Apparatus for the in situ hybridization of slide-mounted cell samples

Apparatus for the in situ hybridization of slide-mounted cell samples contains modules for the three processes required: (a) reagent metering and spreading on a slide (1) which is effected by drawing a quantity of reagent (56-59) into a reservoir (31) and then spraying it on the slide (1) using air pressure. The rate of flow through the spray is ascertained by measurement and the dosage determined by timing the spraying; (b) slide washing; and (c) slide incubation. The slide washing and incubation are performed in the same dual-purpose housing (16). A high humidity level is maintained in the housing during incubation so that the samples do not need to be covered to prevent them drying out. A plurality of slides (1) are mounted in a carrier (11) to be washed and incubated at the same time.

This invention relates to in situ hybridization and in particular to 
apparatus for effecting hybridization of cell samples carried on slides. 
A powerful technique which has been developed for detecting particular 
sequences of nucleic amino-acids in a segment of DNA is known as in situ 
hybridization. This technique uses a chemically or radioactively labelled 
"probe" to hybridize the DNA of tissue samples in situ, the samples which 
become labelled can then be detected by the physical or chemical 
properties of their chemical label or their radioactivity. Because of the 
risk to the health of the operator through the use of radioactive 
materials, chemically labelled probes may be preferred. 
The process of in situ hybridization requires the treatment of samples on a 
microscope slide, for example, with measured small quantities, say 50 
.mu.liters, of reagents such as enzymes. The reagents are applied by 
pipette or other hand-held dispenser to the slide and then spread evenly 
over the surface. These pre-hybridization reagents are then washed off in, 
say, phosphate buffered solution (PBS) and a hybridization "cocktail" of 
further reagents applied in the same way as the first reagents. The 
samples are then covered by a glass cover-slip or a film of inert plastics 
material to prevent them drying out during incubation which may extend for 
a period between say 30 minutes and 16 hours or even more. After that the 
cover-slip or film may be removed, the slide washed in, say, phosphate 
buffered solution (PBS), further reagents applied and incubation carried 
out for a second period. There are many different "recipes" for in situ 
hybridization, some involving several treatments with reagents and several 
incubations. It is evident that the process must be performed carefully by 
skilled personnel and that it is very time-consuming. It is therefore 
desirable to reduce the labour content of the process without compromising 
the care and accuracy of the treatment of the samples. 
It is an object of the present invention to provide a machine which is 
capable of performing in situ hybridization on slide-mounted tissue 
samples. 
According to the present invention there is provided apparatus for the in 
situ hybridization of slide-mounted cells samples in which slides bearing 
samples are brought successively to a station to be sprayed with one or 
more fluid reagents by a spray means and thereafter are subjected to 
incubation to further the process, 
the spray means including a reservoir into which a fluid reagent can be fed 
and a spray head to which the fluid reagent can be passed from the 
reservoir for spraying on a sample on a slide, 
wherein the apparatus includes at least one container for containing a 
fluid reagent, pipe means leading from the or each container to the 
reservoir, valve means selectively effective to block and to permit flow 
along the pipe means, and means for selectively establishing an air 
pressure difference between the or each container and the reservoir, the 
arrangement being such that the air pressure difference can cause fluid to 
flow from a particular container into the reservoir when the corresponding 
valve means is such as to permit flow along the pipe means, the valve 
means being operated to permit a predetermined volume of a fluid reagent 
to be fed to the reservoir. 
The feeding of the fluid into the reservoir and the passing of the fluid 
from the reservoir to the spray head may be effected at least partly by 
air pressure. The air pressure may be provided by a higher than 
atmospheric pressure applied to the fluid to cause it to flow to a region 
held at atmospheric pressure or it may use atmospheric pressure to cause 
the fluid to flow to a region where a pressure below atmospheric pressure 
is maintained. 
The apparatus may include a container for containing fluid subject to 
atmospheric pressure, with the pipe means leading from the container to 
the reservoir, and the valve means selectively effective to block and to 
permit flow along the pipe means, and a suction pump selectively 
connectible to draw air from the reservoir. The apparatus may include a 
plurality of such containers respectively for containing different fluids 
and having associated therewith respective valve means selectively 
effective to block and to permit flow of the fluid from the containers to 
the reservoir. The rate of flow of the fluid along the pipe means may be 
measured under predetermined conditions and the result used to determine 
the time for which the valve means is opened to allow a required quantity 
of the fluid to be fed into the reservoir. 
The spray head may be arranged to receive air under pressure at the same 
time as the fluid is fed to it and the air flow used to produce a fine 
even spray of the fluid. 
