U.S. patent application number 09/993168 was filed with the patent office on 2002-06-27 for aspirating and mixing of liquids within a probe tip.
Invention is credited to Brookes, Ronald F., Ding, Zhong, Jacobs, Merrit N..
Application Number | 20020081747 09/993168 |
Document ID | / |
Family ID | 24030186 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081747 |
Kind Code |
A1 |
Jacobs, Merrit N. ; et
al. |
June 27, 2002 |
Aspirating and mixing of liquids within a probe tip
Abstract
Apparatus and a method for mixing a liquid within a disposable
aspirating probe tip so that most of the liquid is forced to move
past a transition zone between two different inside diameters to
cause rotational mixing. The apparatus and method can be used to
provide agglutination of blood, which in turn can be used for blood
typing. The probe tip can comprise a single integral piece, or two
separate portions. The transition zone can comprise a sharp
demarcation between inside diameters, or a smooth one.
Inventors: |
Jacobs, Merrit N.;
(Fairport, NY) ; Ding, Zhong; (Fairport, NY)
; Brookes, Ronald F.; (N. Chili, NY) |
Correspondence
Address: |
AUDLEY A. CIAMPORCERO JR.
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
24030186 |
Appl. No.: |
09/993168 |
Filed: |
November 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09993168 |
Nov 6, 2001 |
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09510298 |
Feb 22, 2000 |
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Current U.S.
Class: |
436/174 ;
422/400; 436/179; 73/864.11; 73/864.22 |
Current CPC
Class: |
H05K 2203/0165 20130101;
H05K 2203/167 20130101; H05K 2201/10598 20130101; B01L 3/0275
20130101; H05K 3/0097 20130101; B01F 31/651 20220101; G01N 35/10
20130101; Y10T 436/25625 20150115; B01F 31/65 20220101; G01N
2035/106 20130101; H05K 3/225 20130101; B01F 25/50 20220101; Y10T
436/25 20150115; B01F 2101/23 20220101; H05K 3/0052 20130101; B01F
33/00 20220101 |
Class at
Publication: |
436/174 ;
436/179; 73/864.11; 73/864.22; 422/100 |
International
Class: |
G01N 001/10; B01L
003/02; G01N 035/10 |
Claims
What is claimed is:
1. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip; c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause mixing of said
liquids; the improvement wherein the capillary number resulting
from the mixing in said step c) does not exceed about 0.01, said
capillary number being defined as the ratio of liquid velocity
times viscosity and surface tension, so that any tails formed
during said mixing step c) are minimized.
2. A method as defined in claim 1, wherein the capillary number of
step c) does not exceed about 0.001, so that any entrained air
bubble is more readily removed from said liquids as they are
mixed.
3. A method as defined in claim 2, wherein said any bubble is
aspirated into said probe tip in-between said plural liquids, with
a volume that is less than that which prevents mixing of the
liquids in the part of said cavity having the larger of said inside
diameters.
4. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip; c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause mixing of said
liquids; the improvement wherein said cavity parts comprise two
separate but matable tip portions, and said method further includes
the step of mounting a mountable tip portion of one of said inside
diameters onto said tip portion of the other inside diameter
in-between aspiration of liquids, such that carry-over
contamination between liquids is prevented.
5. A method as defined in claim 4, and further including the steps
of removing said tip portion after each additional liquid is
aspirated, and attaching a new tip portion before aspirating into
said probe tip an additional liquid.
6. A method as defined in claim 4, wherein said mountable tip
portion has a larger inside diameter tan that of said tip portion
on which it is mounted.
7. A method as defined in claim 6, wherein said tip portion on
which said mountable portion is mounted, further includes two
inside diameters of significantly different values, so that flow of
said liquids past a demarcation zone between said differently
valued inside diameters also provides rotational mixing of the
liquids.
8. A method as defined in claim 7, wherein the larger of said
differently valued inside diameters is at least as large as the
largest inside diameter of said mountable tip portion.
9. A method as defined in claim 8, wherein said larger of said
differently valued diameters is at least equal to three times the
value of the smaller of said differently valued inside
diameters.
10. A method as defined in claim 6, wherein the largest of said
inside diameter of said mountable tip portion is at least equal to
three times the value of the smaller of said differently valued
inside diameters.
11. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip; c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause mixing of said
liquids; the improvement wherein said inside diameters are each a
measure of a cross-sectional flow-through area of said cavity part,
and the cross-sectional flow-through area of said larger inside
diameter is at least three times the cross-sectional flow through
area of said smaller inside diameter.
12. A method as defined in claim 11, wherein said one liquid is
whole blood and wherein said moving step causes only mixing such
that cells that have agglutinated are less likely to break
apart.
13. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip; c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause mid of said liquids;
the improvement wherein said larger inside diameter is obtained by
i) selecting as a first tip portion a tapered tip at least a
portion of which has an inside diameter that is much larger than
the smaller inside diameter of the probe tip, and ii) joining said
tapered tip to said probe tip having the smaller inside diameter
using a joining collar mounted around said tip portion of step
b).
14. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip; c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause m of said liquids;
the improvement wherein the total amount of liquid provided by said
step b) is such that if all liquid is moved into said part with the
larger inside diameter, the larger inside diameter is greater than
the height of the total moved liquid, but less than twice the
height of the total moved liquid, so that mixing as per step c) is
maximized.
15. A method as defined in claim 14, wherein said mixing is
accomplished without any substantial agitation or shaking of the
probe tip.
16. In a method of mixing a plurality of liquids comprising the
steps of: a) providing a probe tip with an internal cavity having a
plurality of different inside diameters; b) providing by aspiration
a plurality of liquids inside a portion of the probe tip, c) moving
at least most of said liquids back and forth at least several times
between a part of said cavity with a smaller inside diameter and a
part with a larger inside diameter, said larger and smaller
diameters being sufficient to provide a sufficient rotation of
liquid as it moves between diameters to cause mixing of said
liquids; the improvement wherein said step c) comprises moving at
least most of the liquids back and forth at least between said
cavity part with said smaller inside diameter and a part of said
cavity of a larger inside diameter located at opposite ends of said
cavity part of said smaller inside diameter, so that mixing
efficiency is enhanced by rotation of the liquid as it moves past
said opposite ends, rather than a single end of said smaller inside
diameter cavity part.
