WO2003071395A2 - Procedes et appareil pour le mappage a multiples utilisations d'un bio-disque optique - Google Patents

Procedes et appareil pour le mappage a multiples utilisations d'un bio-disque optique Download PDF

Info

Publication number
WO2003071395A2
WO2003071395A2 PCT/US2003/005166 US0305166W WO03071395A2 WO 2003071395 A2 WO2003071395 A2 WO 2003071395A2 US 0305166 W US0305166 W US 0305166W WO 03071395 A2 WO03071395 A2 WO 03071395A2
Authority
WO
WIPO (PCT)
Prior art keywords
disc
optical bio
bio
optical
methods
Prior art date
Application number
PCT/US2003/005166
Other languages
English (en)
Inventor
Mark Oscar Worthington
Andrew Attila Pal
Original Assignee
Burstein Technologies, Inc.
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 Burstein Technologies, Inc. filed Critical Burstein Technologies, Inc.
Priority to AU2003216346A priority Critical patent/AU2003216346A1/en
Publication of WO2003071395A2 publication Critical patent/WO2003071395A2/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0637—Moving support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G01N15/0612—Optical scan of the deposits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells

Definitions

  • This invention relates in general to assay devices, and in particular to methods and an apparatus for an assay device implemented in conjunction with an optical bio- disc. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to methods and an apparatus for multi-use mapping of an optical bio-disc
  • CDs and DVDs enable large amounts of data to be stored and quickly retrieved. Audio, visual, and computer program data are frequently stored on CDs or DVDs in a digital format. The end appearance of stored visual data is not readily apparent upon visual inspection. Thus, to extract visual information from a disc, a CD or DVD reader collects a stream of data that contains an encoding of the visual image. The functional aspects of a CD or DVD can be better understood with a review of CDs.
  • a CD is a fairly simple piece of plastic, about four one-hundredths (4/100) of an inch (1.2 mm) thick. Most of a CD consists of an injection-molded piece of clear polycarbonate plastic. During manufacturing, this plastic is impressed with microscopic bumps arranged as a single, continuous, extremely long spiral track of data. Once the clear piece of polycarbonate is formed, a thin, reflective aluminum layer is sputtered onto the disc, covering the bumps. Then a thin acrylic layer is sprayed over the aluminum to protect it. The label is then printed onto the acrylic.
  • a CD has a single spiral track of data, circling from the inside of the disc to the outside.
  • the fact that the spiral track starts at the center means that the CD can be smaller than 4.8 inches (12 cm) if desired.
  • the data tracks are approximately 0.5 microns wide, with 1.6 microns separating one track from the next, and the elongated bumps that make up the track are each 0.5 microns wide, a minimum of 0.83 microns long and 125 nanometers high.
  • data is described as encoded in pits on a CD instead of bumps.
  • the marks appear as pits on the aluminum side, but on the side the laser reads from, they are bumps. However, the scheme can be reversed.
  • the CD player has the job of finding and reading the data stored on the CD.
  • the drive consists of three fundamental components.
  • a drive motor spins the disc and is precisely controlled to rotate, typically, between 200 and 500 rpm depending on which track is being read.
  • a laser and a lens system focus in on and read the bumps.
  • a tracking mechanism moves the laser assembly so that the laser's beam can follow the spiral track. The tracking system has to be able to move the laser at micron resolutions.
  • the fundamental job of the CD player is to focus the laser on the track of bumps.
  • the laser beam passes through the polycarbonate layer, reflects off the aluminum layer and hits an opto-electronic device that detects changes in light.
  • the bumps reflect light differently than the lands (the rest of the aluminum layer), and the opto-electronic sensor detects that change in reflectivity.
  • the electronics in the drive interpret the changes in reflectivity in order to read the bits that make up the bytes.
  • the tracking system As it plays the CD, has to continually move the laser outward. As the laser moves outward from the center of the disc, the bumps move past the laser faster. This happens because the linear, or tangential, speed of the bumps is equal to the radius times the speed at which the disc is revolving (rpm). Therefore, as the laser moves outward, the spindle motor must slow the speed of the CD. That way, the bumps travel past the laser at a constant speed, and the data comes off the disc at a constant rate.
  • the embodiments of the present invention provide methods and an apparatus for an assay device for multi-use mapping of an optical bio-disc.
  • the following embodiments discusses the techniques for mapping available ports of the optical bio- disc.
  • a user has to identify a port that has been used when the optical bio-disc is initialized.
  • the above embodiment is necessary when the optical bio-disc is designed for multiple uses during different sessions over extended periods of time.
  • the assay device is designed to be able to logically map the available ports and provide logic to the software that maps the optical bio-disc.
  • the optical bio-disc drive first interrogates the bio-disc and looks for signs if a port has been violated.
  • the disc drive looks for the presence of an analyte on the bio-disc.
  • the assay device creates a data file that relates available sample areas on the bio-disc to a customer interface.
  • the sample areas are preferably encoded in the bio-disc so the software can identify a known area.
  • the sample area is also preferably labeled so that a user can interrogate a response from the software.
  • the logic from the encoded sample area matches the visible labels.
  • used areas on a bio-disc are also provided with a visual response that prevents the user from trying to reuse a used area.
  • This invention or different aspects thereof may be readily implemented in, adapted to, or employed in combination with the discs, assays, and systems disclosed in the following commonly assigned and co-pending patent applications: U.S. Patent Application Serial No. 09/378,878 entitled “Methods and Apparatus for Analyzing Operational and Non-operational Data Acquired from Optical Discs" filed August 23, 1999; U.S. Provisional Patent Application Serial No. 60/150,288 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed August 23, 1999; U.S.
  • Fig. 1 is a pictorial representation of a bio-disc system
  • Fig. 2 is an exploded perspective view of a reflective bio-disc
  • Fig. 3 is a top plan view of the disc shown in Fig. 2;
  • Fig. 4 is a perspective view of the disc illustrated in Fig. 2 with cut-away sections showing the different layers of the disc;
  • Fig. 5 is an exploded perspective view of a transmissive bio-disc;
  • Fig. 6 is a perspective view representing the disc shown in Fig. 5 with a cutaway section illustrating the functional aspects of a semi-reflective layer of the disc;
