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WO1997026993A1 - Heating - Google Patents

Heating Download PDF

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Publication number
WO1997026993A1
WO1997026993A1 PCT/GB1997/000195 GB9700195W WO9726993A1 WO 1997026993 A1 WO1997026993 A1 WO 1997026993A1 GB 9700195 W GB9700195 W GB 9700195W WO 9726993 A1 WO9726993 A1 WO 9726993A1
Authority
WO
WIPO (PCT)
Prior art keywords
sheet
heating
carrier
specimen
metallic
Prior art date
Application number
PCT/GB1997/000195
Other languages
French (fr)
Inventor
Ian Alan Gunter
Original Assignee
Bjs Company Ltd.
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
Priority claimed from GBGB9601471.7A external-priority patent/GB9601471D0/en
Priority claimed from GBGB9603717.1A external-priority patent/GB9603717D0/en
Application filed by Bjs Company Ltd. filed Critical Bjs Company Ltd.
Priority to DE69703381T priority Critical patent/DE69703381T2/en
Priority to AT97901166T priority patent/ATE197129T1/en
Priority to DK97901166T priority patent/DK0876218T3/en
Priority to JP52665897A priority patent/JP3705606B2/en
Priority to AU14510/97A priority patent/AU1451097A/en
Priority to EP97901166A priority patent/EP0876218B1/en
Priority to CA002244178A priority patent/CA2244178C/en
Publication of WO1997026993A1 publication Critical patent/WO1997026993A1/en
Priority to GR20000402296T priority patent/GR3034671T3/en

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/45—Magnetic mixers; Mixers with magnetically driven stirrers
    • B01F33/452—Magnetic mixers; Mixers with magnetically driven stirrers using independent floating stirring elements

Definitions

  • the present invention relates to heating and more particularly to the thermal cycling of specimen carriers.
  • specimen carriers in the form of support blocks or platterns are used for various processes where small samples are thermally cycled.
  • a particular example is the Polymerase Chain Reaction method (often referred to as PCR) for replicating DNA samples.
  • PCR Polymerase Chain Reaction method
  • Such samples require rapid and accurate thermal cycling, and are typically placed in a multi-well block and cycled between several selected temperatures in a pre-set repeated cycle.
  • the present invention solves this problem by applying direct electrical resistive heating to a metallic specimen carrier.
  • the invention provides a method of heating a specimen carrier in the form of a metallic sheet and applying a heating current to said sheet.
  • the metallic sheet will be of silver which has a high thermal and electrical conductivity.
  • the sheet will generally have a thin section in the region of 0.3mm thickness, and may be in a form where a matrix of sample wells is incorporated in the sheet.
  • the metallic sheet may be a solid sheet or block of silver (which may have cavities forming wells)
  • a metallised plastic tray which may have impressed wells
  • deposited metal forms a resistive heating element
  • Another alternative is to electro form a thin metal tray (which again may have impressed wells), and to coat the metal with a bio-compatible polymer.
  • the plastic trays are conventionally single use disposable items.
  • the incorporation of the heating element into the plastic trays may increase their cost, but the reduction in cycling time for the PCR reaction more than compensates for any increased cost of the disposable item.
  • the bottom of the composite tray should be unobstructed when fan cooling is employed. If sub-ambient cooling is required at the end of the PCR cycles, either with a composite tray or a block, chilled liquid spray-cooling may be employed.
  • the boiling point of the liquid should be below the low point of the PCR cycle so that liquid does not remain on the metal of the tray or block to impede heating. This also allows for the latent heat of evaporation of the liquid to increase the cooling effect.
  • the heating current may be an AC current supplied from the secondary winding of a transformer. This allows cycling control to be applied to the primary circuit of the transformer (higher voltage, lower current) in a convenient way without encountering problems which arise when operating with high current devices .
