System for performing immunochemical and other analyses involving phase separation

A system for automatically processing liquids in open-ended containers. The containers are moved past a plurality of operating stations that carry out the following operations: washing, reagent-adding, incubation, detection, and storage. The operating stations for washing and reagent-addition include vertically and horizontally movable assemblies for operating upon the containers. The containers are carried in groups in individual carrying blocks which are moved throughout the system.

BACKGROUND AND BRIEF DESCRIPTION OF THE INVENTION 
This invention relates to the automatic processing of liquids in open-ended 
containers for testing purposes, and more particularly provides an 
automated system for detecting Hepatitis B. Surface antigen (HB.sub.s AG) 
in human serum or plasma. 
Many immunochemical reactions are performed by using what is known as a 
solid phase or phase separation system. Such analyses place one of the 
components on a solid support so it may readily be removed from the system 
during analysis. An example is the detection of HB.sub.s AG, wherein the 
antibody to HB.sub.s AG is affixed to solid controlled pore glass 
particles (CPG). Such CPG is mixed in a test tube with a patient's serum. 
The serum in a diseased patient contains antigen to hepatitis, and under 
proper conditions the antigen may be made to react with the antibody on 
the CPG. A typical operation includes the removal of other serum proteins 
from those hepatitis associated proteins which adhere biologically to the 
CPG. This separation involves the use of aqueous washes, such as saline or 
phosphate buffers which allow the extraneous proteins to be removed 
without disturbing the chemical bond between the antigen and the 
hepatitis-associated proteins on the CPG. Identification markers or labels 
are also added in this analysis, but the excess quantity of these reagents 
usually must be removed also by washing or other phase separation 
techniques. 
In performing a typical hepatitis assay, a patient's serum is incubated or 
mixed with CPG so that the required chemical reaction takes place. Next, 
buffer is added to and agitated with the patient's sample, and following 
agitation the CPG settles to the bottom of the test tube. Following 
settling, the liquid in the tube is aspirated or otherwise withdrawn, with 
the CPG remaining in the tube. Such buffer addition-agitation-removal may 
be repeated a number of times to remove most of the extraneous proteins. 
To verify the presence of an antigen adhering to the CPG, a label is added 
to the test tube which may be identified as, for example, by a coloring 
agent, an enzyme multiplication agent, or radioactive tag or a fluorescent 
agent incubation of the tagged label with the antibody antigen complex on 
the CPG forms a "sandwich". After a suitable incubation period, the excess 
label is removed by a wash in generally a number of washing steps. The 
material adhering to the CPG in the tube, as a sandwich, yields an 
indication which may be compared to known positive or negative controls to 
identify the patient's serum. 
The operations outlined above are described in the brochure 
RIAUSURE.sup..RTM. II published by Electro-Nucleonics Laboratories, Inc. 
These operations are tedious and require manual dexterity and continued 
operator attention to be properly carried out. The danger from operator 
fatigue and mistake, leading to false identification of a sample, is 
always present. In addition, variations in test parameters can occur from 
one test to another, leading to varying results. 
It is desirable to optimize any immunochemical analysis by choosing proper 
conditions. While generally it is desirable to decrease the size of the 
CPG and the serum-carrying container (because the kinetics of the 
reactions will be faster and more reliable), the reduction of size creates 
problems in the manual handling of the tests, leading to errors. 
The present invention provides for the automating of the HB.sub.s AG test 
procedure. It proceeds from the RIAUSURE.RTM. procedure of 
Electro-Nucleonics Laboratories, Inc. That procedure provides agitation 
and settling according to a programmed time schedule using small ferrite 
magnets placed inside each of the assay tubes. The test tubes are placed 
on an electro-magnetic assembly containing field coils energized by a 
digital counter, producing a varying field causing the magnets to move 
within the test tube and thereby agitate the serum-buffer-glass system 
within each tube. At an appropriate time, the magnetic field is 
deenergized, allowing the magnets to rest at the bottom of the test tubes, 
leaving a clear, supernatant liquid thereabove. A multiple head aspirater 
is employed to withdraw the supernatant liquid from the test tubes; a 
separate multiple head dispenser is used to introduce liquid along the 
wall of each test tube. The above cycle is completed manually and repeated 
until the tubes are properly washed. An appropriate label is added 
manually to each tube. 
