Note: Descriptions are shown in the official language in which they were submitted.
~09~i~2
1
SAMPLE PIPETTING METHOD
BACKGROUND OF THE INVENTION
This invention relates generally to a non-invasive
automated pipetting method, and more particularly, relates to
determining the level of fluid present in a test sample. Level
sensing is accomplished by moving a pipettor toward a sample
while aspirating air and monitoring for a pressure change
within the pipettor. Controlled aspiration of the fluid sample
is then performed.
. Automated pipetting systems that contact a liquid test
sample with an electrode are known. For example, a
conducting pipette tip or an electrode adjacent to the pipette
tip generates an electrical signal when the conducting pipette
tip or the electrode touches the surface of an electrically
conducting fluid, such as a buffer solution or serum, plasma or
2 0 urine sample. These methods involve invasive procedures
which suffer from the danger of cross-contamination between
test samples. A portion of the first sample clings to the
pipette tip or electrode and the next sample to be pipetted
becomes contaminated with the first sample when the pipette
2 5 tip or electrode contacts the second sample. This cross-
contamination or carry-over can also occur between assay
reagents and between assay reagents and samples. The danger
of cross-contamination or carry-over can be reduced by
extensive washing of the pipette tip or electrode between
3 0 each pipetting step, but such washing steps suffer from
decreased sample throughput on the automated instrument.
Detecting the surface of a fluid is very important for the
precise pipetting of the fluid. Locating the fluid surface
permits the controlled immersion of the pipette tip in the
3 5 fluid. By controlling the depth of immersion of the pipette tip
in the fluid, a consistent amount of fluid will cling to the
outside of the tip resulting in greater consistency in the total
CA 02095152 2002-04-15
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volume dispensed. The use of non-invasive fluid sample surface sensing methods
and devices in conjunction with disposable polymeric pipette tips results in
such
greater control and consistency. Thus, non-invasive fluid sample surface
sensing
achieves two advantages. First, it eliminates the need to wash the pipette tip
between sampling, thereby increasing the throughput of the instrument. Second,
a
non-invasive surface probing method eliminates the potential of sample carry-
over.
A non-invasive fluid surface-sensing system is disclosed in U.S. Patents Nos.
3,474,902 and 3,494,191. This non-invasive fluid surface-sensing system
utilizes a
method that involves blowing air via a. steppes-motor controlled syringe to
detect a
fluid surface level. This level sensing method can be used in automated
pipetting
of biological samples. However, air is often blown into the test sample
causing
bubbles and generating aerosols. In an attempt to minimize bubble creation,
the
pipettor is moved toward the sample very slowly until the sample surface is
detected, and then immediately withdrawn to the end of its travel range. The
syringe
is then fully dispensed to blow all the remaining air out of the syringe.
Finally, the
pipettor is returned to the fluid surface and aspiration is commenced.
An object of the present invention is to non-invasively level sense a fluid
sample without the need of blowing air through the pipette tip. Another object
of the
present invention is to aspirate a fluid sample by immersing the pipette tip
into the
sample at a controlled, minimal depth in order to minimize the amount of
sample
that clings to the outside of the pipette tip. Yet another object of the
present
invention is to detect nonhomogeneity, such as clots, bubbles and foam, in the
fluid
sample. Still other objects of the present invention will be apparent to one
skilled in
the art.
The present invention offers advantages over known methods of level
sensing and aspiration of a fluid sample. Carry-over or cross-contamination
between
samples and reagents is eliminated by employing a non-invasive method in
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which no contact is made between the level sense means, such
as a pressure transducer, and the test sample. The present
invention also has advantages over positive pressure (blowing
air) level sense methods. The possibility of bubbles and
S aerosols is eliminated by the present invention. Also, because
the need to reverse the direction of the syringe pump between
the level sense step and the aspiration step is eliminated, the
instrument throughput is increased. In addition, the present
invention eliminates the necessity of withdrawing the
pipettor from the sample in order to evacuate the syringe
before aspiration again improving the instrument throughput
through the elimination of method steps without the creation
of bubbles and aerosols.
1 5 SUMMARY OF THE INVENTION
This invention provides an apparatus and method of
pipetting a fluid from a container, which comprises (a)
determining the level of the fluid in the container by (i)
determining the ambient air pressure within a pipettor as a
2 0 baseline pressure reading, (ii) aspirating air into the pipettor
as the pipettor moves toward the fluid sample in the
container, (iii) monitoring for an air pressure change in the
pipettor to indicate the surface level of the fluid in the .
container; (b) immersing the pipettor in the fluid and
2 5 aspirating fluid from the container into the pipettor in
controlled steps while aspirating incremental volumes; (c)
monitoring pressure changes after each incremental
aspiration step; and (d) moving the tip of the pipettor from the
sample in such a way to shear off any droplets. The tip of the
3 0 pipettor may be disposable or reusable.
