Study and Validation of a Model
of Fetoplacental Circulation
2.3. Circuito di perfusione con formaldeide
Perfusion Circuit with Formaldehyde 
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Date le difficoltà oggettive riscontrate nell'analisi della placenta,
gli studi fatti in questo campo sono in numero esiguo, e i risultati spesso discordanti.
Le misurazioni vengono generalmente effettuate in vivo, sui soli vasi
del funicolo, mediante strumentazioni per uso diagnostico ad ultrasuoni,
o su placente prelevate post-partum e conservate in formaldeide.
La formaldeide blocca i processi di decomposizione e fissa le dimensioni
degli organi. Agisce piuttosto lentamente
ma penetra in profondità nei tessuti. Alla soluzione di formaldeide
viene generalmente aggiunta glutaraldeide,
che attua le stesse funzioni ma in modo molto più veloce, senza
penetrare negli strati profondi.
Nella quasi totalità degli studi precedentemente effettuati
la fissazione con formaldeide è stata attuata mediante immersione.
Risulta invece necessario compiere prove mediante perfusione di formaldeide,
poiché con la semplice immersione le dimensioni dei vasi non coincidono
con quelle reali nella placenta durante la sua attivitàdata la mancanza di
pressione al loro interno.
In un confronto tra placente fissate per immersione e attraverso perfusione
Burton [41] ha mostrato che la perfusione
genera sagome dei villi più regolari e uniformi, con capillari di
volume maggiore.
Il corretto comportamento della placenta è stato da noi controllato
perfondendo con soluzione fisiologica eparinata per circa 15-20 minuti,
prima di passare alla soluzione di formaldeide.
Il sistema che abbiamo messo a punto per la perfusione con formaldeide
(figura 44) si differenzia in parte da quello usato con il sangue.
2.3. Perfusion Circuit with Formaldehyde 
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Due to the difficulties faced during experiments with placentas, few
writings are available on this matter, and their results are usually conflicting
each other.
The measurements are usually taken in-vivo, only on the umbilical vessels,
with Doppler instruments, or with placentas stored in formalin.
The formaldehyde can stop the decomposition and harden the structure.
It works rather slowly but the results are good also for the deeper tissues.
Some glutaraldehyde is usually added: it is quite faster, also if does
not reach the deep layers. The concentrations used by the researchers can
be different, as a standard formula is not yet known.
Almost all the previous studies were related to placentas fixed with
formaldehyde by simple immersion, for at leas 2 weeks..
We used a system of perfusion for the following reasons:
- using a simple immersion, the vessel gauge is smaller that in the
physiological situation, because the blood pressure is not applied.
- the penetration of formaldehyde into the deep tissues takes place
when the decomposition is already started, thus the results could be not
correct.
Burton [41] compared placentas fixed with
the 2 methods, with and without perfusion, showing that the perfusion allows
to obtain better results.
The correct behavior of the placenta is checked with a perfusion of
isotonic solution, for about 15-20 minutes before starting with formaldehyde.
During the experiment we tracked the following data:
- Flow of perfusion;
- Pressures during the preliminary perfusion with isotonic solution;
- Pressures during the perfusion with the fixing solution;
- Flow of leakage.
Fig.44: Diagram of the circuit for perfusion with formaldehyde.
The developed system (figure 44) is slightly different
from the one used with blood: the differences are described in this chapter.
2.3.1. The Perfusion Circuit 
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The developed circuit, whose diagram is reported
in figure 45, is similar to the one used with blood,
previously described.
The differences regard the connections with the reservoirs of the perfusion
fluid, as the oxygenator is not used, and the fact that the perfusion is
carried out in 2 different ways, initially using isotonic solution, then
using our fixing solution. In particular the switching between these two
ways must be easy to do.
Fig.45: Schema del circuito di perfusione
Diagram of the perfusion circuit.
During the operation of fixing perfusion, the circuit sinks the fluid
from connection 1 of the reservoir, at the bottom of the container. The
roller pump (20) pushes the fluid towards the placenta, with the shunt
closed at point (14). The venous output goes to the reservoir through connection (2).
In the preliminary phase of perfusion, with isotonic solution, the
tubing normally connected to the points (1) and (2) simply sink from the
bath in which the placenta floats.
The heat exchanger is a coil of copper tube connected to the pump (24)
and dipped in the container of the reservoir.
Fig.46: La placenta durante la perfusione preliminare.
In primo piano: le connessioni delle cannule arteriose,
il funicolo ed il sistema di fissaggio.
Dietro la rete di separazione: i tubi per lo scambiatore
di calore, l'uscita collegata alla cannula venosa ed il tubo di aspirazione.
A placenta during the preliminary perfusion.
The connections of the arterial cannulas, the funiculus
and the system of fixing.
Behind the separation net: the tubing for the heat exchanger
(greater diameter), the output connected to the venous cannula (at right,
with the clip) and the sinking tube.
2.3.2. The Oxygenation
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The usage of a bubbling oxygenator for a formaldehyde
solution has not been considered mainly for two reasons:
- Operating with a solution of water and formaldehyde, that does not
have the characteristics of affinity for oxygen proper of the hemoglobin,
and above all that causes the sudden death of the cells, the usage of an
oxygenator has been considered a useless complication.
- The gaseous mixture bubbling in the perfusion fluid, gets out from
the oxygenator carrying out formaldehyde vapors.
So the circuit does not have any system of oxygenation for the fixing solution.
Anyway it was indispensable to equip the bath, collecting the isotonic
solution for preliminary perfusion, with a bubbler for carbon dioxide:
no experiments could have been completed without supplying the appropriate
level of PCO2 that normally flows in the umbilical arteries.
The isotonic solution normally on the market already has CO2 traces.
In order to avoid the sudden vasoconstriction previously described we made
a mix of CO2 and N2 in such proportions to obtain a PCO2 = 40-50
mmHg bubbling into the bath. The gas flows have been controlled by 2 flowmeters.
2.3.3. The Reservoir .
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Due to the elimination of the oxygenator we had to create
a reservoirs for the perfusion fluid.
For the isotonic solution we used the same thermostatic bath in which
the placenta floats.
For the solution with formaldehyde we used a 2.5-liter container of
PET.
On its cover we made the 4 holes for the insertion of the tubes by
means of connectors, as shown in figure 47.
Fig.47: La riserva, lo scambiatore di calore e la relativa
pompa.
The reservoir, the heat exchanger and its pump.
The solution is maintained to the proper temperature by means of a
heat exchanger made by a coil of copper with opportune dimensions, connected
to the circuit of exchange that was used for the system of perfusion with
blood.
The container is semi-transparent, it allows to control the level of
the solution and to measure the leakages.
Fig.48: Collegamenti alla riserva. Si nota in particolare
il morsetto a vite per la regolazione della pressione venosa.
Connections to the reservoir. The screw-clip is used
to control the venous pressure.
2.3.4. The Instrumentation .
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The level control has not been used as, for the reservoir,
the problem is negligible as the situation of perfusion with formaldehyde
is rather stable and does not demand particular attentions and, for the
bath of isotonic solution, the thermostat is already equipped with an alarm
system.
The pressure meter must be zeroed with the described steps, as the
test can be executed with variable flows. During the switching from isotonic
solution to formaline, the viscosity of the perfusion fluid does not change
appreciably.
best viewed with
res. 800 x 600.
Last updated: October 1, 2003