17. A method as defined in claim 16, wherein said liquids are
completely mixed within 7.5 repetitions of said movement back and
forth at a flow rate of about 50 microliters per sec., within about
10 sec.
18. A probe tip for mixing liquids within the tip after aspiration
of the liquids therein to, said tip comprising a wall defining 3
connected cavities of unequal inside diameters one of the
compartments being sandwiched as a middle compartment between the
other two which form end compartments, each two adjacent cavities
being connected by a transition zone wall and said inside diameters
being sufficiently unequal in said adjacent 2 cavities as to cause
rotational mixing of liquids as they move past said transition zone
wall, wherein said transition zone of the one cavity is formed by a
variance of said inside diameter that increases in value as the
middlemost cavity is transited outward into either of said other
two end cavities.
19. A probe as defined in claim 18, wherein one of said end
cavities is defined by a wall portion removably mounted on a wall
defining said middle cavity.
20. A probe as defined in claim 19, wherein the inside diameter of
at least one of said end cavities is at least equal to three times
the value of the smaller of said differently valued inside
diameters.
21. A method of determining the strength of an agglutination
reaction within a hollow container comprising walls capable of
transmitting light at certain predetermined wavelengths, comprising
the steps of: a) providing a mixture of a sample and an
agglutinating reagent within a first cavity of the container, said
cavity having a first inside diameter, b) transferring the mixture
to a second cavity having a second inside diameter substantially
smaller than said first inside diameter, c) scanning the liquid
within said second cavity during said step b) with a beam of light
at said predetermined wavelengths, said 10% portion being that
portion closest to said first cavity, d) after said scan step c),
detecting the amount of light absorbed within or scattered by said
10% portion by said beam, e) transferring said mixture back into
said first cavity, f) repeating steps b)-d) at least once until
some agglutinated material has separated from non-agglutinated
material, and g) calculating the amount of agglutination from the
absorbance or scattering detected in said step d).
22. A method as defined in claim 21, wherein said transfer step
moves the liquid down from the first cavity to said second cavity,
so that gravity assists in said separation of step f).
23. A method as defined in claim 22, wherein said step g) comprises
determining what percentage of the total possible absorbance is
detected at a preselected percent of the volume scanned that is
indicative of agglutinating reactions, as an indication of the %
and therefore the strength, of the agglutination that has
occurred.
24. A method as defined in claim 21, wherein said detecting step d)
uses radiation at about 540 nm, the peak absorption wavelength of
hemoglobin.
25. A method as defined in claim 21, wherein said step d) comprises
detecting the amount of scattered radiation, so that any hemolysis
interference is avoided.
26. A method of agglutinate blood cells in whole blood, comprising
the steps of a) aspirating whole blood into a disposable tip
mounted on a probe, said tip having at least two portions with
significantly different inside diameters, connected to each other
by a transition zone, b) aspirating into the same tip thereafter,
an agglutinating reagent, and c) moving said blood and reagent back
and forth as a total liquid, first entirely into one of said
portions and then entirely into the other of said portions, a
sufficient number of times so as to cause coagulation of the cells
of the whole blood, and then subsequent separation of plasma from
the coagulated cells.
27. A method of separating as defined in claim 26, wherein said
cells are allowed to settle adjacent to an exit orifice of said
tip, and d) thereafter, dispensing said cells out of said tip,
leaving only plasma remaining therein.
28. A method of separating as defined in claim 27, and further
comprising the step of e) dispensing at least a portion of said
remaining plasma from said tip into a reaction well adapted for
carrying out an immunoassay of the plasma.
29. A method of separating as defined in claim 26, wherein said
agglutinating reagent is a polyelectrolyte or an antibody.
Description
FIELD OF THE INVENTION
[0001] The invention relates to apparatus and a method for mixing
two liquids within a tip on an aspirating probe, to ensure a
reaction between the liquids.
BACKGROUND OF THE INVENTION
[0002] It is known from U.S. Pat. Nos. 5,773,305 and 5,114,162 to
mix a fluid sample such as blood and a diluent, inside a probe tip
by first aspirating both liquids into the tip, and then drawing
said liquids further up into the tip into a mixing chamber having
an enlarged inside diameter compared to the rest of the tip. The
mixing can be achieved, for example, by reciprocating the mass of
liquids up and down numerous times.
[0003] In the examples shown in U.S. Pat. No. 5,773,305, the
liquids are retained in the enlarged chamber and simply sloshed
back and forth in that chamber to achieve mixing. FIG. 3 thereof
makes it clear that simply aspirating the liquids into the enlarged
chamber past a step discontinuity created by the enlarged inside
diameter, is ineffective in creating a mixture. That is, a single
movement past the step discontinuity is shown as not mixing the
fluids homogeneously. An air bubble can also be included between
the liquids when first aspirated. Cross-over contamination between
bodies of liquid being aspirated is preferably prevented by
ejecting an inert oil shield around the outside of the tip, FIGS. 7
through 11 thereof.
[0004] Such a construction is generally equivalent to transferring
two liquids from a pipette into a larger diameter container (the
mixing chamber) and attempting mixing by sloshing the liquids
vertically within the container. Although mixing can occur in such
a fashion for relatively large volumes, it is not as effective for
small volumes, e.g., volumes that total 100 to 600 microliters.
That is, in a constant diameter channel, inertial mixing is reduced
if the volumes are small as here. It is this phenomenon that
requires the movement of the liquids back and forth in the mixing
chamber, as much as 20 times, to achieve homogeneous mixing. Such
reiterations of the mix step are time-consuming, and beg for an
improvement.
[0005] Furthermore, it is not the case that cross-contamination is
preventable only by using such an oil shield. That is, in some
cases, the first-aspirated liquid can be removed from the tip
simply be repeated washing with a diluent, or by wiping. In any
event, should washing prove to be unsatisfactory, there has been a
need for a more reliable method of preventing contamination than by
using the oil shield. (The oil shield is not guaranteed to form
completely around the tip just because a plural of dispensing
nozzles are disposed about the circumference of the exterior of the
tip.) Furthermore, some proteins can destroy the shield effect of
the oil.