In the present invention, a card is utilized to hold a number of test tubes 
(e.g., twenty test tubes). The tubes are open-ended at the top thereof, 
and are carried by the card so that the bottom portions thereof extend 
below the card. The card rests on a carrying block, and the lower portions 
of the containers extend loosely into openings in the block. In this 
fashion, the test tubes are carried loosely by the block, facilitating 
later operations, as described below. A number of such blocks are 
positioned on a support table and are moved thereon in step-wise fashion 
around the table. Each tube contains a sample of a patient's serum along 
with magnetic particles, and underlying the table is a series of coils, 
the magnetic fields from which cause agitation of the magnetic particles 
and an appropriate mixing of the liquids within the test tubes. The test 
tube-containing blocks are caused to move in a rectangular pattern around 
the table in step-wise fashion by moving pins. The positions on the table 
provide for incubation, washing, label addition, detection, storage, and 
instrument cleansing. The washing assembly for adding a washing liquid to 
the test tubes and removing that liquid therefrom utilizes supply and 
aspirating heads positioned above the test tubes and movable vertically 
thereover. To process a number of test tubes simultaneously, the supply 
head includes a number of liquid supply tubes that communicate with a 
liquid supply manifold within the head. The aspirating head includes a 
plurality of aspirating tubes communicating with a vacuum chamber within 
the aspirating head. The aspirating and supply heads are movable 
vertically with respect to each other. Preferably, the supply tubes extend 
at an angle with respect to the vertical so as to wash the sides of the 
test tubes, and terminate within the supply head. The aspirating head is 
movable to move the aspirating tubes to positions in which they extend 
through and below the supply head and into the test tubes for aspirating 
liquid therefrom. To facilitate holding the test tubes, the supply head 
includes a test tube engaging portion in the lower part thereof for 
engaging the open ends of the test tubes. That lower portion of the supply 
head is movable vertically within the supply head and is yieldably biased 
to a lower position, moving upwardly upon engagement of the supply head 
with the test tubes. The supply head includes passages therein in which 
the supply tubes are positioned, and other passages for containing the 
aspirating tubes from the aspirating head thereabove. These passages 
terminate in a frusto-conical opening of increasing diameter to facilitate 
the positioning of the open-ended test tubes therein, facilitated by the 
loose holding of the test tubes in each carrying block. 
Provision is made for washing the aspirating tubes to avoid carryover of 
contaminants. 
Label addition is carried out by a label adding head preferably carrying 
two syringes, one for each of two rows of test tubes carried in a card. 
This head is moved vertically and horizontally to receive label and to 
dispense that label into the test tubes. The syringes are driven so that 
there is a slight drawing motion after each dispensing operation to 
prevent droplet leakage from the syringes. 
Following passage of the test tubes through the system, the tubes are 
inspected, for example, by an appropriate radiometric or colorometric 
test. 
The invention will be more completely understood by reference to the 
following detailed description which is to be read in conjunction with the 
attached drawings.

DETAILED DESCRIPTION 
Referring to FIG. 1, a system for automatically carrying out the various 
steps of a HB.sub.s AG test is shown. A number of test tube carrying 
blocks 20 are positioned upon a support surface 22 for movement in 
step-wise fashion in a rectilinear pattern as guided by rails 24. Pusher 
pins 26a, 26b, 26c and 26d are employed, operated in appropriate sequence 
to move the blocks 20. The movement of the blocks is rectilinear, 
generally counterclockwise as viewed in FIG. 1. After each movement of the 
blocks, they all remain at rest for approximately 7.5 minutes and then are 
moved to the next position. Typically, the sequence of pin movement is the 
withdrawal of pins 26a and 26c (respectively moved to the right and to the 
left in FIG. 1), followed by the extension of pins 26b and 26d 
(respectively downwardly and upwardly as viewed in FIG. 1). In this 
fashion, the block designated 20a in the lower right-hand portion of FIG. 
1 is moved from the forward row to the rear row, while the test tube 
carrying block designated 20b in the upper left-hand portion of FIG. 1 is 
moved from the rear row to the forward row. Following this movement of 
these two blocks at the ends of the respective rows, the pins 26a and 26c 
are extended respectively to move all the blocks 20 in the forward row at 
the lower portion of FIG. 1 to the right and all the blocks in the rear 
row in the upper portion of FIG. 1 to the left. The next movement of pins 
occurs approximately 7.5 minutes later, for example, allowing sufficient 
time for the various operations to be performed during the at rest 
positions of the test tube carrying blocks. 
