NEWS
The LC Fittings Kit is configured to connect typical eluent tubing
(1.6mm or 1/16 inch OD) to the U-Series sample arm (3.2mm or 1/8
inch OD). A diagram of the kit connected to a U-Series nebulizer is
shown in Figure 3.
Figure 3. LC Fittings Kit
Sample Tube U-Series Nebulizer
LC Fittings Kit
Results
A prototype MicroMist nebulizer (P/N AR120-1-UFT02) combining
all the benefits of the Glass Expansion U-Series nebulizer
construction with the LC fittings Kit (P/N FT-16.8) was evaluated
on a CyTOF2 instrument by Dr. Rahman. When designing the
prototype MicroMist nebulizer, it was important to take into account
the standard nebulizer operating conditions for single cell analysis
by TOF-ICP-MS. In contrast to a typical ICP-MS concentric
nebulizer, Mass Cytometry requires a very low nebulizer gas flow
(≤ 0.3 L/min) and back pressure (≤ 20 psi) to keep the cells intact
during nebulization. This poses a challenge, as with typical ICP
nebulizers a low nebulizer gas flow and back pressure will produce
a poor aerosol quality resulting in poor transport efficiency (poor
sensitivity). Therefore, the prototype MicroMist nebulizer for
single-cell analysis required a unique construction to achieve an
optimal aerosol quality at a low gas flow to ensure an acceptable
cell transmission efficiency. Cell transmission efficiency is defined
as the proportion of single-cell (or single particle) events that are
acquired by the instrument relative to the overall number of cells
in the sample. When evaluating relative acquisition efficiency, it
is therefore important to use matched samples and to keep the
acquisition time, the cell (or particle) concentration and sample flow
rate constant. For these experiments an HF resistant TruFlo monitor
at the lowest range (P/N 70-803-0892) was utilized to continuously
monitor the sample uptake rate. Single-cell analysis uses a very
low uptake of 30-45 μL/min for most methods and maintaining a
constant sample flow rate is directly related to the performance of
the analysis. For this reason a syringe drive or pneumatic sample
loader is used to maintain a precise sample flow rate. However,
like any ICP application the nebulizer can clog, leaks can occur and
the syringe barrel or pneumatic sample holder can degrade, all of
which can affect the sample flow rate and analytical performance.
Glass Expansion’s TruFlo sample monitor (Figure 4) provides
a real-time digital display of the sample flow rate, so you always
know the actual flow rate to the nebulizer. This enhances the dayto
day reproducibility of results and reduces the need to repeat
measurements.
Figure 4. TruFlo sample monitor
The gas flow rates for the prototype MicroMist nebulizer were
optimized using Tuning Solution (Fluidigm), a high purity solution
containing known quantities of defined elements. At a liquid flow
rate of 45 μL/min, the nebulizer gas flow and make-up gas flow
of the prototype MicroMist nebulizer optimized at 0.17 L/min
and 0.76 L/min, respectively determined using both manual and
routine autotuning protocols. These gas flow conditions match
those utilized with the standard CyTOF nebulizer and provided an
identical signal intensity. Nebulizer performance was then validated
using EQ Calibration Beads (Fluidigm), which are polystyrene
beads containing known concentrations of metal isotopes. EQ
beads acquired using the prototype MicroMist nebulizer showed
identical median signal intensity and CV to those acquired with the
standard CyTOF nebulizer.
To test performance in a typical experimental application, the
prototype MicroMist nebulizer was used to acquire a stained
preparation of peripheral blood mononuclear cells (PBMCs) from
a healthy donor. PBMCs reflect major populations of circulating
immune cells, and high dimensional analyses of these samples
are a powerful tool that can be used to evaluate the immune
status of patients and evaluate disease progression and treatment
responses. Figure 5 shows human PBMCs that were stained
with a basic immunophenotyping panel and acquired using the
standard CyTOF nebulizer and prototype MicroMist nebulizer.
The cell concentration (1 million cells/mL), flow rate (45 μL/min)
and total acquisition time (280 sec) were kept constant. The
resulting data, visualized using viSNE(15) in Cytobank, show that
overall data quality, single cell staining and resolution obtained with
the prototype MicroMist nebulizer were identical to the standard
CyTOF nebulizer (Figure 5). The prototype MicroMist nebulizer
acquired 69,827 cell events in a 280s acquisition window, providing
a cell transmission efficiency of approximately 33% on the CyTOF2.
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