U.S. patent number 3,650,698 [Application Number 04/881,971] was granted by the patent office on 1972-03-21 for apparatus for the automatic determination of the coagulation, aggregation and or flocculation, or the like, rates of fluids, and novel reaction intensifying agent for use therewith.
This patent grant is currently assigned to Technicon Corporation. Invention is credited to Stanford L. Adler.
United States Patent |
3,650,698 |
Adler |
March 21, 1972 |
APPARATUS FOR THE AUTOMATIC DETERMINATION OF THE COAGULATION,
AGGREGATION AND OR FLOCCULATION, OR THE LIKE, RATES OF FLUIDS, AND
NOVEL REACTION INTENSIFYING AGENT FOR USE THEREWITH
Abstract
Apparatus for the determination of the coagulation rate or the
like of fluids, wherein dispensing means are operable to
successively dispense blood plasma samples, in turn and at a
precisely metered rate, onto spaced-apart measured quantities or
spots of a dried suspension of a reaction intensifying agent
carried on an advancing film strip. The intensifying agent may
comprise magnetic iron oxide particles. Subsequent to incubation of
the sample-intensifying agent mixture, additional dispensing means
are operable to dispense a precisely metered quantity of
prothrombin reagent into the film strip. The resulting
sample-reagent-intensifying agent mixture is subjected to rotating
magnetic fields, with the result that the moving magnetic particles
promote mixing and, also, are operative to collect one or more of
the fibrin strands, whereby the mixture, which is substantially
turbid, undergoes a sharp change in its optical transmission
properties, which change is detected by light sensitive means for
the measurement of the prothrombin time of the blood plasma sample
of interest.
Inventors: |
Adler; Stanford L. (Monsey,
NY) |
Assignee: |
Technicon Corporation
(Tarrytown, NY)
|
Family
ID: |
26979631 |
Appl.
No.: |
04/881,971 |
Filed: |
December 4, 1969 |
Current U.S.
Class: |
422/73; 73/64.43;
210/695; 210/745; 356/442; 436/808; 210/222; 210/709; 356/39;
436/44; 436/69 |
Current CPC
Class: |
B01F
13/0809 (20130101); G01N 33/4905 (20130101); Y10T
436/110833 (20150115); Y10S 436/808 (20130101); Y10T
436/117497 (20150115) |
Current International
Class: |
B01F
13/08 (20060101); B01F 13/00 (20060101); G01N
33/49 (20060101); B03c 001/02 (); G01n 011/00 ();
G01n 033/16 () |
Field of
Search: |
;23/253,253TP,259,23B,23A,253A ;210/222 ;73/64.1 ;356/39,208 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Hyland, Clotek System Instruction Manual, D-219/35-0055B, 4 pages
(Oct. 1968)..
|
Primary Examiner: Scovronek; Joseph
Claims
What is claimed is:
1. In apparatus for determining the coagulation rates or the like
of fluids through the measurement of the reaction time thereof with
a coagulation reagent or the like, means to mix a said fluid and
said reagent with a reaction intensifying agent which, upon
activation, will intensify said reaction and provide a readily
detectable change in an optical property of said
fluid-reagent-reaction intensifying agent mixture at the completion
of said reaction, said reaction intensifying agent comprising a
plurality of paramagnetic particles, means to activate said
reaction intensifying agent by subjecting said mixture to a moving
magnetic field, so as to effect relative movement between said
particles and said mixture, and means to monitor said optical
property and to detect said optical property change to thus effect
the measurement of said reaction time.
2. Apparatus as in claim 1 wherein, said reaction intensifying
agent particles are of a material which is substantially chemically
inert with regard to said fluid and said reagent, whereby said
reaction intensifying agent will not interfere chemically with said
reaction.
3. Apparatus as in claim 1 wherein, said activating means are
operable to generate a rotating magnetic field, whereby said
reaction intensifying agent particles will be rotated through said
mixture under the influence thereof.
4. Apparatus as in claim 3 wherein, said reaction intensifying
agent is substantially opaque and generally uniformly distributed
throughout said fluid-reagent-reaction intensifying agent mixture
at the commencement of said reaction to render the same
substantially turbid, and said reaction intensifying agent is
operable to collect generally centrally of said
fluid-reagent-reaction intensifying agent mixture under the
influence of said activating means only at the completion of said
reaction with concomitant change in the turbidity of said
fluid-reagent-reaction intensifying agent mixture.
5. Apparatus as in claim 4 wherein, said means to mix a said fluid
and said reagent with said reaction intensifying agent comprise,
means to successively supply predetermined quantities of said
reaction intensifying agent, means to successively mix a
predetermined quantity of a said fluid with each of said reaction
intensifying agent quantities, and means to successively mix a
predetermined minimum quantity of said reagent with each of said
reaction intensifying agent-fluid quantity mixtures to effect said
reaction.
6. Apparatus as in claim 5 wherein, said means to successively
supply said predetermined quantities of said reaction intensifying
agent comprise, strip-like carrier means having said quantities
disposed thereon in substantially equally spaced manner.
7. Apparatus as in claim 6 wherein, said carrier means are
substantially transparent, and said detecting means comprise light
sensitive means which are operable to direct a beam of light
through said mixture and through said carrier means to thereby
detect said change in the turbidity of said fluid-reagent-reaction
intensifying agent mixture through the detection of the attendant
change in the light transmission characteristics thereof.
8. Apparatus as in claim 6 wherein, said magnetic field generation
means are disposed remotely of said carrier means and said
mixtures.
9. Apparatus as in claim 1 wherein, said means to mix a said fluid
and said reagent with said reaction intensifying agent comprise,
means to successively supply predetermined quantities of said
reaction intensifying agent, means to successively mix a
predetermined quantity of a said fluid with each of said reaction
intensifying agent quantities, and means to successively mix a
predetermined minimum quantity of said reagent with each of said
reaction intensifying agent-fluid quantity mixtures to effect said
reaction.
