the

transfusion. These errors highlight the need for a pre-

transfusion analyzer which does not require pre-

treatment (plasma extraction, and/or centrifugation)

160

Charrière, K., Guitton, A., Ratié, V., Sighi-Dumoulin, L., Bourcier, V., Morel, P., Pazart, L. and Wacogne, B.

Blood-to-blood Immunological Compatibility Test: A Possibility with Fluorescent Immuno-Biochips.

DOI: 10.5220/0007356001600166

In Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2019), pages 160-166

ISBN: 978-989-758-353-7

in order to limit human manipulation and subsequent

errors.

Some research was carried out in order to make

this process ultimately safe. They are mainly based on

gel agglutination techniques which require plasma

extraction (Cid et al. 2006; Longston et al. 1999) or

SPR (Malomgre et al. 2009; Quinn et al. 2000;

Houngkamhang et al. 2013). More recently, Long-

Range Surface Plasmon-Polaritons have been

suggested to detect selectively captured red blood

cells using specific surface chemistry (Krupin et al.

2014). This system can capture red blood cells in a

sample with a very low cell concentration. The initial

dilution of the sample is carried out in a buffer with a

controlled refractive index. Blood samples must then

be prepared which is not an improvement compared

to current crossmatch. Test plates and image

processing have also been proposed for phenotyping

blood groups (Ferraz et al. 2010). Micro fluidics

coupled with optical fibers are reported in

(Ramasubramanian et al. 2009). A while ago, we

proposed the use of functionalized biochips for ABO

and rhesus compatibility tests (Charrière et al. 2015,

Wacogne et al. 2017).

Except the gel agglutination method which

requires samples preparation, the other techniques

could allow for blood grouping but not a direct

compatibility check between the RCC to be

transfused and the patient. The idea developed in this

position paper arose from the fact that incompatible

antibodies present in the patient’s blood bind to red

cells to be transfused and may lead, more or less

rapidly, to the lyses of RCC.

Therefore, our proposal is to use a single biochip

onto which red cells to be transfused are trapped.

Subsequently, patient’s whole blood is applied onto

the biochip and possibly present irregular antibodies

react with the RCC. Finally, fluorescent anti-IgGs

antibodies are used in order to allow rapid optical

detection. Using such an architecture allows detecting

any erythrocytic immunological incompatibilities

without extracting patient’s plasma. In this way, this

very rapid test can be performed by nonspecifically

trained people, with a reduced and non prepared

receiver’s blood sample.

This principle will be described in part 2 of this

communication together with the actual biological

model used to demonstrate a first proof of concept. A

description of the experimental set-up and results

obtained using fluorescence spectroscopy will be the

subject of part 3. In part 4 and in line with the scope

of a position paper, we will comment on the state of

industrial competition and the new devices market

positioning.

2 GENERAL PRINCIPLE AND

ACTUAL IMMUNOLOGICAL

MODEL

2.1 Biochip Principle

The method we propose is depicted in figure 1 in the

case of non-compatible transfusion. Red cells from

the RCC to be transfused are applied to the biochip

previously grafted with antibodies directed to a high-

incidence antigen (figure 1(a)). After rinsing, a RCC

layer is formed at the biochip surface.

Figure 1: Principle of the blood-to-blood immunological

compatibility test biochip.

Patient’s whole blood is directly injected onto this

surface (figure 1(b)). Antigen/antibody reactions

occur when RCC exhibits an antigen which is

complementary of an antibody present in the patient’s

blood (irregular IgGs in this case). After rinsing,

elements which have not reacted with the red cells

layer are evacuated.

Then, a solution of fluorescent anti-IgGs

antibodies is applied to the biochip (figure 1(c)).

These antibodies react with the patient’s irregular

antibodies forming a fluorescent layer at the biochip’s

surface. The latter is eventually detected using

conventional fluorescence techniques.

In this example, irregular IgGs have been

considered. However, incompatibility may be due to

presence of IgM in the patient’s whole blood (case of

the ABO incompatibility). In this case, and this is not

Blood-to-blood Immunological Compatibility Test: A Possibility with Fluorescent Immuno-Biochips

161

shown in this proof of concept communication,

fluorescent anti IgMs antibodies should be employed.

In order to address any incompatibility situation, the

ideal sensor should be adapted to the presence of both

IgGs and IgMs. A way of doing would be to use a

mixture of fluorescent anti-IgGs and anti-IgMs

antibodies. The fluorescent labels could be chosen in

such a way that both exhibit the same excitation

wavelength but different emission spectra. Here, a

simple fluorescence spectrum fitting would

furthermore indicate which kind of immuno-

incompatibility arose.

2.2 Immunological Model

In this model, RCC are captured on the biochip by

means of anti Kell IgMs (figure 2). The KEL:2

antigen (cellano) is present at the red cell surface of

about 99.6% of the population. Therefore, for this

proof of concept, the anti Kell IgMs are considered

universal. In the future, a red cell capture method

which also works for the remaining 0.4% of the

population will have to be defined.

Figure 2: Immunological model used in this proof of

concept.

