Abstract
INTRODUCTION
Numerous test devices are available to analyze body fluids for the presence or absence of a predetermined soluble constituent. Whole human blood includes at least three types of specialized cells. Plasma or serum is separated from the blood cellular material by centrifugation. The cellular material collects at the bottom of the centrifuge tube and the supernatant plasma or serum is decanted.
Significant advances in blood testing in the last 30 years have made it possible to use the same tube the blood is drawn into for analysis. A built-in gel separator in the tube places itself between the two portions of separated blood. Other attempts have been made to shorten the blood preparation stage by developing laboratory automation systems, which decrease the total labor and cost of the blood tests. However, the bottleneck of the process is still the traditional low speed centrifugation. Specimen centrifugation is one of the most time-consuming tasks associated with pre-analytical specimen processing, as samples wait in line for an available centrifuge. 1 The centrifuge productivity does not conform to modern analyzer outputs. This necessitates using a large number of small and large centrifuges in contemporary laboratories.
Currently, according to some reports, over 94% of life science researchers use centrifugation. Blood testing is a very common and much needed procedure, and requires the separation of the serum, or plasma, from the red and white blood cells. Such a separation is traditionally achieved by centrifuging blood collection tubes for several minutes. The current practice is for tubes to spin for approximately 10 to 15 minutes at 1200 g. It is not possible to increase acceleration, as this will destroy the hemolytic blood cells.
The current blood testing method, however, has two major drawbacks. First, the relatively long time needed for blood separation delays delivery of analytical results. Second, due to the long separation time required, large batch sizes are needed in order to maintain high sample throughput during blood processing.
Some hopes were rushed in developing Axial Spin centrifuges. 2,3 The main disadvantage of these methods was the need to use special blood containers, which were different from standard Becton Dickinson Vacutainers. Axial Spin does not work with clotted blood and therefore does not allow for receiving serum instead of plasma. These blood constituent studies are known to perturb some constituent data and require “special processing”. Small Axial centrifuges spin one tube at a time and are not very useful in batch processes.
In the 1920s, an American scientist by the name of Boycott noticed that blood cells settled faster in test tubes that were inclined than in tubes that were vertical. 4 Attempts have been made to combine the “Boycott” effect in swing-out centrifuges. 5 –7 The constructions of these assemblies are not suitable for implementation in standard centrifuges. These embodiments were also not useful in standard batch technology. The Boycott effect has been widely investigated and described. 8,9 Phase separation occurs more rapidly if the test tube walls are inclined at an angle rather than parallel to the direction of centrifugal force. There are two phenomena occurring in inclined tubes. Heavy particles move a distance less than the diameter of the tube towards a wall. Then the particles slide along this wall to the bottom of the tube. This movement produces a flow, which decreases spin time. Heavy particles are located along inclined tube walls; however, opposite this layer, particles will microcirculate during a spin. These opposing phenomena do not allow for perfect whole blood phase separation using a standard fixed angle rotor centrifuge for a short time.
Standard fixed angle centrifuges use the Boycott effect (which requires inclination of the tube walls at a certain angle to the vector of the centrifugation force) in order to make phase separation more efficient. In this case the phase boundary is also inclined and the gel seal is not secure. Becton Dickinson Company does not allow their blood drawing tubes with gel barriers to be spun in fixed angle centrifuges.
A centrifuge and two improved standard holders. These holders connect to standard rotors via their pins. Each holder balanced by using steel plate, which is also used to incline the holder. Displacing means and tubes are loaded inside each holder.
There are two contradictory effects that have yet to be reconciled in a standard centrifuge.
The need to spin a tube with a gel barrier so that the long axis is not aligned with the direction of the centrifugal force for rapid separation;
The need to spin the tube with the long axis aligned with the direction of force to ensure the gel will seal across the phase boundary.
The main target of this innovation was to develop a simple accessory to improve many existing centrifuges.