The slides bearing samples may be mounted in parallel planes in a carrier 
to facilitate handling. The carrier may be indexed past an entry port to 
the spary station where a conveyor brings the slides one at a time beneath 
the spray head. 
The apparatus may include a heatable incubator capable of receiving a 
carrier containing slides. The incubator housing may be closable and means 
may be provided for injecting steam into the incubator to establish 
therein the humidity level just below 100 percent so as to prevent the 
samples from drying out during incubation. The incubator housing may also 
be arranged to contain a washing fluid to enable slides in a carrier to be 
washed, possibly with agitation of the carrier. 
The apparatus may include computer means programmed to control the 
operation of the spray means and the incubation to effect part or all of 
an in situ hybridization process. The program may be adjustable under the 
control of an operator to provide several choices of hybridization 
process.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
A plurality of slides 1 bearing cell samples for in situ hybridization is 
mounted in a carrier 2, which is movable in the up and down directions by 
a displacer 3. The movement of the carrier 3 is arranged to bring the 
slides successively level with an arm 5 which is movable from left to 
right under the control of a slide mover 4 to take a slide from the 
carrier 2 or to return a slide to it. A slide 6 has been moved by the 
slide mover 4 on to a conveyer belt 7 which runs round rollers 8 which are 
rotated by a motor 9. 
A second slide carrier 10, in which are mounted further slides 11, is 
movable under the control of a second slide carrier displacer 12. An arm 
14 controlled by a second slide mover 13 can receive slides from the belt 
7 for transfer to the carrier 10 or can transfer slides from the carrier 
10 to the belt 7. 
Whereas the slide carrier displacer 3 is only capable of moving the carrier 
2 in steps to bring each slide in turn into line with the arm 5, the slide 
carrier displacer 12 can perform the same operation and can also move the 
carrier 10 and the slides mounted in it to a position 15 in which it is 
inside a washer and incubator housing 16. 
The washer and incubator is provided with a heater 17 which is arranged to 
maintain the interior of the housing 16 at a thermostatically controlled 
temperature. Instead of or in addition to the heater 17 a refrigerator 
means (not shown) may be provided to enable the interior of the housing to 
be maintained at a temperature below ambient where such is required for 
the treatment of the slides. For washing the slides in the carrier 15, a 
washing fluid such as, for example, PBS, can be fed through a pipe 18 and 
a valve 19 into the housing 16. The washing fluid flows from a reservoir 
(not shown) of similar design but of larger capacity than the reservoir 31 
for the reagents described below, into which different washing agents can 
be fed in the same way as the reagents are fed into the reservoir 31. In 
order to improve the efficiency of the washing, the carrier 15 may be 
agitated by the displacer 12. Alternatively, the washing fluid may be 
stirred by a rotor or circulated by means of a pump. When the slides have 
been washed, the used washing fluid may be allowed to drain out by opening 
a valve 20 to allow the fluid to pass down the pipe 21 into the drain. 
When it is used as an incubator, the housing 16 is closed by a lid 22, 
which is moved into position by a lid control unit 23. In order to prevent 
the samples on the slides from drying out during incubation, steam from a 
generator 24 is fed through a pipe 25 into the housing 16 to raise the 
humidity inside it to be close to 100 percent at the incubation 
temperature. It is important that the amount of water in the atmosphere in 
the housing 16 should not be too high as this would cause the undesirable 
condensation of water on the slides. 
It may be required to maintain an air pressure above atmospheric within the 
housing 16 during incubation to reduce evaporation from the samples. The 
housing 16 may be constructed to allow that and a suitable pump may be 
provided for the purpose. 
Another modification which could be made to the housing 16 or in the same 
region of the apparatus is to provide some means for directing a current 
of cooling air, preferably of suitable humidity, to cool the slides after 
incubation. 
For applying reagents to the slide 6, a spray head 30 is provided which 
receives a reagent from a reservoir 31 under the control of a valve 32. 
The reservoir 31 is of a closed funnel shape and has input pipes 33, 34, 
35 and 36, through which different reagents can be fed into the reservoir 
and a pipe 37 through which a washing fluid can be fed into it. The 
reagents and washing fluid are drawn into the reservoir 31 by suction 
applied via a pipe 38 which is connected through a valve 39 to a suction 
pump 40. The pipe 37 is preferably so placed that the washing fluid 
flushes round the reservoir 31 as it enters so as to remove residual 
traces of reagent. In order to expel the reagent in the reservoir 31 
towards the spray head 30, air pressure is applied to the reservoir via a 
pipe 41. The pipe 41 is connected through a non-return valve 42, a valve 
43 and a pressure regulator 44 to a pressure pump 45. The suction pump 40 
and the pressure pump 45 are both driven by a motor 46. In addition to 
being connected to a one-way valve 42, the output of the valve 43 is also 
connected through a second one-way valve 47 to a reservoir 48 and from 
that through a valve 49 to a pipe 50 connected to the spray head 30. The 
pipes 33, 34, 35 and 36 are provided with valves 51, 52, 53 and 54 to 
control the flow of reagents from tubes 56, 57, 58 and 59 respectively in 
which they are stored. A valve 55 controls the flow of washing fluid from 
a storage bottle 60 along the pipe 37. 
