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This application claims priority to U.S. Prov. Appl. No. 60/574,000, filed May 24, 2004, the entirety of which is hereby incorporated by reference.
N/A
Heterogeneous immunoassays typically require the separation of sought-for components bound to component-selective particles from unbound or free components of the assay. To increase the efficiency of this separation, many assays wash the solid phase (the bound component) of the assay after the initial separation (the removal or aspiration of the liquid phase). Some chemiluminescent immunoassays use magnetic separation to remove the unbound assay components from the reaction vessel prior to addition of a reagent used in producing chemiluminescence or the detectable signal indicative of the amount of bound component present. This is accomplished by using magnetizable particles including, but not restricted to, paramagnetic particles, superparamagnetic particles, ferromagnetic particles and ferrimagnetic particles. Tested-for assay components are bound to component-specific sites on magnetizable particles during the course of the assay. The associated magnetizable particles are attracted to magnets for retention in the reaction vessel while the liquid phase, containing unbound components, is aspirated from the reaction vessel.
Washing of the solid phase after the initial separation is accomplished by dispensing and then aspirating a wash solution, such as de-ionized water or a wash buffer, while the magnetizable particles are attracted to the magnet.
Greater efficiency in washing may be accomplished by moving the reaction vessels along a magnet array having a gap in the array structure proximate a wash position, allowing the magnetizable particles to be resuspended during the dispense of the wash solution. This is known as resuspension wash. Subsequent positions in the array include additional magnets, allowing the magnetizable particles to recollect on the side of the respective vessel.
Once the contents of the reaction vessel have again accumulated in a pellet on the side of the reaction vessel and the wash liquid has been aspirated, it is desirable to resuspend the particles in an acid reagent used to condition the bound component reagent. In the prior art, a single stream of acidic reagent is injected at the pellet. Because the size of the pellet and limitations on the volume and flow rate of reagent, insufficient resuspension may result. To address this inadequacy, prior art systems have resorted to the use of an additional resuspension magnet disposed on an opposite side of the process path from the previous separation magnets. The resuspension magnet is configured to assist in drawing paramagnetic particles into suspension, though the magnetic field is insufficient to cause an aggregation of particles on the opposite side of the vessel from where the pellet had been formed. In addition, since the prior art approach utilizes a resuspension magnet, there is less motivation to accurately aim the acid resuspension liquid. Any inhomogeneity in the suspended particles is addressed by the resuspension magnet.
It would be preferable to provide a system in which the use of a resuspension magnet is obviated.
An improved acid injection module includes dual, parallel injection probes. A high-precision aiming strategy is employed to ensure that complete, homogenous resuspension of accumulated solid-phase particles is achieved, obviating the need for subsequent resuspension magnet positions.
The dual, parallel injector probe nozzles are spaced by a degree necessary to provide substantially adjacent impact zones on the reaction vessel wall, also referred to as “hit zones” or “hit points.” Through careful control over lateral spacing of the two nozzles, and thus the two hit zones, and by performing an exacting analysis of the various physical tolerances which can effect hit zone location relative to the solid-phase pellet, thorough resuspension can be achieved without use of a resuspension magnet.
Other features, aspects and advantages of the above-described method and system will be apparent from the detailed description of the invention that follows.
The invention will be more fully understood by reference to the following detailed description of the invention in conjunction with the drawings of which:
FIG. 1 illustrates an optimal orientation of resuspension probes relative to a reaction vessel according to the presently disclosed invention;
FIG. 2 illustrates certain physical parameters employed in defining the optimal orientation of the probes of FIG. 1;
FIG. 3 illustrates additional physical parameters employed in defining the optimal orientation of the probes of FIG. 1;
FIG. 4 illustrates additional physical parameters employed in defining the optimal orientation of the probes of FIG. 1;
FIG. 5 illustrates additional physical parameters employed in defining the optimal orientation of the probes of FIG. 1;
FIG. 6 pictorially illustrates system components which contribute vertical tolerances and which must be accommodated in defining the optimal probe orientation of FIG. 1;
FIG. 7 is a vector diagram representation of the tolerance contributors of FIG. 6;
FIG. 8 pictorially illustrates system components which contribute horizontal tolerances and which must be accommodated in defining the optimal probe orientation of FIG. 1;
FIG. 9 is a vector diagram representation of the tolerance contributors of FIG. 8
FIG. 10 is a perspective view of a probe module according to the presently disclosed invention;
FIG. 11 is a front view of the probe module of FIG. 10;
FIG. 12 is a section view of the probe module of FIGS. 10 and 11 taken along lines A-A; and
FIG. 13 is a section view of the probe module of FIGS. 10 and 11 taken along lines B-B.
