WO1991005263A1 - Quantitative immunocytochemistry assay - Google Patents

Quantitative immunocytochemistry assay Download PDF

Info

Publication number
WO1991005263A1
WO1991005263A1 PCT/US1990/005429 US9005429W WO9105263A1 WO 1991005263 A1 WO1991005263 A1 WO 1991005263A1 US 9005429 W US9005429 W US 9005429W WO 9105263 A1 WO9105263 A1 WO 9105263A1
Authority
WO
WIPO (PCT)
Prior art keywords
control
cells
antigen
target molecule
optical density
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1990/005429
Other languages
French (fr)
Inventor
Hector A. Battifora
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
City of Hope
Original Assignee
City of Hope
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 City of Hope filed Critical City of Hope
Priority to EP90914821A priority Critical patent/EP0445277B1/en
Priority to DE69027982T priority patent/DE69027982T2/en
Publication of WO1991005263A1 publication Critical patent/WO1991005263A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones

Definitions

  • This invention relates to the direct quantitative determination by immunocytochemistry of target molecules in tissue specimens.
  • Assays to determine the presence or absence of target molecules in tissue specimens typically entail the running of an appropriate biochemical test on a tissue homogenate. Assays for estrogen receptor (ER) and progesterone receptor (PR) are of value in predicting tumor response and are routinely done in homogenates of breast cancer.
  • ER estrogen receptor
  • PR progesterone receptor
  • Tissue homogenates for quantitative biochemical assays are based on a variable number of normal cells and cells such as cancer cells which express target molecules. Immunocytochemistry has a theoretical advantage over such biochemical assays in that it directly measures the presence or absence of the target molecules in the relevant cells.
  • the staining procedure will indicate differences in intensity of immunoreactivity depending upon multiple variables, such as antibody concentration, timing, type of chromogen used, and the like.
  • the invention replaces the visual estimation procedure of the prior art with a practical technique for utilizing the capability of immunocytochemistry to measure quantitatively the presence or absence of a target molecule in the cells of a tissue or other specimen. More particularly, the invention provides a control or internal standard containing a known amount of antigen (or other target molecules) to be processed concurrently with the tissue specimen. The control is thus subjected to all of the same conditions as the tissue specimen to be assayed. For example, a decrease of 30% in the availability of an antigen to .be detected will equally affect the control and the specimen under examination. Because the control has cells with a known amount of antigen or other target molecules, a compensating factor can readily be determined, and a quantitative assay of the tissue specimen accomplished. DESCRIPTION OF THE INVENTION
  • the control provided by this invention may be a section or slice of a medium, such as gelatin, agar or any other aqueous embedding medium, having embedded therein cells, such as MCF7 (a known breast cancer cell line which expresses ER and PR) or BT474 (a known cell line which expresses a breast cancer oncogene) , expressing a defined amount of a target molecule.
  • a medium such as gelatin, agar or any other aqueous embedding medium, having embedded therein cells, such as MCF7 (a known breast cancer cell line which expresses ER and PR) or BT474 (a known cell line which expresses a breast cancer oncogene) , expressing a defined amount of a target molecule.
  • MCF7 a known breast cancer cell line which expresses ER and PR
  • BT474 a known cell line which expresses a breast cancer oncogene
  • Choice of cell type depends upon the type of antigen or other molecules to be measured. There is a plethora of cells of many types expressing a variety of antigens or other molecules. Transfected cells may be used. Target molecules which may be quantified in a specimen by use of this invention include, for example, proteins expressed by oncogenes, cell growth factors, and any of the various molecules that control cell proliferation.
  • the pathologist receiving, for example, a breast biopsy suspected of containing cancer cells includes a control slice embodying the invention into the cassette to insure that both the tissue sample and the control are concurrently subjected to all ensuing procedures through immunostaining.
  • the optical density of the stained cancer cells in the biopsy sample is compared to that of the cells in the control which acts as an internal standard.
  • the comparison is made by use of a computerized cell analysis system, such as a CAS 200 image analyzer (Cell Analysis Systems, Inc., Lombard, Illinois) .
  • EXAMPLE Cells grown in tissue culture (MCF7 and BT474) , known to express a defined amount of the antigens to be measured, were briefly fixed in paraformaldehyde and suspended in a 3% agar solution (Difco, Detroit, Michigan) at 56°C and allowed to gel. Uniform slices, 3 mm thick, were fixed in formaldehyde for periods of time ranging from 4 to 72 hours and processed together into a single paraffin block. The cross-sectional dimensions of the block were about 2 by about 2.5 cm. Sections cut at 5 microns were im unostained for estrogen receptor (ER-ICA, Abbott, Illinois) and cERB-b2 oncoprotein (Triton Bios., Alameda, California) .
  • ER-ICA estrogen receptor
  • cERB-b2 oncoprotein Triton Bios., Alameda, California
  • the intensity of the immunoreactivity was measured for each quantitation of immunocytochemistry gel with a CAS 200 image analyzer. Progressive reduction in the intensity of the immunoreactivity, which correlated with the lengthening of the fixation time was detected with both antigens. Significantly, such reduction was not noticeable by conventional microscopy in the ER-ICA stains.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Endocrinology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