[0006] In the examples of U.S. Pat. No. 5,174,162, all the liquids
to be mixed are moved completely into the enlarged mixing chamber,
completely out of the chamber, then back into it, and so forth. The
sharp transition at surface 15 causes turbulent mixing, 16, FIG. 2
thereof. This is a more efficient mixing method than that of the
'305 patent. Nevertheless, there are improvements that are needed
in such a mixing system as described in the '162 patent. For
example, no option is described for the geometry of FIG. 2. Nothing
is described regarding any use of air bubbles to separate the
liquids as they are aspirated. As noted however in the '305 patent,
such an air bubble provides an effective prevention against
cross-contamination. Yet, any air bubble must be rapidly eliminated
during mixing.
[0007] Furthermore, the '162 patent is notably deficient in any
teaching to prevent cross-contamination when aspirating liquid 6
immediately after liquid 4, between the two liquids within the bulk
container of liquid 6. Although the oil shield of the '305 patent
might seem to be applicable to the probe of the '162 patent as well
such a shield has disadvantages as noted above. Alternative
protection methods against cross-contamination, besides the
oil-shield method, are thus desirable.
[0008] Yet another disadvantage of the teachings of the '162 patent
is that when the two disparate liquids are moved back and forth
across the boundary 15, unmixed "tails" of one or both liquids can
be left behind as coatings on either the enlarged chamber or the
narrower intake portion. Such residual tails do not get mixed when
the main body of liquids is moved across boundary 15, so that the
tails are undesirable.
[0009] Thus, although substantial development has already occurred
in probes designed to mix two liquids entirely with the probe,
there remains the need for improvements.
SUMMARY OF THE INVENTION
[0010] We have devised a mixing method and a probe tip for doing
the mixing therein, that provide the above-noted needed
improvements.
[0011] More specifically, in accord with one aspect of the
invention, there is provided a method of mixing a plurality of
liquids, comprising the steps of:
[0012] a) providing a probe tip with an internal cavity having a
plurality of different inside diameters;
[0013] b) providing by aspiration a plurality of liquids inside a
portion of the probe tip;
[0014] c) moving at least most of said liquids back and forth at
least several times between a part of said cavity with a smaller
inside diameter and a part with a larger inside diameter, said
larger and smaller diameters being sufficient to provide a
sufficient rotation of liquid as it moves between diameters to
cause mixing of said liquids;
[0015] the improvement wherein the capillary number resulting from
the mixing in step c) does not exceed about 0.01, the capillary
number being defined as the ratio of liquid velocity times
viscosity and surface tension, so that any tails formed during the
mixing step c) are minimized.
[0016] In accord with another aspect of the invention, there is
provided a method of mixing a plurality of liquids comprising the
steps of a) through c) listed above, wherein the improvement
comprises that the cavity parts comprise two separate but matable
tip portions, and the method further includes the step of mounting
a tip portion of one of the inside diameters onto the tip portion
of the other inside diameter in-between aspiration of liquids, such
that carry-over contamination between liquids is prevented.
[0017] In accord with still another aspect of the invention, there
is provided a method of mixing a plurality of liquids comprising
the steps of a) through c) listed above, wherein the improvement
comprises the inside diameters each provide a cross-sectional
flow-through area of the cavity part, and the cross-sectional
flow-through area of the larger inside diameter is at least three
times the cross-sectional flow through area of the smaller inside
diameter, for maximum mixing efficiency.
[0018] In accord with yet another aspect of the invention, there is
provided a method of mixing a plural of liquids comprising the
steps of a) through c) listed above, wherein the improvement
comprises the larger inside diameter being obtained by i) selecting
as a first tip portion a tapered tip at least a portion of which
has an inside diameter that is much larger than the smaller inside
diameter of the probe tip, and ii) joining the tapered tip to the
probe tip having the smaller inside diameter.
[0019] In accord with yet another aspect of the invention, there is
provided a method of mixing a plurality of liquids comprising the
steps of a) through c) listed above, wherein the improvement
comprises providing a total amount of liquid in step b) such that
if all liquid is moved into the part with the larger inside
diameter, the larger inside diameter is greater than the height of
the total liquid, but less than twice the height of the total
liquid, so that mixing as per step c) is maximized.
[0020] In accord with yet another aspect of the invention, there is
provided a method of mixing a plurality of liquids comprising the
steps of a) through c) listed above, wherein the improvement
comprises moving in the step c) at least most of the liquids back
and forth at least between the cavity part with the smaller inside
diameter and a part of the cavity of a larger inside diameter
located at opposite ends of the cavity part of the smaller inside
diameter, so that mixing efficiency is enhanced by rotation of the
liquid as it moves past the opposite ends, rather than a single end
of the smaller inside diameter cavity part.
[0021] In accord with yet another aspect of the invention, there is
provided a method of mixing a plurality of liquids comprising the
steps of a) through c) listed above, wherein the improvement
comprises moving in the step c) at least most of the liquids back
and forth at least between the cavity part with the smaller inside
diameter and a part of the cavity of a larger inside diameter
located at opposite ends of the cavity part of the smaller inside
diameter, so that mixing efficiency is enhanced by rotation of the
liquid as it moves past the opposite ends, rather than a single end
of the smaller inside diameter cavity part.
[0022] In accord with yet another aspect of the invention, there is
provided a probe tip for liquids within the tip after aspiration of
the liquids therein to, the tip comprising
[0023] a wall defining 3 connected cavities of unequal inside
diameters one of the compartments being sandwiched as a middle
compartment between the other two which form end compartments, each
two adjacent cavities being connected by a transition zone wall and
the inside diameters being sufficiently unequal in the adjacent 2
cavities as to cause rotational mixing of liquids as they move past
the transition zone wall,
[0024] wherein the transition zone of the one cavity is formed by a
variance of the inside diameter that increases in value as the
middlemost cavity is transited outward into either of the other two
end cavities.
[0025] In accordance with yet another aspect of the invention,
there is provided a method of determining the strength of an
agglutination reaction within a hollow container comprising walls
capable of transmitting light at certain predetermined wavelengths,
comprising the steps of:
[0026] a) providing a mixture of a sample and an agglutinating
reagent within a first cavity of the container, the cavity having a
first inside diameter,
[0027] b) transferring the mixture to a second cavity having a
second inside diameter substantially smaller than the first inside
diameter,
[0028] c) scanning the liquid within the second cavity during the
step b) with a beam of light at the predetermined wavelengths, the
10% portion being that portion closest to the first cavity;
[0029] d) after the scanning step c), detecting the amount of light
absorbed within or scattered by the 10% portion by the beam,
[0030] e) transferring the mixture back into the first cavity,
[0031] f) repeating steps b)-d) at least once until some
agglutinated material has separated from non-agglutinated material,
and
[0032] g) calculating the amount of agglutination from the
absorbance or scattering detected in step d).
[0033] In accordance with yet another aspect of the invention,
there is provided a method of agglutinating blood cells in whole
blood, comprising the steps of
[0034] a) aspirating whole blood into a disposable tip mounted on a
probe, said tip having at least two portions with significantly
different inside diadems, connected to each other by a transition
zone,
[0035] b) aspirating into the same tip thereafter, an agglutinating
reagent, and
[0036] c) moving said blood and reagent back and forth as a total
liquid, first entirely into one of said portions and then entirely
into the other of said portions, a sufficient number of times so as
to cause coagulation of the cells of the whole blood, and then
subsequent separation of plasma from the coagulated cells.
[0037] As used herein, "probe tip" or "probe tip portion" means any
vessel, disposable or not, into which liquid can be aspirated,
mountable on an aspirating probe, that comprises the features
noted, namely an orifice, an interior chamber spaced from the
orifice, and a passageway connecting the orifice and the chamber.
Thus, the tip or tip portion can be a conventional disposable tip
such as is shown in U.S. Pat. No. 4,347,875 by Columbus, or even a
cup or well with an orifice in the bottom, such as the cup shown in
U.S. Pat. No. 5,441,895 but with an orifice in the bottom. The tip
can be one integral piece or provided in several portions.
[0038] Accordingly, it is an advantageous feature of the invention
that more rapid mixing of two liquids aspirated into the tip, takes
place within the tip than occurs with conventional devices.
[0039] It is a related advantageous feature of the invention that
no additional device is needed beyond the tip that is used anyway
for aspiration, to provide mixing.
[0040] It is another advantageous feature of the invention that, in
some embodiments, carryover contamination between liquids aspirated
is preventable by an inexpensive mechanical device that is less
time consuming than repeated washing.
[0041] A related advantage of the aforesaid mechanical device for
preventing carry-over contamination, is that it renders the tip of
the invention more manufacturable.
[0042] Other advantageous features will become apparent upon
reference to the Detailed Description of the Embodiments, when read
in light of the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a fragmentary elevational view in section of a
probe tip constructed in accordance with the prior art;
[0044] FIGS. 2A-2C are fragmentary elevational views in section,
similar to that of FIG. 1, but illustrating a method of the
invention;
[0045] FIGS. 3-5 are fragmentary elevational views similar to that
of FIG. 2, but illustrating certain preferred embodiments;
[0046] FIG. 6A is a fragmentary elevational view similar to FIGS.
2-5, except it illustrates an alternative embodiment wherein the
second tip portion that is added between aspirations, has a
narrower inside diameter than the first tip portion;
[0047] FIG. 6B is a view similar to that of FIG. 6A, showing the
subsequent steps of mixing,
[0048] FIG. 6C is an elevational view similar to that of FIG. 6A,
but of an alternate embodiment;
[0049] FIGS. 7A-7H are elevational views in section similar to
FIGS. 6A-6C, except showing a further additional embodiment wherein
liquid flowing from the second tip portion to the fast tip portion
is constrained to move into a narrower, rather than wider, diameter
for mixing;
[0050] FIGS. 8 and 9 are elevational views in section similar to
that of FIG. 4, but showing still further embodiments of the
invention; and
[0051] FIGS. 10 and 11 are plots of absorbance versus amount of
liquid scanned by a light beam scanning through the tip, to
illustrate a method of detecting the strength of an agglutinating
reaction.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0052] The invention is hereinafter described in connection with
certain preferred embodiments, wherein mixing of one or two
liquids, one of which is body liquid, is achieved using a
disposable tip with one or two portions of preferred shapes, the
second being preferably separate from and added to the first to
prevent carry-over contamination of a the second liquid after the
first liquid is aspirated, wherein the first liquid is preferably
blood and the second is an agglutinating solution, and mixing is
accomplished at preferred flow and shear rates, preferably to allow
blood typing to occur. In addition, the invention is useful
regardless of how many and what shape portions the tip is divided
into, whether a second portion is separately added or already
present, or is used to prevent contamination or not, what the
liquid compositions are, what order they are added, what the flow
and shear rates within the tip are, and what the end result of the
mixing is; provided that the tip ape induces mixing by forcing the
mixing liquids to move between cavity parts with differing
diameters sufficient to cause rotational mixing of the liquids as
they flow between the cavity parts. That is, it is repeated
movement between the transition in diameters that causes rapid
mixing, rather than sloshing the liquids within a constant inside
diameter. Thus, the reagent interacting with the body liquid can be
reagents for an immunoassay, for example.
[0053] The less successful approach is that of the prior art, shown
here as FIG. 1. This is substantially the teaching of the aforesaid
U.S. Pat. No. 5,773,305. In such an arrangement, an aspiration
probe 12 comprises a narrow cavity or passageway 14 that leads from
an aperture 34 at end 36, to a mixing cavity 18 having a
significantly wider inside diameter than that of cavity 14. A
transition region 28 with relatively sharp demarcations is provided
between the two diameters. A partial vacuum is applied at
passageway 40 to aspirate first a liquid 44, and then a second
liquid 54, into cavity 18, with or without a bubble (not shown)
between them. As shown in the original '305 patent, the first time
both liquids are moved from passageway 14 into cavity 18 fails to
produce complete mixing, since the two liquids still remain
separated The actual mixing is achieved by oscillating the two
liquids, arrows 30, within cavity 18, from an end at transition
zone 28, to the opposite end 32 of cavity 18. Additionally, an oil
shield is taught as useful on the exterior surface 36 of the probe,
to prevent liquid 44 from coating surface 36 and contaminating bulk
liquid 54 when the latter is aspirated. As noted above, such a
techniques requires as much as 20 oscillations, arrows 30, to
achieve mixing.