  • the transformer may comprise a toroidal core having an appropriate mains primary winding and a single bus bar looped through the core and connected in series with the metallic sheet to form a single turn secondary circuit.
  • Figure 1 is a side elevation of a heating apparatus
  • Figure 2 is a plan view of the apparatus of Figure 1.
  • a metallic specimen carrier in the form of a multi-well block (1) measuring 110mm x 75mm and having 96 wells (2) disposed in a grid layout is formed in silver nominally 0.3mm thick. This is attached to bus bars (3) of substantial cross-sectional area.
  • the bus bars loop once through a transformer (toroidal or square), core (4).
  • the core (4) has a primary winding (5) appropriate for the mains voltage employed.
  • the transformer primary current is controlled using a triac device (6).
  • the triac device receives current from an AC source and is controlled by a temperature control circuit (7) which uses a fine wire thermocouple (8) soldered to a central underside region of the block to sense the temperature of the block.
  • the temperature control circuitry may be operated manually or by a personal computer (9).
  • Cooling of the block is by means of a fan (10) mounted under the block, blowing ambient air over the protruding well forms (2), the air being directed by the enclosure in which the block is mounted.
  • the fan is controlled by the same temperature control circuitry that drives the heater triac .
  • the measured performance of the example apparatus gives rates of change of temperature in excess of 6 degrees per second and over/under shoots of less than 0.25 degrees within the typical PCR working range of 50-100 degrees.
  • the described examples use a silver block with cavities, but metalised plastic tray inserts, or electro formed thin metal trays, as previously described, may also be used.
  • the system as described has several important advantages . 1.1
  • the block is heated directly with no requirement for heat transfer from an attached heat source. This is very efficient and taken together with the very low specific heat capacity of silver allows very rapid temperature changes.
  • Direct heating means that there is no thermal lag at all. Temperature control functions are immediate so that the block may be cycled in temperature with little or no over or undershoot. Temperature control is therefore inherently precise.
  • thermocouple is soldered directly to the block so as to provide close temperature measurement and control . Any other temperature measurement device may be used as long as it does not introduce significant sensor lag.
  • the temperature distribution around the surface of the block is dependent on the evenness of heating and the thermal conductivity of the block.
  • the thermal conductivity of silver is very high, and the distribution of heat energy around the block is dependent upon the distribution of the heating current. This may be regulated by varying the geometry of the multi-well block.
  • the large currents required may be easily produced and controlled since the block becomes part of a heavy secondary circuit of the transformer.
  • the cross- sectional area of the winding bars is made considerably larger than the cross-sectional area of the block so that significant heat generation only occurs in the block.
  • the current can be easily controlled in the primary winding (where the current is small), using thyristors, triacs or other devices.
  • the primary winding may be driven by a high frequency, switch mode, controllable power supply. This allows the same degree of control of the current induced in the secondary winding incorporating the block, but the high frequency allows the use of a more compact core in the transformer, and reduces inrush current surges when switching the current on and off.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analyzing Materials Using Thermal Means (AREA)
  • Vaporization, Distillation, Condensation, Sublimation, And Cold Traps (AREA)
  • Resistance Heating (AREA)
  • Sorption Type Refrigeration Machines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

