WO2005014154A1

WO2005014154A1 - Method and apparatus for removing cellular material from faecal stools and container for collecting faecal stools

Info

Publication number: WO2005014154A1
Application number: PCT/GB2004/003434
Authority: WO
Inventors: Ian O'neill
Original assignee: Loots Limited
Priority date: 2003-08-08
Filing date: 2004-08-09
Publication date: 2005-02-17
Also published as: GB0318702D0

Abstract

A method of removing cellular material from the surface of a faecal stool comprises the steps of: a) supporting the stool in a container having a channel along which the stool extends; b) cooling the stool to a temperature below its gel greezing point; c) introducing wash liquid into the container whilst maintaining the stool at a temperature below its gel freezing point; and d) moving the container to cause the liquid to run back and forth along the channel thereby washing the cellular material from the surface of the stool. Further disclosed are an apparatus (4) for removing cellular material from. the surface of a faecal stool and a container for collecting faecal stools (3b) .

Description

METHOD AND APPARATUS FOR REMOVING CELLULAR MATERIAL FROM FAECAL STOOLS AND CONTAINER FOR COLLECTING FAECAL STOOLS

Field of the Invention The present invention relates to a method and apparatus for removing cellular material from faecal stools and a container for collecting faecal stools.

Background Faecal stools are the residue _.of . digestive products .in the gastrointestinal tract- and contain not only bacteria and undigested food residues- but also residual products from the body' s metabolism and structure such as exfoliated epithelial cells from the lining of the gastrointestinal tract . Exfoliated epithelial cells consist of cellular components, such as DNA, RNA and proteins, which potentially provide a multiplicity of approaches to identifying and monitoring unhealthy conditions of the epithelium as exemplified by colorectal cancer, polyps and ulcerative colitis . The facile recovery of such cells makes possible the application of a wide variety of test procedures for qualitative and/or quantitative analysis of, for example, DNA alterations, gene expression and enzyme activity. A. Loktionov et al . , in Clinical Cancer Research, Vol. 4, 337-342, (1998), describe a non-invasive screening test for early detection of colorectal cancer which is based on a quantitative comparison of the DNA concentrations derived from exfoliated epithelial cells isolated from the surface of stools from healthy and tumour-bearing persons. In this isolation procedure, cells on the surface of the stool were predominantly clustered and had varying degrees of intactness and damage. In the present specification by "cells" and "cellular material" we mean all such material irrespective of degree of clustering or intactness. In our earlier patent application, WO 97/09600, we describe a method of isolating cells (especially exfoliated epithelial cells) from the surface of faecal stools, comprising cooling a stool to a temperature (preferably of -80°C to 15°C, preferably -10°C to 15°C, preferably -10°C to 10°C, more preferably 0°C to 5°C) below its gel freezing point and removing the cells from the cooled stool by agitating the stool in an aqueous solution (which may be pre-cooled to a temperature below the gel freezing point, and is preferably ice-cold) . The cooled condition creates a much more physically robust hard gel state that helps to prevent the stool from breaking up or rapidly eroding so that non-surface material is largely prevented from entering the aqueous solution. Stool surface layers provide a particularly rich source of exfoliated cells suitable for isolation. Typically between one-sixth and one third of colonic epithelial cells are exfoliated per day (D. Sidransky et al . (1992), Science 256, 102-105 and W. C. MacDonald, J. S. Trier, N. B. Everett, Gastroenterology (1964) 46 , 405) . It is believed that some of these exfoliated cells, together with mucus, may be envisaged as forming a "sheath" around a stool, containing a low proportion of bacterial cells and other faecal matter. It is further believed that the viability of the cells in the surface layers (i.e. the layer of faecal material within 1.0 mm, and preferably 0.25 mm, of the stool surface) of the stool is facilitated by i) a tendency of the cells to agglomerate into plaques (which helps to prevent apoptosis) , ii) the availability of oxygen at the surface of the stool and iii) the unique ability of colonic epithelial cells to utilise as fuel the butyrate formed locally in high concentrations by microfloral fermentation (J. H. Cummings (1981) Gut 22, 763-799 and W. E. W. Roediger (1980) Gut 21 , 793-798). Oxidation of butyrate is believed to provide fuel for the cells in the absence of blood-borne nourishment following exfoliation. It has been found that isolation of cells from stool surface layers provides significant advantages in yield, purity and ease of recovery over isolation of cells from homogenised stools.