  • Fig. 7 is a graphical representation showing the relationship between thickness and transmission of a thin gold film
  • Fig. 8 is a top plan view of the disc shown in Fig. 5;
  • Fig. 9 is a perspective view of the disc illustrated in Fig. 5 with cut-away sections showing the different layers of the disc including the type of semi-reflective layer shown in Fig. 6;
  • Fig. 10 is a perspective and block diagram representation illustrating the system of Fig. 1 in more detail;
  • Fig. 11 is a partial cross sectional view taken perpendicular to a radius of the reflective optical bio-disc illustrated in Figs. 2, 3, and 4 showing a flow channel formed therein;
  • Fig. 12 is a partial cross sectional view taken perpendicular to a radius of the transmissive optical bio-disc illustrated in Figs. 5, 8, and 9 showing a flow channel formed therein and a top detector;
  • Fig. 13 is a partial longitudinal cross sectional view of the reflective optical bio- disc shown in Figs. 2, 3, and 4 illustrating a wobble groove formed therein;
  • Fig. 14 is a partial longitudinal cross sectional view of the transmissive optical bio-disc illustrated in Figs. 5, 8, and 9 showing a wobble groove formed therein and a top detector;
  • Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the reflective disc and the initial refractive property thereof;
  • Fig. 16 is a view similar to Fig. 12 showing the entire thickness of the transmissive disc and the initial refractive property thereof;
  • Fig. 17 is a pictorial graphical representation of the transformation of a sampled analog signal to a corresponding digital signal that is stored as a one-dimensional array
  • Fig. 18 is a perspective view of an optical disc with an enlarged detailed view of an indicated section showing a captured white blood cell positioned relative to the tracks of the bio-disc yielding a signal-containing beam after interacting with an incident beam
  • Fig. 19A is a graphical representation of a white blood cell positioned relative to the tracks of an optical bio-disc;
  • Fig. 19B is a series of signature traces derived from the white blood cell of Fig. 19A;
  • Fig. 20 is a graphical representation illustrating the relationship between Figs.
  • Figs. 20A, 20B, 20C, and 20D when taken together, form a pictorial graphical representation of transformation of the signature traces from Fig. 19B into digital signals that are stored as one-dimensional arrays and combined into a two- dimensional array for data input;
  • Fig. 21 is a logic flow chart depicting the principal steps for data evaluation according to processing methods and computational algorithms related to the present invention.
  • Fig. 22 is a flowchart illustrating disc initialization according to an embodiment of the present invention.
  • Fig. 23 is a flowchart illustrating a disc program logic according to an embodiment of the present invention.
  • Fig. 24 is an illustration of a bio-disc with an analyte on its mapping area.
  • the present invention is directed to disc drive systems, optical bio-discs, image processing techniques, counting methods and related software, x, y, and z. Each of these aspects of the present invention is discussed below in further detail.
  • Fig. 1 is a perspective view of an optical bio-disc 110 according to the present invention as implemented to conduct the cell counts and differential cell counts disclosed herein.
  • the present optical bio-disc 110 is shown in conjunction with an optical disc drive 112 and a display monitor 114. Further details relating to this type of disc drive and disc analysis system are disclosed in commonly assigned and co- pending U.S. Patent Application Serial No. 10/008,156 entitled “Disc Drive System and Methods for Use with Bio-discs" filed November 9, 2001 and U.S. Patent Application Serial No. 10/043,688 entitled "Optical Disc Analysis System Including Related Methods For Biological and Medical Imaging” filed January 10, 2002, both of which are herein incorporated by reference.
  • Fig. 2 is an exploded perspective view of the principal structural elements of one embodiment of the optical bio-disc 110.
  • Fig. 2 is an example of a reflective zone optical bio-disc 110 (hereinafter "reflective disc") that may be used in the present invention.
  • the principal structural elements include a cap portion 116, an adhesive member or channel layer 118, and a substrate 120.
  • the cap portion 116 includes one or more inlet ports 122 and one or more vent ports 124.
  • the cap portion 116 may be formed from polycarbonate and is preferably coated with a reflective surface 146 (Fig. 4) on the bottom thereof as viewed from the perspective of Fig. 2.
  • trigger marks or markings 126 are included on the surface of the reflective layer 142 (Fig. 4).
  • Trigger markings 126 may include a clear window in all three layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to a processor 166, as shown Fig. 10, that in turn interacts with the operative functions of the interrogation or incident beam 152, Figs. 6 and 10.
  • the second element shown in Fig. 2 is an adhesive member or channel layer 118 having fluidic circuits 128 or U-channels formed therein.
  • the fluidic circuits 128 are formed by stamping or cutting the membrane to remove plastic film and form the shapes as indicated.
  • Each of the fluidic circuits 128 includes a flow channel 130 and a return channel 132.
  • Some of the fluidic circuits 128 illustrated in Fig. 2 include a mixing chamber 134. Two different types of mixing chambers 134 are illustrated. The first is a symmetric mixing chamber 136 that is symmetrically formed relative to the flow channel 130.
  • the second is an off-set mixing chamber 138.
  • the off-set mixing chamber 138 is formed to one side of the flow channel 130 as indicated.
  • the third element illustrated in Fig. 2 is a substrate 120 including target or capture zones 140.
  • the substrate 120 is preferably made of polycarbonate and has a reflective layer 142 deposited on the top thereof, Fig. 4.
  • the target zones 140 are formed by removing the reflective layer 142 in the indicated shape or alternatively in any desired shape.
  • the target zone 140 may be formed by a masking technique that includes masking the target zone 140 area before applying the reflective layer 142.
  • the reflective layer 142 may be formed from a metal such as aluminum or gold.
  • Fig. 3 is a top plan view of the optical bio-disc 110 illustrated in Fig. 2 with the reflective layer 142 on the cap portion 116 shown as transparent to reveal the fluidic circuits 128, the target zones 140, and trigger markings 126 situated within the disc.
  • Fig. 4 is an enlarged perspective view of the reflective zone type optical bio- disc 110 according to one embodiment of the present invention. This view includes a portion of the various layers thereof, cut away to illustrate a partial sectional view of each principal layer, substrate, coating, or membrane.
  • Fig. 4 shows the substrate 120 that is coated with the reflective layer 142.
  • An active layer 144 is applied over the reflective layer 142.
  • the active layer 144 may be formed from polystyrene.
  • polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co-maleic anhydride, may be used.
  • hydrogels can be used.
  • the plastic adhesive member 118 is applied over the active layer 144.