10. Apparatus as in claim 9 wherein, said means to successively
supply said predetermined quantities of said means reaction
intensifying agent comprise, strip-like carrier means having said
quantities disposed thereon in substantially equally spaced
manner.
11. Apparatus as in claim 10 wherein, said carrier means are
substantially transparent.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention.
This invention relates to new and improved apparatus and reaction
intensifying agent for determining the coagulation, agglutinations,
and/or flocculation or the like rates of fluids and, more
specifically, to such apparatus and reaction intensifying agent as
are particularly adapted to the determination of the Prothrombin
Time of blood plasma samples.
2. Description of the Prior Art.
Although a wide variety of methods and apparatus are known in the
prior art for the determination of the Prothrombin Time of blood
plasma samples for essential diagnostic use, it may be understood
that, in general, no prior art method and/or apparatus is known
which can accomplish this essential Prothrombin Time determination
in fully automatic, rapid and consistently highly accurate manner
with minimum utilization of the expensive thromboplastin reagent
required therefor.
More specifically, and considering first the plurality of
substantially manual classical Prothrombin Time determination
methods in the nature of the visually monitored loop or tilt method
as developed by Dr. A. J. Quick, and described in detail, for
example, in the informative publication "Coagulation Procedures"
published by Dade Reagents Inc. of Miami, Florida, in January 1966,
it is believed that the significant disadvantages thereof in the
areas of undue time consumption, inaccuracy as occasioned by
unclear coagulation reaction endpoint indication, technician error,
incompetence, and/or general inattention, and/or unduly high use of
the required, expensive thromboplastin reagent, are so well known
to those skilled in this art as to require no elaboration here.
On the other hand, although a wide variety of automatic or
semi-automatic apparatus have been developed for Prothrombin Time
determination, and are operable on such divergent bases as the
detection of changes in the viscosity of the blood plasma
sample-thromboplastin reagent mixture, or the creation of an
electrically conductive path by the clotted materials, to indicate
the end point or clotting time, these semi-automatic or automatic
prior art apparatus will generally be found to be incapable of
consistently and rapidly providing accurate Prothrombin Time
determination results on a fully automatic basis with minimum use
of the expensive thromboplastin reagent.
Probably the most widespread single source of error with regard to
the operation of all of the Prothrombin Time determining methods
and apparatus of the prior art is the fact that the same do not
provide for sharp and dramatic change in a characteristic or
property of the blood plasma sample-thromboplastin reagent mixture
at the end point of the coagulation reaction which can be readily
discerned and/or automatically detected to clearly indicate the
precise point in time at which said end point occurs, and this
disadvantage may be understood to be due in large measure to the
fact that there are no known means by which said reaction can be
intensified or enhanced to provide this most desirable sharp and
dramatic, readily discernible or automatically detectable
change.
Of further disadvantage with regard to the Prothrombin Time
determining methods and apparatus of the prior art is believed the
fact that the above-discussed disadvantages thereof generally
become even more pronounced when the same are utilized with diluted
blood plasma samples, to thereby render the same generally
unsatisfactory for diagnostic use in establishing controls for
essential anti-coagulant dosage adjustments.
OBJECTS OF THE INVENTION
It is, accordingly, an object of this invention to provide new and
improved apparatus for the rapid, automatic, and consistently
accurate determination of the coagulation, aggregation or
flocculation or the like rates of fluids, and which is particularly
adapted to such determination of the Prothrombin Time of blood
plasma samples.
Another object of this invention is the provision of apparatus as
above which operate to substantially minimize the amount of the
expensive thromboplastin reagent required for a blood plasma sample
Prothrombin Time determination to thus materially reduce the cost
of such determination.
Another object of this invention is the provision of a novel
coagulation reaction intensifying or enhancing agent which provides
for sharp and dramatic change in a readily discernible and/or
automatically detectable characteristic of the blood plasma
sample-thromboplastin reagent mixture at the end point of the
coagulation reaction.
Another object of this invention is the provision of apparatus as
above which are particularly adapted for operation with undiluted
blood plasma samples.
A further object of this invention is the provision of apparatus as
above which are fully and continuously automatic in operation and
provide a readily interpretable, permanent readout of the
determined blood plasma sample Prothrombin Times to thus eliminate
most major sources of technician-occasioned inaccuracy.
A still further object of this invention is the provision of
apparatus as above which require the use of only readily available
components of proven dependability in the fabrication thereof to
thus insure long periods of satisfactory, substantially
maintenance-free apparatus operation.
SUMMARY OF THE INVENTION
As disclosed herein in a preferred embodiment directed toward the
determination of the Prothrombin Time of a plurality of blood
plasma samples, the apparatus of my invention comprise sample
supply means which supply a stream consisting of successive ones of
said blood plasma samples, and blood plasma sample dispensing means
which are operable to successively dispense said blood plasma
samples at a precisely metered rate. A novel coagulation reaction
intensifying or enhancing agent which comprises opaque magnetic
iron oxide particles as the essential ingredient thereof is
supplied in the form of spaced, measured quantities or spots of a
dried suspension of said agent as disposed on suitable carrier
means which take the form of a substantially transparent film
strip.
Automatically operable thromboplastin reagent dispensing means are
provided and function to dispense precisely metered minimum
quantities of said thromboplastin reagent upon demand. Further
included are temperature controlled means to effect blood plasma
sample incubation and bring the same to appropriate coagulation
reaction temperature, rotating magnetic field generation means to
generate a rotating magnetic field through said temperature
controlled means, and light sensitive detection means to
automatically detect the end point of the coagulation reaction and
provide the desired Prothrombin Time determination accordingly.