[0054] In both the '305 patent and the instant invention, movement
of liquids within the tip is achieved while the tip is on a
pipette, by actuation of a piston within a piston cylinder, not
shown, to create a partial pressure or partial vacuum. For example,
the piston can be operated manually.
[0055] In accordance with the invention, FIGS. 2A-2C, the number of
oscillations can be reduced to as few as three, by simply forcing
most of the liquid to flow past the transition zone 128 each time.
This, in turn, is ensured by forcing most of both liquids to flow
from one cavity adjacent the transition zone, into the other cavity
so adjacent, and then back As used herein, "most of the liquids"
being moved means, at least 90% of the liquids.
[0056] More specifically, a probe preferably in the form of a
disposable tip 112, is constructed substantially the same as that
of the prior art, with a narrower cavity 114 leading to a wider
cavity 118 connected to the narrower one by a transition zone 128.
A common axis of symmetry 100 preferably extends through both
cavities.
[0057] In this example, the transition zone is defined by
relatively sharp edges 134 and 136 at the junction with the
respective cavities. "Relatively sharp" means, having a radius of
curvature at the junction that is less than 25 microns. Any radii
greater than that tend to produce a smooth transition between zone
128 and the respective cavities. In fact, a smooth transition
through the use of such greater radii of curvature is preferred,
but not essential, as such a smooth transition gives better results
when blood agglutination for blood typing is the goal of the
mixing. That is, the smooth transition using greater radii of
curvature is less likely to cause the agglutinates to be broken up,
all other things such as bulk flow rates, being equal. An example
of a smooth transition using such greater radii of curvature is
shown in FIG. 4. For example, R.sub.1, and R.sub.2 for FIG. 4 can
be, respectively, 1.2 mm each.
[0058] Since the structure is generally the same as for FIG. 1 of
the prior art, the main distinction, at least with respect to FIGS.
2A-2C, is in the use of probe 112. That is, a first liquid 144 is
aspirated into cavity 114, followed by an air bubble 160.
Thereafter, second liquid 154 is aspirated in so that both are
still in cavity 114, FIG. 2A.
[0059] Next, most and preferably all of both liquids are aspirated
past zone 128 and into cavity 118, FIG. 2B. Transition zone 128
produces sufficient rotation, arrows 170, of the liquids as to
start them to mix. As shown in FIG. 1, however, just this step is
not enough. Next, most and preferably all of the liquids are
ejected from cavity 118 past transition zone 128 and into cavity
114, arrows 172, FIG. 2C. Still further, the process is repeated,
phantom arrows 174, until complete mixing has occurred. Depending
on the liquids involved, only three passages from cavity 114 into
cavity 118 may be necessary for complete mixing, although more can
be used.
[0060] FIG. 3 illustrates certain preferred parameters for optimal
mixing in general. Probe 112 has an aperture 134 and an exterior
surface 136 adjacent to that aperture, similar to that of the prior
art. However, the cross-sectional flow-through area A.sub.2 of
cavity 118, provided by inside diameter D.sub.2, is preferably no
smaller than nine times that of the cross-sectional flow-through
area A.sub.1 provided by inside diameter D.sub.1, of cavity 114.
Furthermore, the diameters D.sub.1 and D.sub.2 are generally
constant so that their respective cavities are cylindrical Thus,
D.sub.2 is preferably at least equal to three times D.sub.1.
[0061] Useful examples of D.sub.1 and D.sub.2 include, e.g., 0.8 mm
and 3.2 mm, respectively, for use with a total height H.sub.2, FIG.
5, of about 3 mm.
[0062] Still further, to aid in the dispersal of air bubble 160,
FIG. 2A, during mixing, the wall surface of at least cavity 118,
and optionally also cavity 114, is selected from materials that are
easily wetted by the liquids in question, that is, produce a low
contact angle at the meniscus. Thus, the materials used for the
surfaces are a function of the liquids to be mixed, as is
well-known. Most preferably, for maximum dispersal of the air
bubble (present to aid in preventing cross-contamination between
liquids during the second aspiration), the capillary number for the
system does not exceed 0.001, where capillary member, as is
conventional, equals liquid velocity of movement, arrow 170,
divided by surface tension of the liquid mixture.
[0063] However, it is not essential that an air bubble be present
to avoid contamination. An oil shield can be used as in the '305
patent, or alternatively, probe 112 can be wiped off before
aspirating the second liquid. In that case, the capillary number
can be larger, but preferably not exceeding 0.01, since above that,
the movement of the liquids between cavities can product "tails" of
liquid remaining in the exit cavity that delay or even ruin the
mixing process.
[0064] If an air bubble is used, a further consideration is that
the size or volume of the bubble must be less than that which will
prevent mixing of the liquids as they flow past the transition
zone. Thus, the air bubble must not be so large that, after
aspiration of the probe contents into cavity 118, FIG. 3, the
bubble (not shown in FIG. 3) continues to totally separate the two
liquids--that is, has a diameter equal to the inside diameter of
cavity 118. Thus, for FIG. 3, the bubble volume must be less than
.pi.(D.sub.2).sup.3/6.
[0065] In the event the mixing is being done for blood typing, a
further factor is important in addition to those noted above. That
is, to prevent the rotational action, arrows 170, FIG. 3, from
significantly breaking apart the desired blood cell agglutination,
the flow velocity in either direction past the transition zone 128
is preferably that which provides a shear rate along the wall which
does not exceed about 20 sec.sup.-1. This, of course, is also a
function of the viscosity of the liquids, of the diameters D.sub.1
, D.sub.2, and of angle alpha.
[0066] Regardless of the end use of mixing, the embodiment of FIG.
3 can also be used by coating either cavity 114 or 118 in dry form,
with the reagent that is to react with the body liquid, so that
only one liquid namely the body liquid, need be aspirated in at
aperture 134. Thus, the agglutinating reagent solution can be
provided during manufacturing by coating either or both cavities
114 or 118. This coating is then redissolved when the whole blood
is aspirated into the appropriate cavity.
[0067] For other uses, other reagents, such as an antibody for an
immuno-assay, may be permanently attached to the cavity walls.