An internal control or standard is provided for the direct quantitative assay by immunocytochemistry of target molecules in tissue specimens and the like. The control is subjected to the same conditions including immunostaining as the tissue specimen. Optical density of the control and the specimen after staining is compared, preferably by a cell analysis computer system.

Description

QUANTITATIVE IMMUNOCYTOCHEMISTRY ASSAY
This application is a continuation of United States application Serial No. 07/412,450 filed 26 September 1989, which is still pending.
This invention relates to the direct quantitative determination by immunocytochemistry of target molecules in tissue specimens.
BACKGROUND OF THE INVENTION Assays to determine the presence or absence of target molecules in tissue specimens typically entail the running of an appropriate biochemical test on a tissue homogenate. Assays for estrogen receptor (ER) and progesterone receptor (PR) are of value in predicting tumor response and are routinely done in homogenates of breast cancer.
Tissue homogenates for quantitative biochemical assays are based on a variable number of normal cells and cells such as cancer cells which express target molecules. Immunocytochemistry has a theoretical advantage over such biochemical assays in that it directly measures the presence or absence of the target molecules in the relevant cells.
To date, however, this advantage has not been realized because current immunocytochemistry is based on a visual estimation of the intensity of the immunostaining reaction product. This procedure has major drawbacks which preclude its use as a precise, reproducible, quantitative assay. Among other things: (1) The various tissue processing steps (fixation time, type of fixative, dehydration, embedding and related procedures) are all responsible for some variation, in particular loss of antigen, which modifies the im unostain signal. These variables are difficult to control as they depend on fixative concentration, temperature, contaminants and, importantly, time.
(2) The thickness of the tissue section is difficult to control—an important factor because the signal strength increases as a function of section thickness.
(3) The staining procedure will indicate differences in intensity of immunoreactivity depending upon multiple variables, such as antibody concentration, timing, type of chromogen used, and the like.
SUMMARY OF THE INVENTION This invention replaces the visual estimation procedure of the prior art with a practical technique for utilizing the capability of immunocytochemistry to measure quantitatively the presence or absence of a target molecule in the cells of a tissue or other specimen. More particularly, the invention provides a control or internal standard containing a known amount of antigen (or other target molecules) to be processed concurrently with the tissue specimen. The control is thus subjected to all of the same conditions as the tissue specimen to be assayed. For example, a decrease of 30% in the availability of an antigen to .be detected will equally affect the control and the specimen under examination. Because the control has cells with a known amount of antigen or other target molecules, a compensating factor can readily be determined, and a quantitative assay of the tissue specimen accomplished. DESCRIPTION OF THE INVENTION
The control provided by this invention may be a section or slice of a medium, such as gelatin, agar or any other aqueous embedding medium, having embedded therein cells, such as MCF7 (a known breast cancer cell line which expresses ER and PR) or BT474 (a known cell line which expresses a breast cancer oncogene) , expressing a defined amount of a target molecule. More specifically, cells grown in tissue culture are suspended in the gelatin or other embedding medium which is allowed to solidify by cooling or polymerization. The solidified medium containing the cell suspension is cut into sections or slices of appropriate thickness, e.g., a thickness of from about 2 to about 5 mm.
Choice of cell type depends upon the type of antigen or other molecules to be measured. There is a plethora of cells of many types expressing a variety of antigens or other molecules. Transfected cells may be used. Target molecules which may be quantified in a specimen by use of this invention include, for example, proteins expressed by oncogenes, cell growth factors, and any of the various molecules that control cell proliferation.
The pathologist receiving, for example, a breast biopsy suspected of containing cancer cells includes a control slice embodying the invention into the cassette to insure that both the tissue sample and the control are concurrently subjected to all ensuing procedures through immunostaining. The optical density of the stained cancer cells in the biopsy sample is compared to that of the cells in the control which acts as an internal standard. Preferably, the comparison is made by use of a computerized cell analysis system, such as a CAS 200 image analyzer (Cell Analysis Systems, Inc., Lombard, Illinois) . EXAMPLE Cells grown in tissue culture (MCF7 and BT474) , known to express a defined amount of the antigens to be measured, were briefly fixed in paraformaldehyde and suspended in a 3% agar solution (Difco, Detroit, Michigan) at 56°C and allowed to gel. Uniform slices, 3 mm thick, were fixed in formaldehyde for periods of time ranging from 4 to 72 hours and processed together into a single paraffin block. The cross-sectional dimensions of the block were about 2 by about 2.5 cm. Sections cut at 5 microns were im unostained for estrogen receptor (ER-ICA, Abbott, Illinois) and cERB-b2 oncoprotein (Triton Bios., Alameda, California) . The intensity of the immunoreactivity was measured for each quantitation of immunocytochemistry gel with a CAS 200 image analyzer. Progressive reduction in the intensity of the immunoreactivity, which correlated with the lengthening of the fixation time was detected with both antigens. Significantly, such reduction was not noticeable by conventional microscopy in the ER-ICA stains.

