WO2002006880A2 - Plastic substrates slide apparatus for infrared microscopy analysis - Google Patents
Plastic substrates slide apparatus for infrared microscopy analysis Download PDFInfo
- Publication number
- WO2002006880A2 WO2002006880A2 PCT/CA2001/001031 CA0101031W WO0206880A2 WO 2002006880 A2 WO2002006880 A2 WO 2002006880A2 CA 0101031 W CA0101031 W CA 0101031W WO 0206880 A2 WO0206880 A2 WO 0206880A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- slide
- infrared
- plastic
- range
- substrate
- Prior art date
Links
- 239000004033 plastic Substances 0.000 title claims abstract description 48
- 229920003023 plastic Polymers 0.000 title claims abstract description 48
- 239000000758 substrate Substances 0.000 title claims abstract description 44
- 238000004458 analytical method Methods 0.000 title claims abstract description 24
- 238000004971 IR microspectroscopy Methods 0.000 title claims abstract description 20
- 230000003287 optical effect Effects 0.000 claims abstract description 22
- 239000004698 Polyethylene Substances 0.000 claims abstract description 14
- 229920000573 polyethylene Polymers 0.000 claims abstract description 14
- -1 polyethylene Polymers 0.000 claims abstract description 11
- 230000005540 biological transmission Effects 0.000 claims abstract description 10
- 229920000306 polymethylpentene Polymers 0.000 claims abstract description 10
- 239000011116 polymethylpentene Substances 0.000 claims abstract description 10
- 230000002380 cytological effect Effects 0.000 claims description 22
- 238000002834 transmittance Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
- 238000010186 staining Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 20
- 239000011521 glass Substances 0.000 description 9
- 238000004476 mid-IR spectroscopy Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 238000009595 pap smear Methods 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- 239000005388 borosilicate glass Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000011218 segmentation Effects 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 238000010191 image analysis Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 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 description 1
- 229910014033 C-OH Inorganic materials 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 229910014570 C—OH Inorganic materials 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910001632 barium fluoride Inorganic materials 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 239000011824 nuclear material Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical group 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
Definitions
- the present invention relates to medical and biological specimen analysis, and more particularly to a plastic substrate slide apparatus and technique for infrared microscopy analysis.
- FTIR Fourier Transform Infrared
- the mid-Infrared (i.e. 2.5 - 25 microns) response of normal and pre-cancerous human tissue comprises a variety of structural and chemical changes which may be used to discriminate between the two. These changes include increases in glycogen content, extensive hydrogen bonding of phosphodiester groups in nucleic acids, tighter physical packing of nucleic acids, phosphorylation of C-OH groups in carbohydrates and proteins, increased disorder of methylene chains in membrane lipids, increased ratio of methyl to methylene, reduction in the hydrogen bond strength in the amide groups of ⁇ -helical segments and an increase in the hydrogen bond strength in the amide groups of the ⁇ -sheet segments.
- Proposals have been made to utilize these mechanisms in cancer screening protocols, such as, the early detection of pre-cancerous lesions of the uterine cervix and carcinomatous breast tissue.
- Pap test a screening test that searches for evidence of precancerous lesions in exfoliated cervical cells.
- the Pap test Involving the manual examination of tens of thousands of cervical epithelial cells, the Pap test is costly to apply and is subject to human error. Nevertheless, it has an enviable record of reducing cervical cancer mortality in the countries where it is applied. As a result considerable effort has been put into developing automated alternatives for the manual Pap test.
- the third step is known as feature extraction.
- feature extraction each of the segmented regions or objects in the image is subjected to a range of mathematical measures that seek to encapsulate the visual appearance in numerical form.
- classification comprises utilizing the numerical features to arrive at some type of conclusion about the object's identity.
- the mid- infrared absorption properties of the cervical epithelial cells may be used to yield a new and useful channel of information in addition to the usual visible-light imaging measures.
- these new mid-IR channels may be used to improve segmentation performance, to create a new set of features for classification, or as discrimination measures on their own. It has been found that restricting the use of the mid-IR channels to regions of the samples that contain nuclear material improves the effective signal-to-noise ratio of the measurement over bulk tissue measurements offering a greater power of discrimination to the technique.
- differential mid-IR measure one channel acting as an interrogator, the other as a reference
- a differential mid-IR measure allows a high-speed real-time scan of cellular material to quickly discriminate between normal and abnormal cells or tissue and may be extended to other important diagnostic properties of the cells.
- the nature of the substrate or carrier holding the specimen poses an acute problem for practical applications of mid-infrared spectroscopy to anatomic pathology.
- the most common substrate for anatomic pathology evaluations is the glass microscope slide. Glass slides are typically manufactured from borosilicate glass to relatively relaxed tolerances.
- the problem with borosilicate glasses (and most other reasonable substitute glasses) is the lack of a suitable transmission window for mid-infrared radiation. As shown in Fig. 4, the optical transmission of borosilicate glass declines rapidly after 1.5 microns. This is well short of the mid-IR region of interest which falls between 7 and 12 microns. Thus, the interrogation signal required by an automated system is essentially absorbed indiscriminately by the glass substrate.