A microcomputer 61 is provided with a plurality of control outputs 62 which 
are respectively connected to the control inputs, labelled C, of the slide 
carrier displacers 3 and 12, the slide movers 4 and 13, the motor 9, the 
heater 17, the lid control unit 23, the steam generator unit 24 and the 
valves 19, 20, 32, 39, 43, 47, 49, 51, 52, 53, 54 and 55. The 
microcomputer 61 contains a program for operating the components of the 
apparatus in the correct order and for the appropriate periods of time or 
to the appropriate amount to effect a required in situ hybridization 
process. The parameters of this process are adjustable by an operator and 
the program is arranged to display a menu of choices on a display unit 63, 
so that the operator can enter by means of keyboard 64 the choices which 
he wishes to make. 
An example of the apparatus shown in the FIGURE is shown in diagrammatic 
form because the shapes and dispositions of the various components can be 
changed considerably without affecting the fundamental operation. For 
example, the slide carriers 2 and 10 need not be of the simple linear form 
shown but could be toroidal form similar to that used for handling 
photographic slides for projection. In fact, the techniques used for 
handling photographic slides could be used for handling the slides in this 
apparatus. Only a single slide carrier need be provided and the carrier 2 
and the mechanisms for moving it and for selecting slides from it could be 
omitted. The conveyor belt 7 could be replaced by a disc rotating about a 
vertical axis having recesses in its periphery for receiving slides and 
being arranged to be rotated in steps by a suitable mechanism to bring the 
slides successively into position beneath the spray head 30. Similarly, 
changes could be made to the configuration of the washer and incubator; if 
desired, these two functions could be performed by separate parts which 
would have the advantage of permitting the washing of some slides while 
other slides are being incubated. 
An in situ hybridization process includes three fundamental tasks, namely: 
1. reagent metering and spreading on the slide; 
2. slide washing; 
3. slide incubation. 
The performance of these three operations by the apparatus shown will now 
be described. 
The reagents to be spread on the slides are stored in the tubes 56 to 59. 
In practice, more than four tubes may be provided and in one example 
fourteen such tubes are provided and there could be even more tubes than 
that if required. In order to apply the reagent in the tube 56 to the 
slide 6, some of the reagent is drawn into the spray reservoir 31 by 
opening the valve 39 so that the pressure in the reservoir 31 is reduced 
and then the valve 51 is opened allowing the reagent to be sucked along 
the pipe 33 into the reservoir 31. At this time all other valves 
controlling pipes connected to the reservoir 31 are closed. When a 
sufficient quantity of the reagent has been transferred to the reservoir 
31, the valves 39 and 51 are closed. Air pressure above atmospheric 
pressure is now applied to the reservoir 31 by opening the valve 43 
allowing pressure of the level set by the regulator 44 to be established 
in the reservoir 31. The same air pressure is also established in the 
reservoir 48 for use in dispersing the reagent as a fine spray by the 
spray head 30 using air flow along the pipe 50 when the valve 49 is 
opened. The air flow also helps to cut off cleanly the flow of reagent. 
Prior to the use of the apparatus for spraying the reagent on to the 
slides, the rate of flow of reagent through the valve 32 under the 
influence of the regulated air pressure established in the reservoir 31 is 
ascertained by measurement and on the basis of this measurement the 
duration of the opening of the valve 32 needed to produce the required 
dosage of reagent is calculated. When the valve 32 is opened, the reagent 
flows through it into the spray head 30 and the reagent is distributed as 
a fine spray over the slide 6 whilst the valve 32 is kept open for the 
required period. If more than one reagent is to be applied to a slide, 
then the required reagents can be drawn into the reservoir 31 by separate 
opening of the valves 51, 52, 53 and 54, as required. The flow 
characteristics of the valves 51 to 54 may be measured in the same way as 
described above for the valve 32 so that the required proportions of the 
different reagents are drawn into the reservoir 31. Preferably, the 
measurements are performed on the apparatus itself because factors such as 
the lengths of the pipes and the bends in them can influence the delivery. 