  • the exposed section of the plastic adhesive member 118 illustrates the cut out or stamped U- shaped form that creates the fluidic circuits 128.
  • the final principal structural layer in this reflective zone embodiment of the present bio-disc is the cap portion 116.
  • the cap portion 116 includes the reflective surface 146 on the bottom thereof.
  • the reflective surface 146 may be made from a metal such as aluminum or gold.
  • FIG. 5 there is shown an exploded perspective view of the principal structural elements of a transmissive type of optical bio-disc 110 according to the present invention.
  • the principal structural elements of the transmissive type of optical bio-disc 110 similarly include the cap portion 116, the adhesive or channel member 118, and the substrate 120 layer.
  • the cap portion 116 includes one or more inlet ports 122 and one or more vent ports 124.
  • the cap portion 116 may be formed from a polycarbonate layer.
  • Optional trigger markings 126 may be included on the surface of a thin semi-reflective layer 143, as best illustrated in Figs. 6 and 9.
  • Trigger markings 126 may include a clear window in all three layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to the processor 166, Fig. 10, which in turn interacts with the operative functions of the interrogation beam 152, Figs. 6 and 10.
  • the second element shown in Fig. 5 is the adhesive member or channel layer 118 having fluidic circuits 128 or U-channels formed therein.
  • the fluidic circuits 128 are formed by stamping or cutting the membrane to remove plastic film and form the shapes as indicated.
  • Each of the fluidic circuits 128 includes the flow channel 130 and the return channel 132.
  • Some of the fluidic circuits 128 illustrated in Fig. 5 include the mixing chamber 134. Two different types of mixing chambers 134 are illustrated. The first is the symmetric mixing chamber 136 that is symmetrically formed relative to the flow channel 130. The second is the off-set mixing chamber 138. The off-set mixing chamber 138 is formed to one side of the flow channel 130 as indicated.
  • the third element illustrated in Fig. 5 is the substrate 120 which may include the target or capture zones 140.
  • the substrate 120 is preferably made of polycarbonate and has the thin semi-reflective layer 143 deposited on the top thereof, Fig. 6.
  • the semi-reflective layer 143 associated with the substrate 120 of the disc 110 illustrated in Figs. 5 and 6 is significantly thinner than the reflective layer 142 on the substrate 120 of the reflective disc 110 illustrated in Figs. 2, 3 and 4.
  • the thinner semi-reflective layer 143 allows for some transmission of the interrogation beam 152 through the structural layers of the transmissive disc as shown in Figs. 6 and 12.
  • the thin semi-reflective layer 143 may be formed from a metal such as aluminum or gold.
  • Fig. 6 is an enlarged perspective view of the substrate 120 and semi-reflective layer 143 of the transmissive embodiment of the optical bio-disc 110 illustrated in Fig.
  • the thin semi-reflective layer 143 may be made from a metal such as aluminum or gold.
  • the thin semi-reflective layer 143 of the transmissive disc illustrated in Figs. 5 and 6 is approximately 100-300 A thick and does not exceed 400 A.
  • This thinner semi-reflective layer 143 allows a portion of the incident or interrogation beam 152 to penetrate and pass through the semi-reflective layer 143 to be detected by a top detector 158, Figs. 10 and 12, while some of the light is reflected or returned back along the incident path.
  • Table 1 presents the reflective and transmissive characteristics of a gold film relative to the thickness of the film.
  • the gold film layer is fully reflective at a thickness greater than 800 A. While the threshold density for transmission of light through the gold film is approximately 400 A.
  • Fig. 7 provides a graphical representation of the inverse relationship of the reflective and transmissive nature of the thin semi-reflective layer 143 based upon the thickness of the gold. Reflective and transmissive values used in the graph illustrated in Fig. 7 are absolute values. TABLE 1 Au Film Reflection and Transmission (Absolute Values)
  • FIG. 8 there is shown a top plan view of the transmissive type optical bio-disc 110 illustrated in Figs. 5 and 6 with the transparent cap portion 116 revealing the fluidic channels, the trigger markings 126, and the target zones 140 as situated within the disc.
  • Fig. 9 is an enlarged perspective view of the optical bio-disc 110 according to the transmissive disc embodiment of the present invention.
  • the disc 110 is illustrated with a portion of the various layers thereof cut away to show a partial sectional view of each principal layer, substrate, coating, or membrane.
  • Fig. 9 illustrates a transmissive disc format with the clear cap portion 116, the thin semi-reflective layer 143 on the substrate 120, and trigger markings 126.
  • trigger markings 126 include opaque material placed on the top portion of the cap.
  • the trigger marking 126 may be formed by clear, non-reflective windows etched on the thin reflective layer 143 of the disc, or any mark that absorbs or does not reflect the signal coming from the trigger detector 160, Fig. 10.
  • Fig. 10 is an enlarged perspective view of the optical bio-disc 110 according to the transmissive disc embodiment of the present invention.
  • the disc 110 is illustrated with a portion of the various layers thereof cut away to show a partial sectional view of each principal layer, substrate, coating,
  • target zones 140 formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone 140 may be made on the thin semi- reflective layer 143 on the substrate 120 or on the bottom portion of the substrate 120 (under the disc). Alternatively, the target zones 140 may be formed by a masking technique that includes masking the entire thin semi-reflective layer 143 except the target zones 140. In this embodiment, target zones 140 may be created by silk screening ink onto the thin semi-reflective layer 143. In the transmissive disc format illustrated in Figs. 5, 8, and 9, the target zones 140 may alternatively be defined by address information encoded on the disc. In this embodiment, target zones 140 do not include a physically discemable edge boundary.
  • an active layer 144 is illustrated as applied over the thin semi-reflective layer 143.
  • the active layer 144 is a 10 to 200 ⁇ m thick layer of 2% polystyrene.
  • polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co- maleic anhydride may be used.
  • hydrogels can be used.
  • the plastic adhesive member 118 is applied over the active layer 144. The exposed section of the plastic adhesive member 118 illustrates the cut out or stamped U-shaped form that creates the fluidic circuits 128.
  • the final principal structural layer in this transmissive embodiment of the present bio-disc 110 is the clear, non-reflective cap portion 116 that includes inlet ports 122 and vent ports 124.
  • Fig. 10 there is a representation in perspective and block diagram illustrating optical components 148, a light source 150 that produces the incident or interrogation beam 152, a return beam 154, and a transmitted beam 156.