Claims

HAT IS CLAIMED IS:
1. A control for use in quantitative immunocytochemistry assays which comprises a section of a solidified embedding medium having suspended therein cells which express a defined amount of, a preselected target molecule.
2. A control as defined by claim 1 in which the target molecule is an antigen, a protein expressed by an oncogene, a cell growth factor, a receptor molecule, or a molecule that controls the proliferation of a cell.
3. A control as defined by claim 1 or claim 2 in which the preselected target molecule expressed by said cells embedded in said solidified embedding medium is an antigen.
4. A control as defined by claim 1 or claim 2 in which said antigen is an estrogen receptor or a progesterone receptor.
5. A control for use in the quantitative immunocytochemistry assay of a breast tissue for estrogen and progesterone receptors which comprises a section of solidified agar having embedded therein MCF7 cells which express a defined amount of estrogen receptors and progesterone receptors.
6. An immunocytochemical method for the direct, quantitative determination of the presence or absence of a target molecule in the cells of a tissue specimen which comprises concurrently subjecting said specimen and a control as defined by claim 1 to all of the same processing steps including immunostaining and thereafter comparing the optical density of the stained tissue specimen cells with the optical density of the control cells. *7. A method as defined by claim 6 in which the comparison of the optical density of the stained tissue specimen cells with the optical density of the stained control cells is made by use of a computerized cell analysis system.
8. A method as defined by claim 6 or claim 7 in which said target molecule is an antigen.
PCT/US1990/005429 1989-09-26 1990-09-26 Quantitative immunocytochemistry assay Ceased WO1991005263A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP90914821A EP0445277B1 (en) 1989-09-26 1990-09-26 Quantitative immunocytochemistry assay
DE69027982T DE69027982T2 (en) 1989-09-26 1990-09-26 QUANTITATIVE IMMUNOCYTOCHEMICAL TEST

