Bxxxx

CYTO 2019

Pengfei Zheng1, Hongtai Gao1, Qiyao Wang1, Chuixin Liao1, Nan Li2*, Xiaobo Wang2

1. R&D, Agilent Biosciences (Hangzhou) Co. Ltd, Hangzhou, People's Republic of China 2. R&D, Agilent Technologies, San Diego, CA, United States

* Contact: Nan Li, Ph.D nancy.li@Agilent.com

Continuous Sampling with High Absolute Counting Accuracy in Flow Cytometry

Visit Agilent Technologies at Virtual Exhibit Hall of CYTO 2020 and visit our website at www.aceabio.com

Introduction

The primary function of the fluidics system in a flow cytometer is to transport the cells or microparticles from the sample tube to the flow chamber where they

interrogate with the laser beam one by one for flow cytometry measurement. Conventionally, the sample and sheath fluidics are driven by air pressure supplied by

a compressor. Thus, a tight seal on the sample tube is required. With this design, the sample volume cannot be accurately monitored. Therefore, the absolute

counting can only be achieved by an indirect method of adding the known concentration reference beads into the sample to derive the true concentration of the

cells in the sample. Recent new flow cytometers use the syringe pumps to drive the sample volume accurately, therefore direct volumetric absolute counting is

achieved with high accuracy. The drawback of current syringe pump driven approach is that it takes a series of aspiration and injection actions to drive the sample

from the sample tube to the flow chamber for analysis (such a cycle of action to aspirate and inject is called one stroke). This process is not continuous and takes

longer to analyze samples of large volume when it is more than one stroke volume of the syringe pump. To conquer this issue, some systems use multiple syringe

pumps (such as BD™ High Throughput Sampler (HTS)) to run alternatively to increase the sampling throughput. However, this increases the complexity of the

system and the carryover is inevitably increased since it takes complex steps to clean the sampling line.

In this study, we explore a new sampling line design with innovative fluidics control method to realize the continuous sampling using one syringe pump. With

the new approach, the volume between the syringe pump and the three-way sampling valve (between the syringe pump and the flow chamber) is significantly

reduced (delta V). The sample injection probe is immersed inside the sample tube all the time. The syringe pump repeatedly aspirates the volume of delta V from

the sample and injects it into the flow chamber, until the final sample volume is injected and analyzed. During this procedure, minimal air gap is introduced into the

sampling line and the sampling acts in a continuous mode, similar to the conventional compressed air driven system. By using the syringe pump, it can still achieve

volumetric absolute count with guaranteed accuracy. Therefore, no costly reference counting beads are needed. The sampling line of a NovoCyte Quanteon flow

cytometer is modified and tested in this study. The signal Mean Fluorescence Intensity (MFI) and Coefficient of Variation (CV) remain almost the same, and the

accuracy of direct volumetric absolute count is maintained. Unlike the conventional aspiration injection method using the syringe pump sampling, the new

approach can continuously drive the sample for analysis, therefore the assay throughput can be improved and flexible sample volume to be acquired can be used,

without sacrificing the data quality of and accuracy of the direct absolute count.

Results and Conclusions

Principle and Method

Procedure of Continuous Sampling

Conclusions and Discussion

1. Power on solenoid valve. The common port (COM) is connected to

normal-closed port (NC).

2. Sample Injection Probe 1 moves downwards to the sample tube.

3. During the course of Sample Injection Probe 1 moving downwards, the

syringe pump aspirates 10 μL air from the tip of the Sample Injection

Probe 1. Also, the shaker mixes the sample.

4. Syringe pump aspirates the sample into the Sample Injection Probe 1 and

sample tubing until the front end of sample in the sample tubing reaches

point①①①①. Define the sample volume of sample in tubing between point①①①①

and COM port of the solenoid valve as ΔV.

5. Power off solenoid valve. The common port (COM) is connected to

normal-open port (NO).

6. Syringe pump pushes the sample in the tubing between point ①①①① and

COM port of the solenoid valve into flow chamber with the sample flow

rate that set by user. Start flow cytometer data acquisition.

7. If ΔV volume of the sample is used up before the Stop Condition is

reached, the solenoid valve switches to the NC port, and instrument will

repeat the step 4 to 6. The Sample Injection Probe 1 is immersed inside

the sample tube all the time.

8. When the Stop Condition is reached or if user stops the test, syringe

pump will push the sample remaining in the sample tubing and Sample

Injection Probe 1 back to the sample tube. Sample Injection Probe 1 will

then move upwards to the reset position.