In this example, red cells are Lub positive and Jka

negative. For the positive test, a solution of irregular

anti-Lub IgG antibodies is applied to the RCC

immobilized on the biochip. In the same manner, a

solution of anti-Jka antibodies is used for the negative

test. When antigen-antibody reactions occur,

antibodies bind to the red cells surface.

After rinsing, FITC coupled anti-IgGs antibodies

are applied to the biochips. They react with the

irregular antibodies possibly present at the RCC

surface. In a positive test, a large amount of FITC

coupled antibodies is present and a strong

fluorescence signal is detected. On the contrary in a

negative test, only non-specific interactions occur

leading to a weak fluorescence signal.

For these experiments, commercially available

irregular antibodies were used (Bio-Rad). Further

experiments will be conducted with whole blood.

3 EXPERIMENTAL SET-UP AND

PRELIMINARY RESULTS

3.1 Biochemical Reactions

Immunological reactions described in figure 2 were

performed using the fluidic system of our SPRi

apparatus (SPRi-Plex imager, Horiba Scientific) at

37°C with a MLB2 (Bio-Rad) running buffer.

Biochips were prepared according to the process

described in (Charriere et al. 2015) without RSA

saturation. Anti-Kell IgMs (dilution 1/10 in acetate

buffer, 10 mM and pH 4.5) were grafted onto

biochips.

200 µL of pouch’s red cells were injected onto the

biochips at 20 µL/min. After rinsing, 200 µL,

irregular antibodies (anti-Lub and anti-Jka, 1/10 in

MLB2, BioRad) were injected at 20 µL/min. FITC

anti-IgGs (Sigma) were injected (200 µL, 20µL/min,

1/25 in MLB2).

Complex red cells / antibodies were finally fixed

using a 0.5% glutaraldehyde solution. Biochips were

wetted with MLB2 buffer and protected with cover

slits before fluorescence measurements.

3.2 Fluorescence Measurement Set-up

FITC was excited at 488 nm using an Oxxius 488-50-

COL-PP laser. Excitation light was injected into a

fluorescence beam-splitter as shown in figure 3.

This beam-splitter (Doric Lenses) is equipped

with excitation and emission filters together with a

dichroic mirror (respectively FF01-488/10-25,

BLP01-488R-25 and FF500-Di01-2536).

Fluorescence spectra were recorded using a QE-Pro

spectrometer (Ocean Optics).

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162

Figure 3: Fluorescence beam-splitter used in these

experiments.

3.3 Additional Fluorescence

This kind of beam-splitter is usually designed to be

used with optical fibres. Because we use it in an open

beam configuration, additional fluorescence signals

are observed due to multiple reflexions in the beam-

splitter (figure 4). This additional fluorescence is

mainly generated by the epoxy resin used to glue

optical elements in the beam-splitter.

Figure 4: Origin of the additional fluorescence.

The shape of the additional fluorescence was

recorded and used as explained in the next section.

3.4 Experimental Results

Overall, 18 measurements were performed with 2

demonstration biochips (7 random places on the

positive biochip and 11 on the negative).

Fluorescence spectra were recorded as above

mentioned. In order to isolate the contribution of the

FITC signal from the whole recorded spectrum a

simple fitting was employed. Indeed, we recorded the

shape of the additional fluorescence and we know the

shape of the FITC emission spectrum. Fitting the

experimental spectra with these 2 shapes allows

separating the contribution of the FITC from the

contribution of the additional fluorescence. Figure

5(a) shows an example of spectrum recorded with the

positive biochip.

In this figure, the green shape corresponds to the

FITC while the grey shape correspond to the

additional fluorescence. It can be seen that the fitting

efficiently reproduces the recorded spectra. The

feature reported on the right of the spectra represents

the standard deviation of the difference between the

experimental spectrum and the fitted one. It is an

estimation of the fitting accuracy. It is clearly

observed that the signal due to the FITC (about 10000

levels) is much greater than the signal of the

additional fluorescence.

In figure 5(b), we present a spectrum obtained

with the negative biochip. Here the shape of the

additional fluorescence is clearly visible.

Figure 5: Fluorescence spectra of: (a) positive biochip, (b)

negative biochip.

As previously mentioned, 18 spectra were

recorded. Table 1 summarizes the levels of FITC and

additional fluorescence obtained at each places of the

biochips.

Blood-to-blood Immunological Compatibility Test: A Possibility with Fluorescent Immuno-Biochips

163

Table 1: Summary of the fluorescence levels obtained with

the 18 measurements on 2 biochips (1 positive, 1 negative).