Summary of the Invention Although the method described in WO 97/09600 is effective for isolating cells from the surfaces of faecal stools, in order to make the method more _.suitable for high throughput screening of e.g. hundreds or thousands of samples per day, problems remain of washing the stools in a standard and reproducible way that maximises the amount of surface cells extracted from the stools but minimises the risk of breaking up the stools. Consequently, in a first aspect, the present invention provides a method of removing cellular material from the surface of a faecal stool comprising the steps of: a) supporting the stool in a container having a channel along which the stool extends; b) cooling the stool to a temperature below its gel freezing point; c) introducing wash liquid into the container whilst maintaining the stool at a temperature below its gel freezing point; and d) moving the container to cause the liquid to run back and forth along the channel thereby washing the cellular material from the surface of the stool. A minority of stools are very soft when excreted, such that on cooling they do not reach the gel freezing point, but do become sufficiently viscous to be washed with minimal entry into the wash liquid of sub-surface material. Thus, for that minority of stools, by "gel freezing point" we mean the temperature at which the stools become sufficiently viscous to be washed with negligible entry of sub-surface material into the wash liquid. The method promotes the reproducible and gentle washing of the stool, which facilitates the quantitative comparison of cellular material between individuals. Human faecal stools are typically elongate and approximately cylindrical in shape. So the channel significantly reduces the likelihood that sections of the stool will break off when the stool is being washed because the channel walls substantially prevent side-to-side rolling motion of the stool and, for a wide range .of rocking angles and speeds, friction largely prevents the stool from sliding along the channel. Preferably the container is moved to cause a wash frequency of from 5 to 40 back and forth cycles per minute, and more preferably from 15 to 25 cycles per minute. The stool may be pre- soaked (for e.g. 3 minutes) in the wash liquid prior to step d) , in which case preferably the container is moved for a total time of from 10 seconds to 1 minute. Without a pre-soak, preferably the container is moved for a total time of from 30 seconds to 5 minutes. With or without pre- soaking it is advantageous to limit the exposure of the stool to the wash liquid so as to avoid localised depletion of wash reagents or over-enrichment of released cellular material. The channel may have a V or U-shaped transverse section. An advantage of the container motion, is that the wash liquid is channelled back and forth along the length of the stool so that, compared with e.g. circular agitation by an orbital shaker of the wash liquid relative to the stool (which we have found in any event tends to break up the stool) , less liquid is required to wash the cells from the surface of the stool. Clearly the less liquid that is used to wash the stool, the higher the resulting concentration of cells in the liquid, which can in turn improve the accuracy of screening tests which may be subsequently performed on the liquid. Also smaller volumes of liquid reduce cost and disposal problems. Preferably from 30 to 150 ml of liquid is used in steps c) and d) . This is generally sufficient to wash a typical 100 g stool while minimising losses due to residual wetting of the container and stool. Most preferably a fixed ratio of liquid to stool is used so that all stools experience a similar degree of wetting. This ratio may be in the range 0.5 to 1.0 ml of liquid per gram of stool. In one embodiment,, _.step d)_. comprises, rocking the container about a horizontal axis perpendicular to the channel so that the liquid runs back and forth under gravity. In another embodiment, step d) comprises displacing the container back and forth in the direction in which the channel extends. Preferably the container is of fixed shape, e.g. by being formed of rigid material. In a second aspect, the present invention provides an apparatus for removing cellular material from the surface of a faecal stool, comprising: mounting means for mounting a container having a channel along which, in use, the stool extends; and motion imparting means for moving the container so that, when wash liquid is introduced into the container, the liquid runs back and forth along the channel thereby washing cellular material from the surface of the stool. In one embodiment, the motion imparting means rocks the container about a horizontal axis perpendicular to the channel so that the liquid runs back and forth under gravity. In another embodiment, the motion imparting means displaces the container back and forth in the direction in which the channel extends. The apparatus may further comprise control means to control e.g. the rocking angle, rocking or displacement frequency and/or total time of movement of the container, preferred ranges