GB2355790A - Detecting cancer - Google Patents

Abstract

Presence of cancer or precancerous cells in samples of biological material is detected by colour screening. Typically, enzyme activity in the material is detected histochemically e.g. using a dye or stain, to indicate the presence of a cancer in a tissue sample.

Description

2355790 -1 METHOD OF DETECTING A CANCER IN A TISSUE SAMPLE The present

invention relates to a method of detecting a cancer in a sample of tissue taken from the human or animal body.

Cancer is a disease which has many forms and develops when the usually ordered relationship of cell division and cell differentiation becomes disordered.

Typically, cancer develops in a single cell and effects the ability of a dividing cell to differentiate. The non-differentiating cell obtains the capability to spread to many areas of the body through the bloodstream or lymphatic system. This non differentiating cell usually produces progeny which behave in a heterogeneous manner and may continue to divide as well as develop the ability to spread around the body. In the event that the above mentioned single cell and its progeny are left unchecked, death of the host commonly follows.

It is commonly accepted that the curability of a cancer is inversely proportional to its size or potency at the time of diagnosis. Consequently, an early diagnosis is critical and people are often encouraged to be screened for certain types of cancer on a routine basis.

One example of an early screening process is the screening for cancer of the cervix in women. It has taken a considerable number of years to develop appropriate guidelines yet such a screening process possesses significant drawbacks insofar as it is expensive to perform on a large scale and the results are not always reliable.

The reliability problem is a consequence of the heavy reliance on the ability of a technician to firstly notice and then interpret the subtle morphological changes when reading a slide coated with a cervical smear.

A further example of screening for a cancer is when careful examination of biopsy material is performed by a skilled pathologist.

However, this screening technique has similar drawbacks to the cervical smear process in that it is expensive when performed on a large scale and is heavily reliant on the pathologist's interpretive abilities of the morphological information contained within the material.

Due to the problems with the presently used techniques of detecting a cancer it is desirable to have a technique which is inexpensive to perform and, particularly, less reliant on the interpretive ability of the technician/pathologist whilst providing accurate and reproducable results.

In addition, as it is critical that a cancer is diagnosed in its early stages on order to improve the curability thereof, it would also be desirable if pre cancerous cells could be identified.

The term pre-cancerous cell(s), used hereinbefore and hereinafter, is intended to relate to at least one or more cells which are in the initial stages of I becoming cancerous but have yet to display any morphological changes which are considered to be indicative of a cancerous cell.

The present invention has been made from a consideration of the above problems.

According to a first aspect of the present invention therefore there is provided a method of detection of a cancer in a sample of biological material comprising colour screening.

According to a second aspect of the present invention therefore there is provided a method of detection of a cancer in a sample of biological material comprising the detection of pre-cancerous cell(s) by colour screening.

The above-described methods are superior to the prior art insofar as they are not as reliant on the ability of the person analysing the results when compared with the known methods.

Indeed it can easily be envisaged that a person would need to be in command of significantly more skill, interpretive ability and knowledge to correctly perform a technique based on interpreting morphological information than would be required in order to successfully perform a method of detection based on interpreting colour.

The above-described methods produce reliable results which do not require any great interpretative ability, consequently, mistakes in diagnosing a cancer should be prevented.

In addition, the above described methods allow pre-cancerous cell(s) to be successfully and reliably diagnosed before any morphological changes have occurred to any of the cells in the sample. This has particular relevance following the removal of a biopsy in order to ensure that the tissue which previously surrounded the biopsy is free of any cancer. Similarly, the above described methods may be used to check that a cancer is not beginning to return to tissues which have been treated for a cancer previously.

Further, the above-described methods are inexpensive and, generally, experimentally straightforward to perform on both a small or a large scale.

It is to be noted that all of the above described methods are to be performed in vitro on a sample of cells which have been removed from the human or animal permanently.

In a preferred embodiment of the invention the colour screening is used to detect enzyme activity. Preferably the enzyme activity is detected by histochemical means, and particularly, by qualitative histochemistry.

The preferred embodiments provide methods which are advantageous over the known methods for a plurality of reasons. In a first instance, although hitherto the activity or presence of an enzyme in a cell or sample of cells has not been acknowledged as being a useful indicator by which the presence of a cancerous cell may be detected, the physiology of many enzymes is well known and a plurality of known tests, further than the tests detailed hereinafter, for the presence of activity of a range of enzymes may be adapted to be used in the present invention.

In a second instance, the enzyme activity may be detected via histochernical means rather than biochemical means. Such detection is advantageous as in living cells the properties of an enzyme in its natural environment, perhaps attached to a solid matrix or bounded by a membrane, are subjected to varied and diverse factors which can be quite remote from the isolated and purified enzyme as studied by conventional biochemistry and enzymology. The use of histochernical means of detection provides information relating to the natural activity of a specific enzyme acting within its personal environment of a particular cell.

Indeed, the realisation that histochernical means may be employed in a method for the detection of a cancer in a sample of tissue is an important feature of the present invention.

