Flow Stability in Life Science Applications

WHITE PAPER

Flow Stability in Life Science Applications

02  A White Paper - Flow stability in Life Science applications

Flow Stability - How smooth is your flow?

In cytometry, hematology and sequencing instruments particularly, maintaining a steady fluid delivery rate is critical to obtaining reliable results. The importance of this requirement is further amplified where very low flow rates are necessary or when working with very small particles (< 10 Îźm). This paper explores flow stability as a characteristic, defines its importance and presents solutions to optimize flow stability using syringe pumps.

Definition

Importance

Flow stability, also sometimes referred to as pulsatility, is a measure of the variation of flow rate over time. A common method of measuring flow stability is by using Coefficient of Variation (CV); a statistical measure of relative variability. For flow stability, the CV is derived from a population of measurements of instantaneous flow readings. CV is then defined as the ratio of the standard deviation of this data population to its mean and is expressed as a percentage. In these terms, a lower CV result signifies higher stability in the flow output.

Flow stability is important in instrument applications where a significant variation in flow has the potential to deliver erroneous results. For example, in flow cytometry instruments where individual cells are being counted, analyzed and/or sorted, unstable delivery of either the sheath fluid or the sample as it approaches its interrogation point can result in coincident events (multiple cells being analyzed together) and large spreads in data.

This data would therefore be treated with low confidence. It then becomes very easy to understand why the fluid delivery system selected for such instruments is a critical decision as it has the largest direct influence on the overall effectiveness and reliability of the instrument.

Where; standard deviation mean number of samples sample

Interchangeable rotary valve

Smart controller

Better flow stability performance can also impact the overall design and operational cost of the instrument. Increased flow stability: > Could allow for lower resolution optics to be used in imaging instruments > Avoid reagents being wasted from repeat test > Reduce time to complete a test reliably Other common life science instruments that depend on reliable flow stability for effective performance include hematology, urinalysis, droplet PCR and DNA sequencing.

Configurable pump design

A White Paper - Flow stability in Life Science applications 03

Improving Flow Stability While several fluid delivery mechanisms exist, syringe pumps are commonly employed for sample delivery in life science instruments. For cost efficiency, stepper motors are often selected in the design of these pumps. However, the pole to pole operation of stepper motors can cause fluctuations in the torque output of the motor which transfers through the drive train of pump and results in variations in the fluid flow, increasing pulsation.

Poles in the motor align to internal stators and rotors. When a coil in the stator is powered, it generates an electric field to which the permanent magnet (rotor) is attracted. The magnetic force of attraction between the poles of the rotor and

stator increases as the poles approach each other. This varying attractive force is what causes fluctuations in the torque output of the motor and is referred to as “pole snapping”.

The solution to pole snapping is to precisely control how power is applied to the poles of the motor. By applying power to neighboring poles proportionally, the motor’s rotor can be turned with a consistent power output. This method of motor control is called micro stepping and it provides a reliable way to minimize any disturbance transfer to the fluid and hence improve flow stability. The combination of stepper motors and micro stepping addresses major concerns of instrument manufacturers; namely

Figure 1 shows a plot of flow stability performance for an IMI Cadent 3 syringe pump over various flow rates and using various syringe sizes. Specific test conditions are described in the next section.

The chart can be used to extrapolate flow stability performance for a given configuration.

16% 14%

Flow Stability, CV

instrument reliability and overall cost.

It is however important to note that any residual disturbance present, even when micro stepping, is accentuated when the syringe pump is operated at very low speeds. Sizing the syringe pump, by selecting the right configuration as well as valve and syringe combinations, to achieve the desired performance requirements therefore also requires careful consideration.

12%

Two conslusions can be drawn from this chart:

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10

100

1000

µL/min

Flow Rate, μL/min 50µL

250µL

1000µL

5000µL

Figure 1 - Cadent 3 flow stability performance using various syringe sizes

multiple materials for optimum chemical compatiblity

multiple port configurations

> At the lower end of the flow rate range, a better flow stability outcome is achieved the smaller the syringe size; 50μL in this case.

However, in system designs, the need for this level of performance should be weighed against the required total dispense volume and time to dispense. A larger syringe size may be preferable. > At the higher end of the flow rate range, the syringe size has little impact on flow stability performance as there is minimal difference in the results.

wide range of size and materials

140 120 100

04 A White Paper - Flow stability in Life Science applications

80 60 40 20 0

The selection and design of other components in the syringe pump also play a role in its flow stability performance. Misaligned parts can result in unpredictable friction effects and inconsistent mechanical power output.

Figure 2 shows an example of these effects where Figure 2A shows flow data over time as the pump is actuated and Figure 2B is the equivalent Fourier transform of the flow data. The Fourier transform result shows the

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Amplitude 3,5

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Amplitude

Flow Rate, μL/min

Flow 140

100 80 60 40

3 2,5 2 1,5 1 0,5

20 0

5

frequencies of the disturbances in the flow and each peak corresponds to a specific component or interface within the pump. This type of analysis allows designers to identify any sources of disturbances and therefore improve the design of the syringe pump to eliminate or optimize their effects.