In operation, said film strip is advanced to a first position on
said temperature controlled means whereat a precisely measured
quantity of a blood plasma sample is added to said reaction
intensifying or enhancing agent to rapidly re-suspend the latter
with the effect of said rotating magnetic field being to promote
initial blood plasma sample-intensifying or enhancing agent mixing
and render the resultant mixture substantially turbid or opaque.
After the expiration of a period of time predetermined to provide
for substantial blood plasma sample incubation, the film strip is
advanced to said reagent dispensing means whereat a precisely
metered quantity of the thromboplastin reagent is added to the
substantially turbid or opaque blood plasma sample-intensifying or
enhancing agent mixture to commence the coagulation reaction with
the effect of said magnetic field on said magnetic iron oxide agent
further promoting thorough blood plasma sample-thromboplastin
reagent mixing. Therefrom, the film strip is advanced to the
operation position of said light sensitive detecting means, which
position generally coincides with the center of said rotating
magnetic field. Thereafter, as the end point of the coagulation
reaction is reached, the substantially turbid or opaque blood
plasma sample-thromboplastin reagent-intensifying agent mixture
will undergo a sharp and dramatic change in optical property
through the collection of the rotating magnetic iron oxide
particles in one or more of the fibrin strands generally centrally
of the mixture to thus render the latter substantially transparent
for immediate detection by said light sensitive detecting means,
and attendant provision of a printed readout of the Prothrombin
Time of the blood plasma sample of interest. Operation of the
apparatus is continuous in the manner described until each of the
blood plasma samples of said successive stream thereof has been
determined.
DESCRIPTION OF THE DRAWINGS
The above and other objects and significant advantages of my
invention are believed made clear by the following detailed
description thereof taken in conjunction with the accompanying
drawings wherein:
FIG. 1 is a flow diagram depicting the new and improved apparatus
of my invention;
FIG. 2 is a top plan view of a portion of the film strip carrier of
the apparatus of FIG. 1 illustrating the disposition of the
measured quantities or spots of the reaction intensifying agent
thereon;
FIG. 3 is a top plan view of a portion of the film strip carrier of
FIG. 2 illustrating the manner in which the reaction intensifying
agent promotes blood plasma sample-thromboplastin mixing; and
FIGS. 4 and 5 illustrate the sharp and dramatic change in the
turbidity of the blood plasma sample-reagent-reaction intensifying
agent mixture attendant the completion of the coagulation
reaction.
DETAILED DESCRIPTION OF THE INVENTION
Referring now to FIG. 1, sample supply means which may, for
example, take the general form of those shown and described in U.S.
Pat. 3,134,263 issued May 26, 1964 to Edward B. M. DeJong, are
indicated generally at 10 and comprise a turntable 12 upon which is
disposed a circular array of blood plasma sample containers 14. A
sample off-take device is indicated at 16 and comprises a sample
off-take probe 18 and probe operating means 20, respectively. A
wash liquid receptacle 22 is disposed as shown adjacent the
turntable 12, while sample supply device drive means are indicated
at 24 and are operative to drive each of the turntable 12 and the
sample off-take probe operating means 20 in the manner described
directly hereinbelow as indicated by the dashed lines extending
therebetween.
In operation, the turntable 12 is intermittently rotated, or
indexed, to present each of the blood plasma sample containers 14
in turn to the sample off-take probe 18, while the latter is in
turn intermittently operated to immerse the inlet end of the
off-take probe 18 in a thusly presented sample container for a
predetermined period of time to aspirate (as described in detail
hereinbelow) a predetermined measured volume of the blood plasma
sample therefrom, to then transfer the said off-take probe inlet
end through the ambient air for immersion in the wash liquid
receptacle 22 for a predetermined period of time to thus aspirate a
predetermined measured volume of ambient air followed by a
predetermined measured volume of said wash liquid therethrough, and
to then again transfer the said off-take probe inlet end through
the ambient air for immersion in the next presented sample
container 14 for a predetermined period of time to thus aspirate
another predetermined measured volume of ambient air therethrough
and commence the aspiration of a predetermined measured volume of
the blood plasma sample from said next presented sample
container.
As a result, it may be understood that a fluid stream consisting of
successive, predetermined measured volumes of said blood plasma
samples as spaced, in each instance, by a segment of air, a segment
of the wash liquid from wash liquid receptacle 22, and a segment of
air, respectively, will be supplied to the sample off-take probe
18.
A compressible tube or peristaltic proportioning pump which, for
example, may take the general form of that shown and described in
U.S. Pat. 3,227,091 issued Jan. 4, 1966 to Jack Isreeli, et al., is
indicated generally in dashed lines at 26 and, as utilized herein,
may be understood to comprise a plurality of resilient,
compressible pump tubes 28, 30, 32 and 34, respectively, each of
which is progressively compressible or occludable by a plurality of
non-illustrated pump rollers, in synchronous manner, to pump fluids
therethrough in the direction from right to left as indicated by
the flow directional arrows in FIG. 1.
The inlet end of compressible pump tube 30 is connected as shown to
the outlet end of the blood plasma sample off-take probe 18 to
provide for the aspiration of the blood plasma sample-wash
liquid-air stream therethrough as discussed hereinabove. The outlet
end of compressible pump tube 30 is connected as shown to one inlet
of a junction conduit 36, and the outlet of the latter is connected
to a blood plasma sample supply conduit 38.
The inlet end of compressible pump tube 28 is left open as shown to
atmosphere, while the outlet end thereof is connected to one inlet
of three-way valve means 40. One outlet conduit 42 of the three-way
valve means 40 extends as indicated to atmosphere, while the other
outlet conduit 44 of said three-way valve means is connected to the
other inlet of the junction conduit 36. Accordingly is believed
made clear that with the three-way valve means 40 in a first
operating position thereof to connect compressible pump tube 28 to
outlet conduit 42, air will simply be pumped through the said valve
for return to atmosphere while, with said three-way valve means in
a second position thereof to connect compressible pump tube 28 to
valve outlet conduit 44, air will be pumped to junction conduit 36
for merger with the blood plasma sample-wash liquid-air stream
being pumped therethrough from compressible pump tube 30.