In addition, as enzyme activity may be detected via quantitative histochernical means only a small sample of cells is required. Consequently, needle and punch biopsies as well as cervical smears provide suitable sample sizes which may be used by the methods of present invention. Moreover, viable results may be obtained from a sample size comprising as little as 20 cells or even less.

The following enzymes in particular may be detected by the methods of the present invention, namely phosphatases, esterases, cytochiomes, oxidases, cholinesterases, monoamine oxidases, glucos-6-phosphatase, acid phosphatases, alkaline phosphatases, ATPase, ADPase, phospholipases, acid deoxyribonuclease, acid ribonuclease, cathepsin, &glucuromidase, 9-Nacetyl glucosamidase, arylsulphatase, phospho-protein phosphatase, MADase, NAIDase, NAIDPase.

In a preferred embodiment of the present invention there is provided at least one means of effecting a dye and/or a stain to the sample being tested.

The preferred means of effecting a dye and/or a stain is by the inclusion of a diagoniurn salt, particularly nuclear fast red, fast red RL, fast black K, fast garnet GBC, fast red LTR, fast violet B, fast blue VIRT, fast black B, 5- chloro-o toluidine, fast red-violet L, fast red RR, fast red RC, fast red TR, HNF, fast red ITR, Mayer's haemalum, methyl green, carmalum and/or; acetylthiocholine iodine; tetranitrio-blue tetrazolium; sodium sulphate saturated with copper thiocholine/copper sulphate saturated ammonium sulphate solution; cobalt chloride/yellow ammonium sulphate; eosin; lead nitrate/yellow ammonium sulphate; sodium salt/yellow ammonium sulphide.

Ideally, the percentage presence of a colour in a sample which has been tested by a method of the present invention is used to determine a result.

The following percentages of presence of a colour may be used to determine a result from a sample:

0-20% normal cells; 50-100% pre-cancerous cells; 80-100% cancerous cells. Preferably the following percentages of presence of a colour may be used to determine a result from a sample:

0-10% normal cells; 70-100% pre-cancerous cells; 90-100% cancerous cells. Most preferably the following percentages of presence of a colour may be used to determine a result from a sample:

0-2% normal cells; 80-100% pre-cancerous cells; 98-100% cancerous cells. Alternatively, a specific colour or colours produced in a sample which has been tested by a method of the present invention may be used to determine a result.

-8 As a further alternative, the result may be determined by the percentage presence of a colour in combination with a specific colour or colours produced by a method of the present invention.

It is advantageous to be able to determine a result of the present invention by the identification of a percentage of presence of a colour and/or by the production of a specific colour as the margin for human error is dramatically minimised over known methods of detection. Consequently, the probiern of mis diagnosis which is commonly associated with the known methods is substantially removed with the methods of the present invention. Furthermore, as significantly less interpretive ability is required to determine a result than required with the known methods, a suitably trained technician may be employed to perform this determination.

In a further preferred embodiment of the present invention enzyme activity is substantially halted following removal of a sample of cells from a human or animal body. Enzyme activity may be substantially halted by freezing of the sample. Preferably the sample is frozen through exposure to liquid nitrogen. Although freezing is the preferred method of substantially halting enzyme activity, where the sample of cells is a smear, such as a cervical smear, it may be satisfactory if the smear if simply dried.

Preferably following the halting of enzyme activity the sample of cells is incubated in a pH controlled medium/fluid.

It is important for the pH of a medium/fluid to be controlled for a plurality of reasons. One such reason is that when the use of diazoniurn salts is employed the coupling of said salts is dependent on the pH. At higher levels of pH both ends of the diazonium salt molecule are able to react whereas at lower pH levels only one end of the salt molecule is available for coupling.

In the case of bis-diazoniurn salt in particular, the salt acts like many mono-diazonium salts and produces a red dye on coupling with --napthol.

In order that the methods of the present invention may be more readily understood several examples thereof will now be described by way of example only. It is to be noted that at least some of the following examples are also described by Pearse et al. (3 d Edition 1968).

Preparation of Sections of Tissue Samples To prepare sections of tissue samples it is necessary to halt enzyme activity, which may involve autolysis, and to harden the sample so that said sample is suitable for sectioning.

A commonly available technique is the cryostat technique whereby liquid nitrogen is used to instantly freeze the sample. This technique provides a tissue sample in which enzyme activity has been halted and which is hard enough to be sectioned.

_10 Alternatively, where the tissue sample is a smear sample it is sufficient if the sample is left to air dry.

With regard to the frozen sections, it is necessary to fix the samples into a form suitable to undergo a method of the present invention. The sample may be sliced into thin sections (2-4mm) and fixed in 10% neutral formalin.

Alternatively, the frozen samples may be cut into thin sections (10-1 5Am) thick and mounted ont o a glass slide without adhesive followed by drying for 2 to 3 minutes at room temperature to ensure adhesion. A further alternative is to use cold microtome sections mounted on coverslips, fresh (or post-fixed) in cold acetone or cold Wolman's fixture.

Once the tissue sample has been fixed it is necessary to incubate the sample in a suitably buffered incubation fluid.