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Time, secs Figure 2A - Flow data over 10 second period

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Frequency Figure 2B - Fourier transform of flow data

Competitive Review 3,5 3

A comparative study was undertaken to analyze flow stability performance on syringe pumps. The subjects of this study included the IMI Cadent™ 3 2,5 syringe pump as well as syringe pumps from 3 other well-known manufacturers.

2 1,5 1

The0,5objectives of this study were to: 0

0 2 4 6 8 10 12 14 16 18 20 > Compare flow stability performance of various syringe pumps products

> Understand how flow stability performance varies at different flow rates > Determine the influence of syringe size/volume on flow stability performance

The test and measurement conditions for this study were as follows: > Each pump was tested under 4 different flow rates using 4 different syringes > Each test was conducted using DI (Deionized) water at ambient temperature > Flow measurements were taken using a flow meter, aligning with the test flow rate (SLI 430, SLI 1000 & SLI 2000) > Flow data was recorded for a total of 90 seconds in each test, starting after acceleration was complete > Data capture was sampled at a rate of 650Hz

While the same test protocol was employed for all test subjects, each product was operated within the limits specified by their manufacturer.

A summary of the test subjects used in this study is shown Table 1.

A White Paper - Flow stability in Life Science applications 05

IMI Cadent 3

Competitor 1

Competitor 2

Competitor 3

Mechanical resolution*

24K half-steps

3K steps

3K steps

24K steps

Maximum speed (secs)

2.4

1

1.2

4.2

Minimum speed (mins)

6,400

50

20

3,200

Speed control

1/32 microstep

1/8 microstep

1/8 microstep

1/32 microstep

Drive mechanism

Rail

Rod

Rod

Rail

Drive screw

Lead screw

Lead screw

Ball screw

Lead screw

Screw pitch (mm)

2

1

2

1

Motor degree

0.9°

1.8°

1.8°

1.8°

Table 1 - Characteristics of test syringe pumps *As published by the original manufacturer

Reliability Test A reliability test was conducted to understand performance over the life time of the product and to understand the ultimate failure mode.

In this test, each pump was paired with a 5mL syringe and was operated to drive the maximum possible load at the maximum speed as defined by the corresponding manufacturer. The precision and accuracy of each pump was measured routinely until the pump was no longer operational (i.e. unable to drive the syringe) or the measurements exceeded the performance limits.

The performance limits were set as follows: > Precision: < 0.5% CV > Accuracy: < 0.3% error A summary of the observed results are shown in Table 2.

IMI Cadent 3

Competitor 1

Competitor 2

Competitor 3

Syringe size

5mL

5mL

5mL

5mL

Operating condition

80psi

60psi

60psi

50psi

Speed (half-steps/sec)

10,000

5,800

6,000

6,000

Life cycles

5M

4M

2.1M

0.1M

Failure mode

Still operational within precision and accuracy limits

Drive mechanism stall

Significant degradation in precision and accuracy

Drive mechanism failure

Table 2 - Life Test parameters and results

> The IMI Norgren Cadent 3 pump continued to operate past 5M cycles within the set performance limits.

> Competitor 2 pump showed significant decline in performance, beyond 2M cycles although it was still operational.

> Competitor 1 pump experienced a stall in its drive mechanism shortly after 4M cycles.

> Competitor 3 pump suffered heavy wear to its linear guide system causing it to fail at 100K cycles. Stalls were observed at very short intervals past this point.

06 A White Paper - Flow stability in Life Science applications

Summary Results Overall summary

Flow Rate with 50 μL/min syringe

Flow Stability, CV

25%

20%

15%

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5%

0%

1

10 Cadent 3

100 Competitor 1

Competitor 2

1000 Competitor 3

Flow Rate, μL/min Figure 3 - Flow stability performance with 50μL syringe

The syringe pumps were tested at various flow rates using 50μL volume syringes and the average CV % for each flow rate was documented. This data is plotted in the chart shown in Figure 3. The flow rate on the x-axis is plotted on a log-scale to account for data skewness. At the lower flow rates (<10μL/min), the average CV % result is relatively higher as all products averaged between 8% and 23%; with the Cadent 3 excelling at the lower end of this range. Figure 4 shows the detailed performance over time for the 1μL/min flow rate As the flow rate increases, flow stability performance of all the test syringe pumps begin to converge at around 3% CV demonstrating that this is not a differentiating factor amongst syringe pump products. Partial or no data shown for any of the test subjects, indicates that the manufacturer’s specifications does not allow performance at the given flow rate.