In FIG. 1, the various operations carried out in the system, to be 
described below, are indicated generally. It will be noted that the test 
tubes carried in the blocks generally undergo incubation during the eight 
positions of movement in the rear row of FIG. 1. It is assumed that each 
of the test tubes is filled with appropriate serum, in the case of the 
HB.sub.s AG test, and that appropriate negative and positive controls are 
included. As shown by the notations in FIG. 1, washing follows incubation, 
followed thereafter by label addition, followed thereafter by further 
incubation, followed thereafter by further washing, with a completion of 
these procedures at the time the block 20 reaches the position of the 
block 20b in FIG. 1. In this position of the block, appropriate detection 
of the test tubes may follow to characterize the serum under tests in each 
test tube. The front row of tube-carrying blocks is designated "storage"; 
no operations on the test tubes are carried out in these positions. 
As noted above, the table includes coils 28 positioned thereunder which 
carry out the appropriate agitation of the contents of the test tubes, 
which include magnetic particles therein. Additionally, in FIG. 1, a wash 
tray 30 is included for washing aspirator needles used in the washing of 
the test tubes prior to label addition, as will be explained below. 
Referring to FIG. 2, a typical test tube carrying block 20 is shown, along 
with a card 32 that carries test tubes 34. The card 32 carries two rows of 
ten test tubes each, but this number is simply representative. The card 
normally snuggly receives the test tubes 34, with their open ends 34a 
extending above the card and their closed, lower ends extending below the 
card and loosely into holes 36 in the block 20. As will be noted from FIG. 
3, there is adequate space between the walls of the test tubes and the 
walls of the openings 36 in the block to permit movement of the card and 
test tubes. This movement facilitates in the registry of the test tubes 
with respect to the washing thereof, as will be explained in more detail 
below. As shown in FIG. 3, the block 20 is arch shaped in its lower 
portion, as at 38. This permits the entire block to pass over a detection 
station, which may include light emitting diodes 40 and photoelectric 
detectors 42, as shown in FIG. 3, which are mounted on the table of FIG. 1 
in the detection station positioned under the block 20 in the position of 
the block 20b as shown in FIG. 1. Thus, a colorometric test may be 
utilized, e.g., for enzyme amplification, in which case, the test tubes 34 
would be of clear material. Other detection systems could be utilized, 
e.g., radiometric involving the counting of radioactivity as is typically 
used in the HB.sub.s AG test procedure. In such case, the test tubes 34 
could be opaque or clear. 
FIG. 4 shows a washing station, such as the washing station prior to the 
label addition station in the system of FIG. 1. That station includes a 
supply head 44 and an aspirating head 46 positioned above the test tubes 
34 in a carrying block 20. The supply head 44 is for supplying a washing 
liquid to the test tubes, while the aspirating head 46 is for the purpose 
of aspirating from the test tubes the washing liquid. Referring to FIGS. 4 
and 5 together, the heads 44 and 46 are carried in tongue and groove rail 
mountings 48 and 50, which permit the removal of these heads for 
cleansing, replacement, etc. The rails 48 and 50 are mounted for vertical 
movement in rails 52 and 54. A motor 56 drives the rail 50 up and down by 
virtue of a threaded drive shaft 58 which passes through the rail 48 and 
threadedly engages the rail 50. In similar fashion, a drive motor 60 
drives the rail 48 upwardly and downwardly through use of a threaded drive 
shaft 62 which passes through the rail 50 and threadedly engages the rail 
48. Appropriate timed vertical movement of the rails 48 and 50 is achieved 
through suitable control of the drive motors 56 and 60. 
As noted above, prior to the washing operation, eight positions of 
incubation of the contents of the test tube take place, involving 
approximately one hour of incubation. During this time of incubation, the 
antigen to hepatitis contained in a diseased patient's serum contained in 
one of the test tubes reacts with the antibody of the CPG particles within 
the test tube. In the washing station, two separate washes with an 
appropriate buffer solution take place. The wash solution is supplied to 
the supply head 44 through an appropriate line 64. During the washing 
cycle, the motor 60 is energized to lower the supply head 44 downwardly. 
Before the washing solution is dispensed from the head 44, the detection 
of test tubes in position beneath the supply head is completed by a light 
generating and detecting unit 66 carried by the supply head 44. To this 
end, the cards 32 carrying the test tubes are typically colored white or 
light reflective, while the blocks 20 on their upper surface are colored 
black or light absorptive. If a card 32 is in place upon the block 20, 
light will be reflected sufficiently and detected by the unit 66, 
indicating the positioning of the test tubes in place to receive the wash 
solution. If a card 32 is not in position, indicating the absence of test 
tubes, then the light absorptive coating on the block 20 would absorb 
sufficient light so that no detection of light by the unit 66 would take 
place, indicating an absence of test tubes and ceasing further operation 
in the washing cycle, specifically preventing the supply of wash fluid 
from the supply head 44. 