A reagent container is indicated at 46 and, for use as disclosed
herein in determining the end point of the blood coagulation
reaction, or Prothrombin Time, of the blood plasma samples from
sample supply means 10, may be understood to contain a suitable
supply of thromboplastin reagent as indicated at 48. A reagent
supply conduit 50 is disposed as shown within the reagent supply
container 46 so that the inlet end of the former is in close
proximity to the bottom of the latter for obvious purpose, and the
outlet end of reagent supply conduit 50 is connected as shown to
the inlet end of compressible pump tube 32, whereby is believed
made clear that operation of the peristaltic pump 26 will result in
the pumping of the reagent 48 from said supply container through
said compressible pump tube in the indicated direction.
The outlet end of compressible pump tube 32 is connected as shown
to one inlet of a junction conduit 52, while the outlet of said
junction conduit is connected to a reagent dispensing conduit 54. A
reagent by-pass and return conduit is indicated at 56, and the
inlet end thereof is connected as shown to the other outlet of the
junction conduit 52, while the outlet end of said reagent by-pass
and return conduit extends as shown into the reagent supply
container 46 for the return of the thromboplastin reagent thereto
as described in detail hereinbelow.
Temperature control means which include a heating coil 58 connected
as shown in the reagent supply conduit 54 adjacent the outlet end
of the latter, and a temperature control bath as indicated in
dashed lines at 60, are included to enable the precise control of
the temperature of the thromboplastin reagent immediately prior to
the dispensing thereof from said conduit outlet end as described in
detail hereinbelow.
A suction conduit for reagent return is indicated at 62 and is
connected as shown to the reagent supply conduit 54 immediately
adjacent the outlet end of the latter. The outlet end of the
suction conduit 62 is connected as shown to the inlet end of
compressible pump tube 34, while the outlet end of the latter is
connected to the inlet end of a reagent return conduit 64 which
extends as shown to within the reagent supply container 46 to
provide for the return of the thromboplastin reagent thereto,
against as described in detail hereinbelow.
A supply roll of a strip of a film substrate 66 which is made from
any material having suitable strength, light transmission, and
chemical inertness characteristics in the nature, for example, of
Mylar, is disposed as shown on rotatable support means as indicated
at 68. As best seen in FIG. 2, measured quantities or spots 70 of a
dried suspension of a reaction intensifying or enhancing agent are
disposed at substantially equally spaced intervals on the upper
surface of the film strip 66. For use as disclosed herein the
automatic, sequential determination of the respective Prothrombin
Times of a plurality of blood plasma samples, said reaction
intensifying agent may be understood to comprise substantially
opaque particles of a paramagnetic material such as magnetic iron
oxide particles of the nature used in the manufacture of magnetic
recording tape, as homogeneously suspended before drying in a
suitable carrier solution. Magnetic iron oxide particles of this
nature are of generally acicular configuration and have a major
dimension in the range of 0.40 to 0.60 microns. Alternatively, such
particles may be constituted by cobalt or nickel oxide
particles.
More specifically, and for use as disclosed herein in intensifying
or enhancing the blood plasma sample-thromboplastin reaction, said
reaction intensifying agent may consist of a suspension of
approximately 50 grams percent magnetic iron oxide particles in
approximately 25 percent PVP which contains approximately 5 percent
glycerine, before drying, and said suspension may be applied to the
film strip 66 for the formation of the spots 70 in any appropriate
manner as, for example, through the use of well-known silk
screening techniques. Preferably, the pH of the reagent
intensifying or enhancing agent is adjusted, if necessary, to
insure that the same will not react with the thromboplastin reagent
upon the mixture thereof.
The particular PVP and glycerine constituents of the magnetic iron
oxide particle suspension, and the listed relative percentages of
said iron oxide particles and said PVP and glycerine constituents
in the reaction intensifying or enhancing agent, may be understood
to be particularly effective in insuring the desired, substantially
instantaneous re-suspension of said iron oxide particles in an
aqueous solution upon the mixture thereof with the respective blood
plasma samples as described in detail hereinbelow. It is, however,
believed clear that other and different suspensions of said
magnetic iron oxide particles in other and different relative
percentaged with regard to said PVP and glycerine constituents,
and/or with other and different suspending agents, may prove
equally useful.
Boundary means in the nature of those indicated at 72 in FIG. 2
will preferably be formed on the film strip 66 in any appropriate
manner as, for example, through embossing of the latter, to effect
the substantial containment in the indicated area of the reaction
intensifying or enhancing agent and the respective blood plasma
samples upon the re-suspension of the former in the latter as
described in detail hereinbelow.
Heat sink means having a substantially level, flat upper surface
are indicated at 74, and heating coil means 76 are disposed as
shown adjacent the lower surface of said heat sink means to provide
for the substantial maintenance of the latter at a desired
temperature as should be obvious. A mirror 78 is disposed as shown
in the heat sink means 74 in such manner that the upper surface of
said mirror is substantially flush with the upper surface of said
heat sink means.
Film strip guide and drive means are provided and comprise an idler
roller 80, a film strip advance drive roller 82, and a film strip
pressure roller 84 cooperatively associated with the latter as
shown. Accordingly, intermittent driven rotation of the film strip
advance drive roller 82 in the indicated counterclockwise
direction, as through operation of a drive motor 86, may be readily
understood to function to intermittently advance the film strip 66
through the unwinding of the latter from the film strip supply
roll, the passage thereof beneath idler roller 80, the passage
thereof over the upper surface of heat sink means 74 in close
contact therewith, and the subsequent passage thereof between the
film strip advance drive roller 82 and the film strip pressure
roller 84, respectively.
