GB2372811A - Staining physiological samples - Google Patents
Staining physiological samples Download PDFInfo
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- GB2372811A GB2372811A GB0203523A GB0203523A GB2372811A GB 2372811 A GB2372811 A GB 2372811A GB 0203523 A GB0203523 A GB 0203523A GB 0203523 A GB0203523 A GB 0203523A GB 2372811 A GB2372811 A GB 2372811A
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- staining
- stain
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- haematoxylin
- alcohol
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- 238000010186 staining Methods 0.000 title claims abstract description 64
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims abstract description 28
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000003745 diagnosis Methods 0.000 claims abstract description 8
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims abstract description 8
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 claims abstract description 6
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims abstract description 6
- WLKAMFOFXYCYDK-UHFFFAOYSA-N [5-amino-4-[[3-[(2-amino-4-azaniumyl-5-methylphenyl)diazenyl]-4-methylphenyl]diazenyl]-2-methylphenyl]azanium;dichloride Chemical compound [Cl-].[Cl-].CC1=CC=C(N=NC=2C(=CC([NH3+])=C(C)C=2)N)C=C1N=NC1=CC(C)=C([NH3+])C=C1N WLKAMFOFXYCYDK-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229940032753 sodium iodate Drugs 0.000 claims abstract 2
- 235000015281 sodium iodate Nutrition 0.000 claims abstract 2
- 239000011697 sodium iodate Substances 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 30
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 8
- 238000009595 pap smear Methods 0.000 claims description 5
- 230000000750 progressive effect Effects 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000002253 acid Substances 0.000 abstract description 4
- 229910052808 lithium carbonate Inorganic materials 0.000 abstract description 4
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 abstract description 3
- 230000001476 alcoholic effect Effects 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 2
- ZXRRHFSTAFVGOC-UHFFFAOYSA-N [AlH3].[K] Chemical compound [AlH3].[K] ZXRRHFSTAFVGOC-UHFFFAOYSA-N 0.000 abstract description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 abstract 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 abstract 1
- 229960002327 chloral hydrate Drugs 0.000 abstract 1
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 8
- 238000012216 screening Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 206010008342 Cervix carcinoma Diseases 0.000 description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 3
- DGOBMKYRQHEFGQ-UHFFFAOYSA-L acid green 5 Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 DGOBMKYRQHEFGQ-UHFFFAOYSA-L 0.000 description 3
- 239000012670 alkaline solution Substances 0.000 description 3
- 201000010881 cervical cancer Diseases 0.000 description 3
- KXTYBXCEQOANSX-WYKQKOHHSA-N fusicoccin Chemical compound O([C@H]1[C@H](O)[C@H](C)[C@@H]\2CC[C@@H](C/2=C/[C@@]2(C)[C@@H](O)CC(=C21)[C@H](C)COC(C)=O)COC)[C@H]1O[C@H](COC(C)(C)C=C)[C@@H](O)[C@H](OC(C)=O)[C@H]1O KXTYBXCEQOANSX-WYKQKOHHSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000007447 staining method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000016942 Elastin Human genes 0.000 description 2
- 108010014258 Elastin Proteins 0.000 description 2
- 210000003679 cervix uteri Anatomy 0.000 description 2
- 230000001934 delay Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 229920002549 elastin Polymers 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229940101209 mercuric oxide Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000010936 aqueous wash Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- MPBRYMWMMKKRGC-UHFFFAOYSA-M carbocyanin DBTC Chemical compound [Br-].C1=CC=CC2=C([N+](=C(C=C(C)C=C3N(C4=C5C=CC=CC5=CC=C4S3)CC)S3)CC)C3=CC=C21 MPBRYMWMMKKRGC-UHFFFAOYSA-M 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000009429 distress Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000007431 microscopic evaluation Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 230000001373 regressive effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012859 tissue stain Substances 0.000 description 1
- HFFLGKNGCAIQMO-UHFFFAOYSA-N trichloroacetaldehyde Chemical compound ClC(Cl)(Cl)C=O HFFLGKNGCAIQMO-UHFFFAOYSA-N 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
At least one stain selected from Haematoxylin HR3, OG9 or EA66 is used to stain samples. Particular utility is in cell staining for cancer diagnosis. Haematoxylin HR3 stain comprising haematoxylin, aluminium potassium phospahte, sodium iodate and chloral hydrate in water is claimed as new as is stain EA66 comprising fast green, eosin, Bismarck brown, phototungstic acid and lithium carbonate in alcohol. Stain OG9 typically contains alcoholic orange G and phototungstic acid in alcohol.