[0068] It is not essential that the probe be al in one piece, or
that contamination be prevented by only an oil shield or by wiping.
Instead, FIG. 4, it is useful to have the probe comprise two
portions, 112A and 112B one of which (112B) has an inside diameter
that is different from, e.g., smaller than, at least part of the
inside diameter of the other portion (112A), and which fits over
the other portion adjacent aperture 134. The purpose is to allow
the portion that over-fits the fast portion, to cover up the
exterior surface 136 adjacent to aperture 134 where residual first
liquid 44 might remain. As shown, inside diameter D.sub.3 of cavity
165 of portion 112B is substantial identical to diameter D.sub.1,
but less than diameter D.sub.2, of portion 112A. In use, liquid 44
is aspirated into portion 112A with portion 112B absent. Portion
112B is then mounted onto portion 112A with a sliding fiction fit.
At this point, liquid 44 is moved down, arrow 180, into portion
112B to the phantom position 182, leaving an amount of air at 160
to form an air bubble in the next step. That step is to move the
probe of combined tip portions so as to insert only portion 112B
into a bulk quantity of liquid 54 (not shown). Aspiration then
causes liquid 44 at position 182, bubble 160, and an amount of the
second liquid to be aspirated in the probe. When the desired amount
of the second liquid is present, the probe is removed from the bulk
liquid 54, and mixing proceeds as described above using repeated
movement of most of the liquids past transition zone 128.
[0069] This construction ensures both that residual first liquid
amounts on portion 112A are prevented from contacting said bulk
liquid 54, and that the results length of portions 112A and 112B
are easily moldable.
[0070] In this embodiment, and any embodiment using a smooth
transition between zone 128 and the two cavities provided by the
radii of curvature R.sub.1 and R.sub.2, angle alpha described above
is measured against the tangent line A-A drawn to a point on the
wail of zone 128 that is between the definition of the wall
provided by the two radii.
[0071] In FIG. 5, another preferred aspect of the probe 112 is
illustrated. That is, cavity 118 of portion 112 or 112A has a
diameter D.sub.2 that is selected to be larger in value than the
height H.sub.2 of the total liquids aspirated thereinto, one those
liquids have been moved into cavity 118. The advantage of this
relationship is that it has been found to enhance the mixing
efficiency. At the same time, however, D.sub.2 should be less than
twice H.sub.2, as otherwise the volume in cavity 118 becomes so
thin that it is in danger of bursting at the middle when pressure
is applied to push the liquid, arrow 200, into cavity 114. Such
bursting will of course prevent transfer of the liquid across the
mixing transition zone.
[0072] Thus, if all of the preferred features are utilized as
described above, it has been found that a whole blood sample and an
agglutinating solution can be thoroughly admixed after only three
cycles of drawing most of the liquids into cavity 118 and returning
most of the liquids to cavity 114.
[0073] As noted above, when the probe comprises two portions, it is
not essential that the inside diameter of the added-on portion
equal the inside diameter of the probe portion that is covered The
remaining embodiments illustrate wherein, in fact, this is not the
case. Parts similar to those previously described bear the same
reference numeral to which the distinguishing mark ` or " is
appended.
[0074] Thus, in the embodiment of FIG. 6A, the second portion 112B'
has an inside diameter D.sub.3 for cavity 165' that is considerably
smaller than inside diameter D.sub.2 of cavity 118'. In effect,
probe 112' is now divided into two separable portions 112A' and
112B' having cavities 118' and 165' which between them provide the
transition zone 128' that causes mixing. That is, zone 128' is
formed by an external angle alpha (shown in FIG. 5) which is 270
.degree..
[0075] In use, liquid 44' is aspirated into portion 112A' by
itself. Portion 112B' is then affixed to portion 112A' as shown,
FIG. 6A, and liquid 44' is pushed down into portion 112B', arrow
200. The probe is then moved so that portion 112B' is inserted into
a bulk quantity of liquid 54', preferably with an air bubble 160'
at aperture 134', FIG. 6B. Aspiration, arrow 202, causes all of
liquid 44', bubble 160', and liquid 54' to move through cavity
165', past transition zone 128', and into cavity 118', thus
starting mixing by rotation, arrows 170'. The oscillating movement
of all the liquid via arrows 200 and 202 is then repeated as many
times as is needed to complete the mixing.
[0076] Alternatively, FIG. 6C, the transition zone provided by the
add-on portion 112B', when placed around exterior surface 136A'
adjacent aperture 134A', can be a smooth transition zone 128' in
the manner of the embodiment of FIG. 4. In such a case, care needs
to be taken to ensure that a proper match of the inside diameters
of portions 112A' and 112B' occurs at aperture 134A', so that
indeed the transition in inside diameters is a smooth one. At the
same time, however, the smaller inside diameter remains with probe
portion 112B', rather than portion 112A', except where they match
substantially exactly at aperture 134A'.
[0077] The opposite of FIGS. 6A and 6B is illustrated in FIGS.
7A-7H. That is, the inside diameter of the added-on, second tip
portion is substantially larger, at the transition zone, than the
inside diameter of the first tip portion already used to aspirate
liquid. Additionally, this embodiment illustrates that the two
cavities adjacent the transition zone need not be cylindrical, but
can be tapered instead along their axis of symmetry 100, FIG.
7B.
[0078] Thus, FIG. 7A, probe portion 112A" comprises a conical
cavity 118" extending from an aperture 134A", to an upper portion
132A" that connects to a pump, not shown, the inside diameter of
cavity 118" increasing with increasing distance from the aperture.
To allow the two portions 112A" and 112B" to join together, the
exterior surface 136A" adjacent to aperture 134A" is enlarged, also
with a tapered shape, such as by securing a cork collar to the rest
of the portion 112A". The inside diameter at aperture 134A" is
relatively small, e.g., about 1 mm.
[0079] The second probe portion 112B", FIG. 7B, has an upper
portion 132B" shaped to frictionally mate with surface 136A", that
is, with an enlarged inside diameter. Portion 112B" tapers down to
a lower portion at aperture 134B" producing a cavity 165" having an
inside diameter that is greatly reduced from said enlarged inside
diameter, and in fact, preferably is about the same as that of
aperture 134A".