Photosensitive detection means are indicated generally at 88 and
may be seen to comprise a suitable, generally cylindrical opaque
housing 90 having a focusing lens 92 disposed therein as shown. A
light source is indicated at 94, and light detection means which
may, for example, take the form of a photoelectric cell, are
indicated at 96, and each of said light source and said
photoelectric cell are positioned within the housing 90 as shown in
such manner that the light beam will be focused as indicated by the
focusing lens 92 for impingement upon the mirror 78 and reflection
therefrom for re-focusing by the focusing lens 92 for impingement
upon the active surface of the photoelectric cell 96.
Reaction timer and readout printing means are indicated
schematically at 98 and are operatively associated as shown with
the photoelectric cell 96 to provide for reaction timing and
readout printing on a tape 99 as described in detail
hereinbelow.
A bar magnet of appropriate field strength is indicated at 100 and
is supported as shown for rotation at appropriate rate through
operation of drive motor means 102 to thereby establish a rotating
magnetic field, the lines of force of which will, of course, extend
through and above the heat sink means 74. Preferably, the bar
magnetic 100 is disposed relative to the mirror 78 in such manner
that the respective centers thereof are in substantial vertical
alignment as seen in FIG. 1, whereby it may be understood that the
center of the rotating magnetic field established by rotation of
the bar magnet 100 will be generally coincident with the center of
the mirror 78.
Multi-element snap-action valve means are indicated generally in
dashed lines at 104 and may, for example, take the general form of
those shown and described in the co-pending application for United
States Patent of Carl V. Johnson et al., entitled "New and Improved
Method and Apparatus for Simultaneously Controlling the Flow of
Fluids in a Plurality of Flow Paths", Ser. No. 864,262, filed Sept.
22, 1969 and assigned to the assignee hereof. Each of the reagent
by-pass and return conduit 56, the reagent dispensing conduit 54,
and the suction conduit 62 pass as shown through the valve means
104 for control by the latter of the respective fluid flows
therethrough and, to this effect, it may be understood that at
least the portion of each of said conduits which passes through
said valve means is constituted by a compressible tube or conduit
as indicated respectively at 106, 108 and 110.
The valve means 104 comprise a valve actuator member 111 having
projection bar members 112, 114, and 116 formed thereon with said
bar members being respectively operatively associated with the
compressible conduit portion 106, 108 and 110. In operation for use
as disclosed in detail hereinbelow, it may be understood that the
valve means 104 are arranged so that the valve actuator 111 is
pivotally moveable, in extremely rapid, snap-action manner between
a first position thereof wherein bar member 114 contacts and
substantially compresses or occludes compressible conduit portion
108 against a non-illustrated platen to substantially prevent fluid
flow therethrough, while neither of bar members 112 or 116 contacts
compressible conduit portions 106 and 110 and thus does not
interfere with the respective flow of fluids therethrough, to a
second position of said valve actuator wherein bar members 112 and
116 respectively contact and substantially compress or occlude
compressible conduit portions 106 and 110 against said platen to
substantially prevent fluid flow therethrough, while bar member 114
does not contact compressible tube portion 108 and thus does not
interfere with the flow of fluids therethrough. Accordingly, it is
believed made clear that with said valve actuator 111 in said first
position thereof, fluid flow through reagent dispensing conduit 54
will be substantially prevented, while fluid flow through the
respective reagent by-pass and return conduits 56 and suction
conduit 64 will be unaffected. Conversely, with said valve actuator
111 in said second position thereof, it is believed clear that
fluid flow through the respective reagent by-pass and return
conduit 56 and suction conduit 64 will be substantially prevented,
while fluid flow through reagent dispensing conduit 54 will be
unaffected.
A blood plasma sample dispensing probe is indicated at 120 and the
inlet end thereof is connected as shown to the outlet end of blood
sample supply conduit 38. The blood sample dispensing probe 120 is
supported as shown adjacent the outlet end thereof from the arm 122
of blood sample dispensing probe operating means 124. The blood
sample probe operating means 124 are operated from drive motor
means as indicated at 126A for oscillatory movement to move the
inlet end of the blood sample dispensing probe 120 in an arc
between the depicted first position thereof wherein said outlet end
is in substantial vertical alignment with a measured quantity or
spot 70 of the reagent intensifying or enhancing agent on the upper
surface of film strip 66 when the latter is in a specific position
thereof as described in detail hereinbelow, and a second position
of the probe as depicted in phantom in FIG. 1 wherein said
dispensing probe outlet end is in general vertical alignment with a
blood plasma sample and wash liquid collection receptacle 126A
which leads as indicated to waste.
Programmer means which may take any appropriate form in the nature,
for example, of cam-operated electrical programmer means are
indicated schematically at 128 and are operatively connected as
indicated by the dashed lines to each of the sample supply means
drive motor 24, the three-way valve means 40, the multi-element
valve means 104, the blood plasma sample dispensing probe operating
means drive motor 126, the film strip advance drive roller drive
motor 86, the photoelectric cell 96, and the timer and readout
printing means 98, respectively.
OPERATION
For typical use in the automatic, sequential determination of the
respective Prothrombin Times of a plurality of blood plasma
samples, it may be understood that each of the blood plasma sample
containers 14 would contain an undiluted blood plasma sample from a
different patient, each of which will have been previously treated
in classical manner with a fixative reagent in the nature of sodium
oxalate or sodium citrate immediately upon withdrawal from the
patient, and will preferably be maintained at approximately
4.degree. to 5.degree. C. in the relevant blood plasma sample
container 14 to inhibit clotting factor deterioration through the
use of non-illustrated cooling means which may be included in or
operatively associated with the sample supply device 10.