The supply head 44 is shown in more detail in FIG. 7. It includes a lower 
test tube-engaging portion 44a which is slidable vertically and which is 
yieldably biased by a spring 70. The lower portion of the supply head 44 
is frusto-conical, as at 44b, to facilitate in the positioning of the test 
tubes 34 properly in position with respect to the supply head. As that 
supply head is lowered, the test tubes bear against the head portion 44a, 
causing it to move upwardly against the biasing action of the spring 70. 
When the supply head 44 has engaged the test tubes 34, each test tube has 
positioned in the upper, open end thereof a liquid supply tube 72. That 
supply tube is angled with respect to the vertical (typically an angle of 
four degrees) so that the wash liquid impinges against the sides of the 
test tubes 34. The washing action is enhanced by such side impingement of 
liquid on the test tubes. The liquid supply tubes 72 loop upwardly and 
downwardly, as at 72a. It has been found that by so looping the liquid 
supply tubes, problems of dripping can be avoided. The supply tubes 72 are 
supplied with liquid from a chamber within the supply head 44 that is 
supplied with liquid from the liquid supply line 64. O-rings 74 may be 
used to provide an appropriate seal between the supply head 44 and the 
tubes 72 where the latter enter into the liquid supply chamber within the 
head 44 (FIG. 6). 
During the washing operation, the aspirating head 46 is moved downwardly by 
appropriate energization of the motor 56 so that aspirating tubes 76 
carried thereby extend downwardly through and below the supply head 
(through passages 78 in the supply head 44) to the positions shown in FIG. 
7 in dashed lines. In these positions of the aspirating tubes 76, the 
tubes exhaust the liquid within the test tubes 34. The movement of the 
aspirating head 46 is such that the lower tips of the aspirating tubes 76 
are just above the settled CPG in the test tubes 34. The aspirating of 
liquid takes place only after sufficient settling time has occurred so 
that the CPG and the magnets in the test tubes 34 have settled, preventing 
the aspiration of the CPG and magnets through the aspirating tubes. The 
aspirating tubes 76 communicate with a vacuum chamber within the 
aspirating head 46, which in turn communicates with an exhaust line 80 
shown in FIG. 4. 
In a typical HB.sub.s AG test, 200 microliters of serum and 3 microliters 
by volume of CPG and 10 microliters by volume of magnets may be included 
in each of the test tubes 34. 1,000 microliters of washing solution may be 
used in each of two separate washes while the test tubes are in the 
washing station. Following the washing and aspiration of washing solution 
from the test tubes, the block 20 is moved to the next station in FIG. 1, 
which is for label addition. Prior to the next washing of a succeeding 
block 20, wash tray 30 shown in FIG. 1 is moved into position beneath the 
supply head 44 and aspirating head 46. The washing tray 30 contains two 
channels 30a and 30b which are supplied with liquid via supply and exhaust 
lines 82. Typically, liquid is pumped into the channels 30a and 30b in 
intermittent fashion so that a wave type movement of liquid within the 
channels occurs. The lower ends of the aspirating tubes 76 extend into 
these channels 30a and 30b and are completely washed thereby. During the 
washing of the aspirating tubes 76, vacuum is applied by the line 80 so as 
to aspirate liquid through the tubes 76 to ensure a complete cleaning 
thereof. This washing of the aspirating tubes removes all contamination, 
so that material from one test cannot contaminate another. Detection of 
the complete washing of the aspirating tubes 76 may be achieved by use of 
a probe 88 positioned in the aspirating head 46 within the vacuum chamber 
thereof opposite the end of each of the aspirating tubes 76. The spraying 
of cleansing fluid onto the probe 88 may complete an electrical circuit so 
that the passage of cleansing fluid is sensed. Failure to sense the flow 
of cleansing fluid can actuate a suitable alarm. The probe 88 may be used 
to sense the flow of washing liquid through the aspirating tubes during 
the washing of the test tubes as well as the washing of the aspirating 
tubes. 
The wash tray 30 is movable horizontally back and forth by a drive motor 
(not shown) which moves the wash tray along a support rail 90. 
