On 2017 Oct 13, Gerhard Nebe-von-Caron commented:

looks like the paper slipped through the review process somehow. Whilst the journal published an erratum to clarify some volumes used, they did not check for coherence of the methodology.

Ultra Rainbow beads were supplied in a dropper bottle, which was inconvenient for volume control. The dropper lid was part of the bottle design and could not be removed. The volume of a drop (50ul) was specified by the datasheet.

Considering that it would be well known to anyone skilled in the art that the accuracy of counting is dependent on the accuracy of the volume of the materials used as clearly described in the spherotech method datasheet http://www.spherotech.com/Updated STN 8-21-07/STN-15 Rev B.pdf "Procedure To obtain accurate absolute cell counts, the SPHEROTM AccuCount Particles are used in conjunction with flow cytometry. The SPHEROTM AccuCount Particles have a concentration of approximately 1x106 particles/mL. The actual concentration is listed on the Technical Data Sheet for the product. The first step during sample preparation is to add the monoclonal antibody to 100μL of the test sample. The sample is then incubated, lysed, washed, and resuspended in 1 to 2 mL of phosphate buffer saline, 0.1M, pH 7.4. If staining and lysing are not necessary, add a known volume of test sample to the 1 to 2 mL of phosphate buffer saline. Washing the AccuCount Particles with the sample prior to analysis is discommended because a reduction in the number of reference particles will occur. Next, add exactly 50μL of the AccuCount Particle to the suspension. The precision during pipetting of the AccuCount Particles is absolutely critical. The sample is then analyzed by flow cytometry. The bead and cell population are gated on the fluorescence and/or side scatter channel. Record the number of events for the AccuCount Particles and the test sample. The absolute cell count is then determined with the following equation. ..."

Considering that the authors claim that they dispensed the particles by reverse pipetting but then claimed the poor reproducibility of the spherotech beads because they used the drops for direct dispensing is ironic. If I remember right the AccuCount beads are screwtop and the normal Ultra Rainbow beads are usually used for detector linearity check for which the dropper dispensation is perfectly adequate, for absolute counting this is inadequate and the authors should have been aware of that. If they wanted to use the beads they had in the fridge they should have just pre dispensed the approximate volume and then pipetted accurately.

and if Fig 1 confuses you - it's probably because they got the axis labels the wrong way round

On 2017 Oct 13, Gerhard Nebe-von-Caron commented:

looks like the paper slipped through the review process somehow. Whilst the journal published an erratum to clarify some volumes used, they did not check for coherence of the methodology.

Ultra Rainbow beads were supplied in a dropper bottle, which was inconvenient for volume control. The dropper lid was part of the bottle design and could not be removed. The volume of a drop (50ul) was specified by the datasheet.

Considering that it would be well known to anyone skilled in the art that the accuracy of counting is dependent on the accuracy of the volume of the materials used as clearly described in the spherotech method datasheet http://www.spherotech.com/Updated STN 8-21-07/STN-15 Rev B.pdf "Procedure To obtain accurate absolute cell counts, the SPHEROTM AccuCount Particles are used in conjunction with flow cytometry. The SPHEROTM AccuCount Particles have a concentration of approximately 1x106 particles/mL. The actual concentration is listed on the Technical Data Sheet for the product. The first step during sample preparation is to add the monoclonal antibody to 100μL of the test sample. The sample is then incubated, lysed, washed, and resuspended in 1 to 2 mL of phosphate buffer saline, 0.1M, pH 7.4. If staining and lysing are not necessary, add a known volume of test sample to the 1 to 2 mL of phosphate buffer saline. Washing the AccuCount Particles with the sample prior to analysis is discommended because a reduction in the number of reference particles will occur. Next, add exactly 50μL of the AccuCount Particle to the suspension. The precision during pipetting of the AccuCount Particles is absolutely critical. The sample is then analyzed by flow cytometry. The bead and cell population are gated on the fluorescence and/or side scatter channel. Record the number of events for the AccuCount Particles and the test sample. The absolute cell count is then determined with the following equation. ..."

Considering that the authors claim that they dispensed the particles by reverse pipetting but then claimed the poor reproducibility of the spherotech beads because they used the drops for direct dispensing is ironic. If I remember right the AccuCount beads are screwtop and the normal Ultra Rainbow beads are usually used for detector linearity check for which the dropper dispensation is perfectly adequate, for absolute counting this is inadequate and the authors should have been aware of that. If they wanted to use the beads they had in the fridge they should have just pre dispensed the approximate volume and then pipetted accurately.

and if Fig 1 confuses you - it's probably because they got the axis labels the wrong way round