[0080] The use of this embodiment is similar to that described for
FIGS. 6A-6B. Thus, portion 112A" by itself is inserted into a bulk
quantity of liquid 44" and an aliquot is aspirated, FIG. 7A. Next,
probe portion 112B" is fitted over the surface 136A" of the collar,
FIG. 7B. After that, liquid 44" is pushed or ejected from portion
112A" into the cavity of portion 112B", FIG. 7C.
[0081] Next, FIG. 7D, the combined probe has aperture 134B" of
portion 112B" inserted into a bulk quantity of liquid 54", and that
and an air bubble 160", FIG. 7E, is aspirated into cavity 165".
[0082] The stage is now set for the actual mixing steps. That is,
FIGS. 7F-7H, all of the liquid is aspirated and ejected back and
forth past the transition zone created by the narrower inside
diameter at aperture 134A". FIG. 7F, it is first drawn into cavity
118", arrow 202", to produce the condition shown in FIG. 7G. It is
then ejected back into cavity 165", FIG. 7H, arrow 200", so that
rotational mixing occurs. This process is repeated as necessary,
until the two liquids become homogeneous, or as homogenous as is
possible, given the nature of the liquids.
[0083] In all of the embodiments above wherein a second probe
portion 112B is fitted onto the first portion 112A prior to
aspirating a second liquid, another alternative, following such
second aspiration and aspiration of all liquids into the first
portion, is to remove the second portion and to fit onto the first
portion in the place of the second, a clean third portion of equal,
smaller, or larger inside diameter, for the purpose of aspirating
into the probe yet another, third liquid in a manner similar to the
aspiration of the second liquid.
[0084] Additional mixing transitional zones between unequal inside
diameters can be provided--that is, it is not essential that there
be only two adjacent compartments of varying inside diameters.
Indeed, a probe tip that comprises three such compartments serially
connected, FIGS. 8-9, has proven to be most efficient in mixing, of
all the embodiments described herein. Most preferably, in such an
arrangement the middlemost compartment has the smallest inside
diameter at the transition zone. Parts similar to those previously
describe bear the same reference numeral, to which the
distinguishing superscript suffix'" has been appended.
[0085] Thus, FIG. 8, like the design of FIG. 4, probe 112'"
comprises an upper portion or cavity 118'" that is mounted onto the
permanent probe (FIG. 4), and a lower cavity 114'" integrally
connected to cavity 118'" by a transition zone wall 128 , the
inside diameter D.sub.2 of cavity 118 being larger than D.sub.1,
and preferably at least equal to three times D.sub.1. An additional
cavity 165 is provided at exterior portion 136'" of cavity 114,
with aspiration occurring at arrow 210, also as described for FIG.
4. However, cavity 165'" is integrally connected to cavity 114'" in
that all 3 cavities are formed from a common wall, preferably one
that is molded. Further, inside diameter D.sub.3 of cavity 165'" is
significantly larger than inside diameter D.sub.1, creating a
transition zone 220 not present in the embodiment of FIG. 4. The
value of D.sub.3, like that of D.sub.2, is selected to cause
rotational mixing when most, and preferably all, of the liquids
aspirated into tip 112'", is moved from cavity 114'" into cavity
165'" past transition zone 220. Hence, like D.sub.2, D.sub.3' is
most preferably at least equal to three times D.sub.1. D.sub.3 can
be the same as or different from D.sub.1.
[0086] Although it is not essential, the inside diameter of cavity
165'" can be narrowed to D.sub.3 at the end into which liquid is
first aspirated, arrow 210.
[0087] Additionally, as shown in FIG. 9, cavity 165'" of tip 112'"
can be formed by the wall of tip portion 112B'", removable as in
the embodiment of FIG. 4, so that portion 112B'" can be added after
the first liquid is aspirated, and portion 112B'" covers any first
liquid remaining on exterior surface 136'". Aspiration of a second
liquid then occurs as shown by arrow 210'", FIG. 9. However, unlike
the FIG. 4 embodiment, inside diameter D.sub.3' at the junction of
the cavities 114'" and 165'", is greater than the inside diameter
D.sub.1, rather than equal thereto as in FIG. 4, creating a
transition zone 220'" similar to zone 220, FIG. 8, effective to
cause liquids to rotationally mix as they move from cavity 114'"
into cavity 165'". In this example, D.sub.3' at the transition zone
equals D.sub.1+twice the value of T, where "T" is the thickness of
the wall providing exterior surface 136'". In such an example,
D.sub.3' may or may not be at least equal to three times D.sub.1,
depending on the value of T.
[0088] As in the case of the embodiment of FIG. 8, the inside
diameter of cavity 165'" can be narrowed to D.sub.3" a the end into
which liquid is first aspirated.
[0089] It is the embodiments of FIGS. 8 and 9 that have proven to
be most efficient in mixing, that is, in producing complete mixing
in the fewest cycles of repeated back and forth movement past the
transition zones. For example, the embodiment of FIG. 9 produced
complete mixing of two liquids totaling 20 microliters in only 7.5
cycles of such back and forth movement, at a flow rate of 50
microliters per sec., in about 10 sec.
Agglutination Reactions
[0090] As noted above, a preferred use of this mixing action is to
produce sufficient blood cell agglutination as to allow blood
typing. To that end, one of the liquids is, of course, whole blood
and the other is a solution of agglutinating reagent, aspirated
into the tip, in either order. Any such solution can be used. A
highly preferred example comprises a 3% bovine serum albumin in a
0.1 molar phosphate buffered saline solution containing anti-B IgM
clones formulated from tissue culture supernatant (1, 20, and 31
.mu.g/ml concentrations) plus 0.004% FD&C blue dye number 1.
All concentrations are % by weights.
[0091] It is not necessary that the detection of a strong, weak, or
negative reaction of such blood typing be done outside of the
mixing tip. Instead, it can be achieved by detecting the amount of
agglutination separation within the tip, and thus the strength of
the blood typing reaction.