Description
"STAIN" The present invention relates to stains for physiological samples, and in particular relates to physical stains.
A wide variety of stains and staining techniques are known for the staining of physiological samples.
Staining is commonly required in order to achieve contrast within different parts of the tissues, which may consist of particular types or mixtures of various types of cells, to facilitate microscopic analysis thereof.
Staining is therefore frequently used not only in research but also to provide diagnosis of pathological conditions. For example, where a malignant tumour (such as cervical cancer) is suspected a sample, such as a sample of exfoliated cells, may be taken, stained as appropriate, and viewed by an experienced cytopathologist. The
particular staining technique used is selected in order to differentiate between the malignant or cancerous cells, and the normal cells of the body (for example in cervical cancer, the normal cells exfoliate from the uterus and/or cervix will be examined). Early diagnosis is of course extremely beneficial in providing treatment before malignancy becomes advanced.
A histochemical stain relies on chemical interaction between a component in the stain and a component in the tissue. A physical stain relies only upon the affinity of the cell or a part of a cell to absorb and retain a particular dye. Some tissues, for example elastin fibres, have an affinity for a particular stain and will retain such a stain despite repeated differentiation.
In several countries routine screening for cervical cancer takes place on a regular basis. Currently the staining technique used for the cervical cells is known as Papanicolaou staining method. Several problems with the cervical smear screening programme have lead to delays of several weeks or even longer before the results are available, and such delay is generally viewed as unacceptable.
For cervical smear screening the doctor or nurse will remove a sample of the cell from the cervix and fix the cells onto a microscope slide. In the
United Kingdom usually one smear is taken, but this
may vary from country to country, and for example, in Sweden two samples are taken from each patient.
The samples are then sent to a screening facility, which is generally provided in a specialised unit normally located in a local hospital. Papanicolaou staining of the cervical cells is generally conducted by machine and the process takes approximately 20 minutes. Up to 100 slides can normally be stained at any one time.
These staining machines generally cost about 6600 plus VAT and this cost inhibits local doctors' practices from being able to provide a staining facility at the clinic attended by the patient.
After staining the slides are each screened individually, with possible positive slides being re-examined by more experienced personnel.
Generally any potentially positive result is screened at least three times before being passed as "positive". Even at the initial level, screening will take approximately 15-20 minutes for each slide. The staining of the sample is critical in achieving good contrast in the sample and facilitating the task of the screener.
If a positive result is recorded, or if the smear is not deemed to be normal, a repeat smear is requested without delay. The repeat smear will be screened in the normal way as a safeguard against a false positive result. The current delays in examination and diagnosis of this second smear, particularly
where an initial positive has been obtained, are unacceptable and are not in the best interest of the patient for whom early treatment of any condition is extremely desirable. It would be highly advantageous for a simple, rapid and reliable staining method to be possible so that the stained slides for any particularly urgent sample could either be screened on site by a doctor or trained nurse immediately after the smear has been taken from the patient, or stained and sent directly to a cytopathologist for diagnosis.
The present invention provides a modification of the
Papanicolaou staining technique which allows staining to be achieved in a greatly reduced time span; for example less than 10 minutes and possibly even less than 5 minutes. The modified staining technique of the present invention permits good quality staining of the specimen which is essential to enable the cytoscreener to scan the sample and perform an accurate diagnosis.
Conventional Papanicolaou staining may be carried out as described in the literature, for example in "Histopathologic Technic and Practical Histochemistry"by R D Lillie, 3rd edition. Briefly, the fixed sample is stained using a haematoxylin for approximately 5 minutes followed by a series of washes in water and alcohol. The specimen is then stained in OG6, washed again in alcohols and finally stained using EA50. Again, excess stain will be removed by washes.
The present invention provides a method of staining a physiological sample, said method including a step of staining a sample with at least one stain chosen from the group comprising; 1. Haematoxylin HR3; 2. OG9 ; 3. EA66.
Desirably all of stains (1) to (3) above are used.
However, this is not essential to the present invention as benefits may be obtained by using only one of stains (1) to (3).
Preferably stains (1) to (3) are used sequentially.
Alternatively stains (1) to (3) are used together.
Preferably where stain (1) is used, progressive staining with Haematoxylin HR3 is carried out for between 10 and 50 seconds.
More preferably where stain (1) is used, progressive staining with Haematoxylin HR3 is carried out for approximately 30 seconds.
Preferably where stain (2) is used, staining with
OG9 is carried out for between 2 to 30 seconds.
More preferably where stain (2) is used, staining with OG9 is carried out for approximately 5 to 10 seconds.