DISCLOSURE OF THE INVENTION
A new centrifugation assembly (US Patent #6,234,948) promises to deliver faster and better centrifugation, and greatly improve the throughput of the blood separation process. 10 The invention is a new tube holder called Fast Spin, which may be easily set up and removed from standard centrifuges. This invention provides an assembly and a method for rapid phase separation in liquids in general and for blood phase separation in particular. The invention can be implemented in ordinary swing-out rotor centrifuges, in high-speed centrifuges, or in laboratory automation systems.
The method comprises spinning blood sample tubes while they are inclined, in order to make use of the Boycott effect for more rapid phase separation. In the second stage of separation, the tubes spin while their longitudinal axes are aligned with the direction of the centrifugal force to allow proper sealing of the gel separator.
The position of the common centre of gravity of the holders and of the tubes placed therein varies during the separation process. The first common center of gravity is above the pivoting axis of each holder. By virtue of this provision the centrifugal force tries to pivot the holders and the tubes opposite to the ordinary horizontal direction. A particular method of stopping the centrifuge prevents this attempt and inclines the holders and tubes in an ordinary direction. This method of stopping has various constructions in order to maintain the degree of inclination of the holders.
Two experimental lightening holders and two displacing means (closed cylindrical container). There are 18 tubes and one displacing means inside each holder. A simple detachable stopping means is adjusted to each holder.
After completing the first stage of the separation using the Boycott effect, the common center of gravity is automatically displaced in the second position, i.e. under the pivoting axis of the holder. During the second stage, the centrifugal force pivots the holders and tubes into a horizontal position so they can align with the vector of the centrifugal force and thus the complete gel seal can take place. Stopping means does not prevent this pivoting movement. At the end of the second stage the centrifuge is stopped and the holders and tubes are returned back into their initial positions.
The location of the center of gravity can be varied during centrifugation. This is made possible using a closed cylindrical container filled with fluid and inserted into the holder. The container may contain a free mass placed therein which can move along the container. The outside diameter of the mass is less than the inside diameter of the container, leaving a gap between them. This gap allows fluid to flow through there and thus sets the mass displacing time. The mass and thus the center of gravity are displaced by centrifugal force toward the opposite end of the container. The container then must be turned over to prepare it for the next run. The next iteration of this design will contain a spring to return the mass from its lower post-spin position to the initial pre-spin position.
There is an option of decapping the tubes while they are in the holder. The decapper comprises a removable insert fixed on the upper part of the holder, a support plate for supporting the tubes and a spring. The tubes can be moved by centrifugal force toward the lowermost position. Their caps lean against the partition of the insert so as to be removable from the tubes. The decapper is equipped with safety devices, such as a support plate and springs to secure the tubes from shocking. The springs return the tubes into their uppermost position. The decapper is an inexpensive option for the Fast Spin Holder. This device works automatically at the end of a separation spin.
The present Fast Spin does not require any changes in the construction of the standard swing-out rotor and therefore can be used easily in various conventional centrifuges. The assembly can be easily mounted on a standard centrifuge as well as conveniently removed. All Fast Spin centrifuge parts and accessories work automatically during a spin and do not require operator interaction.
Fast Spin technology is not a categorical alternative to super-speed and ultracentrifuges. Instead, the machines equipped with a Fast Spin Dual Rotor will produce separation of different fluids more rapidly and qualitatively. This rotor, when built, will be used for specimens other than blood. This development requires calculations and balancing according to specific laboratory demands.
EXPERIMENTAL
In 1993–1994, the author was involved in the development and testing of a Dual Spin Centrifuge, which was an improvement of DuPont Axial Spin Technology. Early tests were carried out in a Fast Spin development stage with a goal of confirming or disproving some technical preconceptions. The main problems were in balancing the rotors and synchronizing the holders in their turn from inclined to horizontal position during a spin. After these problems were solved we started to determine a spin time sufficient to fully separate blood. Experiments showed a two minute spin cycle. We plan to decrease the spin time to one minute after further system improvement.