[0092] Turning to FIG. 8, this detection is preferably done by
scanning for absorbance or light scattering at a position in
narrower tip portion 114". (Any other embodiment of the invention
can also be used.) That is, at the position of arrow 300, an
appropriate optics such as a conventional fiber optics is used to
deliver light of a predetermined wavelength that is then
transmitted into the tip. The amount of light that is absorbed,
measured approximately 10 minutes after mil has been completed, is
then detected as shown schematically by arrow 302, or the amount of
light scattered is detected as shown by arrow 304. The results
differ depending on how much agglutination has occurred, as shown
below. If absorbance is used, suitable wavelengths include 540 nm,
and/or 830 nm. The former is particularly useful since that is the
peak absorption of hemoglobin. Detection of the amount of light
scattered, as at 304, is particularly useful to avoid interference
from any hemolysis.
[0093] FIGS. 10 and 11 illustrate the method using absorbance and
an illuminating wavelength of 540 nm. In the case of FIG. 10, the
liquid is passed down from portion 118'" to portion 114'", after 10
minutes have passed after it has been mixed sufficiently. At the
zero to about 18% of the volume that passes, the amount of
absorbance rises from zero due to the passage of air. After that,
only liquid is passed by the scanner, and the first part of that
liquid is very absorbent, regardless of whether the reaction is
negative, weak, or strong. However, after about 50% of the liquid
has been scanned, the results deviate depending on the amount of
agglutination achieved. A strong reaction clumps the red cells so
well that after about 65%, the volume is essentially free of cells
and is clear. A weak reaction has less absorption, but still much
more than the strong, after 65% of the volume scanned.
[0094] Alternatively, the liquid can be moved upward from portion
165'" into narrower portion 114'" and on upward into portion 118'",
to do the scanning. The results are shown in FIG. 11.
Differentiation of the results occurs when from zero to 18% of the
volume has been scanned. That is, the first portion to flow past
the scanner is the liquid portion free of red cells, in the event
of a strong reaction, because almost all of the cells have
coagulated together. But in the case of the weak reaction, some red
cells are still unagglutinated and remain in that first portion of
the volume, as shown by the middle curve of FIG. 11.
[0095] It is not only blood typing agglutination that is useful as
an agglutination reaction in the tip of the invention.
Agglutination caused by a coagulating reagent allows separation of
the cellular fraction of whole blood from the plasma, to occur in
the tip. That is, when the agglutinating reagent is selected from
conventional coagulating reagents such as a polyelectrolyte, eg,
polylysine, or an antibody such as anti-glycophorin, the mixing
within the tip as described above will not only cause coagulation
of all the red cells, but it will also lead to a physical
separation of those coagulated cells from the plasma. The cells
settle to the bottom of the tip, e.g., tip portion or cavity 165'"
of FIG. 9. At this juncture, those cells can then be expelled by
dispensing them out of the orifice of the tip, leaving only plasma
remaining behind. That plasma can then be dispensed onto a suitable
platform for testing, for example, into a well or cup adapted for
immunoassay, such as is described in U.S. Pat. No. 5,441,895.
[0096] The following are non-limiting working examples of the
mixing steps of this invention:
Example No. 1
[0097] A probe was constructed having two capillaries with
different inner diameters. The smaller capillary had an inner
diameter of 0.557 mm. The larger capillary had an inner diameter of
2.29 mm. The length of the smaller capillary was 41 mm, which holds
up to 10 micro-liter of fluid The larger capillary had a length of
30
[0098] A type B blood of 4 micro-liters was aspirated from the
bottom end of the small capillary by the pump. The pump then
continued to withdraw 1 micro-liters of air in the small capillary.
4 micro-liters of the agglutinating reagent described above was
aspirated thereafter and the air bubble separated the two liquids
in the smaller capillary.
[0099] The pump was then driven to move all the fluids across the
transition zone between the small and large capillary with a flow
rate of 0.5 micro-liter/second. Once in the larger capillary, a
spherical air bubble was created by the surface tension, and the
two liquids started to encounter and mix. As the pump drove the
fluids to flow down into the smaller capillary with a flow rate of
0.5 micro-litter/second, the bubble was eliminated.
[0100] The mixture of the two fluids was oscillated between the two
capillaries with a constant flow rate of 0.5 micro-liters/second.
The agglutinated structure formation was visible at the end of the
fist cycle of this motion. Phase separation was very significant at
the end of the second cycle in the small capillary, with clear
supernatant in the up portion and the agglutinated cell structure
in the bottom portion. Some very small agglutinated cells were
still visible in the supernatant at this stage. The phase
separation was completed by the end of the third cycle, with almost
zero cell structure left in the supernatant.
[0101] The total time period for the three cycles was 2 minutes.
Weaker reactions can be expected to take longer.
[0102] Once complete mixing has been achieved, it is then
necessary, of course, to achieve a determination of the blood type
from the agglutinated results. Although that is not part of this
invention, one method of doing this is to make a visual observation
of light transmittance through the mixture to determine the amount
of agglutination within a fixed time of the agglutination reaction.
A chart is used for comparison, and the user estimates the blood
type from the amount of clumping or agglutination observed in
whichever probe portion that the combined liquids are in at the
time.
Example2
[0103] A probe was constructed similar to shown in FIG. 9, except
that the tip portion 165'" was cut off at line C-C to create a
cone-shaped tip portion having an inside diameter at D.sub.3 of
about 2.54 mm, and at cut line C-C of about 1 mm, with a cone angle
of about 20 degrees and a length of 10 mm. Tip portion 114'" had a
length of about 15 mm and an inside diameter D.sub.1 of about 1 mm.
Tip portion 118 '" had a diameter D.sub.2 of about 4.7 mm. Blood in
an amount of 10 microliters was aspirated into the entire tip
ensemble through cone portion 165'", after which the cone was wiped
clean. Then 10 microliters of reagent were aspirated into the tip
in the same manner, producing a total liquid volume of 20
microliters. This total volume was then moved back and forth so as
to proceed entirely into portion 118'" and then entirely into
portion 165'", and so forth, until mixing was complete. This
required 7.5 repetitions (cycles) at a flow rate of 50 microliters
per sec. Total displacement of fluids was 40 microliters in each
direction of motion, and the time required for complete mixing was
about 15 sec.
[0104] The invention disclosed herein may be practiced in the
absence of any element which is not specifically disclosed
herein.
[0105] The invention has been described in detail with particular
reference to preferred embodiments thereof but it will be
understood that variations and modifications can be effected within
the spirit and scope of the invention.
* * * * *