Any suitable incubation method may be employed and one such method is the open ring technique. This technique uses a fixed sample or smear on a glass slide, the sample is subsequently covered by an open perspex ring which is just large enough to encircle the sample. The perspex has a depth of approximately 3mm and is held in engagement with the slide by a film of vaseline.

The perspex is then filled with an incubation fluid and inserted into an incubation box, hot room, or any other incubation means, which is set to _11approximately body temperature ie. approximately 37'C.

Example 1 - Standard Coupling Azo Dye Technique for Acid Phosphatase (1) Incubate sample of tissue at 37'C in the incubation fluid comprising: 1020mg of sodium --naphthyl phosphate in 20ml of 0. 1 m veronal acetate buffer at pH 5.0 (alternatively 0. 1 M acetate buffers may be used).

(2) Add 1.5g polyvinyl pyrididone and allow to dissolve.

(3) Add approximately 20mg of the stable diazotate of o-amino azototase or diethyl sulphamino-o-anisidine (fast red TTR), followed by shaking and filter onto dry sample sections.

(4) Incubate for 30 seconds-dog postate; or 30 to 60 minutes-rat liver.

(5) Wash in running tap water for 2 minutes.

(6) Counter stain in Mayers Haemalium and stand for 1 to 2 minutes.

(7) Wash in running water.

(8) Mount if necessary in glycerine jelly.

Result Sites of acid phosphatase activity are coloured reddish-brown. The nuclear appear deep blue.

-12Example 2 - Modified Coupling Azo Dye Technique for Alkaline Phosphatase (Cold formalin or fresh frozen sections; freeze-dried paraffin sections) Frozen Sections Method.

(1) Fix thin slices of tissues in 10 per cent neutral formalin at 40 for 10-16 hours. Alternatively use fresh frozen cold microtome sections, mounted on cover slips.

(2) Cut frozen sections 10-15,u thick and mount on clean slides without adhesive.

(3) Allow to dry in air for 1-3 hours to ensure adherence.

(4) Dissolve 10-20mg, sodium -naphthyl phosphate in 20ml 0.1 M stock "tris" buffer (pHlO). Add 20 mg of the stable diazotate of 5-chloro-o toluidine and stir well. Filter on to the slides sufficient to cover each section adequately and incubate at room temperature (17-22') for 15 60 minutes. (Alternatively, use the same quantity of the stable diazotate of 2-benzoylamino-4-methoxy toluidine, or of p-p'- diaminodiphenylamine, and proceed in the same manner).

(5) Wash in running water for 1-3 minutes.

(6) Counterstain in Mayer's haernalum, 1-2 minutes.

(7) Wash in running water for 30-60 minutes.

(8) Mount in glycerine jelly.

The pH of the medium is lowered both by the addition of the substrate and also by the various diazotates.

-13Result The sites of alkaline phosphatase activity are coloured brown (or coloured black with alternative salt). Nuclei are dark blue.

Paraffin Sections (Cold acetone-fixed; paraffin embedded) Method (1) Bring sections to water via absolute acetone after removing the paraffin with light petroleum.

(2) Cover with freshly made and filtered substrate-diazonium salt mixture as above.

(3) Incubate for 30 minutes to 4 hours or for up to 12 hours depending on the salt used.

(4) Wash in water, counterstain as above and blue in running water.

(5) Mount in glycerine jelly.

Results The sites of alkaline phosphatase activity appear dark reddish-brown. Localization is excellent. Nuclei appear blue.

Example 3 - Azo Dye Methods for Leucocyte Phosphatase (Formal i nmethanol) Method (1) Prepare smears from capillary or sequestrene-venous blood.

(2) Allow the freshly made smears to dry in air for a few minutes.

(3) Fix for 30 seconds in ice-cold 10 per cent f ormalin in methanol (1 Oml of 40 per cent formaldehyde and 90ml of methanol).

(4) Prepare incubating solution At or B and filter directly on to the smears. I ncu bate 5-10 minutes at 22' (A) or 30 minutes (B).

(5) Wash running water.

(6) Counterstain with Mayer's haemalum for 1 1/2-3 minutes or with 2 per cent chloroform-washed methyl green for 2 minutes.

(7) Wash in running water.

(8) Mount in glycerine jelly.

Result Neutrophil granulocytes (40-90 per cent) show alkaline phosphatase activity as a brown, red or blue deposit.

With temperature above 220, diffusion and abnormal crystallization effects may be noted.

tlt is possible to remove the aluminium sulphate, present in most stable diazotates, by the addition of a few ml of a saturated aqueous solution of purpurin. The red aluminium lake is retained by a Whatman No.

paper. This procedure appreciably increases the speed of the reaction.

Removal of inhibitory zinc salts by means appreciably increases the speed of the reaction. Removal of inhibitory zinc salts by means of chelators such as dithizone unfortunately removes the diazotates also.

tTo 25ml 0-1m-"tris" buffer (pH 9.2) as 20mg sodium --naphthyl _15acid phosphate and 30 mg. Fast garnet GBC salt.

To 5ml "stock" naphthol AS-MX phosphate add 10mg. Fast violet LB salt or Fast blue RR salt.

