Artel VMS Volume Measurement System: Measuring Volumes in REMP STBR 384 Plates

By Parshley, R., Albert, K. | Application Note

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Introduction

This application note discusses how to use the Artel VMS to measure the amount of volume occupied by contents within REMP STBR 384 tube plates1. The volume of sample transferred into and out of these plates can be critical to the success of assays or validation of procedures. One specific example is in the preparation of mother/daughter plates from compounds pulled from library stores. It is not uncommon to find a compound storage tube containing less volume than expected, which may cause incorrect volumes delivered to mother plates used in screening tests. A simple approach for determining volumes in these prepared mother plates is needed2.

STBR plates are commonly employed in compound management processes, where these plates store important compounds for downstream assay testing. By relying only on theoretical volume amounts (i.e., original volume transferred into the source tubes minus subsequent volume removal(s)) and without implicitly measuring the exact amount of volume occupied by each well/tube, assay screens might, in turn, produce erroneous data or at minimal, they could be a waste of time and materials. Using the VMS and its pressure-based volume measurement technology, it is possible to make direct measurements of volumes in compound source plates. The goal of this application note is to demonstrate: (1) how to incorporate an STBR 384 tube plate into the VMS software by calibrating using cylinders of known volume; and (2) how to measure the volumes of contents in the tubes for tracking purposes.

Materials & Methods

The following were employed for this application note:

  • VMS with operating software
  • Multiple REMP STBR 384 tube rack plates
  • REMP 384 shim plate
  • 23- and 43-μL volume cylinders
  • 20-200 μL Rainin 8-tip LTS manual pipette and tips
  • Aqueous red dye (MVS Range A)

Shim Plate Definition

Shim plates are used to facilitate the pressure-based measurement by being placed under the test plate to help minimize plate bending/warping. When using STBR tube racks, the REMP 384 shim plate is required – the raised portions of the REMP 384 shim plate align with the bottom of the tubes, so that the tubes are not pushed into the plate during the measurement process. To load the shim plate into the VMS software: (1) on the Home screen, click the Configure menu icon; (2) click on the Shim Plates icon (Figure 1); (3) enter the name for the REMP 384 shim in an open/available field; and (4) click Save. Once the shim plate is defined, it can be placed on the VMS plate carrier tray (Figure 2).

Figure 1.

Before using the STBR plate for volume measurements, the shim plate must be added to the software. The shim can be named appropriately as STBR 384 or REMP 384 (left, center). The characteristics of this shim enable the bottom of each tube in the plate to be supported during the measurement process (right).

Figure 2.

Before using the STBR plate for calibrations and measurements, place the REMP 384 shim on the extended plate carrier tray of the VMS. The STBR plates sit directly on top of the shim plate. Note – it is very important for the STBR test plate to be fully-flush with the A1 corner of the VMS carrier tray. Due to the raised portions of the shim plate, if the test plate is not flush, the plate is positioned incorrectly and mechanical, alignment and/or read errors will result.

STBR Plate Calibration – VMS Calibration Mode.3

Next, the STBR plate needs to be defined and calibrated within the VMS software. When setting up the STBR plate, click on the Configure icon from the Home screen to get to the list of previously defined “Calibration” plates. Click on an available plate type entry (down arrow) and fill out the STBR plate information, with emphasis on requiring the REMP 384 shim plate and with plate geometry listed as 16 rows x 24 columns (Figure 3, left). Now click on the Calibration tab and subsequently click the Run Plate icon to start the calibration process (Figure 3, right). The calibration employed the use of known 43-μL volumetric cylinders (manually placed in to columns 1, 2, 12 and 13) and the rest of the tube plate was purposely left empty to represent 0-μL “blank” measurements (Figure 4, left). Once the Run Plate icon is clicked, the per column calibration information is entered within the Plate Calibration Definition interface to align with the volume cylinders (Figure 4, right). For the purposes of this application note, only one STBR plate was employed for the calibration and the data were used to overwrite any existing calibration data for this plate type. The empty well volumes and the filled well volumes are used to establish a calibration curve that matches a known volume with a specific pressure for each plate type.

This calibration curve is subsequently used to accurately estimate the volume of any samples measured in all future STBR test plates-of-interest.

Figure 3.

Defining and incorporating the STBR test plate when in Calibration Mode. The information for the Plate Geometry tab is shown on the left. Once the Calibration tab is clicked, the user can click Run Plate for the STBR calibration plate (right).

Figure 4.

The STBR plate used as the calibration plate was filled with 43-μL volumetric cylinders, which were placed in columns 1, 2, 12 and 13 (left). After clicking Run Plate, the user then defines the specific columns filled with known volume values (right).

Test Plates – Measurement Mode.3

Once the STBR plates are properly calibrated and entered into the VMS software, STBR test plates can be measured indefinitely (or until the calibration data are cleared for that plate ID).

Test Plate 1.

The same plate employed for the STBR Calibration Mode (above) was subsequently run in Measurement Mode, whereby only data for columns 1, 2, 12 and 13 were collected. From the home screen, the Plate Type drop down menu was used to select the STBR 384 plate (Figure 5, left). In the Test Method interface, only columns 1, 2, 12, and 13 were selected for measuring volumetric data (Figure 5, center). From the Home screen, after clicking the Run Plate icon, the software interface allows the user to enter Plate ID information, comments, and select a Device ID (when appropriate). The Run Plate software interface also has instructions for the shim and test plates (Figure 5, right). The entered Plate ID characters will eventually be included in the file name of the auto-exported test report that is saved to the defined Results folder. The Plate ID text can also be entered into text field using a compatible barcode reader (Figure 6).