Preferably where stain (3) is used, staining with EA66 is carried out for between 10 to 80 seconds.
More preferably where stain (3) is used, staining with EA66 is carried out for approximately 30 to 40 seconds.
In the conventional Papanicolaou staining Harris
Haematoxylin is used as the initial stain.
Haematoxylin by itself is not a powerful tissue stain. The function of the Haematoxylin is to stain the nucleus of the cells. Conventionally, the tissue samples are overstained with Haematoxylin so that both DNA in the nucleus and also the RNA in the cytoplasm become stained. The sample is then destained by washing excess Haematoxylin out of the sample through a series of washes until only the nuclei in the sample retain the stain (regressive staining technique). The washes are conducted using weak acid alcohol solutions.
The differentiation is controlled microscopically.
After the washing is complete and only the nucleus of each cell remains stained, the nuclei appear as a pale grey/blue colour. As soon as the sample is immersed in a weak aqueous alkaline solution, such as lithium carbonate, the stain in the nuclei appears as a deep blue/purple colour. This process is termed"blueing".
After staining with Haematoxylin, the tissues are then counter-stained with a contrasting colour, such as eosin. Eosin would stain all other tissue elements, for example red blood cells, muscle, collagen fibres, elastin fibres etc, in varying shades from red to pale pink. The cellular nuclei would remain a crisp dark blue colour.
With the Papanicolaou staining method, the
Haematoxylin is the basic stain and OG9/EA66 are the counter-stains. The nuclei of the exfoliated cells should be clearly visible to the screener in a good, well-balanced, stained sample.
The Harris Haematoxylin used in the prior art process has the further disadvantage of requiring mercuric oxide as a ripener. Mercuric oxide is undesirable (being a poisonous substance) and also because it forms a scum on top of the Haematoxylin stain necessitating filtering before use. This is time-consuming, especially where an urgent result is required.
Haematoxylin HR3 according to the present invention comprises: Haemtoxylin-3 grams
Aluminium Potassium Sulphate-50 grams
Sodium Iodate-0. 2 grams
Chloral Hydrate-50 grams Water-1000 ml (approx)
Different quantities may be produced by admixing the ingredients specified above in the ratio indicated. In the present invention the Haematoxylin is only in contact with the tissue long enough to stain the nucleus ; other tissue elements remain unaffected (progressive staining technique). Thus the need for washing off excess stain is avoided.
In the present invention the nucleus would still need to be"blued"to make it stand out clearly.
"Blueing"would occur in the conventional way by contacting the sample with a dilute aqueous alkaline solution, for example a solution of lithium carbonate, to make the stained nucleus stand out clearly.
The ingredient OG9 as used in the present invention is comprised of 0.6 to 1.5% preferably 0.6 to 0.9% by weight, especially 0.8% by weight, of orange G in 95% alcohol. For example a suitable formulation would be as follows:
10ml 10% alcoholic orange G ; 190ml absolute alcohol;
6mg of phosphotungstic acid.
The stain EA66 is a modified version of the conventional stain EA50 and may comprise light green
SF or fast green FSC, eosin and Bismarck brown.
Desirably equal amounts of light green SF or fast green FSC and eosin are present together with a small amount of Bismarck brown and phosphotungstic
acid. The amounts of green (whether light green SF or fast green FSC) must be carefully balanced with the eosin.
A suitable formulation of EA66 is given below:
7ml of 0.6% fast green in 95% alcohol
43ml of eosin in 95% alcohol 5ml of 0.5% Bismarck brown in 95% alcohol
5ml of 10% phosphotungstic acid in 95% alcohol
40ml of 95% alcohol
5 drops of aqueous saturated lithium carbonate
A solution of phosphotungstic acid in 95% alcoholic
solution should be prepared used lOg phosphotungstic acid per 100ml of 95% alcohol (or equivalent quantities thereof). 5ml of this phosphotungstic acid solution may then be added per 100ml of EA66 (i. e. the phosphotungstic acid solution constitutes 5% by volume of the final product). Phosphotungstic acid is important for imparting transparency to the green dye, enabling all cells of the sample to be viewed by the screener, even if some of the cells are layered on top of each other in the smear.
A further embodiment of the present invention provides a kit to stain physiological samples (in particular cervical smears or other samples which may contain cancerous or pre-cancerous cells), said kit comprising one or more of the following stains: 1. Haematoxylin HR3;
2. OG9 ; and 3. EA66.
Desirably the above components should be packaged separately. Optionally washes of water and/or alcohol may also be included.