The invention also provides an apparatus and method of
detecting non-homogeneity in a fluid sample, such as the
presence of foam or bubbles on the surface of the sample,
and/or the presence of clots on the surface or in the bulk of
3 5 the test sample. This method comprises (a) determining the
ambient air pressure within a pipettor as a baseline pressure
reading; (b) aspirating air into the pipettor as the pipettor
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4
moves towards a sample in a container; (c) monitoring for an
air pressure change in the pipettor to indicate the surface
level of the fluid in said container; (d) immersing the pipettor
in the fluid and aspirating a volume of fluid from the
S container into the pipettor; (e) monitoring pressure changes
after said aspiration of step (d); (f) comparing measured
pressure change to predetermined normal aspiration pressure
windows; and (g) observing pressure values falling outside
said predetermined values.
BRIEF DESCRIPTION OF DRAWING
Figure 1 is a graph of a representative sample of normal
calf serum using the level sensing method of this invention
wherein Pressure (+1 psi to -1 psi represented as 10 bit
binary) is plotted against a sequential number of pressure
readings taken at periodic intervals. The change in pressure
occurs as the pipette tip touches the surface of the sample
and the air aspiration is immediately halted.
Figure 2 is a graph of representative pressure changes
2 0 during aspiration of a sample of normal calf serum using the
level sensing and aspiration method of this invention to
aspirate 1000 p.L of sample, wherein Pressure (+1 psi to -1
psi represented as 10 bit binary) is plotted against Aspiration
Data Points. All of the pressure measurements are within the
2 5 predetermined aspiration pressure windows indicating a
homogeneity in the sample.
Figure 3 is a graph of a representative sample of normal
calf serum containing foam on the surface using the fluid
level sensing and aspiration method of this invention to
3 0 aspirate 1000 p.L of sample, wherein Pressure (+1 psi to -1
psi represented as 10 bit binary) is plotted against Aspiration
Data Points. At least one of the measured pressure values is
outside a predetermined aspiration pressure window
indicating a heterogeneity in the sample, i.e. the presence of
3 5 foam.
Figure 4 is a graph of a representative sample of normal
calf serum containing large bubbles on the surface using the
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fluid level sensing and aspiration method of this invention to
aspirate 1000 p.L of sample, wherein Pressure (+1 psi to -1
psi represented as 10 bit binary) is plotted against Aspiration
Data Points. At least one of the measured pressure values is
5 outside a predetermined aspiration pressure window
indicating a heterogeneity in the sample, i.e. the presence of
bubbles.
Figure 5 is a graph of a sample of water containing a
non-dairy creamer to simulate clots in the sample using the
fluid level sensing and aspiration method of this invention to
aspirate 1000 p,L of sample, wherein Pressure (+1 psi to -1
psi represented as 10 bit binary) is plotted against Aspiration
Data Points. At least one of the measured pressure values is
outside a predetermined aspiration pressure window
indicating a heterogeneity in the sample, i.e. the presence of a
clot.
Figure 6 is a illustration of an apparatus which level
senses and aspirates two samples simultaneously followed by
dispensing of the two samples into a sample reaction tray.
DESCRIPTION OF PREFERRED EMBODIMENT
The novel level sensing and aspiration apparatus and
method of the present invention is based upon measurable
pressure changes within a pipettor during the level sensing
2 5 and aspiration steps. When compared to conventional level
sensing and aspiration apparatuses and methods, the present
invention was surprisingly found to more rapidly and
accurately level sense and aspirate a fluid sample.
As illustrated in Figure 6, the pipettor apparatus of the
3 0 present invention is comprised of a disposable pipette tip 10
capable of making an air tight connection with a pipette tip
holder means 12 having a bore and a tube connection means 14
capable of making an air tight coupling between tubing 16 and
the pipettor such that air can be drawn through the disposable
3 5 pipette tip 10 into the pipette tip holder means 12 and then
into tubing 16. Tubing 16 is connected to a pressure
measurement means 18 and a means 20 for aspirating air
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CA 02095152 2002-04-15
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through the pipettor such that said connections are air tight. Finally,
pipette tip
holder means 12 is attached to a means (not shown) for moving the pipettor
vertically along the Z-axis.
Pipette tip holder means 12 are well know in the art. For example, manual
and automated pipettors are readily available which have a tip probe that
permits the
formation of an air tight seal between the pipettor and a disposable pipette
tip and at
the same time, permit the easy removal of the disposable pipette tip. In
addition, a
limit-switch can be incorporated to detect the presence or absence of a
pipette tip on
the tip probe, One skilled in the art would be able to prepare such a pipettor
without
undue experimentation. Also, tube connection means 14 are well known in the
art,
such as luer-lock, compression fitting tube adaptors and the like, and can be
readily
adapted by one skilled in the art for use in the present invention without
undue
experimentation.
Standard disposable pipette tips (10), such as polypropylene and the like, and
standard tubing (16), such as Tygon~, vinyl, polypropylene, polyethylene,
metal and
the like, can be used in the present invention and are readily available for
purchase.
The disposable pipette tips (10) can be stored in racks which are accessible
by the
pipettor.
Pressure measurement means 18 measures the air pressure within the pipettor
either continuously or periodically during the invention method. A preferred
pressure measurement means is a pressure transducer (electrode). The pressure
transducer is interfaced to a host computer system through a Datem dcm300
Digital
I/O Board (available from Datem Limited, Ontario, Canada). The pressure
transducer provides a 10-bit binary output (0 to 1023) with an ambient air
pressure
measuring at approximately the center of the range (512). Full scale pressure
(+1
psi) translates to 0 and full scale vacuum (-1 psi) translates to 1023.
Means 20 for aspirating air through the pipettor must be capable of precisely
controlled movements. For example, during level sense, the aspiration means
(20)
must be capable
209512
of stopping immediately after the pipette tip reaches the fluid
surface. A preferred aspiration means (20) is a syringe,
preferably a 1500 p.L syringe, mechanically connected to a
stepper motor and home limit-switches capable of controlling
the movement of the syringe piston and causing the syringe to
aspirate and dispense air through tubing 16. The stepper
motor and home limit-switches are interfaced to the host
computer through the Daterri dcm340 Stepper Motor Control
(available from Datem Limited, Ontario, Canada).
Means for moving the pipettor vertically along the Z-
axis is an electro-mechanical assembly that is capable of at
least moving the pipettor along the Z-axis (vertically)
relative to the XY-plane of the sample .container (22) and may
also be capable of moving the pipettor along the X and Y axes.
Preferably, a stepper-motor and home limit-switches are used
for positioning- the pipettor along the Z-axis. Again, the
stepper-motor and home limit-switches are interfaced to the
host computer through the Datem dcm340 Stepper Motor
Control. Preferably, a Magnori .XY Table (available from Magnon
2 0 Engineering, Fontana, California) or the like is used for
positioning the pipettor. ~ The Magnon XY Table includes both
the mechanical hardware and the electronic controls
necessary for positioning the pipettor. A Bitbus*
communication port (available from Intel, Santa Clara,
California) is used to interface the XY table controller to the
host computer.
Multiple pipettor assemblies can be interfaced in order
to pipet multiple samples simultaneously. For example, Figure
6 illustrates dual pipettor assemblies. Each pipettor
3 0 assembly comprises a disposable pipette tip 10, pipette tip
holder means 12 connected through tubing 16 to a pressure
measurement means 18 and an aspiration means 20. The two
pipettor assemblies are interfaced with a means for moving
bath pipettors vertically along the Z-axis such that two
3 5 samples can be level sensed and aspirated simultaneously or
sequentially. These dual pipettor assemblies permit the
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dispensing of two samples into a reaction tray having dual
reaction wells (24).
SAMPLE PIPETTING METHOD
The sample
pipetting
method of
the present
invention
involves level sensing of the sample's fluid surface and
sample aspiration. The following is a detailed summary
of the
present invention using the pipettor apparatus shown
in Figure
6:
LEVEL SENSE
1. The ambient air pressure is measured in the pipettor
by a pressure transducer while the syringe is
in its
fully dispensed position and the pipettor is
located at
the top of its stroke in the Z axis. The value
becomes
a baseline to which all other pressure readings
are
compared. By using this value as a baseline,
any
effect on the pressure measurements due to changes
in the atmospheric pressure are eliminated.
2. The pipettor is moved down toward the test sample
until the pipette tip i.s even with the top of
the test
2 0 sample tube.
