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.

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Last updated: October 1, 2003