- the present invention provides an apparatus and technique for a microscope slide or carrier suitable for application of mid-IR spectroscopic techniques, such as, discrimination between normal and pre-cancerous cells prepared in the form cells or cell groupings in a specimen placed on the slide.
- the present invention provides a microscope slide comprising a plastic substrate having mid-infrared transmissive characteristics in the range of 7 to 12 microns.
- the present invention provides a slide apparatus for infrared microscopy analysis of a biological specimen
- the slide apparatus comprises: (a) a plastic slide substrate for receiving the biological specimen; (b) the plastic slide substrate has optical transmission characteristics in the mid- infrared range.
- the present invention provides a slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 975 to 1000 cm -1 .
- the present invention provides a slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 1155 to 1180 cm "1 .
- the present invention provides slide a apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 975 to 1000 cm "1 .
- the present invention provides slide a apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 1155 to 1180 cm "1 .
- Fig. 1 shows in diagrammatic form a plastic slide apparatus suitable for infrared microscopy analysis according to the present invention
- Fig.2 shows in graphical form the optical transmittance characteristics for the plastic slide formed from polyethylene (PE) according to one embodiment of the invention
- Fig.3 shows in graphical form the optical transmittance characteristics for the plastic slide formed from polymethyl pentene (TPX).
- Fig.4 shows in graphical form the optical transmittance characteristics for a conventional borosilicate glass specimen slide.
- Fig. 1 shows in diagrammatic form a plastic slide 10 suitable for infrared microscopy analysis in accordance with the present invention.
- the slide 10 comprises a substrate or carrier indicated by reference 12. Cells indicated by reference 20 are deposited on the slide substrate 12. The cells 20 may be covered by a plastic cover slip 14.
- the plastic cover slip 14 is formed from the same material as the substrate 12. As will be described in more detail below, the plastic slide 10 provides optical transmission in the mid-infrared region in the range of 7 to 12 microns.
- the cells 20 may be stained for the usual visual analysis (i.e. the Papanicolaou procedure for epithelial cell observations) and mounted in the usual mounting medium in an automated analysis system for example.
- visual analysis i.e. the Papanicolaou procedure for epithelial cell observations
- the cells in the specimen 20 that respond to the stain comprise naturally- derived, encapsulated protein and this is what the stains will generally bind to.
- the hematoxylin will bind to DNA or RNA in the cell nucleus
- the Orange G stain will selectively bind to keratin in the cytoplasm, etc.
- an infrared absorbance signal at 975 cm “ 1 to 1000 cm “1 and again at 1155 cm “1 to 1180 cm “1 may be used to discriminate between normal and abnormal cells.
- the infrared band between 975 cm “1 to 1000 cm “1 corresponds to the symmetric stretching vibration of the dianionic phosphate monoesters of phosphorylated proteins and nucleic acids.
- the infrared band at 1155 cm “1 to 1180 cm “1 is associated with the stretching vibrational modes of the C-O groups serine, threonine, or tyrosine amino acid residues of cellular proteins.
- the substrate 12 for the plastic slide 10 is manufactured from polyethylene (PE).
- PE polyethylene
- the cellular sample 20 to be analyzed is fixed using known techniques and then deposited onto the surface of the plastic slide 10.
- Fig. 2 shows in graphical form optimal transmittance characteristics for polyethylene.
- polyethylene has an optical transmission in the mid-IR 'windows' or bands of 975 - 1000 cm "1 and 1155 - 1180 cm "1 which is never less than 50%.
- the substrate 12 for the plastic slide 10 is made from polymethyl pentene (TPX). As shown in Fig. 3, the substrate 12 of polymethyl pentene shows an optical transmission of more than 20% in the mid-IR bands of 975 - 1000 cm “1 and 1155 - 1180 cm “1 .
- TPX polymethyl pentene
- a cell or cells 20 deposited on the plastic slide 10 are analyzed according to the following method.
- the cell or cells 20 of interest on the slide 10 are brought into focus using conventional light microscopy by either automatic or manual means.
- the microscope stage is translated to a blank region that contains no cells at all.
- a background reading is obtained for the infrared absorption through the substrate 12 of the plastic slide 10 using an infrared microscopy apparatus (not shown).
- the background infrared bands of interest lie between 970 cm “1 and 1000 cm “1 and again between 1145 cm “1 and 1190 cm “1 .
- the slide 10 carrying the sample or specimen is rapidly scanned with the infrared microscope (not shown) across all of the cells in the specimen.
- Scan data is collected in the infrared bands of 970 cm “1 and 1000 cm “1 and 1145 cm “1 and 1190 cm “1 .
- the absorbence is corrected for the presence of the background, and the following ratios are calculated:
- the plastic slide according to the invention provides apparatus which increases the signal-to-noise ratio of the relatively weak mid-IR absorbencies and makes possible the practical realization of mid-infrared microscopy analysis for cytological specimens.