A method and apparatus for heating specimens in wells of a metallic specimen carrier. The specimen carrier is heated by applying resistive heating directly to the carrier. An AC source and transformer may be used where the specimen carrier is in series with a single turn secondary winding of the transformer.

Description

" Heating "
The present invention relates to heating and more particularly to the thermal cycling of specimen carriers.
In many fields specimen carriers in the form of support blocks or platterns are used for various processes where small samples are thermally cycled.
A particular example is the Polymerase Chain Reaction method (often referred to as PCR) for replicating DNA samples. Such samples require rapid and accurate thermal cycling, and are typically placed in a multi-well block and cycled between several selected temperatures in a pre-set repeated cycle.
Previous methods of heating such specimen carriers have involved use of resistance wire coiled around the wells, use of Peltier effect devices or hot air methods. However such methods are difficult to control to the precision required, necessitate slow cycle times and can give rise to thermal over shoot.
The present invention solves this problem by applying direct electrical resistive heating to a metallic specimen carrier. Thus the invention provides a method of heating a specimen carrier in the form of a metallic sheet and applying a heating current to said sheet.
Preferably the metallic sheet will be of silver which has a high thermal and electrical conductivity. The sheet will generally have a thin section in the region of 0.3mm thickness, and may be in a form where a matrix of sample wells is incorporated in the sheet.
While the metallic sheet may be a solid sheet or block of silver (which may have cavities forming wells) an alternative is to use a metallised plastic tray (which may have impressed wells) , in which deposited metal forms a resistive heating element.
Another alternative is to electro form a thin metal tray (which again may have impressed wells), and to coat the metal with a bio-compatible polymer.
These measures enable intimate contact to be achieved between the metallic heating element and the bio¬ compatible sample receptacles. This gives greatly improved thermal performance in terms of temperature control and rate of change of temperature when the actual temperatures of the reagents in the wells is measured.
The plastic trays are conventionally single use disposable items. The incorporation of the heating element into the plastic trays may increase their cost, but the reduction in cycling time for the PCR reaction more than compensates for any increased cost of the disposable item.
The bottom of the composite tray should be unobstructed when fan cooling is employed. If sub-ambient cooling is required at the end of the PCR cycles, either with a composite tray or a block, chilled liquid spray-cooling may be employed. The boiling point of the liquid should be below the low point of the PCR cycle so that liquid does not remain on the metal of the tray or block to impede heating. This also allows for the latent heat of evaporation of the liquid to increase the cooling effect.
The heating current may be an AC current supplied from the secondary winding of a transformer. This allows cycling control to be applied to the primary circuit of the transformer (higher voltage, lower current) in a convenient way without encountering problems which arise when operating with high current devices .
The transformer may comprise a toroidal core having an appropriate mains primary winding and a single bus bar looped through the core and connected in series with the metallic sheet to form a single turn secondary circuit.
An embodiment of the invention will now be described by way of example with reference to the accompanying diagrammatic drawings in which:
Figure 1 is a side elevation of a heating apparatus, and
Figure 2 is a plan view of the apparatus of Figure 1.
A metallic specimen carrier in the form of a multi-well block (1) measuring 110mm x 75mm and having 96 wells (2) disposed in a grid layout is formed in silver nominally 0.3mm thick. This is attached to bus bars (3) of substantial cross-sectional area. The bus bars loop once through a transformer (toroidal or square), core (4). The core (4) has a primary winding (5) appropriate for the mains voltage employed. The transformer primary current is controlled using a triac device (6). The triac device receives current from an AC source and is controlled by a temperature control circuit (7) which uses a fine wire thermocouple (8) soldered to a central underside region of the block to sense the temperature of the block. The temperature control circuitry may be operated manually or by a personal computer (9). Cooling of the block is by means of a fan (10) mounted under the block, blowing ambient air over the protruding well forms (2), the air being directed by the enclosure in which the block is mounted. The fan is controlled by the same temperature control circuitry that drives the heater triac .
The measured performance of the example apparatus gives rates of change of temperature in excess of 6 degrees per second and over/under shoots of less than 0.25 degrees within the typical PCR working range of 50-100 degrees.
The described examples use a silver block with cavities, but metalised plastic tray inserts, or electro formed thin metal trays, as previously described, may also be used.
The system as described has several important advantages . 1.1 The block is heated directly with no requirement for heat transfer from an attached heat source. This is very efficient and taken together with the very low specific heat capacity of silver allows very rapid temperature changes.
1.2 Direct heating means that there is no thermal lag at all. Temperature control functions are immediate so that the block may be cycled in temperature with little or no over or undershoot. Temperature control is therefore inherently precise.
1.3 Since there are no obstructions or thermal barriers attached to the block, simple forced-air cooling of the back of the block (which may be shaped to increase its surface area), provides rapid and controllable cooling.
1.4 The fine wire thermocouple is soldered directly to the block so as to provide close temperature measurement and control . Any other temperature measurement device may be used as long as it does not introduce significant sensor lag.
1.5 The temperature distribution around the surface of the block is dependent on the evenness of heating and the thermal conductivity of the block. The thermal conductivity of silver is very high, and the distribution of heat energy around the block is dependent upon the distribution of the heating current. This may be regulated by varying the geometry of the multi-well block.
The large currents required may be easily produced and controlled since the block becomes part of a heavy secondary circuit of the transformer. The cross- sectional area of the winding bars is made considerably larger than the cross-sectional area of the block so that significant heat generation only occurs in the block. The current can be easily controlled in the primary winding (where the current is small), using thyristors, triacs or other devices. Alternatively, the primary winding may be driven by a high frequency, switch mode, controllable power supply. This allows the same degree of control of the current induced in the secondary winding incorporating the block, but the high frequency allows the use of a more compact core in the transformer, and reduces inrush current surges when switching the current on and off.

