Development of a *two hit* in vivo autologous platelet transfusion model in healthy volunteers

Transfusion-related acute lung injury (TRALI) is the leading cause of
transfusion-related morbidity and mortality. The incidence is high and reported
up to 8% in specific patient groups. TRALI is thought to be a *two hit* event.
The first event is the underlying condition, often sepsis or an infection, of
the patient resulting in priming of neutrophils. The second event is the
transfusion of a blood product, after which either antibodies present in the
blood product or pro-inflammatory mediators present in stored cell-containing
blood products (e.g. Red Blood Cells (RBCs) or Platelets (PLTs)) or aged
erythrocytes and/or platelets themselves activate the primed neutrophils,
resulting in pulmonary oedema. Opposed to the traditional view that TRALI has a
good prognosis, evidence is accumulating that TRALI has a significant impact on
morbidity and outcome, at least in specific patient groups such as critically
ill patients. The association of transfusion with adverse outcome resulted in
blood product and donor management strategies aimed at decreasing the risk of
acquiring TRALI. Plasma products originating from female donors and
specifically multiparous donors have up to 40% presence of antibodies directed
against leukocyte antigens and are associated with the onset of TRALI. From
this point of view the Netherlands and the UK have started from 2006 and 2003
respectively a male only plasma donation policy. Excluding female donors for
plasma donation seems to have reduced, but not prevented the occurrence of
TRALI. Additional research is needed to determine whether the use of fresh (at
present RBCs are stored up to 35 days and PLTs up to 7 days in The Netherlands)
cell-containing blood products may be an additional measure to reduce TRALI. We
recently demonstrated in an in vivo animal model that the supernatant of stored
PLTs induce mild lung injury in the presence of a *first hit* of
lipopolysaccharide (LPS). However, clinical studies on the impact of
transfusion of aged platelets (PLTs) on respiratory complications have yielded
conflicting results. The discrepancy between results from pre-clinical and
clinical studies on the effect of storage time of cell-containing blood
products and the onset of mild lung injury calls for a randomized trial.
Although recent studies showed a relative high incidence of TRALI, still the
numbers are low and the presence or absence of a *first hit* is hard to measure
in the clinical setting which in total makes it difficult to perform a clinical
randomized trial. For this reason we propose to develop a *two hit* in vivo
autologous PLTs transfusion model, i.e. a mild TRALI model in healthy
volunteers. When the model is developed it has to confirm the hypothesis that
stored PLTs products induce mild lung injury in the presence of a *first hit*
in the human setting (i.e. a mild form of TRALI).

The advantages of such a model are the following;
1) an autologous transfusion model makes it possible to investigate the effect
of storage time on the onset of lung injury as the effect of anti-body mediated
TRALI is excluded; 2) the *first hit* is standardized; 3) this model will help
us to investigate pathways involved in onset of stored blood induced lung
injury and may enable us to test preventive or therapeutic measurements aimed
at improving storage conditions. The latter aspect of the model will become
very important in the near future and results from these studies may prevent
impeding a continuous reliable blood supply when a policy of fresh blood only
is proposed. Addition of a control arm to our study in which volunteers receive
saline as *first hit* will allow us to study the effect of stored platelets in
a *naïve* circulation that has not been primed by LPS. We propose the
development of a *two hit* in vivo autologous PLTs transfusion model, i.e. a
mild TRALI model in healthy volunteers to confirm that stored PLTs are
associated with the onset of mild lung injury in the presence of a *first hit*
of LPS in the human situation.

To develop a *two hit* in vivo autologous PLTs transfusion model in healthy
male volunteers

Open label, randomised intervention trial

Group 1 (6 volunteers): *First hit* Lipopolysaccharide (LPS) 2ng/kg i.v. +
*Second hit* Saline
Group 2 (6 volunteers): *First hit* LPS i.v. + *Second hit* Fresh Platelets
Transfusion (2 day storage)
Group 3 (6 volunteers): *First hit* LPS i.v. + *Second hit* Stored Platelets
Transufions (7 day storage)
Group 4 (6 volunteers): *First hit* Saline 10 ml i.v. + *Second hit* Saline
Group 5 (6 volunteers): *First hit* Saline 10 ml i.v. + *Second hit* Fresh
Platelets Transfusion (2 day storage)
Group 6 (6 volunteers): *First hit* Saline 10 ml i.v. + *Second hit* Stored
Platelets Transufions (7 day storage)

Methods: All subjects will be screened (medical history, physical examination,
ECG, blood examination, spirometry, DLCO, chest x-ray) by the research
physician of our hospital and of Sanquin Blood Bank prior to involvement in the
experiment. All included healthy volunteers (n=36) will donate 1 unit of whole
blood at Sanquin Blood Bank which will be processed into 1 unit of PLTs
(approximately 150 - 400 ml). Processing and storage will be according to
Sanquin Blood Bank protocol. Prior to transfusion stored PLTs will be
biotinylated (Vitamin B8) to allow their identification with flow cytometry. In
short stored PLTs will be labelled with Sulfo-NHS biotine of Pierce (6-20µg/
ml). Subsequently on the study day healthy volunteers receive a *first hit* of
either E. coli lipopolysaccharide (LPS) 2 ng/kg i.v. (n=18) or NaCl 0.9% 10 ml
intraveneously ( n=18). One and a half hours after the first hit, circulating
volume will be calculated using intravenous indocyaninegreen.
Two hours after the *first hit* they receive an autologous transfusion of 1
unit of fresh (2 day storage) biotinylated PLTs or an autologous transfusion of
1 unit aged biotinylated (7 days of storage) PLTs or an equivalent volume of
saline 0.9% infusion. The transfusion itself will be performed in one hour.
During the experiment subjects will be monitored for blood pressure and
arterial oxygenation using an indwelling arterial line. Blood samples will be
drawn from an indwelling artery line prior to the *first hit*, prior to the
indocyanine green infusion, 5, 10 and 20 minutes after indocyanine green
infusion, prior to the transfusion, 10 minutes, 0,5, 1, 2, 4 and 6 hours after
transfusion. Furthermore, 6 hours after transfusion spirometry and DLCO
measurement will be repeated. A chest x-ray and a directed broncho-alveolar
lavage (BAL) will be performed 6 hours after transfusion. The BAL will be
performed by an experienced pulmonologist according to the Dutch pulmonologist
guidelines (NVALT Guidelines, 2004). In the BAL-fluid and plasma samples
markers of inflammation, neutrophil activation and coagulation activation are
measured to confirm whether we have developed a model of TRALI. Two to four
days and three months after the study day a venous sample of 4 ml will be
collected to monitor platelet kinetics and to measure prevalence of biotin
antibodies.
Furthermore cardiac output wil be measured continously with a nexfin and
EEG-dopler measurements will be perfomed during the studyday, to investigate
the neurovascular coupling during endotoxemia.