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41245089A 1989-09-26 1989-09-26
US412,450 1989-09-26

Publications (1)

Publication Number Publication Date
WO1991005263A1 true WO1991005263A1 (en) 1991-04-18

Family

ID=23633028

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/005429 Ceased WO1991005263A1 (en) 1989-09-26 1990-09-26 Quantitative immunocytochemistry assay

Country Status (5)

Country Link
EP (1) EP0445277B1 (en)
AU (1) AU628678B2 (en)
CA (1) CA2042018A1 (en)
DE (1) DE69027982T2 (en)
WO (1) WO1991005263A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5761945A (en) * 1993-10-18 1998-06-09 Vandenbroucke; Jack-Eric Quick automated tool changer roll forming apparatus
WO2000062064A3 (en) * 1999-04-14 2001-01-11 Cytologix Corp Quality control for cytochemical assays
EP1131628A4 (en) * 1998-10-21 2004-05-12 Steven Jay Smith PROTEIN QUANTIFICATION WITH DENSITOMETRY BY CELLULAR IMAGING
US6855490B2 (en) 1999-04-14 2005-02-15 Medical Discovery Partners Llc Method for attaching biological molecules to a glass surface
WO2009085575A3 (en) * 2007-12-28 2009-09-17 Spring Bioscience Corporation Calibrator quality control cell device for immunohistochemistry assay and methods of use thereof
CN112219119A (en) * 2017-12-06 2021-01-12 基因泰克公司 Synthetic controls for immunohistochemistry

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8785150B2 (en) * 2006-06-30 2014-07-22 University Of Southern California Quantifiable internal reference standards for immunohistochemistry and uses thereof
US20110306064A1 (en) * 2006-06-30 2011-12-15 University Of Southern California Quantifiable Internal Reference Standards For Immunohistochemistry And Uses Thereof
US20140113385A1 (en) * 2010-10-22 2014-04-24 University Of Southern California Compositions and Methods for Identifying Single Antigens or Other Molecules in Cell Preparations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816410A (en) * 1986-10-29 1989-03-28 Coulter Corporation Control slide for immunoassay kit and method of making same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816410A (en) * 1986-10-29 1989-03-28 Coulter Corporation Control slide for immunoassay kit and method of making same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0445277A4 *
WICK et al., Immunofluorescence Technology-Selected Theoretical and Clinical Aspects, Published 1982 by Elsevier Biomedical Press, Amsterdam (N.Y.), see pages 86-90. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5761945A (en) * 1993-10-18 1998-06-09 Vandenbroucke; Jack-Eric Quick automated tool changer roll forming apparatus
EP1131628A4 (en) * 1998-10-21 2004-05-12 Steven Jay Smith PROTEIN QUANTIFICATION WITH DENSITOMETRY BY CELLULAR IMAGING
WO2000062064A3 (en) * 1999-04-14 2001-01-11 Cytologix Corp Quality control for cytochemical assays
US6281004B1 (en) 1999-04-14 2001-08-28 Cytologix Corporation Quality control for cytochemical assays
US6855490B2 (en) 1999-04-14 2005-02-15 Medical Discovery Partners Llc Method for attaching biological molecules to a glass surface
US7011940B1 (en) 1999-04-14 2006-03-14 Medical Discovery Partners Llc Quality control for cytochemical assays
WO2009085575A3 (en) * 2007-12-28 2009-09-17 Spring Bioscience Corporation Calibrator quality control cell device for immunohistochemistry assay and methods of use thereof
WO2009085573A3 (en) * 2007-12-28 2009-09-17 Spring Bioscience Corporation Quality control cell device for immunohistochemistry assay and methods of use thereof
WO2009085576A3 (en) * 2007-12-28 2009-09-24 Spring Bioscience Corporation Positive control cell pellets for immunohistochemistry and methods of use thereof
CN112219119A (en) * 2017-12-06 2021-01-12 基因泰克公司 Synthetic controls for immunohistochemistry
JP2021505878A (en) * 2017-12-06 2021-02-18 ジェネンテック， インコーポレイテッド Synthetic control for immunohistochemistry
TWI820061B (en) * 2017-12-06 2023-11-01 美商建南德克公司 Synthetic controls for immunohistochemistry
US12019069B2 (en) 2017-12-06 2024-06-25 Genentech, Inc. Synthetic controls for immunohistochemistry