We tested prototypes for two cases: separation of whole blood and separation of clotted blood. We spun tubes with whole blood in a standard centrifuge with new holders at 3000 RPM for 2 minutes. The acceleration time for this model is 15 seconds. Separation was complete in all tubes. The count of white blood cells and red blood cells was zero. These provisional studies proved our technical conception and we intend to continue our tests.
We processed blood drawing tubes with clotted blood samples in the improved holders spinning for 2 minutes at 3000 RPM (instead of the usual 10 min). The result was successful and the shape of the gel seal was satisfactory in all cases. A comparative processing of clotted blood samples in ordinary swing-out holders in the same centrifuge for 3 minutes at 3000 RPM was not successful.
In our tests we used only conventional blood constituent studies, which exist in standard laboratories. We have not yet implemented wide laboratory studies. In the future we must continue comparing tests for potassium and other cellular or chemical analyses that are sensitive to centrifugation. However, we have now received enough evidence to show that Fast Spin Centrifuge works successfully.
LABORATORY AUTOMATION PROBLEMS
During the last ten years many companies were rushing to bring heavy and expensive total laboratory automation (TLA) to the US market, due to its success in Japan. Standard TLA includes traditional testing equipment (centrifuges, analyzers, e.g.) as well as special transport tracks.
Manufacturers wanted to sell expensive equipment to laboratories. They did not take into account that Japan has a large population density and its 170 Automation Laboratories serve a large amount of patients. The US laboratory market is not suitable to TLA due to small population density and the presence of a large number of small- and medium-sized laboratories across North America.
Beckman Coulter Inc. achieved some success in TLA after their merger. 11 This technology requires arranging tubes in a line on a transport track before testing. Other attempts were made to bring the testing process closer to the physician's office. Becton Dickinson and Company distributed their QBC™ AUTOREAD™ Plus Centrifugal Hematology System and QBC STAR™ Centrifugal Hematology System. 12 Some reports show the change this has had on clinical laboratory tests over the past five years. Some companies divide their big TLA into different parts, each of which is called a Module. They use standard equipment and standard technology but remove the heavy transport tracks.
The clinical-laboratory-testing market is exceptionally competitive and there will likely be continued pressure on the prices of products and services provided by companies. 13 Some strategic alliances between leading hospitals and healthcare systems across the country were built to reduce this risk. To increase profits they closed local small- and medium-sized laboratories and instead built big main laboratories equipped with expensive equipment.
Health services companies using centralization and globalization are forced to transport tubes over long distances, causing a delay in the time it takes a result to reach a physician's office. (For example: two companies send specimens from San Francisco to San Jose or Dublin and physicians do not receive test results for two days.) According to some investigations and publications, the US Health System is not ready to increase inexpensively and quickly the amount of mass blood testing in case of an infectious disease attack. Fast Spin intends to solve this problem rapidly and cheaply.
Laboratory automation is available in many forms and can be tailored to almost any laboratory budget. 1 The new modular generation has fewer transport tracks, with analyzers located closer to the actual processing stations rather than going to separate stations. 14 Since new powerful analyzers have appeared in laboratories, only tube sorting, sample balancing, load/unload time (robotics or manual) and recapping have remained. Different attempts have been made to solve these problems.
There are a well-known number of different approaches to pre-analytical sample treatment. Axial Spin Technology 2,3 offers single sequence tube processing. This method connects well with existing analyzers, but is time and cost deficient in loading/unloading and recapping operations. In US Patent No. 5,551,941, 15 a bulk loading method was shown. This approach to automated centrifugation allowed tube loading and unloading using gravity. Unfortunately, this machine is very complicated and was not implemented in laboratories.
The traditional batch loading method is very useful in standard swing-out centrifuges. This method was improved using our Fast Spin centrifuge. A new arrangement of some well known parts allows the use of this machine as a simple pre-analytical module.
FAST SPIN TECHNOLOGY IN LABORATORY AUTOMATION
The pre-analytical stage is the most labor-intensive part of the overall testing process. Fast Spin technology is best suited to laboratory automation and will improve the overall laboratory automation process. The Fast Spin Module will include original hardware and software systems.
