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 PDF

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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
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WO
WIPO (PCT)
Prior art keywords
mucus
mucolytic
cells
mucolysis
sputum
Prior art date
Application number
PCT/EP1996/004856
Other languages
German (de)
English (en)
Inventor
Alfred Böcking
Alexander Berg
Original Assignee
Boecking Alfred
Alexander Berg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boecking Alfred, Alexander Berg filed Critical Boecking Alfred
Priority to AU75666/96A priority Critical patent/AU7566696A/en
Publication of WO1997019351A1 publication Critical patent/WO1997019351A1/fr

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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).

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  • 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.
PCT/EP1996/004856 1995-11-20 1996-11-06 Procede pour preparer des secretions bronchiques contenant des cellules pour un diagnostic cytologique WO1997019351A1 (fr)

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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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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