Claims

CLAIMS ;
1. A method of heating a specimen carrier of the kind comprising a plurality of specimen sites such as wells, which carrier is in the form of a metallic sheet and the method comprising applying a current to said sheet so as to provide resistive heating of said sheet so as to heat specimens carried by said carrier.
2. A method according to Claim 1 in which the heating is applied as an alternating current providing resistive heating, and is controlled to provide repeated cycles of heating.
3. A method according to any preceding claim in which said metallic sheet is a solid block of silver.
4. A method according to any preceding claim in which said sheet is a metallised plastic tray.
5. A method according to any preceding claim in which said sheet is an electro-formed thin metal tray.
6. A method according to any preceding claim in which said metallic sheet includes a plurality of wells to contain a plurality of specimens.
7. Apparatus for carrying out the method of any preceding claim comprising a specimen carrier of the kind carrying a plurality of specimen sites such as wells, which carrier is in the form of a metallic electrically conductive sheet, power supply means, and a transformer having a primary winding connected to said power supply means, and a secondary winding directly connected to said conductive sheet.
8. Apparatus according to claim 7 in which said secondary winding is a single turn winding.
9. Apparatus according to any of Claims 7 or 8, comprising temperature control means connected to regulate flow of heating current through said secondary winding at a rate which maintains a controlled heating temperature within said specimen carrier.
10. Apparatus according to Claim 9 comprises fan cooling means arranged to direct cooling air to a rear side of said specimen carrier and operatively connected to said temperature control means .
11. Apparatus according to any of Claims 7 to 10 in which said metallic sheet is a solid block of silver.
12. Apparatus according to any of Claims 7 to 10 in which said metallic sheet is a metallised plastic tray.
13. Apparatus according to any of Claims 7 to 10 in which said metallic sheet is an electro-formed thin metal tray.
PCT/GB1997/000195 1996-01-25 1997-01-23 Heating WO1997026993A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
DE69703381T DE69703381T2 (en) 1996-01-25 1997-01-23 HEATING A SPECIMEN CARRIER
AT97901166T ATE197129T1 (en) 1996-01-25 1997-01-23 HEATING OF A SAMPLE CARRIER
DK97901166T DK0876218T3 (en) 1996-01-25 1997-01-23 Heating a sample carrier
JP52665897A JP3705606B2 (en) 1996-01-25 1997-01-23 heating
AU14510/97A AU1451097A (en) 1996-01-25 1997-01-23 Heating
EP97901166A EP0876218B1 (en) 1996-01-25 1997-01-23 Heating of a specimen carrier
CA002244178A CA2244178C (en) 1996-01-25 1997-01-23 Method of heating a specimen carrier
GR20000402296T GR3034671T3 (en) 1996-01-25 2000-10-26 Heating

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9601471.7 1996-01-25
GBGB9601471.7A GB9601471D0 (en) 1996-01-25 1996-01-25 Heating
GBGB9603717.1A GB9603717D0 (en) 1996-02-22 1996-02-22 Heating
GB9603717.1 1996-02-22

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/815,696 Continuation-In-Part US6635492B2 (en) 1996-01-25 2001-03-23 Heating specimen carriers

Publications (1)

Publication Number Publication Date
WO1997026993A1 true WO1997026993A1 (en) 1997-07-31

Family

ID=26308521

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/000195 WO1997026993A1 (en) 1996-01-25 1997-01-23 Heating

Country Status (11)

Country Link
EP (1) EP0876218B1 (en)
JP (1) JP3705606B2 (en)
AT (1) ATE197129T1 (en)
AU (1) AU1451097A (en)
CA (1) CA2244178C (en)
DE (1) DE69703381T2 (en)
DK (1) DK0876218T3 (en)
ES (1) ES2151241T3 (en)
GR (1) GR3034671T3 (en)
PT (1) PT876218E (en)
WO (1) WO1997026993A1 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000025920A1 (en) * 1998-10-29 2000-05-11 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Ultrathin-walled multiwell plate for heat block thermocycling
WO2001072424A1 (en) * 2000-03-24 2001-10-04 Bjs Company Ltd. Heating specimen carriers
DE10028323A1 (en) * 2000-06-07 2001-12-20 Evotec Biosystems Ag Microtiter plate or chip for containing biological or chemical samples, comprises a flat plastic sheet containing wells, a supporting core made from high melting point material surrounding each well being embedded in plastic sheet
DE20101734U1 (en) * 2001-02-01 2002-06-13 EVOTEC BioSystems AG, 22525 Hamburg sample carrier
GB2370112A (en) * 2000-12-15 2002-06-19 Hybaid Ltd Multiwell sample plates
WO2003000419A3 (en) * 2001-06-21 2003-05-08 Hybaid Ltd Sample well plate
WO2003022439A3 (en) * 2001-09-10 2003-05-30 Bjs Company Ltd Zone heating of specimen carriers
US6635492B2 (en) 1996-01-25 2003-10-21 Bjs Company Ltd. Heating specimen carriers
WO2012080746A1 (en) 2010-12-17 2012-06-21 Ian Gunter Methods and systems for fast pcr heating
WO2013175218A1 (en) 2012-05-24 2013-11-28 Bjs Ip Limited Clamp for fast pcr heating
WO2014140596A1 (en) 2013-03-15 2014-09-18 Bjs Ip Limited Fast pcr heating

