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Teamwork Enhances the Flow

Breaking the boundaries in flow cytometry


Teamwork Enhances the Flow
Breaking the boundaries in flow cytometry
Joyce Laird
BD’s FACSAria flow cytometer.

BD Biosciences, a business segment of Becton, Dickinson and Co. (BD; San Jose, CA) already produced a successful line of flow cytometry–based cell sorter systems. However, BD engineers wanted to push the envelope further to meet the increased needs of enhanced technologies. One small device was key to their success and it turned out to be the biggest challenge of the entire project.

It took a team effort on the part of BD and suppliers, Creative Metal Products (CMP; San Jose, CA) and Small Precision Tools (SPT; Petaluma, CA) to develop the solution.

To understand the situation BD faced, it pays to review what makes flow cytometry unique. Traditionally, cell sorters work by breaking up a saline jet stream containing blood cells into several thousand drops. From those drops, the individual cell types are sorted for laboratory analysis. This is key to learning about specific cell abnormalities and blood diseases, for research into immunology and genetics, and for the development of pharmaceuticals.

“These instruments work by measuring the fluorescence properties of cells, which can be specified by labeling them with a fluorescently conjugated antibody or an organic dye that is specific to a particular cell,” Bob K. Doust, BD’s program manager for R&D, explains. “The labeled cells flow in single-file fashion and are subjected to a laser beam. Every time a cell passes through the laser, because of its attached dye, it emits a characteristic fluorescence signal. Specific timing calculations tell the system when the cell will arrive in a droplet where it can be sorted out. It does this at a rate of tens of thousands of cells per second.”

What is different about the new system BD developed is that in contrast to all existing flow-based sorting systems that collect the fluorescent light-tags in air, the new instrument channels the stream into a cuvette and the light from the particles never sees air. The system is more efficient because the cuvette provides much higher levels of sensitivity for a given laser, according to BD.
“Many different cytometers in the past have had cuvette capabilities but they generally were not sorters. They were analyzers,” Pierce Norton, BD senior scientist says.

The new BD system analyzes the cell while it’s in the cuvette and then sorts it out into an open airstream. Analyzing in the cuvette provides high sensitivity with low-power lasers.

Since positioning is important, the location of the orifice center to the side walls of the handle must be maintained within microns.

One Tiny Part—One Big Challenge

The new cuvette-based approach put constraints on the tiny nozzle that is key to separating the flow into precise droplets. The nozzle was the most important component of the entire architecture since the nozzle location and the shape and geometry of its orifice were critical to drop formation.

BD’s previous sorters used a ceramic nozzle that worked very well, but it could not meet the challenges associated with the new approach. So the company began testing new design configurations and materials.

“We developed designs and tried various metals and alloys,” Vijay Kumar, associate staff specialist on the BD project, says. “The design often worked well, but the surface of most materials at the micron level was too rough. At this size, even imperceivable imperfections catch cells or fluid. The fluid must move in a straight, predictable fashion.”

“The nozzle also had to be configured so the customer could easily remove and replace it for cleaning or changing sizes. It had to align perfectly every time,” Norton adds. “That had never been achieved before.”

BD found a solution by moving to an electroforming process using a nickel base with a gold-plated surface. Electroforming lent itself very well to forming a small hole with a funnel radius that was needed for the nozzle orifice geometry.

Functionally, this nozzle worked well; however, there were concerns about its durability when compared with the traditional ceramic nozzle.

“We originally used electroforming technology because it had the capabilities to easily make the dimensions and geometry that we needed,” Norton says. “We also wanted the nozzle to be conductive because we have to deliver an electric charge to the flow that goes to the nozzle, optimally very close to the nozzle orifice where the stream breaks off into droplets. However, by definition, when it’s conductive and there’s an ionic solution and a charge, it becomes a corrosive situation. The gold plating had intrinsic imperfections that allowed the solution to eventually corrode the nickel below.”

