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The BDIS Turbo Sort ® Option

What it is

The Becton Dickinson Turbo Sort Option ® (TSO) allows you to greatly increase sorting speeds on the FACS Vantage and FACStar Plus flow cytometers. We have been using this option here at TSRI on our FACS Vantage since August 1995. We updated the peak sense and hold board in January 1998 with the Vantage SE version.

Several modifications have been made to the instrument to handle the higher pressures and increased drop drive frequencies necessary to achieve higher sorting speeds.


Fluidic Modifications

  • Maximum system pressure is increased from 20 psi to 60 psi. This includes changing the fluidic controls from mechanical valves to electronically controlled solenoids. The assembly is housed in a small add-on cabinet usually placed below the optical bench.
  • Modified sample collection assembly to allow for either standard or 15 ml sample collection test tube holders.

Electronic Modifications

  • Analysis dead time may be decreased down to approximately 5 usec to minimize coincidence aborts and undetected passenger cells.
  • Drop drive frequency maximum increased from 50,000 drops/sec to 99,000 drops/sec.
  • Drop delay range is extended from 37 drops to 89 drops and the internal clock resolution increased from 0.25 drops to 0.1 drops.
  • Increased drop delay resolution (Video scale - Vantage only).
  • Deflection envelope choices modified for 1.0, 1.2, 1.3, 1.4, 1.5, 2.0, and 3.0 drop packets.
  • Sort Control board (Card Cage Slot #6) modified and a new three-position switch added above Phase Gating switch to enable 2 new modes:
    1. UP - Enhanced Normal-R (nullified sort conflicts are not counted).
    2. MIDDLE - standard modes.
    3. DOWN - Enhanced Enrich (dead time abort ignored, No Sort decisions from overlapping sort packet envelopes are active.

Drop Deflection Modifications


Initial Impressions

Installation/Modifications

The Tubro Sort Option kit we received needed some careful tuning after installation. We found that a couple of minor modifications we required (a capacitor and resistor were added to the system) to tune up the side streams, charge plates were modified (lowered), and careful adjustment of the drop drive frequency/amplitude is essential to avoid problems with satellite drops at higher drop rates. Properly tuned and optimized, however, Turbo Sort really works well for us. The additional pulse shaping adjustments and charge plate voltage control are used often to tune it properly when changing nozzles, pressures, and drop packet sizes. TSO is not as simple to setup as the standard Vantage in terms of drop stability, etc., but my guess is most operators would probably pick it up quickly.

What we think - so far

We really like it. So far, we have been able to at least double the flow rate for most sorting experiments. Many sorts that would usually need 8 to 10 hours of Vantage time are being done in 3 to 4 hours without any loss in sorting efficiency. Most sort samples we see here at TSRI are usually hard enough to obtain that we need to be rather conservative in terms of what a tolerable sample loss is, so we like to account for at least 90% of what is really there and not lose more than about 20% of any sorted subset due to coincidence aborts. If you can tolerate higher losses, you can sort very, very fast with TSO. At moderately fast speeds, TSO allows a 10% subset to be sorted at nearly 2.2 times the rate than on a standard FACS Vantage. To illustrate, compare sort recovery values for standard and TSO Vantage parameters.

The relative yields and purity remain essentially the same (reasonable recovery and 98%+ purity). The pressures used vary from 18 to 35 psi or more to handle the TSO "sweet spots" that appear to be around 38,000, 48,000, 61,000, and 66,000 drops/second using the 70 micron nozzle. Care is taken when adjusting the dead time and timing the deflection packets.

We have done test sorts at up to 94,000 drops/second with the 50 micron nozzle, and it seems that the ability of various cell types to survive the higher pressures and velocities is probably going to be the limiting factor. We are currently working our way up the pressure/speed scale (as time permits us to test various cell preparations), but initially it seems that most common cells will behave fairly well after sorting at pressures up to around 28 or 30 psi, where some of the more fragile cell types begin to show some negative effects (increased numbers of "dead" upon analysis). We hope to have results soon from some proliferation assays being done on cells sorted at high velocity. Stay tuned.... it looks like two and three laser sorting will work just fine too (even though BD won't support it yet with TSO), as long as you are sober about

We've already sorted mouse spleen cells at 10,000+ cells/sec (61,000 drops/sec) in dual laser mode (FITC, PE, APC) with excellent results in terms of yield, viability, and purity. We don't have the BD Sort Enhancement Module, so I cannot comment on how TSO may work with it (probably not well - according to the BD manual)... In summary, our sorter logs for August and September are telling the story - the older FACStar instruments were fired up only twice during a month instead of used daily, and I'm having trouble getting "test bed" time on the Vantge with TSO now that our users are seeing how quickly we can sort their samples.

Updates

It looks like the additional losses to the delay circuit in dual lasr mode amount to an additional 1 -> 2 percent more than in single laser more under our standard sorting conditions.

January 1998 - recplaced the peak sense and hold board (#13) with a new redesigned circuit board (Vantege SE version) developed to eliminate the losses that occured in dual laser mode. The improvement is significant, no huge differences in sorting efficiency when in multi-laser mode. The new board allows for up to 3 laser beams in 3 different spatial positions, although we are still using two spatial positions.

Joe Trotter, Director TSRI Flow Cytometry
asaluk@scripps.edu

Turbo Sort - ® Becton Dickinson Immunocytometry Systems 1995


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