In one example of the apparatus, the valves 39, 43 and 49 which control air 
flow are solenoid valves of conventional construction, for example, REEDEX 
types V22 IP5-2PNS and V32 IP5-2BNS. The former type is used for the valve 
49 and the latter type for the valves 39 and 43. The suction pump 40 is 
arranged to provide a pressure of -10.8 psig relative to atmospheric 
pressure, and the regulator 44 is arranged to control the positive 
pressure to a value of 25 psig above atmospheric pressure. The valves 51 
to 55 are solenoid pinch valves of the type ALPHA NC P/N 161 P011 with the 
pipes 33 to 37 being formed of flexible plastics tubing. The valve 32 is a 
LEE 3/2 solenoid valve LFAA 1201618 H. The spray head 30 is of the type 
LEE INSTAC liquid dispenser TCDA 6201110K. Of course, other suitable types 
of valve and spray head may be used. 
It has been found convenient for the tubes 56 to 59 and the bottle 60 to be 
carried on a detachable rack with the pipes 33 to 37 held in fixed 
positions. 
The washing of slides is effected by placing a carrier in which the slides 
are mounted in the housing 16, filling the housing with a washing fluid 
such as, for example, PBS, which is maintained at a required temperature 
by the heater 17. The carrier with the slides in it may be agitated by the 
displacer 12 to improve the effectiveness of the washing. Several 
different washing fluids may be used and separate valve controlled inputs 
for the different fluids may be provided. 
When slides are to be incubated, they are placed in the housing 16 on a 
carrier and the lid 22 of the housing closed. Steam from the generator 24 
is fed into the housing 16 as the temperature in it is raised so as to 
maintain the humidity of the atmosphere at about 95 percent, thereby 
reducing to a low value the moisture loss due to evaporation from the 
tissue samples on the slides. Initially, to denature the DNA content of 
the cells of the samples, they are heated to 100.degree. C. and then 
rapidly cooled to room temperature. This rapid cooling could be achieved 
by means of water circulating in a jacket in the housing 16 or air blown 
around carrier 15 whilst in housing 16. 
A typical slide-mounted sample may require a dosage of between 10 and 50 
.mu.liters of the reagent to be applied in order to achieve efficient 
penetration of the cell membranes of the sample. It is necessary to 
maintain the stipulated volumes of reagents because if insufficient 
reagent is applied then the hybridization will be incomplete or possibly 
ineffective. On the other hand, the reagents required for hybridization 
are expensive and if excessive quantities are used this will be 
economically wasteful. Generally, the reagents take the form of small 
volumes of active material dissolved in a buffer solution such as PBS 
which has a density and viscosity similar to that of water. However, the 
hybridization "cocktail" contains reagents which are diluted in a 10 
percent solution of dextran sulphate which has a higher density and 
viscosity than of those of water. The flow characteristics of the valves 
are therefore measured for both buffer solutions and dextran sulphate. In 
each case, the quantities passed by the valves for periods of up to 2 
seconds in quarter second steps are measured and corresponding calibration 
data stored in the microcomputer. It was found that for volumes of reagent 
less than 6 .mu.liters, a simple linear factor was inadequate, but this is 
not significant because for most hybridization procedures the minimum 
quantity of reagent used is 10 .mu.liters. 
As the apparatus is required to spray different reagents at different 
times, it is necessary to be able to wash out residual traces of one 
reagent from both the reservoir 31 and the spray head 30 before drawing 
the next reagent or group of reagents into the reservoir 31. This washing 
out is usually done with PBS, although other washing fluids may be used. 
Tests have shown that if the reservoir is flushed more than once with PBS 
after one reagent, then the contamination of the following reagent is 
insignificant. 
Various modifications can be made to the apparatus described apart from the 
alteration of the number of storage tubes for reagents and the associated 
valves and pipes and the addition of alternative washing fluids and the 
containers and feed pipes for them. For example, instead of using air 
pressure to transfer amounts of reagent or washing fluid to the reservoir 
gravity feed may be used. The air pressure feed of reagent from the 
reservoir to the spray head may be replaced by gravity feed although air 
dispersion of the reagent to form a spray could still be used. As another 
alternative to gravity feed a pump, such as a peristaltic pump, may be 
used. A storage place may be provided for carriers containing slides so 
that they can be prepared and placed in the apparatus for processing in 
their turn. As mentioned above, other configurations of slide carrier than 
the one shown may be used with suitable modification to the displacers. 
The apparatus may be of modular construction, with separate modules for 
reagent spraying, slide transport, slide washing and slide incubation 
connectible together in different arrangements. Refrigeration means may be 
provided to keep cool all or part of the apparatus if required.