The presently disclosed concept finds particular applicability to automated laboratory analytical analyzers in which paramagnetic particles are drawn into a pellet on the side of a reaction vessel as part of a separation and wash process. In particular, in an analyzer in which chemiluminescence is utilized for determining analyte concentration, the accumulated particles must be thoroughly resuspended to obtain an accurate reading. One approach in such systems is to resuspend the accumulated, washed particles in acid prior to introducing a base, and thus triggering the chemiluminescent response, at an optical measuring device such as a luminometer. However, it is noted that the presently disclosed concept is also applicable to any environment in which thorough resuspension of accumulated particles is required.
FIG. 1 illustrates a reaction vessel (also referred to as a cuvette), a probe nozzle, and the ideal orientation of the probe with respect to the cuvette. Note that two probes are employed, though only one is visible in the profile illustrated of FIG. 1. Linear distance values are given in millimeters. As shown, the ideal distance below the cuvette top plane where the liquid stream hits the cuvette wall, referred to as the hit point, is 25.98 mm. In the illustrated embodiment, this hit point is 5.74 mm above the centerline of a magnet array which forms the solids pellet and represents an empirically determined ideal locus of the hit point for achieving thorough particle resuspension. The probe is angled 6.9 degrees from vertical, with the probe tip being located 0.92 millimeters behind the cuvette centerline and 2.304 mm above the cuvette top plane. These values are obtained, as described below, by calculating the worst-case tolerance errors which could effect the hit point and by finding the locus where, even assuming all tolerances have a maximum deviation, the hit point will still be above the magnet centerline.
One practical aspect not accounted for in the configuration described in FIG. 1 is the effect of gravity on the liquid stream itself. The ideal hit point illustrated in FIG. 1 is calculated by extending the axis of the probe towards the cuvette wall. Because of the effect of gravity, the actual hit point is slightly below the one illustrated in FIG. 1. That distance is calculated in the following.
With respect to FIG. 2, given values include:
Pump flow rate | V = 1300 μl/s | |
Probe inner diameter | id = 0.65 mm | |
Probe inclination from vertical | φ = 6.9° | |
Vertical probe tip to hit point | h = 27.868 mm | |
Axial probe tip to hit point | l = 28.071 mm | |
The speed v_{0 }of the liquid at the probe tip can be derived from the pump flow rate and the needle inner diameter:
With reference to FIG. 2, the horizontal distance between the probe tip and the cuvette wall can be calculated from:
s=√{square root over (l^{2}−h^{2})}
s=3.37 mm=3.37 ·10^{−3}m
With reference to FIG. 3, the arc of the liquid stream can now be calculated:
The first part of the term is equal to h and the second part gives the difference between the ideal shown in FIG. 1 and actual hit point.
Overall, there are four tolerance chains which can affect the hit point:
In the following, every tolerance chain is treated individually. Eventually, the total tolerance is estimated by adding the results of the individual tolerance chains.
The calculations for the individual tolerance chains are performed by executing the following steps:
Identification of related parts and their respective tolerances, providing a graphical description of the tolerance chain;
Graphical vector analysis of the tolerance chain;
Generation of a table of dimensions, tolerances, maximum dimensions, minimum dimensions;
Calculation of the ideal closure dimension;
Calculation of the arithmetic maximum and minimum closure dimensions and the arithmetic tolerance;
Identification of mean values from asymmetric tolerance zones and means values of shape and positional tolerances;
Generation of closure dimension as distribution average;
Identification of deviation σ/variance σ^{2 }for every dimension and calculation of the total error according to the theorem of error propagation; and
Evaluation of statistical closure dimension and tolerance.
The dimensions of all parts are considered to have normal, Gaussian distributions with a deviation of ±3σ. This means that 99.73% of all parts are inside the tolerance zone. This assumption is realistic for lot sizes of 60 to 100 parts and greater. The shape and position tolerances have a folded normal distribution.
For statistical calculation of the hit point tolerance, a mathematical description of the hit point depending upon linear position and angle of the probe is necessary. The arc of the liquid stream is omitted at this point for simplicity, but is factored in subsequently.
A simplified arrangement of a probe module and cuvette is shown in FIG. 4. The draft or outward curvature of the cuvette wall is omitted. h is the distance between the cuvette top plane and the hit point on the inner wall of the cuvette. The width of the cuvette is assumed to be constant. cw thus gives half the width of the cuvette such that cw=2.73 mm.
The draft angle β, not taken into account in the foregoing, is 0.5°.
FIG. 5 illustrates the offset produced by the cuvette wall draft. The value h_{r }has to be deducted from h to get the actual value of the hit point h_{real}.
(Eq. 1). Substituting the projected values from FIG. 1 into x, y, and φ as control gives the correct value for h_{real}, 25.98 mm.
Height tolerances are now considered with respect to FIG. 6, which illustrates all parts which add tolerances in height. These parts include a washer plate on which is mounted the acid injection probe module, the probe module, a cuvette transport ring segment in which the cuvettes are disposed, and a transport ring on which the ring segments are disposed. The transport ring is supported by a taper roller bearing and opposing circlips. Both the washer plate and the taper roller bearing/circlips are supported upon an incubation ring.
For the worst case in terms of height, it is assumed all tolerances are at their maximum, so that clearance between the washer plate and the cuvette is minimal. The hit point is thus lowered towards the bottom of the cuvette. To achieve this, parts of the left side of FIG. 6 must be at their minimum thickness whereas the parts on the right side must be at their maximum thickness. These requirements are illustrated in FIG. 6 by the large arrows.
The vector diagram of FIG. 7 shows all dimensions with their maximized or minimized direction. M0 is the so-called closure dimension, or the vertical gap between the probe tip and the cuvette upper plane. In the equations, this value is referred to as y. The const. vector sums the two constant values shown in FIG. 6, the thickness of the cuvette top plane and the vertical distance between the probe tip and the washer plate.
In the following table, all factors with the respective maximum and minimum values and resulting tolerance zones are provided:
Max. | Min. | |||
vector | Dimension | dimension G_{o} | dimension G_{u} | Tolerance zone |
+const. | 3.596 | 3.596 | 3.596 | 0 |
+M1 | 0 | 0.1 | −0.1 | 0.2 |
−M2 | 90 | 90 | 89.95 | 0.05 |
+M3 | 6.15 | 6.17 | 6.13 | 0.04 |
+M4 | 1.75 | 1.75 | 1.69 | 0.06 |
+M5 | 15 | 15.2 | 15 | 0.2 |
+M6 | 1.2 | 1.2 | 1.14 | 0.06 |
+M7 | 52 | 52.04 | 51.96 | 0.08 |
+M11 | 0 | 0.2 | −0.2 | 0.4 |
+M8 | 0 | 0.2 | −0.2 | 0.4 |
+M9 | 5 | 5.1 | 4.9 | 0.2 |
+M10 | 3 | 3.1 | 2.9 | 0.2 |
The nominal closure dimension M_{OH}:
The arithmetic maximum closure dimension y_{max}:
The arithmetic minimum closure dimension y_{min}:
The arithmetic closure dimension with tolerance zone is thus:
Some statistical calculations are necessary to account for component fluctuations. The mean values from asymmetric tolerance zones M2, M4, M5 and M6 are now defined. For M2:
Similar calculations for M4, M5, and M6 yield:
μ_{4}=1.72
μ_{5}=15.1
μ_{6}=1.17
As for M1, M8, and M11, shape and positional tolerances are distributed with a folded normal distribution. Mean values and deviations must therefore be calculated with the following equations. A deviation of 3σ is thereby assumed.
The closure dimension μ_{0H }is calculated as a distribution average:
The deviation σ_{0H }of the closure dimension:
The statistical closure dimension with tolerance zone is:
M_{0H}=y=μ_{0H}±(T_{SH}/2)=1.974±0.405
Axial tolerances are now considered. FIG. 8 illustrates the components which contribute tolerances in the axial direction. The worst case is reached if the probes are displaced towards the inside of the incubation ring and the cuvette is displaced away from the probes. The large arrows in FIG. 8 illustrate these conditions.
The vector diagram of FIG. 9 the various contributing factors with the respective direction. M0 is the closure dimension, here the horizontal gap between the probe tip and the cuvette centerline. In the equations that follow, this value is identified as x.
The const. vector is the constant value shown in FIG. 8 and represents the horizontal distance between the probe tip and the cuvette centerline. The tolerance of this separation can be neglected due to the construction of a preferred instrument.
In the following table, all of the contributors with their maximum and minimum dimensions and tolerance zones are provided.
Max. | Min. | |||
Vector | Dimension | dimension G_{o} | dimension G_{u} | Tolerance zone |
−const. | 0.3 | 0.3 | 0.3 | 0 |
−M11 | 23.03 | 23.13 | 22.93 | 0.2 |
+M12 | 226 | 226.02 | 225.98 | 0.04 |
−M13 | 0 | −0.05 | 0.05 | 0.1 |
−M14 | 215.87 | 215.92 | 215.82 | 0.1 |
+M15 | 14.12 | 14.22 | 14.02 | 0.2 |
The nominal closure dimension M_{0A }is given by:
The arithmetic maximum closure dimension x_{max }is given by:
The arithmetic minimum closure dimension x_{min }is given by:
From these values, the arithmetic closure dimension with tolerance zone is given by:
Some statistical calculations are necessary to account for component fluctuations. The mean values for shape and position for tolerance M13 are now defined.
M13: σ_{13}=0.033 μ_{F13}=0.027 σ_{F13}=0.02
Closure dimension μ_{0A }is given as a distribution average:
−0.3−23.03+226−0.02−215.87+14.12=0.9
The deviation σ_{0A }of the closure dimension is determined from:
The statistical closure dimension with tolerance zones is given by:
M_{0A}=x=μ_{0A}±T_{SA}/2=0.9±0.162
Injector inclination tolerances are now addressed. The tolerance of the bores in the washer plate is M16=±0.05°. The parallelism of the axis of the probe bore and the axis of the injector outer diameter is M17=0.05 mm. With the length of 18 mm this results in an angle tolerance of:
| ||||
Max. | Min. | |||
Vector | dimension | dimension G_{o} | dimension G_{u} | Tolerance zero |
M16 | 6.9° | 6.95° | 6.85° | 0.1° |
M17 | 0 | 0.05° | −0.05° | 0.1° |
The nominal angle φ_{0 }is given by:
The arithmetic maximum angle φ_{max }is given by:
The arithmetic minimum angle φ_{min }is given by:
The closure dimension with tolerance zone is thus given by:
Some statistical calculations are necessary to account for component fluctuation. The mean values for shape and position for tolerance M17 are now defined.
M17: σ_{17}=0.033° μ_{F17}=0.027° σ_{F17}=0.02°
The average angle distribution μ_{0φ} is given by:
The deviation of the angle error is given by:
The statistical angle error with tolerance zone is thus given by:
The worst case calculation for hreal can now be calculated by setting the arithmetic maximum values for x_{max}, y_{max}, and φ_{max }into Eq. 1, above.
The arithmetic minimum can be calculated using the analog:
Thus, the arithmetic derivation of the hit point with tolerance zone is given by:
The hit point μ_{h }as distribution average with μ_{0H}=1.974, μ_{0A}=0.9, μ_{0φ}=6.986° and employing Eq. 1:
μ_{h}=25.812
The statistical deviation σ_{h }of the hit point, depending upon the variables σ_{0A}, σ_{0H}, σ_{0φ}, can now be calculated using Eq. 1. Using partial derivatives at the distribution average:
With μ_{0H}=1.974, μ_{0A}=0.9, μ_{0φ}=6.986° and σ_{0H}=0.135, σ_{0A}=0.054, and σ_{0φ}=0.017°, the result is:
σ_{h}=0.435
T_{SH}=6σ_{h}=2.61
The statistical error of the hit point with tolerance zone is thus given by:
In the embodiment in which the pellet is formed by a magnet array, the tolerance of the array relative to the cuvettes must also be accounted for. The magnets, in a preferred embodiment, are fixed in a ring which is suspended under the transport ring. Most of the tolerance of the magnets is addressed in the height tolerances previously calculated. Thus, there are only the following tolerances to be accounted for:
M18—slide bearing;
M19—magnet ring (i.e. the position of the magnet assembly in the magnet ring);
M20—magnet assembly (i.e. the tolerance of the fixture into which the magnet assembly is fixed); and
M21—the slide bearing support.