  • the return beam 154 is reflected from the reflective surface 146 of the cap portion 116 of the optical bio-disc 110.
  • the return beam 154 is detected and analyzed for the presence of signal elements by a bottom detector 157.
  • the transmitted beam 156 is detected, by a top detector 158, and is also analyzed for the presence of signal elements.
  • a photo detector may be used as a top detector 158.
  • Fig. 10 also shows a hardware trigger mechanism that includes the trigger markings 126 on the disc and a trigger detector 160.
  • the hardware triggering mechanism is used in both reflective bio-discs (Fig. 4) and transmissive bio-discs (Fig. 9).
  • the triggering mechanism allows the processor 166 to collect data only when the interrogation beam 152 is on a respective target zone 140.
  • a software trigger may also be used. The software trigger uses the bottom detector to signal the processor 166 to collect data as soon as the interrogation beam 152 hits the edge of a respective target zone 140.
  • FIG. 10 further illustrates a drive motor 162 and a controller 164 for controlling the rotation of the optical bio-disc 110.
  • Fig. 10 also shows the processor 166 and analyzer 168 implemented in the alternative for processing the return beam 154 and transmitted beam 156 associated the transmissive optical bio-disc.
  • Fig. 11 there is presented a partial cross sectional view of the reflective disc embodiment of the optical bio-disc 110 according to the present invention.
  • Fig. 11 illustrates the substrate 120 and the reflective layer 142.
  • the reflective layer 142 may be made from a material such as aluminum, gold or other suitable reflective material.
  • the top surface of the substrate 120 is smooth.
  • Fig. 11 also shows the active layer 144 applied over the reflective layer 142.
  • the target zone 140 is formed by removing an area or portion of the reflective layer 142 at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer 142.
  • the plastic adhesive member 118 is applied over the active layer 144.
  • Fig. 11 also shows the cap portion 116 and the reflective surface 146 associated therewith.
  • the path of the incident beam 152 is initially directed toward the substrate 120 from below the disc 110.
  • the incident beam then focuses at a point proximate the reflective layer 142. Since this focusing takes place in the target zone 140 where a portion of the reflective layer 142 is absent, the incident continues along a path through the active layer 144 and into the flow channel 130.
  • the incident beam 152 then continues upwardly traversing through the flow channel to eventually fall incident onto the reflective surface 146. At this point, the incident beam 152 is returned or reflected back along the incident path and thereby forms the return beam 154.
  • Fig. 12 is a partial cross sectional view of the transmissive embodiment of the bio-disc 110 according to the present invention.
  • Fig. 12 illustrates a transmissive disc format with the clear cap portion 116 and the thin semi-reflective layer 143 on the substrate 120.
  • Fig. 12 also shows the active layer 144 applied over the thin semi- reflective layer 143.
  • the transmissive disc has the thin semi-reflective layer 143 made from a metal such as aluminum or gold approximately 100-300 Angstroms thick and does not exceed 400 Angstroms. This thin semi- reflective layer 143 allows a portion of the incident or interrogation beam 152, from the light source 150, Fig.
  • a top detector 158 to penetrate and pass upwardly through the disc to be detected by a top detector 158, while some of the light is reflected back along the same path as the incident beam but in the opposite direction.
  • the return or reflected beam 154 is reflected from the semi-reflective layer 143.
  • the reflected light or return beam 154 may be used for tracking the incident beam 152 on pre-recorded information tracks formed in or on the semi-reflective layer 143 as described in more detail in conjunction with Figs. 13 and 14.
  • a physically defined target zone 140 may or may not be present.
  • Target zone 140 may be created by direct markings made on the thin semi-reflective layer 143 on the substrate 120. These marking may be formed using silk screening or any equivalent method. In the alternative embodiment where no physical indicia are employed to define a target zone (such as, for example, when encoded software addressing is utilized) the flow channel 130 in effect may be employed as a confined target area in which inspection of an investigational feature is conducted.
  • Fig. 13 is a cross sectional view taken across the tracks of the reflective disc embodiment of the bio-disc 110 according to the present invention. This view is taken longitudinally along a radius and flow channel of the disc.
  • Fig. 13 includes the substrate 120 and the reflective layer 142.
  • the substrate 120 includes a series of grooves 170.
  • the grooves 170 are in the form of a spiral extending from near the center of the disc toward the outer edge.
  • the grooves 170 are implemented so that the interrogation beam 152 may track along the spiral grooves 170 on the disc.
  • This type of groove 170 is known as a "wobble groove".
  • a bottom portion having undulating or wavy sidewalls forms the groove 170, while a raised or elevated portion separates adjacent grooves 170 in the spiral.
  • the reflective layer 142 applied over the grooves 170 in this embodiment is, as illustrated, conformal in nature.
  • Fig. 13 also shows the active layer 144 applied over the reflective layer 142.
  • the target zone 140 is formed by removing an area or portion of the reflective layer 142 at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer 142.
  • the plastic adhesive member 118 is applied over the active layer 144.
  • Fig. 13 also shows the cap portion 116 and the reflective surface 146 associated therewith. Thus, when the cap portion 116 is applied to the plastic adhesive member 118 including the desired cutout shapes, the flow channel 130 is thereby formed.
  • Fig. 14 is a cross sectional view taken across the tracks of the transmissive disc embodiment of the bio-disc 110 according to the present invention as described in Fig. 12, for example. This view is taken longitudinally along a radius and flow channel of the disc.
  • Fig. 14 illustrates the substrate 120 and the thin semi-reflective layer 143.
  • This thin semi-reflective layer 143 allows the incident or interrogation beam 152, from the light source 150, to penetrate and pass through the disc to be detected by the top detector 158, while some of the light is reflected back in the form of the return beam 154.
  • the thickness of the thin semi-reflective layer 143 is determined by the minimum amount of reflected light required by the disc reader to maintain its tracking ability.
  • the substrate 120 in this embodiment, like that discussed in Fig.
  • Fig. 14 includes the series of grooves 170.
  • the grooves 170 in this embodiment are also preferably in the form of a spiral extending from near the center of the disc toward the outer edge.
  • the grooves 170 are implemented so that the interrogation beam 152 may track along the spiral.
  • Fig. 14 also shows the active layer 144 applied over the thin semi-reflective layer 143.