The three-way valve means 40 would be arranged to operate, under
the control of programmer 128, to be in the second position thereof
to introduce air to the blood plasma sample-wash liquid-air stream
flowing through junction conduit 36 only when the wash liquid
segments are flowing therethrough to thus air segment each of said
wash liquid segments to materially improve the cleansing action
thereof, while insuring the flow of each of the aspirated blood
plasma samples as a continuous stream to the blood plasma sample
dispensing probe 120. The reagent supply container 46 would, of
course, contain a supply of the thromoplastin reagent 48 sufficient
for the Prothrombin Time determination of each of the blood plasma
samples carried from the sample supply means 10.
In addition, the film strip advance drive roller motor 86 would be
arranged to operate the film strip advance drive roller 82, under
the control of programmer 128, to alternatively, intermittently
advance the film strip 66 to two positions thereof relative to the
focal point of lens 92, the outlet end of reagent dispensing
conduit 54, and the outlet end of the blood plasma sample
dispensing probe 120, respectively, and to enable the dwelling of
said film strip in each of said positions for different
predetermined periods of time as described in detail hereinbelow.
More specifically, it may be understood that said film strip
advance drive roller would initially be operated to advance the
film strip to the position thereof depicted in FIG. 1 wherein a
leading measured quantity or spot 70 of the reaction intensifying
or enhancing agent (as now thoroughly mixed and re-suspended with a
blood plasma sample and a precisely metered amount of the
thromboplastin reagent in the manner described in detail
hereinbelow) would be disposed as indicated at position C on the
mirror 78 at the exact focal point of the focusing lens 92, while
the next succeeding or trailing measured quantity or spot 70 of
said reaction intensifying or enhancing agent would be disposed as
indicated at position A directly below the outlet end of blood
plasma sample dispensing probe 120 when the same is in the depicted
blood plasma sample dispensing position thereof as shown in FIG. 1.
Accordingly it is believed made clear that the spacing S (FIG. 2)
between said measured quantities or spots 70 of the reaction
intensifying or enhancing agent on the upper surface of the film
strip 66 is predetermined to substantially coincide with the
distance between said lens focal point on mirror 78 and the point
on heat sink 74 which is in substantial vertical alignment with the
outlet end of the blood plasma sample dispensing probe 120. In said
second position to which the film strip 66 is intermittently
advanced by operation of said film strip advance drive roller 82,
it may be understood that the former would be positioned so that
the measured quantity or spot 70 of the reaction intensifying or
enhancing agent which had been disposed at position A would now be
advanced for disposition in substantial vertical alignment with the
outlet end of reagent dispensing conduit 54 at position B as
indicated in FIG. 1, it being believed clear that with the film
strip 66 thusly disposed, no measured quantities or spots 70 of the
reaction intensifying or enhancing agent will be disposed at either
of positions A or C.
The multi-element valve means 104 would be arranged to operate,
again under the control of programmer 128, to be in said second
position thereof to enable reagent flow from reagent supply conduit
32 through compressible conduit portion 108 to reagent dispensing
conduit 54 only for a period of time predetermined to substantially
coincide with the period of time in which the film strip 66 is
dwelled in a said second position thereof to position a measured
quantity or spot 70 of the reaction intensifying or enhancing agent
in substantial vertical alignment with the outlet end of said
reagent dispensing conduit. At all other times, it may be
understood that the multi-element valve means 104 would be arranged
to be in said first position thereof to permit fluid flow only
through compressible conduit portions 106 and 110. With further
regard to the reagent dispensing conduit 54, it may be understood
that temperature control bath 60 would be arranged to maintain the
temperature of the temperature control coil 58 at approximately
37.degree. C. to thus insure the dispensing of the thromboplastin
reagent at substantially this temperature.
The blood plasma sample dispensing probe operating means 124 would
be arranged to operate, through drive motor 126A under the control
of programmer 128, to position the blood plasma sample dispensing
probe 120 as depicted in the blood plasma sample dispensing
position thereof only for a part of the time when the film strip 66
is dwelled in a said first position thereof with a measured
quantity or spot 70 of the reagent intensifying or enhancing agent
being disposed as depicted in FIG. 1 at position A, and to at all
other times position said blood plasma sample dispensing probe in
the position depicted in phantom in FIG. 1 whereby the remainder of
each of the blood plasma samples that is not dispensed for test,
and the respective inter-sample wash liquid segments, will be
dispensed into receptacle 126 for flow therefrom to waste.
Finally, heating coil means 76 would be arranged to operate to
maintain the temperature of the heat sink means 74 at approximately
37.degree. C.
In operation, and assuming steady state operational conditions to
have been reached and the film strip 66 to have just been advanced
to a first position thereof to dispose succeeding measured
quantities or spots 70 of the reagent intensifying or enhancing
agent at positions A and C as depicted in FIG. 1, it may be
understood that the blood plasma sample dispensing probe 120 will
just have been moved to the depicted position thereof and that a
blood plasma sample will just be commencing to flow therefrom as a
continuous stream to fall upon and mix with the measured quantity
or spot 70 of the reaction intensifying or enhancing agent now
disposed at position A with resultant almost instantaneous
re-suspension of the magnetic iron oxide particles therein. This
almost instantaneous re-suspension, and concomitant commencement of
the thorough mixing of said iron particles in said blood plasma
sample will be significantly enhanced by the effect of the rotating
magnetic field generated by the rotation of the bar magnet 100 upon
said magnetic iron oxide particles as should be obvious.