The modified components Haematoxylin HR3, OG9 and
EA66 themselves as described above also form a further aspect of the present invention.
Yet another aspect of the present invention provides a use of the modified components described above for staining physiological samples. In particular the modified components may be used to stain physiological samples suspected of containing precancerous or cancerous cells, for example in cervical smear screening and for staining fine needle aspirate smears to detect cancerous and precancerous cells in breast screening clinics.
A yet further embodiment of the present invention provides a method of diagnosis of a cancerous condition, said method comprising obtaining a sample of cells from the patient, staining said sample using one or more of the following reagents: 1. Haematoxylin HR3; 2. OG9; 3. EA66; and
examining said stained sample to determine whether cancerous or pre-cancerous cells are present therein.
In one embodiment the Papanicolaou staining technique of the present invention comprises the following steps: 1. Haematoxylin HR3; 2. aqueous wash (dilute alkaline solution, for example containing lithium carbonate); 3. alcohol wash (to ensure dehydration of the sample and to prevent dilution of the next staining stage); 4. OG9; 5. alcohol wash (to avoid contamination of the following staining stage); and 6. EA66.
In general each of the above stages will take less than 3 minutes and the wash stages may indeed be very quick, comprising merely agitating the slide onto which the sample has been fixed in the wash media for less than 30 seconds, for example 1-15 seconds, in particular less than 10 seconds, especially 5-10 seconds.
Step (1) comprising staining in Haematoxylin HR3 will generally be adequately achieved in 2 minutes or less, for example 15-90 seconds, in particular 20-60 seconds, for example approximately 40 seconds.
The same time scale is appropriate while staining using OG9.
With regard to EA66 adequate staining will normally be achieved in 1-3 minutes, in particular 1-2 minutes.
After the staining process is complete a cover slip will normally be used to protect the sample before examination using an optical microscope.
Advantageously, the modified Papanicolaou staining technique of the present invention may be used by a doctor or nurse or a general practitioner's surgery or clinic to provide an"on-the-spot"diagnosis of the stained sample when viewed by an experienced screener or cytopathologist. The short time span required to achieve adequate staining by the present invention is sufficiently short to be acceptable during the procedure of surgery without causing undue delay to the surgeon or distress to the patient.
Claims (17)
1. A method of staining a physiological sample, said method including a step of staining a sample with at least one stain chosen from the group consisting of; Haematoxylin HR3, OG9 and
EA66.
2. A method of staining as claimed in claim 1, wherein the method includes staining the sample with haemotoxylin HR3 and/or EA66 and OG9.
3. A method of staining as claimed in claims 1 or
2, wherein progressive staining with
Haematoxylin HR3 is carried out for between 10 and 50 seconds.
4. A method of staining as claimed in claims 1,2 or 3, wherein progressive staining with
Haematoxylin HR3 is carried out for approximately 30 seconds.
5. A method of staining as claimed in any preceding claim, wherein staining with OG9 is carried out for between 2 to 30 seconds.
6. A method of staining as claimed in claims 1 in any preceding claim, wherein staining with OG9 is carried out for approximately 5 to 10 seconds.
7. A method of staining as claimed in any preceding claim, wherein staining with EA66 is carried out for between 10 to 80 seconds.
8. A method of staining as claimed in any preceding claim, wherein staining with EA66 is carried out for approximately 30 to 40 seconds.
9. A stain for staining a physiological sample, the stain comprising; - Haematoxylin-3 grams - Aluminium Potassium Sulphate-50 grams - Sodium Iodate - 0. 2 grams - Chloral Hydrate-50 grams - Water-1000 ml
10. A stain for staining a physiological sample, the stain comprising; - 7ml of 0.6% fast green in 95% alcohol - 43ml of eosin in 95% alcohol - 5ml of 0.5% Bismarck brown in 95% alcohol - 5ml of 10% phosphotungstic acid in 95% alcohol - 40mil of 95% alcohol
5 drops of aqueous saturated lithium carbonate.
11. A kit to stain physiological samples, said kit comprising one or more of the following stains:
A. Haematoxylin HR3;
B. OG9 ; and C. EA66.
12. A kit as claimed in claim 11 wherein the physiological sample to be stained is a cervical smear or similar samples which may contain cancerous or pre-cancerous cells.