Figure 5.

Before running a test plate, be sure to select the proper Plate Type from the Home tab’s drop down menu (left). Specific columns were selected when testing the STBR plate with the 43-μL cylinders, i.e., only data from columns 1, 2, 12, and 13 were collected (center). Before running the test plate, enter the name for the Plate ID (i.e., the resulting saved file name contains this ID) and follow the instructions (right). Note – the information for the Plate ID was entered via a barcode scan (see Figure 6).

Figure 6.

When using a compatible barcode reader, information stored within a plate barcode can be automatically entered into the Plate ID text field.

Test Plate 2.

Volume measurement data were collected for a test plate containing 23-μL cylinders manually placed into columns 1 and 2 of a second STBR test plate.

Test Plate 3.

Using a calibrated 8-tip manual pipette, aqueous red dye was pipetted into columns 2 through 15 of a third STBR test plate. Target volumes were dispensed in increments of 5 μL, where two full columns (32 replicates) were used for each volume. For example, 20 μL was transferred into columns 2 and 3; 25 μL was transferred into columns 4 and 5; 30 μL was transferred into columns 6 and 7; and this continued up to 50 μL transferred into columns 14 and 15 (Figure 7).

Figure 7.

An 8-tip handheld pipette was employed to dispense aqueous red dye into columns 2 through 15 within a third STBR test plate.

Results

Calibration Results.

As shown by Figure 8, the calibration lines for the 8 individual pressure sensors for the two volumes (43 μL; 0 μL) are very linear over the range.

Figure 8.

Calibration results for the STBR tube rack for a high volume (4 columns of 43-μL cylinders) and a low volume (20 columns of empty tubes).

Measurement Results.

Software Interface with Test Plate 1.

The same plate employed for the STBR calibration was also read as a test plate in volume measurement mode. Figure 9 represents the on-screen data and image that appear immediately after the plate is measured.

Figure 9.

Measurement results for the STBR test plate 1 for the four columns filled with the 43-μL cylinders (left). Note that only a portion of the volume data are shown and the user can hover over any test point to observe the volume value for that specific tube, as shown when hovering over well A13 (right).

Table 1. Results for Test Plates 1 & 2 Containing Volume Cylinders

Table 2. Results for Hand Pipetted Test Plate 3

Results

Table 1 summarizes the results of the experiment.  The microliter values at each time point represent the average amount of water either absorbed or evaporated from each well at a given time point compared to the starting volume.  Positive values represent an increase in volume while negative values represent a loss of volume.  The starting volume of each well was approximately 50 µL.

Using the Artel VMS, small changes in volume were measured over the course of the 8-hour experiment as illustrated in Figure 1. The water-filled wells in the covered plate lost an average of 4.2%, while the uncovered side lost 14.8%.  The covered, DMSO-filled side gained 3.2%, and the uncovered, DMSO-filled side gained 16.2%, due to the hygroscopic nature of the solvent.  There were also two other observations worth noting: The first is an obvious “edge-effect” on each side of the uncovered plate.  More water was lost around the edge wells than in the center – over 2%.  The second observation involved the interface columns between the DMSO and Water-filled wells (columns 12 and 13). Average water gain in column 12 (DMSO) was 3% greater than the mean average of the entire DMSO side, while average water loss in column 13 (Water) was 5% greater than the mean average of the entire water side.  Interestingly, this effect was even more pronounced in the covered plate. Column 12 (DMSO) lost 9% more volume while column 13 (Water) gained 9% more volume that the mean averages of their respective sides. These observations are illustrated in Figure 3.  The proximity of the water to the DMSO appears to increase evaporation and absorption. The plastic cover used during the experiment did not produce the airtight seal that a heat or adhesive seal provides.  Instead, the plastic cover seemed to provide a microenvironment wherein the DMSO in column 12 was able to act as a desiccating chamber for the water-filled wells in column 13.  Some condensation formed on the underside of the cover over the entire water section, but seemed to be more prevalent over the two middle columns.    This also seems to show that the proximity of the hygroscopic DMSO in column 12 to the water in column 13 affected the evaporation of the water.  The evaporated water did not all end up being absorbed by the DMSO; some also condensed on the cover.

Conclusions

Modern labs increasingly rely on automation for managing their liquid transfer and plate filling
needs. These instruments are susceptible to tip clogging, aspiration and dispensing errors, as well as volume variation due to non-optimized liquid class settings. The Artel VMS is an effective quality control and pipetting error detection tool that is versatile, quick, and accurate. Gathering volume information from the individual wells of microplates in an established workflow can now be quickly and effectively completed using the Artel VMS. This information can be used for quality control of dispensed plates or verification of pipetting steps for multistage reactions.


References

  1. REMP STBR 384 product information can be found at: http://www.brooks.com/products/life-science-systems/consumables/remp-microtube-technology/384-way-microtubes (assessed August 2016).
  2. Artel Technical Poster: “Pressure-Based Volume Measurement Technology for In-Process Measurement of Microplate Contents” http://www.artel-usa.com/resource-library/pressure-based-volume-measurement-technology-process-measurement-microplate-contents/ (assessed August 2016).
  3. VMS User Guide, Artel controlled document #15A6747

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