A blood-mimicking fluid with cholesterol as scatter particles for wall-less carotid artery phantom applications

The BMF mixture fluid was first prepared by varying the percentage compositions of distilled water, propylene glycol (Sigma Aldrich, Germany), and polyethylene glycol (Merck KGaA, 64271 Darmstadt, Germany) in order to find the correct combination that gives an acceptable ternary fluid. The process was carried out as follows:

(i) First, a glass beaker the size about twice the volume of the BMF was washed with distilled water and wiped clean with a tissue paper. The beaker of this size was selected to avoid the overflowing of the fluid while stirring. This was followed by placing a magnetic stirrer inside the beaker.

(ii) 90% weight/weight (w/w) of distilled water was measured using a measuring cylinder, and 5% w/w of propylene glycol (PG) was weighed as well as 5% w/w of polyethylene glycol (PEG) in a fume cupboard using an electronic balance (multi-function balance, A&D Company Ltd, Japan). Distilled water was first poured into the beaker, followed by PG and PEG. The combined components were then placed on a magnetic stirrer hot plate (Favorit, 41200 Klang, Malaysia) for stirring.

(iii) The stirring plate was set to operate at 700 revolutions per minute (rpm) for 20 minutes at a temperature of 37⁰C, after which a vacuum pump was used to degas the fluid for about 30 minutes.

(iv) The procedures above were repeated four more times but with different percentages of distilled water and PEG, while the amount of PG remained fixed at 5% w/w composition. The five (5) samples as well as 100% w/w distilled water were then tested for their densities, viscosities, speed of sound, and attenuation properties. The results are tabulated in Tab. 2.

The serial number 8 in the table has a density (1.065 g/cm³) close to that of cholesterol. However, it was not suitable as a mixture fluid because the speed of sound (1670.2 m/s) and viscosity (12.40 mPa.s) diverged considerably from the internationally acceptable values (Tab. 1), even though the attenuation value was within a good range. This led to the replacement of PEG with D(+)-Glucose (DG) (Merck KGaA, Darmstadt, Germany) with a higher density (1.54 g/cm³), which the steps in (i) to (iv) above were repeated, with the results shown in Tab. 3.

(v) A linear relationship was established using the statistical package for social sciences (SPSS, Version 21) software between the density and the amount of DG in the fluids, resulting in the equation:

Density = 0.00426 (amount of DG) + 0.993

With equation 1, 17.37% w/w of DG by weight was required to mix with 5% w/w of PG and 77.63% w/w of distilled water to produce exactly a mixture fluid with a density of 1.067 g/cm³, a value required to match the density of cholesterol scatters. This allows the scatter particles to remain neutrally buoyant in the fluid mixture.

(vi) Finally, a sieving net with the mesh size of approximately 10 was used to sieve the cholesterol particles (≥90% purified, Merck KGaA Darmstadt, Germany) before mixing them with the fluid. Different concentrations of cholesterol particles were added to the mixture fluid until the right amount required for the particles to stay suspended in the fluid was achieved. The BMF thus prepared was then mixed with about 4 drops (0.2 ml) of benzalkonium chloride (Sigma Aldrich, Germany) solution to serve as a preservative.

A TMM wall-less phantom of the CCA was prepared with a lumen diameter of 8.0 mm, as described by Ammar et al. ⁽²⁴⁾. The TMM composition was made up of 84% w/w distilled water, 0.53% w/w of silicon carbide (Logitec, Glasgow, UK), 0.96% w/w of aluminum oxide, 3.0 μm size (Sigma Aldrich, Germany), 0.89% w/w of aluminum oxide, 0.3 μm size (Sigma Aldrich, Germany), 0.92% w/w of gelatin (gel strength 300, Type A, Sigma Aldrich, Germany), 1.5% w/w of konjac powder (dietary supplement, NOVA Nutritions, Scotch Plains, New Jersey USA), 1.5% w/w of carrageenan powder (Sigma Aldrich, Germany), 0.7% w/w of potassium chloride (Sigma Aldrich, Germany), and 9.0% w/w of glycerol (Vetec^TM reagent grade, Sigma Aldrich, Germany). About 5 cm³ of benzalkonium chloride solution was added to 2 liters of the TMM as an anti-fungal agent. The prepared TMM was cast inside a plastic (acrylic) rectangular box with a straight metal rod placed horizontally inside at a depth of 15 mm. When the TMM had cooled to about 40°C, the metal rod was removed gradually to provide a lumen which was attached to rubber tubes for connection to a pump.