3. The pipettor is then moved downward toward the
sample fluid surface. Simultaneously with the
pipettor's movement down toward the sample fluid
surface, air is aspirated through the pipettor
by
2 S drawing air into the syringe and the pressure
inside
the pipettor is measured with the pressure
transducer. Inherently in the process of aspirating
air into the pipettor, the pressure in the pipettor
will
be measurably lower than ambient pressure.
3 0 4. When the pressure inside the pipettor suddenly
drops,
the tip of the pipettor has contacted the surface
of
the sample (see Fig. 1 ). Immediately, the movement
of both the syringe and the pipettor is stopped.
Preferably, the pipettor must be stopped so that
the
3 5 tip of the pipettor is not more than 0.125 or
1/8 inch
below the surface of the sample. By stopping
within
0.125 inches, the amount of sample fluid clinging
to
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CA 02095152 2002-04-15
9
the outside of the pipette tip can be more easily minimized.
The continuous pressure measurement will actually be recorded as a
series of periodic measurements rather than one continuous measurement
(see Fig. 1 ). It is well known that inherently, a pressure measurement
system, such as described herein, must process the measured pressure value
to record the value in a binary output and such data processing requires a
minimum amount of time. Thus, the pressure is recorded shortly after each
data processing cycle.
SAMPLE PIPETTING
5. The pipettor is lowered further into the sample a short distance
(approximately 0.055 inches) to avoid starving the pipettor of sample
during sample aspiration.
6. At least about 100 ~L of sample is aspirated into the pipettor by
withdrawing the syringe the appropriate distance. Immediately after the
syringe has stopped moving, the pressure in the pipettor is measured (see
Fig. 2, Aspiration Data Point 1). The pressure is again measured after the
pressure has reached equilibrium, i.e. a steady state pressure (about 0.2
seconds) (see Fig. 2, Aspiration Data Point 2).
7. The two pressure measurement values are then compared with the
aspiration pressure windows. If either or both pressure values are outside
the windows, the sample is nonhomogeneous and the pipetting is halted
(see Figs.. 3-5). If both values are within the windows, the pipetting is
continued with Step 8.
The aspiration pressure windows are calculated from the ambient
pressure measurement taken in Step 1 above by adding empirically
determined values to the ambient pressure measurement (see Table 1 ).
The empirically determined values are obtained using
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WO 92/08545 PCT/US91/08375
standard experimental methods known in the art, i.e. a
variety of normal, homogeneous samples are pipetted
using the sample pipetting method herein with a
variety of pressure transducers. Pressure
5 transducers are known to differ in their individual
performance characteristics and by establishing a
normal range, i.e. aspiration pressure windows,
variations between different transducers and
between different normal samples can be eliminated
10 from being a factor in the sample pipetting method. A
nonhomogeneous sample is a sample that has at least
one pressure measurement that falls outside the
aspiration pressure windows which are the ranges
within which the pressure measurements of normal,
homogeneous samples will fall.
The empirically determined values are simply
obtained by subtracting the ambient pressure (at the
time of the normal sample pressure measurements)
from the highest and lowest pipettor pressure
2 0 measurements obtained during the sample pipetting
of the normal samples. Thus, these empirical values
when added to the ambient pressure in some future
sample pipetting will automatically be adjusted for
any variation in the ambient pressure at the time of
2 5 the future sample pipetting.
8. The pipettor is then lowered further into the sample a
short distance (approximately 0.055 inches) to avoid
starving the pipettor of sample during sample
aspiration.
3 0 9. About 150 p.L of sample is aspirated into the pipettor
by withdrawing the syringe the appropriate distance.
The pressure in the pipettor is measured after the
pressure has reached equilibrium, i.e. a steady state
pressure (about 0.2 seconds) (see Fig. 2, Aspiration
3 5 Data Point 3).
10. The pressure measurement value is then compared
with aspiration pressure windows. If at least one
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CVO 92/08545 PCT/US91/08375
11
pressure value is outside the corresponding window,
the sample is nonhomogeneous and the pipetting is
halted. If the value is within the windows, the
pipetting is continued with Step 11.
11. Steps 8-10, i.e. an aspiration cycle (see Fig. 2,
Aspiration Data Points 4-8), are repeated, if
necessary, until the total desired amount of sample is
aspirated into the pipettor (from about 100 p,L to
about 1000p.L).
12. The pipettor is moved up a short distance
(approximately 0.055 inches) such that the pipettor
tip is at the current sample surface. About 50 pL of
sample is dispensed into the sample to eliminate any
backlash in the syringe prior to dispensing the test
sample into a reaction vessel. This backlash is well
known to occur in mechanical systems due to
looseness in the parts or imperfect gear meshing.