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4574328B2 (en) * 2004-11-10 2010-11-04 キヤノン株式会社 Sample temperature controller
US7632464B2 (en) * 2006-06-29 2009-12-15 Bio-Rad Laboratories, Inc. Low-mass sample block with rapid response to temperature change

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0058428A2 (en) * 1981-02-18 1982-08-25 Eisai Co., Ltd. An enzyme immuno-assay for simultaneously measuring a plurality of samples and test vessel for carrying out this method
EP0603411A1 (en) * 1992-07-01 1994-06-29 KATOH, Keiichi Ceramic heating/cooling device
WO1995001559A2 (en) * 1993-07-02 1995-01-12 Evotec Biosystems Gmbh Sample holder and its use
US5410130A (en) * 1994-04-20 1995-04-25 Ericomp, Inc. Heating and temperature cycling

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0058428A2 (en) * 1981-02-18 1982-08-25 Eisai Co., Ltd. An enzyme immuno-assay for simultaneously measuring a plurality of samples and test vessel for carrying out this method
EP0603411A1 (en) * 1992-07-01 1994-06-29 KATOH, Keiichi Ceramic heating/cooling device
WO1995001559A2 (en) * 1993-07-02 1995-01-12 Evotec Biosystems Gmbh Sample holder and its use
US5410130A (en) * 1994-04-20 1995-04-25 Ericomp, Inc. Heating and temperature cycling

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6635492B2 (en) 1996-01-25 2003-10-21 Bjs Company Ltd. Heating specimen carriers
EP1000661A1 (en) * 1998-10-29 2000-05-17 Hans-Knöll-Institut für Naturstoff-Forschung e.v. Ultrathin-walled multiwell plate for heat block thermocycling
WO2000025920A1 (en) * 1998-10-29 2000-05-11 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Ultrathin-walled multiwell plate for heat block thermocycling
JP4965786B2 (en) * 2000-03-24 2012-07-04 ビージェイエス カンパニー リミテッド Method and apparatus for heating specimen carrier
WO2001072424A1 (en) * 2000-03-24 2001-10-04 Bjs Company Ltd. Heating specimen carriers
JP2003527873A (en) * 2000-03-24 2003-09-24 ビージェイエス カンパニー リミテッド Method and apparatus for heating specimen carrier
DE10028323A1 (en) * 2000-06-07 2001-12-20 Evotec Biosystems Ag Microtiter plate or chip for containing biological or chemical samples, comprises a flat plastic sheet containing wells, a supporting core made from high melting point material surrounding each well being embedded in plastic sheet
GB2370112A (en) * 2000-12-15 2002-06-19 Hybaid Ltd Multiwell sample plates
DE20101734U1 (en) * 2001-02-01 2002-06-13 EVOTEC BioSystems AG, 22525 Hamburg sample carrier
WO2003000419A3 (en) * 2001-06-21 2003-05-08 Hybaid Ltd Sample well plate
US6949725B2 (en) 2001-09-10 2005-09-27 Ian Alan Gunter Zone heating of specimen carriers
WO2003022439A3 (en) * 2001-09-10 2003-05-30 Bjs Company Ltd Zone heating of specimen carriers
WO2012080746A1 (en) 2010-12-17 2012-06-21 Ian Gunter Methods and systems for fast pcr heating
US9168530B2 (en) 2010-12-17 2015-10-27 Bjs Ip Ltd. Methods and systems for fast PCR heating
WO2013175218A1 (en) 2012-05-24 2013-11-28 Bjs Ip Limited Clamp for fast pcr heating
US9579657B2 (en) 2012-05-24 2017-02-28 Bjs Ip Ltd Clamp for fast PCR heating
US10315198B2 (en) 2012-05-24 2019-06-11 Bjs Ip Ltd Clamp for fast PCR heating
WO2014140596A1 (en) 2013-03-15 2014-09-18 Bjs Ip Limited Fast pcr heating

Also Published As

Publication number Publication date
EP0876218B1 (en) 2000-10-25
EP0876218A1 (en) 1998-11-11
ES2151241T3 (en) 2000-12-16
DE69703381T2 (en) 2001-04-12
CA2244178C (en) 2008-10-14
DE69703381D1 (en) 2000-11-30
DK0876218T3 (en) 2001-01-08
JP3705606B2 (en) 2005-10-12
JP2000505889A (en) 2000-05-16
AU1451097A (en) 1997-08-20
CA2244178A1 (en) 1997-07-31
GR3034671T3 (en) 2001-01-31
PT876218E (en) 2001-03-30
ATE197129T1 (en) 2000-11-15

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