Fast Spin is not just a rapid separator. We propose a powerful pre-analytical station based on the Fast Spin Centrifuge equipped with a decapper and a bar code system. Fast Spin technology simplifies the process of connecting tubes with the analyzer. In our case the centrifuge and analyzer are not connected by tracks since the module is small in size with extra capacity.
The Fast Spin module might be integrated not only in larger TLA systems; but also in small labs for sample processing. The result would save time and money. Fast Spin modules will use a standard batch tube processing method. This arrangement will decrease the number of intermediate loading /unloading steps.
Fast Spin Technology is aimed at solving the problems of long separations, loading and unloading tubes and cap removing in a sample preparation module. The Fast Spin Centrifuge separates specimens and removes caps simultaneously. This processing decreases the pre-analytical operation time. A laboratory automation system equipped with a Fast Spin module will have only a small number of loading and unloading operations and a few transportation problems.
The Abbott FE500 is designed to handle 500 tubes per hour. 14 Our module will be able to operate 2,500 tubes in the same amount of time, due to rapid centrifugation and cap removal being united in one device.
We are able to build a laboratory automation system with a big processing potential using our Fast pre-analytical module, simple robot and improved standard analyzer. This system will be managed by a laboratory information system (LIS), which commences at blood drawing stations and cooperates with clinical software systems. These information systems will use the barcode system and support the linkage between suppliers, provider health systems and the laboratory in a common effort focused on resource utilization, cost effectiveness, quality outcomes and best practices.
Fast Spin technology will not solve all laboratory problems. The Fast Spin pre-analytical module will provide significant time and cost savings. There will be a significant processing improvement using the Fast Spin pre-analytical module in combination with an improved analyzer and a new laboratory information system. We need cooperation with any laboratory to apply a new implementation strategy for net processing improvement.
RESULTS
The new technology called “Fast Spin” will be useful both in ordinary centrifuges and laboratory automation systems. It utilizes a three-phase approach. During the first stage, it allows for rapid separation due to spinning the sample tubes in an inclined position. (This enables it to make use of the “Boycott” effect for more rapid separation). In the second stage, the centrifugal force turns the holders and tubes into a horizontal position. In the last stage, after stopping the centrifuge, the holders and tubes return to the original position.
An add-in component can provide a means for removing the caps from the tubes while they are still residing within the bucket. Such a device can save labor as well as valuable testing time.
The set of Fast Spin holders may be utilized in conjunction with standard centrifuges and may be easily removed from the centrifuge. The combination of the Fast Spin Centrifuge and the cap removing device allows for a Fast pre-analytical module. This inexpensive machine will provide great benefits to every laboratory.
The main advantages of Fast Spin Technology are:
It can be widely used in clinical automation equipment that involves specimen centrifugation. Specifically, it can be mounted on the centrifuge(s) of the Sample Preparation Module.
A 70% reduction in blood separation time, which allows for higher sample throughput and expedites the entire blood processing process.
Use the same blood constituent studies as with conventional swinging bucket separation.
Full compatability with any conventional swing-out rotor centrifuge.
Easily assembled and mounted on the centrifuge.
Do not need precise sample balancing like in a standard swing-out rotor.
Consists of simple parts and materials.
Ease of maintenance.
Good gel seal and complete separation of blood specimens is achieved.
Separating and cap removing are conducted in one step.
Super-speed centrifuges and ultracentrifuges equipped with a Fast Spin Dual Rotor will produce separation of different fluids more rapidly and qualitatively.
In some cases Fast Spin assembly in combination with low-speed centrifugation can replace more expensive machines.
Uses standard equipment in a new technology.
Is suitable for use in every laboratory and with laboratory automation.
The cost is small regarding the potential benefit.
APPENDIX
For more technical information please refer to US Patent No. 6,234,948 found at http://www.uspto.gov/patft/index.html