  • the plastic adhesive member or channel layer 118 is applied over the active layer 144.
  • Fig. 14 also shows the cap portion 116 without a reflective surface 146.
  • Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the reflective disc and the initial refractive property thereof.
  • Fig. 16 is a view similar to Fig. 12 showing the entire thickness of the transmissive disc and the initial refractive property thereof.
  • Grooves 170 are not seen in Figs. 15 and 16 since the sections are cut along the grooves 170.
  • Figs. 15 and 16 show the presence of the narrow flow channel 130 that is situated perpendicular to the grooves 170 in these embodiments.
  • Figs. 13, 14, 15, and 16 show the entire thickness of the respective reflective and transmissive discs.
  • the incident beam 152 is illustrated initially interacting with the substrate 120 which has refractive properties that change the path of the incident beam as illustrated to provide focusing of the beam 152 on the reflective layer 142 or the thin semi-reflective layer 143.
  • the return beam 154 carries the information about the biological sample. As discussed above, such information about the biological sample is contained in the return beam essentially only when the incident beam is within the flow channel 130 or target zones 140 and thus in contact with the sample. In the reflective embodiment of the bio-disc 110, the return beam 154 may also carry information encoded in or on the reflective layer 142 or otherwise encoded in the wobble grooves 170 illustrated in Figs. 13 and 14. As would be apparent to one of skill in the art, pre-recorded information is contained in the return beam 154 of the reflective disc with target zones, only when the corresponding incident beam is in contact with the reflective layer 142.
  • Such information is not contained in the return beam 154 when the incident beam 152 is in an area where the information bearing reflective layer 142 has been removed or is otherwise absent.
  • the transmitted beam 156 carries the information about the biological sample.
  • the information about the biological test sample is directed to a processor 166 for signal processing.
  • This processing involves transformation of the analog signal detected by the bottom detector 157 (reflective disc) or the top detector 158 (transmissive disc) to a discrete digital form.
  • the signal transformation involves sampling the analog signal 210 at fixed time intervals 212, and encoding the corresponding instantaneous analog amplitude 214 of the signal as a discrete binary integer 216. Sampling is started at some start time 218 and stopped at some end time 220.
  • sampling frequency also called the sampling rate
  • bit depth is the number of bits used in each sample point to encode the sampled amplitude 214 of the analog signal 210. The greater the bit depth, the better the binary integer 216 will approximate the original analog amplitude 214.
  • the sampling rate is 8 MHz with a bit depth of 12 bits per sample, allowing an integer sample range of 0 to 4095 (0 to (2 n - 1), where n is the bit depth.
  • This combination may change to accommodate the particular accuracy necessary in other embodiments.
  • the sampled data is then sent to processor 166 for analog-to-digital transformation.
  • each consecutive sample point 224 along the laser path is stored consecutively on disc or in memory as a one- dimensional array 226.
  • Each consecutive track contributes an independent one- dimensional array, which yields a two-dimensional array 228 (Fig. 20A) that is analogous to an image.
  • Fig. 18 is a perspective view of an optical bio-disc 110 of the present invention with an enlarged detailed perspective view of the section indicated showing a captured white blood cell 230 positioned relative to the tracks 232 of the optical bio-disc.
  • the white blood cell 230 is used herein for illustrative purposes only. As indicated above, other objects or investigational features such as beads or agglutinated matter may be utilized herewith.
  • the interaction of incident beam 152 with white blood cell 230 yields a signal-containing beam, either in the form of a return beam 154 of the reflective disc or a transmitted beam 156 of the transmissive disc, which is detected by either of detectors 157 or 158.
  • Fig. 18 is a perspective view of an optical bio-disc 110 of the present invention with an enlarged detailed perspective view of the section indicated showing a captured white blood cell 230 positioned relative to the tracks 232 of the optical bio-disc.
  • the white blood cell 230 is used herein for illustrative purposes only. As indicated above, other objects
  • FIG. 19A is another graphical representation of the white blood cell 230 positioned relative to the tracks 232 of the optical bio-disc 110 shown in Fig. 18.
  • the white blood cell 230 covers approximately four tracks A, B, C, and D.
  • Fig. 19B shows a series of signature traces derived from the white blood cell 210 of Figs. 19 and 19A.
  • the detection system provides four analogue signals A, B, C, and D corresponding to tracks A, B, C, and D.
  • each of the analogue signals A, B, C, and D carries specific information about the white blood cell 230.
  • a scan over a white blood cell 230 yields distinct perturbations of the incident beam that can be detected and processed.
  • the analog signature traces (signals) 210 are then directed to processor 166 for transformation to an analogous digital signal 222 as shown in Figs. 20A and 20C as discussed in further detail below.
  • Fig. 20 is a graphical representation illustrating the relationship between Figs. 20A, 20B, 20C, and 20D.
  • Figs. 20A, 20B, 20C, and 20D are pictorial graphical representations of transformation of the signature traces from Fig. 19B into digital signals 222 that are stored as one-dimensional arrays 226 and combined into a two- dimensional array 228 for data input 244.
  • Fig. 20A there is shown sampled analog signals 210 from tracks A and B of the optical bio-disc shown in Figs. 18 and 19A.
  • Processor 166 then encodes the corresponding instantaneous analog amplitude 214 of the analog signal 210 as a discrete binary integer 216 (see Fig. 17).
  • the resulting series of data points is the digital signal 222 that is analogous to the sampled analog signal 210.
  • digital signal 222 from tracks A and B (Fig. 20A) is stored as an independent one-dimensional memory array 226. Each consecutive track contributes a corresponding one-dimensional array, which when combined with the previous one-dimensional arrays, yields a two-dimensional array 228 that is analogous to an image.
  • the digital data is then stored in memory or on disc as a two- dimensional array 228 of sample points 224 (Fig. 17) that represent the relative intensity of the return beam 154 or transmitted beam 156 (Fig. 18) at a particular point in the sample area.
  • the two-dimensional array is then stored in memory or on disc in the form of a raw file or image file 240 as represented in Fig. 20B.
  • the data stored in the image file 240 is then retrieved 242 to memory and used as data input 244 to analyzer 168 shown in Fig. 10.
  • Fig. 20C shows sampled analog signals 210 from tracks C and D of the optical bio-disc shown in Figs. 18 and 19A.