Thus, as the mechanical system is moved in the
reverse direction in order to move the syringe in the
2 0 reverse direction, the backlash or slack in the system
must be taken up before the syringe will begin
moving. By dispensing about 50 p.L of sample back
into the sample tube, the backlash is removed and the
dispensing of the sample into the reaction vessel will
2 5 be more accurate.
13. The pipettor is then moved slowly up about 0.5 inches
to prevent shearing off of a small amount of sample
from inside the pipettor tip. The pipettor is then
moved to the home position from which the pipettor
3 0 can be moved to a dispensing location and is ready to
dispense an accurate amount of sample into a
reaction vessel.
In the level sensing method, the pipettor is moved
3 5 downward toward the fluid sample's surface simultaneously
with aspiration of air into the pipettor. Preferably, the
syringe used in this method is a 1500 p.L syringe. When using
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WO 92/08545 PCT/US91/08375
12
a 1500 p.L syringe to aspirate 1000 pL of sample, only 500 p.L
is available for level sensing. Thus, the pipettor's movement
down to the sample's surface must be coordinated with the
500 pL of air aspiration. The preferred syringe speed for the
level sensing method is 160 ~L per second and the speed of
the pipettor is adjusted so that the pipettor reached the end
of its stroke (i.e. the limit of the pipettor's downward
movement) simultaneously with the syringe reaching 500 p.L
of air aspirated if no sample surface was detected. However,
the pipettor's downward speed should preferably be less than
the maximum speed so that the pipettor can be stopped such
that the end of the pipette tip is no more than 0.125 (1/8) inch
below the surface of the sample. The syringe begins
aspiration prior to the downward movement of the pipettor
from the point where the pipette tip is even with the top of
the sample container. The delay in the pipettor's movement,
preferably for about 0.2 seconds, gives the air pressure time
to reach steady state.
The aspiration method begins after the sample surface
2 0 has been level sensed and the pipette tip is located no more
than about 0.125 inches below the sample's surface. Both the
syringe and the pipettor are stationary. The pipettor is moved
down a short distance, preferably about 0.055 inches, to
prevent the pipettor from being starved for sample during
2 5 aspiration. If the pipettor is starved for sample, air will be
aspirated along with sample and an erroneous amount of
sample will have been aspirated. At least 100 ~L of sample is
then aspirated (first aspiration). It is believed that about 100
p.L of sample is the minimum amount of sample that can be
3 0 accurately aspirated by this protocol using the apparatus
shown in Figure 6. This amount of sample provides a
consistent basis for comparison between various samples
which permits the detection of non-homogeneity in a sample,
such as bubbles, foam and the like, and other problems with
3 5 the system, such as air leaks in the apparatus and the like.
Immediately after the syringe stops during the first
aspiration of the sample, a pressure reading is taken
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13
(Aspiration Data Point 1 ). If this pressure reading is outside
the aspiration pressure window for Aspiration Data Point 1,
the sample is non-homogeneous. For example, a sample with
high viscosity, such as a sample with clots or thick foam, will
cause a higher vacuum to be created within the pipettor than a
normal homogeneous sample. A sample with bubbles or a leak
in the system will result in a lower vacuum within the
pipettor than a normal homogeneous sample and may even read
at ambient pressure. A second aspiration pressure reading
(Aspiration Data Point 2) is taken within the pipettor after a
short delay, preferably at least about 0.2 seconds, which is
sufficient time for the pressure within the pipettor to reach a
steady state. As with the first pressure reading, a pressure
reading outside the aspiration pressure window corresponding
to Aspiration Data Point 2 is an indication of non-homogeneity
or problems with the system. Whenever any pressure reading
is outside the corresponding aspiration pressure window, the
aspiration process is stopped automatically.
The remainder of the sample that is to be aspirated into
2 0 the pipettor is accomplished by sample aspiration cycles
wherein a set volume (or less if that is all that is needed to
obtain the desired amount of total sample aspirated),
preferably at least about 100 ~L, is aspirated in each cycle.
More preferably, a volume of about 150 p,L is aspirated during
2 5 each aspiration cycle. Thus, seven aspiration cycles would be
needed to completely aspirate a total of 1000 p.L of sample.