After the expiration of a time period predetermined to enable the
dispensing of the desired amount of the blood plasma sample, which
time period may, for example, be of approximately 1.5 to 2 seconds
duration, it may be understood that the blood plasma sample
dispensing probe operating means will be actuated to move said
probe to said second position thereof directly over wash-liquid
collection receptacle 126 to enable the flow of the remainder of
the blood plasma sample, and the succeeding air segment, air
segmented wash liquid segments, and air segments, through said
blood plasma sample dispensing probe to insure a thorough cleansing
of the latter and prevent the contamination of the succeeding blood
plasma sample by the residue of the just dispensed blood plasma
sample, to obvious advantage.
Preferably, the film strip advance drive roller 82 will, at this
point, be maintained stationary for approximately 55 seconds to
dwell the film strip 66 in this first position thereof for that
period of time to bring the thusly dispensed blood plasma sample
and the measured quantity or spot 70 of the reaction intensifying
or enhancing agent up to the preferred test temperature of
approximately 37.degree.C. and commence the required incubation of
said blood plasma sample.
At the expiration of this dwell period, it may be understood that
the film strip advanced drive roller 82 will be operated to advance
the film strip 66 to said second position thereof wherein the now
thoroughly mixed blood plasma sample and reagent intensifying or
enhancing agent measured quantity 70 will be disposed at position B
directly beneath the outlet end of reagent dispensing conduit 54.
Concomitantly, the multi-element valve means 104 will be rapidly
shifted to the second position thereof to enable the commencement
of reagent dispensing, in precisely metered amount, through the
reagent dispensing conduit 54 at approximately 37.degree. C. As the
thusly dispense reagent joins the blood plasma sample and the now
re-suspended magnetic iron oxide particles to commence the desired
blood plasma sample-thromboplastin reagent reaction, it may be
understood that the advantageous thorough mixture thereof will
continue to be enhanced by the action of the rotating magnetic
field on said magnetic iron oxide particles. With a dwell period of
approximately 2 seconds for the film strip 66 in this second
position thereof, it may be understood that a reagent dispensing
time ranging from approximately 11/2 to 2 seconds may be utilized.
Too, for purposes of determining the Prothrombin Time of the blood
plasma sample of interest, it may be understood that the time of
shifting of the multi-element valve means to said second position
thereof may be taken as time 0 and communicated from the programmer
128 to the timer and readout printing the means 98 since this time
substantially coincides with the time the thromboplastin reagent
first contacts said blood plasma sample to commence the coagulation
reaction.
At the expiration of the period of time predetermined to be
sufficient to enable the precise dispensing of the desired quantity
of the thromboplastin reagent through reagent dispensing conduit
54, it may be understood that multi-element valve means 104 will be
rapidly shifted to return to said first position thereof wherein
further reagent flow from reagent supply conduit 32 to reagent
dispensing conduit 54 will be prevented by the abrupt closure of
compressible conduit portion 108, and the supply of reagent being
pumped through compressible pump tube 32 by-passed instead through
reagent by-pass and return conduit 56 for return to reagent supply
container 46. This shifting of the multi-element valve means to
said second position thereof will, in addition, be effective to
open compressible conduit portion 110 with attendant creation of
considerable suction in suction line 62 through the operation of
compressible pump tube 34 to thus insure that any thromboplastin
reagent remaining in the outlet end portion of the reagent
dispensing conduit 54 after the completion of reagent dispensing is
sucked therefrom through the suction 62 for return to reagent
supply container 46 on reagent return conduit 64 to thus further
insure the precise dispensing of exactly the desired amount, only,
of said reagent and substantially prevent any waste of the latter
to obviously significant advantage.
At the expiration of this approximately 2 seconds dwell period, the
film strip advance drive roller 82 is operated to again advance the
film strip 66 to a said first position thereof wherein the blood
plasma sample-reagent-reagent intensifying or enhancing agent
mixture under discussion will be advanced to position C which, as
discussed hereinabove, substantially coincides with the focal point
of focusing lens 92 on mirror 78. Although appropriate spacing
between the respective measured quantities or spots 70 of the
reaction intensifying or enhancing agent on the film strip 66
relative to the spacing between said focusing lens focal point and
the outlet end of blood plasma sample dispensing probe 120 when the
latter is in the dispensing position thereof may be utilized to
insure that the mixture of interest comes to rest precisely at
position C, it may be understood that a further input from the
photoelectric cell 96 to the programmer 128 may, in addition, be
utilized to insure this occurs. More specifically, it may be
understood that since this blood plasma sample-reagent-reaction
intensifying agent mixture is at this point still substantially
opaque or turbid due to the wide distribution of the substantially
opaque magnetic iron oxide particles therein, the movement of the
said mixture into position C will function to break the beam of
light from light source 94 to said photoelectric cell, and that
this occurrence may be utilized for the provision of a control
signal to programmer 128 to immediately discontinue operation of
the film strip advance drive roller drive motor 86 as should be
obvious.
As the blood plasma sample-reagent-reaction intensifying agent
mixture of interest assumes position C, it may be understood that
the very thorough mixing thereof through movement of the magnetic
iron oxide particles under the influence of the magnetic field
generated by the rotating bar magnet 100 will now be very
significantly enhanced. More specifically, and referring now to
FIG. 3 wherein a number of the acicular magnetic particles are
depicted to better illustrate the thoroughness of this mixing, it
may be understood that each of said particles will be caused by the
action of said magnetic field to both rotate about its own axis and
to rotate about the center of the mixture, as indicated in each
instance by the rotational direction arrows, to significantly
promote the desired blood plasma sample-thromboplastin reaction as
should be obvious.
As the coagulation reaction proceeds it may be understood that
polymerization of the fibrinogin in the blood sample into fibrin
strands will result, and that these fibrin strands will be in
essence collected by the multitude of rotating magnetic iron oxide
particles which will be continually rotated therethrough. As this
fibrin strand collection continues to occur, strand collection
continues to occur, the said fibrin strands will become interwoven
until such time as the same are in essence collected by the
magnetic iron oxide particles into one or more relatively large
globules or agglomerates thereof to indicate that the reaction end
point or blood plasma sample clotting time has been reached.