  • Processor 166 then encodes the corresponding instantaneous analog amplitude 214 of the analog signal 210 as a discrete binary integer 216 (Fig. 17).
  • the resulting series of data points is the digital signal 222 that is analogous to the sampled analog signal 210.
  • digital signal 222 from tracks C and D is stored as an independent one-dimensional memory array 226. Each consecutive track contributes a corresponding one-dimensional array, which when combined with the previous one-dimensional arrays, yields a two-dimensional array 228 that is analogous to an image.
  • the digital data is then stored in memory or on disc as a two- dimensional array 228 of sample points 224 (Fig. 17) that represent the relative intensity of the return beam 154 or transmitted beam 156 (Fig. 18) at a particular point in the sample area.
  • the two-dimensional array is then stored in memory or on disc in the form of a raw file or image file 240 as shown in Fig. 20B.
  • the data stored in the image file 240 is then retrieved 242 to memory and used as data input 244 to analyzer 168 Fig. 10.
  • the computational and processing algorithms of the present invention are stored in analyzer 168 (Fig. 10) and applied to the input data 244 to produce useful output results 262 (Fig. 21) that may be displayed on the display monitor 114 (Fig. 10).
  • a first principal step of the present processing method involves receipt of the input data 244.
  • data evaluation starts with an array of integers in the range of 0 to 4096.
  • the next principle step 246 is selecting an area of the disc for counting. Once this area is defined, an objective then becomes making an actual count of all white blood cells contained in the defined area.
  • the implementation of step 246 depends on the configuration of the disc and user's options.
  • the software recognizes the windows and crops a section thereof for analysis and counting.
  • the target zones or windows have the shape of 1x2 mm rectangles with a semicircular section on each end thereof.
  • the software crops a standard rectangle of 1x2 mm area inside a respective window.
  • the reader may take several consecutive sample values to compare the number of cells in several different windows.
  • step 246 may be performed in one of two different manners.
  • the position of the standard rectangle is chosen either by positioning its center relative to a point with fixed coordinates, or by finding reference mark which may be a spot of dark dye.
  • a dye with a desired contrast is deposited in a specific position on the disc with respect to two clusters of cells.
  • the optical disc reader is then directed to skip to the center of one of the clusters of cells and the standard rectangle is then centered around the selected cluster.
  • the user may specify a desired sample area shape for cell counting, such as a rectangular area, by direct interaction with mouse selection or otherwise.
  • a desired sample area shape for cell counting such as a rectangular area
  • this involves using the mouse to click and drag a rectangle over the desired portion of the optical bio-disc-derived image that is displayed on a monitor 114.
  • a respective rectangular area is evaluated for counting in the next step 248.
  • step 248 is directed to background illumination uniformization.
  • Background illumination uniformization offsets the intensity level of each sample point such that the overall background, or the portion of the image that is not cells, approaches a plane with an arbitrary background value Vbackground- While Vbac ground may be decided in many ways, such as taking the average value over the standard rectangular sample area, in the present embodiment, the value is set to 2000.
  • V at each point P of the selected rectangular sample area is replaced with the number (Vba ck groun d + (V - average value over the neighborhood of P)) and truncated, if necessary, to fit the actual possible range of values, which is 0 to 4095 in a preferred embodiment of the present invention.
  • the dimensions of the neighborhood are chosen to be sufficiently larger than the size of a cell and sufficiently smaller than the size of the standard rectangle.
  • the next step in the flow chart of Fig. 21 is a normalization step 250.
  • a linear transform is performed with the data in the standard rectangular sample area so that the average becomes 2000 with a standard deviation of 600. If necessary, the values are truncated to fit the range 0 to 4096.
  • This step 250, as well as the background illumination uniformization step 248, makes the software less sensitive to hardware modifications and tuning.
  • the signal gain in the detection circuitry such as top detector 158 (Fig. 18) may change without significantly affecting the resultant cell counts.
  • a filtering step 252 is next performed.
  • the number of points in the neighborhood of P, with dimensions smaller than indicated in step 248, with values sufficiently distinct from Vb ack gr o un d is calculated.
  • the points calculated should approximate the size of a cell in the image. If this number is large enough, the value at P remains as it was; otherwise it is assigned to Vba ck gr o un d -
  • This filtering operation is performed to remove noise, and in the optimal case only cells remain in the image while the background is uniformly equal Vbackground-
  • An optional step 254 directed to removing bad components may be performed as indicated in Fig. 21. Defects such as scratches, bubbles, dirt, and other similar irregularities may pass through filtering step 252.
  • defects may cause cell counting errors either directly or by affecting the overall distribution in the images histogram.
  • these defects are sufficiently larger in size than cells and can be removed in step 254 as follows. First a binary image with the same dimensions as the selected region is formed. A in the binary image is defined as white, if the value at the corresponding point of the original image is equal to Vbackground, and black otherwise. Next, connected components of black points are extracted. Then subsequent erosion and expansion are applied to regularize the view of components. And finally, components that are larger than a defined threshold are removed.
  • the component is removed from the original image by assigning the corresponding sample points in the original image with the value Vbackground-
  • the threshold that determines which components constitute countable objects and which are to be removed is a user-defined value. This threshold may also vary depending on the investigational feature being counted i.e. white blood cells, red blood cells, or other biological matter.
  • the next principal processing step shown in Fig. 21 is step 256, which is directed to counting cells by bright centers.
  • the counting step 256 consists of several substeps. The first of these substeps includes performing a convolution. In this convolution substep, an auxiliary array referred to as a convolved picture is formed. The value of the convolved picture at point P is the result of integration of a picture after filtering in the circular neighborhood of P. More precisely, for one specific embodiment, the function that is integrated, is the function that equals v-2000 when v is greater than 2000 and 0 when v is less than or equal to 2000.