An aspiration cycle includes the following: (1 ) moving the
pipettor downward into the sample a short distance,
preferably about 0.055 inches, in order to avoid starving the
3 0 pipettor of sample; (2) aspirating a volume of sample into the
pipettor, preferably about 150 p.L; and (3) after a short delay,
preferably at least about 0.2 seconds, taking a pressure
reading within the pipettor (Aspiration Data Points 3-8).
Again, if a pressure reading is outside the corresponding
3 5 aspiration pressure window, the sample is non-homogeneous
and the aspiration is stopped.
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WO 92/08545 PCT/US91 /08375
14
The aspiration pressure windows are empirically derived
values. A number of normal homogeneous fluid samples are
aspirated using the present invention method using different
lots of pressure measurement means. Thus, the normal
fluctuations in the pressure readings (Aspiration Data Points)
due to variations in the samples and pressure measurement
means will establish a normal range of pressure values that
can be expected to be observed when aspirating normal
homogeneous samples. The range at each Aspiration Data
Point extends from the lowest pressure reading value to the
highest pressure reading value obtained when the normal
homogeneous samples were aspirated. To eliminate the effect
of variations in the atmospheric pressure, the ambient
pressure (at the time of the aspiration) is subtracted from the
lowest and highest pressure reading values obtained at each
Aspiration Data Point. For example, the differential values at
each Aspiration Data Point obtained from ten samples of
normal calf serum are listed in Table 1. The aspiration
pressure windows in future aspirations can then be calculated
from these differential values by adding the ambient pressure
measured at the beginning of the level sensing steps (step 1
above). The accuracy of these ranges (i.e. the differential
values) will be dependant upon the number of normal samples
used to establish the aspiration pressure windows. Preferably
2 5 at least ten (10) samples should be used to establish the
TABLE 1
Aspiration Pressure Windows
AspirationHigh PressureLimit Low Pressure
Limit
Data Point
1 AmbientPressure+ 60 Ambient Pressure+ 180
2 AmbientPressure+ 60 Ambient Pressure+ 180
3 AmbientPressure+ 120 Ambient Pressure+ 240
4 AmbientPressure+ 170 Ambient Pressure+ 290
5 AmbientPressure+ 210 Ambient Pressure+ 330
6 AmbientPressure+ 240 Ambient Pressure+ 370
7 AmbientPressure+ 280 Ambient Pressure+ 420
8 AmbientPressure+ 300 Ambient Pressure+ 450
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aspiration pressure windows.
If any pressure reading (Aspiration Data Point) taken
during the aspiration of a sample is outside of the
corresponding aspiration pressure window, the sample is non-
5 homogeneous and the aspiration is halted. For example, on the
sixth pressure reading, if the ambient pressure is 512, the
aspiration pressure window values according to the values in
Table 1 would be from 752 (512 + 240) to 882 (512 + 370). A
sample pressure reading value less than 752 or greater than
10 882 would indicate that the sample is non-homogeneous.
After the total amount of sample desired has been
aspirated, the pipettor is moved upward a short distance,
preferably about 0.055 inches, so that the end of the pipette
tip is within 0.125 inches below the surface of the sample.
15 This permits some of the sample, preferably about 50 p,L, to
be dispensed back into the sample. Because mechanical
systems have a looseness in the mechanical connections, such
as slippage between gears and the like, a backlash is observed
when the mechanism driving the syringe piston is reversed in
2 0 direction from its former movement. For example, when
approximately 50 pL of sample is dispensed back into the
sample, some of the mechanical movement readjusts the
tension between the parts before the syringe piston begins to
move. Thus, less than 50p.L of sample is actually dispensed
2 5 back into the sample.
After dispensing aspirated sample back into the sample,
a short delay, preferably at least 0.5 seconds, permits much of
the sample fluid clinging to the outside of the pipette tip to
run off. The pipettor is then moved a short distance upward,
3 0 preferably at least about 0.5 inches, at a very slow rate,
preferably no more than 2.88 inches per second. This permits
the removal of the pipette tip from the sample without
shearing off or loosing a small amount of sample from inside
the end of the pipette tip. The pipettor can then be returned to
3 5 the home position at normal speed. From the home position
the pipettor is moved to a new location where dispensing of
the aspirated sample occurs. Alternatively, the reaction tray
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zo95 ~ ~z
16
is moved in place of .the sample container and the pipettor is
again moved downward.
A sample must be at least partially liquid to be
aspirated. Thus, a sample can be a liquid biological fluid such
as blood, serum, plasma, urine, cerebrospinal fluid, ascites
fluid, cell growth media, tissue and swab extracts, fluids
resulting from sample processing in DNA cycli2ers, .and the
like. The biological sample can contain particles such as cells
and the like. The particles can also include microparticles or
other small particles used in assay procedures. A sample can
also be a non-biological fluid such as water samples, or any
chemical or solid which can be at least dispersed in a liquid
suspension.