Type Spectra N° FITC Add. std

Positive

QEP003631_11 12525 757 0.61

QEP003631_12 7470 766 0.39

QEP003631_13 8419 702 0.64

QEP003631_14 11261 797 0.37

QEP003631_15 12355 732 0.58

QEP003631_16 13935 702 0.7

QEP003631_17 1784 575 2.27

Negative

QEP003631_02 0 666 1.79

QEP003631_03 0 697 1.72

QEP003631_04 198 697 1.31

QEP003631_05 198 697 1.31

QEP003631_06 270 657 1.78

QEP003631_20 577 672 1.15

QEP003631_21 537 668 1.51

QEP003631_22 8 664 1.65

QEP003631_23 24 663 1.78

QEP003631_24 6 732 1.73

QEP003631_25 74 750 2.06

From these data, it can be seen that the level of

additional fluorescence is relatively constant (700±50

levels). Also, FITC levels of the positive biochip is

always much larger (except for the QEP003631_17

spectrum). It is however possible to define a threshold

above which a biochip is considered positive. This is

shown in figure 6 where a threshold at 1300 levels is

reported.

Figure 6: Definition of a positive threshold.

This figure shows that the experimental set-up can

be drastically simplified as a simple photodiode can

be used to detect positive biochips. This would make

the final device cost-effective and would ensure its

use in a large number of situations. Practically, the

same fluorescence beam-splitter as the one presented

in figure 3 can be used. This time, the expensive and

centimetre size spectrometer will be replaced by a

millimetre size photodiode.

However, the use of fluorescence spectroscopy

may prove to be extremely useful in the case of weak

antigen-antibody affinity or weak concentration of

irregular antibodies. In some cases indeed, even a

very low antibody concentration can lead to severe

consequences for the patient (for example the anti-

Jka). The interest of fluorescence spectroscopy is

illustrated in figure 7 which reproduces results

obtained with the QEP003631_20 spectrum. This

demonstrates that a simple fitting can extract a weak

level of FITC signal from a spectrum possibly

recognized as negative. Importantly, it must be noted

that figure 7 does not represent a weak affinity/avidity

situation but a case of non-specific interaction at the

place where the spectrum was recorded.

Figure 7: Extracting a weak FITC response using spectral

fitting.

To conclude this experimental section, this work

represents the preliminary results demonstrating that

the immuno-biochip technology can be used to

perform a complete blood compatibility test without

plasma extraction. Further experiments are still

required with a large range of irregular antibodies

before considering a clinical trial on a larger scale.

Also, issues concerning reduced labor and error risk

can be addressed by designing an immuno-combined

medical device as the one proposed in (Charrière et

al. 2018).

4 MARKET POSITIONING

In countries with a safe transfusion system (80% of

wealthy countries and 60% of averagely wealthy

countries), the compatibility check is carried out

either in the laboratory or at the patient's bedside, both

in some cases.

In the laboratory, ABO grouping of RCC and the

patient are carried out by analysers (Bio-Rad,

Diagast, HTZ, Dia Pro, Grifols). The current

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techniques for carrying out a crossmatch or an

irregular antibody screening are either manual, with

blood reagents and samples being mixed in tubes or

being placed on gel columns before centrifuging (e.g.

Across Gel® Cross Match, from Dia Pro or ID-Card

50531 from Bio-Rad), or automated (e.g. the Qwalys

analysers from Diagast). The analysers require

additional time-consuming manual operations, (blood

centrifugation for example) which may increase the

risk of errors. Furthermore, they are oversized for

technical platforms or small size laboratories.

To mitigate these risks, some countries, including

France, have formalised a final ABO compatibility

check at the patient's bedside (ABO compatibility

charts from Bio-Rad, Diagast). This check requires

qualified and regularly trained staff, is limited to

ABO compatibility and cannot prevent certain human

errors in terms of allocation, realization or

interpretation.

During the work presented here, a market research

carried out by a specialist firm and companies with a

potential interest, found that the biochip technology

is of major interest. There is nothing similar in this

enormous market. This market research also showed

that the final control of just ABO compatibility on a

biochip would not currently be sufficient to penetrate

the world market because of practices in place in most

countries. The companies we approached strongly

advised increasing the added value of this biochip by

broadening its application to carry out crossmatch,

what we did. Given this international perspective, the

two initial patents we published (Pazart et al. 2001-1,

2011-2) were also broadened and recorded in Europe,

North America and in “BRIC” countries.

5 CONCLUSION

We have presented the proof of concept of a biochip

potentially able to perform a blood-to-blood

immunological compatibility test in a simple fluidic

environment. It relies on a single biochip onto which

red cells to be transfused are trapped. Subsequently,

patient’s whole blood is applied onto the biochip and

possibly present incompatible antibodies react with

the RCC. Eventually, fluorescent anti-IgGs

antibodies are used in order to allow rapid optical

detection.

Fluorescence spectroscopy experiments showed

that irregular antibodies can easily be detected. Here,

we used solutions of irregular antibodies. The next

steps will consist in multiplying the types of irregular

antibodies and to perform experiments with whole

blood obtained from donors. Potentially, a simple

photodiode based detection can be used. This would

allow using a compact device which can be used

either by trained or non trained medical staff.

However, the use of fluorescence spectroscopy in a

more complex set-up can probably lead to the

detection of weak but potentially dangerous

incompatibilities.

ACKNOWLEDGMENTS

This work is funded by the Etablissement Français du

Sang Bourgogne Franche-Comté, contract “X-ult”,

April 2015.

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