9. Consequent sampling line washing procedure will follow.

CYTO 2020

Poster: P182

Flow Chamber

Sample Tube

Sample Injection Probe 1

Sheath Fluid

Waste

Solenoid valve

Syringe pump

Sample Tubing

COM

NCNO

①①①①

Sample Injection Probe 2

Shaker

The fluidics system of a NovoCyte® Quanteon flow cytometer is modified

with the schematic illustration shown above. In order to reduce the time

consumed by aspirating sample, the inner volume of sample injection

probe 1 and sample tubing should be as small as possible. In order to save

sample, the inner volume of sample injection probe 2 also should be as

small as possible and the dead volume of the solenoid valve should be

close to zero.

Continuous Sampling Mode

Normal Sampling Mode

Comparison of Signal Mean Fluorescence Intensity (MFI)

and Coefficient of Variation (CV)

Sampling ModeE2 MFI

FSC-H

E2 MFI

SSC-H

E2 MFI

FSC-A

E2 MFI

SSC-A

E2 CV

FSC-H

E2 CV

SSC-H

E2 CV

FSC-A

E2 CV

SSC-A

Normal

Sampling Mode123,294 799,643 150,628 1,022,886 0.74% 0.78% 0.74% 0.90%

Continuous

Sampling Mode123,314 801,696 150,732 1,026,012 0.74% 0.79% 0.74% 0.91%

Deviation +0.02% +0.26% +0.07% +0.31% / / / /

1. In both mode, SPHERO™ Ultra Rainbow Fluorescent Particles are used for testing. Tthe Sample Flow Rate is 14 μL/min and Stop Condition is 12,000 Events in E1.

2. In Normal Sampling Mode, syringe pump will aspirate V volume of sample into instrument in one stroke depending on the capacity of the syringe pump (V=100 μL in this test). In the

case of “# of Events” and “Time” as the Stop Condition, since the concentration of the sample is unknown, the full V volume will be aspirated. If the Stop Condition is reached before

the V volume of sample is used up, this test is stopped. The rest of sample will be discarded as waste or be recovered (if the instrument has sample recovery function). If the Stop

Condition is not reached by one stroke (V volume of sample is used up), the syringe pump will take another stroke and continue sample acquisition until the Stop Condition is reached.

3. In Continuous Sampling Mode, syringe pump will aspirate ΔV (= 20 μL) volume of sample into instrument in one stroke. If ΔV volume of sample is used up but the Stop Condition has

not reached, the system will repeatedly aspirate ΔV sample until the Stop Condition is reached.

4. The data show that with Continuous Sampling Mode, the signal quality (i.e. Mean Fluorescence Intensity (MFI) and Coefficient of Variation (CV) ) remains unchanged from the Normal

Sampling Mode.

Normal Sampling Mode

Continuous Sampling Mode

Sample Flow Rate = 14 μL/minStop Condition: 90,000 Events in R1

Sample: SPHEROTM AccuCountParticles

SPHEROTM is a trademark of

Spherotech, Inc.

Comparison of Absolute Counting

Mode Normal Sampling Mode (events/μL) Continuous Sampling Mode (events/μL)

Test 1 959 968

Test 2 954 962

Test 3 951 968

Average 955 966

CV 0.42% 0.36%

Target Value(events/uL) 967

Deviation from the Target Value -1.28% -0.10%

A syringe pump-based continuous sampling method is achieved with this presented work. The method is validated to be effective and efficient based on

the following results:

1. The signal quality, qualified by the Mean Fluorescence Intensity (MFI) and Coefficient of Variation (CV), remains the same compared to normal

sampling mode using fluorescent calibration beads.

2. The Volumetric Absolute Counting results are the same compared to the normal sampling mode using the absolute counting beads.

The advantages of this method include:

1. Reduces the sampling time, especially for high volume of sampling.

2. Reduce the sampling overhead volume usually associated with the syringe pump-based system.

3. Still maintains the advantages of the volumetric absolute counting enabled by the accurate volume dispensing capability using syringe pump.

Signal Quality

Volumetric Absolute Counting

1. The same sample is prepared and tested using two sampling modes for

comparison.

2. The target absolute counting value is certified by the beads supplier.

3. The sample is run under each mode for three times and the Volumetric

Absolute Counting results are obtained by the average of three tests.

4. The Volumetric Absolute Counting from the continuous sampling mode

is no difference from the normal sampling mode.

For Research Use Only. Not for use in diagnostic procedures.

Fluidics System Schematics