The following examples are illustrative of the invention
I 5 and are in no way to be interpreted as limiting the scope of
the invention, ,as defined in the claims. It will be appreciated
that one skilled in the art can conceive of many other devices
and methods of use to which the present inventive concepts
can be applied.
2 0 EXAMPLE 1
SAMPLE DETECTION BY VACUUM
The surface of a sample (level sensing) was detected
according to the method of the invention described
hereinabove. A vacuum was created within the pipettor shown
2 5 in Figure 6 by aspirating air into the pipettor (with a syringe)
as the pipettor was moved toward the sample surface. The
pressure was monitored during this process with a pressure
transducer and the data was stored in the variable named
"p_array". The sample was goat plasma. The pipettor control
3 0 software was executed under the control of the Soft-Scopes
286 Debugger program (available from Concurrent Sciences,
Moscow, Idaho). This software package has the ability to
monitor execution of the program and examine the values of
variables during processing. After level sensing was
3 5 completed, the first 30 pressure readings were determined by
examining the values stored in the "p array". A slight delay
exists between each pressure reading due to the processing of
CA 02095152 2002-04-15
17
the data by the computer. The length of this delay is dependent upon the
software
and computer hardware. A longer delay may be incorporated into the software if
desired. The reading number and pressure obtained are tabulated in Table 2.
The values in Table 2 are 10-bit binary where full scale vacuum (-1 psi) has
a value of 1023 and full scale pressure (+1 psi) has a value of 0. The change
in
pressure at reading number 14 indicates that the surface has been reached,
i.e. the tip
of the pipettor has touched the sample surface. Immediately, the pipettor's
movement was halted and the aspiration was stopped. The pressure within the
pipettor then returned to equilibrium with ambient pressure.
These data verified the ability of the system to detect the surface of the
sample while drawing a vacuum (i.e. aspirating air). The pressure values were
saved
during level sensing. The vacuum spike occurred when the sample was contacted.
The data are graphed in Figure 1, wherein pressure (+1 psi to -1 psi
represented as
10-bit binary) is plotted against pressure readings.
EXAMPLE 2
ASPIRATION PRESSURE TEST WITH NORMAL CALF SERUM
The aspiration pressure windows were verified using normal calf serum as
follows. Ten samples of normal calf serum were individually aspirated and the
pipettor aspiration pressure data was saved. All ten samples were visually
verified to
contain no foam, bubbles, dots or other non-homogeneity. All ten samples were
level sensed within 0.125 (1/8) inches of the sample surface, and thus fell
within the
allowed window of 0.125 inches from the sample surface. The aspiration data
from
all ten samples is summarized in Table 3. A representative sample is graphed
in
Figure 2, wherein pressure (+1 psi to -1 psi represented as 10-bit binary) is
plotted
against aspiration data points. One calf serum sample, shown as the solid line
between closed squares, was plotted. Also plotted was the low limit windows,
graphed as the solid line between crosses, and the high limit windows, shown
as the
solid line between asterisks.
~~iJ51~2
WO 92/08545 PCT/US91 /08375
18
TABLE 2
Number Value Number Value Number Value
1 575 11 576 21 582
2 579 12 575 22 583
3 579 13 592 23 582
4 578 14 679 24 583
5 575 15 594 25 583
6 575 16 580 26 582
7 575 17 582 27 582
8 574 18 582 28 582
9 575 19 582 29 582
575 20 582 30 582
Reading Pressure Reading Pressure Reading Pressure
T_~BLE 3
SAMPLE Aspiration
Data
Points
No. 0 1 2 3 4 5 6 7 8
*
1 582 718 718 771 821 859 893 925 949
2 584 709 717 768 816 856 893 925 949
3 583 709 708 769 819 857 893 925 951
4 581 710 708 771 817 858 893 925 950
5 583 712 712 773 822 861 898 928 954
6 583 717 717 771 817 856 893 925 950
7 583 709 709 774 821 861 896 926 952
8 583 710 710 773 821 861 895 929 952
9 584 706 710 770 816 857 893 924 949
10 584 712 712 775 821 861 897 928 955
*AmbientPressure
SUBSTITUTE SHEET
2~~~15
JVO 92/08545 PCT/US91/08375
19
EXAMPLE 3
DETECTION OF FOAM DURING ASPIRATION
The effect of foam on the surface of the sample was
tested as follows. Three samples of normal calf serum each
were pipetted into separate containers. All three samples
were visually verified to contain no foam, bubbles, clots or
other non-homogeneity. Foam was created in each sample by
vigorously shaking the test sample. The fluid level was
sensed according to the method of the invention described
hereinabove. All three samples which contained foam resulted
in a sample surface detected at the surface of the foam.