All of the above contribute to movement in the same direction.
Min. | Tolerance | |||
Vector | dimension | Max. dimension G_{o} | dimension G_{u} | zone |
M18 | 4 | 4.1 | 4.05 | 0.05 |
M19 | 7.4 | 7.45 | 7.35 | 0.1 |
M20 | 0 | 0.05 | −0.05 | 0.1 |
M21 | 1 | 1.1 | 0.9 | 0.2 |
The nominal closure dimension M_{0M }is given by:
M_{0M=ΣM}_{i }
4+7.4+1=12.4
The arithmetic maximum closure dimension P_{0M }is given by:
P_{0M=ΣG}_{0i }
4.1+7.45+0.05+1.1=12.7
The arithmetic minimum closure dimension P_{0M }is given by:
P_{0M=ΣG}_{0i }
4.05+7.35−0.05+0.9=12.25
The arithmetic closure dimension with tolerance zone is thus given by:
Mean values from asymmetric tolerance zone M18 is given by:
μ_{18}=4.075
The closure dimension μ_{0M }as a distribution average is found according to:
5.075+7.4+1=12.475
The deviation σ_{0M }of the closure dimension is given by:
The statistical closure dimension with tolerance zone is thus:
The nominal distance between the magnet centerline and the cuvette top plane at the acid injection position is 31.72 mm. This value can be calculated with the nominal dimensions listed above:
3.9+12.4+6.35+5+3+1.067=31.717
(3.9 being the distance between the upper magnet and the magnet ring, 6.35 being the magnet width).
The deviation σ_{h }and the tolerance zone T_{SH }of the hit point relative to the cuvette top plane was estimated above as 25.98±1.305 mm. The nominal measure between hit point and magnet centerline is thus:
h_{total}=31.717−25.98=5.737
The total deviation σ of the difference between hit point and magnet centerline is thus calculated by:
√{square root over (0.435^{2}+0.042^{2})}=0.437
T_{s}=6σ=2.622
The statistical error of the hit point versus magnet centerline with tolerance zone can then be written as:
Once 0.25 mm is added to compensate for the arc of the liquid stream, the acid injection is calculated to hit the cuvette wall not deeper than 4.167 mm above the magnet centerline.
One embodiment of a probe housing 100 is illustrated in FIG. 10. This housing, which supports dual probe nozzles 102 is mounted in order to direct a parallel stream of liquid, preferably acid, above a pellet of particles such as paramagnetic particles which have accumulated on the interior wall of a reaction vessel such as a cuvette. By following the tolerance analysis procedure detailed above, the hit point for both acid streams can be assured to be above the pellet, regardless of variations in the physical components of the system.
The linear dimensions in FIGS. 11, 12 and 13 are all given in millimeters. A front view of the probe housing 100 is provided in FIG. 11, showing the mutually adjacent nozzles which produce parallel streams of resuspension liquid. In FIG. 12, a cross-section taken along lines A-A in FIG. 11, it can be seen that ideally a source of resuspension liquid is coupled to the back of the probe housing. As can be seen in FIG. 13, a cross-section taken along lines B-B of FIG. 11, the liquid source feeds both nozzles 102 in generating the parallel streams, five millimeters apart.
On the back of the probe housing 100 is a mounting recess 110 for interfacing to a resuspension liquid-supplying conduit (not shown). Secure attachment of the conduit to the housing 100 is preferably through interlocking threads or other means known to one skilled in the art. Preferably a buffer zone 112 exists between the forward end of the conduit once installed in the recess 110. Liquid from the conduit passes into the buffer zone and then into each of two channels 114 which lead to respective probes 116 and the probe nozzles 102 themselves. In the illustrated embodiment, the probes 116 and nozzles 102 are 0.65±0.02 mm in diameter.
Having described preferred embodiments of the presently disclosed invention, it should be apparent to those of ordinary skill in the art that other embodiments and variations incorporating these concepts may be implemented. Accordingly, the invention should not be viewed as limited to the described embodiments but rather should be limited solely by the scope and spirit of the appended claims.