Search for a Solution

BD engineers tried many noncorrosive materials. All had different challenges relating to manufacturability, holding the critical tolerances needed, or the required finish smoothness. BD fielded each new design concept to CMP, its prime vendor for machining and assembly.

“The role of CMP has always been to machine the handle and assemble it with whichever nozzle we directed,” Norton says. “But on this project, Kenneth Hutchinson, owner of CMP, also helped source out other material and manufacturing vendors. As Vijay delved into different materials, the manufacturing process had to change, impacting the design and the handle shape. CMP would test manufacture those changes.

“Vijay knew what absolutely could not be compromised as far as the geometry,” Hutchinson says. “We knew how to make machined parts. We teamed to find an alternative process to electroforming. This nozzle assembly is just a little thing, but you can’t run the machine without it.”

Yttria-stabilized zirconia was used to create a nozzle with an orifice smaller than the diameter of an average human hair, and with exacting roundness and concentricity.

It soon became obvious that traditional machining could not meet the extreme tolerances and repeatability needed on such a small part. However, CMP’s prototypes were the basis for the next step.
The solution came about at the MD&M West show in 2003.

“I met Travis Ayers, plant manager for Small Precision Tools, the very last hours of the show,” Hutchinson says. “We discussed the BD part. I mentioned this to Vijay, who suggested that we provide SPT with the drawing for the prototype nozzle. That was the beginning.”

“We’d been using SPT for the last 10 years for the original ceramic nozzle, but felt that the needs of this part were beyond the realm of ceramic molding. It was a lucky happenstance that Ken ran into him at the show,” Kumar adds.

The New Solution

“The fact that we injection mold microceramic shapes is unique,” Ayers says. The geometry is so small and the material is so hard that if you tried to fabricate it, it would be virtually impossible to produce.

Ceramic injection molding (CIM) basically joins several sciences. Ceramic powder less than one micron in diameter is blended with a system of polymers that is then injection molded. The mixture is about 2/3 ceramic and 1/3 plastic. The plastic acts as a carrier in order to get the tiny ceramic particles into a very complex shape in the mold. When the part comes out of the mold, the binder is burned off. Finally the parts go into a high-temperature sintering kiln, where they shrink by 20 to 30%, become very dense, and obtain the final shape. It is a way to form a highly durable and complex shape in an economical fashion.

Before committing to a mold, SPT prototyped the nozzle in TiN-SiN cermet, using micro-EDM processes. Once the design was final, yttria-stabilized zirconia, also known as YTZP, was chosen for its submicron grain structure, excellent surface finish, biocompatibility, and wear characteristics. The nozzle required an orifice smaller than the diameter of an average human hair, and with exacting roundness and concentricity.

Final Production Challenges

“Our nozzle orifice size pushes the edge of machining and molding technology,” Kumar states. “The electroforming process used for the original nozzle was basic semiconductor technology, where it was easier to work in such small dimensions.”

“The internal geometry of the orifice must start from a wider diameter and taper down with a very smooth radius—like a very smooth funnel,” Doust says. “How quickly the orifice tapers is critical because that causes the jet to be formed coming out of the nozzle. That profile was difficult for machining or molding because we had to make sure that the orifice geometry was normal to the rest of the nozzle. Fluid going through it must come out straight to hit the target several inches out.”

CMP receives the ceramic nozzles and assembles them to handles they machine. Since positioning is important, the location of the orifice center to the sidewalls of the handle must be maintained within microns. CMP measures each assembled part and grinds material away in an iterative way until it comes into precise BD specification.

Wrapping It All Up

Once the three companies partnered, it took a little less than a year to perfect the new ceramic nozzle. There are currently two main sizes in production, 70 and 100 µm, and two experimental sizes in development.

The process has been running smoothly for a year. The new nozzle assembly integrated into machines in the field without needing retrofit.

Copyright ©2007 Medical Product Manufacturing News
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