However, these samples were later rejected on the basis of
aspiration pressure data. The aspiration data from all three
samples is summarized in Table 4. The data from one of the
calf serum samples containing foam (shown as the solid line
between closed squares) is graphed in Figure 3, wherein
pressure (+1 psi to -1 psi represented as 10-bit binary) is
plotted against aspiration data points. Also plotted was the
low limit windows, graphed as the solid line between crosses,
and the high limit windows, shown as the solid line between
asterisks.
EXAMPLE 4
DETECTION OF BUBBLES DURING ASPIRATION
The effect of large bubbles on the surface of the sample
2 5 was tested as follows. Three samples of normal calf serum
each were pipetted into separate containers. All three
samples were visually verified to contain no foam, bubbles,
dots or other non-homogeneity. Large bubbles were created in
each test sample by blowing air on the surface of the sample
3 0 using a Pasteur pipet and a dropper bulb. The fluid level of
each sample was then sensed according to the method of the
invention described hereinabove. The aspiration data from all
three samples is summarized in Table 5. It was found that the
first two samples with bubbles were correctly level sensed,
3 5 i.e. the bubbles present in each of these two samples had no
effect on the level sense. Apparently, the pipette tip broke
the bubbles as the pipettor moved toward the sample surface.
I t ~ ~ ';';'' ; i~ c t-; ccT
..: ;J ..~
CA 02095152 2002-I04-15
20
TABLE 4
SAMPLE Aspiration Data Points
No. 0* 1 2 3 4 5 6 7 8
1 582 825 752 **
2 582 823 757 **
3 582 839 758 **
*Ambient Pressure
** The sample was rejected on the basis of the initial aspiration data point
and
aspiration was halted.
TABLE S
SAMPLE Aspiration Data Points
No. 0* 1 2 3 4 5 6 7 8
1 584 728 728 789 838 879 913 944 971
2 583 712 712 774 819 861 896 926 953
3 583 584 582 **
*Ambient Pressure
**The sample was rejected on the basis of the initial aspiration data point
and
aspiration was halted.
TABLE 6
SAMPLE Aspiration Data Points
No. 0* 1 2 3 4 5 6 7 8
1 582 1020 1022 **
2 578 937 699 **
3 577 1020 1020 **
*Ambient Pressure
**The sample was rejected on the basis of the initial aspiration data point
and
aspiration was halted.
CA 02095152 2002-04-15
21
The bubbles of the third sample hindered the sample surface level sense. The
bubbles and not the sample surface were detected when the bubbles were
contacted
by the pipettor. This sample was rejected on the basis of the aspiration
pressure data.
The third sample aspiration data is graphed in Figure 4 (shown as the solid
line
between closed squares), wherein pressure (+1 psi to -1 psi represented as 10-
bit
binary) is plotted against aspiration data points. Also plotted was the low
limit
windows, graphed as the solid line between crosses, and the high limit
windows,
shown as the solid line between asterisks.
EXAMPLE 5
DETECTION OF CLOTS DURING ASPIRATION
A test was performed in order to verify the ability of the method of the
invention to detect, clots in a test sample. Clots were simulated by partially
mixing a
non-dairy creamer with water creating small lumps. Three attempts were made to
aspirate this mixture by the method of the invention described hereinabove.
The
aspiration pressure data is summarized in Table 6.
All three samples were rejected on the basis of the initial aspiration data
point. The first and third samples also were out of limits on the steady state
pressure
following the initial aspiration. Figure 5 is a graph of a representative
sample
(shown as the solid line between closed squares), wherein pressure (+1 psi to -
1 psi
represented as 10-bit binary) is plotted against aspiration data points. Also
plotted
was the low limit windows, graphed as the solid line. between crosses, and the
high
limit windows, shown as the solid line between asterisks.
The embodiments described and the alternative embodiments presented are
intended as examples rather than as limitations. Thus, the description of the
invention is not intended to limit the invention to the particular embodiments
disclosed, but it is intended to encompass all equivalents and subject matter
within
the spirit and scope of the invention as described above and as set forth in
the
following claims.