for some of these parameters being provided above in respect of the first aspect of the invention. The control means may further control a sequence of stationary and mobile periods for the container, so that e.g. stationary exposure of the stool to the wash liquid allows the liquid to penetrate the stool and loosen its surface layers prior to active washing when the container is moved. With such an apparatus, stool washing can be performed in a repeatable, standardised manner, and with a relatively high stool sample throughput. These are significant advantages if the wash liquid is to provide the material for a screening test based on e.g. a quantitative comparison of amounts of epithelial cell components. The apparatus may further comprise cooling means for cooling the stool to a temperature (preferably of -80°C to 15°C, preferably -10°C to 15°C, preferably -10°C to 10°C, more preferably 0°C to 5°C) below its gel freezing point. In one embodiment the apparatus further comprises a filter means such as a sieve, so that, when wash liquid is removed from the container, the cells are retained in the liquid but food residues from the stool are filtered from the liquid. The apparatus may further comprise a metering device for introducing a metered dose (e.g. 50 to 150 ml, or an amount which is related to the stool weight) of wash liquid into the container, and/or a wash liquid pre-cooler for cooling the wash liquid to a temperature below the gel point of the stool before the wash liquid is introduced into the container. This temperature is preferably about 0°C. The apparatus may further comprise at least one and preferably a plurality of detachable sample receptacles into which wash liquid is transferable from the container, so that e.g. when stool washing is completed the wash liquid may be poured from the container into the receptacles. These can then be detached from the apparatus and transferred to an automated liquid-handling system for high throughput processing of the liquid samples contained therein. A third aspect of the invention provides a fixed shape container for collecting faecal stools which has an upper receiving mouth and a lower stool collection channel, the internal walls of the container narrowing from the mouth to the channel so that when a stool _.is deposited through the mouth the stool is encouraged to lie lengthways along the channel. The mouth can be of any suitable shape, e.g. rectangular or circular. The length of the channel is preferably in the range from 10 to 25cm, which corresponds to the size of a typical stool. The container may be used in the first or second aspect of the invention. As faecal stool is generally regarded as disgusting material that people are reluctant to handle, even indirectly, the fixed shape container has several advantages as compared with, e.g. a flexible plastic specimen bag for collecting stool. For example, the mouth of the container cannot collapse as a stool is being deposited in the container, thereby making it easier for a user to excrete into the container. Also the container can be handled without, apart from the weight of the stool, any sensation that a stool is contained therein. Furthermore the funnelling effect of the narrowing container walls guides a formed stool (which on defecation can be quite malleable) to a position in which it extends along the channel. This reduces or eliminates the need for the user to directly or indirectly manipulate the stool into that position prior to stool washing. Soft stools, and even diarrhoea, are also encouraged to align along the channel by the narrowing of the container walls. A further advantage of the container is that it can serve both as the receptacle into which the stool is excreted and as the container in which the stool is washed. So the stool does not need to be transferred to a different washing container, which avoids damage to the stool surface caused by such a transfer. Such damage would interfere with the accuracy of e.g. screening tests which may be performed on the stool. If the stool is excreted into the container at home and then transported to e.g. a clinic for washing, the container has the further advantage of holding the stool reasonably securely in the channel so that transportation damage to the stool surface is reduced or avoided. To avoid user embarrassment during transportation at least a majority of the container may be opaque (i.e. a stool inside the container is not visible through the opaque portions of the container, even if the material of the container transmits light to some degree) , or an opaque transport casing may be provided for transporting the container. However, desirably at least a portion of the container permits viewing to the inside of the container so that e.g. after delivery of the container for stool processing a health professional can check the stool for gross abnormalities such as blood before the stool is processed and without opening the container. The container may further have installation means (which may be detachable from the container) for removably installing the container on a toilet (e.g. below the toilet