WO1997019351A1 - Procede pour preparer des secretions bronchiques contenant des cellules pour un diagnostic cytologique - Google Patents
Procede pour preparer des secretions bronchiques contenant des cellules pour un diagnostic cytologique Download PDFInfo
- Publication number
- WO1997019351A1 WO1997019351A1 PCT/EP1996/004856 EP9604856W WO9719351A1 WO 1997019351 A1 WO1997019351 A1 WO 1997019351A1 EP 9604856 W EP9604856 W EP 9604856W WO 9719351 A1 WO9719351 A1 WO 9719351A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mucus
- mucolytic
- cells
- mucolysis
- sputum
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000028327 secretion Effects 0.000 title claims abstract description 19
- 230000002380 cytological effect Effects 0.000 title claims abstract description 5
- 210000003097 mucus Anatomy 0.000 claims abstract description 40
- 206010036790 Productive cough Diseases 0.000 claims abstract description 12
- 210000003802 sputum Anatomy 0.000 claims abstract description 12
- 208000024794 sputum Diseases 0.000 claims abstract description 12
- 230000000510 mucolytic effect Effects 0.000 claims abstract description 11
- 238000001085 differential centrifugation Methods 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 6
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 claims description 20
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000000834 fixative Substances 0.000 claims description 8
- 238000003745 diagnosis Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 239000003172 expectorant agent Substances 0.000 claims description 2
- 229940066491 mucolytics Drugs 0.000 claims description 2
- 229920000715 Mucilage Polymers 0.000 claims 1
- 239000000853 adhesive Substances 0.000 claims 1
- 239000000470 constituent Substances 0.000 abstract description 2
- 230000002452 interceptive effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 26
- 239000013049 sediment Substances 0.000 description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 5
- 238000010186 staining Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010006417 Bronchial carcinoma Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 208000003362 bronchogenic carcinoma Diseases 0.000 description 1
- 230000001680 brushing effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N cystine group Chemical group C([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000007431 microscopic evaluation Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Definitions
- the invention relates to a method for preparing cell-containing bronchial secretions and / or sputum for cytological diagnosis.
- Coughed-up secretion from the bronchi obtained by suction or brushing contains cells that have been stripped from the bronchial mucosa. These cells can be examined for diagnostic purposes. For example, when a bronchial carcinoma or its precursors are present, tumor cells or their precursors are released, on which microscopic analysis of the carcinoma or its precursors is possible. The microscopic diagnosis is carried out either manually on the light microscope by pathologists, pulmologists, pathology or cytology assistants, or automated by cyto-automatons.
- the cells to be examined are surrounded by mucus, which makes diagnosis more difficult, since it can lead to a superposition of several cells and it is not possible to separate fragments of dead cells which interfere with the diagnosis .
- the bronchial secretion fixed by an alcohol solution be homogenized with a kitchen mixer or ultra-high speed at high speed. This process is intended to mechanically cut the mucus filaments into short fragments, which the subsequent cell disrupt diagnostics less. It has also been proposed to carry out a centrifugation step after the mechanical shredding of the mucus threads. However, such a centrifugation does not lead to a separation of the cells from the shortened mucus threads, rather a concentrated mixture of cells and mucus is obtained.
- the present invention has for its object to provide a simple and safe method of the type mentioned above, with which the cells in the secretion from the mucus can be largely separated.
- the method according to the invention has the following steps:
- mucolytic is understood to mean any reagent which is able to split disulfide bridges in such a bronchial secretion. Substances containing thiol groups can be used particularly advantageously for this purpose. Chemical mucolysis leads to a shortening of the mucus threads as well as to an extensive dissolution of the network of the mucus threads among themselves.
- the separation of the sedimented cells from the centrifugate or supernatant mentioned in feature c) of claim 1 does not necessarily include a complete separation of the cells from any residues of the liquid medium.
