Week 8

Week 8

After doing numerous tests, we have narrowed down the critical pressure of the glass tube. With this information, and the pending information from the leaking rate experiment that is till being run, we will be able to provide a shelf life for the VacuStor tube. Now we need to determine the critical pressure of the plastic VacuStor tube as well. In addition, we will potentially explore the leaking rate at lower temperatures for the possibility of storing them at -4 or -20 degrees Celsius.

Critical Pressure Experimentation

As of now, we have determined the critical pressure to be -25 kPa under atmospheric pressure, or 76 kPa. Anything past this critical pressure will not draw enough sample for proper analysis. The minimum sample required is 90 microliters, so 76 kPa internal pressure is enough to draw that much. However, the optimal pressure is any pressure lower than -30 kPa below atmospheric pressure, or 71 kPa. This ensures 100% sample transfer required for proper assay analysis and accurate results. The pressure determined seems to be accurate

Once we obtain the leaking rate time to reach this pressure, we will know the shelf life of the tube at room temperature. However, we also know pressure and leaking rate of pressure is not only dependent on permeability, but also temperature. Hence, we are considering testing the leaking rate of the tube at different temperatures as well.

Effect of Temperature on Leaking Rate

A possible experiment I am considering is to determine the effect of temperature on the leaking rate of the VacuStor tube. This may be helpful because if we can get a longer shelf life from keeping the tube at a lower temperature, it might be better to advise the tube be stored at lower temperatures if possible. So, in order to test this, we will evacuate a tube and then see how pressure and the vacuum inside is affected by the temperature.

I have begun to compile the already conducted experimental data and will hopefully begin to start my report.

Week 7

Week 7

As we narrowed the critical pressure threshold for the glass VacuStor tube, we noticed a slight complication. Depending on the speed at which we pierced the cap of the tube to transfer the liquid, we got differing amounts of sample transfer. Piercing the tube faster resulted in more sample transfer. We realized that this is due to pressure leak during the puncture, but before the bevel has entered the tube. The sample only begins to be drawn into the tube when the bevel fully enters the tube. However, if the bevel is longer than the cap thickness, there is pressure leakage until the bevel enters wholly into the tube. Because there is such a huge pressure difference between the internal pressure and the external pressure, we have to find a needle that has a bevel shorter than the cap thickness. Thus, we slightly modified the experiment by manufacturing new needles with capillaries that have a shorter bevel.

Modified Needles with Capillary Tubes

We used the same method to make these needles as the previous needles. However, we used 30 Gauge 1/2 inch needles instead of 21 Gauge 1 inch needles. Using the UV epoxy, we attached the capillaries to the needles. This was the result:

Modified Capillary Tube with Needle

This needle has a much shorter bevel as compared to the previous needles we used, and is also much thinner and shorter. The following pics show a comparison of the two needles (the green needle is the old needle):

Comparison of Bevel Length

Comparison of Length of Needle

This should aid in the sample transfer to provide more accurate results. Hence, this week I made these needles, which will be the needles that we will use for the remainder of experimentation.

Week 6

Week 6

As we began collecting data on the leaking rate of the plastic tube as well, we have begun preliminary testing of  the critical pressure. We finished constructing the experimental setup and tried varying internal pressures to see if the setup worked.

Critical Pressure Threshold Experimentation

We finally finished fabricating the experimental setup for the critical pressure testing. This is how the experimental setup looked like:

Experimental Setup for Critical Pressure Experimentation

The protocol simulates how the capillary tube will pierce the tube with reagent in it. First, we will the tube with 'reagent'. In this case, we used water, as shown:

VacuStor Tube with Mock Reagent (Water)

Then, we fit it onto the rubber stopper, which has a half-drilled hole to place the tube in. Then, we attach the vacuum tube to the tube and evacuate the tube at the desired pressure we want. We can evacuate the tube to varying extents to create differing levels of internal vacuum. Finally, we cap the tube, and then remove the tube out of the setup. Then, we fill the capillary with fluid, and then pierce the tube with the capillary. Depending on the pressure, we can get no sample transfer, partial sample transfer, or full sample transfer. Since we wanted to make sure this setup worked, we chose pressures that we knew the outcome would be, and here were the results:

Left tube was tube with high vacuum; Right tube was tube with low vacuum

As you can see, one had no sample transfer, and one did. We weighed the tubes before adding the water, after adding the water, and after the sample transfer, and used the density of liquid in order to determine how much sample transfer there was.

Week 5

Week 5

This week we mainly began building our experimental setups for determining the critical pressure for the tube and for determining the leaking rate for the plastic tube. We had already constructed a setup for the glass tube to figure out the leaking rate, so we created the exact setup for the plastic tube.

Leaking Rate - Plastic Tube

We created the same setup for the leaking rate of the plastic tube as the setup for the glass tube since both are approximately the same size. We sealed the gauge to the tube using a vacuum epoxy and then we evacuated the tube and sealed the tube with a rubber cap through teh use of the setup we developed. Here are both tubes with their respective gauges monitoring the internal pressure:

Leaking Rate gauges attached to both the glass and plastic VacuStor tubes

Top View of both gauges attached to their respective tubes

We will be monitoring both tubes now to determine which of these materials will be most effective in maintaining the vacuum, and thus, having the longest shelf life.