Example 4 - Naphthol AS-Phosphate Azo Dye Method (Freeze-dried or cold acetone-fixed; paraffin sections) Method (1) Bring sections to water via xylene and acetone.

(2) Incubate for 5-30 minutes at 22' in 40ml stock solution (vide supra) containing 40mg of the chosen diazoniurn salt (Fast red-violet LB salt, Fast blue RR, red PIC and red TR are recommended).

(3) Wash briefly in water and mount in PVP medium.

Result Intensely coloured red or blue azo dyes indicate. sites of alkaline phosphatase activity.

Example 5 - Naphthol AS-Phosphate HNF Method (Freeze-dried paraffin sections; fresh cold microtome sections) Reagents Naphthol AS, AS-Bl, AS-MX, AS-CL or AS-TR phosphates may be used. HNF (Hexazotized New Fuchsin) is prepared as follows:

Solution A. Dissolve 4g New Fuchsin in 100ml 2N HCl at 600, filter. This solution is stable for some weeks.

-16Solution B. Dissolve 4g NaN021 in 100ml distilled water. Should be freshly prepared every 2 days.

Preparation of Incubating Medium Add 0.2ml freshly prepared HNF solution to 40ml 0.2M Tris-HCI buffer and adjust the pH to 8.8 to 9.2 by adding 1 N NaOH. To this add 5-1 Omg of the chosen substrate in 0.2ml dimethy1formamide. Filter and use immediately.

Method (1) Bring paraffin sections to water via xylene and acetone.

(2) Incubate sections in the freshly prepared medium for 2-60 minutes.

(3) Wash briefly in water and mount in a suitable watery medium.

Result Alkaline phosphatase appears in different shades of red. With the same substrate the colour may differ appreciably in different sites.

Prevention of Gas Bubbles in AZO Dye Methods Method (1) Following incubation by any of the recognized techniques, immerse the slides in a dilute solution of gelatine for 2 minutes, with gentle 20 movement. (2) Remove slides and stand them on their long edge to drain. (3) Leave to dry for 20-60 minutes. (4) If required for critical microscopy or photomicrography apply a coverslip after application of a few drops of a suitable aqueous mounting medium.

Example 6 - Standard Naphthol AS Phosphate Method (Freeze-dried or cold acetone fixed; paraffin sections) Preparation of Incubating Medium Dissolve 4mg naphthol AS-BI phosphate in 0.25ml dimethyl-formamide and add 25ml. 0.2 m-acetate buffer, pH 5.2-5.6. add 35mg of a suitable diazonium salt (Red-violet LB salt) and 2 drops of 10%. MnCl2 shake and f ilter.

Method (1) Incubate sections, after removal of wax, for 1/2-6 hours at 370.

(2) Wash in running wash.

(3) Counterstain with Mayer's haernalum, 1 % minutes or with carmalum for a similar time.

(4) Wash in running water.

(5) Mount in glycerine jelly or in Burstone's PVP mounting.

Resu It Sites of acid phosphatase activity appear in various shades of red or blue. Nuclei appear blue or red.

Example 7 - --Naphthyl Acetate Method for Esterase The following example is suitable for use with frozen sections; smears -18cold formalin, cold acetone, paraffin sections, cold microtome formalin post fixed sections Standard Method (1) Cut frozen sections 10-1 5gm thick and mount on clean slides. Dry in air to ensure adherence.

(2) Incubate for 1 to 15 minutes at room temperature in the following medium: Dissolve 10 mg -naphthyl acetate in 0.25ml acetone and add 20ml O.1M phosphate buffer (pH 7.4). Shake thoroughly until most of the initial cloudiness disappears. Add 50-100mg fast blue B salt, shake, and filter directly on to the sections. These should be dry if frozen sections but brought to water and left wet if paraffin sections.

(3) Wash in running water, 2 minutes.

(4) Counterstain in Mayer's haernalum, 4-6 minutes.

(5) Wash in running water for at least 30 minutes.

(6) Mount in glycerine jelly.

Result Esterase activity appears black. Nuclei appears dark blue, (Lipase, AchE and ChE can also hydrolyse a-naphthyl acetate and appear black). HPR Method Preparation of Incubation Medium. For preparation of hexazotized pararosanilin. Add to 7.5ml 0.2 M-phosphate buffer stock solution (Na2HP04) 2.5ml distilled water and 0.25ml substrate solution (1 % naphthyl acetate) in acetone. Shake and add 0.8ml of HPR.

The pH of this mixture is about 6.5, at which it can remain. It should be filtered immediately before use. Incubation (1) Incubate for 10-30 minutes at room temperature.

(2) Wash briefly in water.

(3) Counterstain nuclei in Mayer's haernalum for 4-6 minutes.

(4) Wash in running water for 30 minutes.

(5) Dehydrate in alcohols, clear in xylene and mount in DPX or other synthetic resin.

Result Esterases appear brick red. Nuclei appear blue.

Example 8 - Indoxyl Azo Method for Esterases Preferably used with frozen sections or cold formalin sections Preparation of Tissues Fix small tissue blocks for 12-24 hours in cold 4% formol-calcium.