13. A kit as claimed in claim 11 or 12 wherein the stains are packaged separately.
14. Use of Haemotoxylin HR3 in the staining of a physiological sample.
15. Use of OG9 in the staining of a physiological sample.
16. Use of EA66 in the staining of a physiological sample.
17. A method of diagnosis of a cancerous condition, said method comprising obtaining a sample of cells from the patient, staining said sample using one or more of the following reagents:
A. Haematoxylin HR3;
B. OG9;
C. EA66; and examining said stained sample to determine whether cancerous or pre-cancerous cells are present therein.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0103605A GB0103605D0 (en) | 2001-02-14 | 2001-02-14 | Stain |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB0203523D0 GB0203523D0 (en) | 2002-04-03 |
| GB2372811A true GB2372811A (en) | 2002-09-04 |
| GB2372811B GB2372811B (en) | 2005-01-26 |
Family
ID=9908700
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0103605A Ceased GB0103605D0 (en) | 2001-02-14 | 2001-02-14 | Stain |
| GB0203523A Expired - Fee Related GB2372811B (en) | 2001-02-14 | 2002-02-14 | Stain |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0103605A Ceased GB0103605D0 (en) | 2001-02-14 | 2001-02-14 | Stain |
Country Status (1)
| Country | Link |
|---|---|
| GB (2) | GB0103605D0 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103207104A (en) * | 2013-03-29 | 2013-07-17 | 中国科学院华南植物园 | Method for staining iron element in plants |
| CN104714030A (en) * | 2015-03-05 | 2015-06-17 | 杭州欣叶生物科技有限公司 | Urine detection kit for cervical cancer and using method of urine detection kit |
| CN105954085A (en) * | 2016-05-20 | 2016-09-21 | 北京九州柏林生物科技有限公司 | Mercury-free hematoxylin staining fluid |
| IT201900001117A1 (en) | 2019-01-25 | 2020-07-25 | Fondazione St Italiano Tecnologia | Contrast solution for the characterization of biological samples by electron and correlative microscopy |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103471892A (en) * | 2013-09-22 | 2013-12-25 | 厦门大学附属第一医院 | Method for preparing pulled type cervical smear |
| CN112781963B (en) * | 2020-12-30 | 2024-04-30 | 深路医学科技(武汉)有限公司 | Papanicolaou staining solution and preparation method and staining method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6047960A (en) * | 1983-08-26 | 1985-03-15 | Wako Pure Chem Ind Ltd | Staining method and staining test solution of cell in cytology |
| US4550016A (en) * | 1982-09-30 | 1985-10-29 | Harris Cynthia J | Composite cytologic counterstain formulation |
-
2001
- 2001-02-14 GB GB0103605A patent/GB0103605D0/en not_active Ceased
-
2002
- 2002-02-14 GB GB0203523A patent/GB2372811B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4550016A (en) * | 1982-09-30 | 1985-10-29 | Harris Cynthia J | Composite cytologic counterstain formulation |
| JPS6047960A (en) * | 1983-08-26 | 1985-03-15 | Wako Pure Chem Ind Ltd | Staining method and staining test solution of cell in cytology |
Non-Patent Citations (5)
| Title |
|---|
| Chemical Abstract No: 100:188234 & Mikroskopie 40 (9-10), 1983, pages 264 - 267. * |
| Chemical Abstract No: 102:200737 & JP 60 047 960 A * |
| Chemical Abstract No: 105:168275 & Morphol. I. Orv. Sz. 26(3), 1986, pages 218 - 220. * |
| Chemical Abstract No: 105:75237 & Morphol. I. Orv. Sz. 26(2), 1986, pages 144 - 147. * |
| Chemical Abstract No: 84:27722 & Acta Histochem. 54(1), 1975, pages 78 - 83. * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103207104A (en) * | 2013-03-29 | 2013-07-17 | 中国科学院华南植物园 | Method for staining iron element in plants |
| CN103207104B (en) * | 2013-03-29 | 2015-12-23 | 中国科学院华南植物园 | Ferro element colouring method in a kind of plant |
| CN104714030A (en) * | 2015-03-05 | 2015-06-17 | 杭州欣叶生物科技有限公司 | Urine detection kit for cervical cancer and using method of urine detection kit |
| CN105954085A (en) * | 2016-05-20 | 2016-09-21 | 北京九州柏林生物科技有限公司 | Mercury-free hematoxylin staining fluid |
| IT201900001117A1 (en) | 2019-01-25 | 2020-07-25 | Fondazione St Italiano Tecnologia | Contrast solution for the characterization of biological samples by electron and correlative microscopy |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2372811B (en) | 2005-01-26 |
| GB0203523D0 (en) | 2002-04-03 |
| GB0103605D0 (en) | 2001-03-28 |
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| PCNP | Patent ceased through non-payment of renewal fee |
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