  • the next substep performed in counting step 256 is finding the local maxima of the convolved picture in the neighborhood of a radius about the size of a cell. Next, duplicate local maxima with the same value in a closed neighborhood of each other are avoided. In the last substep in counting step 256, the remaining local maxima are declared to mark cells.
  • some cells may appear without bright centers. In these instances, only a dark rim is visible and the following two optional steps 258 and 260 are useful.
  • Step 258 is directed to removing found cells from the picture.
  • the circular region around the center of each found cell is filled with the value 2000 so that the cells with both bright centers and dark rims would not be found twice.
  • Step 260 is directed to counting additional cells by dark rims.
  • Two transforms are made with the image after step 258.
  • substep (a) the value v at each point is replaced with (2000-v) and if the result is negative it is replaced with zero.
  • substep (b) the resulting picture is then convolved with a ring of inner radius R1 and outer radius R2.
  • R1 and R2 are, respectively, the minimal and the maximal expected radius of a cell, the ring being shifted, subsequently, in substep (d) to the left, right, up and down.
  • substep (c) the results of four shifts are summed. After this transform, the image of a dark rim cell looks like a four petal flower.
  • maxima of the function obtained in substep (c) are found in a manner to that employed in counting step 256. They are declared to mark cells omitted in step 256.
  • the last principal step illustrated in Fig. 21 is a results output step 262.
  • the number of cells found in the standard rectangle is displayed on the monitor 114 shown in Figs. 1 and 5, and each cell identified is marked with a cross on the displayed optical bio-discderived image. Additional computer science methodologies and apparatus directed to extracting and visualizing data from bio-discs and/or optical analysis discs are discussed in commonly assigned U.S. Patent Application Serial No.
  • the embodiments of the present invention are methods and an apparatus for an assay device for multi-use mapping of an optical bio-disc.
  • numerous specific details are set forth to provide a more thorough description of embodiments of the invention. It will be apparent, however, to one skilled in the art, that the embodiments of the present invention may be practiced without these specific details. In other instances, well known features have not been described in detail so as not to obscure the invention.
  • the present invention provides an independent mapping step when the bio-disc is first initialized. Since the possibility of changing the disc configuration each time the disc is removed from the drive, it is necessary to provide a user with a mapping procedure each time the drive is initialized by the disc.
  • one way to initialize a disc is to follow a sequence of commands that include a pseudo code on the disc.
  • This code describes all of the possible testing positions on the disc.
  • a manufactured area in each of the sample positions respond to a violation of the sample boundary that defines the potential addition of an analyte to that position.
  • the initial scan of the disc provides a software routine with a map of used and unused areas of the disc.
  • Pseudo-Code And Mapping The above embodiment is illustrated in Fig. 22, where at step 100 the pseudo code is included on the disc. At step 110, a check is made to see if there have been any violations of the sample boundary of the disc. If there is a violation, then at step 120 a manufactured area on the disc responds to the violation. If not, then at step 130 the initial scan of the disc generates a software routine with a map of all used and unused areas on the disc.
  • An example of using pseudo code variables for a mapping of "use" is shown below, where A, B, etc. are the variables along with their Cartesian co-ordinates.
  • a chemistry is added to the channel that responds to the presence of any potential analyte. Since the response to the analyte is optical, it can be identified by a detector configuration in the disc drive. The response to the analyte provides a positive or negative use to the disc usage map.
  • the chemistry may also, according to another embodiment of the present invention, react over a period of time to identify that the mapped locations usage has expired, which provides the viability of that position for disc usage.
  • a micro-controller performs a target inquiry of the drive using a program run from the bio-disc.
  • Fig. 23 illustrates a disc program logic that includes the above mentioned target inquiry.
  • the disc drive performs a target inquiry of the drive using a program run from the disc with the help of a micro-controller.
  • a check is made to see if the drive is a Binary Coded Decimal (BCD) drive. If, at step 210 it is found that the drive is a non-BCD drive, then at step 220 there is a video disclaimer and at step 230, the disc is ejected.
  • BCD Binary Coded Decimal
  • step 210 reveals that the drive is a BCD drive
  • step 240 the program logic performs a command like BTI.inf.
  • the pseudo code variables are read from the disc into the system, which is on board (refer to the pseudo-code and mapping section above).
  • mapping described in the pseudo-code and mapping section above is performed.
  • the scanning results are compared with the pseudo-code to produce a usage map for the disc.
  • step 280 an instructional video is displayed to the user.
  • Fig. 24 is an illustration of a bio-disc with an analyte (300) placed at a location on its mapping area (310). The arrows illustrate the use of a program disc logic, like the one explained above to analyze the analyte.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne des procédés et un appareil pour un dispositif de dosage pour assurer le mappage à multiples utilisations d'un bio-disque optique afin de détecter la présence d'un analyte sur ce dernier. En cas de détection d'un analyte, le dispositif crée un ficher de données qui relie les zones échantillons disponibles sur le disque à une interface client. Des zones échantillons sont codées dans le disque de telle sorte que le logiciel peut les identifier, et sont de préférence, étiquetées de telle sorte qu'un utilisateur peut demander une réponse à partir de ce logiciel. Les zones utilisées donnent également une réponse visuelle, ce qui empêche l'utilisateur d'utiliser de nouveau une zone utilisée. Un utilisateur doit identifier un port qui a été utilisé lors de l'initialisation de ce disque, ce qui est nécessaire lorsque le disque est conçu pour de multiples utilisations pendant diverses sessions sur des laps de temps prolongés. Ce dispositif est prévu pour mapper de manière logique les ports disponibles et assurer une logique pour le logiciel.
PCT/US2003/005166 2002-02-19 2003-02-19 Procedes et appareil pour le mappage a multiples utilisations d'un bio-disque optique WO2003071395A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003216346A AU2003216346A1 (en) 2002-02-19 2003-02-19 Methods and an apparatus for multi-use mapping of an optical bio-disc