Also Published As

Publication number Publication date
EP0445277A4 (en) 1991-10-16
CA2042018A1 (en) 1991-03-27
AU6500290A (en) 1991-04-28
DE69027982D1 (en) 1996-09-05
DE69027982T2 (en) 1996-12-19
AU628678B2 (en) 1992-09-17
EP0445277A1 (en) 1991-09-11
EP0445277B1 (en) 1996-07-31

Similar Documents

Publication Publication Date Title
US5610022A (en) Internal control for immunocytochemistry assay
Pertschuk et al. Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Spγ in frozen sections or cytosol‐based ligand‐binding assays
Penault‐Llorca et al. Optimization of immunohistochemical detection of ERBB2 in human breast cancer: impact of fixation
True Quality control in molecular immunohistochemistry
Bilous et al. Current perspectives on HER2 testing: a review of national testing guidelines
US5846749A (en) Quantitative measurement of tissue protein identified by immunohistochemistry and standardized protein determination
Pectasides et al. HER-2/neu status of primary breast cancer and corresponding metastatic sites in patients with advanced breast cancer treated with trastuzumab-based therapy
JP2002537561A (en) Methods and apparatus for isolating and analyzing cellular protein components
JP2014501388A (en) Protein profiles of cancer and other pathological entities by selective response monitoring (SRM)
Briffod et al. Immunohistochemistry on cell blocks from fine-needle cytopunctures of primary breast carcinomas and lymph node metastases
Esteban et al. Improvement of the quantification of estrogen and progesterone receptors in paraffin-embedded tumors by image analysis
EP0445277B1 (en) Quantitative immunocytochemistry assay
Charpin et al. c-erbB-2 oncoprotein detected by automated quantitative immunocytochemistry in breast carcinomas correlates with patients' overall and disease-free survival
Thomsen et al. Estrogen receptor-α quantification in breast cancer: concordance between immunohistochemical assays and mRNA-in situ hybridization for ESR1 gene
Ciocca et al. Colocalization of estrogen and progesterone receptors with an estrogenregulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue
Jonmarker Jaraj et al. Intra-and interobserver reproducibility of interpretation of immunohistochemical stains of prostate cancer
Rampaul et al. HER-2 in breast cancer—methods of detection, clinical significance and future prospects for treatment
Lörz et al. Proliferating cell nuclear antigen counts as markers of cell proliferation in head and neck cancer
Lowry et al. Immunohistochemical methods for semiquantitative analysis of collagen content in human peripheral nerve
Katoh et al. Immunoperoxidase staining for estrogen and progesterone receptors in archival formalin fixed, paraffin embedded breast carcinomas after microwave antigen retrieval
JP5039264B2 (en) System for internal qualitative and quantitative verification of marker indices
Siñczak-Kuta et al. Evaluation of HER2/neu gene amplification in patients with invasive breast carcinoma. Comparison of in situ hybridization methods
Hepburn et al. Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA
JP2000508074A (en) Immunohistochemical detection assay for cancer growth status
Cavaliere et al. Estrogen and progesterone receptors in breast cancer: comparison between enzyme immunoassay and computer‐assisted image analysis of immunocytochemical assay

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 2042018

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1990914821

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1990914821

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1990914821

Country of ref document: EP