Benefits: none.

Blood donation:
1. The donation of the blood transfusion will take place at the Sanquin Blood
Bank. The blood donation can be accompanied by some pain and the chance of a
bruise. The blood transfusions are processed by Sanquin according to their
standard protocol for clinical blood products.

Blood products:
1. Blood transfusions originate from the test person himself and will not bare
risk of for example virus transmission. The possibility exists that the
transfusion will induce mild transient lung injury. The test person might
perceive this as as mild stuffiness.
2. During storage of the blood product samples from the blood product will be
taken to measure the storage related changes in the blood product. The sampling
finds place under sterile conditions.
3. Prior to transfusion stored PLTs will be biotinylated (Vitamin B8) to allow
their identification according to previously published protocols.In short
stored PLTs will be labelled with Sulfo-NHS biotine of Pierce (6-20µg/ml).
Preparation will be done under sterile conditions. Cultures will be taken of
transfused products to confirm sterile conditions. Three months after the study
day a venous blood sample will be collected to detect development of biotine
antibodies. This data will be used to investigate antibody prevalence after
exposure to biotin. The presence of absence of antibodies has no clinical
relevance and repeated intravenous exposure to biotin does not produce adverse
effects.

The experiment:
1. The test person will undergo two times a chest X-ray. The radiation exposure
is considered to be minor.
2. During the experiment an artery line will be inserted in one of arteries of
the arms of the test person. The placing of the artery line is performed by an
experienced doctor. There is a small chance this procedure results in bruises
or blood clot formation in the blood vessel. During the last years no major
complications occurred with this procedure in our center.
3. The administration of endotoxin can lead to mild flu-like symptoms, which
can include a slight increase in body temperature, muscle pain and/or fatigue.
4. The bronchial lavage (lung rinse) is performed by an experienced
pulmonologist. The main source of discomfort during this procedure is a dry
cough or mouth. However, these complaints are suppressed by the lidocaine
spray. In addition, there may be onset of fever within 24 hours.
5. Blood sampling during the experiment will be from the artery line and will
not result in additional punctures. The total amount of blood that is sampled
during the experiment is 150 ml. The 150 ml sampling is next to the 150 - 400
ml of blood donation. However, depending on the experimental group the test
person is in, the majority of the 150 - 400 ml donation will be returned during
the experiment by the platelet transfusion. The human body can easily handle
these changes in blood volume. It is not allowed to donate blood or participate
in another study within the three months prior to this study or during this
study.
6. The pulmonary function measurement is estimated as no burden or risk for the
healthy volunteers.
7. Indocyaninegreen will be administrated through the same i.v. as the LPS.
Therefore we estimate that the use of indocyanine green is a limited extra
burden for the healthy volunteers.

Risks assessment:
1. Infusion of E. coli LPS with a dose of 4 ng/kg has previously been proven to
be safe in healthy adult volunteers in our institution. We will use a lower
dose of 2 ng/kg which will induce neutrophil priming but will not result in a
SIRS reaction.
2. The use of an autologous transfusion human volunteer model has also been
proven to be safe.
3. Furthermore transfusions will be prepared and transfused using the standard
clinical protocols by Sanquin and our hospital.

The combination of these two models is expected only to cause mild temporary
side effects because of the following reasons:
1. Pre-clinical studies show only mild lung injury after a *first hit* of LPS
2mg/kg and a *second hit* of transfusion of stored blood products.
2. In the present model a 1000 fold lower dose of LPS (2ng/kg) will be used
compared to the pre-clinical model which we assume will make the model less
severe.
3. We developed a comparable model with 2 ng/kg LPS with consecutive
transfusion of aged Red Blood Cells. This model was proven to be safe in
healthy volunteers.
4. Studies in healthy human volunteers showed that biotinylated (Vitamin B8)
RBCs can be safely administered without any side-effects. Although in a healthy
volunteer study 1 out of 8 subjects developed a transient positive test for
antibody to biotin, at 11 months post transfusion antibodies had disappeared.
Repeated intraveneous adminsitration of biotinylated PLTs had no adverse
effect. Thus, biotinylation of PLTs is considered safe.
5. The use of indocyaninegreen has been extensively studied in healthy
volunteers and in critical ill patients, these studies did not show any
negative side effects. However indocyanine green contains iodine, therefore
people with active thyroid diseases, an allergy or hypersensivity for
iodine(products) are excluded from the study.
6. Nexfin and EEG-dopler measurements are non-invasive and painless.