Corner case performance 1μL/min using 50μL Syringe

Flow rate, μL/min

2,5

2

1,5

1

0,5

0

0

0,1

0,2

0,3

0,4 Cadent 3

0,5 Competitor 1

0,6

0,7

0,8

0,9

1

Competitor 3

Time, secs Figure 4 - Flow stability performance at 1μL/min with 50μL syringe over time

The syringe pumps were tested at a flow rate of 1μL/min with a 50μL volume syringe over a period of 90 seconds and data was collected at a 650Hz sample rate. The results of this test are plotted in Figure 4. This data shows the IMI Cadent 3 syringe pump to significantly outmatch its competitors in flow stability performance with an average CV of 8%. Competitor 3 was the second best performing product at an average CV of 16% followed by Competitor 1 at 23% CV. Competitor 2 shows no data in this test because the manufacturer’s specification does not allow for operation at 1μL/min.

20%

A White Paper - Flow stability in Life Science applications 07 15%

10%

Detailed results

5%

0%

The charts below present a graphical representation of flow stability performance for each pump tested under a matrix as described.

50 250 of different conditions 1000 5000 Each graph represents a target flow rate, the x-axis denotes the syringe size used and the y-axis denotes the average CV%. Competitor 2 Competitor 3 Cadent 3 Competitor 1 25%

Flow Stability @ 1μL/min

25%

Flow Stability @ 20μL/min

25%

20%

20%

Flow Stability, CV

Flow Stability, CV

20%

15% 15%

10% 10%

5% 5%

0% 0%

50 50

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1000

Cadent 3

250 Competitor 1

1000 Competitor 2 Competitor 3

Cadent 3

Competitor 1

Competitor 2

15%

10%

5%

5000

0%

5000

Cadent 3

25%

Competitor 3

50 50

250

1000

Cadent 3

250 Competitor 1

1000 Competitor 2 Competitor 3

Cadent 3

Competitor 1

Competitor 2

Flow Stability @ 100μL/min

At 20μL/min, a wider data sample can be gathered from all pumps, 20% although once again, each of Competitor 1, 2 & 3 show some limitations in their ability to achieve this flow rate with the range of syringe sizes.

15% These limitations largely stem from restrictions in speed control. Of note, with10%this data set is the large spread in results obtained when using the various syringe sizes for each competitor pump, while the Cadent 3 pump consistently delivers a CV less than 10% in all conditions.

5%

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Competitor 3

250 CadentFlow 3 Competitor 1 Stability

1000

5000

2 Competitor 3 @ Competitor 1000μL/min

25%

25% 25%

25% 20%

Flow Stability, CV

20%

Flow Stability, CV

Competitor 2

5000

25%

When tested at 1μL/min, the Cadent 3 is found to be the only pump 20% capable of achieving this flow rate using the entire range of syringe sizes.

15% Competitor 1 and 3 can only do so only using a 50μL syringe, while 15% Competitor 2 is not able to achieve this at all. Even with a 50μL syringe, 10% the 10% Cadent 3 shows a significant performance advantage with a CV rating 5% Flow stability overall, can be seen to deliver the best results when of 8%.

5% using smaller syringe sizes.

20%

20% 15%

15% 15%

15% 10%

10% 10%

5% 5% 0% 0%

Competitor 1

1000

Syringe Size, μL

20%

0%

250

Syringe Size, μL

25%

0%

50

Competitor 3

50 50 Cadent 3 Cadent 3

25% 25%

250 250 Competitor 1 Competitor 1

1000 1000 Competitor 2 Competitor 3 Competitor 2 Competitor 3

5000 5000

Syringe Size, μL

Here, the data collected from each pump shows a convergence in 20% performance results with all pumps showing close results in all conditions, 20% except Competitor 2 which exhibits the poorest performance by a factor 15% 15%least 2. The use of a ball screw in the design of Competitor 2 pump is of at the source of increased noise in its actuation which directly impacts its flow stability performance. While Competitor 3 shows a mild dominance in this test, the overall product lifetime makes it a less attractive product choice.

5%

0%

50

250 Cadent 3

Competitor 1

1000 Competitor 2

5000 Competitor 3

Syringe Size, μL

With the goal of delivering fluid at 1000μL/min, both Competitor 1 and 3 produce very similar results as the Cadent 3 pump. Competitor 2 once again shows the most inferior performance by a factor of 2 in most test conditions.

Conclusion The data presented in this paper demonstrates the IMI Norgren Cadent 3 pump’s high performance capability with respect to stable flow over an extended range of flow rates. This is of significant value to instrument designers looking to have flexibility in their instrument operation. This, coupled with its ability to achieve life cycles in excess of 5M, with minimal degradation of precision and accuracy performance over this time, makes the IMI Norgren Cadent 3 an ideal choice for any life science instrument requiring world class standards.

IMI Precision Engineering operates four global centres of technical excellence and a sales and service network in 75 countries, as well as manufacturing capability in the USA, Germany, China, UK, Switzerland, Czech Republic, Mexico and Brazil.

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