Transfer to gum-sucrose for 24 hours at 4'. Wash for 5 minutes in water, blot dry and cut cryostat sections at 5-10gm.

Preparation of the Incubating Medium (pH 6.1) Dissolve 1 mg 0-acetyl-5-bromoindoxyl (alternatives are the 4-chloro-5 bromo and 4-chloro-5-bromo-7-methyl-substituted indoxyl acetates) in 0.1 ml ethanol and add to 5ml hexazotized pararosanilin stock solution mixed with 0.1 25ml freshly prepared 4 per cent sodium nitrite solution and allow to stand -20for 1 minute before adding to the rest of the medium. Finally, add 11 mg calcium chloride and adjust the pH to 6.1 by adding sufficient 0.1 N NaOH.

Filter before use.

Method (1) Incubate sections at 370 for 15-20 minutes.

(2) Wash well in distilled water.

(3) Counterstain nuclei with 2 per cent chloroform-washed methyl green for 5 minutes.

(4) Wash briefly in tap water.

(5) Dehydrate in alcohols, clear in xylene and mount in a suitable synthetic resin.

Result A dark brown indoxylazo dye is deposited in sites of esterase activity.

Nuclei appears deep green.

Preparation of Incubating Medium (pH 8.8) Dissolve 1mg 0-acetyl-5-bromoindoxyl in O.1mI ethanol and add to 9.5ml Tris-HCI buffer (pH 8.8). Add 11 mg calcium chloride (optional) and 1 Omg. Fast blue RR salt. Adjust pH to 8.8 with 0. 1 N NaOH. Filter before using.

Method (1) Incubate sections at 37'for 15-30 minutes.

(2) Wash well in distilled water.

(3) Counterstain nuclei in carmalum for 5 minutes.

(4) Wash briefly in tap water.

(5) Mount in glycerine jelly.

Result Nuclei appears red Esterase activity appears brown.

Example 9 - Method for PhospholiDase Q Cryostat sections post-fixed in cold formulin are preferred.

Preparation of Solutions and Reagents Stock Tris Buffer. Dissolve 36.3g reagent grade Tris in 400ml distilled water and add 100ml 3N acetic acid and make up to 1000ml.

Stock Incubating Solution A. Add 1 00ml stock Tris buffer to 1 50ml water. Dissolve 3g cobalt acetate (4H20)with stirring and add 0. 1 N NaO H to restore the original pH. Dissolve 24g sucrose and bring to boil volume to 270ml.

Preparation of Lysolecithin. Dissolve 2.5g lecithin in 1000ml ethyl ether. Add 10mg lyophilized Agkistrodon piscivorus piscivorus venom dissolved in 110ml 8mM CaC'2,with vigorous stirring. Continue stirring for a few minutes and allow to stand overnight. Separate precipitate by centrifuging, and wash 3 times with ethyl ether and then with acetone.

Dissolve the product in hot absolute ethanol. Centrifuge and recrystallize from ethanol. Commercial lysolecithin may be used if available.

Lysolecithin Stock Solution. Dissolve a small quantity of lysolecithin in 50ml water by heating to 40'. Dialyse overnight against distilled water.

Estimate organic phosphorus content of the solution and adjust to 20 4moles/ml.

Nuclear Counterstain. 0. 1 g Nuclear Fast red in 1 00ml of 5% aluminium sulphate.

Incubating Medium. Add 5ml lysolecithin stock to 40-45ml stock solution A.

Method (1) Incubate for 30-60 minutes.

(2) Rinse twice in 0.9 per cent NaCl.

(3) Immerse in dilute ammonium sulphide at 4' for 15 seconds.

(4) Rinse twice in 0.9 per cent NaCl.

(5) Counterstain in Nuclear Fast red, 2 minutes, and wash briefly.

(6) Dry in air and mount in a synthetic resin.

Result Brownish-black deposits indicate activity of phospholipase B. Example 10 - The Acetylthiocholine Method for Cholinesterases Suitable for use with frozen sections, cold formulin, cold microtome, free floating sections.

Preparation of Stock Solutions Copper sulphate (CuS04.5H20) 0.3g Glycerine 0.375g Magnesium chloride (MgC'2.6H20) 1.0g -23 Maleic acid 1.75g NaOH (4 per cent) 30ml Na2SO4 (40 per cent; hot saturated) 170 ml This solution keeps indefinitely (pH 6.0) Preparation of Incubating Medium Dissolve about 20mg acetylthiocholine iodine in a few drops of water.

Add 10 ml stock solution.

Method (1) Incubate sections for 10-60 minutes at 37'.

(2) Rinse in 3 changes of saturated Na2SO4.

(3) Treat with dilute yellow ammonium sulphate, 2 minutes.

(4) Wash briefly and mount in glycerine jelly.

Result Sites of cholinesterase activity appear in shades of brown.

Examr)le 11 - The Acetyl - and Butrylthiocholine Method for Cholinesterases It is preferable for fresh solution to be used for this method.

Preparation of Solutions Storage Solutions (1) DFP Distilled Water 4.5ml per cent Na2SO4 9.Oml 1 uM-DFP 1.5ml -24The 14M-DFP solution was prepared within 30 minutes of the time of use by serial dilution of a 0.1 M stock solution in propylene glycol.

(2) Storage Solution C Distilled water 4.5ml 40% Na2SO4 10.5) m 1 (3) Storage Solution B-D, Distilled water 9.Oml Storage solutions are kept at 35' and used at this temperature.

Incubating Solution The following Table gives the composition of the final incubation media for localization of AChE and ChE.

Solution Enzyme Cu-GI H 0 Mal Na 2S04 M9Cl 2 CuThCh AThCh BuThCh Localized mi. MI. MI. MI MI mi MI B AChE 0.6 2.1 1.5 9.0 0.6 trace 1.2 - C ChE 0.6 0.6 1.5 10.5 0.6 trace - 1.2 D Control 0.4 1.4 1.0 6.0 0.4 trace - 0.8 All solutions allowed to incubate 15 minutes after addition of CuThCh; substrates (AthCh and BuTuCh) added immediately prior to filtering and introducing slides.

The solutions from which the final incubating media are made up are composed as follows:

(1) Copper-glycerine (Cu-Gl): 3.75g glycine 2.5g CUS04, 51-120, distilled water 1 00ml. (2) Maleate buffer (Mal) 9.6g sodium hydrogen maleate, 52.2ml NaOH,

distilled water to 100ml.

(3) Sodium sulphate (Na2SO4): 40 per cent (W/v) NA2SO4 adjusted to pH 6.0, stored at 37'.

(4) Magnesium chloride (MgC'2): 9.52gMgC'2 in 100ml distilled water.

(5) Acetylthiocholine (AthCh): 23mg acetylthiocholine iodine, 1-2ml distilled water, 0.4ml m-CuS04, 5H20; centrifuged and the supernatant decanted for use.

(6) Butyrylthiocholine (BuThCh): 43mg butyrylthiocholine iodine, 1.8ml distilled water. 0.6ml O"M-CUS04, 5H20; centrifuged, etc., as above.

(7) Copper thiocholine (CuThCh): This is initially prepared by alkalinizing a solution of AthCh in copper-glycerine solution to pH 12.0 with KOH, allowing to stand overnight at room temperature, collecting the precipitate and washing with water. Subsequently, the incubating solutions, after use, are filtered, allowed to stand at 37) for 204 days to permit spontaneous hydrolysis of the substrate, and the precipitate is collected and washed with water.

Method (1) Following a minimal period of storage in the appropriate storage solutions, transfer the mounted sections into incubating solutions for 20 5-60 minutes at 37' (average time, 30 minutes). (2) Transfer to rinsing solution 1 (20 per cent Na2SO41 saturated with CuThCh) for 5 minutes.

(3) Transfer to rinsing solution 11 (10 per cent Na2SO4, saturated with CuThCh) for 1 minute.

(4) Transfer to rinsing solution III (water, saturated with CuThCh) for 1 minute.

(5) Treat with CuS-saturated ammonium sulphide solution (dilute yellow ammonium sulphide treated with a few drops of CUS04, shaken and filtered) for 20 seconds.

(6) Rinse rapidly in water.

(7) Fix for 30 minutes in 10 per cent neutral formalin, saturated with CuS.

(8) Dehydrate in CuS-saturated alcohols and clear in CuS-saturated xylene.

(9) Mount in Canada balsam.

Result A dark brown precipitate indicates sites of chlorinesterase activity. In sections incubated with solution B this indicates AchE, in those incubated with solution C, this indicates ChE. Control sections treated with DFP and incubated with solution D should be blank.

Example 12 - Naphthoic Hydrazine Method for Monoamine Oxidase Suitable for use with, particularly, cold microtome, fresh frozen sections mounted on glass slides.

Preparation of Stock Solutions (1) 0. 1 M-hydrazine hydrochloride.

(2) 40% NaS04 (w/v), adjusted to pH 8.6 with NaOH.

(3) 0.2 M-Na2HP04 (buff e r).

-27(4) 2-Hydroxyl-3-naphthoic acid hydrazide (add to final medium).

(5) M-isonicotinyl-2-isopropyl hydrazine phosphate (Marsilid).

(6) 0.1 M-tryptamine hydrochloride. (7) 1 N NaOH.

Preparation of Working Solutions (1) Pre-incubating medium 3.Oml water, 1.5ml hydrazine, 3.Oml buffer 7.5ml Na2SO4 (pH should be 7.6).

(2) Control pre-incubating medium. As above with 0.1 5ml Marsilid.

(3) Rinse 4.5ml water 0.3ml buffer and 7.5ml Na2SO4' (4) Incubating medium 4.35ml water, 0.15ml NaOH, 3.Oml buffer, 7.5ml Na2SO4. Heat to 80-90' and saturate with naphthoic acid hydrazide.

Cool, filter and add 1.0ml typtamine. Saturate with 02 immediately before use.

(5) Developing medium 300mg. Fast blue B salt, dissolve in 10ml water with 5ml phosphate buffer, pH 7.4. Filter and use at once.

Method (1) Immerse sections in pre-incubating medium for 1 hour at 22(,.

(2) Rinse in buffered sodium sulphate medium.

(3) Blot, dry, and immerse in incubating medium for 2 hours at 37'. During incubation a streamof 02must be passed through the medium.

(4) Rinse in distilled water.

(5) Develop in Fast blue B solution for 3 minutes at room temperature.

(6) Rinse is distilled water.

(7) Fix in 10% formalin for 1 hour.

(8) Mount in glycerine jelly.

Result A purplish-blue colour indicates sites of MAO activity. Control sections pre-incubated in Marsilid and incubated in the presence of this inhibitor should show no colour. Control sections incubated in the absence of substrate should likewise show no colour.

Example 13 - Calcium Method for Adenosine Triphosphate Suitable for use with, particularly, unfixed frozen sections.

Preparation of Incubating Medium This must be prepared immediately before use, heated to 37' for 1 hour filtered, and finally adjusted to pH 9.9 with molar KOH..

0.1 M-glycine &0.4 M-KCI) in saturated sodium acetate 12ml Saturated sodium acetate 1 2ml 0.36 M-CaCl2 3.6ml 1 M-KOH 0.6ml 0.04 M-sodiurn adenosine triphosphate ([\IaPO4) 6ml Distilled water 1 3.8ml Saturated sodium phosphate (NaP04) 0.3ml Method (1) Incubate free-floating sections (10-15 um) for 5 minutes to 3 hours. (2) Wash in three changes of 1 %. CaCl2 in 75 per cent ethanol.

(3) Transfer to 2%COC'2 for 3 minutes.

(4) Develop in 1 % yellow ammonium sulphide.

(5) Wash, dehydrate and mount in a suitable synthetic medium.

Result ATPase activity shows as a blackish-brown deposit.

Example 14 - Calcium Method for Adenosine Triphosphate Suitable for use with, particularly, unfixed frozen sections; free- floating or attached to cover slips.

Preparation of Incubating Medium This solution must be freshly prepared.

0.1 M-sodium barbiturate (2.062g/100mi) 20ml 0.18 M-CaC12 (1.998g/l 00ml) 1 Oml Distilled water 30ml Adenosine triphosphate (disodium salt)t 152ml As soon as the ATP is dissolved, adjust the pH to 9.4 with 0.1 M-NaOH and make up to 100ml with distilled water. Filter if turbid. Method (1) Incubate sections for 5 minutes to 3 hours at 37'.

(2) Wash in three changes of 1 % CaC12.

(3) Transfer to 2% CoC12, 3 minutes.

(4) Wash in distilled water for 1 minute.

(5) Develop in dilute yellow ammonium sulphide.

(6) Wash, counterstain nuclei if desired (carmalum) or background (eosin).

Dehydrate, clear, and mount in a suitable synthetic medium.

tFinal concentration, 0.005 M.

tFinal concentration, 0.005 M.

Result Black deposits indicate ATPase. Localization is good. (Whenever formalin fixation is employed, in conjunction with this method, it is advisable -to use formaldehyde made by dissolving paraformaldehyde in appropriate concentration. Methanol (contaminant in formalin) inhibits ATPase).

Example 15 - Lead method for Adenosine Triphosphate Suitable for use with, particularly, unfixed frozen sections, free- floating or mounted, or formalin-fixed frozen sections. Preparation of the Incubating Medium Final concentration 20ml 125mg % ATP (disodium salt) 8.3 X 10_4 M ml 0.2 M "Tris"-maleate buffer (pH 7.2) 8 X 10_2 M 3ml 2 % Pb(N03)2 3.6 X 10_2 M Ml 0.1 M-MgS04 1 X 10_2 M 2ml Distilled water Add constituents in the above order and adjust the pH if necessary.

Method (1) Incubate fresh and formalin-fixed sections for 5-60 minutes.

(2) Rinse in distilled water.

(3) Develop in 1 % yellow ammonium sulphide, 1 minute.

(4) Rinse again in water.

(5) Mount in glycerine jelly.

Result Brownish-black deposits indicate ATPhase activity.

Example 16 - Method for Sodium Activated Adenosine Triphospate Suitable for use with, particularly, frozen sections, cold from Aldehyde.

Preparation of Solutions 0.01 M ATP. Dissolve the barium salt of ATP in distilled water acidified to pH 3.1 with 0.1 N HCL Preparation of Medium Final Concentration 1.5ml 0.01 M ATP (Ba salt) 3mM 1.4ml distilled water 0.6ml Tris buffer, pH 7.8, 0.2M 24mM 0.5ml 1 % lead nitrate (0.03M) 3mM 1.0ml magnesium sulphate (0.015 M) 3mM The final pH should be 7.2. Centrifuge to remove precipitate. Place half of the medium into each of two containers.

To one, add 15mg NaCl 1 00mm and 5mg KCI 30mM Method (1) Incubate free floating sections in medium, containing Na and K and in control medium (Na/K free), for 10-15 minutes at 37' (2) Rinse briefly and treat with yellow ammonium sulphide. (3) Rinse and mount in glycerine jelly.

Result Black deposits indicate enzyme activity.

