2.2. Protocollo di prova con sangue Test Protocol with Blood |
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2.2. Test Protocol with Blood |
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2.2.1. Instrumentation . |
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The instrument must be zeroed following 2 different procedures for the 2 channels:
Fig. 31: Theoretic pressure loss in the arterial cannulas
versus m.
The real diagram are depending, other than by the viscosity
m
and by the flow, by the arterial pressure too, for the elasticity
of the cannulas. As the geometry of the cannulas is quite variable
(they are manufactured without particular control on the accuracy of the inner diameter) we
we were not able to define an accurate
DP, we measured this value at the end of each experiment.
It must be subtracted from the value read by the instrument. Moreover the instrument does not allow
the zeroing if the measured pressure is higher than 80 mmHg.
For these reasons the transducer has been put in higher position,
90 cm over the level of the placenta, and the zeroing, not definitive,
has been done with a reference flow of 100 cc/min.
Fig.32: Model of the transducer.
Both the transfer functions have 2 complex poles, but the second circuit has a lower bandwidth. Without air fcut is about 80 Hz for the transducer, so the response is almost flat up to about 50 Hz, but air bolls reduce fcut to 20 Hz, jeopardizing the signal.
2.2.2. Fluid for Perfusion |
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Erythrocytes | Leukocytes | Platelets | Hemoglobin | Haematocrit |
1012 / l | 109 / l | 109 / l | g/100ml | % |
6.5±1.5 | 7±3 | 500±300 | 11.5±2.5 | 31±9 |
To minimize the difference between fetal and bovine blood, we analyzed
its properties at each working session, measuring the haematocrit and
the viscosity.
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1012/l |
109/l |
109/l |
g/100ml |
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2.44± 0.46 | 4.3± 2.8 | 204± 44 | 11.1± 1.8 | 34.1± 9.8 |
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2.71± 0.57 | 3.9± 2.4 | 266± 87 | 11.6± 1.9 | 36.4± 9.3 |
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2.77± 0.51 | 4.3± 2.8 | 244± 66 | 11.7± 1.6 | 36.9± 7.3 |
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2.89± 0.58 | 4.2± 1.9 | 254± 102 | 12.2± 2.4 | 37.8± 9.1 |
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2.98± 0.43 | 4.2± 1.6 | 260± 109 | 12.4± 1.6 | 34.4± 6.8 |
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3.02± 0.52 | 4.2± 2.5 | 261± 93 | 12.3± 1.8 | 38.1±8.3 |
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3.17± 0.45 | 4.3± 1.2 | 275± 88 | 12.6± 1.7 | 39.1± 6.2 |
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3.31± 0.53 | 4.3± 1.2 | 263± 91 | 13.1± 1.7 | 40.3± 7.5 |
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3.35± 0.68 | 4.4± 1.5 | 269± 98 | 12.9± 2.4 | 40.5± 7.8 |
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3.43± 0.53 | 4.3± 1.6 | 268± 81 | 13.2± 1.9 | 41± 6.9 |
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3.60± 0.55 | 4.6± 2.1 | 290± 86 | 13.4± 2.9 | 41.6± 8.1 |
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3.66± 0.51 | 4.8± 1.4 | 264± 144 | 13.6± 1.8 | 43.2± 8.6 |
Available data about the blood viscosity are conflicting and not helpful: the authors used different methods whose results cannot be compared, not only for fetal blood, but even in the case of maternal blood.
The main results obtained in the last 70 years on pregnant women
are listed in table 4. The values of the first column were obtained by
Cohen e Thomson through calculations based on the hematocrit, the second
and the third column were obtained by Hamilton and Buchan, who
used a Ostwald viscometer and a spinning viscometer [40].
In table 5 data about normal fetuses are compared with values of pregnant / not
pregnant women (by Buchan [40]).
Tab.4: Viscosity during pregnancy
Compared with a woman, the fetal
hematocrit is higher, and its erythrocytes are less deformable.
The 2 effects give a higher viscosity of the whole blood.
Anyway, if analyzed with a normalized value of hematocrit,
the fetal blood viscosity is rather low,
due to the low concentration of fibrinogen and of the others
blood proteins.
The values are also dependant from the gestational age. The following table is the only published study.
[40]:
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Hematocrit |
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Deformablity erythrocytes |
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Viscosity plasma cP |
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Viscosity blood cP |
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A spinning viscometer was offered by
Giuseppe Cambiaghi S.p.A. in Rho (MI, Italy), but it has not been used
because requiring at least 50 cm3 of blood, probably too much
for a newborn. For the same reason we did not want to repeat these measurements
with our capillary viscometer.
As our method was comparable to the one used by
Cohen and Thomson, we used to dilute the bovine blood
if the measured viscosity was greater than 4,5 cP, up to this value.
To reduce the differences between fetal and bovine blood we considered
also the pH value: it affects the metabolism and the oxygen saturation curve.
As the pH value is related to PCO2, its measurement must be performed
when PCO2 is 40 mmHg.
Fig.37: Placenta at UV rays after perfusion with blood and fluorescent yellow coloring agent.
During some of the sessions we added few
mg of uranin (fluorescent yellow coloring agent) to the bovine blood, in order to mark
leakages and quality of the perfusion by UV rays (365 nm, 4 W). The above picture evidences
that the perfusion reached all the districts of the placenta.
2.2.3. Preparation of the Circuit . |
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Fig.40: The circuit ready to be connected
to the placenta, working with the shunt (near the venous and arterial connections).
2.2.4. Preparation of the Placenta . |
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Fig.41: A placenta after the insertion of the venous cannula (click to enlarge).
During this step air should never be introduced. The arterial cannulas must be previously filled with physiologic solution using 2 syringes, that are kept connected during the insertion into the umbilical cord. A light force on the syringe pistons allows to verify that the solution flows into the chorionic arteries, evidencing the correct insertion of the cannulas. This force must be low: to create a pressure as high as 100 mmHg, only 2.67 N (it is the force due to the gravity for an object weighting 272 grams) are enough:
piston surface = 2 cm2
F = 200 mm2 *1,333 * 10-2 N * mm-2 = 2,67 N.
At the end, the cannulas can be fixed with 2 clamping strips.
About 10 cc of physiologic solution with heparin should be injected into each artery
to divide the fetal blood inside the placenta
from the bovine blood that is to be introduced by the perfusion circuit.
Doing so, some blood should come out from the venous cannula, but in lower
quantity, for the general constriction due to the absence of blood pressure after the
withdrawal.
Fig.42: The umbilical cord with its cannulas. The venous blood is evident (click to enlarge).
Some authors [38] noticed
a seepage of water through the membrane dividing
the fetal and the maternal circulation (this fact happened also
during our experiments): this membrane, to allow the
exchange of substances, is permeable to molecules with low molecular weight.
With a seepage of few cc/minute we did not notice
fluorescent colouring agents with molecular weight higher than
5000 crossing the membrane.
All the mentioned steps must be completed in less than 10 minutes.
2.2.5. Connection to the Perfusion Circuit . |
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Fig.43: A placenta being perfused.
If the pressure is too high, the vessels can break, as evidenced by
blood flowing into pool or under the chorionic membrane. If the mentioned level is exceeded,
The blood flow has to be reduced, also recalculating
the pressure losses due to the cannulas.
The placenta, just released, is generally vasoconstricted. The vasoconstriction decreases
slowly during the perfusion with blood characterized by high PCO2; for
this reason, at the beginning of the perfusion, the physiologic flow rate
has to be reached gradually.
None of the researchers that made experiments of
in-vitro perfusion has ever been able to obtain a physiologic
pressure with input flow greater than 120 cc/minute:
see table 7 for the values available in the literature [38]:
Q cc/min | P mmHg | |
Gagel | 120 | 120 |
Kranz | 78~ 120 | 110 |
Nesbitt | 60~ 90 | |
Vermeulen | 90~ 200 | 90~ 250 |
Before connecting the venous cannula to (11) the fetal blood that is inside the placenta
must be discharged waiting few minutes, during which the flow of the venous output can be measured.
The air chambers are activated opening (21) and (22) and trimming
the resistances (5) and (6) when the needed flow is reached:
measurements can be done only after that the levels of the chambers is stable.
If the flow has to be increased for any reason, the pressures have to be monitored
with (21) and (22) closed: otherwise the air chambers introduce strong
delays, making the trimming of the pump velocity difficult.
2.2.6. Data Measurement . |
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Apparent total leakage: DV = Vi-Vf
Leakage of corpuscular part: DVc = Hti*Vi-Htf*Vf
Real leakage of blood: DVs = DVc/Htm
Seepage: DVt = DV-DVs
Tables with data obtained during our experiments are listed in Ch 2.5 of this thesis.
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