seat) with the receiving mouth opening upwardly and the collection channel extending horizontally beneath the mouth. This allows the user to produce the stool in the usual manner, which promotes user acceptability. Typically, for hygiene reasons, at least the stool- contacting parts of the container are disposable. However, in one embodiment the internal walls of the container serve as a former for a bag which is reversibly fixable inside the container, whereby the bag assumes the shape of the mouth and channel. In this embodiment the bag is typically disposed after use, but the container can be reused. Preferably the container is made of plastics material such as polypropylene. Desirably the container has smooth internal surfaces which reduce damage to the surface of the stool as the stool passes from the mouth to the channel. In one embodiment the container has cooling means, such as an outer jacket or bath, for cooling a stool deposited in the container. _.This me_a.ns_.that the stool can be cooled immediately it is produced, which helps to preserve the surface layer cells and makes the stool robust for physical transport. The cooling means may be of material that can be pre-cooled in a domestic refrigerator or freezer. This is particularly convenient when the stool is produced at home, allowing the donor to defecate at a convenient time and in a minimally stressful setting and thereby promoting user acceptability. The cooling means may enable the stool to be cooled to a temperature below its gel freezing point. The cooling means may be detachable from the container so that the cooling means can be pre-cooled before fitting to the container. Also, if the container is disposable, the cooling means can still be reused. Anaerobic bacteria in stools produce damaging radicals when exposed to atmospheric oxygen (C. F. Babbs (1990) Free Radical Biology and Medicine, 8_, 191-200) . Such oxygen also oxidises the mucus on the surface of stools and encourages the growth of aerobic bacteria. So preferably the container is hermetically sealable to reduce the exposure to atmospheric oxygen of a stool deposited therein. The seal may be achieved by means of a lid or cover (e.g. having a snap-fit or screw fitting) for the mouth. A sealed container rapidly becomes anaerobic due to the high concentration of anaerobic bacteria in the stool, and this apparently largely prevents the growth of aerobic bacteria on the stool surface, helping to preserve the stool. We have found that sealed containers allow stools to be stored at 0°C for a number of weeks apparently without significant degradation. To promote user acceptability, the seal should also eliminate any odour from the stool escaping from the container, particularly if the stool is produced at home and then transported before washing. The container may also_. have a liquid .port which, allows wash liquid to be removed from the container while maintaining the stool in the container. For example the diameter of the port may be sized to prevent a typical- sized stool from exiting via the port, or a foraminous screen over the port may serve the same function. The port may include closure means such as a valve or a cap which allows the liquid to be poured or sucked out of the container but at other times maintains an airtight seal. The same or another port may be used to introduce wash liquid into the container. A further aspect of the invention provides a kit of parts comprising the container of the previous aspect with the installation means and/or the cooling means and/or the transport casing. Another aspect of the invention provides a method of screening for unhealthy condition (s) of the epithelium (such as colorectal cancer, polyps and ulcerative colitis) in a plurality of subjects (which are typically human subjects, but may be other animal species such as dogs and cats), the method comprising the steps of: a) providing to the subjects respective containers (e.g. according to the third aspect of the invention) for defecation of a stool sample thereinto at the convenience of the subject (which is typically when the subject is "at home" or in a domestic setting) ; b) collecting the filled containers from the subjects at a central location (which may be a doctor's surgery or a hospital out-patients clinic) ; c) removing a specimen of cellular material from the surface of each stool sample (e.g. according to the first aspect of the invention) ; and d) testing each specimen for unhealthy condition (s) of the epithelium (e.g. as described by A. Loktionov et al . , referenced above) . Brief Description of the Drawings The present invention is now described in respect of a specific embodiment and with reference to the accompanying drawings in which: Figs, la and b show schematically and respectively a cross section through and a plan view of a receptacle for collecting a faecal stool, and Fig. lc shows a plan view of an alternative form of the receptacle; Fig. 2 shows the same cross section as Fig. la except that a lid is fixed to the receptacle; Fig. 3a shows a schematic cross section of the receptacle of Fig. la fixed inside a transport unit, and

Fig. 3b shows the receptacle of Fig. lc fixed in another version of the transport unit; Fig. 4 shows schematically a stool washing apparatus with the receptacle of Fig. la positioned in a sample rack; and Fig. 5 shows the apparatus of Fig. 4 with the sample rack pivoted to pour wash liquid out of the receptacle.

Detailed Description Figs, la and b show respectively a cross section through and a plan view of a transparent plastic receptacle

1 having a V-shaped cross-section for collecting a faecal stool 3 produced by a user. The receptacle has a mouth 5 and a straight stool collection channel 7 (which extends perpendicularly to the plane of the paper in Fig. la) . The receptacle is removably installed inside a toilet bowl 9 by a support web 11 which is attached over the rim of the toilet and has a central hole 12 to receive the receptacle so that channel 7 is horizontal and mouth 5 is directly above channel 7. The smoothly sloping walls 14 of the receptacle guide the stool to the bottom of channel 7 and encourage the deposited stool to align with the longitudinal direction of the channel . Fig. lc shows an alternative form of_. the receptacle which has a U-shaped channel cross-section, and which is capable of receiving larger specimens . Once the stool is collected in the receptacle, a lid 13 (see Fig. 2) is used to close mouth 5 and is releasably fixed to the receptacle by a snap-fit mechanism 16. A sealing ring 15 seals the receptacle from the external atmosphere . Lid 13 includes a carrying handle 17, thermally insulated upper wall 19 and wash liquid entry/exit port 21. The port is sealed by closure cap 23 and has a foraminous screen 25 at its inner end which prevents the stool from entering the port when (as described below) wash liquid is decanted from the receptacle, and a coarse sieve 25a to retain particles of e.g. greater than 2 mm in diameter. The receptacle is lifted by handle 17 from web 11 and fixed inside a transport unit 27 by a snap-fit mechanism 29 (see Fig. 3a) . The transport unit has thermally insulated walls 31 and contains a cooling block 33 of pre-cooled ice or chemical cooling medium contained under a shaped divider 35. The cooling block is brought into close proximity with the outer wall of receptacle 1. Ring 15a maintains the position of channel relative to the cooling block. The cooling block cools stool 3 to a temperature preferably in the range from 0 to 5°C, which is below the gel freezing point of the stool. Fig. 3b shows the receptacle of Fig. lc fixed in another version of the transport unit. In this version divider 35 forms an air space around the receptacle. The air space and divider 35 prevent the cooling block cooling the stool so rapidly as may damage the outer surface layers of the stool. The stool is then transported in the transport unit to e.g. a clinic where stool washing is performed. The walls of the transport unit and lid are. opaque .so that the stools are not visible inside the unit, and lid 13 prevents odours from escaping the unit. Channel 7 also reduces movement of the stool during transportation which prevents significant damage to the surface of the stool. At the clinic, the receptacle 1 and lid 13 are removed from the transport unit. As the receptacle is transparent visual inspection of the stool is possible without removing the lid. At this stage the stool sample may be rejected if, for example, it is too small or runny. also any gross evidence of disease, such as blood, may be noted. However, if the stool is acceptable, the receptacle and lid are positioned into the sample rack 39 (i.e. receiving means) of a stool-washing apparatus 37 shown in Fig. 4. Collection channel 7 is aligned perpendicular to axis 41 about which rack 39 is pivotable and which at this stage is horizontal. Cooling means (not shown) , such as a cold air jet, refrigerator unit or coolant bath, keeps or brings the stool to a temperature in the range 0 to 5°C so that it is below its gel freezing point. Apparatus 37 has a reservoir 43 of chilled wash liquid, such as a buffered aqueous solution containing N- acetylcysteine, bovine serum albumin and sodium butyrate which has been found (see WO 97/09600) to improve recovery of exfoliated cells. When the stool is sufficiently cool, closure cap 23 is removed and a metered dose of wash liquid 44 is injected into the receptacle via an injector 45 and port 21. The liquid may alternatively be injected through an air-tight septum at port 21. Afterwards injector 45 is swung away from the sample rack. Rocking means (not shown), which is e.g. an electric motor and associated drive, then rocks the sample rack and receptacle backwards and forwards about axis 41. A control unit (also not shown) which controls the rocking means defines a wash _.programme consisting of rocking angle,- rocking frequency and total exposure and rocking time experienced by the stool. As receptacle 1 moves, gravity causes wash liquid 44 in the receptacle to run back and forth along channel 7 so that intimate contact with the stool is achieved and surface cells from the stool are washed into the liquid. To a significant extent friction prevents the stool from moving during the rocking procedure . When the wash programme ends, the sample rack is pivoted about pivot 47 to bring axis 41 into a vertical position (see Fig. 5) . Almost all of wash liquid 44 is then decanted out of receptacle 1 via port 21, foraminous screen 25 substantially preventing larger food residues or other broken off pieces from the stool exiting with the liquid. Coarse sieve 25a removes smaller pieces from the liquid. Finer downstream sieves (not shown) further limit the maximum particle size in the liquid. The liquid is then be divided into standard volumes and received into a plurality of removable (e.g. clip-on) sample receptacles (not shown) . The sample receptacles may be a strip of tubes each of e.g. 0.1-0.5 ml volume as can be removed and clipped into a standard microplate format used for clinical analyses. Alternatively the sample receptacles may be individual tubes each of e.g. 1-2 ml volume. The sample rack is then returned to its horizontal position and receptacle 1 is removed and safely disposed of. In this way, the apparatus converts a typical total 100-500 g mass of stool and wash liquid into aliquots of cell-suspension of typically only 50-1000 mg (i.e. a thousand-fold reduction in scale) . The aliquots can be made representative by preventing the cells from settling out during the decanting process the aliquots. One method of preventing settling out is to provide mixing channels (not shown) through which the liquid flows on its way to the sample _.receptacles .. Alternatively a. stirrer (also not shown) can be provided to stir the wash liquid. Fine sieves (e.g. of water wettable material such as nylon, and/or having a mesh size in the range 50-400 μ , preferably 100-250 μm, more preferably 125-200 μm) may be provided over the entrances to the receptacles to further filter the liquid. The excess liquid flows to waste. The samples may be used for e.g. DNA assays as described in WO 97/09600 and A. Loktionov et al . , Clinical Cancer Research, Vol. 4, 337-342, (1998). The entire procedure, from when the receptacle is positioned in the sample rack to when the wash liquid exits the receptacle, can be automated, with standard wash programme settings being used. Also no manual contact with or manipulation of the stool required, which promotes good hygiene. So the present invention allows a large number of stool samples to be processed in a safe and reproducible manner.

In an experimental study, stools were collected into re- sealable bags and stored at 0°C from no more than a few minutes after excretion. The bags were transparent, which allowed visual inspection of the stools during testing. For stool washing, a wash solution was poured into each bag and the bag was located inside a rigid rocking channel of a stool washer apparatus, the channel being 40-45mm wide and perforated so as to permit intimate cooling by surrounding ice-water held in an insulated container equipped with a submersible pump. Rocking caused a gravity-imposed back and forth circulation of the wash solution along the channel and also promoted cooling of the stool by the surrounding ice- water. A frequency of 3 seconds per rocking cycle was chosen. The wash solution was as previously described by A. Loktionov et al . (referenced above), but the stool wash was passed through, a_..250. μm instead, of a_125 μm sieve. -Cell clean-up was performed robotically on a TECAN™ Genesis RSP 150 and then DNA was released by mechanical cell disruption in the presence of 0.9%SDS as lysing agent. Following dilution to prevent quenching, fluorimetric DNA assay was performed with a TECAN™ Spectrafluor Plus using PicoGreen (Molecular Probes™) to yield the stool DNA index (SDNAI, defined as nanograms of DNA per ml of stool wash) . The effects of pre-soaking, rocking angle, mechanical and bacterial damage, stool age, and wash volume were studied, and the results are given below.

Effect of pre-soaking on SDNAI The following SDNAI data from the stool washer (using a +/-45deg rocking angle) demonstrate the advantage of a period of pre-soaking. Presoaking appears to promote a more or less equilibrated suspension state of cellular material in the wash solution, which thereby helps to prevent the chance dynamic release of cellular material.

Effect of Rocking Angle If too small a rocking angle was used or the channel became obstructed by the stool, the wash liquid tended to form non-flowing pools that failed to contact the stool surface so that unacceptable variations in washing condition were experienced. However, under favourable conditions, reproducible and relatively constant results could be obtaine_.d over, a range, of rocking angles. For example in three different experiments the same stool was given a 3 minute pre-soak followed by a 1 minute of wash at different rocking angles in a visibly free flow of liquid in a channel 40-45 mm wide and using a wash liquid to stool ratio of 0.7 ml per gram. The rocking angles for the three experiments were +/-30, 45 and 60° from the horizontal and the respective SDNAI measurements were 263, 254 and 260 ng/ml .

Effects of Damage to Stool Surface Layer and of Stool Age Sometimes stored stools were found to be accidentally squashed, or stored in improperly sealed containers as evidenced by smalls amount of white bacterial over-growth on the stools. The following data (using different stools all washed for 1 minute after a 3 min pre-soak at a wash solution/stool weight ratio of 0.7) demonstrate the importance of protecting the stool surface layer during storage. However, they also demonstrate that under favourable conditions storage durations of up to several weeks have no particular effect on measured SDNAI values. The squashed stools had SDNAI values significantly above the 700 ng/ml value which is considered to be the upper limit for healthy persons.

^s indicates the stool was squashed ^b. indicates the .stool . had bacterial -over-growth

Effect of Wash Solution Volume Each of a series of undamaged stools from one donor was washed (with a 3 min pre-soak and a 3 min wash at +/- 45° rocking angles) with both a fixed 50 ml volume of wash solution and a variable volume of wash solution based on a 0.5 ml/gram wash/stool ratio. For successive stools of the series, washing first was alternated between the fixed and variable volume. The variable volume based on the 0.5 ratio resulted on average in the use of a 64% greater volume of wash solution which visibly appeared to wet the whole stool better and produced a 21% greater DNA concentration. The second washes gave a 54% higher on average DNA yield than the first washes (largely irrespective of whether the fixed or variable wash solution volume was used first) which was presumably due to the pre- softening effect of the first washes. In general therefore the fixed ratio wash took better account of the stool weight without significantly influencing the DNA result. As shown above, stool age had an insignificant effect on the results.

Claims

Claims :

1. A method of removing cellular material from the surface of a faecal stool comprising the steps of: a) supporting the stool in a container having a channel along which the stool extends; b) cooling the stool to a temperature below its gel freezing point; c) introducing wash liquid into the container whilst- maintaining the stool at a temperature below its gel freezing point; and d) moving the container to cause the liquid to run back and forth along the channel thereby washing the cellular material from the surface of the stool .

2. A method of removing cellular material according to claim 1, wherein step d) comprises rocking the container about a horizontal axis perpendicular to the channel so that the liquid runs back and forth under gravity.

3. A method of removing cellular material according to claim 1, wherein step d) comprises displacing the container back and forth in the direction in which the channel extends .

4. A method of removing cellular material according to any one of claims 1 to 3, wherein the container has a fixed shape .

5. An apparatus for removing cellular material from the surface of a faecal stool, comprising: mounting means for mounting a container having a channel along which, in use, the stool extends; and motion imparting means for moving the container so that, when wash liquid is introduced into the container, the liquid runs back and forth along the channel thereby washing cellular material from the surface of the stool.

6. An apparatus for removing cellular material according to claim 5, wherein the motion imparting means rocks the container about a horizontal axis perpendicular to the channel so that the liquid runs back and forth under gravity.

7. An apparatus for removing cellular material according to claim 5, wherein the motion imparting means displaces the container back and forth in the direction in which the channel extends .

8. An apparatus for removing cellular material according to any one of claims 5 to 7, further comprising cooling means for cooling the stool to a temperature below its gel freezing point.

9. An apparatus for removing cellular material according to any one of claims 5 to 8 , further comprising filter means which filters food residues from the stool but retains the cellular material in the wash liquid when the liquid is removed from the container.

10. An apparatus for removing cellular material according to any one of claims 5 to 9, in which the container is mounted at the mounting means .

11. A container for collecting faecal stools which has a fixed shape and has an upper receiving mouth and a lower stool collection channel, the internal walls of the container narrowing from the mouth to the channel so that when a stool is deposited through the mouth the stool is encouraged to lie lengthways along the channel.

12. A container according to claim 12, further having installation means for removably installing the container on a toilet with the receiving mouth opening upwardly and the collection channel extending horizontally beneath the mouth.

13. A container according to claim 11 or 12, further having cooling means for cooling a stool deposited in the container.

14. A container according to any one of claims 11 to 13, which is hermetically sealable to reduce the exposure to atmospheric oxygen of a stool deposited therein.