The densities of the BMF fluids were measured using a portable density meter (DMA 35, Anton Paar GmbH, Austria) at 37⁰C. The strip attached to the meter was pressed down and dipped inside the fluid, and then released to draw up the fluid inside the strip by suction pressure, while the density reading was recorded automatically by the meter in just few seconds.

About 700 cm³ of each BMF was required for the viscosity measurements using the electronic rotational viscometer (ERV) (Software Version 1.2, Fungilab, Barcelona, Spain). The spindle L1 was selected for viscous liquid; it was fixed to the ERV and then lowered into the fluid, while the ERV was switched on. The spindle rotated inside the liquid for a few minutes until a steady value of viscosity was recorded due to the viscous resistance from the fluid^(25,26).

The speed of sound was measured by the pulse echo (PE) method using the ultrasonic echoscope GAMPT-scan machine (German Society for Applied Medical Physics and Technology, GAMPT Ultrasonic Solutions, Hallesche Straße, Merseburg, Germany). The arrangement for this measurement is shown in (Fig. 1), where the time of flight (TOF) between the highest two peaks of the transmitted and about 90% of the received signal (energy) at a frequency of 5 MHz was measured. For good accuracy, the protective layer thickness of the probe was first calculated and added to the distance between the walls of the vessel containing the liquid. This was done by using two acrylic plates, 40 mm and 80 mm thick, with the same speed of sound of 2700 m/s. The pulse echo method using A-scan GAMPT technique was able to provide time of flight for the two plates which was used to calculate the thickness of the protective layer of the probe from the equation: d p 1 = ( t 1 t 2 d a 2 ) ‒ d a 1 2 ( 1 ‒ t 1 t 2 )

Fig. 1.

where dp₁ is the thickness of the transducer protective layer, da₁ is the thickness of the acrylic plate (40 mm), is the thickness of the acrylic plate (80 mm), t₁ is the time of flight through the plate (40 mm), and t₂ is the time of flight through the plate (80 mm). The values for the speed of sound for the liquids were calculated from the equation: speed of sound = 2 ∗ ( X + d p 1 ) t

where x is the distance between the walls of the vessel containing the mixture liquid and t is the time of flight.

The amplitudes of the highest two similar peaks were also measured, and the attenuation coefficients (α) were calculated from the attenuation equation: a = 2 × 0.868 X ln ⁡ A 2 A 1

where A₁ and A₂ are the amplitudes of the two highest but similar wave peaks, respectively, and x is the distance.

The backscatter power of the BMF was measured at different radio-frequency signals by calculating the average power spectrum through applying the fast Fourier transform (FFT) generated by the A-scan GAMPT at 5 MHz. This was done to find out if the BMF simulates real human blood.

A digital clinical Hitachi ultrasound scanning machine (HI VISION Avius, Hitachi Medical Corporation, Tokyo, Japan) connected to a linear array transducer (probe) EUP-L74M with frequencies ranging from 5 to 13 MHz was used to obtain an image of the wall-less flow phantom. The phantom was placed on a flat table and connected to a centrifugal multi-flow pump (German Society for Applied Medical Physics and Technology, GAMPT mbH, Hallesche Straße, Merseburg, Germany) with the aid of plastic tubes to pump the BMF. The fluid was pumped through the lumen at a flow rate of 1500 ml/min (for both steady and pulsed flow), and B-mode images of the lumen and the BMF were displayed on the screen of the scanning machine. The measurements of peak systolic velocity (PSV), end-diastolic velocity (EDV), mean or average velocity (V_m), pulsatility index (PI) and resistivity index were carried out by color Doppler and pulsed-wave (PW) Doppler systems. The angle of beam (insonation angle) was set at the center of the flow at 60⁰ with the required sample volume and entrance length of about 8 cm. To obtain a good image of the phantom and BMF flow, the transmitting frequency was adjusted on the scanner and set at 5 MHz, while the display depth, focus position, image size, sample gate, pulse repetition frequency (PRF), sample volume and color brightness settings were all adjusted until the normal settings were achieved.