- "Mucus-containing centrifugate" in the sense of the invention is that part of the centrifugate which contains parts of mucus and / or mucolyzed mucus residues which interfere with cytodiagnostics.
- a particularly advantageous mucolytic agent is thio glycolic acid or its salts, the thioglycolates.
- the thioglycolic acid is expediently buffered to a pH of about 6.3 to 8.3. This range corresponds to the pH of the blood (7.3 ⁇ 1.0).
- Ammonia or an ammonia / ammonium chloride solution can be used as a buffer.
- the mucus is comminuted to a mucus thread length of at most 30 ⁇ m by chemical mucolysis.
- the mucolysis is often so complete that a thread structure of the mucus can no longer be seen in the light microscope.
- the method according to the invention can be carried out directly with coughed up bronchial secretions or sputum.
- the secretion is fixed with an alcohol-containing fixative before adding the mucolytic.
- Such a fixation is particularly useful or necessary if the patient takes the sample himself by coughing up at home. He will then add a fixative to the sample and send the fixed sample to the cytodiagnostic laboratory.
- a fixative that can expediently be used in the process contains the following components:
- fixative The above list of the components of the fixative is not exhaustive; additional components may be included.
- an antibiotic such as rifampicin can be added, for example.
- Polyethylene glycols in the molecular weight range mentioned are commercially available under the trade name Carbowax R. Loading a composition which contains 50% by weight of ethanol and 2% by weight of polyethylene glycols is particularly preferred.
- the solution obtained after chemical mucolysis which contains the cell-mucus mixture, can be subjected directly to the differential centrifugation according to feature b) of claim 1.
- This intermediate centrifugation differs from differential centrifugation according to the feature b) mentioned in that during the intermediate centrifugation both the cells and the mucus have a higher density than the mucolysis solution and therefore sediment together.
- aqueous sucrose solution is particularly suitable as the liquid medium for differential centrifugation.
- a sucrose solution is inexpensive and toxicologically harmless.
- concentration is generally set to 40 to 50% by weight, preferably 40 to 45% by weight, of sucrose in water.
- sucrose solution it is also possible to use any other solution which is chemically inert to the secretion and the reagents used.
- the bronchial secretion or sputum coughed up by a patient is mixed with 50 to 100 ml of fixative and mixed.
- this fixing step is carried out by the patient at home, and the sample thus fixed is sent to the cytodiagnostic laboratory.
- 1 ml of the mucolytic prepared under 1. is added to 10 ml of the fixed sputum solution, the mixture is mixed on a shaker for 30 s and left to stand at room temperature for 30 min. The sample is then centrifuged at 1,600 g for 10 minutes. This is the intermediate centrifugation to concentrate the cell-mucus mixture in the sample.
- the supernatant is pipetted down to 2 ml, the sediment is shaken for the remaining 30 s. 120 ⁇ l of the shaken-up sample are coated on 10 ml of 45% sucrose solution and centrifuged at 1,500 g for 30 min. In this differential centrifugation, the cells are separated from the mucolyzed mucus and other disturbing, extracellular admixtures due to their higher density, and they are deposited as sediment. After pipetting off the supernatant sucrose solution, which contains the mucus and other disruptive constituents, the cell sediment can be spread out on slides, stained and cytodiagnosed with the light microscope. A single-layer and dense storage of the diagnostically relevant cells is obtained. For example, hematoxylin-eosin staining or Papanicolaou staining can be used for staining.
- the cell sediment can be examined in a known cytodiagnostic machine.
- cytodiagnostic machine E.g. the Cyto-Rich R system from Röche Image Analysis Systems can be used (see James W. Geyer et al., Diagnostic Cytopathology 1993, Vol. 9, No. 4, 417-422).