FIG. 8 shows the details of the label addition station of FIG. 1. A label 
dispensing head 92 is carried by a mounting member 94 for horizontal 
movement along a rail 96. A motor 98 is mounted on the rail 96, and 
includes a threaded drive shaft 100 which threadedly engages the mounting 
member 94. Suitable energization of the motor 98 causes the mounting 
member 94 to move horizontally back and forth along the rail 96. 
The label dispensing head 92 is also mounted for vertical movement through 
the mounting of the rail 96 on a vertical post 102 for vertical movement 
on that post. A motor 104 mounted on the post 102 drives a threaded drive 
shaft 106 which threadedly engages the rail 96. Suitable energization of 
the motor 104 moves the rail 96 vertically. 
The label dispensing head 92 includes a pair of syringes 108 having piston 
members 110 therein. The piston members 110 are driven by a suitable drive 
motor 112 acting through drive shaft 114 that threadedly engages coupling 
116 that joins together the two pistons. 
In operation, the motor 98 is first energized to move the label dispensing 
head 92 to the dashed line position shown at the right of FIG. 8. In this 
position, that label dispensing head is positioned over liquid label 
supply container 118. Next the drive motor 104 is energized, lowering the 
rail 96 and the dispensing head 92 so that syringe tubes 120 extend into 
the label supply container 118. Next the motor 112 is energized to move 
the pistons 110 to the left in FIG. 9, drawing label solution from the 
container 118 into the syringes 108 and thereby filling the syringes. Next 
the motor 104 is energized to raise the rail 96 and to remove the syringe 
tubes 120 from the supply solution. To avoid dripping, the motor 112 may 
be energized to drive the syringe pistons 110 slightly to the right (the 
dispensing direction) in FIG. 9 and then slightly to the left (the drawing 
direction in FIG. 9) to suck in liquid droplets at the end of the syringe 
tubes 120 to prevent droplet leakage from the ends of the syringe tubes. 
Next the motor 98 is energized to move the label dispensing head 92 in 
position above the first two test tubes 34 on the block 20 (the right most 
test tubes in FIG. 8). The motor 104 is next energized to lower the rail 
96 so that the syringe tubes 120 are positioned appropriately within the 
test tubes 34. The motor 112 is then driven so that the syringe pistons 
110 move to the right a measured distance to dispense a measured amount of 
label solution (typically 100 microliters). Following the dispensing 
operation, the motor 112 is energized to draw the syringe pistons 110 
slightly to the left so as to suck in any droplets at the ends of the 
syringe tubes 120 and to prevent droplet leakage from those tubes. The 
motor 104 is energized to raise the head 92, the motor 98 is then 
energized to move the label dispensing head 92 to the next pair of test 
tubes 34, and the operation just described repeats. 
Following the addition of label solution to all the test tubes, the block 
20 moves through the incubation positions shown in FIG. 1. Following 
incubation, during which the labelled antibody added to each test tube 
undergoes reaction with the contents of the tube, the contents of each 
tube are again washed with a buffer solution as in the washing that took 
place following the first incubation as described above. The washing 
apparatus may be the same as that described above in connection with the 
washing following first incubation. Cleansing of the aspirating tubes 
through use of a wash tray may or may not take place, as desired. 
Typically, in the HB.sub.s AG test, elaborate cleaning of the aspirating 
tubes is not necessary, because contamination is not present within the 
test tubes as it is during the first washing operation described above. 
Following the final washing, which takes place at two washing stations, 
the CPG within the test tubes is ready for examination to determine the 
results of the test. Detection may take place when the test tube carrying 
block 20 is in the position of the blok 20b shown in FIG. 1, as described 
above. With reference to FIG. 10, the block 20 may include a tag 122 
thereon which is magnetized, e.g., as at 124 as shown in FIGS. 10 and 11, 
for suitable detection of the test tube carrying block as it passes 
through the various stations within the system. 
A presently preferred embodiment of the invention has been described above. 
Obviously, modifications could be made. Electrical coils beneath the test 
tube support table have been shown, in conjunction with magnetic particles 
within the test tubes. The coils could be replaced with an oscillating 
magnetic mat, if desired. Cooling coils could also be added, as well as 
thermastatically controlled heating elements. Controls for the detection 
of low level of reagents, washing solutions, and overfilling of various 
reservoirs can be utilized, as desired. Beginning of test and end of test 
detections may be used, as well as optical/electrical devices at the 
various liquid dispensing stations to sense liquid levels, liquid 
presence, and the like. 
Further, while the device has been described in connection with a hepatitis 
test, any immunological test may be performed involving some or all of the 
various operations described above. 
Accordingly, the invention should be taken to be defined by the following 
claims.