Mont Blanc: where red meets white: The effect of altitude on thrombin generation

Thrombin generation (TG) is increasingly being recognized as a versatile
diagnostic tool in the field of thrombosis and haemostasis. Many groups
established that measuring TG is more sensitive to detect conditions of hyper-
and hypocoagulabilty, compared to the routine coagulation tests performed in
the hospital (e.g. aptt and INR). In plasma TG is most accurately measured with
the Calibrated Automated Thrombogram assay (CAT) as developed by Prof. Dr.
Hemker and co-workers. This method has been designed for application to
platelet free and platelet rich plasma. However, application of this method to
samples of whole blood leads to highly erratic signals, due to variable
quenching of the fluorescent signal by haemoglobin and red cells that sediment,
cluster and retract with in the clot. At Synapse b.v. we found that the
problems caused by the presence of red blood cells can be overcome by
dispersing the blood in a porous matrix and by using a new substrate that is
not quenched by erythrocytes. By these modifications of the plasma CAT assay,
quenching and problems related to unequal distribution of red blood cells were
acceptably reduced. The method can be applied in a 96 well flat bottom plate
like the original CAT method. The technical validation of the method revealed
that the assay is of sufficient precision to enable measurement of TG and a
study the contribution of blood cells (i.e. platelets, leukocytes and red blood
cells) to thrombin generation. We also developed a transportable point-of-care
(POC) test able of measuring TG in a drop of blood. A reliable TG assay in
whole blood is of importance because it enables to go one step closer to
physiology than present practice is. Apart from this conceptual advantage there
is the practical advantage that no centrifugation step is required. In the past
many studies have been executed to study the effect of oxygen deprivation due
to altitude on hemostasis. Contrasting results were found though most of the
studies found a prothrombotic effect on hemostasis. Two major conceptual errors
were made with studies performed in the past. The first error is that on
altitude the change in barometric pressure has an effect on blood drawing as
less blood will be in the tube although the anticoagulant will be the same.
With other words, the ratio blood:anticoagulant is no longer correct and
thereby induces an artificial anticoagulant effect. Another conceptual error
often made is that exercise, e.g. due to climbing or walking to a different
height induces an increase in Von Willebrand Factor and thereby in factor VIII.
This will introduce an exercise-increased hemostatic response not related to
altitude. Both influences have blurred the studies performed till now. We have
designed a study in which the exercise-induced increased hemostatic response is
isolated and we make use of a point of-care machine using capillary blood
without the need for vena-puncture. In fact, this will be the first *clean
study* to performed in the field of altitude induced hemostatic changes. The
primary goal of this study is to investigate the effect of hypoxia due to high
altitude on coagulation and haemostatic parameters.

Primary Objective: The primary objective is to investigate the effect of
hypoxia on blood coagulation using different techniques, among others the very
sensitive TG assay in whole blood and plasma.
Secondary Objective: The secondary objective is to look at the effect of
physical activity during hypoxia on coagulation parameters between two groups:
the physically inactive *cable car group*, who will climb the Mont Blanc by
cable car, and the physically active *climbing group*, who will climb the Mont
Blanc by walking.

This study is a comparative case-control study, in which we compare the effect
of oxygen deprivation on coagulation. For this study we will travel to the Mont
Blanc area where two groups will be formed. One group existing of 15 healthy
volunteers will actively climb the Mont Blanc by walking, while the other group
of 15 healthy volunteers will use the cable car. Blood will be taken every
other day by experienced researchers at different heights (at sea level, at
1000m, at 2000m, at 3100m and at 3850m) via venapuncture in the control group
(non-exercise group). In the exercise group blood will be drawn at sea level,
at 1000m, at 2000m, at approximately 3000 meter and at approximately 4000m. In
total 90ml of blood will be taken (5 times 18ml). Blood will also be taken 5
times with a fingerstick (two times 15µl, in total 150µl). The duration of the
study will last 11 days.
All blood drawing procedures will be done by certified persons holding at least
a nursing degree. Each group will be accompanied by at least 3 experienced
medical doctors.
All medical equipment will be delivered to the mountain refuges via helicopter
where needed. During the 11 day experiment, a helicopter will be stand by for
any needs.

Due to the climbing there will be reduced oxygen in the air, so the volunteers
will experience hypoxia.

Serious precaution is taken to prevent any other discomfort/burden: Experienced
guides (With E. Bekker as leader) will be hired to escort both groups to the
indicated areas. In case of symptoms preceding acute mountain illness or other
discomfort, the subject will be withdrawn immediately. The subject will be
escorted immediately to the basic camp at 1000m (Finhaut, Swiss) with a
helicopter and there he/she will receive the proper attention and transported
back to the Netherlands. All adverse events reported spontaneously by the
subject or observed by the investigator or his staff will be recorded. Both
groups will be escorted by at least 2 experienced medical doctors
(cardiologists, haematologists, aneasthologists).