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Microscoopes, Condenser (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2001275617A AU2001275617A1 (en) | 2000-07-17 | 2001-07-16 | Plastic substrates slide apparatus for infrared microscopy analysis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US21855700P | 2000-07-17 | 2000-07-17 | |
US60/218,557 | 2000-07-17 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002006880A2 true WO2002006880A2 (en) | 2002-01-24 |
WO2002006880A3 WO2002006880A3 (en) | 2002-10-24 |
Family
ID=22815567
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2001/001031 WO2002006880A2 (en) | 2000-07-17 | 2001-07-16 | Plastic substrates slide apparatus for infrared microscopy analysis |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2001275617A1 (en) |
WO (1) | WO2002006880A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103105670A (en) * | 2013-02-06 | 2013-05-15 | 李宏 | Cell slide glass for microscopic observation |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5095213A (en) * | 1988-11-03 | 1992-03-10 | Syntex (U.S.A.) Inc. | Method of using an opaque plastic microscope slide for epi-fluorescent microscopy |
CA2103446A1 (en) * | 1991-06-25 | 1992-12-26 | James E. Gagnon | Spectroscopic sample holder and method for using same |
US5463223A (en) * | 1994-01-24 | 1995-10-31 | Patwong Technologies, Inc. | Disposable all purpose micro sample holder |
EP0839336A1 (en) * | 1995-07-19 | 1998-05-06 | Morphometrix Technologies Inc. | Automated scanning of microscope slides |
US5945674A (en) * | 1997-07-30 | 1999-08-31 | Vysis, Inc. | Method of identifying cellular types in a biological sample supported on an absorptive substrate by infrared spectroscopy |
US5812312A (en) * | 1997-09-04 | 1998-09-22 | Lorincz; Andrew Endre | Microscope slide |
-
2001
- 2001-07-16 WO PCT/CA2001/001031 patent/WO2002006880A2/en active Application Filing
- 2001-07-16 AU AU2001275617A patent/AU2001275617A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103105670A (en) * | 2013-02-06 | 2013-05-15 | 李宏 | Cell slide glass for microscopic observation |
Also Published As
Publication number | Publication date |
---|---|
WO2002006880A3 (en) | 2002-10-24 |
AU2001275617A1 (en) | 2002-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6181414B1 (en) | Infrared spectroscopy for medical imaging | |
Wood et al. | An investigation into FTIR spectroscopy as a biodiagnostic tool for cervical cancer | |
US6750964B2 (en) | Spectral imaging methods and systems | |
US5945674A (en) | Method of identifying cellular types in a biological sample supported on an absorptive substrate by infrared spectroscopy | |
Shetty et al. | Raman spectroscopy: elucidation of biochemical changes in carcinogenesis of oesophagus | |
US6718053B1 (en) | Method and apparatus for automated image analysis of biological specimens | |
US7783098B2 (en) | Method and apparatus for automated image analysis of biological specimens | |
Baker et al. | Investigating FTIR based histopathology for the diagnosis of prostate cancer | |
US6690817B1 (en) | Spectral bio-imaging data for cell classification using internal reference | |
EP3009832A1 (en) | A cytological system and method for analyzing a biological sample by raman spectroscopy | |
US20140070102A1 (en) | Terahertz Spectroscopy Characterization with High Spectral and Spatial Resolution for Biological and Chemical Sensing and Method of Use | |
US20120083678A1 (en) | System and method for raman chemical analysis of lung cancer with digital staining | |
JPH11502935A (en) | Method for detecting cellular abnormalities using Fourier transform infrared spectroscopy | |
Bird et al. | A protocol for rapid, label-free histochemical imaging of fibrotic liver | |
US20050250091A1 (en) | Raman molecular imaging for detection of bladder cancer | |
Kümmel et al. | Rapid brain structure and tumour margin detection on whole frozen tissue sections by fast multiphotometric mid-infrared scanning | |
US7316904B1 (en) | Automated pap screening using optical detection of HPV with or without multispectral imaging | |
Levenson et al. | Digital spectral imaging for histopathology and cytopathology | |
Maggioni et al. | Hyperspectral microscopic analysis of normal, benign and carcinoma microarray tissue sections | |
Levenson et al. | Spectral imaging for brightfield microscopy | |
Mansoor et al. | Fine‐needle aspiration of follicular adenoma versus parathyroid adenoma: The utility of multispectral imaging in differentiating lesions with subtle cytomorphologic differences | |
WO2002006880A2 (en) | Plastic substrates slide apparatus for infrared microscopy analysis | |
Romeo et al. | Introduction to spectral imaging, and applications to diagnosis of lymph nodes | |
Ploem | Appropriate technology for the quantitative assessment of the final reaction product of histochemical techniques | |
Commoner | Quantitative determination of the pigment content of single cells by means of a new microspectrophotometer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |