AU646406B2 - Laser tissue welding with dye enhanced solders - Google Patents

Description

WO 91/04073 PCT/US90/05125 646406 Laser Tissue Welding With Dye Enhanced Solders BACKGROUND OF THE INVENTION The present invention relates to tissue welding with energy absorbing dye and more particularly to tissue welding using a source of energy, such as a laser, with energy absorbing dye and a soldering agent such as fibrinogen applied to the welding site.

Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Traditionally, physicians have used suture materials to appose tissue until the natural healing properties of the body allowed scar formation over the wound. Welding of tissue with lasers removes the necessity for the use of sutures, thus allowing primary repair without the creation of excessive scar and foreign body reaction. Successful tissue welding using-a laser as an energy source has been reported in several surgical fields 2, 9, 21, 22, 23).

Techniques generally rely upon precise primary apposition of the tissue edges and avoidance of thermal injury to this and other surrounding tissue. The possibility of early SU STITUTE SHEET WO 91/04073 PCT/US90/05125 2 disruption due to the diminished strength of welds immediately after creation has prevented more widespread use of this technology.

Laser tissue welding techniques have been employed which can produce an anastomosis which causes less turbulence avoids foreign body reaction and granuloma formation, and is faster healing than a conventional sutured anastomosis However, in the event of weld failure, edges of tissue to be approximated are often thermally damaged and cannot be exposed again to laser energy. In addition, although laser welds usually have sufficient strength to withstand systolic blood pressure, they initially are weaker than their sutured counterparts and are more prone to rupture 4).

Fibritogen in conjunction with additional fibrin glue activators, including thrombin and calcium, has been used to enhance microvascular weld strength Laser energy appears to induce protein glue cross-linking and thus creates stronger welds. However, successful application of the laser for tissue welding especially in conjunction with a biologic bonding material requires a high threshold power, typically above 7.6 watts/cm 2 and often results in significant injury to surrounding tissue. It would therefore be advantageous to develop a technique of tissue welding which requires lower laser energy output and which reduces the amount of collateral thermal injury, while still achieving a strong if not stronger weld than obtained using suturing techniques.

SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 3 SUMMARY OF THE INVENTION In accordance with the present invention, a method of welding a tissue of a subject is provided comprising contacting the tissue to be welded with an energy absorbing material and a tissue soldering agent in sufficient quantities to permit a tissue weld to be formed, and imparting energy absorbable by the energy absorbing material to the energy absorbing material in an amount sufficient and under conditions so as to cause heating of the tissue and welding of the tissue. Preferably, the imparting of energy comprises imparting electromagnetic energy such as laser energy to the energy absorbing material. Also preferably, the contacting of the tissue comprises contacting the tissue with a mixture of energy absorbing material such as dye and a tissue soldering agent such as fibrinogen. The laser energy may be from a laser consisting of a phased array of gallium-alluminum-arsenide semiconductor diodes, or an argon ion laser, for example. When the laser is the phased array of diodes, the energy absorbing material preferably comprises indocyanine green (ICG) dye, whereas when the laser is an argon ion laser, the dye is preferably fluorescein isothiocyanate (FITC). The soldering agent may comprise huxtan or animal fibrinogen, but can be any polypeptide. The soldering agent may be a serum protein, albumin, or fibronectin.

The present invention also provides a tissue soldering composition for use in welding tissue comprising an energy SUBSTITUTE SHEET WO 91/04073 PCT/US0/05125 4 absorbing material and a soldering agent in relative amounts sufficient to effect welding of tissue when applied to tissue and when a sufficient amount of energy is imparted to the composition.

By providing an exogenous dye at the welding site, a lower threshold power can be used, and consequently a smaller laser. Also, using a smaller laser will result in less tissue damage. Moreover, the use of a dye enhances selective delivery of energy to target tissue, thus reducing the amount of collateral thermal injury to exposed tissue in general. Mixing a soldering agent such as fibrinogen with the exogenous dye prior to application to a welding site further enhances the weld strength of the resultant weld.

Moreover, use of soldering agents such as fibrinogen requires neither precise apposition of tissue edges nor excessive heating of host tissue, thus allowing greater room for error during the weld. If initial attempts with the soldering agent fail reapplication of the soldering agent allows repeated trials without destroying the host tissue.

Bonds created with soldering agents are structurally stronger than primary lasce welds. The fact that the addition of the laser energy enhancing dye to the soldering agent, such as fibrinogen, allow selective heating of the solde without injury to underlying host tissue is especially important for tissue which does not contain endogenous pigments. Use of exogenous compounds can photosensitize the solders on the tissue to allow enhanced SUBSTITUTE SHEET WO 91/04073 PCT/U590/05125 5 and selective laser energy uptake. As in photodynamic therapy or diagnostic fluorescence imaging (10,11,12) a laser and dye combination can be chosen such that the laser's output frequency matches an absorption peak of the dye closely, thereby providing efficient and target-specific laser energy delivery.

SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 6 DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, a method of bonding or welding a tissue in a subject is provided comprising contacting the tissue to be welded with an energy absorbing material and a tissue soldering agent in sufficient quantities to permit a tissue weld to be formed, and imparting energy absorbable by the energy absorbing material to the energy absorbing material in an amount sufficient and under conditions so as to cause heating of the tissue and welding of the tissue. Preferably, energy is imparted by directing an electromagnetic energy source such as a laser to the energy absorbing material. The contacting of the tissue comprises preferably contacting the tissue with a mixture of energy absorbing material and a tissue soldering agent. The energy absorbing material is preferably a dye. The dye preferably has a dominant absorption peak at a particular wavelength and the laser preferably has a dominant wavelength corresponding to that dominant absorption peak of the dye. The energy source also preferably emits energy having a frequency component in the visible spectrum to thereby aid in directing the energy source toward the tissue to be welded. One preferred laser/dye combination is wherein the laser comprises a phased array of gallium-aluminum-arsenide semiconductor diodes having a dominant wavelength of about 808 nm and wherein the dye comprises indocyanine green (ICG). Another SUBSTITUTE SHEET WO 91/'04073 PCT/US90/05125 7 preferred laser/dye combination is wherein the laser is an argon ion laser having a dominant wavelength of about 488 nm and wherein the dye is fluorescein isothiocyanate (FITC).

The soldering agent may be human or animal fibrinogen, but may be any polypeptide. The soldering agent may comprise a fibrin glue comprising fibrinogen combined with additional fibrin glue activators such as thrombin, with or without calcium, as set forth in U.S. Patent No. 4,627,879, incorporated by reference herein. The tissue soldering agent may comprise a cryoprecipitated suspension comprising fibrinogen and Factor XIII and a fibrin glue activator comprising thrombin. The soldering agent may be for example serum protein, albumin or fibronectin. The laser may be a continuous laser or a discontinuous pulse or chopped laser.

In accordance with another aspect of the invention, a tissue soldering composition is provided for use in welding tissue comprising an energy absorbing material and a soldering agent in relative amounts sufficient to effect welding of tissue when applied to tissue and when a sufficient amount of energy is imparted to the composition. The composition may be dye and fibrinogen. The dye may be indocyanine green (ICG) or fluorescein isothiocyanate (FITC), for example.

The soldering agent may be human or animal fibrinogen, but can be any polypeptide, and may be for example serum protein, albumin or fibronectin. The composition may also comprise thrombin and calcium.

SUBSTITUTE SH1EET WG ?1/04073 PCT/US90/05125 8 MATERIALS AND METHODS A. INTRODUCTION Two laser welding studies according to the invention will be des--ibed below. In the first study, the energy source was a laser diode having a dominant wavelength at about 808 nm and the energy absorbing material was idocyanine green dye (ICG) dye hvIng a maximum absorption at about 805 nm. In the second study, the energy source was an argon ion laser having a dominant wavelength within the range of 488-514 nm and the energy absorbing material was fluorescein isothiocyanate (FITC) dye having a maximum absorption at a wavelength corresponding closely to the argon laser dominant wavelength.

B. LASER DIODE/ICG DYE 1. Fibrinogen Preparation Human fresh frozen plasma was transferred to test tubes and placed in a freezer at -800 C for at least 12 hours. The tubes were then thawed at 4°0 and centrifuged at 1000 X G for 15 minutes. The supernatant was decanted leaving the precipitate, fibrinogen. The viscosity of this solution allowed easy manipulation with forceps. Fibrinogen can be stored at -800 C for up to 1 year For further details regarding the preparation of fibrinogen, attention is directed to U.S. Patent No. 4,627,879 which is incorporated by reference herein.

SUBSTITUTE SnFF WVO 91/04073., 0IPC/US90/05125 9 2. Dye Preparation Indocyanine green dye (Becton Dickinson, Baltimore, Maryland) was mixed with normal saline to make a saturated solution. The half life of the solution was approximately hours. The dye has a maximum absorption at a wavelength of about 805 nm with an absorption coefficient 2 x 105 M-cm 3. Laser System Welding was performed with a System 7200 diode laser module coupled into a hand held focusing optic (Spectra-Physics, Mountain View, CA). This laser consists of a phased array of gallium-aluminum-arsenide semiconductor diodes. The major wavelength output of the laser diode is 808 1 nm.

Additional bands of laser energy occur in the visible red spectrum and allow the operator to visualize the spot size of the laser during creation of the weld.

The foc'ssing optic allowed for a greater working distance, providing greater visibility of the anastomotic area. With the addition of the focusing optic the beam diameter is 2 mm at a distance of 4 cm. Laser power was measured at the output of the focusing optic with a Model 201 laser power meter (Coherent Science Division, Palo Alto, CA) and was maintained at 4.8 watts/cm 2 The distance between the focusing lens system optic and welded tissue was maintained at approxiately 4 cm during laser application.

4. In vivo Bursting Pressure Measurements Newly created welds were evaluated in New Zealand White rabbits anesthetized with ketamine (35 mg/kg) and xylazine SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 10 mg/kg). After celiotomy a 16 gauge ridged, duck bill valve vessel cannula (DLP Inc., Grand Rapids, MI) was inserted into the distal abdominal aorta and secured in place with 3-0 silk sutures for measurement of intravascular pressure on a Datascope 2000 pressure monitor (Datascope Corporation, Paramus, NJ). Next, the abdominal aorta was cross-clamped and incised distal to the clamp to create a 7 mm longitudinal aortotomy. Small branch vessels were clamped as needed to prevent backflow bleeding. A polypropylene suture was placed at each end of the incision and retracted in order to hold the tissue in place during welding. The field was irrigated free of all blood with sterile saline. Fine forceps were used to appose the edges of the aortotomy as needed.

For non-soldered welds (n 11), the ICG was applied topically to the welding site through a 27 gauge needle immediately prior to laser exposure. For the laser soldered welds (n 11), a drop of ICG.was mixed with human fibrinogen and then placed with forceps onto the welding site prior to laser exposure. Only the minimum amount of ICG necessary to stain the fibrinogen was used; this allowed precise placement of the dye on the vessel surface.

Aortotomies were welded using a power density of 4.8 watts/cm 2 for the diode laser. When desiccation of the fibrin glue or tissue was noted, the laser energy was redirected to the next target site. The end point of welding was judged to have taken place when coaptation of the edges was observed and the tissue desiccation and SUBSTITUTE SHEET WO 91 /04073 PCr/US90/05125 11 retraction had just begun, or either the dye or the fibrin glue has clotted on the surface and no anastomotic defects were visible.

At the completion of the weld, the branch vessels were opened and backbleeding into the aorta was permitted for 3 minutes. Next, these branch vessels were clamped again and an infusion pump was used to advance normal saline through the previously placed angiocatheter into the distal aorta and the pressure at the time of weld rupture was recorded.

In an additional five rabbits, the above protocol was followed exactly using the ICG-fibrinogen combination; however, just prior to measurement of bursting pressure, 25,000 IU of urokinase (Abbott, Chicago, IL) in 0.5cc normal saline was infused into the aorta for five minutes. This dose of urokinase was the amount needed to completely lyse of clotted rabbit blood within 5 minutes Bursting pressure was then measured.

Control Survival Studies Twenty two New Zealand White rabbits were prepared as above with a pair of 7 mm longitudinal abdominal aortotomies in each animal. The superior incision was closed with running polypropylene suture and the inferior incision was Welded closed. Each-animal underwent ICG enhanced fibrinogen soldering. A threshold power density of 4.8 watts/cm 2 was used for the diode laser. Following the welds, the inferior aortic cross-clamp was removed. If the welded anastomosis leaked, the hole was repaired with SUBSTITUTE SHEET WO 91/04073 PCr/US90/05125 12 additional fibrinogen and laser exposure. After a leak-free anastomosis was created, the proximal aortic clamp was removed and the aorta was examined for evidence of thrombosis or leakage. At intervals between 1 and 90 days, the animals were sacrificed and weld sites harvested.

Welded tissue was preserved in a 10% formaldehyde solution buffered to normal pH for HPS, Masson trichrome, or muscle specific actin staining. In addition, selected specimens were preserved in a 0.5% gl~teraldehyde and 2% paraformaldehyde in 0.05M cacodylate solution (pH 7.38) for transmission and scanning electron microscopic evaluation.

6. Results a. In vivo Bursting Pressure Measurements The ICG dye enhanced welds required a laser application time of approximately one minute to close a 7 mm incision.

Occasionally retreatment was required for residual weld defects noted after release of the cross-clamps.

Immediately after welding, the bursting pressures of welds created without fibrin glue (262 29 mm Hg, n 11) were significantly less than welds soldered with the fibrin glue (330 75 mm Hg, n 11)(p less than Suture closures did not burst at the highest pressures measured, up to 300 mm Hg; however, leakage through needle holes of the suture and small defects in the repair were noted at pressures above 145 mm Hg. The fibrinogen welds exposed to urokinase were not significantly weaker than nonperfused fibrinogen welds (290 74 mm Hg, n SUBSTITUTE

SHEET

WO 9104073 PCIT/US90/05125 13 b. In vivo ICG Welded Aorta and Suture Control Survival Studies Among the animals followed for 1 to 90 days, no evidence of anastoinotic rupture, thrombosis, or aneurysms occurred in any of the sutured or fibrinogen-ICG solders. Immediately after welding, the applied fibrinogen formed a smooth flow surface onto which.red blood cells and platelets adhered.

By the second day after surgery, sheets of fibrin were identified crossing the aortotomy weld and the thrombotic response had not increased significantly more than that seen from the specimens harvested immediately after soldering.

Within 10 days a new intimal surface had regenerated over the fibrin. By the second week after operation, histiocytes were seen engorgad with fibrinogen as the process of resorption started. By the third week after operation, the internal and external elastic lamina remained disrupted, and the initially acellular fibrin was densely packed with proliferating cells which had eosinophilic cytoplasms characteristic of myofibroblasts extending from just below the intimal surface to the adventitia. This identification was confirmed using a Smooth Muscle Specific Actin stain (Enzo Diagnostics, New York, specific for myofibroblasts. These cells appear to have proliferated and laid the matrii for the rapid regeneration of tissue at the weld site. No foreign body reaction was note,. As the healing response continued, fibrin deposited closest to the intimal surface appeared to be resorbed by histiocytes faster than JBSTITUTE

SHEET

WO 91/04073 Pcr/US90/05125 14 fibrin laying on the adventitial surface. By 90 days post operatively, the fibrin had been almost completely removed and only normal appearing myointimal 1ells remained between the external elastic lamina and the intima. Outside the external elastic lamina, occasional foci of fibrin surrounded by histiocytes remained; however, in general only fibrous tissue laid down after the welding was present. No foreign body reaction was evident.

The suture closed aortotomies demonstrated ischemic changes at the point of suture pressure on the aortic tissue. The suture closures did not appear to induce thrombus adherence to any greater extent than the laser welded closures; however, tissue reaction to the sutures resulted in a fibrotic build-up which distorted the intimal flow surface by post-operative day 25. Comparable areas of soldered aorta at the same time after operation demonstrated complete re-endothelialization, with the aortotomy site completely covered with intimal cells, and it was difficult to distinguish this site from the surrounding normal rabbit lumen.

C. ARGON ION LASER/FITC DYE 1. Dye Preparation Ten milligrams of isomer I FITC (Sigma Inc. St. Louis, MO), 10 ml 0.9% sodium chloride irrigation solution at pH 4.5-7 (Abbott Laboratories, Chicago, IL) and 1 ml of 8.4% sodium bicarbonate at pH 8.4 (Fisher Scientific, Fair Lawn, N.J.) were mixed, resulting in an orange solution. Two to threo SUBSTITUTE

SHEET

WO 91/04073 PCI/US90/05125 15 drops (0.1 cc) of this solution were added topically to the weld site prior to exposure of the tissue to the argon laser. In control animals, a similar quantity of 0.9% sodium chloride was applied topically to the weld site.

2. Laser System Welding was performed with a System 1000 surgical argon laser with a 1 mm spot size applicator (Coherent Medical Division, Palo Alto, CA). The laser was operated in the multiline mode. At low laser power the dominant wavelength is 488 nm.

The applicator was modified by mounting a focusing optic on the output end of the applicator (Becton Dickenson FACS IV part No. 53.10006-01, Becton Dickenson Immuno-cytometry Systems, Mountain View, CA). The focusing optic allowed for a greater working distance, providing greater visibility of the anastomotic area. With the addition of the focusing optic the beam diameter is 1.5 mm diameter at 6 cm. Laser power was measured at the output of the focusing optic with a Model 201 laser power meter (Coherent Science Division, Palo Alto, CA). The distance between the focusing optic and the welded tissue was maintained at approximately 6 cm during laser application.

3. In Vitro Dose-Response Curve The adventitia of freshly harvested rabbit aorta was exposed to increasing output laser power ranging from 100 to 500 mW (power fluence range, 5.66 to 12.04 watts/cm For one group of exposures, a single drop of FITC was topically SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 16 applied to the specimen through a 27 gauge needle. In a second group, no FITC was used. The tissue was exposed to the laser system described above for intervals of 15, 60, 90, and 120 sec, and gross tissue changes were noted.

The specimens were placed in 10% formalin and stained with hematoxylin, phloxin, and safranin (HPS).

4. In Vivo Bursting Pressure measurements Ten New Zealand white rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg). After celiotomy a 16 ga\uge ridged, duck bill valve vessel cannula (DLP Inc., Grand Rapids, MI) was inserted into the distal abdominal aorta and secured in place with 3-0 silk sutures for measurement of intravascular pressure on a Datascope 2000 pressure monitor (Datascope Corporation, Paramus, Next, the abdominal aorta was cross-clamped and incised distal to the clamp to create a 7mm longitudinal aortotomy.

A 6-0 poLypropylene suture was placed at each 6nd of the incision and retracted to hold the tissue in place during welding. The field was irrigated free of all blood with sterile saline. Pine forceps were used to oppose the edges of the aortotomy as needed. For the FITC welds, the dye was topically applied to the welding site through a 27 gauge needle. Only the minimum amount of FITC necessary to stain the welded area was used. Although it was impossible to control precisely the lateral spread of the dye once applied to the tissues, one to two drops of dye were usually adequate. Aortotomies were welded using power densities of SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 17 3,8 watts/cm 2 with FITC and 7.6 watts/cm 2 without FITC.

These power densities represented the minimum power levels at which reliable welds could be consistently obtained.

When desiccation of the aorta was noted, the laser energy was redirected to the next target site. The end point of welding was judged to have taken place when coaptation of the edges was observed and the tissue desiccation and retraction had just begun.

At the completion of the weld, an infusion pump was used to advance normal saline through arterial pressure tubing and the previously placed angiocatheter into the distal aorta.

To minimize the rnumber of animals sacrificed, we were able to perform two to three welds and bursting pressure determinations on the aorta of each animal by progressively moving the aortic <Cross clamp distally after each bursting pressure determination.

S. In Vivo welded Aorta and Suture Control Survival Studies Twenty-five additional New Zealand White rabbits were prepared as above with a pair of 7 mm longitudinal abdominal aortotomies in each animal. The superior incision was closed with running 6-0 polyproylene suture, and the inferior incision was welded closed. Eighteen animals underwent F'IC-enhanced welding? seven rabbits underwent laser welding without addition of FITC. A threshold power density of 3.8 watts/cm 2 was used for the non-FITC welds.

Following the welds, the inferior aortic cross-clamp was removed. If the welded anastomosis leaked, the hole was SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 18 repaired by additional laser exposure. After a leak-free anastomosis was made, the proximal aortic clamp was removed and the aorta was examined for evidence of thrombosis or leakage. At intervals between 1 and 75 days, the animals were sacrificed and weld sites harvested. Velded tissue was preserved in a 10% formaldehyde solution buffered to normal pH for PHS or Masson trichrome staining.

6. Results a. In Vitro Dose-Response Curve For FITC stained in vitro rabbit aorta, the threshold for tissue blanching was 15 sec of 100 mW exposure. As the time of exposure and laser power density increased, the tissue acquireda reddish hue followed by furth6e desiccation, contraction, and eventual charring at approximately 30 sec of 300 mW exposure. No holes were cre&ted until 90 sec of exposure to 400 mW laser energy. In the unstained aorta, no tissue effects were observed until 15 sec of 300 mW laser energy was applied. The initial tissue desiccation rapidly progressed to charring, and by 90 see of exposure to this power, a hole had been created. The progression of tissue changes in the FITC-stained group in comparison to the unstained controls is demonstrated in Table 1 reproduced at the end of this specification. As compared with FITC-stained aorta, three times greater laser energy was needed to induce the same tissue changes on nonstained specimens. Also, the progression of tissue changes from initial tissue effects of the laser to charring and eventual hole creation was more gradual in tissue stained with FITC.

SUBSTITUTE

SHEET

WO 91/04073 PCT/7US9O/05125 19 b. In Vivo Bursting Pressure Measurements Btth FITC and non-FITC welds required laser application time of approximately 90 sec to close a 7 mm incision.

'Occasionally a retreatment was required for residual weld defects noted after release of the cross-clamps. Mean bursting prissures immediately after weld creation were 164 mm Hg for FITC group compared with 147 mmHg for the non-FITC-welded group. Suture closures did not burst at the highest pressure used (300 mm Hg); however, leakage through needle holes of the suture and small defects in the repair were noted at pressures above 125 mm Hg.

c. In Vivo FITC-Welded Aorta and Suture Control Survival Studies Among the animals followed for 1 to 75 days, no evidence of anastomotic rupture, thrombosis, or aneurysms occurred in any of the suture or FITC welds. The weld site of FITC-enhanced argon ion laser welds was characterized by myointimal proliferation with a regenerated intim surface.

Collagen deposit was noted at the weld site. One of the welds performed without FITC developed a small anastomotic dilation by 2 weeks postoperatively. No other abnormalities were noted in this group. Histologically, carbonaceous debris was usually visible on the surface tissue directly exposed to the argon energy without FITC. After treatment without FITC, the argon ion laser induced a 50-100 um region of amorphous collagen surrounding the point of welding, A 300 um region of tissue manifested the loss of nuclear detail and cellular integrity characteristic of thermally SUBSTITUTE

SHEET

'WO 91/040873 PCT/US 0/05125 20 damaged areas. No deposition of carbonaceous debris was evident on the FITC weld specimens. The suture closed aortotomies demonstrated ischemic changes at the point of suture pressure on the aortic tissue. These closures did not appear to induce thrombus adherence to any greater extent that the laser-welded closures. By 2 weeks after laser welding, a new endothelial surface had repopulated the intimal surface. Similar changes had occurred in the suture-closed aortotomies, although patches of denuded endothelium often remained.

D. DISCUSSION OF STUDIES All three elements in the described welding system i.e. an energy source, an energy absorbing material and a tissue soldering agent meet the requirements of simplicity and safety for application in the clinical setting. Idocyanine green, fibrin glue, and diode lasers have been proven safe in clirAcal application (8,14,15). Indocyanine green has a large absorption coefficient at the 808 nm output of the diode laser. The diode laser is an inexpensively manufactured solid state semiconducting electronic device with heretofore limited medical applications due to low energy output capacities (less than 400 mW). When combined with topically applied indocyanine green dye, lasers at this wavelength can cause substantial and rapid tissue effects.

The diode.laser, for example, because of its low cost, small size, simple power requirements and durability, will replace ,ther solid state lasers and popular gas discharge lasers in SUBSTITUTE SHEET WO 91/0/4013, PCr/US90/05125 21 many applications. Finally, fibrin glue has been used as a hemostatic agent without significant complications. In elective surgery, fibrinogen can be harvested from the subject. Addition of thrombin and calcium have been required to achieve hemostasis intra-operatively (16,17).

We were unable to create durable anastomoses using this clotting system. Following laser exposure, however, a durable fibrin coagulum is achieved even without the use of thrombin and calcium. Laser "spot welding" may be useful for repair of anastomotic leaks as well as during creation of sutured anastomoses.

We have observed that argon ion laser welding of biliary tissue is facilitated by the bile and blood staining of the tissues Here we employ an exogenous compound to photosensitize vascular tissue for laser welding. Under identical experimental conditions, the use of FITC enabled welds to be formed at one-half the threshold laser power density as compared with non-FITC welds.

The thr(ashold laser power density for welding without FITC is it agreement with previous reports By using FITC, this threshold can be decreased. The lower energy required for welding minimized thermal damage to surrounding, healthy tissue. Additionally, because of the topical application of FITC, the energy deposition is localized to the area of the weld. The circumscribed zone of coagulation injury was apparent on the adventitial surface of the vessel without underlying thermal damage, SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 22 The specificity of energy deposition is further enhanced by use of argon ion laser power outputs below 0.2 watts. In this range, an all-lines mode argon laser energy output is predominantly a 488 nm wavelength. This wavelength closely corresponds to the maximum absorption for FITC. At higher powers other wavelengths contribute to the total output and can be nonspecifically absorbed by surrounding tissue, leading to thermal injury.

Blood also appeared to act as an engaging endogenous chromophore. Welds were easier to create and appeared more durable if the aortic tissue was blood stained. In the bursting strength experiments, total elimination of blood from the field by copious irrigation prior to welding made welding without FITC much more difficult. With use of FITC, the lack of blood in the field was much less important. The FITC dye also helps to improve visualization of the weld site because of its bright green fluorescence as observed through standard argon ion laser safety goggles used to filter out laser light (Laserguard, Glendale, CA).

Extrinsic dye to enhance selective delivery of laser energy to tissue has been used in photoradiation of cancers, ophthalmologic vessel ablation, and diagnostic fluorescence imaging (0O, 11, 12, 24). Use of exogenous dyes on target tissues may prove superior to use of endogenous chromophores by allowing greater control of local chromophore concentration and energy delivery.

SUBSTITUTE SHEET WO 91/04073,7 I'/UIS90/0S1 23 A variety of mechanisms may be responsible for fibrinogen soldering. The inability of urokinase to prevent welding with fibrin glue suggests that enhanced clotting of rabbit blood with exogenous fibrinogen is not the dominant mechanism of soldering. More likely, heated fibrinogen undergoes covalent crosslinking independent of the coagulation cascade and is transformed into an insoluble, durable biologic glue holding together edges of the host tissue. The amorphous coagulum is rapidly infiltrated with myofibroblasts and covered with host endothelial cells.

Fibrin may be stimulating this rapid regeneration of vessel wall As the fibrin glue is resorbed by histiocytes, myointimal proliferation continues until a new media has been created. The internal and external elastic lamina, on the other hand, are not reconstituted. As has been reported by others (19,20), no foreign body reaction is evident. In effect, the fibrin solder consists of a biodegradable scaffold upon which the divided vessel heals. Use of dye enhanced fibrLn( n also reduces injury to tissue surrounding the weld and may allow more rapid proliferation of myofibroblasts and the deposition of the extracellular uwatrix necessary for regeneration of the vascular tissue at the welding site, The foreign body response to suture material (19) and the irregular flow surface caused by the sutures are avoided. Other advantages include superior bursting strength, less red blood cell death and less plaque adherence to the intimal vessel wall. As our techniques SUBSTITUTE SHEET WO 91/04073 lCr/US90/05125 24 have improved, the laser soldering method has also proven faster than sutured and conventional laser weld anastomoses, especially in smaller sized vessels.

Despite theoretical advantages, laser tissue welding has been hindered by technical obstacles and an inability to achieve consistent bonding. Use of dye enhanced protein solders obviates these difficulties and make clinical applications of laser welding technology feasible.

In the case of the J08 nm diode laser energy output, the output wavelength lies within the "optical window" of the vessel wall thus failing to show any tissue effects even at the highest energy outputs available (9.6 watts/cm2). In this way, despite elevation of fibrinogen temperature during welding, the underlying vessel wall is not damaged. The solder-dye mix also provides a convenient means to apply the dye in a controllable fashion.

While two different laser/dye combinations have been described, namely an 808 nm wavelength near infrared diode laser with indocyanine green (ICG) dye, and an argon ion laser with fluorescein isothiocyanate (FITC) dye, other energy sources and energy absorbing materials may be used.

The energy source need not be a laser, but could be any type of energy source such as for example an X-ray source, radiowave source, microwave source or non-coherent visible light source. The energy absorbing material need no£ be a dye, but could be any energy absorbing substance in any form which is used to absorb energy from energy sources. As in SUBSTITUTE SHEET WO 91/04073 PCr/US90/05125 25 photodynamic therapy or diagnostic flourescence imaging, a laser and dye combination can be chosen such that the laser's output wavelength matches the absorption peak of the dye, thereby providing efficient and target-specific laser energy delivery.

In the case oi the 808 un diode laser the output wavelength lies within the "optical window" of the vessel wall. That is, the laser fails to show any tissue effects even at the highest power outputs available (9.6 wtts/cm In this way despite elevation of fibrinogen temperature during welding, the underlying vessel wall is minimally damaged. The soldaring agent-dye mix also provides a convenient means to apply the dye in a controlled manner.

Other dye/laser combinations which have been successfully tested are FITCdextran/argon ion laser, methylene blue, acridine orange, or rose bengal/dye laser, and Kodak Q-switch II/Nd:Yag (1064 nm) laser. Other possible dye/laser combinations include, for example, hematoporphyrin derivative/argon ion laser and tetramethyl rhodamine isothiocyanate/krypton ion laser.

It is preferable to use a dye that is water soluble, such as the FITC dye disclorvd above. The dye and energy absorbing material are preferably mixed before application to the tissuet otherwise the dye will tend to run freely to areas which are not desired, such as inside an anastomosis.

The soldering agent may be any substance which after exposure to the energy source above, especially in SUSTITUTE SHFFT WO 91/04073 PCT/US90/0S125 26 conjunction with one of the energy absorbing materials, is transformed into a biological glue which will hold tissue together. The solders may be synthetic or naturally occurring, biodegradeable or permanent, and may require coibination with solvents and gels in order to acquire the appropriate consistency for the welding process. In effect, the solder constitutes a biodegradable scaffold upon which the divided tissue heals. Use of dye enhanced solders also reduces injury to tissue surrounding the weld and may allow more rapid proliferation of myofibroblasts and the deposition of the extracellular matrix necessary for regeneration of the vascular tissue at the weld site. The foreign body response to suture material and the irregular flow surface caused by the sutures are avoided.

The soldering agent may have as a base composition human or animal proteins, such as bovine proteins. Serum proteins such as albumin or fibronectin may also be used. The tissue to be welded may already havea natural soldering agent such as collagen or elastin. These and other additives may change the viscosity of the overall soldering agent composition and make it easier to apply.

One may also wish to add one or more drugs to the energy absorbing material/soldering agent mixture so that the mixture may also serve as a slow release drag delivery system. One such drug may be a growth factor to improve wound and tissue healing.

SUBSTITI1TF S.FFT WO 91/04073., PCT/US90/05125 27 The present invention provides new means of apposing tissue to allow more natural healing. The techniques developed are quicker and in some instances safer than sutured repair.

The potential applications of the present invention include: primary tissue soldering, especially in vascular, colon, fallopian tube, tracheal, esophagus and neural anastomoses, repair of sutured anastomoses when leakage occurs, especially in cardiovascular surgery when the protein's clotting mechanism is disrupted, management of oozing surfaces, for example injured liver, renal, or splenic tissue following trauma, repair of organs and endothelial layers within said organs, such as a colon lining, and secondarily to strengthen a sutured area. Additional uses for the invention will occur to those skilled in the art and will become more widespread once the present technique becomes more widely available.

The method according to the present invention can be practiced on any living subject, human or animal. The preferred subject is a human being undergoing surgical operations.

The relative quantities of the energy absorbing material and soldering agent Which contact the tissue should be sufficient to the effect welding of the tissue. It is possible that the tissue already has a natural soldering agent present so that such agent need not be added exogenously.

SUBSTITUTE SHEET WO 91/04073 PCT/US90/05125 28 Contacting the tissue with a soldering agent thus includes presence of a soldering agent endogenously and/or adding a soldering agent exogenously.

Similarly, the energy imparted to the energy absorbing material should be sufficient to effect heating of the tissue to result in welding. For example, when the energy is being imparted using a laser, factors such as the temperature and size of the tissue area to be welded will vary, and therefore the amount of energy absorbing material, soldering material and energy level of the laser required should necessarily take these factors into account.

As used herein, the term welding includes welding in the conventional sense, as well as bonding and soldering.

Although a preferred embodiment of the invention has been disclosed and described herein, it should be understood that the present invention is in no sense limited thereby and its scope is to be determined only by that of the appended claims.

SUBSTITUTE

SHEET

WO091/04073 SO0 2 PCT/US90/05125 29 TABLE 1. Dose-Response Curve* Seconds Milliwatts 15 30 60 90 120 'Argon laser with FITC 100 1 2 2 2 2 200 2 2 2 2 2 300 3 4 4 4 4 400 4 4 4 5 500 5 5 5 5 Argon laser without FITC 100 0 0 0 0 0 200 0 0 0 0 0 300 3 3 4 5 400 5 5 5 5 500 5 5 S 5 no observable effect; 1, blanching; 2, red; 3, curling of tissue after dessicationt pergoration 4, carbonaceous deposition; SUBSTITUTE SHEET WO 91/04073 WO 9104073PCT/US90/05125

REFERENCES

1. White RA, Kopchok G, Donayre C, White G, Lyons R, Fujitani R, Klein SR, Uitto J: Argon laser-welded arteriovenous anastomoses. J Vasc Surg 1987; 6:447-53.

2. Oz MC, Bass LS, Popp HW, Chuck RS, Johnson JPI Trokel SL, Treat VIR: In vitro comparison of THC:YAG and argon ion lasers for welding of biliary tissue. Lasers Surg Med 1989; 9:248-253.

3. Basu St 'Wang S, Robertazzi R# Grubbs PE, Jacobowitz I, Rose D, Acinapura AJ, Cunningham JN Jr: in vitro bursting strength studies of laser-welded tissue and comparison with conventional anastomosis. J Vasc Surg 1988;7:420-2.

4. Quigley MR, Bailes JE, Kwaan HC, Cerullo UI Brown JT, Fitzsim~mons J: Comparison of bursting 61rength between suture-and laser-anastomosed vessels. Microsurgery 1985;6:- 229-232.

Grubbs PE Jr., Wang S. Marini C, Basu St Rose DM# Cunningham Jt* Enhancement of CO 2 laser microvascular anastomoses by fibrin glue. i Surg Res 1988145t112-119.

6. Oz MC, Chuck RS, Johnson JPt Parangi S, Bass US, bNowygrod R, Treat MR. Photosensitizer enhanced diode laser vascular welding. Surg Forum 1989 (in press).

SUBSTITUTE SHEET WO 91/04073 W091/4073PcT'/US9O/05125 31- 7. Gestring GFp Lerner R: Autologous fibrinogen for tissue adhesion, hemostasis, and embolization. Vasc Surg 1983;17:- 294-304.

8. Pang Do Sciabassi RJ, Horton JA: Lysis of intraventricular blood clot with urokinase in a canita model: part 2. Neurosurg 19.:547, 1986,0 9. Poppas DP, Schlossberg SM, Richmond IL, Gilbert ';)Ap Devine CJ: Laser welding in urethral surgery: Improvled results with a protein solder. J Urology 1988;139:415-417.

Doucaherty TJ, Wieshaupt KR, Boyle MG Photoradiation therapy of human tumors. In Regan JD* Parrish JA eds. "The Science of Photomedicine." New York: Plenum Press, 1982: 625-638.

11. Prout GR, Lin CW, Benson Nseyo UO,. Daly JJ, Griffen PP, Rinsey J# Tian M* Lao Y, Mian Y, Chen X, Ren F, Qiao S: Photodynamic therapy with hematoporphyrin derivative in the treatment of superficial transitio~nal cell. carcinoma of the bladd1er. NEJM 198701l7%1251-1255.

12. Wolfe DR: Fluorescein angiography basic science and engineer.ng. Ophthalmology 1986;93:1617-1620.

SUBSTITUTE SHEET WO 91/04073 IC/US9O/05125 32 13. Anderson RR, Parrish JA: Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation. Science 1983;220:524-7.

14. Benchimol A, Li Y, Legler JF, Diizond EG: Rapidly repeated determinations of the cardiac output with the indicator-dilution technic. Am J Card 1964;13, 790-797.

15. Puliafito CA, Deutsch TF, Boll J, To K: Semiconductor laser endophotocoagulation of the retina. Arch Opthalmol 19871105:424-427.

16. Dregdale, A, Rose EA, Jeevanadum V, Reemtsma K, Bowman POO Maim JR: Preparation of fibrin glue from single-donor fresh-frozen plasma. Surgery 1985797:750-754.

17. Wolner E. Fibrin gluing in cardiovascular surgery: Thorac Cardiovasc Surgeon 1982;30:236-237.

lP,. Powanda MCI Moyer ED: Plasma proteins and wound healing. Surg Gynecol obstre, 19817153:749-755.

19. Pearl RM# Wustrack KO, Harbury C, Rubenstein E# Kaplan EN: microvasculat anastomosis using a blood product sealantadhesive. Surg Gyr~ecol Obstat 19771144:227-231.

SUBSTITUTE SHEET W0O91/04073 PC.T/US9O/OS 125 33 Baker JW, Spotnitz WD, Matthew TL, Fechner RE, Nolan SPt Mediastinal Fibrini glue! hemostatic effect and tissue response in calves. Ann Thorac Surg 1989;47:450-452.

.921. White IRAI Kopchok G, Dornayre C, Abergel RP, Lyons R, Klein SRO Dwyer RM, Uitto J: Comparison of Laser-welded and sutured arteriotomies. Arch Surg 1986; 121:1133-1135.

2 2. White RA: Technical frontiers for the vascular surgeon: laser anastomotic welding and angioscropy-a'~sisted intraluminal instrumentation. J. vaec. Durg. 1987; :673- 680.

23. White FV: Laser tissue repair with the C0 2 laser. Proc SPIE 1989; 1066 (in press).

24. ohnishi Y, Yamana Y0 Ishibashi. T: Effect of argon laser photoradiation on monkey retina treated with hematoporphyrin derivative-FlUoreecein angiographic and light microscopic study. Jpn U OphthalmoJ. 1987; 31:160-179.

Oz MC, Chuck RS, Johnson JP, Parangi St Bass IS, Nowygrod R, Treat MR: Indocyanine Green Dye Enhanced Vascular Welding With the New Infrared Diode Laser, Vasc.

Surg. 1989 (in press) 26. Chuck RS, Oz MCI Delohoxy TM, Johnson JP, Bass LS, Nowygrod R, Treat MR: Dye-Enhanced Laser Tissue welding, Lasers Surg. Med. 1989 (in prs) SUBSTITUTE SHEET

Claims (6)

1. A method of welding a tissue of a subject comprising: contecting the tissue to be welded with an exogenous energy absorbing material having an absorption peak at a particular wavelength and which enhances energy absorption by the tissue at said wavelength relative to energy at other wavelengths, and an exogenous tissue soldering agent in sufficient quantities to permit a tiss'ue weld to be formed; and imparting energy having a characterising wavelength corresponding to the particular wavelength of the energy absorbing material and absorbable by the energy absorbing material to the energy absorbing material in an amount sufficient and under conditions so as to cause hou-ting of the tissue and welding of the tissue.

2. The method according to claim 1, wherein the imparting of energy comp'-ises imparting electromagnetic energy to the energy absorbing material.

3. The method according to claim 1, wherein the contacting of the tissue comprises contacting the tissue with a preformed mixture of energy absorbing material and a tissue soldering agent.

4. The method according to claim 1#,wherein the impTting of energy comprises imparting laser energy to the energy absorbing material. s. The method accordiry to claim 1, wherein the contacting of the tissue comprises contacting the tissue with a dye. 61 The method according to claim It, wherein the contacting of the tissue comprises contacting the tissue with a dye having a dominant absorption peak at a particular WO 91/04073' PCI7US90/05125,

35- wavelength a,;d wherein imparting of energy comprioes imparting laser energy having a dominant wavelength corresponding to the dominant absorption peak of the dye. 7. The method according to claim 1,/wherein the imparting of energy comprises imparting of energy having a frequency component in the visibl3 sprctrum to the energy absorbing material. 8. The method according to claim 1, wherein the imparting of energy comprises imparting energy having a dominant wavelength of about 808 nm to the energy absorbing material. 9. The method according to claim 8, wherein the contacting of the tissue comprises contacting the tissue with a mixture of indocyanine green (XCG) dye and a soldering agent. The method according to claim I, wherein the imparting of energy comprises imparting of energy having a dominant wavelength of about 488 nm to the energy absorbing material. 11. The method according to claim 10, wherein the contacting of *hhe tissue comprises contacting the tissue with a mixture of fluorescein isothiocyanate (FXTC) dye and a soldering agent. 12. The method according to claim I, wherein the contacting of the tissue comprises contacting the tissue with a soldering agent comprising human or animal fibrinogen. SUBSTITUTE SHEET WO 91/04073 Pcr/US90/05125 36 13. The method according to claim 1, wherein the contacting of the tissue comprises contacting the tissue with a soldering agent comprising fibrin glue which comprises fibrinogen combined with at least one fibrin glue activator. 14. The method according to claim 13, wherein the fibrin glue activator comprises thrombin, The method according to qlaim 13, wherein the fibrin glue activator comprises calcium. 16. The method according to claim i, wherein the contactner of the tissue comprises contacting the tissue with a tissue soldering agent comprising a cryoprecipitated asaspension which comprises fibrinogen and Factor X111, and a fibrin glue activator comprising thrombin. 17. The method according to claim I, wherein the contacting of the tissue comprises contacting the tissue with a soldering agent comprising a polypeptide. 18. The method according to claim 1, wherein the contacting of the tissue comprises contacting the tissue with a soldering agent comprising a serum protein. 19. The method according to claim It wherein the contacting of the tissue comprises contacting the tissue with a soldering agent comprising albumin. 20. The method according to claim I# wherein the contacting of the tissue comprises contacting the tissue with a voldering agent comprising fibronectin. SUBSTITUTE SHEET

37- 21. The method according to claim 1, wherein the imparting of energy comprises imparting laser energy continuously to the energy absorbing material. 22. The method according to claim 1, wherein the imparting of energy comprises imparting laser energy discontinuously to the energy absorbing material. 23. The method according to claim 1, wherein the contacting of tissue comprises contacting the tissue with an energy absorbing material and a tissue soldering agent applied into or on the surface of the tissue. 24. A tissue soldering composltion for use in welding tissue comprising an exogenous energy absorbing material having an absorption peak at a particular waivelength and which enhances energy absorption by the tissue at said wavelength relative to energy at other 'wavelengths, and an exogenc,,s tissue soldering agent in relative amounts sufficient to effect welding of the tisgue when applied to the tissue and when a sufficient amount of energy is imported t the composition. 2S. A tissue, soldering composition according to claim 24, comprising dye and fibrinogen. 26, A tissue soldering composition according to claim 24, wherein said energy absorbing material is indocyanina grean (ICG) dYe. 27. A tissue soldering composition according to claim 24, wherein said energy absorbing material is fluoroscain isothiocyanate (FXTC) dye. 28, A tissue soldering composition according to claim 24, wherein said soldering agent comprises human or animal fibrinogen. 14XW* I6" WO 91/04073,, PC/US90/OS125 38 29. A tissue soldering composition according to claim 24, wherein said soldering agent comprises a poiypeptide. A tissue solderP composition according to claim 24, wherein said soldering agent comprises a serum protein. 31. A tissue soldering composition according to claim 24, wherein said soldering agent comprises albumin. 32. A tissue soldering composition according to claim 24, wherein said soldering agent comprises fibronectin. 33. A tissue soldering composition according to claim 24, wherein said soldering agent comprises thrombin and calcium. SUBSTITUTE SHEET INTERNATIONAL SEARCH REPORT international Application NJo PGT/US9O/05125 I ICLASSIFICATION OF SUB.JECT M~ATTER (it several classn~iatin symbols apoly. ndicato all) According to Intern~tional Patent Classification lIPC) or to both National Classitcleaton and IPC IPC 5: A61N 5/06 A61B 19100; US: 128/_3A-1, 397, 398, 898; 606/8 It. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System I Classiication Symbols us 128/395, 397, 398, 898 Documentation Searched other than Minlimum Documentation to the Extent that loch Documents are Included in the Fields Searched 0 Ill. DOCUMENTS CONSIOERED TO BE RELEVANT 14 2a, j Citation of Diicument, 14 with indication, where appropriate, ol the relevant passage$ I I Relevant to Claim No. It X Lasers in surgery and medicine Vol. 9, Vol. May, 1989, 1,2,415,12J-14 Saur, "The first sutureless End-to-End Bowel 17,18,20,21,23- Anastcmosis t pages 70-73 25 28-30,32,33 X The Journal of Urology; Vol. 139, February 1988, 1-6112-20,231 Poppos et al, :Laser Welding in Urethane Surgery: 24, 28-33 Improved Results with a protein solder" pages 415-417 X Journal of Surgical Research, Vol. 45, Vol. July -16112-18I20, 1988, Grubbs, Jr. et al, "Enhancement of CO 2 Laser 2-25,28-30,32, Anastomosts by Fibrin Glue" pages 112-119 nd33 27 0 Spatial categories of cited documntst I$ document dooinng the goneral sate at the artoshich Is not considered to be of Paricular rolevanc* elqt document but Published on ot after the Intetnationil tlona data "L0 dotument which~ ty throw doubts an gtlatity clainfla) or which Is Cited to establish the publication date Wi another Citation Or other satiat 1 1411n (As speified) 00 document Wallrin; to an ao$ disclocure, use, exhibition or Other means 1101 document publshed Piliot to the International fing data but later than the priority data Claimed later document Psublished Alter the international filing date Or 01iority date and not In tonliet with the application but Cited 10 undetstaild the ptindipIeor atiliory undelyinig the Invention "X document of ourllcult relevant#., the claimed Invention Cannot be COnsidered novel Or Cannot be ctinedtred to inv~o An Inventive Step "Y document of particutar relevance., the claimed Invention Cannot be Considered to Involve an Inventive ti00 when the 00o1ument it Combined with One or More Other such docu- Moenti, Such Combination being obvious to A Pere*n Skilled li the A". 04*' document member ot the cuin patent family Otofthe Actual Complae 1theIltillonsachIot f a oo th I T U SE 1t IntenatI~nal SearthIngo Authority I jSgnatU1 of Auhtio Ifo ISA/US 1).qvid Sh~y r Form PCISA121O (stecocd cheat) May 1980) Inlternational Application No. PCT/US9IO5125 iFURTHER INFORMATION CONTINUED FROM THE SECOMS SH4ET I y I USIA 3,769,963 (Goldinan)06 November 1973 column 2 lines 10-32 USIA 4,774,339 (Haugland) 27 September 1988 see column 1, lines 51-65 USIA 4,805,623 (Jobsis) 21 February 1989 see column 23, lines 6-9 UJSIA 4,457,992 (Bhattacharjee) 03 July 1984 see column 5, lines 40-45 and column 6, lines 30-33 Journal of Vascular Surgery, Vol. 6, No. 5, November 1987, White et al, "Argon laser-welded arteriovenous Mrintn~n~qr1n m AA7-Aqq i-11,21-23,026, 27 ,6,10,11,21-23 27 5-9,26 5-9,26 P-9,10,11,21-23 26,27 V.Q[ OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UfUAFICNAIIE1I This International search report has not boen itutablI10W~ in respect of certain claims under Article 17(2) lot the following reasons: III Claim numbers *because they rolate to subject matter t not required to be searchod by this Authority, namelyz Claim numbers ,because they retlt to Osta of the International application that do not ceni.ly with the Prooocribed require. ments to such an extent that no meaningful International search can be Carried out I. SPIcifIlcly.: Claim numbers because ovy ore O~pandmnt clalrna not drafted In acoordarice wi tll sa,'v- and th~ird sentences of PCT Rule 0.4(a). vt.Q: OBSERVATIONS WHERE UNITY OF INVENTION 13 LACKINVn This International Searching Authority found multiple Inventions in this Infornational application as followst I.CQ As1 all required additional search fees wete timely paid by the applicant, this International searcl taport caoers all searchable claims of the Internatlonal application. 2,C] As only some of the required adlditional search foet were tmInd, paid by the applicant. this Intornational search report covers only those claims Of the International Application for which fest were Paid, specifically itlaimot 3.[3 No required additional search lees wea timely paid by the applicant. Consequsnily, this itiernationaltoarch report is tastited to the Invention first mentioned In the clainist it Is coveted by claim numbort A s Aal se aafhali claims9 c ould be s$earth ad with out 4 fott lttilfyil an a dditional fee, the International Statchn@l Authority d id n ot i Ivi oayn vt ot any additlonal tee D the additional search ltes were accompanied by applicant's lirstesv. 0 NO prtlzit accompanhied the payment of additional seitch fess. V&rr PCtmlotJt ouprw"n 61,1041 0 (t STANDARD ABSTRACT AND ABRIDGEMENT WORKSHEET P/01/005 PATENT A U -A Where space Is insufficient, us& and ACTION BY continue overleaf In space provided 21 Applifron No. Sra o LODGEMENT/ J ACCEPTANCE 464 01 24E. it different from earliest date at INID 32) EXAMINER 31 Priority Document No(s) 32 Date Of illIng 7 outrVCd (earliest first) [,S)Cutyod *4~44*4* ~j)Title (if title on application form unsuitable) 6iCognate SApplication Nos, SAdditional To Application No, Appli(.aton No D7 rief Summary Of Inventive Concept And Other Noteworthy Disclosures (or Identify a suitable extract In the space provided) *44 Extract Inventive Concept From Specification At Page -jo Lines -io. Claim No. Or Claims Explanatory* 1 A 1 1 Extract I IOn Pago -0 I Lines -op Drawing Sheet No. M.B.S.S. or S.S$r, No. I. AU-B31 ACTION BY tj. I. 56Related Art (in chronological ordor, most recent first) Publn cc Levell Patent No. App, No. If AU Class Marks if AUI I1 Brief Summary Of Inventive Con.~ept (or Identify a suitable extract In the space provided below) 6 11-6.. Extract Inventive Concept From Specification At Page -1 Lines Claim No, Or Clalims Lodged On Amended By Items 0 1HIVJ I Explanatory Extract On Page-Do. Linesp 4P Lodged on I /Amended -By Items-0 Drawin Sheet No. Lodged On Amended ByItems- Z~iuiL~i Overflow Space For Continuation From Overleaf Or Above (Identify entries by INID No.) I .6,166. 61 616,666 6, 6161*66*1 EXAMINER Examiner's FOR FORM FS Roquest Application ACCEPTANCE Spocilicalion Drawings