Critical Pressure Experimental Setup

We have started building the critical pressure experimental setup, but were unable to finish it this week. Hopefully, we will be able to finish it next week. However, this is what we have so far:

Central portion of Critical Pressure Experimental Setup

In theory, we will have a similar setup to the leaking rate setup, but the gauge will be on the outer tube instead of the VacuStor tube. This is because we only need to know the pressure inside the VacuStor just before we cap it, after which we will puncture the cap with the capillary tubes we fabricated to see how much liquid the vacuum will draw. Once we find the minimum pressure that can draw enough fluid into the tube, we can use this as our reference for extending the shelf life.

Week 4

Week 4

This week we manufactured the capillary tube with the piercing needle for testing, which will likely happen next week. In addition, the materials we ordered for our experiments arrived, which also means we will likely begin experimentation next week.

Capillary Tube with Piercing Needle Fabrication

In the schematic shown from Week 1, our design requires a piercing needle with a capillary tube. We need the needle to pierce the cap of the VacuStor tube so that the vacuum in the tube is maintained until blood collection. To do this, we took capillary tubes and cut them so that they hold around 100 microliters. Then, we took 21 Gauge 1-inch needles and sawed off the hub where you would put the syringe. We then used an epoxy to stick the capillary tube to the opening of the hub of the needle, and cured the epoxy with the capillary and needle under ultraviolet light.

The result was this:

Capillary Tube with Piercing Needle

We made a few of these to set up for next week's experiments. In addition, the pressure gauges, along with other materials we purchased, have arrived, so we can begin experimentation. We will likely follow a similar experimental setup as the rise time setup, but instead we need to determine a method to have the gauge measure the pressure within the tube, but be able to change the pressure of the tube as well so that we can determine at the lowest pressure difference through which we can still draw fluid through into the tube. We will likely use a three way valve to facilitate this, though a major part of next week will be hashing out how to go about doing this and hopefully running some trials.

Week 3

Week 3

We finished developing a basic theoretical analysis on the shelf life of the VacuStor tube, and have started an experiment with the rise time of the tube. We first had to fabricate an experimental setup for this experiment, and now we have begun taking experimental data regarding the rise time, and we do so each day to monitor the internal pressure of the tube.

Theoretical Analysis

The shelf life of the tube is determined based on how long it can retain the vacuum. It is extremely difficult to create an impermeable tube and seal, especially one that can be mass manufactured. Hence, we want to be able to limit the permeability of gas to the best of our ability. There are two factors in determining the shelf life of the tube. One, which was already looked into, is the threshold or critical pressure. Since we know for sure the pressure in the tube will rise, it is a matter of not only what pressure that, once surpassed, makes the tube unusable, but also how quickly it reaches this pressure.

The diffusion of gas is described by Fick's First Law, and we used this equation to determine the mass transfer flux rate, which is the flux density times the surface area of the tube, or:

J is the mass transfer flux rate, which is the total rate of diffusion of gas across the entire tube. j is the flux density from Fick's First Law. A is the surface area, P is the permeability, p_out­ and p_in­ are the pressures outside and inside the tube respectively, A is the surface area of the tube, and δ is the thickness of the tube.This mass transfer flux rate is also the leaking rate, which we want to minimize. Hence, in order to do so, we want to have a tube with a small surface area and a large thickness with a small permeability constant, which is determined by the number of holes in the polymer structure of the tube. 

The models are helpful in understanding what occurs during the experiment and what variables to focus on most when attempting to test the tube, which is what we started doing this week.

Rise Time

The rise time is the time the tube takes to equilibriate from the low pressure vacuum to standard pressure. To test this quantitatively, we created the following setup:

Rise Time Experimental Setup

 The vacuum gauge was sealed to the VacuStor with an epoxy, as shown:

Vacuum Gauge and VacuStor Tube

This allows the vacuum gauge to monitor the pressure inside the tube. Then, having evacuated the tube, we added a leakage port so that the surrounding pressure would be equal to atmospheric pressure. We then monitor the pressure of the inside of the tube daily so that we can determine the rise time of the tube. This will obviously take multiple weeks until results can be analyzed, but hopefully this experiment will provide useful results.

Week 2

Week 2

For those of you who would like to see in depth the scope of this project, I have uploaded my proposal.

A vital step before beginning experimentation is to develop a theory behind the concepts that you wish to experiment on. This is crucial because it narrows the huge number of variables to a select few that will impact the outcome the most. For this project, the most crucial factors are assessing the vacuum needed for proper sample transfer, the shelf life and permeability of gas, or the leaking rate. These were modeled mathematically with certain assumptions. So far we have developed a theoretical analysis for determining the vacuum threshold for which there will be proper sample transfer of blood from finger to the VacuStor tube.

Theoretical Analysis

Figure 1

Figure 1 depicts the evacuated VacuStor tube with the reagent inside the tube. There is 200 microliters of reagent prior to blood collection. After blood collection, 20 microliters of blood are added to the 200 microliters of reagent in the tube. We have to factor in the occupied space by the reagent and the reagent and blood when determining the vacuum required for sample transfer. Hence, we used different models to develop an equality to determine the critical pressure to ensure sample transfer. This equation is:

where Pa,c is the critical pressure, above which the sample will not transfer. Vr, Vs, and Vt are the volume of reagent, sample, and the tube itself respectively. Pout is the outside pressure, Pc is the capillary pressure to be overcome for blood draw, and Pw is the water vapor pressure.

Table 1 tabulates these values for a glass tube and a plastic tube and shows the critical pressures at 10,000 ft and at sea level.

Table 1

Once again, these are just models to narrow our experimental focus. We need to confirm these theories through our experiments which we hope to start soon. We hope to develop a model for the permeability of gas as well, which can affect the shelf life of the tube. In addition, we are going to start testing the rise time, or the time it takes for the vacuum within the tube to equilibrate with the outside pressure.