More specifically, and referring now to FIGS. 4 and 5, it may be
understood that FIG. 4 depicts the blood plasma
sample-reagent-reaction intensifying agent mixture as the same
initially assumes position C and clearly illustrates that the said
mixture is, at this point, substantially turbid or opaque due to
the substantially even distribution of the multitude of magnetic
iron oxide particles therein. However, as the coagulation reaction
progresses to the end point or clotting time thereof, as discussed
directly hereinabove, it may be understood that the interaction
between the fibrin strands and said magnetic iron oxide particles,
and collection thereof in one or more relatively large globules as
indicated at 73 in FIG. 5 will result in a rapid and dramatic
change in the optical characteristics of the said mixture to those
depicted in FIG. 5, wherein the said fibrin-magnetic iron oxide
particle globules will be collected generally centrally of said
mixture to result in sharp and dramatic change in the light
transmission properties thereof from the substantially turbid or
opaque to the substantially translucent or transparent.
This sharp and dramatic change in the turbidity or opaqueness of
the mixture will, of course, be immediately detected by the
photoelectric cell 96 through the sharp and dramatic re-opening of
the light path thereto for the beam of light from light source 94,
as should be obvious, with resultant instantaneous provision
thereby of an appropriate signal to the timer and readout printing
means 98 to stop said timer (it being recalled that said timer was
started at time 0 by the shifting of multi-element valve means 104
into said second position thereof to commence thromboplastin
reagent dispensing) and provide a printed readout in seconds upon
the tape 99 of the Prothrombin Time of the blood plasma sample of
interest, to be followed by appropriate resetting of the timer and
readout printing means 98 in preparation for the determination of
the Prothrombin Time of the succeeding blood plasma sample.
Concomitantly with the movement of the blood plasma
sample-reagent-reaction intensifying agent mixture under discussion
into position C, it is believed clear that the succeeding measured
quantity or spot 70 of the reaction intensifying or enhancing agent
or film strip 66 will, of course, have been moved into position A
for addition of the succeeding blood plasma sample thereto through
blood plasma sample dispensing probe 120, and commencement of the
approximately 55 second incubation period of said succeeding blood
plasma sample.
Operation of the apparatus of my invention is, of course,
automatically continuous as described until the Prothrombin Time
for each of the blood plasma samples supplied from sample supply
device 10 has been determined. For a typical application of this
nature, 60 of said blood plasma samples may be positioned at one
time on the sample supply means turntable 12, and approximately
only one hour will be required for the determination of the
Prothrombin Times of all of said blood plasma samples.
Of particular advantage with regard to the new and improved,
automatic coagulometer apparatus of my invention is believed the
fact that, as applied to the determination of the respective
Prothrombin Times of a plurality of blood plasma samples, the same
provides for absolute minimization in the amount required of the
expensive thromboplastin reagents. More specifically, it is
believed clear that, through the use of the proportioning pump 26,
the rapidly acting multi-element valve means 104, the reagent
dispensing conduit 54, the reagent by-pass and return conduit 56,
and the suction conduit 62, the precisely metered dispensing of
only that quantity of the thromboplastin reagent which is required
for the test purposes is insured, and that waste of said reagent is
substantially prevented. Thus, for example, the apparatus of my
invention may be understood to make possible the precise and
automatic determination of the Prothrombin Time of an undiluted
blood plasma sample in a ratio of approximately 0.02 ml. of
thromboplastin reagent to each 0.01 ml. of blood plasma sample as
opposed, for example, to blood plasma sample Prothrombin Time
determination made in accordance with the principles of the prior
art which require approximately 0.2 ml. of thromboplastin reagent
per 0.1 ml. of blood plasma sample. Thus may be readily appreciated
that the apparatus of my invention make possible a very substantial
and significant reduction of approximately 90 percent in the amount
of expensive thromboplastin reagent required per blood plasma
sample Prothrombin Time determination.
The significant advantages attendant the fully automatic and
extremely accurate operation of the apparatus of my invention with
regard to the elimination of technician errors and the like are
believed so clear as to not require further elaboration here.
Although disclosed hereinabove in the form of a preferred
embodiment which is directed toward the automatic, successive
determination of the respective Prothrombin Times of a plurality of
blood plasma samples, it is believed clear that the apparatus of my
invention would have significant utilization for different
purposes. More specifically, it is believed clear that through
suitable modification the said apparatus could readily be adapted
to the determination, for example, of the partial thromboplastin
time, or PTT, of the blood plasma samples to enable the use thereof
for the more specific isolation of the factor or factors causing
deficiencies in the clotting time of a patient's blood. In
addition, and again with suitable modification, it is believed
clear that the apparatus of my invention may be utilized in the
determination of the end point of a polymerization type reaction,
the end point of which is evidenced by an abrupt change in
viscosity, in a wide variety of liquids other and different than
blood plasma samples. Thus, for example, the apparatus of the
invention could be utilized to determine the end point of the
flocculation reaction as would occur in pregnancy testing and/or
the end point of the agglutination reaction as would occur in
testing for rheumatoid arthritis.
Too, although disclosed herein by way of example for the
determination of the Prothrombin Times of undiluted blood plasma
samples, it may be understood that the apparatus of the invention
is applicable for such determination in reliable and accurate
manner for blood plasma samples diluted down as far as 5 percent to
thus enable the use of the apparatus to establish anti-coagulant
dosage controls for essential therapeutic use.
While I have shown and described the preferred embodiment of my
invention, it will be understood that the invention may be embodied
otherwise than as herein specifically illustrated or described, as
that certain changes in the form and arrangement of parts and in
the specific manner of practicing the invention may be made without
departing from the underlying idea or principles of this invention
within the scope of the appended claims.
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