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35849002P 2002-02-19 2002-02-19
US60/358,490 2002-02-19

Publications (1)

Publication Number Publication Date
WO2003071395A2 true WO2003071395A2 (fr) 2003-08-28

Family

ID=27757746

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/005166 WO2003071395A2 (fr) 2002-02-19 2003-02-19 Procedes et appareil pour le mappage a multiples utilisations d'un bio-disque optique

Country Status (2)

Country Link
AU (1) AU2003216346A1 (fr)
WO (1) WO2003071395A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010040371A1 (fr) * 2008-10-07 2010-04-15 Bio Explorer B.V. Dispositif et procédé d'analyse automatique de micro-organismes dans un échantillon

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010040371A1 (fr) * 2008-10-07 2010-04-15 Bio Explorer B.V. Dispositif et procédé d'analyse automatique de micro-organismes dans un échantillon
US11226347B2 (en) 2008-10-07 2022-01-18 Biotrack Holding B.V. Method for simultaneously and automatically analyzing microorganisms in a sample using filter cytometry

Also Published As

Publication number Publication date
AU2003216346A1 (en) 2003-09-09

Similar Documents

Publication Publication Date Title
US7428200B2 (en) Method for triggering through disc grooves and related optical analysis discs and system
US20050032052A1 (en) Methods and apparatus for extracting data from and optical analysis disc
AU2003209372A1 (en) Method for triggering through disc grooves and related optical analysis discs and system
US7200088B2 (en) System and method of detecting investigational features related to a sample
US6965433B2 (en) Optical biodiscs with reflective layers
US7221632B2 (en) Optical disc system and related detecting methods for analysis of microscopic structures
US7061594B2 (en) Disc drive system and methods for use with bio-discs
US20050002827A1 (en) Optical discs including equi-radial and/or spiral analysis zones and related disc drive systems and methods
US20070280859A1 (en) Fluidic circuits for sample preparation including bio-discs and methods relating thereto
US20050037484A1 (en) Optical bio-discs including spiral fluidic circuits for performing assays
US20050084422A1 (en) Fluidic circuits for sample preparation including bio-discs and methods relating thereto
US7077996B2 (en) Methods and apparatus for blood separation and analysis using membranes on an optical bio-disc
US20040241381A1 (en) Microfluidic structures with circumferential grooves for bonding adhesives and related optical analysis discs
US20080094974A1 (en) Optical disc system and related detecting methods for analysis of microscopic structures
US20040226348A1 (en) Magnetic assisted detection of magnetic beads using optical disc drives
US20050023765A1 (en) Bio-safety features for optical analysis disc and disc system including same
JP2005523684A (ja) バイオドライブ用のセグメント領域検出器とその関連方法
US20050170490A1 (en) Processes for manufacturing optical analysis discs with molded microfluidic structures and discs made according thereto
WO2003071395A2 (fr) Procedes et appareil pour le mappage a multiples utilisations d'un bio-disque optique
US20050221048A1 (en) Manufacturing processes for making optical analysis discs including successive patterning operations and optical discs thereby manufactured
WO2003007293A2 (fr) Systeme d'analyse de disque optique comprenant des procedes associes destines a l'imagerie biologique et medicale

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 OM PH PL PT RO RU SD SE SG SK SL TJ TM TN TR TT TZ UA UG UZ VN YU ZA ZM 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 ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WA Withdrawal of international application
NENP Non-entry into the national phase

Ref country code: JP