Example 17 - Method for-Glucose-6-PhosDhate Suitable for use with, particularly, cold montome free-floating sections.

Method (1) Incubate 10-15 gm sections for 5-15 minutes at 32' in a substrate mixture consisting of 20ml of a 125mg % solution of potassium glucose-6-phosp hate, 20ml of 0.2 M - "Tris" buffer (pH 6.7), 3ml of 2 % lead nitrate, and 7ml distilled water.

Alternatively Incubate 10,um cryostat sections, mounted on slides and post-f ixed in 3% neutral f ormalin for 1 hour at 0', for 15 minutes at 40' in a solution containing:

mM Tris buffer, pH 6.7.

mM Lead nitrate.

6.4 mM Glucose-6-phosphate (K salt).

(2) Wash in distilled water.

(3) Develop in dilute yellow ammonium sulphide.

(4) Wash in water.

* (5) Post-fix in 6% neutral formaldehyde.

(6) Mount in glycerine and ring the coverslip with nail polish.

Result Brownish-black deposits indicate sites of glucose-6-phosphatase activity.

Example 18 - Method of Alkaline Nucleoside Phosphatase Suitable for use with, particularly, cryostat sections, briefly post- fixed in formalin at 00 Preparation of Incubating Medium Make up a solution containing 5ml 100 mM sodium barbital, 3ml 180 mM CaCl2, 7ml water, 5 mM uridine cliphosphate or inosine cliphosphate or uridine triphosphate, 5 ml 0. 1 N NaOH. Adjust pH with 1 N NaOH or 1 N HCI to 9.2 add water to bring volume to 25ml.

Method (1) Incubate sections for 5-20 minutes at 40' (2) Wash briefly in water.

(3) Apply Cobalt-Sulphicle procedure.

(4) Rinse, dehydrate, clear and mount.

Result A black or brown deposit indicates enzyme activity.

It is to be understood that the above described specific examples are by way of illustration only. Many modifications and variations are possible.

Claims (18)

1 A method of detection of a cancer in a sample of biological material comprising colour screening.

2. A method of detection of a cancer in a sample of biological material comprising the detection of pre-cancerous cell(s) by colour screening.

3. A method according to claim 1 or claim 2, wherein the colour screening is used to detect for evidence of enzyme activity.

4. A method according to claim 3, wherein the evidence of enzyme activity is detected by histochemical means.

5. A method according to claim 3 or claim 4, wherein the evidence of enzyme activity is detected by qualitative histochemical means.

6. A method according to claims 3 to 5, wherein the detection of enzyme activity is of at least one enzyme selected from a phosphatase, an esterase, a cytochiome, an oxidase, a cholinesterase, a monoamine oxidase, a glucos-6-phosphatase, an acid phosphatase, an alkaline phosphatase, ATPase, ADPase, a phospholipase, acid deoxyribonuclease, acid ribonuclease, cathepsin, 9-glucuromidase, 9N-acetyl glucosamiclase, an arylsulphatase, a phospho-protein phosphatase, MADase, NADase, NADPase

7. A method according to any preceding claim, wherein there is provided at least one means of effecting a dye and/or a stain to a sample of biological material.

8. A method according to claim 7, wherein the dye and/or stain is a ?0 diazonium salt; acetylthiocholine iodine; tetra n itrio-blu e tetrazolium; sodium sulphate saturated with copper thiocholine/copper sulphate saturated ammonium sulphate solution; cobalt chloride/yellow ammonium sulphate; eosin; lead nitrate/yellow ammonium sulphate; sodium salt/yellow ammonium sulphide.

9. A method according to claim 8, wherein the diazonium salt is nuclear fast red, fast red RL, fast black K, fast garnet GBC, fast red LTR, fast violet B, fast blue VRT, fast black B, 5-chloro-o-toluidine, fast redviolet L, fast red RR, fast red RC, fast red TR, HNF, fast red ITR, Mayer's haemalum, methyl green, carmalum.

10. A method according to any preceding claim, wherein percentage C presence of a colour is used to determine a result.

11. A method according to any preceding claim, wherein the following percentage presence of colour is used to determine a result:

0-20% normal cells; 50-100% pre-cancerous cells; 80-100% cancerous cells.

12. A method according to claims 1 to 10, wherein the following percentage presence of colour is used to determine a result:

0-10% normal cells; 70-100% pre-cancerous cells; 90-100% cancerous cells.

13. A method according to claims I to 10, wherein the following percentage presence of colour is used to determine a result: 0-2% normal cells; 80-100% pre-cancerous cells; 98-100% cancerous cells

14. A method according to any preceding claim, wherein at least one specific colour is produced in order to determine a result.

15. A method according to any preceding claim, wherein the percentage presence of colour in combination with a specific colour(s) produced is used to determine a result.

16. A method according to any preceding claim, wherein enzyme activity is substantially halted following removal of a sample from a human or animal body.

17. A method according to claim 16, wherein enzyme activity is substantially halted by freezing the sample.

18. A method according to claim 16 or claim 17, wherein once the enzyme activity has been substantially halted the sample is incubated in a pH controlled medium/fluid.