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
L'invention concerne un procédé pour préparer des sécrétions bronchiques et/ou expectorations contenant des cellules afin de réaliser un diagnostic cytologique. Ce diagnostic comporte les étapes suivantes: a) addition d'un mucolytique et mucolyse chimique du mucus, b) centrifugation différentielle dans un liquide dont la densité se situe entre la densité des cellules contenues dans les sécrétions bronchiques ou les expectorations et la densité du mucus, c) séparation des cellules sédimentées du centrifugat contenant le mucus. Le procédé de l'invention permet de séparer sensiblement complètement les cellules à examiner des composants muqueux perturbateurs.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU75666/96A AU7566696A (en) | 1995-11-20 | 1996-11-06 | Method of preparing cell-containing bronchial secretions for cytological analysis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19543252.5 | 1995-11-20 | ||
DE1995143252 DE19543252A1 (de) | 1995-11-20 | 1995-11-20 | Verfahren zum Vorbereiten von zellhaltigen Bronchialsekreten für eine zytologische Diagnostik |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997019351A1 true WO1997019351A1 (fr) | 1997-05-29 |
Family
ID=7777942
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1996/004856 WO1997019351A1 (fr) | 1995-11-20 | 1996-11-06 | Procede pour preparer des secretions bronchiques contenant des cellules pour un diagnostic cytologique |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU7566696A (fr) |
DE (1) | DE19543252A1 (fr) |
WO (1) | WO1997019351A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046258A2 (fr) * | 1997-04-11 | 1998-10-22 | Beth Israel Deaconess Medical Center, Inc. | Utilisation de chondroitinase dans la fabrication d'un medicament destine au traitement et a la prevention de secretions mucoides |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10241254B3 (de) * | 2002-09-06 | 2004-01-22 | Bach, Gerd, Dr. Med. | Verfahren zur Aufbereitung von durch Biopsie entnommener Schleimhautteilchen für die zytologische Diagnostik |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5112758A (en) * | 1988-05-09 | 1992-05-12 | Epitope, Inc. | Treating body fluids for diagnostic testing |
EP0586024A1 (fr) * | 1987-04-01 | 1994-03-09 | Gen-Probe Incorporated | Techniques pour la préparation des spécimens par les dosages bartériens |
-
1995
- 1995-11-20 DE DE1995143252 patent/DE19543252A1/de not_active Withdrawn
-
1996
- 1996-11-06 AU AU75666/96A patent/AU7566696A/en not_active Abandoned
- 1996-11-06 WO PCT/EP1996/004856 patent/WO1997019351A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0586024A1 (fr) * | 1987-04-01 | 1994-03-09 | Gen-Probe Incorporated | Techniques pour la préparation des spécimens par les dosages bartériens |
US5112758A (en) * | 1988-05-09 | 1992-05-12 | Epitope, Inc. | Treating body fluids for diagnostic testing |
Non-Patent Citations (2)
Title |
---|
CHEMICAL ABSTRACTS, vol. 92, no. 15, 14 April 1980, Columbus, Ohio, US; abstract no. 124452, V. FAILI ET AL.: "The solubilisation of glycoprotein mucins and slimes." page 313; column 2; XP002002063 * |
PAHLAVI MED. J., vol. 9, no. 1, 1978, pages 341 - 366 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046258A2 (fr) * | 1997-04-11 | 1998-10-22 | Beth Israel Deaconess Medical Center, Inc. | Utilisation de chondroitinase dans la fabrication d'un medicament destine au traitement et a la prevention de secretions mucoides |
WO1998046258A3 (fr) * | 1997-04-11 | 1999-03-11 | Beth Israel Hospital | Utilisation de chondroitinase dans la fabrication d'un medicament destine au traitement et a la prevention de secretions mucoides |
US6200564B1 (en) | 1997-04-11 | 2001-03-13 | J. Thomas Lamont | Pharmaceutical compositions containing chondroitinase and uses therefor |
Also Published As
Publication number | Publication date |
---|---|
AU7566696A (en) | 1997-06-11 |
DE19543252A1 (de) | 1997-05-22 |
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